Beruflich Dokumente
Kultur Dokumente
Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Edited by
Arie J. Zuckerman
Royal Free and University College Medical School, London, UK
Jangu E. Banatvala
The Guy’s, King’s and St Thomas’ School of Medicine, London, UK
John R. Pattison
Royal Free and University College Medical School, London, UK
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Contents
Contributors
Preface
It is now thirteen years since the First Edition of molecular biological techniques and their applica-
Principles and Practice of Clinical Virology was tion to clinical problems, are reflected in a new
published. A comparison of the first and fourth chapter on ‘Diagnostic Approaches’, which presents
editions testifies to the rapid expansion in virology an overview of the value and limitations of estab-
during the intervening years, including major devel- lished and more recently developed techniques. Ad-
opments in technology, the application of these to vances in serological techniques as well as in virus
clinical practice and advances in the treatment of identification are emphasised. This chapter also in-
viral infections with an increasing number of anti- cludes an important section on assays for determin-
viral drugs. Indeed, such has been the progress in ing antiviral drug resistance.
the field of clinical virology even within the period Most of the chapters reflect advances in patient
between the third and fourth editions that we have management, including—where appropriate—
asked a number of new authors to contribute chap- antiviral chemotherapy. As expected, the chapters
ters. These include the chapters on rhinoviruses, on hepatitis viruses and human retroviruses have
viruses associated with acute diarrhoeal disease, been expanded considerably in the light of continu-
and human polyomaviruses. Of the remaining ing and rapid advances in these fields. Both chap-
chapters, virtually all have been revised substan- ters now include a component by authors with
tially. The chapter on the herpesviridae has been everyday practical experience in the management
expanded considerably, particularly in relation to and treatment of patients with these infections.
Human herpes viruses 6, 7 and 8; new authors have In comparison with the third edition, more col-
contributed to these sections. The chapter on hepa- oured plates have been included, which we hope our
titis viruses reflects the considerable expansion of readers will appreciate. As in prevous editions, we
information relating to hepatitis E and C as well as have attempted to limit the reference lists to key
the role of such newly recognised agents as GB and publications.
the new human virus, TTV.
Advances in diagnostic methods, particularly A. J. Zuckerman
J. E. Banatvala
J. R. Pattison
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Principles and Practice of Clinical Virology was first As expected, rapid developments continue to
published in 1987; a third edition within seven years occur in the field of human retroviruses, particular-
of the first attests to the continuing and rapid pro- ly the human immunodeficiency viruses, and this
gress in the field of clinical virology. chapter reflects the accumulation of new and im-
All the chapters have been revised and some com- portant data in this area. The chapter on human
pletely rewritten, reflecting the increasing know- prion disease has been almost entirely rewritten to
ledge of viruses or groups of viruses included in the reflect many of the substantial advances in prion
various chapters. Thus, the chapter on hepatitis research.
viruses now contains sections on hepatitis A and E Most of the authors stress the developments and
viruses as well as individual contributions on hepa- application of molecular biological techniques
titis B, D and C. The previous edition contained two which are leading not only to improved methods of
chapters on viral haemorrhagic fevers but this sec- diagnosis but also to an increased understanding of
tion now includes separate chapters for the viral pathogenesis.
flaviviruses, alphaviruses, Bunyaviridae, arena- Rather than burden readers with a large number
viruses and filoviruses. The chapters on arena- of references, we have aimed to include most of the
viruses and filoviruses have been contributed by key ones.
new authors. Finally, we are grateful for the helpful comments
New authors have also contributed the chapter which we received from many of our readers; some
on herpes simplex virus infections and the chapter of their suggestions have been incorporated into the
on the more newly-recognized herpes viruses now third edition.
includes a section on human herpes virus 7.
A. J. Zuckerman
J. E. Banatvala
J. R. Pattison
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
In the preface to the first edition of Principles and much more information on human immuno-
Practice of Clinical Virology we stated that it was deficiency viruses but also on HTLV-1 and -2. The
our intention, with new editions, to remain up to chapter on hepatitis has been separated into two
date as the subject advanced. Such is the pace of sections: the first on hepatitis A and the viruses
development in clinical virology that plans were causing non-A, non-B hepatitis, two of which have
laid for a new edition within a few months of the been identified as hepatitis C and E, and the second
first being published, and this second edition is on hepatitis B and D (delta agent).
appearing only two and a half years after the first. As before, we were aware when organizing the
Each chapter has been revised, many extensively, to book that no single arrangement is entirely satisfac-
take account of progress in the understanding of the tory. We have chosen to arrange the chapters on the
epidemiology, pathogenesis, diagnosis, manage- basis of individual viruses or groups of viruses. Gen-
ment and prevention of virus infections. Perhaps eral chapters on virus structure, taxonomy and
the greatest single recent contribution to the subject pathogenesis are not included, but the information
has been made by the application of molecular bio- on these aspects necessary for an understanding of
logical techniques, and each of our authors has the practice of clinical virology is included in the
highlighted the contribution of this rapidly deve- individual chapters.
loping discipline to clinical virology. Such factors as the likely discovery of yet new
Human herpesvirus 6 is now the subject of a new viruses, improvements in the rapidity and sensitiv-
chapter, and we have also added a new chapter on ity of diagnostic techniques, and the development of
haemorrhagic fevers to include much new informa- new vaccines, which are likely to involve recombi-
tion on their pathogenesis. As expected, the exten- nant techniques and progress in antiviral therapy,
sive accumulation of new information relating to will ensure that thought will be given to revision
infection by human retroviruses has resulted in ex- and preparation of another edition.
tensive updating of this chapter, not only to include
A. J. Zuckerman
J. E. Banatvala
J. R. Pattison
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
There has been a spectacular increase during the are concerned with rapid laboratory diagnosis lead-
last thirty years in our knowledge of virology. This ing to appropriate patient management which
has taken place to such an extent that virology can might involve specific therapy and/or infection con-
now be regarded as an umbrella term encompassing trol measures at a hospital, a national and occa-
a variety of distinct but related disciplines. There sionally at an international level.
are fundamental connections with biochemistry, In organizing the book we were aware that there
genetics and molecular biology, and each of these is no single arrangement that is entirely satisfactory.
aspects would be worth a treatise in itself. Clinical We have chosen to arrange the chapters on the
virology is that aspect which is concerned with the basis of individual viruses or groups of viruses. Gen-
cause, diagnosis, treatment and prevention of virus eral chapters on virus structure, taxonomy and
infections of man. It too has acquired a substantial pathogenesis are not included but the information
body of knowledge and accumulated experience on these aspects necessary for an understanding of
over the past thirty years and this book is intended the practice of clinical virology is included in the
to be an authoritative account of the present situ- individual chapters.
ation. Formerly virological diagnosis was time con- Clinical virology is a subject which continues to
suming, retrospective and rarely influenced the evolve. This is usually for one of two reasons, either
management of the patient. During the past 10—15 the need to apply new technology or the need to
years the picture has changed dramatically. Newly study new diseases or epidemiological situations.
recognized diseases such as AIDS and some haem- We have therefore invited authors who are special-
orrhagic fevers which have very serious conse- ist investigators into each of the viruses to contri-
quences for individuals and populations have been bute up-to-date, stimulating accounts of the prac-
shown to be due to viruses. In the clinical virology tice of clinical virology and provide a framework for
laboratory there has been a change in emphasis the assimilation of imminent advances. One chapter
towards rapid diagnostic techniques. Finally, effec- has already had to be significantly updated during
tive anti-viral chemotherapy is a reality at least for the time of preparation of the book and it is our
some virus infections and there has been an expan- intention, with new editions, to remain up-to-date
sion in the use of immunoprophylaxis. Thus the as the subject advances.
current principles and practice of clinical virology
A. J. Zuckerman
J. E. Banatvala
J. R. Pattison
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Plates
PLATE I xvii
Figure 2A.3M‘Cold sores’ at the pustular and crusting stages Figure 2A.4MRose Bengal staining of herpes simplex virus den-
dritic ulceration in a grafted cornea. (Kindly provided by R. E.
Bonshek and A. B. Tullo)
US28 TRS1
US27
UL4
US11
US9
US6 UL16
US3 TRL UL18
US
US2
IRS1
UL32
UL33
IRL HCMV UL36
Nov 97 UL37
PG & VE UL44
UL127 UL45
UL123 UL46
UL48
UL122 UL UL48/49
UL115 UL54 UL49
UL114 UL55
UL112/113 UL57 UL56
UL65
UL110 UL69
UL105 UL70
UL101/102 UL72
UL100 UL99 UL89 UL75
UL85/6 UL78
UL98 UL82 UL80A
UL97 UL94 UL84
UL83
Figure 2C.4M‘Proteins of CMV which have been mapped to date. UL = unique long region; US = unique short region; TR = terminal
repeat; IR = inverted repeat. The genome is linear within the virus but has been circularized for convenience. Open reading frames of known
function are coloured according to the following code: orange = transactivators; pink = DNA replication; green = capsid and/or assembly;
red = tegument; pale blue = envelope; dark green = immune evasion; dark blue = miscellaneous
PLATE II xviii
Figure 2C.7M‘The ways viruses can interfere with presentation of HLA–peptide complexes at the plasma membrane. RIB = ribosome; ER
= endoplasmic reticulum; V = virus-encoded protein; PRO = proteosome; PM = plasma membrane; TAP = transporter associated with anti-
gen presentation. Peptides derived from virus-infected cells are generated in the proteosome and actively transported by TAP into the lumen
of the ER. A ribosome is shown producing a protein with a signal peptide, which folds in the ER to produce the HLA class I chain. This
should normally associate with peptide and be transported to the plasma membrane. Misfolded HLA molecules can be re-exported from
the lumen of the ER back into the cytosol, where they are degraded by the proteosome. Virus-encoded genes interfere with this process as
follows: the proteins may be inherently insusceptible to proteosome digestion (EBNA of EBV) or may be modified to reduce their diges-
tion (pp65 acts on MIE protein of human CMV). Proteins may block the function of TAP (ICP47 of HSV; US6 of human CMV). Proteins
may bind mature class I molecules within the ER and so sequester them (E3–19K protein of adenoviruses; US3 protein of HCMV; m152
protein of MCMV). Two proteins of HCMV (US2 and US11) facilitate the re-export from the ER to the cytosol of mature HLA class I mol-
ecules. If all of these mechanisms are completely successful, the level of HLA display at the PM will be insufficient to prevent NK cells
recognizing the cell as being abnormal and so destroying it. Proteins encoded within HCMV (UL18) or MCMV (m144) are thus presented
at the plasma membrane to act as a decoy for NK cells
Figure 2D.7M‘Photomicrograph of a May–Grunwald–Giemsa-stained peripheral blood film from acute infectious mononucleosis. An
atypical mononuclear cell is illustrated. (x 1000)
PLATE III xix
Figure 2E.6M‘The classical roseola infantum rash in a 10-month-old child. (Reproduced with permission from Stammers, 1988)
Figure 2F.4M‘Expression of the latent ORF K12/T0.7 mRNA in KS spindle (endothelial tumour) cells by in situ hybridization, as report-
ed in Stürzl et al. (1997). The majority of spindle cells expresses this transcript which is indicative of latent infection (see text). KS = KS
tissue; P = surrounding tissue. Bottom panel: dark field. (Courtesy M. Stürzl, Munich)
PLATE IV xx
Figure 24.4M‘The slapped-cheek appearance commonly seen in children following B19 infection. (Reproduced with the permission of Dr
A. du Vivier, King’s College Hospital, London)
Figure 24.5M‘The maculopapular rash on the trunk of an adult infected with B19 virus
Figure 24.6M‘The lacy or reticular appearance of the rash on the limb of a girl infected with B19 virus. (Reproduced with permission from
Parvoviruses: Medical and Biological Aspects, Pattison, J. R. in Field’s Virology, 2nd edition 1990, p. 1769)
PLATE V xxi
Figure 25A.4M‘Global seroprevalence of (a) HTLV–I and (b) HTLV–II. Lettering refers to HTLV–II subtypes; numbering is percent of
population sample infected with HTLV–II; symbol size relates to size of population; IDVU = intravenous drug user; CSW = commercial
sex worker. (Courtesy of the Audiovisual Department at Imperial College School of Medicine, St Mary’s Hospital)
Figure 25A.7M‘Perivascular lymphocytic infiltration in the central nervous system. (Courtesy of Dr Margaret Esiri)
PLATE VI
Figure 26.1M‘Mutations of the prion protein gene. Mutations causing inherited human prion disease and polymorphisms in human, mouse and sheep. Above the line of the
human sequence are mutations that cause prion disease. Below the lines are polymorphisms, some but not all of which are known to influence the onset as well as the phe-
xxii
notype of disease. (Data compiled by Paul Barnborough and Fred E. Cohen; from Prasiner, S. B. (1997) Science, 278, 245–251)
PLATE VII
Figure 26.3M‘Structures of prion proteins. (A) NMR structure of Syrian hamster (SHa) recombinant (r) PrP(90–231). Presumably, the structure of the α-helical form of
rPrP(90–231) resembles that of PrPC. rPrP(90–231) is viewed from the interface where PrPSc is thought to bind to PrPC. The color scheme is: α-helices A (residues
144–157), B (172–193) and C (200–227) in pink; disulphide between Cys179 and Cys214 in yellow; conserved hydrophobic region composed of residues 113–126 in red;
loops in grey; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassing strand S2; the arrows span residues 129–131 and 161–163,
as these show a closer resemblance to ß sheet (James et al., 1997). (B) NMR structure of rPrP(90–231) is viewed from the interface where protein X is thought to bind to
PrPC. Protein X appears to bind to the side-chains of residues that form a discontinuous epitope; some amino acids are in the loop composed of residues 165–171 and at
the end of helix B (Gln168 and Gln172 with a low density van der Waals rendering), while others are on the surface of helix C (Thr215 and Gln219 with a high density van
der Waals rendering) (Kaneko et al., 1997). (C) Diagram showing the flexibility of the polypeptide chain form PrP(29–231 (Donne et al., 1997). The structure of the por-
tion of the protein representing residues 90–231 was taken from the coordinates of PrP(90–231) (James et al., 1997). The remainder of the sequence was hand-built for illus-
tration purposes only. The color scale corresponds to the heteronuclear {1H}-15N NOE data: red for the lowest (most negative) values, where the polypeptide is most flex-
ible, to blue for the highest (most positive) values in the most structured and rigid regions of the protein. (D) Plausible model for the tertiary structure of human PrPSc (Huang
et al., 1995). Color scheme is: S1 ß strands are 108–113 and 116–122 in red; S2 ß strands are 128–135 and 138–144 in green; α helices H3 (residues 178–191 and H4
(residues 202–218 in grey; loop) (residues 142–177 in yellow. Four residues implicated in the species barrier are shown in ball-and-stick form (Asn108, Met112, Met129,
xxiii
Ala133). (a) and (b) from Prusiner, S.B. et al. (1998) Cell, 93 337–348; (c) from Donne et al., 1997 (d) from Prusiner, S.B. (1997) Science, 278, 245–251)
PLATE VIII xxiv
Figure 26.4M‘Neuropathology of human prion diseases. Sporadic CJD is characterized by vacuolation of the neuropil of the grey matter,
by exuberant reactive astrocytic gliosis, the intensity of which is proportional to the degree of nerve cell loss, and rarely by PrP amyloid
plaque formation (not shown). The neuropathology of familial CJD is similar. GSS(P102L), as well as other inherited forms of GSS (not
shown) is characterized by numerous deposits of PrP amyloid throughout the CNS. The neuropathological features of vCJD are unique
among CJD cases because of the abundance of PrP amyloid plaques that are often surrounded by a halo of intense vacuolation (A) Sporadic
CJD, cerebral cortex stained with haematoxylin and eosin showing widespread spongiform degeneration. (B) Sporadic CJD, cerebral cor-
tex immunostained with anti-GFAP antibodies demonstrating the widespread reactive gliosis. (C) GSS, cerebellum with most of the GSS
plaques in the molecular layer (left 80% of micrograph), and many but not all are periodic acid–Schiff (PAS) reaction positive. Granule
cells and a single Purkinje cell are seen in the right 20% of the panel. (D) GSS, cerebellum at the same location as (C) with PrP immuno-
histochemistry after the hydrolytic autoclaving reveals more PrP plaques than seen with the PAS reaction (E) Variant CJD, cerebral cortex
stained with haematoxylin and eosin shows the plaque deposits uniquely located within vacuoles. With the histology, these amyloid
deposits have been referred to as ‘florid plaques’. (F) Variant CJD, cerebral cortex stained with PrP immunohistochemistry after hydrolyt-
ic autoclaving reveals numerous PrP plaques often occurring in clusters as well as minute PrP deposits surrounding many cortical neurons
and their proximal processes. (Bar in (a)–(c) and (e) = 50µm; Bar in (d) and (f) = 100 µm.) (Photomicrographs prepared by Stephen J.
DeArmond. Variant CJD specimens provided by James Ironside, Jeanne Bell and Robert Will. From Prusiner, S.B. et al. (in press) The
prion diseases. In Alzheimer Disease. Lippincolt Williams and Wilkins, Philadelphia)
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Diagnostic Approaches
Deenan Pillay
Birmingham Public Health Laboratory and University of Birmingham, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
2 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 1.1 Virus isolation quicker results by use of the many rapid techniques
that have been reported.
Advantages Disadvantages
Labelled
* Labelled
* Labelled
* Labelled
*
anti-IgG anti-IgM anti-IgM antibody
Serum
antibody *
Labelled
competitor
antibody
Rheumatoid Ag
factor
Ag Ag Ag Anti-IgM Antiviral Ab
Figure 1.2 ELISA formats: (a) indirect IgG assay; (b) indirect IgM assay; (c) rheumatoid factor interference in IgM assay (indirect); (d)
IgM capture assay; (e) competitive assay
DIAGNOSTIC APPROACHES 5
Table 1.4 Serology
Advantages Disadvantages
Specific IgG assays good indicator of prior infection Retrospective (e.g. rising CFT titres) CFTs insensitive,
Capture IgM assays good indicator of recent infection especially to assess previous infection
Allows retrospective diagnosis if no acute clinical specimen Cross reactivity and interference
obtained Insensitive for diagnosing some congenital infections (e.g.
Rapid CMV)
Automated Not appropriate for immunocompromised
Diagnosis of unculturable or poorly culturable viruses Spurious results possible following receipt of blood products
Can utilize non-invasive clinical samples e.g. saliva, urine
specificity ; (1 9 prevalence)
Negative predictive value Proportion of patients with
negative test results who are (1 9 sensitivity) ; prevalence ; specificity ; (1 9 prevalence)
correctly diagnosed
DIAGNOSTIC APPROACHES 11
where appropriate) or the clinical background. Asymptomatic Symptomatic
Thus, an expanded gold standard, including posi-
Time
As discussed above, the nature of viral disease has
had to be redefined in the light of qualitative and Figure 1.6 The relevance of PCR sensitivity for CMV diagnosis
quantitative molecular data. Increasingly, it is poss- following transplantation. A very sensitive assay for detection of
ible to detect the presence of infectious agents at low persistent/latent viruses, such as CMV, may not provide clini-
copy number, in the absence of symptoms. This cally useful information. This is because low levels of viraemia
may occur without causing disease. With regard to CMV, the
makes the interpretation of positive results prob- threshold for detection of virus in whole blood should have a
lematic, and requires close clinical—virological li- higher level
aison. Three approaches are possible:
1. Qualitative detection of viral genome at a site 1994). These persistent infections can be detected
which is normally virus free. A good example is by PCR in saliva and blood of many seropositive
the diagnosis of viral encephalitis, in which de- individuals, so that a qualitative PCR is not
tection of HSV, CMV, VZV or enterovirus helpful in diagnosis of symptoms specifically as-
genome is diagnostic (Jeffrey et al., 1997). Indeed, sociated with these infections. By contrast, appli-
it has been very difficult traditionally to propa- cation of a quantitative PCR to such clinical
gate herpesviruses in cell culture from cerebro- specimens demonstrates that febrile seizures are
spinal fluid (CSF) samples. It is unclear whether specifically associated with higher systemic viral
this is a reflection of a low level of virus, or loads (Clark et al., 1997). Quantitative molecular
whether there is a preponderance of disrupted, and antigenaemia data on CMV disease in trans-
non-infectious virus produced from brain tissue plant patients (Boeckh et al., 1996) and those
into the CSF. with the acquired immune deficiency syndrome
2. Qualitative detection of virus without an exquisite (AIDS) (Spector et al., 1998) also demonstrates
level of sensitivity. This is important for instances the usefulness of this approach. Clear diagnostic
in which low-level viraemia may occur in the definitions of diseases will depend on the assay
absence of disease, and which does not predict itself and the patient group concerned. Large
disease. An example is CMV infection following prospective studies are therefore required in each
transplantation. The sensitivity of a diagnostic case. Standardization within commercial assay
qualitative PCR must be chosen with care in systems will help in this respect.
order that results are predictive of disease (Fig-
ure 1.6).
3. Quantitative detection of virus at a level asso-
ciated with disease. For many persistent virus Prediction of Disease/Staging of
infections with transient or continual low level Infection
viraemia, the onset of disease is related to a
higher viral replication rate. This provides the Pre-emptive therapy entails the initiation of ther-
rationale to identify levels of viraemia, which are apy in those at highest risk of disease, and is an
predictive of disease. HHV-6 and HHV7 infec- approach which is used commonly for CMV infec-
tions can be used as examples. These herpes- tion in bone marrow and solid organ recipients. The
viruses are acquired commonly in early child- capacity of a positive laboratory test to predict
hood, and primary infections are associated with disease must be established by detailed prospective
erythema infectiosum and febrile seizures in a surveillance protocols, in order to generate positive,
small proportion of infants, with the majority of and negative predictive values (Table 1.8). Since the
infections remaining asymptomatic (Hall et al., natural history of viral infections (relationship be-
12 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Treatment Assay limit of
tween replication and disease) may be influenced by sensitivity
Advantages Disadvantages
mutations—that is, they cannot exclude different patient population is required, since they may ex-
mutations existing on separate genomes. Despite perience life-threatening viral infections, which may
the cost of these techniques, and the software and present atypically. Ongoing antiviral prophylactic
expertise required for data analysis, they are cur- therapy may also distort the nature and timing of
rently being introduced into routine practice for presentation. Table 1.10 highlights a typical set of
HIV drug resistance testing. protocols for the monitoring of transplant recipi-
ents. Precise protocols will depend on the patient
group concerned, availability of laboratory facilities
and, of course, budgetary constraints. Nevertheless,
RECOMMENDED INVESTIGATIONS in the context of high risk patients, such as those
receiving long-term chemotherapy or transplants,
It is the role of the clinical virologist to decide on the the overall cost of virological investigations will be
most appropriate investigations for any given clini- relatively small.
cal scenario. In the light of assay developments and
identification of new viruses, clinical protocols re-
quire constant updating. There is an increasing em- REFERENCES
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DIAGNOSTIC APPROACHES 17
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
The Herpesviridae
Graham M. Cleator and Paul E. Klapper
University of Manchester, Manchester, UK
The Herpesviridae are a large family of enveloped this latent state only a small subset of the viral genes
double-stranded DNA viruses. To date, more than are expressed. Reactivation, with expression of viral
150 members of the family have been identified. proteins and production of progeny virus, may oc-
These viruses have been found in almost every spe- cur at intervals to produce recurrent infection. This
cies in which they have been actively sought, includ- allows virus transmission to new, susceptible hosts.
ing both warm- and cold-blooded species, verte- Individual members are well adapted to their natu-
brate and invertebrates. Some species appear to be ral host and exhibit little or no ability to cause
the host of only one member of the Herpesviridae, cross-species infection. The viruses encode many
while others harbour multiple viruses (e.g. nine her- enzymes involved in nucleic acid metabolism, and
pesviruses have been found in cercopithecine spe- the replication and assembly of progeny virus oc-
cies, eight herpesviruses in equids, and eight human curs in the host cell nucleus. The host cell is ulti-
herpesviruses are known). mately lysed as a result of virus infection. Sev-eral
Viruses included in the family Herpesviridae have members of the Herpesviridae have strong
a double-stranded DNA genome of between 80 and oncogenic associations, most notable among the
150 million daltons molecular weight. The genome, human herpesviruses being the associations of
tightly wound in the form of a torus, is enclosed Epstein—Barr virus with nasopharyngeal carcin-
within an icosadeltahedral capsid (triangulation oma and Burkitt’s lymphoma, and Human herpes-
number T : 16), 100—110 nm in diameter. The cap- virus 8 with Kaposi’s sarcoma and abdominal cav-
sid is composed of 12 pentavalent capsomers (one at ity B cell lymphomas in patients with the acquired
each apex) and 150 hexavalent capsomers. Each immune deficiency syndrome (AIDS).
capsomer has a deep central indentation. An envel- The International Committee on the Taxonomy
ope surrounds the nucleocapsid from which numer- of Viruses divided the Herpesviridae family into
ous glycoprotein spikes project; these provide im- three subfamilies, the Alpha-, Beta- and Gammaher-
portant antigenic determinants of the individual pesvirinae (Roizman et al., 1995), broadly on the
members of the group. Between the nucleocapsid basis of differences in the biological properties of
and the envelope is an amorphous electron dense the various viruses. Inevitably, a classification sys-
area—the tegument. The overall diameter of the tem based upon biological properties creates
enveloped particle is between 120 and 300 nm, de- anomalies. It is acknowledged that this classifica-
pending upon the particular virus and the prepara- tion system is imprecise, being based upon artificial,
tion method utilized for examination by electron subjective criteria. It is conceivable that, in the fu-
microscopy. Following infection of their natural ture, classification will exploit more objective par-
host, the viruses establish a latent infection, in ameters, such as the conservation of genes and gene
which form they persist for the life of the host. In clusters and their relative position in the genome;
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
20 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 2.1 Human herpesviruses
Trivial name
and G;C Genome
Official name Subfamily Genus abbreviation Site of latency (moles %) (kb pairs)
Human herpesvirus 1 Alpha- Simplexvirus Herpes simplex Sensory nerve ganglia 68 152
virus type 1
(HSV-1)
Human herpesvirus 2 Alpha- Simplexvirus Herpes simplex Sensory nerve ganglia 69 152
virus type 2
(HSV-2)
Human herpesvirus 3 Alpha- Varicellovirus Varicella Sensory nerve ganglia 46 125
zoster virus
(HVZ)
Human herpesvirus 4 Gamma- Lymphocryptovirus Epstein—Barr Leucocytes, epithelial 57 172
virus (EBV) cells
Human herpesvirus 5 Beta- Cytomegalovirus Human B lymphocytes 60 229
cytomegalovirus
(CMV)
Human herpesvirus 6 Beta- Roseolovirus HHV-6 T lymphocytes (CD4 ; ), 43 160
epithelial cells
Human herpesvirus 7 Beta- — HHV-7 T lymphocytes (CD4 ; ) 35 145
Human herpesvirus 8 Gamma- Rhadinovirus Kaposi’s sarcoma- B lymphocytes, — 170
associated epithelial cells
herpesvirus
(KSHV)
the presence and distribution of nucleotides that are herpesvirus 3. The subfamily Betaherpesvirinae con-
subject to methylation; and genome sequence ar- tains three genera: Cytomegalovirus, exemplified by
rangements (Roizman, 1996). The last of these dif- Human herpesvirus 5 (Human cytomegalovirus);
ferences is particularly distinctive: six different cytomegalovirus); Muromegalovirus, exemplified by
structural forms of virion DNA have been identified Mouse cytomegalovirus; and Roseolovirus as exemp-
among members of the Herpesviridae (reviewed by lified by Human herpesvirus 6. The subfamily Gam-
Roizman, 1996), differing in the presence and loca- maherpesvirinae contains two genera: Lymphocryp-
tion of reiterations of terminal sequences of greater tovirus, exemplified by Human herpesvirus 4
than 100 bp. However, at the present time detailed (Epstein—Barr virus); and Rhadinovirus, exemplified
molecular biological data are available for only a by Ateline herpesvirus 2 and also Human herpesvirus
limited number of the herpesviruses. As more infor- 8.
mation on the genomes of a wider number of mem- As mentioned previously, the present classifica-
bers of the Herpesviridae emerges, a more precise tion, being based upon a summary of biological
classification system may evolve. properties, is somewhat inexact. Within the human
A formal binomial nomenclature is not applied herpesviruses an example is the problem surround-
presently n classification of the Herpesviridae. The ing the classification of Human herpesvirus 6 (HHV-
International Committee on the Taxonomy of Vi- 6). All HHV-6 strains analysed can be segregated
ruses has decided that herpesviruses will be de- into one of two subtypes. Type B is the major
scribed by serial number and the family or subfam- aetiologic agent of exanthem subitum (Dewhurst et
ily in which the natural host of the virus is classified al., 1993), while type A has no clear association with
(e.g. Bovine herpesvirus 1, Cercopithecine herpesvirus human disease. The subtypes differ in in vitro cell
1). Each subfamily is divided into a series of genera. tropism, reactivity with monoclonal antibodies and
The sub-family Alphaherpesvirinae contains two T cell clones, and in nucleotide sequence. Thus the
genera: Simplexvirus, exemplified by Human herpes- question arises: should these virus continue to be
virus 1; and Varicellovirus exemplified by Human classified under the same name or are they distinct
THE HERPESVIRIDAE 21
viruses? The classification scheme for the Herpes- monkey, the virus may cause an ascending myelitis
viridae will doubtless evolve to accommodate such that may progress to a fulminant demyelinating
anomalies in the future. encephalopathy.
There are presently eight members of the Herpes-
viridae known to infect humans (Table 2.1). The
viruses are distributed worldwide and no animal REFERENCES
reservoirs of infection are known for any of the
human herpesviruses. The official names, Human Dewhurst, S., McIntyre, K., Schnabel, K. et al. (1993) Human
herpesvirus 6 (HHV-6) variant B accounts for the majority of
herpesvirus 1—8, are, with the exception of Human symptomatic primary HHV-6 infections in a population of US
herpesvirus 6, 7 and possibly 8, seldom utilized and infants. Journal of Clinical Microbiology, 31, 416—418.
they are more usually known by their common Roizman, B. (1996) Herpesviridae. In Fields Virology (eds B.N.
names (Herpes simplex virus type 1 and type 2, Fields, D.M. Knipe, P.M. Howley et al.), pp 2221—2230. Lip-
Varicella zoster virus, Human cytomeglovirus, Ep- pincott-Raven, Philadelphia.
Roizman, B., Deroisiers, R.C., Fleckenstein, B. et al. (1995) Her-
stein—Barr virus). One further member of the Her- pesviridae: In: Sixth Report of the International Committee on
pesviridae—Cercopithecine herpesvirus 1, common Taxonomy of Viruses (eds F.A. Murphy, C.M. Fauquet,
name ‘B’ virus—is known to occasionally infect D.H.L. Bishop et al.). Archives of Virology suppl. 10, 586 pp.
humans. Transmitted by the bite of an infected
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
2A
Herpes Simplex
Graham M. Cleator and Paul E. Klapper
University of Manchester, Manchester, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
24 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
(Zhou et al., 1994, 1995). These studies suggest the
involvement of four proteins (VPs 5, 19c, 23 and 26)
aggregated as pentons and hexons, which are asso-
ciated with polypeptide triplexes to form the nuc-
leocapsid. The interior of the capsid is accessible via
transcapsomeric channels (‘tubes’) formed by the
polypeptide arrangement of the pentons, hexons
and holes at the base of each triplex. These openings
are postulated to play a role in the transport of
genomic DNA and scaffolding proteins during cap-
sid morphogenesis.
Between the capsid and envelope is the tegument.
An amorphous structure as visualized in thin-
section EM, but with a fibrous appearance on nega-
tive staining. The tegument contains at least 12
proteins that are believed to have important func-
Figure 2A.1 Enveloped virus particle, HSV-1 tions in the early stages of virus replication follow-
ing penetration of the host cell by the virion.
functional organization has been published (Roiz- The virus envelope has a typical trilaminar ap-
man and Sears, 1996). The genetic maps of HSV-1 pearance and is thought to be derived from patches
and HSV-2 are largely colinear but differ in restric- of host cell nuclear membrane modified by the in-
tion endonuclease cleavage sites and in the appar- sertion of virus glycoprotein spikes. Numerous such
ent sizes of viral proteins. Up to 70 polypeptides are spikes may be observed on the surface of the envel-
thought to be encoded by the genome, and 33 of ope, with lengths ranging from 8 to 24 nm (Stannard
these have been identified as structural (virion) pro- et al., 1987).
teins.
The viral capsid (100—110 nm in diameter) is a
closed shell in the form of an icosadeltahedron REPLICATION
(T : 16) with 162 capsomers arranged as 12 pen-
tamers (vertices) and 150 hexamers (face and edges). An appreciation of the replicative cycle of HSV is
Using high resolution cryo-EM and computer im- fundamental to an understanding of HSV disease
age reconstruction techniques the three-dimen- and its control.
sional structure of empty capsids of HSV-1 has been Initial attachment and penetration of the host cell
determined to a resolution of approximately 26 A is mediated via the glycoprotein spikes found on the
Figure 2A.2 The HSV genome. There are two covalently linked components designated ‘L’ (long) and ‘S’ (short). The four possible
isomeric forms of the virus genome are created through inversions of the L or the S sequences. The L and the S components each consist
of unique sequences bracketed by inverted repeats. The repeats of the L component are designated ab and ab; those of the S, ac and ca.
The number of ‘a’ sequence repeats at the L—S junction and the L terminus is variable. The structure of the ‘a’ sequence is highly
conserved but consists of a variable number of repeat elements; the detail shown is for HSV-1 strain F
HERPES SIMPLEX 25
Table 2A.1 HSV glycoproteins
gB U 27 Yes Forms a dimer, gB is essential for viral entry. Induces neutralizing antibody
*
gC U 44 No Involved in cell attachment
*
gD U6 Yes Required after attachment of virus to cell, to allow virus entry into the cell
gE U8 No Complexes with gE. Binds Fc portion of antibodies
gG U4 No Involved in entry, egress and spread from cell to cell
gH U 22 Yes Forms complex with gL (see below); role in entry, egress and cell—cell spread
*
gI U7 No gI and gE form a complex for transport to plasma membrane
gK U 53 Yes Required for efficient egress (viral exocytosis)
*
gL U 1 Yes Forms complex with gH which is required for transport of gH and gL to plasma
*
membrane and for viral entry mediated by gH
gM U 10 No
*
? Viral glycoproteins are named sequentially by letter as glycoprotein A, glycoprotein B, etc. The missing sequence letters (e.g. gA, gF) reflect earlier
misidentification of precursors of glycoprotein species as the actual virion glycoprotein. A further glycoprotein, gJ, has been predicted from DNA analyses
(open reading frame U 5) but has not been identified. there are a further two (the products of genes U 20 and U 34), and possibly more, non-glycosylated
* *
viral proteins inserted in the membrane.
@ Gene or transcriptional unit: U : unique short sequence of the genome; U : unique long sequence.
*
A Information from in vitro experimentation with HSV-1. All glycoproteins are essential in ‘wild-type’ virus. In cell culture only some functionality can be
dispensed with. Requirement of glycoprotein, non-essential in routine cell culture, can be demonstrated under specialized conditions, e.g. gC is essential for
attachment to the apical surface of polarized MDCK cells; gI is essential for basolateral spread of virus in polarized cells.
surface of the virus envelope. These glycoprotein host macromolecular metabolism is rapidly and ef-
spikes have been extensively investigated (Table ficiently shut down. Host DNA synthesis ceases,
2A.1) and the major antigenic differences between protein synthesis declines rapidly, ribosomal RNA
HSV-1 and HSV-2 relate to the type-specific epi- synthesis is reduced and the glycosylation of host
topes found on certain of these glycoproteins (Be- proteins ceases. There are at least 12 tegument pro-
rgstom and Trybala, 1996). teins, and while the function of several of these is not
Studies with deletion mutants, purified glyco- understood, they are all believed to act directly or
proteins, peptides and monoclonal antibodies show indirectly to produce early shut-off of host macro-
that in vitro, at least five of the 10 virion glyco- molecular synthesis and to contribute to the early
proteins are essential for virus entry to a host cell events of replication. The virion host shut-off pro-
and for egress from the infected cell. It has not been tein (VHS), for example, causes non-specific degra-
possible to define either a single virus glycoprotein dation of mRNA, leading to shut-off of macro-
or a single cellular receptor as the sole means of molecule synthesis after infection. Conversely, some
virus attachment. Experiments in which the genes tegument proteins are believed to facilitate attach-
encoding particular glyco-proteins were individ- ment of the nucleocapsid to nuclear pores and facili-
ually deleted failed to prevent each of the resulting tate release of viral DNA. An important tegument
virus constructs from attaching to non-polarized protein is -trans-induction factor (-TIF), which is
cells. It now seems likely that the virus is capable of essential to the initiation of transcription of viral
attachment to more than one cell surface receptor, DNA (see below); two further tegument proteins
as blockade of one of the known cell receptors, (the products of U 46 and U 47) are reported to
* *
heparan sulphate, does not prevent attachment of modulate its activity.
virus. The nucleocapsid is transported through the
Attachment to the host cell activates a process, cytoplasm to a nuclear pore. There is evidence from
mediated by the virion surface glycoproteins, that monoclonal antibody studies that the virus nuc-
induces fusion of the virion envelope and cell leocapsid proteins exhibit ‘molecular mimicry’ of
plasma membrane. This process probably involves host cell proteins, permitting the capsid to utilize
several, if not all, of the virion surface glycoproteins the cell cytoskeleton for transport to the nuclear
and is accomplished very rapidly. Fusion results in pore. On arrival at the nuclear pore, the capsid
the introduction of tegument proteins and viral degrades and the virus DNA is released into the
nucleocapsid into the cell cytoplasm. Subsequently nucleus of the cell, where it circularizes immediate-
26 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ly. To initiate transcription the circularized DNA escapes from the infected cell by transit through the
must bind a host cell protein (OCT-1) to a cis-acting cisternae of the rough endoplasmic reticulum of the
site; the tegument protein -TIF binds an addi- Golgi apparatus and cytoplasmic transport ves-
tional factor designated C1 (and possibly others). icles. Productive infection of a cell results in the
This -TIF—C1 complex then binds to the OCT- destruction of the host cell through the major struc-
1—DNA complex (Roizman and Sears, 1996). -TIF tural and biochemical changes induced by the viral
acts in trans to induce (or ‘immediate-early’) replication.
genes—the first set of viral genes to be transcribed.
Viral gene expression is coordinately regulated and
sequentially ordered in a cascade manner PATHOGENESIS
( ; ; ). The five gene products are regulatory
proteins; their expression is required for the produc-
tion of all subsequent polypeptide groups. The pro-
Terminology
teins serve to trans-activate and gene expression
A primary infection refers to the first experience of
and to turn-off both and early gene expression
HSV-1 or HSV-2 infection by a susceptible individ-
(other domains of the viral genome are transcribed
under ‘’ conditions including the ‘latency-asso- ual. If a person already infected with one type of
ciated transcript’ LAT-1, which will be discussed virus (for example HSV-1) becomes infected with
below). Viral DNA is transcribed throughout the the other virus type (e.g. HSV-2), the infection is
replicative cycle by host RNA polymerase II. described as an initial infection. During primary
The expression of the genes results in the pro- infection and during initial infection the virus estab-
duction of enzymes involved in nucleic acid metab- lishes a latent infection of sensory nerves at the local
olism (ribonucleotide reductase, thymidine kinase, dorsal root ganglion. Subsequent reactivation of
thymidilate synthetase, alkaline DNase, dUTPase, latent virus results in recurrent infection. If someone
etc.) and in DNA synthesis (DNA polymerase, heli- already infected with HSV becomes infected with
case, primase, etc.). Peak rates of synthesis of -gene the same strain of virus but at different site to the
usual site of recurrent infection (e.g. transfer of
products are observed 5—7 h postinfection and their
HSV-1 from a cold store to the eye), infection at the
appearance in the infected cell coincides with the
commencement of viral DNA synthesis. HSV DNA new site is described as an endogenous reinfection.
is believed to replicate by a rolling circle mechanism Finally, it is possible that a person already infected
yielding a concatomer that must be cleaved subse- with HSV may be reinfected with the same type of
quently to package genome lengths of DNA within virus. Infection in this case is described as
the nucleocapsid. exogeneous reinfection.
The synthesis of nucleocapsid and all other struc-
tural proteins occurs when gene expression is
induced by -gene products. Nucleocapsids as- Overview
semble and encapsidate viral DNA within the host
cell nucleus. A model to describe the cleavage of the Humans are the only natural host of HSV. While
concatomer of replicating DNA, packaging of a animal studies have provided, and continue to pro-
genome length of viral DNA within the nucleocap- vide, significant insights into the pathogenetic
sid and restoration (repair) of the concatomer of mechanisms of HSV infection of humans, it is im-
DNA has been proposed (summarized in Roizman portant to appreciate that no animal model can
and Sears 1996). Virus glycoproteins undergo ex- exactly mimic human HSV disease.
tensive post-translational modification during tran- The transmission of HSV requires direct contact
sit through the Golgi apparatus; they then become between a susceptible individual (usually antibody
inserted in the nuclear and other cellular mem- negative) and a person actively shedding the virus.
branes. The tegument proteins also migrate to the Transfer of virus is achieved by infection of mucosal
cell nucleus and form patches underneath the modi- surfaces or by entry through abrasions or cuts in the
fied nuclear membrane. Mature nucleocapsids bud skin. Virus replication occurs at the site of infection
through the nuclear membrane at these points and and produces a short-lived viraemia. This primary
acquire their envelope and tegument. The virion infection is usually inapparent, but in a minority of
HERPES SIMPLEX 27
cases may lead to localized and even systemic symp- that does not code for a translated gene product).
toms. The site of infection is determined mainly by The product of the gene from which the LAT RNA
the route of transmission of the virus from the infec- is excised has not been identified in latently infected
ted to the susceptible host. For HSV-1 it is the cells. A further point of interest is that latently
buccal cavity and oropharynx, and for HSV-2 the infected neurons seem to harbour more than one
genital mucosa. HSV-1 and HSV-2 may, however, viral genome per cell, and in animal experiments the
infect at either of these sites. numbers of copies per cell appears to increase with
During the primary infection, virus comes into time. A host-dependent origin of DNA replication
contact with the cutaneous receptors of local sen- has been identified within the viral genome (Sears
sory nerves. Virus is believed to attach to and pen- and Roizman, 1990) and thus the increase in the
etrate the nerve cell via these receptors. Once inter- number of viral genomes might be explained in
nalized the nucleocapsid moves to the perikaryon, terms of host cell enzymes effecting replication of
utilizing the normal cellular retrograde axoplasmic the viral DNA, independent of viral protein expres-
flow. Viral DNA is released, enters the nucleus and sion, in latently infected neurons. However, an
immediately circularizes. A latent infection of the understanding of the molecular events involved in
nerve cell is then established. In the latent state the the maintenance of latency remains elusive. The
virus is believed to exist as extrachromosomal cir- latent state appears to be effected by a repression of
cularized DNA (analogous to plasmids). No virions genes or a lack of a host cell protein involved in
can be detected and no viral antigens appear to be -gene transactivation, or both. As yet, no viral
expressed on or within the latently infected cell. The gene, including several of those found to be essential
host immune response to infection rapidly elimin- for productive viral infection in vitro and animal
ates virus and virus-infected cells from peripheral experiments, has been found to be essential for
sites but does not recognize latently infected nerv- maintenance of the latent state. One of many the-
ous tissue as harbouring virus, as no viral antigens ories (Roizman and Sears, 1996) is that within neur-
are expressed. onal cells a further host cell protein—octamer-
The establishment of latency is central to the binding protein OCT-2 (and spliced variants there-
success of HSV as a human pathogen. Latency per- of)—acts to inhibit the binding of the OCT-1—TIF
mits persistence of the virus in the presence of a fully complex (see Replication, above), thereby preven-
developed immune response and allows lifelong in- ting transactivation of genes (Latchmann, 1996).
fection of the host. As a result of periodic reactiva- Removal of OCT-2 allows the binding of the OCT-
tion of latent virus and the production of recurrent 1—TIF complex and viral genome expression (i.e.
infection, virus shedding occurs at intervals virus reactivation). The concentration of research
throughout life. into the mechanisms of latency will undoubtedly
The underlying molecular mechanisms involved allow an understanding of the process to emerge.
in the establishment and maintenance of latency are The site of virus latency is related to the site of
the subject of intensive study. Because current anti- primary infection: for HSV-1 the trigeminal ganglia,
viral chemotherapy is effective only when the virus and for HSV-2 the sacral ganglia, are the most
is replicating, it cannot eradicate latent virus. An common sites. Other dorsal root ganglia, including
understanding of the pathogenetic mechanisms in- the superior cervical, vagal and geniculate ganglia,
volved in the establishment, maintenance and reac- may also harvour virus. Although primary HSV-1
tivation of latent infection is therefore fundamental infection of the genitalia and primary HSV-2 infec-
to improved management of HSV disease. tion of the oropharynx are often reported, recurrent
Latently infected neurons apparently contain no infection of the genitalia by HSV-1 and recurrent
viral protein but do contain numerous virus-speci- orofacial infection by HSV-2 are rare. Infrequent
fied RNA transcripts—the so-called latency-asso- reactivation is not thought to be due to a failure by
ciated transcripts (LATs). Mutant viruses that do HSV-1 to establish latent infection in cells of the
not produce LAT (i.e. LAT\) are, however, also sacral ganglia or of HSV-2 to fail to establish latent
capable of establishing latency. Current thinking is infection in cells of the trigeminal ganglion, since
that these transcripts have no functional role in the respective genomes of the viruses have been
latency and that they are merely introns (i.e. a seg- detected at these sites. The inhibition seems to be at
ment of RNA spliced out from the gene transcript the level of effective viral gene expression in relation
28 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
to the particular environment of these sites. Sensory in the immune response to HSV infection.
nerve ganglia may not be the only sites of latency of
HSV. Using molecular techniques for the detection
Humoral Immunity
of viral nucleic acid, evidence for latency within the
cornea, brain and other (non-neuronal) sites is ac- Western blot and radioimmune precipitation ex-
cumulating. periments show that serum from patients recover-
Reactivation of latent virus produces recurrent ing from severe HSV infection may contain anti-
infection. A variety of non-specific ‘triggers’ for this body to all 33 structural (virion) proteins of HSV.
process have been described, including sunlight, The actual number and quantity of antibodies de-
cold, infection, stress, menstruation, ingestion of tected correlate with the severity of infection, and in
certain foods, etc. However, the molecular basis of mild infection only a restricted number of anti-
such ‘triggering’ is not understood. A further con- bodies may be detectable.
ceptual difficulty is that productive herpes simplex The immune response is directed principally to
infection is cytolytic. If the usual process of virus the glycoprotein spikes (Table 2A.1) found on the
replication were followed in nervous tissue, an indi- virion envelope and on the surface of virus-infected
vidual experiencing frequent reactivation over a cells. Neutralizing and cytolytic antibodies may be
period of years might be expected to experience loss detected; glycoprotein D being the most potent in-
of sensation through the progressive loss of sensory ducer of neutralizing antibody. In a primary infec-
nerves. The damage might also be compounded tion, an IgM subclass response is detected just be-
through the action of the immune system because in fore, or at the same time as, an IgG and IgA immune
a productive infection virus antigens are expressed response. The response is relatively short-lived but
on the infected cell surface, rendering the infected IgM antibody may also be detected during recur-
cell ‘visible’ to the immune system. Various models rence, rendering serological differentiation of pri-
have been proposed (Ho, 1992; Roizman and Sears, mary from recurrent infection difficult. Both IgG
1996) to explain this process, although none are and IgA antibody persist, although the IgG anti-
entirely satisfactory. It seems likely that virus repli- body response is of greater magnitude. Repeated
cation in nervous tissue is somehow restricted, with HSV recurrences lead to a gradual boosting of anti-
no expression of virion glycoproteins on the cell body levels but individual reactivation events, or
surface (thereby permitting the cell to escape im- even reinfection, may not result in a significant in-
mune-mediated cytolysis) and no irreversible dam- crease in circulating antibody. The presence of
age (cell lysis) to the host neuron. Studies of HSV-1 serum antibody may not protect from reinfection,
infection of cultured human embryonic dorsal root and neither the quantity nor the range of reactivity
ganglion cells (Lycke et al., 1988) suggest that egress (i.e. the lack of antibody to a particular virus pro-
of virus from sensory nerves is accomplished via tein) appears directly to influence either the fre-
anterograde axoplasmic flow in transport vesicles. quency or severity of reactivation events.
The released virus infects adjacent skin cells, repli- The early immune response to HSV is relatively
cates and infects further cells by cell-to-cell passage, type specific but ‘later’ antibody is more broadly
leading to the distinctive lesions of recurrent HSV cross-reacting. Monoclonal antibody studies show
infection. that both type-common and type-specific epitopes
may be found on virion glycoproteins. As a major
part of the humoral immune response is directed to
virion glycoproteins, the change from type specific-
Immune Response to Infection ity with time presumably reflects temporal changes
in the relative preponderance of antibodies to type-
Recovery from HSV infection and the control of specific and subsequently type-common epitopes
reactivated virus infection involves all components on the virion glycoproteins.
of the immune system acting in concert as an im-
mune ‘network’ (Jerne, 1976). The host’s genetic
Cell-Mediated Immunity
make-up, macrophages, specific T cell populations,
complement, specific antibodies, and lymphokine The cellular immune response to infection involves
and cytokine responses all have an important role a complex interaction of natural killer cells, macro-
HERPES SIMPLEX 29
phages, T lymphocytes and associated cytokines. caused epithelial lesions contain a disproportion-
Most adults can be shown to be HSV seropositive ately high number of CD4 ; cells and relatively
and are therefore presumed to harbour latent virus. few CD8 ; cells. It appears that the product of the
About 45% of this population give a history of 47 gene of HSV renders cells resistant to the activ-
herpes labialis. Some may recall only one recur- ity of CD8 ; lymphocytes by retaining MHC class
rence, while others report frequent episodes. If 1 molecules in the cytoplasm, resulting in a lack of
seropositive persons with no history of herpes la- peptide presentation on the cell surface (York et al.,
bialis are monitored, silent virus shedding may be 1994). Undoubtedly other HSV-induced mechan-
shown to occur at intervals. Differences in humoral isms operate to interfere in antigen presentation
immune status fail to explain these observations. and processing and the combination of molecular
The appearance or non-appearance of clinical biological and immunological investigation prom-
symptoms and the severity of these symptoms are ises to continue to provide fascinating information
perhaps most easily explained in terms of differen- on the adaptation of these viruses to their natural
ces in the cell-mediated immune responsiveness host.
(CMI). Thus in patients with known cellular im-
mune deficiencies, herpes labialis occurs frequently
and is a very much more severe and prolonged Pathogenesis in Immunocompromised
disease. Unfortunately, in individuals without overt Patients
cellular immune deficiencies, determination of rela- Congenital deficiencies in humoral immunity (hy-
tive differences in CMI between those with few re- pogammaglobulinaemia or even agammaglo-
currences and those with frequent recurrences is not bulinaemia) do not appear to be significant risk
easily accomplished. factors for serious primary or recurrent HSV dis-
ease. Congenital deficiencies in CMI may, however,
Virus-induced Modulation of the Immune be associated with severe HSV disease. The risk of
Response serious disease varies with the particular immune
deficit; for example, patients with congenital
In in vitro cell culture experiments and in animal athymic aplasia (DiGeorge syndrome) do not ap-
experiments HSV-1 has been shown capable of pre- pear to be at particular risk, while those with severe
venting apoptosis in differentiated cells. Premature combined immune deficiency, such as the Wis-
death of a virus-infected cell presents one of a range kott—Aldrich syndrome, are. These differences em-
of possible host responses to prevent the spread of phasize the complex interplay of the immune net-
virus to other, as yet uninfected cells. The products work in controlling HSV infection. Severe HSV
of gene mapping in the long unique component of disease is also observed in infants, children and
the viral genome, particularly the 34.5 gene (Chou adults with deficiencies in CMI induced by ‘ther-
and Roizman, 1994), appear to be involved in this apy’. These include recipients of cytoxic chemother-
process. The viral glycoproteins expressed on the apy (e.g. for cancer) and recipients of major organ
surface of the infected cell provide further modula- grafts. The severity of HSV disease in these patients
tors of the immune defence. The complex of glycop- is related to the type and degree of immunosuppres-
roteins E and I (Table 2A.1) produces an Fc recep- sion. Severe primary and recurrent HSV infections
tor which binds monomeric IgG molecules. also occur in patients with AIDS.
Glycoprotein E, by itself, provides a further type of
Fc receptor binding polymeric IgG molecules.
Glycoprotein C has been shown to bind the C3b EPIDEMIOLOGY
component of complement, possibly through
mimicry of the C3b receptor CR1. The effect of HSV has a worldwide distribution and is endemic in
these molecules is presumably to interfere in anti- all human population groups examined. There ap-
body interaction with infected cells and virions, and pear to be no animal reservoirs for the infection.
complement-mediated and antibody-dependent The rate of infection and the timing of primary
cytotoxic T cell destruction of virus-infected cells. A infection differs for HSV-1 and HSV-2, reflecting
further effect of HSV is to prevent the induction of the differences in the major modes of transmission
CD8 ; T lymphocytes. It is known that HSV- of the two viruses.
30 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
major mediator of seroprevalence is the frequency
Primary HSV-1 Infection of direct person-to-person contact. In crowded
areas (e.g. disadvantaged inner-city areas) sero-
Primary HSV-1 infection occurs when a susceptible prevalence is highest; in affluent, rural areas, sero-
individual comes into close, intimate contact with prevalence is lowest. On a worldwide basis how-
an individual suffering recurrent infection. Thus in- ever, overall rates of 90—95% seroprevalence are
fants become infected when their parents or rela- still commonplace in adult populations.
tives kiss them; adolescents who escape infection in
infancy are usually infected later by kissing. Infec-
tion in an infant is likely to be missed or dismissed
as ‘teething’. In adolescents infection is more Primary HSV-2 Infection
commonly symptomatic but rarely severe. Because
the majority of primary infections are asympto- HSV-2 transmission is apparently less efficient than
matic, epidemiological data collected by observa- that observed for HSV-1. The principal route of
tion of clinically apparent disease provide only a transmission is via sexual activity. Although some
partial measure of its true incidence. For accurate infants acquire HSV-2 infection, in most cases pri-
data collection serological studies must be em- mary infection is delayed until the onset of sexual
ployed. activity in adolescence and early adulthood. At this
The broad principles of the seroepidemiology of time the majority will have already experienced
primary HSV-1 infection were established in the primary infection with HSV-1 (i.e. infection with
classic study of Burnet and Williams (1939) and HSV-2 usually causes an initial rather than a pri-
have since been confirmed in numerous studies. mary infection). Because of the shared antigenicity
Burnet and Williams showed that in the first 6—9 of HSV-1 and HSV-2, HSV-1 immunity may be
months of life infants escape infection by virtue of partially protective and not all those exposed to
passively transferred maternal immunity. After- HSV-2 will necessarily become infected. Also, since
wards infants become infected and develop HSV transmission usually requires sexual contact, the
IgG antibody. The majority of these seroconver- number of exposures to the virus will inevitably be
sions occur during the first 5 years of life and those lower than the number of exposures to HSV-1 infec-
who escape infection in infancy undergo serocon- tion.
version during adolescence or in early adulthood. A A major problem in determining the sero-
relationship between the age of acquisition of the epidemiology of HSV-2 infections has been the lack
virus and socioeconomic status was also found. of well-characterized methods to differentiate HSV-
Populations associated with a low socioeconomic 1 and HSV-2 antibody. Only very recently have
environment collectively exhibited earlier acquisi- relevant assays become available on a commercial
tion of HSV infection than more affluent popula- basis (see diagnosis below). At the present time
tions, although in both groups 90—95% infection seroepidemiological studies of HSV-2 are thus in-
rates were observed by early adulthood and pri- complete. However, the data collected to date sug-
mary HSV-1 infection was a rare event in those of gest that the major influence on acquisition of HSV-
30 years of age. 2 infections is, as might be expected for a sexually
In recent years seroepidemiological studies have transmitted virus, the number of sexual partners.
shown that in developed countries there has been a Rates of up to 95% seroprevalence have been re-
lowering in the overall prevalence of HSV-1 anti- ported in some female commercial sex workers. In
body (rates of 70% or lower are often reported the general population there are wide differences in
among 30—40—year-old adults). In these countries, seroprevalence between different patient groups
in children, in adolescents and, particularly in those and even between apparently similar social groups
from a high socioeconomic class, in young adults, a in different cities. Women are generally infected at
significant lowering in seroprevalence is evident. an earlier age than men, and rates of infection in
However, even within individual countries there are women are higher for all age groups up to and
wide variations in seroprevalence; for example, in- including those of 45 or more years of age. Studies
ner-city residents generally exhibit higher serop- among genitourinary medicine clinic patients sug-
revalence rates than those from rural areas. The gest that only in homosexual men do the rates of
HERPES SIMPLEX 31
infection with HSV-2 match those found in women; infection can only be accomplished by isolation of
although, even in this context, it is not until age strains of virus from two sites and demonstration of
40—45 that equivalent rates are observed. The ma- genetic polymorphism by restriction enzyme analy-
jority (80% or more) of those found to be HSV-2 sis of their respective DNAs.
antibody positive cannot recall primary infection
with the virus and either do not recall, or are un-
aware of, recurrent genital herpes, emphasizing
that, as with primary HSV-1 infection, the majority CLINICAL FEATURES
of primary infections are mild and clinically ‘silent’.
The changing seroepidemiology of HSV-1 infec- Oropharyngeal and Orofacial Infection
tions in developed countries and changes in sexual
practices have led to suggestions that the epidemiol- In a symptomatic primary HSV infection involving
ogy of genital herpes is changing. In some UK the oropharynx, gingivostomatitis is the most
stud-ies 40—60% of isolates of HSV from first-epi- common symptom. The incubation period ranges
sode genital herpes have been found to be due to from 2 to 12 days, with a median of 4 days, and the
HSV-1. These data have not been corroborated in duration of clinical illness is 2—3 weeks. The spec-
other centres where more usually only 10—20% of trum of severity ranges from the trivial, involving
first episode genital HSV isolates are found to be the buccal and gingival mucosa, to severe, painful,
HSV-1. ulceration of the mouth, tongue, gingivae and fau-
ces. In severe disease, shallow ulcers on an
erythematous base evolve from vesicles and often
Recurrent HSV-1 and HSV-2 Infection coalesce, particularly on the mucosa of the cheeks
and under the tongue. The ulcers are observed on
Both silent and overt (i.e. symptomatic) recurrences the hard rather than the soft palate—a feature that
may help differentiate herpetic ulcerations from
of HSV-1 and HSV-2 infection occur. Thus without
those caused by the Coxsackieviruses (‘herpan-
continuous monitoring of a cohort (by virus isola-
tion studies) accurate data on rates of recurrence gina’). In young children the skin around the mouth
cannot be obtained. Only 38—45% of an adult is frequently involved. Submandibular lympha-
population (in whom seroprevalence rates of denopathy and fever (39—40°C) are common and
90—95% are reported) will give a history of recur- may produce febrile convulsions in children. A
rent herpes labialis. moderate lymphocytosis (up to 700 mm\) and
The frequency of recurrence of genital HSV-2 mild neutropenia are frequently observed. Elevated
infection is thought to be higher than that observed liver enzymes are noted in occasional cases. Acute
in HSV-1 herpes labialis, although the number of gingivostomatitis is a self-limiting disease and the
lesions produced per episode and their duration are resolution begins abruptly. The lesions become
generally shorter. The rate of recurrence is believed painless and inflammation subsides. Intraoral ul-
ceration progresses to healing, although crusting of
to be slightly higher in men than in women, with
lesions, as seen in resolving cutaneous HSV infec-
rates of up to 2.7 and 1.9 episodes per 100 patient-
days, respectively. Overall about 60% of those in- tion, is not usually observed. The patient becomes
fected with HSV-2 infection will report recurrent afebrile and symptoms regress rapidly, although
infection. lymphadenopathy may persist for several weeks.
Other symptoms that may be associated with
primary infection include sore throat, mild conjunc-
tivitis, nausea and vomiting, myalgia and abdomi-
Endogenous and Exogenous nal discomfort. Dehydration and anorexia may re-
Reinfections sult through mouth discomfort and difficulty in
swallowing associated with the pain and oedema of
Reinfection, recognized by the appearance of the mucosal membrane infection. Older patients
lesions at another body site, for example infection of may experience a pharyngitis associated with a
the finger (herpetic whitlow), can occur at any age. mononucleosis syndrome (up to 20% atypical lym-
Differentiation of endogenous and exogenous re- phocytes). Tonsillar ulceration is observed fre-
32 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
quently in pharyngitis, together with submandibu- involved. The lesions may extend to the perineum,
lar lymphadenopathy. upper thigh and buttocks. In men, vesicular lesions
The oral disease can be associated with lesions with an erythematous base are observed on the
elsewhere. Herpetic dermatitis, nasal herpes, ocular glans of the penis or on the penile shank. Perianal
herpes, herpetic whitlows and even genital herpes and anal infections producing proctitis are common
are not infrequent complications of primary HSV in homosexual men.
infection of the mouth. These probably represent Sacral radiculomyelitis is sometimes observed,
endogenous reinfections caused by autoinoculation with urinary retention and attendant neuralgia.
from the site of primary infection. Secondary microbial infections often follow pri-
Recurrent infection, triggered by a variety of ap- mary genital HSV infection and their occurrence
parently non-specific stimuli, such as fever, stress, necessitates appropriate antimicrobial therapy. Pri-
cold, menstruation or ultraviolet radiation, appears mary (maternal) genital herpes occurring at or
as a fresh vesicular eruption termed ‘herpes labialis’, around the time of birth may produce severe
‘herpes febrilis’, ‘fever blisters’ or ‘cold sores’. The neonatal infection. Aseptic meningitis associated
most frequent site is at the border of the lips but with primary genital HSV-2 infection is occa-
lesions may appear elsewhere—for example, on the sionally observed.
chin, cheek and on, or inside, the nose. Their ap- In recurrent genital herpes a limited number of
pearance is usually preceded by a prodrome of vesicles are produced and their appearance is
tingling or itching at the site where recurrence will usually associated with a localized irritation rather
occur. In the immunocompetent, the area of in- than with significant pain. In comparison to herpes
volvement is usually small and vesicles progress to labialis, recurrent lesions in the perineal area tend
the pustular and crusting stage within 3—4 days to be more numerous and can persist longer than
(Figure 2A.3, see Plate I). The time for complete their oral counterparts. New vesicles often appear
healing is variable (5—12 days, mean 7.5 days). during the course of a recurrence, and delay healing.
Asymptomatic oral shedding of HSV can occur The mean healing time in recurrent genital herpes
and is occasionally preceded by a prodrome similar may be up to 15 days, compared to an average of 7.5
to that associated with overt herpes labialis. In the days in recurrent oral disease. Complications (neur-
immunocompetent complications of recurrent in- ological or systemic) associated with recurrent in-
fection are rare. fections are rare.
Recurrent intraoral ulcers are caused only rarely
by HSV. In the few instances where reactivation of
HSV is responsible, lesions are limited to the gingi-
vae and the hard palate. Severe cellular immune Ocular Infection
deficiency can, however, lead to extensive ulcer-
ation of the mouth, and involve the pharynx and The early symptoms of herpes keratitis are of a
oesophagus. unilateral or bilateral conjunctivitis, with pre-
auricular lymphadenopathy. The most common
first presentation is, however, of a unilateral, char-
acteristic, dendritic (branching) ulcer (Figure 2A.4,
Genital Infection see Plate I). The normal course of the disease is 3
weeks, but if ulcers are large, healing can be slow.
In comparison to primary infection of the oro- Mild systemic disturbances, blepharitis and cir-
pharynx, primary genital infection is often a more cumocular herpetic dermatitis are commonly pres-
severe clinical disease that may last up to 3 weeks. ent during the primary infection. Infection may oc-
Fever, dysuria associated with urethritis and cystitis cur as a part of a primary infection but most cases
(with urinary retention in a proportion), localized are thought to represent an endogenous reinfection
inguinal adenopathy and malaise is common. A caused by autoinoculation from the site of a recur-
prominent feature of primary genital herpes is pain, rent infection. Almost all cases are due to HSV-1.
which, especially in women, may be severe. In New HSV infections of the eye are estimated to
women the lesions are located principally on the occur at a rate of about 50 000 per year in the UK
vulva but the vagina and cervix are almost always and up to 300 000 per year in the USA.
HERPES SIMPLEX 33
A single recurrence of ophthalmic infection is neal latency of HSV occurs (they might reflect low-
observed in between 20 and 30% of patients within grade virus persistence rather than latency) and
2 years of the first infection. Subsequent recurrences further work will be required to allow firm con-
are less common but if a second recurrence does clusions to be drawn.
occur then further recurrences will be observed in The influence of the immune response in control
40—45% of these cases. There are several forms of of ocular infection is an additional area of interest in
recurrent ophthalmic infection that may occur in the pathogenesis of ocular HSV infection. Because
combination. Dendritic or larger ‘geographic’ ul- of the unique anatomy and physiology of the eye,
cers are usually the first manifestation of recurrence, the local immune response to infection may differ
with the patient complaining of ocular irritation, from that occurring at, for example, cutaneous sites.
lacrimation, photophobia and sometimes blurring The lack of, and/or relative overproduction of, indi-
of vision. Infection is usually confined to the superfi- vidual components of the immune response un-
cial layers of the cornea and stromal involvement is doubtedly influences disease pathogenesis at this
absent or only relatively mild. Days or weeks after site.
the recurrent infection the corneal epithelium may Recurrent iridocyclitis and occasionally panuvei-
ulcerate to form a nondescript ovoid ulcer (i.e. post- tis may be caused by HSV. Iridocyclitis, produced
infectious or metaherpetic keratitis). Virus replica- by intraocular inflammation, is observed often in
tion does not appear to be directly responsible for association with active herpes keratitis. It may be
the production of this ulcer. If the ulceration is due to direct involvement of the virus or may be
prolonged (weeks and months), collagenolytic ac- secondary to the irritative effects of keratitis.
tivity may appear, leading to stromal melting and Ophthalmic disease associated with intrauterine
perforation. This form of disease requires careful and neonatal infection with HSV (see below) can
management if these serious sequelae are to be present at keratoconjunctivitis or later as chor-
avoided. ioretinitis, but cataract, corneal ulceration, anterior
Repeated and severe epithelial disease recurrence uveitis, vitritis, optic atrophy, nystagmus, strabis-
or chronic epithelial keratitis leads to involvement mus, microphthalmia and retinal dysplasia have all
of the deeper layers of the cornea, producing inter- been reported in association with such infection.
stital or disciform keratitis. Neovascularization and Ocular manifestations are more common in those
scarring may ultimately lead to loss of vision. HSV infected in utero than in those infected intra- or
infection is second only to accidental injury as the postpartum.
major cause of blindness in developed countries.
The observation of recurrent infection of the cor-
nea raises questions as to extraneuronal sites of
latency of HSV. Restriction endonuclease analysis Neonatal Herpes
of viral DNA isolated from successive ocular recur-
rences suggests the same strain of virus is involved The reported incidence of neonatal herpes varies
in each recurrence. Repeated autoinoculation of the from extremes of 1 case in 2500 live births (Ala-
virus from a cold sore seems unlikely and virus bama, USA; Whitley 1993) to 1.65 per 100 000 live
latency in the ophthalmic division of the trigeminal births reported in a UK survey (Tookey and Peck-
ganglion and spread to the cornea in the tear film ham, 1996). This variation reflects both the wide
might not explain the recurrence of virus infection variation in HSV-2 seropositivity found in different
in the same region of the cornea in each episode. geographical and socioeconomic groupings and the
Thus extraneuronal virus latency within the cornea difficulties in diagnosis of the condition. Symptoms
has been suggested. Using the polymerase chain at presentation can range from the very severe,
reaction it has proved possible to demonstrate HSV associated with high mortality, to the relatively
DNA within corneal cells of both normal and dis- mild, which none the less can be a cause of signifi-
eased corneal tissues obtained at keratoplasty. cant residual morbidity. Congenital infection is
Moreover, infectious virus has been isolated using rare: most neonatal herpes is a result of perinatal
cocultivation techniques from corneas obtained infection. A few cases occur as a result of trans-
from patients with stromal herpes keratitis. These mission of oral herpes from the mother, her relatives
observations do not in themselves prove that cor- or hospital staff in the immediate postdelivery per-
34 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
iod. Both HSV-1 and HSV-2 can cause neonatal toms. Herpes encephalitis occurs in all age groups
herpes but the majority of cases are due to HSV-2. and approximately the same number of cases occur
In general HSV-1 infection poses a lower risk for in men as in women. There is no seasonal variation
long-term sequelae, although this distinction is not in incidence and cases occur sporadically. Precise
absolute. estimates of the incidence of HSE are not possible
Primary and also initial infection during the first because of underreporting, but estimates of inci-
or second trimester of pregnancy is not associated dence range from about 1 case per million popula-
with significant risk. When infection occurs during tion per year to 1 case per 200 000. Most cases are
the third trimester there is a risk of transmission to caused by HSV-1, with HSV-2 causing 2—6.5% of
the baby during vaginal delivery. Risk is highest cases. In the immunocompromised, ‘classical’ her-
when infection occurs around the time of labour pes encephalitis appears to occur at the same rate as
(Smith et al., 1998). in the immunocompetent, although in AIDS pa-
Primary maternal genital herpes poses the tients an atypical, subacute encaphalitis associated
greatest threat to the neonate, although recurrent with either HSV-1 or HSV-2 has been identified in
genital herpes is also a risk. Most mothers bearing 1—2% of cases studied at autopsy, usually in associ-
children who develop neonatal herpes do not have ation with a concurrent cytomegalovirus infection
genital lesions at delivery. of the CNS (Cinque et al., 1996).
American studies (Whitley, 1990) suggest that The onset of disease may be either abrupt or
neonatal herpes may manifest as disease localized insidious. In the early stages, signs and symptoms of
to skin, eye and mouth; encephalitis with or without the infection may be incomplete. Electroencaphalo-
skin, eye and mouth involvement; and disseminated graphic examination (EEG) will usually reveal non-
disease with multiorgan involvement including specific slow-wave activity during the first 5—7 days
CNS, lung, liver, adrenals, eyes and skin. Symptoms of illness. Later, more characteristic paroxysmal
appear 4—5 days postpartum, with most peripheral sharp waves or triphasic complexes with a temporal
and disseminated infections being evident at 11—12 predominance can be found. Low-density lesions
days and most CNS disease appearing by 17—18 are demonstrable by X-ray computed axial tomo-
days. In the absence of therapy, mortality in dis- graphy (CT) in about 70% of cases, within 5 days of
seminated disease exceeds 80%, and exceeds 50% in the onset of neurological illness. Proton magnetic
those with CNS symptoms only. Both forms of resonance imaging (MRI) has the potential to detect
disease are associated with severe morbidity. Babies abnormalities that may not be revealed by routine
exhibiting only skin, eye and mouth infection may CT. MRI can show frontobasal and temporal
suffer permanent ocular damage, and most will, if lesions as hypointense lesions on T -weighted im-
followed for a sufficient period of time, eventually ages and hyperintense lesions on proton density
show some sign of neurological impairment. All and T -weighted images at an earlier stage than
HSV infections identified in the neonatal period changes can be detected by CT.
therefore warrant antiviral therapy. The histopathological changes associated with
severe HSE consist of acute inflammation that
evolves to produce haemorrhagic and necrotizing
lesions. The lesions are characteristically located in
Herpes Simplex Encephalitis the temporal lobes and orbital surface of the frontal
lobes, but adjacent frontal, parietal and occipital
The most common presentation of herpes simplex lobes and the cingulate gyri may also be involved.
encephalitis (HSE) is a focal encephalopathic pro- Clinically, herpes encaphalitis ranges in severity
cess with signs and symptoms that localize to the from a mild encephalitis of low mortality and mor-
frontotemporal and parietal areas of the brain. As bidity to severe necrotizing encaphalitis associated
the disease progresses, symptoms increase in sever- with both high mortality (70—90% in the absence of
ity and there is progressive decrease in conscious- specific antiviral chemotherapy and expert neur-
ness, leading, in severe cases, to coma and death. In ological care) and high morbidity (with only 10% of
the majority of patients there is a history of a ‘flu- survivors returning to normal neurological function
like’ illness occurring at some time in the 2 weeks in the absence of antiviral chemotherapy). Herpes
preceding the appearance of neurological symp- encaphalitis is, however, a treatable disease and
HERPES SIMPLEX 35
prompt initiation of chemotherapy using the anti- localization reflects the particular neurochemical
viral drug acyclovir, combined with intensive neur- and perhaps local neuroimmunological environ-
ological nursing care, results in a reduction of mor- ment of the limbic region (Damasio and Van
tality to less than 20%, with up to 40% of survivors Hoesen, 1985); however, such localization is not
returning to apparently normal neurological func- observed in all cases and virus may spread to in-
tion. In some patients there may be residual neur- volve other regions of the brain.
ological deficit: this may be severe, with impair-
ment, or complete loss, of short-term memory being
the most commonly observed sequelae. Disease in the Immunocompromised
In contrast to several other enveloped viruses
(including Varicella zoster virus (VZV), Epstein— Patients who are immunocompromised through
Barr virus and Human cytomegalovirus), HSV does deficits in CMI are at risk of severe HSV infection,
not seem to be a significant cause of perivascular with the possibility of life-threatening, disseminated
leucoencephalopathy (‘postinfectious encephalitis’). infection. Oral HSV infection can progress to exten-
However, this disease has been reported to occur in sive necrotic mouth ulceration, with bacterial
a minority of patients recovering from acute herpes superinfection or haemorrhage in those with
encephalitis. Between 5 days and 3 weeks after ap- thrombocytopenia. Equally severe, progressive
parent recovery from acute encephalitis the patient genital HSV infections may occur. Antoinoculation
suddenly develops a relapse and a further bout of may result in initial infection at sites distant from
encephalitic illness. In the small number of cases the original focus. Cutaneous dissemination can
examined by brain biopsy no virus antigen has been take place and may be clinically indistinguishable
detected and histopathologically lesions are similar from varicella. Visceral dissemination is also poss-
to those seen in postinfectious encephalitis triggered ible with hepatitis, pneumonia and encephalitis.
by other virus infections (i.e. significant demyelina- Primary, recurrent and exogenous reinfections all
tion and immune cellular infiltration). Viral DNA is pose a significant threat to such patients. In organ
usually not detected in the cerebrospinal fluid (CSF) transplant recipients, progressive disease may in-
and most patients survive and recover to at least volve the oesophagus, respiratory tract and even
their level of disability following acute herpes en- the gastrointestinal tract—the severity of disease
cephalitis. observed in such patients being linked with the
The pathogenesis of herpes encephalitis remains degree of immunosuppression. In patients with hu-
enigmatic. The structure of the vasculature within man immunodeficiency virus (HIV) infection, recur-
the brain, together with the meninges surrounding rent HSV infection may occur with high frequency
the brain, represents, in the physiologically normal at multiple sites; widespread cutaneous distribution
host, a significant barrier to virus entry to brain may occur; and markedly prolonged times to heal-
parenchyma. Haematogenous spread of virus to the ing of lesions are observed. Prolonged viraemia in
brain is usually prevented by these blood—brain and such patients leads to multiorgan distribution, in-
blood—CSF barriers. To gain access to the brain the cluding dissemination to the CNS.
virus must circumvent the physiological/anatomic
barriers. Virus may achieve this by transit from the
periphery within nerve cells. Various pathways
have been proposed, although in all probability no Herpes Simplex Dermatitis; Herpetic
one route explains all cases of herpes encephalitis. Whitlows; Traumatic Herpes; Herpes
Once within the brain the spread of virus repre- Gladiatorum
sents a fascinating and largely unexplained area of
neurovirology. At least in the early stages virus Herpetic dermatitis is a complication of primary
infection is principally of neurons, with only rela- infection. Perioral or periorbital herpes simplex
tively occasional involvement of astrocyte and glial regularly accompanies more severe primary gingi-
cells. The frontal and temporal lobes together with vostomatitis or primary/initial herpes keratitis. In
the associated limbic structures of the brain appear babies, seeding of the virus can involve large areas
to be the primary targets of the infective process of the face and, when transferred to the anogenital
(Esiri, 1982) and has led to the suggestion that this area by scratching, may involve the whole napkin
36 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
area. In primary HSV vulvovaginitis, vesicles read- involvement (Stevens—Johnson syndrome). Myco-
ily appear on the perineum and thigh. plasma pneumoniae and HSV are the infectious
In patients with atopic eczema or with Darier’s agents most commonly associated as precipitants.
disease the normal resistance of the skin to HSV Up to 65% of patients with recurrent erythema
infection is reduced and primary, recurrent and multiforme give a preceding history of herpes la-
possibly both endogenous and exogenous reinfec- bialis, on average 11 days before the lesions of the
tion may produce eczema herpeticum. Eczema her- disease appear. Infectious virus cannot be cultured
peticum as a primary infection carries a small but from the lesions but immunofluorescence studies
significant mortality through progression to severe have shown HSV glycoprotein B located around
generalized disease. keratinocytes in the viable epidermis, polymerase
Under normal circumstances, HSV does not chain reaction (PCR) studies have detected HSV
readily penetrate healthy skin but when the dermis DNA in cutaneous lesions, and in situ hybridization
is breached, a portal of entry is created. Health care has been used to demonstrate HSV nucleic acids
personnel are at particular risk of perforation inju- within the epidermis.
ries. Direct inoculation of virus into the fingers of Erythema multiforme does not respond directly
those who constantly manipulate in the oral cavity to antiviral chemotherapy (HSV replication is ap-
(dentists, dental nurses, intensive care doctors, parently a precipitant of the disease but not a direct
nurses and anaesthetists) leads to herpetic whitlow, cause of the pathology) but cases of recurrent HSV-
an inflammation of the nail folds. A similar condi- associated erythema multiforme can be prevented
tion is seen in children and adolescents who transfer by prophylactic use of acyclovir. Some cases of
their own oral virus through nail biting. In other idiopathic recurrent erythema multiforme (i.e.
situations, workers such as hairdressers, dress- where antecedent herpes labialis is not suspected)
makers and laboratory personnel can be infected by also respond to prophylactic use of acyclovir. This
accidental stab injuries with contaminated needles is perhaps consistent with a preceding ‘silent’ recur-
or broken glassware (traumatic herpes). rence of HSV.
Herpes gladiatorum and ‘scrum pox’ are condi-
tions spread among wrestlers through bites or ‘mat
burns’, and rugby players through bites and facial
scraping. The appearance of herpetic vesicles at DIAGNOSIS
‘unusual’ sites can sometimes be explained by an
inquiry into the individuals athletic pursuits! The ability of HSV to establish latent infection in-
A distinct form of cutaneous infection ‘zosteri- evitably complicates diagnosis. A clinical role for
form herpes simplex’ is an infrequent presentation the virus in causation of disease is not established
of herpes simplex but is recognized when the dis- simply because it is recovered from a patient. To
tribution of HSV lesions accords with a dermatome achieve meaningful diagnosis a close collaboration
and otherwise resembles zoster. Unlike zoster, between clinic and laboratory is always necessary.
nerve root pain is not a feature.
HO N
N NH 2
Valaciclovir (ValtrexTM, ZelitrexTM)
OH The oral bioavailability of acyclovir is relatively low
and for this reason a prodrug ester of acyclovir—
valaciclovir—was developed (Figure 2A.5b). Val-
aciclovir hydrochloride is rapidly adsorbed from
the gastrointestinal tract and rapidly and almost
(d) N completely converted to acyclovir and -valine by
O N
first-pass intestinal and/or hepatic metabolism. The
CH 3 O N
N NH 2 mode of action and clinical spectrum of activity of
valaciclovir is identical to that of acyclovir. The
plasma concentrations of acyclovir achieved after
O CH 3 oral administration of valaciclovir are believed to
be equivalent to those achieved by intravenous ad-
O ministration of acyclovir and are 3—5 fold greater
than are achieved with oral administration of acyc-
lovir.
Figure 2A.5 Antiviral drugs. (a) Acyclovir: 2—amino-
1,9—dihydro-9—[(2—hydroxyethoxy)methyl]-6H-purin-6—one
(mol. wt 225.21). (b) Valaciclovir: -valine, 2—((2—amino-
1,6—dihydro-6—oxo-9H-purin-9—yl)methoxy)-ethyl ester, mono- Penciclovir (DenavirTN)
hydrochloride (mol. wt 360.80). (c) Penciclovir: 9—[4—hydroxy-
3—(hydroxymethyl) butyl] guanine (mol. wt 253.26). (d) Famcic- Penciclovir is an acyclic nucleoside analogue (Figue
lovir: 2—[2—(2—amino-9H-purin-9—yl]-1,3—propanediol-acetate 2A.5c) whose mode of action is essentially identical
(mol. wt 321.3)
to that of acyclovir. However, the half-life of the
either altered substrate specificity (i.e. acyclovir is active antiviral (penciclovir triphosphate) is sub-
no longer recognized as a substrate for virus speci- stantially longer than that of acyclovir triphos-
fied enzyme) or with loss of thymidine kinase (TK) phate. In in vitro experiments the half-life of pencic-
activity—the latter group being of lesser import- lovir triphosphate in HSV-1—infected cells is about
ance because loss of TK activity is associated with 10 h, and in HSV-2 infected cells is about 20 h.
42 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Recurrent Infection
Specific Management
Recurrent HSV infections inconvenience the host
but are rarely serious in the immunocompetent in-
Primary or Initial Infection
dividual. Patients with only occasional recurrences
Primary or initial infection with HSV is, in the often develop their own preferred self-treatment; for
majority of cases, inapparent or only associated example, the dabbing of perfume or alcohol on cold
with mild symptoms, and therapeutic intervention sores, or the use of acyclovir cream. Frequent or
is not considered. For symptomatic infection, man- severe recurrences may require oral or systemic
agement is tailored to the severity of symptoms. antiviral chemotherapy. The most effective episodic
Bicarbonate- or chlorhexidine-containing mouth- treatment requires a 5 day course of oral antiviral
washes combined with simple analgesia may be therapy initiated as soon as prodromal symptoms
adequate in gingivostomatitis. Saline douches may are observed. Acyclovir (200 mg 5 times daily), fam-
provide relief from herpes vaginitis. Severe cases ciclovir (125 mg twice daily) or valaciclovir (500 mg
merit the introduction of specific antiviral chemo- twice daily) can be used. In a recurrent episode, the
therapy. Acyclovir and penciclovir creams are earlier the treatment is started the more effective it
available for direct application to skin lesions; how- proves in reducing the severity and duration of the
ever, a more effective treatment is oral administra- recurrent episode.
tion of acyclovir (given in 250 mg doses five times Since the maintenance of latent virus infection
daily), valaciclovir (1 g per day) or famciclovir (250 g appears to be independent of virus replication, anti-
three times daily) for 5—10 days. There is some viral chemotherapy cannot ‘cure’ recurrent virus
evidence that antiviral treatment of primary or in- infection. Patients with frequent recurrences
itial infections may impact upon the subsequent should, however, be considered for continuous sup-
frequency of reactivation. Severe primary or initial pressive therapy; acyclovir (400 mg twice daily
infections are associated with a higher frequency of 200 mg p.o. 3—5 times a day), valaciclovir (500 mg
(symptomatic) reactivation. twice daily) or famciclovir (125 mg twice daily) are
Secondary bacterial and fungal infection is believed to be effective. Usually a 6—12 month per-
common in severe symptomatic primary and initial iod is chosen for continuous therapy and the necess-
HSV infection and requires appropriate antimic- ity for therapy is then reassessed.
robial therapy.
Steroid treatment in general increases the sever-
Ocular Infection
ity of disease and, where patients are receiving high
doses of steroids (e.g. in treatment of eczema), the The majority of cases of ocular HSV infection are
appearance of HSV infection is an indication for thought to result from an endogenous reinfection
temporary cessation or reduction of steroid dosage. (i.e. transfer of virus from the site of a recurrent
In other cases of iatrogenic immunosuppression infection to the eye). Misdiagnosis of the condition
(e.g. for organ transplantation), HSV infection is an has led frequently to the administration of corticos-
indicator for temporary reassessment of the dosage teroids to the eye. Unfortunately such treatment
of the immunosuppressive therapy. Primary HSV may exacerbate and prolong the infection. En-
infection in the immunocompromised host always dogenous reinfections or primary infections of the
gives cause for concern because of the possibility of eye should not be treated with steroids. Conserva-
the development of generalized multiorgan infec- tive treatment is warranted, with application of spe-
tion. Serious HSV infections require not only the cific antiviral chemotherapy (acyclovir ophthalmic
prompt initiation of specific antiviral chemotherapy ointment) and possibly debridement of the cornea.
but also full medical and nursing intervention. Gen- Oral or systemic antiviral therapy (see above)
eralized infection in the immunocompromised pro- should be given in severe disease. Secondary bacter-
duces osmotic imbalances, endocrine dysfunction, ial infection may occur, necessitating appropriate
44 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
antimicrobial therapy. Recurrent ocular HSV infec- at the time of delivery. Prediction of infection by
tions are managed in a similar fashion except that routine cultures performed during pregnancy is not
the indication for antiviral chemotherapy is more possible and caesarean section in prevention of in-
pronounced. fection is of doubtful benefit and should not be
The treatment of the late stage of herpetic eye reserved only for those with known primary or
disease (stromal involvement) is more controversial, ongoing recurrent infection within the birth canal at
in that some authors believe that antiviral treat- the time of delivery. Where vaginal delivery cannot
ment may accentuate rather than alleviate stromal be avoided, intravenous administration of acyclovir
disease. Steroid therapy is almost certainly in- to both mother and infant are probably appropriate
dicated in stromal herpes keratitis and possibly also (Smith et al., 1998).
in iridocyclitis, as most damage is believed to result Antiviral therapy for HSV must be administered
from an inflammatory reaction rather than the di- to the neonate early in the course of the disease
rect action of virus replication in corneal tissues. before irreversible damage occurs. In at least 50%
Severe stromal scarring necessitates corneal trans- of cases the child and the mother lack apparent
plantation. lesions and the majority of mothers have no history
of genital herpes. Recurrence of herpetic lesions or
relapse of neurological or retinal disease is unfortu-
CNS Infection
nately frequent in infants with neonatal herpes who
Treatment of herpes encephalitis requires the early have received a course of parenteral acyclovir.
administration of antiviral chemotherapy (Cinque Treatment of neonatal herpes using increased dos-
et al., 1996). Antiviral chemotherapy should also be ages of acyclovir and prolonged (21 days) intra-
given in herpes meningitis, as the sequelae of this venous therapy has been attempted on an empirical
infection are not defined but do include the possible basis. Prolonged oral administration of acyclovir
development of recurrent meningitis. The currently has also been utilized, although at present there is
accepted antiviral treatment for herpes encephalitis no evidence from appropriately controlled studies
is a 10 day intravenous course of acyclovir at 10 mg to confirm or refute the benefit of either of these
kg\ given every 8 hours. Acyclovir therapy should strategies.
be approached with caution in patients with im-
paired renal function because build-up of excessive
serum concentrations of acyclovir has been asso-
ciated with (reversible) neurotoxicity. REFERENCES
Specific antiviral chemotherapy is not the only
important factor in treatment of CNS infection. Bergstrom, T. and Trybala, E. (1996) Antigenic differences be-
Brain oedema is believed to be the major cause of tween HSV-1 and HSV-2 glycoproteins and their importance
for type-specific serology. Interology, 39, 176—184.
mortality in herpes encephalitis, hence reduction in Burnet, F.M. and Williams, S.W. (1939) Herpes simplex: a new
intracranial pressure is an important consideration point of view. Medical Journal of Australia, 1, 637—641.
in the overall treatment regimen and necessitates Chou, J. and Roizman, B. (1994) Herpes simplex virus 1 34.5
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clinical virology laboratory (Cinque et al., 1996). in the homologous domain of the genes expressed during
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Cinque, P., Cleator, G.M., Weber, T. et al. (1996) The role of
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high, although these associations are not absolute and the localisation of herpes simplex encephalitis. Journal of
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tological study of the distribution of viral antigen within the
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HERPES SIMPLEX 45
Jerne, N.K. (1976) The immune system: A web of v-domains. Revello, M.G., Baldanti, F., Sarasini, A. et al. (1997) Quantitation
Harvey Lectures, 70, 93—110. of herpes simplex virus DNA in cerebrospinal fluid of patients
Koskiniemi, M., Klapper, P.E., Forsgren, M. et al. (1999) Lab- with herpes simplex encephalitis by the polymerase chain
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Linde, A., Klapper, P.E., Monteyne, P. et al. (1997) Specific Smith, J.R., Cowan, F.M. and Munday, P. (1998) The manage-
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Lycke, E., Hamark, B., Johansson, M. et al. (1988) Herpes phological entities projecting from the virion envelope. Jour-
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McGeoch, D.J., Dolan, A., Donald, S. et al. (1986) Complete virus infection in the British Isles. Paediatric and Perinatal
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herpes simplex virus type 1. Nucleic Acids Research, 14, Whitley, R.J. (1990) Herpes simplex viruses. In Fields Virology
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McGeoch, D.J., Dalrymple, M.A., Davison, A.J. et al. (1988) The New York.
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genome of herpes simplex virus type 1. Journal of General Journal of Medical Virology, suppl. 1, 13—21.
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detection of intrathecal synthesis of anti-herpes simplex IgG Zhou, Z.H., Prasad, B.V.V., Jakana, J. et al. (1994) Protein
antibodies: comparison between an antigen-mediated im- subunit structures in the herpes simplex virus A-capsid deter-
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
2B
Varicella Zoster
Judith Breuer, David R. Harper and Hillar O. Kangro
St Bartholomew’s and the Royal London School of Medicine and Dentistry, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
48 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2B.1 Structure of the VZV particle (virion). When intact, the particle is spherical and approximately 200 nm in diameter.
Amorphous forms may be seen using traditional electron microscopy. (From The Sourcebook of Medical Illustration. Parthenon Press)
Figure 2B.2 Gene map showing the organization of the VZV genome and the location of genes (hatched) encoding the viral
glycoproteins, the major capsid antigen (MCA), the DNA polymerase (POL), the thymidine kinase (TK), the thymidylate synthetase
(TS) and the assembly proteinase/protein (AP). The unique long (U ) and unique short (U ) regions are identified, as are the terminal
*
repeat (TR and TR ) and inverted repeat regions (IR and IR ). TR and IR are 88 bp long, while TR and IR (black boxes) are
* * * *
7300 bp long
ities between the HSV and VZV genomes would be ping genes does allow for still further ORFs to be
compatible with a common ancestry, and a model identified. Approximately half of the genes have
for this has been proposed (Davison and McGeoch, been, at least tentatively, identified (Table 2B.1)—
1986). The VZV genome is divided into two main mostly by analogy with HSV. However, five of the
coding regions: a unique long region (approx. VZV genes, including a gene for a thymidylate syn-
105 000 bp) which is flanked by inverted repeat thetase, are not found in HSV, while three HSV
elements (at 88 bp, far shorter than those of HSV), genes have no VZV equivalent.
and a unique short region (approx. 5200 bp), also In common with other herpesviruses, the syn-
flanked by inverted repeat sequences (7300 bp). The thesis of VZV proteins broadly involves three
short region can be found in either of two orienta- phases, designated immediate-early (alpha), early
tions relative to the long region, producing two (beta) and late (gamma). More than 30 virus-coded
isomeric forms of the genome which occur in equal polypeptides can normally be detected in VZV-
proportion. Inversion of the long region is rare. By infected cells, ranging in molecular weight from 7K
contrast, in HSV and CMV both regions invert at to over 200K. Among these, at least six groups of
equal frequency, resulting in four isomeric forms. glycoproteins can be identified, with molecular
An inherent size variation of the genome of ap- weights ranging from 38K to 118K, which corre-
proximately 2% (2500 bp) has been demonstrated spond to the gene products of VZV glycoproteins E,
in clinical isolates, concentrated in five variable re- B, H, I, C and L (Table 2B.2). VZV gE mediates IgG
gions. Restriction endonuclease cleavage patterns Fc-binding in a complex with VZV gI, while gB is
show some DNA polymorphism between clinical present in virions as a disulphide-linked complex of
isolates, and strain differences in circulating viruses 140K molecular weight. In addition to the glyco-
are now becoming apparent which may make such proteins, a range of non-glycosylated proteins have
analyses a useful tool in epidemiological studies (see been identified. These include the main protein
below). component of the nucleocapsid (major capsid anti-
Sixty-nine unique open reading frames (ORFs) gen or MCA, 155K), an assembling proteinase/as-
have been identified on the VZV genome, of which sembly protein complex produced from gene 33,
three are repeated, two are spliced and two are and the IE62 protein (175K)—an immediate-early
located entirely within other genes. Over 70 differ- translational activator analogous to the HSV ICP4
ent RNA transcripts have been demonstrated in protein which is present in the virions. Many of the
VZV infected cells but the majority of these appear other proteins of VZV are involved in virus replica-
to contain more than one ORF. The genes that have tion. These include the thymidine kinase and
been identified to date account for virtually the thymidylate synthetase enzymes, several virus-spe-
whole genome, although the presence of overlap- cified protein kinases, a range of DNA-binding pro-
50 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 2B.1 VZV genes for which functional products have been identified
Translation
Gene HSV homologue Protein identity (%) product (kDa) Function
teins, including the viral DNA polymerase, and five (M-6—P) oligosaccharides, present on the surface of
transcriptional regulators produced from ORFs 4, VZV particles, to M-6—P specific receptors on the
10, 61—63 (Table 2B.1). host cell (Zhu et al., 1995).
The tegument proteins prepare the cell to pro-
duce virus and separate from the nucleocapsid dur-
ing transportation of virus particles to the nucleus.
Replication Once within the the nucleus, the linear DNA
genome circularizes.
VZV attaches to the outer membrane of the cell, In common with other herpesviruses, the replica-
and this is followed by membrane fusion and entry tion cycle is coordinately regulated. There are three
of the viral core into the cell. This process is me- basic stages of gene expression and protein syn-
diated primarily by the glycoproteins projecting thesis: immediate early (IE), early (E) and late (L).
from the virus particles, although some tegument The first viral proteins (IE) can be detected 4—6
proteins may also play a role in viral penetration. hours post-infection. Structural proteins (L) such as
Attachment is initiated by binding to heparan sul- the major capsid protein and viral glycoproteins are
phate proteoglycan on the cell surface. This binding not detected until 18 hours post-infection. Viral
is followed by attachment of mannose 6—phosphate protein synthesis appears to reach maximum levels
VARICELLA ZOSTER 51
Table 2B.2 VZV
gE gE 73 N-linked
81 O-linked
90 Sialyiation
98
(gpl) (22)
gB gB 100 N-linked
126 O-linked
130 Sialylation
140 Proteolysis
66, 68
(gpII) (45) Sulphation
gH gH 79 N-linked
98 O-linked
118
(gpIII) (25)
gI GI 35 60
(gpIV) (23)
gC gC 58 100
(gpV) (23)
gL gL 18 20
(gpVI) (18)
at 46—48 hours post-infection in some cell culture proteins are bound. In the normal course of events
systems. it appears that a second membrane derived from
IE transcripts are translated in the cytoplasm and TGN forms a transport vesicle around the mature
the IE (regulatory) proteins are then transported virions which then leave the infected cell by a pro-
back into the nucleus where they induce the E gene cess of exocytosis. In some cells, however, the
expression and down-regulate further IE gene tran- virions appear to be aberrantly processed and are
scription. The E proteins include those which are transported to digestive lysosomes, which possibly
required for VZV DNA replication, such as viral accounts for the very low level of cell-free infectious
DNA polymerase, viral thymidine kinase and pro- virus found in VZV infected cell cultures (see be-
tein kinases. As with the IE proteins, the E proteins low).
are not generally present in the virus particles and
are only detected in virus-infected cells.
The L proteins can be subdivided into two cate-
gories: the early-late proteins which do not require Growth in Cell Culture
the synthesis of viral DNA, and the late-late pro-
teins which are dependent on new viral DNA syn- Varicella zoster virus will replicate, with varying
thesis. DNA synthesis occurs by the movement of degrees of success, in cultures of most cells of human
the DNA polymerase around the circularized and several of simian origin. Cells from non-pri-
genomic DNA, producing head-to-tail polymers of mates are generally resistant to infection but the
the viral genome (concatamers). This is referred to virus has been adapted to embryonic guinea-pig
as a ‘rolling circle’ mechanism. After translation of cells and passage in these cells was an early stage of
late mRNA, the structural proteins that will form the attenuation of the virus used in the current live
the viral capsid are transported back to the nucleus vaccines. The behaviour of VZV in cell culture can
and are assembled around a core of the viral assem- be regarded as being intermediate between that of
bly protein. The newly transcribed DNA genome is HSV, which will grow in nearly all cell cultures, and
inserted into the assembled capsid, concurrent with CMV, which will grow only in few cell types of
the loss of at least some of the assembly proteins human origin. In other respects, VZV behaves more
(Harper et al., 1995). Capsids acquire a ‘temporary like CMV. For example, it grows slowly even in the
envelope’ from the inner nuclear membrane and most sensitive cell systems, with the cytopathic ef-
move to the trans-Golgi network of membranes fect (Figure 2B.3) taking from 3 days to over 2 weeks
(TGN) where the final stages of assembly take place. to appear. This process may be mediated by apop-
The nucleocapsid acquires an envelope containing tosis rather than direct cell killing. VZV remains
the viral glycoproteins and to which the tegument even more strongly cell-associated than CMV, and
52 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
reveals that these are multinucleated, resulting from
virus-induced cell fusion. Staining of the affected
cell sheet reveals another feature of VZV
cytopathology, namely that many of both the
mononuclear and the multinucleated cells have ir-
regularly shaped intranuclear inclusions (Figure
2B.4). With time, the infection ultimately spreads to
involve the whole cell sheet.
Figure 2B.4 (a) Stained VZV-infected human embryo lung fibroblasts. Note the rounded and multinucleated cells (arrow). (H&E) (b)
High-power view with many affected cells showing typical intranuclear inclusions
ces have also been observed in the amino terminal occur, indicating that these two viruses share
region of ORF 10 (a tegument protein) which may common antigens. It has not been shown conclus-
be useful for identifying strains of Japanese origin, ively on which of the viral proteins the responsible
including the Oka vaccine strain. However, the bio- epitope (or epitopes) is situated. The gB glycop-
logical significance of such minor antigenic differen- roteins of VZV and HSV have cross-reacting epi-
ces is currently unknown. topes but it has been difficult to demonstrate any
Some serological cross-reaction with HSV does significant cross-reactivity with other proteins, in
54 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
spite of the sequence homologies between the two
viruses. Thus, HSV and VZV cannot be regarded as
closely related antigenically. The cross-reactivity
manifests most prominently when an individual
previously infected with one of the viruses becomes
infected with the other virus. This would appear to
be somewhat analogous with the theory of ‘original
antigenic sin’ relating to influenza viruses. This pos-
tulated that when individuals become infected with
a new influenza virus they not only develop antibo-
dies to the new strain but also boost the levels of
antibody to any different but antigenically related
strains they have previously encountered. This the-
ory cannot entirely explain the heterologous reac-
tions between VZV and HSV since it is known that
a small number of children, with no previous expo-
sure to VZV but who are experiencing primary
HSV infection, go on to develop low levels of anti-
body transiently which react with VZV. The con-
verse has also occasionally been observed with pa-
tients experiencing primary VZV infection. It is not
known whether this cross-reactivity extends to cel-
lular immune responses or whether it confers any
cross-protection between the two viruses.
It is also known that there is appreciable cross-
reaction between VZV and simian viruses which
cause varicella-like illnesses (see below). The level of
cross-reactivity between these viruses is such that
administration of live human varicella virus will
prevent the development of clinical illness in ani-
mals subsequently infected with the simian virus.
Following infection with VZV, antibodies are
produced to the various structural and non-struc-
tural proteins of VZV. Up to 30 protein bands can
be detected with convalescent sera by radioim-
munoprecipitation or immunoblotting. The pre-
dominant immunogenic components of the virus
appear to be the glycoproteins, the major capsid
protein and the assembly protein complex (Harper Figure 2B.5 Typical immunoblot patterns for IgG and IgM
et al., 1988). Both IgG and IgM antibodies react antibodies obtained with early convalescent sera from varicella
with these proteins (Figure 2B.5). Typically, sera and zoster cases. The viral proteins were separated on a 9%
polyacrylamide gel, electroblotted on to nitrocellulose and reac-
from cases of zoster react more strongly and reveal ted with the sera. IgG and IgM antibodies were detected with
a wider range of proteins compared to varicella. In I-labelled sheep antihuman IgG or IgM. The bands revealed
addition, gE, gB and gH have been shown to be by zoster sera are more numerous and more intense
potential targets for the cell-mediated immune re-
sponse against VZV. Firm data on other glyco-
proteins are still lacking in this area. Interestingly, Pathogenicity for Animals
the translational activator IE62 also appears to
be a target for cellular responses (Arvin et al., 1991), Animals other than humans are reputed not to be
while the assembly proteinase/protein is a susceptible to VZV but there are reports of success-
poor target. ful infection of primates such as gorillas and chim-
VARICELLA ZOSTER 55
panzees, and also marmosets. It has also been Nevertheless, it is widely believed that transmission
shown that guinea-pigs can be infected with virus occurs from this site, probably from symptomless
which has been passaged in embryonic tissue ob- oral lesions which are present before the skin erup-
tained from these animals, leading to virus replica- tion appears. Whatever the case, VZV undoubtedly
tion in the nasopharynx, viraemia and an immune is transmissible via an airborne route and does not
response, but without a rash. Hairless guinea-pigs require close personal contact. The clinical attack
have been reported to develop a papular rash fre- rate of varicella in outbreaks ranges typically from
quently after inoculation but this model remains 80 to 90% of susceptible individuals, which com-
challenging to use. Some progress has recently been pares well with other viruses transmitted via the
made on studies of VZV latency (see below) using respiratory route, e.g. 90% for measles.
VZV-infected rats in which latent virus appears to The proposed model for the pathogenesis of
behave in a fashion similar to that seen in human varicella is shown in Figure 2B.6. It is presumed, as
ganglia. However, no in vivo reactivation is ob- with the vast majority of human virus infections,
served in this model. that the respiratory tract is the portal of entry.
The lack of a good animal model of human VZV Again it is presumed that after an initial phase of
infections has hampered our understanding of the replication at the site of entry, the virus spreads to a
pathogenesis of these diseases. In passing it should distant site, the lymphoid system, where a second
be mentioned that outbreaks of varicella-like illness phase of replication takes place. What is certain is
have been reported in several specimens of monkeys that after a period of about 14 days the virus arrives
from primate centres throughout the world and at its main target organ, the skin, and the final phase
viruses which are similar to VZV have been isolated of replication occurs there. It is likely that the virus
from these monkeys. These viruses, which are im- spreads to and replicates in many other organs of
munologically indistinguishable from one another, the body, particularly the lung. In most cases, the
are partially related to VZV, showing 70—75% infection at these sites does not manifest clinically,
DNA homology across the genome, and are cur- presumably because little or no damage results
rently being evaluated in an attempt to establish a from it. However, in those cases when extensive
useful model of VZV infection in humans. Studies of infection involves organs such as the lung or brain,
simian varicella virus continue to identify similari- serious disease can result.
ties to human VZV (Pumphrey and Gray, 1995) but There is considerable clinical evidence in support
the applicability of data obtained from such a of this proposed model for the pathogenesis of
model directly to humans is limited. varicella, and leucoviraemia has been demonstrated
during the incubation period in healthy, as well as
in immunocompromised, patients. For a full dis-
cussion of this subject see Grose (1987).
Pathogenesis Histological examination of the skin lesions of
both varicella and zoster shows focal degenerative
Our knowledge about the pathogenesis of VZV- changes in the epidermis. The affected cells are
induced diseases, particularly the primary infection, swollen and many of these contain well defined
is still limited due to the difficulty in growing the eosinophilic intranuclear inclusions. Multinuc-
virus and the lack of a suitable animal model. leated cells, also with intranuclear inclusions, are
Surprisingly little is known about the source and characteristically seen at the base of the lesion (Fig-
route of transmission of the virus. The skin lesions ure 2B.7). The histology of the skin lesions is thus
are certainly teeming with infectious virus, even at essentially similar to the cytopathology seen in cell
the maculopapular stage. Airborne transmission culture. The lesion extends and its centre fills at first
from skin lesions is therefore highly likely, especial- with clear fluid, which then becomes cloudy due to
ly since VZV DNA is readily detected by poly- the influx of inflammatory cells.
merase chain reaction (PCR) in the air surrounding Termination of the infection at this site is in-
patients with VZV infection. It is virtually impossi- dicated by the drying up of the pustules, which is
ble to isolate infectious virus at any stage from the followed by separation of the scabs and regener-
upper respiratory tract of cases of varicella, al- ation of the epithelium. Termination must be
though viral DNA may be detectable by PCR. brought about by both the humoral and the cellular
56 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2B.6 Proposed model for the pathogenesis of varicella. (Reprinted by permission from C. Grose (1981) Pediatrics. 68, 735—737;
copyright 1981)
Figure 2B.7 VZV skin lesions. (a) Low-power view showing inflammatory exudate into the vesicle. (b) High-power view showing
multinucleated cells (arrow). Many of the affected cells have typical intranuclear inclusions
latency-associated transcripts (LATs) and se- regions of the genome are actively transcribed dur-
quences required for neurovirulence are contained ing latency. Transcripts appear to be produced from
within these regions. The absence of LAT genes has ORFs 21, 29, 62 and 63 and there is evidence that
initiated the search for alternative regulators in (unlike the situation with HSV) a protein—the
VZV. In vitro studies have shown that while the ORF 63 product—is produced during latency
majority of the VZV genome is inactive during (Mahalingam et al., 1996).
latency, at least two and possibly three discrete Intense destructive inflammatory changes occur
58 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2B.8 Section of a posterior root ganglion obtained from a patient with herpes zoster. Note the intense inflammatory exudate
and degenerate neurons. (H & E)
in the involved ganglion during reactivation (Figure of the disease or they may develop the disseminated
2B.8) and this may be reflected in the severe pain form of the disease. The term ‘disseminated’ implies
which frequently is experienced in association with that the virus spreads through viraemia from the
zoster. It has been suggested from studies in the rat affected dermatome to infect the skin or other organ
model that the site of latency is in satellite cells, at some distal site, producing lesions which are
rather than in neurons, and this would require a similar to those of varicella in their appearance and
lytic cycle of infection initially to infect the sensory distribution. In recent years it has also been shown
neurons during reactivation. This could explain the that the appearance of zoster correlates with in-
observed damage in the ganglia and the frequent creasing immunodeficiency in patients infected with
prodromal pain experienced during zoster. How- human immunodeficiency virus (HIV), which pri-
ever, more recent studies have shown that latent marily affects cellular immunity.
VZV is present in neurons (Kennedy et al., 1998) Most cases of zoster occur spontaneously but
and the reason for ganglionic damage remains un- trauma and stress are well-known triggers of reac-
resolved. The failure of the host defence mechan- tivation. It has been suggested that re-exposure to
isms to contain the virus in the ganglia after such varicella may trigger zoster but this is based only on
prolonged periods of time is not understood. In anecdotal reports.
immunocompetent individuals it is probably due to
the decline of the effectiveness of previously ac-
quired cell-mediated immunity to VZV with ad-
vancing years. Inadequacy of cell-mediated immun- EPIDEMIOLOGY
ity is likely to be of critical importance since
patients with Hodgkin’s and similar diseases are Cases of varicella are seen throughout the year but
more likely to experience zoster. These patients are they occur more frequently in the winter and early
also more likely to experience more than one attack spring months. The seasonal incidence of the dis-
VARICELLA ZOSTER 59
Figure 2B.9 Age prevalence of antibodies to VZV. The antibodies were detected by both RIA and indirect immunofluorescence
procedures. The numbers of each age group tested are given at the ends of the columns. (These results were obtained in a collaborative
study with Dr J. Cradock-Watson, Withington Hospital, Manchester)
ease is therefore roughly similar to that of other hood. Seroprevalence studies generally show that
systemic diseases such as measles and rubella, and less than 10% of young adults are still susceptible to
the respiratory viral infections, but quite unlike that varicella (Figure 2B.9), although the rate of increase
of the enteroviruses. Because of the comparatively of seropositivity during preschool and school years
small variation in the seasonal incidence of may differ depending on local variations in expo-
varicella, it is preferable to regard it as a disease sure to the virus. Recent surveillance reports from
which shows variation in seasonal endemicity the UK (Royal College of General Practitioners)
rather than an epidemic disease. Annual variation and the USA indicate that the age-specific pattern
in incidence also occurs with higher than average of varicella may have changed in recent years. First-
incidence in 3—4 year cycles. Herpes zoster, in con- ly, there seems to be a trend towards higher inci-
trast, occurs sporadically and evenly throughout dence of varicella in preschool children, which may
the year. Some of the cases of varicella which occur be related to an increased use of daycare and play-
away from the peak periods are due to contacts with group facilities, leading to greater exposure at a
zoster. younger age. Secondly, the incidence and overall
Varicella is one of the most common communi- proportion of cases in adults (aged over 15 years)
cable diseases worldwide and is predominantly a appears to have increased during the past 20 years
disease of childhood. However, different parts of the (Fairley and Miller, 1996). Before 1975, only 10% of
world can show significant variations in the age reported cases of varicella in the UK were in adults,
distribution of infection. In temperate areas it is one but this had risen to approximately 20% by
of the classic diseases of childhood, with the highest 1989—1990. This is also reflected in increased con-
incidence occurring in the age group 4—10 years sultations for varicella in general practices, in-
(Figure 2B.9). Infections before this age are relative- creased hospitalization rates and more deaths
ly uncommon and include cases in the preschool among adults. It is now estimated that there are
years and also the rare congenital and neonatal approximately 3000 hospital admissions for
infections, which will be discussed below. In general, varicella per year in the UK, of which 40% are
varicella is highly communicable in temperate adults. There is evidence of a similar trend in the
countries, with a reported attack rate of up to 96% USA with a five-fold increase in hospital admission
in close contacts (i.e. household or play-friend), and rates for varicella in adults in the past two decades
therefore most people become infected before adult- (Choo et al., 1995). The reasons for this shift in
60 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
incidence to older age groups are not known but same as that reactivating as zoster some years later
smaller families, having the first child at an older and that Oka vaccine strain can be distinguished
age, and immigration of adults from countries with from wild-type viruses in the USA (Adams et al.,
different varicella epidemiology (see below) may be 1989). Most RFLPs are generated by variations in
contributing factors. the numbers and sequence of repeats in the five
It is not known what role different environmental (GC)-rich repeat regions, R1—R5, in the genome.
factors or different virus strains (see below) may RFLP differences between the Oka vaccine strain
play in determining the epidemiology of varicella and wild type American strains have been described
but the patterns of infection and disease can be very (Adams et al., 1989), while PCR across a Bgl 1
different in tropical regions compared to those of restriction site in gene 54 and a Pst 1 site in gene 38
temperate regions. In many tropical countries, has simplified the identification of Oka which, un-
varicella is predominantly a disease of adults, with a like VZV in USA, is Pst 1 9 Bgl 1 ;. The Oka
mean age of 20—25 years, or even as high as 38 years strain also differs from all UK strains tested but is
reported from St Lucia. Studies from the Indian genetically indistinguishable from about 3% of wild
subcontinent, South-East Asia and the Caribbean type Japanese strains. It is probable that, as with
have shown that between 25 and 60% of adults over herpes simplex, geographical variations reflect se-
15 years old are susceptible to varicella. Interesting- lection by host factors. However, it is interesting to
ly, there is also evidence of shifting epidemiology in note that people from the Indian subcontinent, Sin-
these regions. Thus, the peak incidence of varicella gapore and Africa, despite their genetic differences,
in Singapore has shifted from 5—14-year-olds in may carry similar strains of VZV (J. Breuer, unpub-
1982, to 15—24-year-olds in 1990 (Ooi et al., 1992). It lished data).
appears that very little transmission occurs
amongst the young children in these areas, even
though most of them live in crowded conditions
and are in frequent contact with infected adults of CLINICAL FEATURES
their family. The reason for this is not understood
but a number of theories have been put forward, Varicella: The Primary Infection
including that it is due to competition with other
viruses infecting the respiratory tract. Another pos- The incubation period of varicella is approximately
sibility is that in these countries exposure in the 2 weeks but a range of 7—23 days has been quoted. A
early months of life is so common that infants ex- shortened incubation period can be encountered,
perience asymptomatic infection due to the pres- particularly in immunocompromised patients. A
ence of maternally transferred antibodies. Latency potential source of error in these estimates will be
perhaps does not always follow such infection and the infections that are acquired from asymptomatic
some individuals thus become susceptible later in cases.
life. The illness usually commences with the appear-
It is important in hospital practice in temperate ance of the rash but occasionally there are pro-
climates to be aware of the fact that staff who were dromal symptoms that resemble an influenza-like
born in tropical countries are more likely to be illness. These symptoms appear a few days before
susceptible to varicella. the rash and are seen more frequently in adults than
in children.
The rash is characteristically centripetal in dis-
tribution and is seen mainly in areas that are not
Molecular Epidemiology exposed to pressure, such as the flanks, between the
shoulder blades and in the axillae. It is generally
Using restriction fragment length polymorphisms sparse in the antecubital and popliteal fossae and is
(RFLPs) and PCR, strains of VZV in the USA have rarely seen on the palms or the soles. This distribu-
been shown to be related, and distinct from strains tion is markedly different from the more centrifugal
circulating in Japan (Takada et al., 1995). Restric- distribution of the smallpox rash, a distinction
tion analysis has also been used to prove that the which used to be of considerable diagnostic import-
virus causing chickenpox in an individual was the ance. Other differentiating features were that the
VARICELLA ZOSTER 61
Figure 2B.10 Varicella pneumonia. Area of consolidated lung with typical multinucleated cells. (H & E)
lesions of smallpox are rounder and deeper than the Streptococci group A and staphylococci are most
more superficial and irregular shaped lesions of frequently involved and may be life threatening in
varicella. the immunocompromised. Of those children requir-
The skin lesions progress fairly rapidly through ing hospitalization for pneumonia associated with
the stages of macules and papules to vesicles which VZV, over 30% may have bacterial pathogens.
rapidly break down with crust formation. The ves-
icle with its surrounding area of erythema is the
Viral Pneumonia
most characteristic feature of the rash. The lesions
appear in a series of crops so that all stages in their Symptomatic varicella pneumonia occurs in 1 per
genesis can be seen at any one time. This is very 200 000 cases of varicella in children, rising to 1 per
different from smallpox, where the lesions were al- 200 in immunocompetent adults, although radio-
ways at the same stage. Patients with varicella are logical changes may be found in 10 times this numb-
generally considered to be infectious from a couple er. In pregnant women the incidence of pneumonitis
of days before the rash until new vesicle formation is increased to 9%, while 10% of smokers develop
has ceased and existing vesicles have crusted. This the disease. Those most at risk are the immunocom-
usually occurs 5—7 days after onset but may be promised, including children with leukaemia, in
longer in the immunocompromised. The crusts sep- whom up to 32% develop pneumonitis with a mor-
arate usually within 10 days, revealing healthy skin, tality of up to 25% (Feldman and Lott, 1987).
but a minor degree of scarring is common. The Adults with malignant disease of the lymphoreticu-
general constitutional symptoms of the illness are lar system who have received organ transplants,
typically mild, particularly in children. and patients taking systemic steroids, are also at
increased risk.
Pneumonia complicating varicella begins 1—3
Complications of varicella (range 1—6) days after the onset of rash and the
clincial features include dry cough, dyspnoea,
tachypnoea with chest pain; haemoptysis and cy-
Sepsis
anosis occur less frequently. Over 90% of patients
Secondary bacterial infection is by far the most with chest symptoms will develop pneumonitis and
common complication of varicella, especially in all such patients should be admitted to hospital for
children, and causes increased scarring of the skin. antiviral therapy. Histological examination of an
62 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
area of lung affected by VZV shows alveoli filled children and more commonly in adults. Cerebral
with oedema fluid, a few foamy macrophages and symptoms are more common in adults, while cere-
other round cells. The absence of an extensive out- bellar ataxia occurs in children. In the majority of
pouring of polymorphonuclear leucocytes is a fea- cases symptoms begin 4—8 days after onset of rash.
ture which distinguishes this disease from bacterial Cerebrospinal fluid (CSF) examination may be nor-
pneumonia. The presence of multinucleated giant mal or reveal a lymphocytosis, with a high cell
cells with intranuclear inclusions (Figure 2B.10) is count and protein level being more likely in en-
pathognomonic, although somewhat similar cells cephalitis than in cerebellar disease. Typical cases of
are seen in measles pneumonia. It is important to encephalitis presenting with headache and vomiting
realize that these multinucleated cells are not al- and proceeding to coma are rarely seen but carry a
ways seen, but large swollen mononuclear cells, high mortality. By contrast, most patients with
similar to cytomegalic cells, but without the domi- cerebellar disease recover fully, although it may
nant intranuclear inclusion, are always present. Cli- take some months. Other neurological disorders
nical diagnosis is confirmed by chest X-ray changes including meningitis, transverse myelitis and
and direct immunofluorescence of vesicle fluid and Guillain—Barré syndrome have also been associated
nasopharyngeal secretions using a fluorescein-con- with the disease.
jugated monoclonal antibody or specific IgM anti- As with varicella pneumonia, central nervous sys-
bodies in the serum. tem (CNS) involvement occurs much more fre-
The illness runs a fulminating course if not quently in immunocompromised patients and is an-
treated early with high dose intravenous acyclovir other important factor contributing to the
and is the single most common cause of varicella- increased mortality of the disease in these patients.
associated death in the immunocompromised. Sur-
viving patients may recover completely but others
develop fibrosis of the lungs with permanent respir- Other Unusual Manifestations and
atory impairment. Complications of Varicella
As mentioned under Pathogenesis above, VZV in-
Haemorrhagic Chickenpox fection may involve virtually any organ of the body
but, apart from the skin and occasionally the lung
Haemorrhagic symptoms sometimes occur during and brain, it is unusual for infection at other sites to
the course of varicella and usually make their ap- manifest clinically. Nevertheless, myocarditis, ar-
pearance on the second or third day of the rash. thritis, hepatitis and both renal and ureteric damage
Haemorrhage typically occurs into the skin but associated with varicella have been reported.
epistaxis, melaena or haematuria may be additional Reye’s syndrome is a serious and frequently fatal
presenting features. The haemorrhage may be so form of encephalopathy which is secondary to liver
severe as to be life threatening. This complication is damage occurring in children with varicella or in-
more commonly seen in immunocompromised pa- fluenza and is associated with ingestion of aspirin.
tients, such as renal transplant recipients in whom a
The incidence of this has fallen with strictures on
thrombocytopenia and/or consumptive coagulo-
the use of salicylates in children.
pathy may also develop. Other clinical features in
these cases often include hepatitis and gastrointes-
tinal bleeding or distention. Treatment is with high-
dose intravenous antivirals (acyclovir or analogues) Varicella in Pregnancy
and intensive care. Intravenous immunoglobulin,
although it has not formally been evaluated, is also From Figure 2B.9 it can be seen that approximately
commonly used. The addition of steroids is more 10% of women of childbearing age are still suscep-
controversial. tible to varicella but this figure may be as high as
20% in communities with a high proportion of
immigrants from India and Africa. The incidence of
Encephalitis
varicella in adults aged 15—44 years has been es-
Varicella meningoencephalitis necessitating admis- timated at 3 cases per 1000, which would result in
sion to hospital, occurs in 3—4 cases per 100 000 about 2000 cases per year in pregnant women in
VARICELLA ZOSTER 63
Table 2B.3 Clinical features of the congenital varicella
syndrome Varicella in the Late Stages of Pregnancy
The clinical presentations of both varicella and her- Typical herpesvirus particles can be seen in profu-
pes zoster are usually so typical that laboratory sion in fluid taken from early vesicles of either
confirmation is rarely required. Notwithstanding, varicella or zoster. The particles can also be seen in
in one series of shingles diagnosed clinically in the emulsions of material scraped from the base of
community, 15% turned out to be due to HSV lesions. The particles are more difficult to see when
infection or non-herpetic (J.Breuer, unpublished the specimens are taken late in the disease. EM will
data). Where the distinction between HSV infection not distinguish between VZV and HSV infection
and herpes zoster is difficult, such as in a generaliz- unless combined with immunological techniques.
ed rash occurring in an immunocompromised pa-
tient or where atypical lesions occur, laboratory
Cytology
confirmation should be sought. Similarly, labora-
tory diagnosis may also be useful for some of the Smears of scrapings of the base of the lesions,
rarer CNS and occular complications affecting im- stained by Papanicolaou’s method or, for quick-
munocompromised patients. The VZV antibody ness, methylene blue, will reveal characteristic
status of an individual is also commonly required multinucleated giant cells (Figure 2B.11), also
now that treatment and prophylactic measures are known as Tzanck cells. Although not a routine
readily available, and also because of the increasing procedure, microscopic examination of biopsies of
problem of nosocomial varicella outbreaks which the lesion will also reveal these giant cells as well as
require prompt intervention. the other characteristics of the histology which have
VARICELLA ZOSTER 67
been described above. Again, it is unfortunate that able for isolation attempts. Supernatants from
neither cytology or histology will distinguish be- emulsion of affected organs such as the lung, ob-
tween HSV- and VZV-induced lesions. tained at postmortem examination or by biopsy,
can also be used. Virus can rarely, if ever, be re-
covered from crusted lesions, the upper respiratory
Immunofluorescence Cytology tract or blood.
A more specific diagnosis can be made if immuno- With virus-containing specimens, the character-
fluorescence or immunoperoxidase examination of istic cytopathic effect of VZV will ultimately be
the smears is carried out. Even cells from crusted produced (Figure 2B.3). Occasionally, clinical iso-
lesions contain viral antigen in abundance, allowing lates only show a restricted cytopathic effect on
easy detection. This method is therefore particular- primary isolation and require ‘blind passage’ on to
ly useful at a time when EM or virus isolation (see fresh fibroblast cultures before the classical
below) may not be reliable. Direct detection of VZV cytopathic effect develops.
in cells scraped from the base of a vesicle is now It is possible to increase the speed of isolation by
easily achieved using a fluorescein isothiocyanate centrifugation-enhanced infection (‘shell-vial’ cul-
conjugated monoclonal antibody. This simple ture). In addition, immunological staining tech-
method has replaced indirect immunofluorescence. niques utilizing monoclonal antibodies can be used
to detect viral antigens in cell cultures within 24—48
hours of inoculation, before cytopathic effect be-
comes apparent. (Figure 2B.12).
Detection of Viral DNA
Virus Isolation
Serological Diagnosis
This remains the most definitive method for diag-
nosing VZV infections. It is important that the cell A number of different methods are currently avail-
culture used can easily be maintained for up to 21 able for the serological diagnosis of VZV infection,
days because this may be required for some isola- but perhaps the most important use of this technol-
tions. It is for this reason that human fibroblast ogy is the determination of immune status of pa-
cultures are used in most laboratories. tients prior to the administration of prophylactics.
Vesicle fluid or material swabbed from the base of The serological diagnosis of varicella using acute
fresh lesions are the specimens which are most suit- and convalescent sera is easily accomplished but is
68 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2B.12 Immunofluorescence staining of a cell sheet of human embryo lung fibroblast cells infected with VZV. An indirect
method with monoclonal antibody was used
less reliable for herpes zoster. Sera obtained in the and HSV will have shown significant rises in associ-
early stages of varicella are either devoid of or con- ation with a particular illness; however, without
tain only low levels of specific antibody, whereas additional information, such as virus isolation data,
these antibodies are present in high titre in sera it may be impossible to determine which of these
taken in the convalescent period. While significant viruses was responsible for the infection.
rises in antibody titre in paired sera can be demon-
strated in cases of zoster, this is in general only
Complement Fixation (CF)
possible if the first serum is taken soon after the
onset of the rash, for the reason that pre-eruption This test is now all but obsolete for the diagnosis of
sera will always contain some specific antibodies VZV as it is too slow, not sensitive enough for the
and the titres rise very rapidly after onset. In cases of determination of immune status and more suscep-
zoster it is not uncommon to see a drop in the tible to cross-reaction between VZV and HSV than
pre-eruption antibody level around the time of on- other tests.
set so that the antibodies may barely be detectable
in sera taken within the first 2 days after appearance
Immunofluorescence (IF)
of the rash. Testing for avidity of IgG antibodies
provides a means of distinguishing between pri- This method provides a sensitive determination of
mary and anamnestic antibody responses. serological status to VZV but has now been super-
The sharing of antigens between VZV and HSV seded by enzyme immunoassays in most diagnostic
(discussed above) sometimes makes the interpreta- laboratories. In IF tests, serial dilutions of sera are
tion of serological results difficult. It is quite fre- reacted with VZV-infected culture cells, and any
quently found that levels of antibodies to both VZV specific antibodies attaching to these cells are detec-
VARICELLA ZOSTER 69
ted with a fluorescein-conjugated antihuman IgG l00% of convalescent sera, and it can be detected for
serum. There are two variants of this basic test. One about 3 months from the onset of the illness. Tests
is the standard IF procedure in which sera react for specific VZV IgM are therefore useful for diag-
with acetone-treated culture cells and is therefore nosing recent infection when the only sera available
capable, at least theoretically, of detecting antibo- are those taken late or after the termination of the
dies to all virus-induced proteins. The fluorescent illness.
antibody to membrane antigen (FAMA) technique It is, unfortunately, not possible to use these tests
uses glutaraldehyde-fixed cells and is designed to to distinguish reliably between primary and recur-
detect only antibodies to viral antigens that appear rent VZV infection, since specific IgM antibodies
on the surface of infected cells. Consequently are also induced in most cases of herpes zoster. In
FAMA should, in theory, specifically detect those these patients, however, the amount of IgM anti-
antibodies which are concerned with protection. body is generally lower than that found in varicella
cases and is also of shorter duration (Figure 2B.13).
The VZV IgM tests are likely to prove valuable
Enzyme Immunoassays (EIAs)
for determining the nature of congenital varicella
These are generally regarded as being the most infections.
sensitive methods for detecting specific antiviral
antibodies, and are therefore the preferred method
for determining immune status, but they have no
advantage over CF for routine diagnostic purposes. MANAGEMENT
Assays of the indirect solid phase kind are used
most frequently but a competitive type assay has Varicella
also been described which should, theoretically, not
be affected by cross-reacting antibodies to HSV. Varicella in healthy individuals is generally mild
Several EIAs are now commercially available and complications are rare. Where treatment is in-
and are for practical reasons the most commonly dicated, the drug of choice is acyclovir, a nucleoside
used method for determining immune status. analogue which blocks viral replication. VZV is less
susceptible to acyclovir than is HSV and requires
approximately a tenfold higher concentration of the
Neutralization
drug for effective inhibition. The inhibitory concen-
Theoretically this should be the method of choice tration (ID50) of acyclovir for VZV in cell culture is
for determining VZV immune status, since it usually in the range 2—20 mol L\ depending on
measures antibodies concerned with protection. the cell type and virus strain used. It is possible to
However, current procedures are technically very obtain adequate inhibitory concentrations in the
difficult to carry out, due mainly to the difficulty of blood if a dose of 10 mg kg\ (or 500 mg m\ for
obtaining a consistent supply of challenge virus, children 12 years old) is given intravenously over
since virus infectivity is so highly cell associated. It a 1 hour period every 8 hours. This dosage main-
is also an insensitive test. For these reasons, it has tains plasma levels in the range 10—90 mol l\.
no role in routine diagnosis. Duration of treatment is normally 5—10 days, de-
pending on the severity and progression of the dis-
ease, but a minimum of 7 days is recommended for
Detection of VZV-Specific IgM Class
immunocompromised patients and adults with vis-
Antibodies
ceral complications. Oral acyclovir is poorly ab-
This class of antibody can be detected using the sorbed and the recommended dose of 800 mg 5
indirect IF or immunoassay procedures in which times a day, will give blood levels of 4—8 mol l\,
the labelled antibody used in the system is directed which are only just at the ID50 concentration for
against human IgM class antibodies. Procedures in VZV. Studies in children (Dunkle et al., 1991) and
which the IgM antibodies are captured on to the adults (Wallace et al., 1992) have shown that anti-
solid phase (MACRIA or MACEIA) can also be viral treatment must be started within 24 hours of
used. VZV IgM may not initially be detectable in the onset of rash to be effective. In immunocom-
sera from patients with varicella but is present in petent patients, the duration of rash and fever are
70 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2B.13 Detection and duration of the specific IgM response in patients with varicella and herpes zoster. MACRIA was used for
the antibody determinations
reduced by treatment with acyclovir but none of the have not yet been licensed for the treatment of
studies has had the power to show whether treat- varicella.
ment reduces the risk of complications. A detailed review and recommendations for the
The nucleoside analogues famciclovir (Famvir2+) management of varicella in different patient groups
and valaciclovir (Valtrex2+), both of which are bet- have recently been prepared by the UK Advisory
ter absorbed orally (50—70%) than acyclovir (20%), Group on Chickenpox for the British Society for
metabolize to produce blood levels of active drug the Study of Infection (Carrington and McKen-
equivalent to intravenous acyclovir. However, these drick, 1998).
VARICELLA ZOSTER 71
Varicella in Children whether they need intravenous acyclovir and anti-
biotics. For detailed algorithms for the manage-
For the typical childhood case, no treatment is
ment of varicella in adults see Wilkins et al. (1998).
required, apart, perhaps, for soothing lotions for
itching and antibiotics if there is any question of
secondary infection. There is some evidence that Varicella in the Immunocompromised
secondary cases of varicella acquired within a fam- Patient
ily are more severe and treatment of such cases,
particularly adolescents, is advocated by some, al- Varicella in the immunocompromised can be a seri-
though current UK consensus is not to use oral ous, even fatal, illness and consequently its manage-
acyclovir routinely in healthy children. Children on ment in these patients is different from that in a
inhaled or intranasal steroids are not considered to previously healthy individual. Immunocom-
be at special risk but such cases should be consider- promised patients, including those on systemic ster-
ed individually. No great pressure is usually exerted oids (including for up to 3 months previously),
on parents even to quarantine affected children and, should be aware of their immune status and, where
indeed, there are some who advocate that it is pre- possible, i.e. in all except those with lymphoreticu-
ferable for children to contract the disease to ensure lar malignancies, be immunized with live attenuated
that immunity is acquired at an early age. Oka vaccine. Where this is not possible, patients
should be counselled against contact with patients
with varicella or zoster as well as being advised to
Varicella in Adults seek medical help immediately if contact occurs.
Where the patient has no immunity and significant
One in 200 adults will develop clinical pneumonitis exposure has occurred, measures should be taken to
and approximately 1 in 2000 will require intensive prevent or attenuate the infection (see below). Sig-
care. Those most at risk include smokers and pa- nificant exposure has been defined arbitrarily by the
tients with severe chronic lung conditions. Acyc- American Academy of Pediatrics and by the UK
lovir commenced within 24 hours of the onset of Joint Committee on Vaccination and Immuniz-
symptoms does reduce viral shedding and new ation. Broadly there is consensus that significant
lesion formation by 0.5 days, and accelarates rash contact constitutes living in the same house as a
healing by 1—2 days (Wallace et al., 1992). Pregnant case of zoster or chickenpox, indoor contact with a
women are also at increased risk of pneumonitis but case of zoster or chickenpox for a period of time (15
acyclovir is currently not licensed for use in preg- minutes or more in the UK) and face-to-face con-
nancy. However, it has been used successfully in tact with a case of chickenpox.
numerous pregnant women to treat serious VZV There are no data on the efficacy of acyclovir
disease without ill-effect and there is no evidence to prophylaxis in the immununocompromised.
date that it is teratogenic, although it is known to
cross the placenta and can be detected in urine from
infants of mothers who have been treated. It is not
known at present whether treatment with acyclovir Herpes Zoster
has any beneficial effect on fetal varicella syndrome.
Current UK recommendations are therefore to The main aims of therapy in acute herpes zoster
treat varicella presenting within 24 hours in other- occurring in a previously healthy individual, are to
wise healthy adult smokers, patients with chronic heal the rash rapidly, alleviate acute pain, prevent
lung conditions (including adults on inhaled ster- postherpetic neuralgia and reduce the risks of oph-
oids) and pregnant women in the second half of thalmic and neurological complications.
pregnancy (Carrington and McKendrick, 1998).
Adults presenting more than 24 hours after the
Antiviral Drugs
onset of the rash should have their clinical progress
assessed. Those who appear to be deteriorating, for Several drugs are currently licensed for use in the
example with recurrent fever or progressive rash or UK for treatment of acute shingles, including topi-
who develop chest symptoms or signs, should be cal idoxuridine, acyclovir, famciclovir and valacic-
admitted for chest X-ray, gases and assessment as to lovir. Idoxuridine was the first antiviral drug to be
72 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
used for this purpose. It is too toxic to be used Treatment of Established ZAP
systemically but can be administered topically as a
During the acute attack of zoster, mild opiate anal-
40% suspension in dimethyl sulphoxide. This form
gesics are recommended. If pain persists beyond 4—6
of treatment is cumbersome and has now been re-
weeks after the rash, patients can be tried on low
placed by systemic or oral treatment with the newer
dose tricyclic antidepressants such as amitriptyline.
antiviral compounds.
Other recommendations for pain relief include topi-
Patients given high-dose acyclovir (800 mg orally
cal measures such as ice packs, capsaicin, acupunc-
5 times per day) for acute shingles have been shown,
ture and occasionally local injection of sensitive
in placebo controlled studies, to have faster resol-
nerves. Patients with intractable pain should be
ution of the rash (by up to 2 days), have less acute
referred for specialist pain management advice.
pain, reduced viral shedding (by 1—3 days) and fewer
ophthalmic complications. Several studies have
also shown a reduction in the incidence, severity Ophthalmic Zoster
and duration of persistent ZAP in those most at
risk, i.e. over the age of 55 years, when acyclovir is Management of patients with ophthalmic zoster
given within 72 hours of the onset of rash. Valacic- should include topical acyclovir applied to the eye
lovir (a prodrug of acyclovir) and famciclovir (a and oral acyclovir (800 mg 5 times per day), what-
prodrug of penciclovir) give higher blood levels of ever the patient’s age. Treatment should however be
the active drug, and thus allow easier treatment started as soon as possible to be effective and prefer-
regimens, on account of their improved oral bi- rably within 72 hours of onset. It is currently not
oavailability. In addition, penciclovir, and its de- known how effective acyclovir or the newer anti-
rivative famciclovir, have a significantly longer in- virals are in treating chronic complications such as
tracellular half-life compared to acyclovir. A dosing anterior uveitis and stromal keratitis. Early referral
schedule of famciclovir of 250 mg three times a day of patients with ophthalmic zoster to an ophthal-
is as effective as high-dose acyclovir for treatment of mologist is desirable and topical steroids should in
zoster. The newer prodrugs also have the potential no case be administered without specialist consulta-
to prove more effective than acyclovir in reducing tion.
the severity and duration of ZAP (Beutner et al.,
1995; Cirelli et al., 1996). Herpes Zoster in Immunocompromised
Other new drugs which have been developed for Patients
the treatment of zoster include sorivudine
(BVaraU) and brivudin (BVDU). Both these agents The more severe, and particularly the disseminated,
are particularly active against VZV when taken forms of herpes zoster are seen in those who are
orally, and result in accelerated rash healing; how- immunocompromised and may be life or sight
ever, neither appear to affect prolonged ZAP threatening. Patients at high risk should be
(Gnann et al., 1998). Furthermore, when adminis- educated to recognize shingles and to seek medical
tered in combination with 5—fluorouracil, advice early. Highly immunocompromised patients
sorivudine has caused the deaths of a number of should initially receive intravenous acyclovir, fol-
patients. lowed by oral acyclovir if necessary. Treatment
should be continued until the lesions crust, approxi-
mately 5—7 days later. Less severely immunocom-
promised patients with localized shingles can be
given oral acyclovir.
Adjunctive Treatment
Oral prednisolone in addition to acyclovir slightly
Antiviral Drug Resistance
reduces acute symptoms but does not protect
against prolonged ZAP more than acyclovir alone. VZV resistance to acyclovir has been described in
Other treatments, such as sympathetic nerve blocks immunocompromised patients, in particular those
and amitriptyline given acutely, have been reported infected with HIV. There is currently no evidence
anecdotally to reduce pain and require proper trials that such resistant virus strains are transmissible
to assess adequately. but they can present a considerable challenge to
VARICELLA ZOSTER 73
treatment and are therefore of much concern. Al- Antiviral Drugs
most all the resistant strains which have been char-
acterized to date have had reduced thymidine Acyclovir is now routinely used for prophylaxis
kinase (TK) function as a result of mutations in the against HSV infections in immuncompromised pa-
TK gene (Talarico et al., 1993). These mutations tients and may also provide some benefit against
have been either deletions or point mutations lead- VZV. However, studies have not yet been per-
ing to a truncated TK protein, or single point muta- formed to demonstrate the efficacy of such a strat-
tions leading to amino acid substitutions. Muta- egy. Oral acyclovir has been shown to prevent or
tions involved in resistance to acyclovir have been modify varicella in young immunocompetent
demonstrated in different positions in the gene and household contacts given a 7 day course (40 mg
are not only restricted to the ATP or nucleotide kg\ in divided doses) beginning 7—9 days after the
binding sites on the protein. Some acyclovir-resis- contact with the index case, i.e. during the presumed
tant strains have also been shown to be cross-resis- phase of secondary viraemia. Administration within
tant to other TK-dependent drugs, including pen- 7 days following contact, i.e. during primary vir-
ciclovir and sorivudine. However, such resistant aemia, was not as effective, possibly because early
strains are generally found to be sensitive to foscar- priming of T cells is reduced.
net, a broad-spectrum antiviral drug which directly
inhibits viral DNA polymerase and thus provides
an alternative treatment. AIDS patients with acy-
clovir-resistant VZV infections have been success- Passive Immunization
fully treated with foscarnet (40 mg kg\ every 8
hours in a 1 hour infusion over 10 days) in an open Human immunoglobulin preparations with high
study (Safrin et al., 1991) but the optimal dose and titres of antibody to VZV are an established means
duration of treatment are not yet defined. Further- of attempting to prevent varicella. Such prepara-
more, a small number of drug-resistant VZV iso- tions were originally prepared by cold ethanol pre-
lates have been identified which have an altered cipitation from the blood of patients recovering
DNA polymerase. from shingles and were consequently designated
zoster immunoglobulin (ZIG). Current prepara-
tions are obtained by processing preselected sera
from blood donors with high titres of antibody to
VZV. These preparations are referred to as varicella
PREVENTION zoster immune globulin (VZIG) or human anti-
varicella zoster immunoglobulin. VZIG prepara-
The prevention of varicella forms an important part tions are frequently in short supply but antibody
of the overall management of VZV disease in those concentrations in the blood as high as those with
who are at risk of contracting the severe forms of the VZIG have been obtained with intravenous normal
disease. An increase in adult susceptibility to human immunoglobulin (iv-NHIG) preparations
varicella (see above, Epidemiology) also has serious and these may be used empirically as an alternative
implications for hospital infection control, since when VZIG is not available. However, nothing is
medical staff without immunity may become infec- known about the effectiveness of iv-NHIG. NHIG
ted following contact with zoster patients and can, given intramuscularly has been shown not to be
sometimes with disastrous consequences, transmit effective and plays no role in prevention of varicella.
the infection to patients who are immunocom- VZIG is recommended for any susceptible ‘at
promised. risk’ individual (Table 2B.4) who has significant ex-
Many immunocompromised patients will cope posure to varicella or herpes zoster. It should be
normally with varicella but the possibility of admin- administered as soon as possible after contact, pre-
istering VZIG and/or antiviral drugs prophylacti- ferrably within 96 hours, although some studies
cally to these patients should always be considered have shown VZIG to have a beneficial effect when
if they come in contact with VZV. At the moment given up to 10 days after contact. The immune
there is, unfortunately, no means of preventing her- status of the patient should be assessed by testing
pes zoster. for specific antibodies in the serum before adminis-
74 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 2B.4 Underlying or associated conditions which place munocompromised patients. Feldman and Lott
patients at risk of contracting the severe forms of varicella (1987) reviewed the impact of varicella in 280
Leukaemias, Hodgkin’s disease and other neoplasms of the children with cancer and the effectiveness of various
lymphoreticular system, whether or not treatment is being forms of management. In their study group, passive
given immunization significantly reduced both the inci-
Other cancers which are being treated with cytotoxic drugs or dence and mortality of pneumonitis compared to
other regims which are immunosuppressive
Primary immunodeficiency syndromes
untreated children. Even so, pneumonitis developed
Bone marrow transplant recipients irrespective of their own or in 11% of children who received VZIG, requiring
the donors’ VZV status intensive additional antiviral chemotherapy.
Diseases requiring systemic steroids at a dosage equivalent to It is important, particularly in a hospital environ-
at least 2 mg prednisone per kg per day ment, to be aware of the shortcomings of VZIG and
Susceptible pregnant women in close contact with VZV
Newborn infants of women who contracted varicella 7 days
to be alert to the possibility that an inoculated
before or after delivery patient might develop varicella and become a
Premature infants whose mothers have no history of varicella source of infection for others.
or any infant whose birth weight was 1000 g It is also recommended that VZIG be given to
susceptible pregnant women in close contact with
VZV infection at any stage of the pregnancy in the
tration of VZIG whenever possible. Since tests with hope that it will reduce the risk of transmission of
the appropriate sensitivity (see above, Diagnosis) infection to the fetus, and also to ameliorate any
are generally only available in specialist labora- potentially serious VZV disease which can occur in
tories, the administration of VZIG should not be pregnant women. Maternal varicella can still occur
delayed past 7 days after the initial contact while despite VZIG prophylaxis but a large prospective
waiting for the test result. A convincing history of study has shown that, even in such cases, the risk of
varicella or zoster is a reasonably reliable indicator fetal infection during the first 20 weeks of preg-
of immunity and usually obviates the need to ad- nancy, and subsequent fetal. damage, may be reduc-
minister prophylactics. Nevertheless, it is recom- ed (Enders et al., 1994). Should a woman contract
mended that an antibody test is performed to con- varicella perinatally it is important to administer
firm the immune status in immunocompromised VZIG to the newborn baby (Table 2B.4).
patients even if a past history of VZV infection is The recommended dosage for VZIG prepara-
given. Recipients of bone marrow transplants tions available in the UK is as follows: 0—5 years,
should be assumed to be susceptible regardless of 250 mg; 6—10 years, 500 mg; 11—14 years, 750 mg;
past history of varicella because of their severe de- and 15 years or more 1000 mg. A second dose can be
gree of immunosuppression. given after 3 weeks if necessary.
The true efficacy of VZIG has not been estab-
lished in well-controlled trials and differences in the
results of different studies may reflect different po-
tencies of the preparations used. It is known that Active Immunization
VZIG gives incomplete protection against infec-
tion. In a study carried out in the UK it was shown Although individual cases of varicella may be pre-
that of 27 seronegative children who were in contact vented or modified by VZIG or with antiviral
at home, 18 (67%) became infected—14 (52%) with drugs, control of varicella in the community can
symptoms—in spite of receiving VZIG. Therefore, only be achieved by widespread vaccination. Active
the rationale for administering VZIG to those at immunization also has the advantage in individual
risk is not so much to prevent infection but to ‘at risk’ patients by offering long-term protection.
prevent the serious forms of the disease with visceral Varicella vaccines based on the attenuated Oka
involvement. There are numerous studies, based on strain of VZV have been available since 1974 when
case-series, showing the beneficial effects of VZIG it was first developed in Japan (Takahashi et al.,
in reducing morbidity and mortality compared to 1974). The original vaccine was derived from VZV
historic controls or untreated patients. Unfortu- isolated from vesicles of a 3—year-old child with
nately, there are also reports of correctly adminis- typical varicella and was attenuated by serial pas-
tered VZIG failing to prevent fatal varicella in im- sage in guinea-pig cells and human embryo lung
VARICELLA ZOSTER 75
cells. Since its development, the live attenuated Increasing awareness of the seriousness of varicella
varicella vaccine has been shown to be safe and in adolescents and adults, as well as the growing
clinically effective for the prevention of varicella in number of susceptible individuals at risk of severe
healthy children and adults, as well as in immuno- disease, have encouraged serious consideration of
compromised children with leukaemia or other ma- the wider use of the vaccine in the community for
lignancies. control of varicella. The vaccine could undoubtedly
The potential benefits of varicella vaccine in im- be of significant benefit in individual adolescents
munocompromised children are considerable and and adults without a past history of varicella, es-
the vaccine is licensed in several European coun- pecially those at increased risk of contracting or
tries for this purpose, although in the UK it is so far spreading the infection, such as healthcare workers,
only available on a named patient basis. The experi- and such a policy has recently been recommended
ence in immunocompromised patients (Kangro, by the World Health Organization.
1990) has shown that: Routine childhood immunization against vari-
cella is also a distinct possibility and is already rec-
∑ The vaccine is generally well tolerated in children ommended in some industrialized countries. Var-
with cancer, provided the recommended guide- icella vaccine was licensed in the USA in 1995 for use
lines about its use are adhered to. Between 5 and in susceptible healthy persons over the age of 12
15% of recipients develop a very mild varicella months and routine vaccination of healthy children
rash, depending on the level of immunosuppres- has been practised in Japan and Korea since 1987.
sion. It is worth noting that transmission of vac- The benefits of mass vaccination of healthy children
cine virus has been documented from the vac- are currently debated, and supported by the positive
cinees who develop a rash. results of extensive safety, efficacy and cost-effective-
∑ Between 70 and 95% of the vaccinees seroconvert ness analyses. The experience in healthy children has
but the levels of antibody are appreciably lower shown that 95% or more of susceptible recipients
than those observed after wild virus infection or develop humoral and cellular responses following a
in healthy vaccine recipients. Antibody responses single dose of the vaccine. Less than 5% of recipients
persist in the majority of vaccinees for at least develop a mild, varicella-like rash after vaccination
6—10 years but some vaccinees lose detectable but fever is rare. Excellent protective efficacy has
antibody after periods of time which vary from 6 been demonstrated in both uncontrolled and con-
months up to 3 years. For this reason, it is advis- trolled studies showing 100% protection against
able to monitor regularly the immune status of severe varicella and at least 86% protection against
vaccine recipients, and if necessary administer a any form of disease following close contact (Izurieta
booster vaccination. et al., 1997). Antibody levels decline with time but
∑ The vaccine confers significant protection, even long-term follow-up studies spanning 20 years in
in those with waning antibody responses. Break- Japan and 10 years in USA have shown that over
through infections have been reported in 10—15% 90% of vaccinated children retain protective im-
of vaccinees after close contact but they have munity. Interestingly, the immune response to
always been mild. varicella vaccine is relatively poor in adults, com-
∑ The vaccine is potentially useful for postexposure pared to healthy children, and it has been suggested
prophylaxis, although it is generally not licensed that this is related to a common defect which predis-
for this purpose. Antibody responses usually do poses adults to more severe disease following natu-
not appear until 3—5 weeks after vaccination but ral infection with VZV. Concerns have been raised
cell-mediated immune responses develop within 4 about the duration of protection against VZV in the
days after vaccination in approximately 50% of absence of natural exposure to the virus and the
recipients and it has been shown in controlled possibility of a shift in the epidemiology of varicella
trials to confer protection after contact. into older age groups as a result of a childhood
∑ The vaccine virus can undoubtedly establish immunization programme. Trials with combined
latent infection in some vaccinees and reactivate varicella— measles—mumps—rubella vaccines are cur-
to cause zoster. All the evidence to date shows rently under way and, if successful, will undoubtedly
that this occurs less frequently and is less severe make the acceptance of routine immunization of
than with wild-type virus. children easier.
76 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
There is also considerable interest in the possibil- pes zoster in human immunodeficiency virus-infected patients:
ity of vaccinating seropositive adults with the aim of results from a randomized, controlled clinical trial. Collab-
orative Antiviral Study Group/AIDS Clinical Trials Group,
preventing zoster. This arises from the observation Herpes Zoster Study Group. Antimicrobial Agents and Chemo-
that the vaccine effectively can boost both antibody therapy, 42, 1139—1145.
levels and cell-mediated immunity in elderly pa- Grose, C. (1987) Varicella-zoster virus: pathogenesis of the hu-
tients (Sperber et al., 1992). man disease, the virus and viral replication, and the major
viral glycoproteins and proteins. In Natural History of
Varicella-Zoster Virus (ed. R.W. Hyman), chap. 1, CRC Press,
Boca Raton FL.
REFERENCES Hanshaw, J.B., Dudgeon, J.A., and Marshall, W.C. (1985)
Varicella-zoster infections. In Viral Diseases of the Fetus and
Adams, S.G., Dohner, D.E. and Gelb, L.D. (1989) Restriction Newborn, p 161. W.B. Saunders, Philadelphia.
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
2C
Cytomegalovirus
Paul D. Griffiths
Royal Free and University College Medical School, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
80 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2C.1 Typical electron microscope appearance of cytomegalovirus. (Photograph kindly prepared by Mr J.A. Bishop)
trivial name, e.g. gpUL75 (gH) is glycoprotein H, proteins, but recent work suggests that the trans-
the product of gene number 75 in the unique long forming activity is distinct (Wang and Razzaque,
region. 1993). While such experiments are interesting, it
Productively infected cells produce linear must be emphasized that, at present, there is no
genomes from concatameric precursors. Cleavage is evidence that CMV is naturally oncogenic.
accomplished by an endonuclease (terminase) co- The DNA can be digested with restriction en-
incident with this packaging. pUL89 is part of this donucleases so that, following gel electrophoresis,
complex, and there are thus similarities between the oligonucleotide patterns characteristic of distinct
cleavage/packaging of herpesvirus DNA and that of CMV strains are produced. This technique cannot
bacteriophage T4, which are supported by sequence prove that two strains are identical but if strains fail
conservation of the terminase genes. The physical to show different patterns after digestion with at
state of the CMV genome at other replicative stages least two restriction endonucleases then it is very
remains poorly defined. For example, it is not likely that they are identical (Huang et al., 1980).
known whether, during latency, the CMV genome Use of restriction enzyme analysis can provide use-
is integrated into host cell DNA or whether, like ful epidemiological information, but there is no evi-
Epstein—Barr virus (EBV), it exists in an episomal dence to suggest that any one strain is associated
form. with any particular type of clinical presentation.
Some areas of the genome are homologous with Genetic changes also occur in the genome with-
regions of human chromosomal DNA, which has out acquisition of new cleavage sites for commonly
practical importance for selection of CMV DNA used restriction enzymes. The polymerase chain re-
probes. Distinct transforming regions of DNA have action (PCR) followed by sequencing can be used to
been identified. One is adjacent to the major im- explore genetic variation at the fine molecular level
mediate-early region which encodes transactivator for particular regions of interest.
CYTOMEGALOVIRUS 81
Figure 2C.2 Cascade genome expression of herpesviruses. Genes labelled (immediate-early), (early) or (late) are transcribed into
messenger RNA and then translated into proteins. Inhibitors of protein synthesis (cycloheximide), transcription (actinomycin D) or
DNA replication (cytosine arabinoside (ara-C) or ganciclovir (GCV)) can be used to interrupt genome expression, as discussed in the
text
Figure 2C.3 Proteins encoded within the major immediate-early region of CMV. Exons are numbered 1—7. TA : transcriptional
activation; PA : polyadenylation; ie : immediate-early; l : late
The cascade synthesis of CMV is similar to that with the M 72 000 protein to synergistically in-
P
of HSV but is temporally regulated in a different crease expression of its own, and heterologous, pro-
manner. While - and -protein synthesis occurs at moters, probably through activation of endogenous
roughly the same times in cells infected with HSV or transcription factors and/or by bridging between
CMV, DNA synthesis in HSV-infected cells follows their binding sites and the TATA binding protein
promptly the appearance of -proteins, whereas, in (Lukac et al., 1997). The M 55 000 protein has
P
CMV-infected cells, it is delayed. DNA synthesis minor stimulatory effects, either alone or in combi-
also progresses slowly so that the production of nation with either of the other two larger proteins.
CMV -proteins and their incorporation into The smallest protein (M 18 000) is expressed during
P
daughter virions occurs many hours later than in replication in differentiated monocytes (Kerry et al.,
HSV-infected cells. 1995).
The largest of the these proteins downregulates
its own synthesis and so is autoregulated (Stenberg
and Stinski, 1985). This downregulation is mediated
Proteins by a distinct region in the C-terminus, which is
shown stippled in Figure 2C.3. This region is also
The CMV genome is sufficiently large to encode present in a late protein embedded within this re-
over 200 proteins of average size, and sequencing of gion of M 40 000, and it is likely that activation of
P
one strain has identified 204 predicted open reading its promoter at late times leads to downregulation
frames (Chee et al., 1990). Of these, approximately of the major immediate-early region. IE86 also
189 may be unique proteins, since others are found binds cellular p53 which may decrease p53-induced
in duplicate in the repeat regions of the genome. apoptosis. IE86 also interacts with the protein
One expression unit has been studied in great product of UL84, which is a transdominant inhibi-
detail: that encoding the major immediate-early tor of IE86. Some of the recently described latency-
(MIE) proteins of CMV. As shown in Figure 2C.3, associated transcripts found in the bone marrow of
this genetic unit is expressed via differential splicing normal donors code for proteins which are recog-
to produce four -proteins of distinct M . The pro- nized by infected humans (Kondo et al., 1996). Their
P
tein of M 86 000 (IE86) interacts with the basal function is obscure but, by analogy with HSV, they
P
transcriptional machinery, especially the TATA may play important roles in the establishment or
binding protein, to enhance formation of preiniti- regulation of the latent state. Expression was found
ation complexes (Wu et al., 1993). It also cooperates in multiple tissues of transgenic mice containing a
CYTOMEGALOVIRUS 83
Table 2C.1 Human CMV envelope glycoproteins virion formation. For example, surface glyco-
proteins are potentially important because of their
Complex in envelope Constituent proteins Mapped ORFs
interaction with the immune system. Neutralizing
gCI gB homodimer gpUL55 epitopes have been described on gB (Basgoz et al.,
gCII gM (IMP) gpUL100 1992) and gH (Urban et al., 1996). gH requires
Others? gpUL115 (glycoprotein L) to facilitate surface ex-
gCIII gH gpUL75
gL gpUL115 pression. Much of the neutralizing activity of serum
gO gpUL74 samples can be absorbed by recombinant gB, sug-
gesting that this protein contains dominant neu-
ORF : open reading frame; IMP : intergral membrane protein. tralizing sites (Britt et al., 1990). gB forms the active
Table 2C.2 Eleven loci required for human CMV replication component of some vaccine preparations which
have reached the stage of phase I clinical testing.
DNA-pol UL54 Genetic variation at one of the neutralizing sites has
pol-associated protein UL44 been found in immunocompromised patients with
ssDNA BP UL57
Helicase-primase UL70
chronic infection, suggesting that these viruses may
UL105 be antibody-escape mutants evolving under selec-
UL101—102 tive pressure from the humoral immune response
Transactivators UL36—37 (Darlington et al., 1991).
IRS1 (or TRS1) Note that an alternative nomenclature system
IE1/2
Unknown functions UL84 terms gB ‘glycoprotein complex I’ (gCI) and gH
UL112—113 ‘glycoprotein complex III (gCIII) (Gretch et al.,
1988). The protein products of individual genes
After Pari et al. (1993). which combine to form these complex glyco-
proteins are summarized in Table 2C.1.
lacZ gene under the control of the MIE region A variety of other proteins have been identified
which correlated well with the cell types in which and mapped to the genome (see Figure 2C.4, see
CMV replication is found in vivo (Baskar et al., Plate I), important examples of which will be men-
1996). Thus, in summary, expression and regulation tioned briefly. A total of 11 proteins are required in
of the gene are remarkably complex, with the poten- trans to effect replication (Table 2C.2). Many of
tial for exquisite control of virus replication. these perform similar functions to HSV proteins,
Other immediate-early genes are of interest. except that CMV does not have an identified origin
Genes UL36—37 encode transactivators which are binding protein and requires transactivators.
essential for DNA replication and which represent a pUL80A and pUL80.5 encode the protease and
molecular target for antiviral chemotherapy. Like- assembly protein, respectively. Protease is respon-
wise, TRS1 is required for DNA replication. pUL69 sible for cleaving the original polyprotein at three
is present in the tegument of the virion, as is distinct sites. The assembly protein facilitates for-
ppUL82. Although the latter is a late protein, in mation of B-capsids and entry of DNA, being lost
combination, these two proteins appear to play a from the capsid in the process. Four genes (US27,
similar role to the -transinducing factor of HSV US28, UL33, UL78) are homologous to G-protein-
which is released into the cell during the process of coupled receptors and so may be involved in signal
uncoating and is then able to interact with a cellular transduction. The US28 gene facilitates entry of
transcription factor to upregulate the major im- HIV into CD4 ; cells which are otherwise not
mediate-early promoter. susceptible to HIV infection (Pleskoff et al., 1997).
Early proteins mainly provide essential enzymic In addition, open reading frames with predicted
functions within the cell; for example, pUL54, DNA similarities to known proteins have been identified
polymerase and pUL97; a protein kinase which from the sequenced genome of Ad169. It will be
phosphorylates antiviral nucleosides such as gan- important to determine rigorously that these pro-
ciclovir (GCV) or acyclovir (ACV), so starting their teins are actually expressed and that they have the
anabolism to the functional nucleoside triphos- predicted function.
phate inhibitors of pUL54. Note also the 22 extra genes depicted outside the
Late proteins generally play a structural role in main circle. These are not present in Ad169 but are
84 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
found in other strains of CMV with a lower passage Ad169 strain does not contain predicted proteins
history (Cha et al., 1996). Presumably, they have with homology to gE or gI of HSV which form the
been lost during the process of adapting CMV to Fc receptor of that virus. The CMV Fc receptor has
grow in fibroblast cell lines which led to the strain high affinity for human IgG, but not other human
termed Ad169. This strain is popular with re- immunoglobulin isotypes, and has low affinity for
searchers because it grows rapidly to relatively high rabbit IgG or mouse IgG (Keller et al., 1976). The
titre and releases a lot of extracellular virions. Wild Fc receptor is found in the Golgi apparatus, which
strains of CMV lack these properties and so are enlarges to form a perinuclear inclusion body in the
difficult to work with in the laboratory. As a result, concavity of the reniform nucleus. It has been sug-
one has to question whether Ad169 is fully represen- gested that HSV produces an Fc receptor so that
tative of CMV strains in vivo and the presumed loss antibody attached by its Fab portion to a virus
of these 22 genes illustrates this issue. None of the 22 protein can be bound by its Fc portion back onto
genes has homology with known herpesvirus pro- the virion. This would have the effect of preventing
teins. Many appear to encode glycoproteins, and immune effector mechanisms which require an in-
vaccine candidates containing these genes are being tact Fc portion after opsonization. Whether such a
studied. mechanism is operative for CMV remains to be
defined. However, the production by CMV of Fc
receptors in vivo might allow opsonized bacteria or
fungi to gain access to cells which they cannot nor-
Growth In Vitro mally infect; this might explain why CMV infection
is often associated with secondary bacterial and
The only cells fully permissive for CMV replication fungal infections. A similar process might operate
in vitro are human fibroblasts. This finding is in for human immunodeficiency virus (HIV) coated in
complete contrast to that in vivo where, at postmor- non-neutralizing antibody (McKeating et al., 1990).
tem examination, cells infected with CMV are Unlike HSV, CMV does not switch off host mac-
found in tissues of epithelial origin (kidney, liver, romolecular synthesis but actually stimulates cellu-
bile ducts, salivary gland, gut epithelium, lung par- lar DNA, RNA and protein synthesis. One cellular
enchyma, pancreas) as well as in endothelial cells. function which is stimulated as a result is thymidine
This observation again suggests that the virus kinase activity. It is tempting to suggest that CMV
propagated in the laboratory should not be as- has learned to increase cellular uptake of thymidine
sumed to be an authentic model of the wild-type by switching on the host enzyme responsible, while
virus. The cell surface proteins which act as recep- HSV has used a different tactic, the production of a
tors for CMV have not been identified, although novel thymidine kinase to achieve the same objec-
several candidates have been proposed. tive. Likewise, CMV induces cellular topoisomerase
In fibroblast cell cultures, encapsidation occurs II and may package this enzyme into extracellular
in the nucleus and the virus envelope is then ac- virions. Recent results have also shown that human
quired by budding through the inner nuclear mem- complement is bound to the virion but not ac-
brane to the Golgi apparatus. Enveloped virions tivated. Host cell complement regulatory proteins
are found within vesicles in the cytoplasm and these were detected in virions and might explain this phe-
appear to fuse with cellular membranes to allow nomenon (Spiller et al., 1997). Thus, like HIV (re-
egress of the mature virus particles. Dense bodies viewed in Ott, 1997), the mature virion may contain
also mature in the Golgi apparatus and are released host proteins of potential importance for under-
from the infected cell in the same way as virions so standing pathogenesis.
that they contain virus-specific glycoproteins.
At late times after infection, CMV induces the
appearance in infected fibroblasts of an Fc receptor.
It is not clear if this is a virus-encoded protein or EPIDEMIOLOGY
whether it represents upregulation of a host gene.
Expression of the Fc receptor is blocked by inhibi- Cytomegalovirus must be acknowledged as one of
tion of DNA synthesis so, if it is virus-encoded, it the most successful human parasites. It has learned
should be a late gene product. The sequence of the to survive in its human host by infecting both verti-
CYTOMEGALOVIRUS 85
Figure 2C.5 The age-specific prevalence of complement-fixing serum antibodies against cytomegalovirus. The number of women in
each age group is shown above each column of the histogram. (Reprinted with permission of Blackwell Science Ltd from Griffiths and
Baboonian, (1984)
cally and horizontally; the virus can be transmitted apart from child-to-child transmission which has
by either route during primary infection, reinfection been documented in play groups. However infected,
or reactivation; at all times the virus causes minimal children can transmit CMV to their parents (Pass et
disability, allowing infected individuals to remain al., 1986) and so represent a potential threat to a
active and so maintain the maximum opportunity future sibling should the mother be pregnant.
of encountering susceptible contacts; the virus is In populations of poor socioeconomic back-
excreted from multiple sites, so contact of varying ground, the vast majority of children have experi-
degrees of intimacy can lead to transmission. enced primary CMV infection by the onset of pu-
Infection may be acquired during delivery, fol- berty. In countries with good social circumstances,
lowing ingestion of infected maternal genital secre- roughly 40% of adolescents have been infected, and,
tions or soon afterwards by ingestion of breast milk as shown in Figure 2C.5, this increases by approxi-
containing CMV (Stagno et al., 1980). These two mately 1% per annum thereafter (Griffiths and Ba-
means of perinatal transmission combine to infect boonian, 1984). Such primary infection can lead to
between 2 and 10% of infants by the age of 6 vertical transmission if the individual is pregnant
months in all parts of the world. when infected.
Throughout the rest of childhood, close contact is The prevalence of CMV IgG antibodies in organ
known to be required for transmission, although transplant recipients reflects their socioeconomic
the precise route of infection is not known. As a grouping. The same applies to individuals who ac-
result, CMV may transmit readily within family quired HIV infection via blood (or blood products),
groups. The possibilities for infection must be in- heterosexually or through contaminated needles
creased where individuals are crowded together in used for intravenous drug use. In contrast, HIV-
unhygienic circumstances, and this probably ex- positive male homosexuals have a very high preva-
plains why CMV infection is most common in so- lence of CMV IgG antibodies (typically 95%).
cieties which are socially disadvantaged. Infection is At whatever age primary infection occurs, the
transmitted less well in the general community, virus is not eradicated from the host but persists for
86 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
life. Occasionally, however, CMV reactivates from which may or may not trap CMV must surely be
its latent state and infectious virions appear in the simplistic. Recent ultrastructural studies of the pla-
saliva and/or urine. Reactivations of CMV are en- centa emphasize the importance of macrophage-
tirely asymptomatic but form an important means mediated defence against potential virus infections
by which CMV can spread horizontally. Reactiva- (reviewed in Burton and Watson, 1997).
tions can also lead to vertical transmission of CMV.
This finding came as something of a surprise, since
it was assumed, by analogy with rubella, that a
Perinatal Infection
woman who possessed antibody against CMV be-
fore becoming pregnant would be immune to in-
Perinatal infection is acquired predominantly from
trauterine transmission. However, not only can re-
one of two sites: infected maternal genital secretions
activation of latent maternal infection lead to
or breast milk.
congenital infection but more fetuses are infected
During delivery, the fetus is surrounded by copi-
worldwide by this route than are infected as a result
ous quantities of genital secretions which may con-
of primary maternal infection. Congenital CMV
tain high titres of CMV as a result of recurrent
infection thus has its highest incidence in the poor-
maternal infection. Under these conditions, infec-
est communities of the world, since most women in
tion has been described as occurring ‘during pas-
poor societies are infected before reaching child-
sage through the Sea of Cytomegalovirus’.
bearing age. Note, however, that primary CMV
Breast milk, especially colostrum, has also been
infection in the mother represents a greater risk to
shown to be a good source of CMV. Although virus
the fetus that recurrent maternal infection (Fowler
titres are relatively low, large quantities of milk are
et al., 1992).
imbibed, so a heavy viral inoculum can be ingested.
Finally, ‘immune’ hosts can also be reinfected
Clinical studies have demonstrated that it is not just
with another or, possibly, the same strain of CMV.
the presence of CMV in breast milk which is re-
Epidemiologically, it is important to distinguish be-
quired but that this milk must also be fed. Women
tween reinfection and reactivation of latent infec-
whose only site of CMV excretion was from the
tion but, in clinical practice, the term ‘recurrent’
breast were studied. Perinatal infection occurred
infection is often used to cover both possibilities.
only when breastfeeding took place, not when such
women gave bottle feeds (Stagno et al., 1980).
Having ingested CMV, infection might be estab-
ROUTES OF INFECTION lished in the neonate by infection of buccal, pharyn-
geal, respiratory, salivary gland or oesophageal mu-
cosa or by initiation of infection in the small or even
Intrauterine Infection
large bowel mucosa. The latter possibilities seem
unlikely since CMV is likely to be inactivated by
As is the case with rubella, intrauterine infection is
environments containing acid and bile salts.
assumed to follow maternal viraemia and subse-
quent placental infection, although this has not
been proven formally. Due to the lack of maternal
illness it has not been possible to identify a series of Postnatal Infection
pregnant women with primary CMV infection and
show that viraemia is a risk factor for congenital The absence of symptoms associated with postnatal
infection. If viraemia is responsible for trans- CMV infection makes it impossible to implicate
mission, then it must be determined whether cell- with certainty the routes of transmission, although
free virus or leucocyte-associated virus is required evidence exists to support salivary transmission.
for placental infection. Intrauterine transmission of Saliva containing CMV has been recovered from
CMV occurs in only one-third of pregnant women toys at daycare centres and this would seem to be an
with primary infection but we remain ignorant of ideal means by which the virus could be transmitted
how the majority prevent the virus from infecting among young children unable to conform to basic
the fetus. It may be that the placenta acts as a form standards of hygiene (Pass et al., 1986). Likewise,
of barrier, but representation of this organ as a sieve occasional cases of CMV mononucleosis are seen in
CYTOMEGALOVIRUS 87
young adults and, by analogy with EBV, the infec- replenish heat-labile clotting factors and was used
tion has been dubbed a ‘kissing disease’. as soon as possible after donation, a procedure
The prevalence of CMV IgG antibodies in devel- which would increase the likelihood of transferring
oped countries increases at 1% per annum from viable virus. Third, the patients were being carefully
puberty to middle age (Figure 2C.5). It is often followed up so that the appearance of the new syn-
stated that these infections result from sexual expo- drome in convalescence was likely to be recognized.
sure but this remains unproven. Certainly, they re- Although CMV can be transmitted by blood
sult from contact with an infected individual, but transfusion, it is clear that this is an uncommon
whether this contact takes place at the level of oral event, since only 1—5% of blood units taken from
or genital mucosa is a matter of speculation. Evi- seropositive donors leads to infection of
dence can be found to support the concept of ve- seronegative recipients. To date, it has not been
nereal transmission since CMV is found in semen possible to determine which donors have a high risk
and on the cervix. However, this is only circumstan- of transmitting the virus. Attempts to culture CMV
tial evidence; we do not talk of brain-to-brain trans- from fresh donor blood have been unsuccessful at
mission of poliomyelitis or urinary transmission of all centres except one, and the methods used at that
mumps just because these viruses may be found at centre have not been successful elsewhere. This fail-
particular sites. Sexual contact is almost invariably ure to isolate CMV has led to the suggestion that
preceded by oral—oral contact. Thus, even if CMV is the virus exists in the blood of healthy donors in a
shown to be transmitted by intimate contact, it may latent state, presumably within leucocytes, and that
have resulted from salivary rather than from ve- CMV is reactivated following transfusion when
nereal exposure. This issue is important because we these cells encounter an allogeneic stimulus. This
may need to target CMV vaccines to particular suggestion is plausible but remains controversial,
mucosal sites in order to prevent infection. not least because of the failure to isolate CMV from
One setting in which sexual transmission of CMV blood donor leucocytes using cocultivation tech-
does occur, however, is from male homosexuals niques. The nature of the leucocytes in which CMV
who have a high prevalence of CMV infection. Con- may establish latency remains unknown, although
tacts who are initially seronegative have a high risk increasing attention is focusing on the monocyte/
of primary infection during follow-up. At least one macrophage (Söderberg-Naucler et al., 1997). CMV
study has implicated rectal intercourse as an inde- can be grown from the peripheral blood of im-
pendent risk factor for CMV seroconversion. This munocompromised patients and pp65 antigen is
suggests that CMV may be transmitted by this found in polymorphonuclear leucocytes and mac-
route with the rectal mucosa providing a less effi- rophages. However, this is clearly a different
cient barrier to the high titres of virus found in pathogenetic situation from that found in healthy
semen than is provided by the stratified squamous blood donors and could be the result of the phago-
epithelium of the vagina. cytic scavenging activity of these cells.
Renal transplant 9 9 21 0 0
9 ; 34 21 (62) ? 15 (44) ?
; 9 51 19 (37) ? 3 (6)
; ; 71 49 (69) ? 18 (25) ?
Bone marrow 9 9 63 4 (6) 2 (3)
transplant 9 ; 23 6 (26) ? 1 (4)
; 9 25 12 (48) ? 11 (44) ?
; ; 66 33 (50) ? 8 (12)
Liver transplant 9 9 4 1 (25) 1 (25)
9 ; 3 1 (33) 1 (33)
; 9 18 2 (11) 2 (11)
; ; 19 11 (58) ? 6 (32)
either parenchymal cells or infiltrating leucocytes marrow transplants and from patients with HIV
are prime suspects. Interestingly, the same tech- infection, reveals some interesting parallels, despite
niques showed that donor virus could also infect the different organs involved in CMV disease (Table
seropositive individuals and cause CMV disease 2C.4). Thus, primary infection is a major risk factor
(Grundy et al., 1988). Thus, recipient natural im- during pregnancy and for recipients of solid organ
munity acquired prior to immunosuppression can- transplants but not for bone marrow transplant or
not prevent CMV infection, a finding which has patients with the acquired immune deficiency syn-
implications for the development and evaluation of drome (AIDS). Viraemia has been repeatedly
CMV vaccines. Several studies have shown the shown to be a risk factor following solid organ or
same results following all types of solid organ trans- bone marrow transplantation (Meyers et al., 1990)
plants, so all organs from seropositive donors and recent results show the same is true for AIDS
should be regarded as potentially infectious. patients (Bowen et al., 1997; Dodt et al., 1997; Shin-
In contrast, typing of virus strains showed that kai and Spector, 1997). A high CMV virus load was
the virus causing disease after bone marrow trans- initially shown to be important in neonates with
plant is derived from the recipient and not from the congenital infection (Stagno et al., 1975) and more
donor (Winston et al., 1985). Seropositive donors recently in renal transplant (Cope et al., 1997b),
have been reported to transfer immunity to recipi- liver transplant (Cope et al., 1997a), bone marrow
ents adoptively (Grob et al., 1987). This has only transplant (Gor et al., 1998) and AIDS patients
been reported in recipients of T-cell-depleted mar- (Bowen et al., 1996). Furthermore, multivariate stat-
row, so it may be speculated that this process re- istical analyses show that, once virus load in urine
moves the cells containing CMV while leaving in- has been controlled for as a marker of poor progno-
tact immunocommitted non-T cells which can sis in renal transplant recipients, other recognized
function in the recipient (Table 2C.3). risk factors of viraemia and donor/recipient seros-
tatus are no longer statistically associated with
CMV disease (Table 2C.5). This demonstrates that
high CMV load is the determinant of CMV disease
PATHOGENESIS and that viraemia and donor/recipient serostatus
are markers of CMV disease simply because of their
Risk Factors for CMV Disease statistical association with a high virus load. Similar
multivariate studies in liver transplant and bone
Comparison of the results from pregnancy, from marrow patients confirm that high CMV load in
recipients of solid organ transplants, from bone blood explains the association with donor/recipient
CYTOMEGALOVIRUS 89
Table 2C.4 Risk factors for CMV disease
Primary infection ; ; 9 9
Viraemia No data ; ; ;
! Load ; ; ; ;
; Indicates that the factor has been shown to correlate with risk of CMV disease.
Table 2C.5 Univariate and multivariate assessment of prognostic variables for CMV disease after
renal transplant
Univariate Multivariate
Viral load (per 0.25 log) 2.79 (1.22—6.39) 0.02 2.77 (1.07—7.18) 0.04
Viraemia 23.75 (3.69—153) 0.0009 34.54 (0.75—1599) 0.07
Recipient seropositive 0.22 (0.05—0.95) 0.05 0.92 (0.002—446) 0.98
Figure 2C.6 Cartoon illustrating the kinetics of CMV production leading to disease in a target organ. The tap represents production
of virions by virus-infected cells. The drain represents the ability of immune responses to control CMV accumulation
CMV infections in the second or third month fol- a latent state in most individuals for most of the
lowing transplantation. Recurrent CMV infections time. Abrogation of these responses permits CMV
in adults seem to be age-dependent. Studies of preg- replication with full expression of its potential
nant and non-pregnant women as well as male pathogenicity in some cases. Since severe CMV dis-
homosexuals have shown that up to 10% of ease is restricted to individuals with impaired cell-
seropositive individuals may be excreting CMV mediated immunity, it can be concluded that this
from saliva or urine, or from the cervix in females. arm of the immune response provides most protec-
Excretion rates are, however, very low after the age tion against disease. Nevertheless, the ability of the
of 30 years, suggesting that a host response required humoral system to provide a supportive role, es-
for suppression may ‘mature’ at about this age. pecially in keeping CMV viral loads below critical
Earlier reports showed that virus excretion from thresholds, should not be dismissed.
the cervix increased as pregnancy progressed. This
was interpreted as being a response to some ‘im-
Humoral Immunity
munosuppressive’ effect of pregnancy. A later study,
however, showed that CMV excretion was actually Antibodies of IgG class are produced promptly dur-
suppressed during early pregnancy, so the increase ing primary infection and persist for life. IgM class
seen in virus isolation towards the end of pregnancy antibodies are produced on primary but not recur-
only brought the rate up to the level seen in non- rent infection of immunocompetent individuals and
pregnant women (Stagno et al., 1975). All studies to persist for 3—4 months. During primary infection
date have been cross-sectional rather than longi- immunocompromised patients may fail to produce
tudinal, so person-to-person differences in CMV IgM antibodies, although one-third have IgM re-
excretion could account for the results seen. sponses detectable with recurrent infections. With
intrauterine infection, IgM antibodies are produced
by the fetus; IgG class responses are also present,
but only become detectable as passively acquired
Host Defences maternal IgG antibody is catabolized.
There is evidence to support the concept that the
In combination, the host defence responses in those humoral immune response is beneficial to the host.
with normal immunity keep CMV suppressed into Thus, immunocompromised patients who fail
CYTOMEGALOVIRUS 91
promptly to produce neutralizing antibodies run a have reacted to multiple virus proteins and that
high risk of developing disseminated CMV infec- their sera will precipitate more of any given virus
tion. Likewise, renal transplant recipients experi- protein than will asymptomatic infants. These re-
encing reactivation are unlikely to develop life- sults are therefore compatible with heightened im-
threatening illnesses, although patients with pri- mune responsiveness leading to disease production.
mary infection will mostly be symptomatic. Reinfec-
tions produce disease severity intermediate between
Cell-Mediated Immunity
that of primary and reactivated infections. Similar-
ly, intrauterine CMV infection represents less of a For studies of cell-mediated immunity (CMI), the
risk to a fetus if it has been transmitted by means of lymphocyte blastogenic response to CMV antigen
recurrent maternal rather than primary maternal has been used. This test measures only the recogni-
infection (Fowler et al., 1992). Women with primary tion, not the effector function, of T lymphocytes but
CMV infection who transmit the virus in utero have has the practical advantage over other techniques
higher levels of total IgG but lower levels of neu- of not requiring syngeneic target cells.
tralizing antibodies and lower avidity than women Most, but not all, seropositive adults have a posi-
who do not transmit (Boppana and Britt, 1995). tive test result, whereas few congenitally or
While all of this information is compatible with the perinatally infected infants can respond. This failure
concept that immune responsiveness can be protec- recovers with time and there is a direct correlation
tive, it does not prove that antibody is the beneficial between cessation of viruria and acquisition of im-
component. For example, cytotoxic T cells may be mune responsiveness at 3—5 years of age (Pass et al.,
protective and the ability to mount this response 1983).
promptly may correlate with the ability to mount a This defect is known not to be one of generalized
humoral immune response. Likewise, the postu- T cell immunosuppression, for three reasons. First,
lated humoral defect in women experiencing pri- CMV-infected infants who also acquire HSV infec-
mary infection during pregnancy may be a relative tions generally mount good responses against HSV
failure of T helper responses rather than of B cells. but not against CMV. Second, these infants re-
There is evidence to show that enhanced humoral spond normally to both killed and live vaccines.
immune responsiveness in the fetus correlates with Third, the suppressor (CD4) and helper (CD8) T cell
poor prognosis. It has been shown that the cord lymphocyte subsets remain entirely normal. The
blood levels of specific IgM, total IgM and rheuma- precise defect in such children is not known but it is
toid factor are positively correlated with sympto- tempting to speculate teleologically that CMV has
matic rather than asymptomatic congenital infec- learned to specifically suppress the function of the
tion. This effect has been shown to be independent cell designed to respond against it. Recent work,
of virus titre at birth and so is not simply secondary summarized in Figure 2C.7 (see Plate II), has identi-
to a high virus load in symptomatic infants. Al- fied complex but plausible mechanisms, which, in
though this suggests that fetal antibody is respon- combination, allow the CMV-infected cell to evade
sible for immunopathology, it could equally well be lysis by T cytotoxic and natural killer (NK) cells
that there is another response of the fetus which is (reviewed in Riddell and Greenberg, 1997). Immedi-
damaging and which happens to correlate with the ately after uncoating, the tegument protein ppUL83
humoral immune response. (pp65) is available in the cell to phosphorylate the
The beneficial or adverse effects of humoral im- IE72 isoform of the major immediate-early protein
munity could be discerned better if reactivity and so prevent its presentation as a target epitope.
against a particular virus-coded protein could be At immediate-early times, pUS3 retains class I HLA
shown to correlate with a good or bad prognosis. molecules within the endoplasmic reticulum (ER).
Many CMV proteins are recognized by the humor- Once the virus-infected cell has moved into the
al immune system (Landini, 1992), and work to date early phase of CMV gene expression, pUS2 and
has not been able to identify such a pattern in pUS11 act to re-export class I HLA molecules back
congenitally and perinatally infected infants. The from the lumen of the ER into the cytoplasm, where
ability of serial sera to immunoprecipitate they are degraded in the proteasome. At both early
radiolabelled virus-coded proteins has simply and late times, pUS6 blocks the activity of the trans-
shown that symptomatic infants are more likely to porter associated with antigen presentation so that
92 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
epitopes derived from CMV proteins cannot be possible that CMV (and other herpesviruses) may
presented as targets. All of these functions might, in act as cofactors to accelerate HIV disease but this
combination, decrease surface HLA display to such hypothesis remains unfashionable and the reader is
an extent that the cell becomes a target for NK cells referred elsewhere for more details (Griffiths, 1998).
which recognize the absence of these normally ubi-
quitous molecules. To prevent this, CMV encodes
another protein, pUL18, which is structurally
CLINICAL FEATURES
strongly homologous to class I HLA molecules and
acts as a decoy to prevent NK cells attacking the
CMV-infected cells. Congenital Infection
From the above description, it will be apparent
that CMV has evolved a series of genes—coloured Seven per cent of congenitally infected babies are
dark green in Figure 2C.4 (Plate I)—which act in a born with symptoms. They are said to have
coordinated way to abrogate cell-mediated immune ‘cytomegalic inclusion disease’ and their prognosis
responses specific for the virus. Thus, many re- is poor. The remaining 93% appear to be normal at
sponses may be initiated but be unable to detect birth but a proportion develop sequelae on follow-
their cellular targets during the initial round of up (Stagno, 1990).
replication. However, when the virus leaves the first
cell to initiate infection in a second cell, it is vulner-
Those Symptomatic at Birth
able to display of protein-epitopes before down-
regulation of HLA can be effected in that cell. Ac- Classic presentation is with intrauterine growth re-
cordingly, the dominant effector CMI is directed tardation, jaundice, hepatosplenomegaly, throm-
against ppUL83 (pp65), revealed when the input bocytopenia and encephalitis, with or without
virion is uncoated (reviewed in Riddell and Green- microcephaly. It is often difficult, even for experi-
berg, 1997). enced paediatricians, to differentiate solely on clini-
cal grounds between the several agents causing
chronic intrauterine infection; laboratory tests for
CMV, rubella, syphilis and toxoplasmosis are
Possible Interactions with HIV therefore of importance. Most of the pathology out-
side the central nervous system (CNS) is self-limit-
The multiple mechanisms by which HIV may inter- ing, although severe thrombocytopenic purpura,
act with herpesviruses are reviewed elsewhere (Grif- hepatitis, pneumonitis and myocarditis are occa-
fiths, 1998). Many studies have shown in vitro that sionally protracted and life threatening. The CNS
CMV infection (or transfection of particular genes) involvement may present as microcephaly, en-
can transactivate HIV (reviewed in Ghazal and Nel- cephalitis, seizures, apnoea or focal neurological
son, 1993). Under some circumstances, CMV can signs. Such congenital malformations as inguinal
also downregulate HIV replication. Overall, CMV hernia in males, first branchial arch abnormalities,
is more likely to stimulate HIV when the latter has anophthalmia, Peters’ anomaly, diaphragmatic
an integrated provirus and when CMV is not ac- eventration or cerebellar hypoplasia have all been
tively replicating (Moreno et al., 1997). reported. However, these occur sporadically and
All of the postulated mechanisms of interaction may be coincidental. There is therefore no evidence
would require close contact between HIV and that CMV acts as a teratogen to impair normal
CMV, either in the same cell or in neighbouring organogenesis. Most of the clinically apparent se-
cells. Studies of human tissues have shown that quelae can be attributed to destruction of
CMV and HIV frequently coinfect the same tissues preformed target organ cells.
and that individual cells can be found infected with In 20% of cases (1% of all those congenitally
both viruses. infected) the damage caused by the virus is so severe
Epidemiological studies report that the presence that the patient dies during infancy. If a neonate
of active CMV infection or high quantities of CMV survives, it is almost certain to have serious abnor-
are associated with more rapid progression of HIV malities for the rest of his or her life, especially if
infection to AIDS or AIDS to death. It is therefore abnormalities are seen by computerized axial to-
CYTOMEGALOVIRUS 93
Figure 2C.8 Histological preparation of an inner ear structure from a fatal case of congenital CMV infection showing cell—cell
extension of CMV accompanied by an inflammatory response. (Courtesy of Dr S. Stagno, Alabama)
mography (Boppana et al., 1997). On follow-up, young children but there is now sufficient evidence
clinically apparent extraneural damage rarely per- to prove that the hearing loss can occur in a child
sists but brain damage may manifest as micro- born with normal hearing. In addition, a plausible
cephaly, mental retardation, spastic diplegia or seiz- pathogenesis for progressive hearing loss is appar-
ures, and perceptual organ damage as optic ent. Figure 2C.8 shows a histological preparation of
atrophy, blindness or deafness. Any of these abnor- inner ear structures from a fatal case of congenital
malities may occur alone or in combination. These CMV infection, demonstrating virus spread by the
defects may appear in children who do not have cell-to-cell route to produce a focus of infection
signs of CNS involvement at birth (including cere- surrounded by inflammation. It is tempting to sug-
brospinal fluid (CSF) examination). Milder forms of gest that this represents the infectious process in the
CNS damage, such as defects in perceptual skills, inner ear causing progressive damage to the organ
learning disability, minor motor incoordination or of Corti and decreased ability to perceive sound.
emotional lability, may be noticed as the child be-
comes older.
Perinatal Infection
Those Asymptomatic at Birth
Long-term follow-up of such children has revealed Despite continued excretion of virus for many
that, compared with uninfected controls, approxi- months, the vast majority of perinatally infected
mately 15% are likely to have hearing defects or infants do not appear to develop acute symptoms.
impaired intellectual performances. The mean intel- The initial titres of CMV in the urine are significant-
ligence quotient (IQ) of infected children has been ly lower than those found in neonates with congeni-
reported to be significantly lower than controls. tal CMV infection or disease (Stagno et al., 1975),
Other investigators have not noted these defects but which is compatible with the virus load hypothesis
their studies have often, although not always, failed of CMV disease induction. However, occasional
to follow children for a sufficiently long time, or cases of infantile pneumonitis have been attributed
have failed to use matched controls. There has been to perinatal CMV infection. This would appear to
scepticism about the reliability of hearing tests in be an uncommon event, although CMV is a fre-
94 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
quent pathogen in those few infants who do develop Table 2C.6 CMV diseases in the immunocompromised
pneumonitis in the first three months of life.
Solid Bone marrow
Symptoms transplant transplant AIDS
Fever/hepatitis ;; ; ;
Postnatal Infection Gastrointestinal ; ; ;
Retinitis ; ; ;;
Most primary or recurrent infections are asympto- Pneumonitis ; ;;
matic. Indeed, most of those identified by prospec- Immunosuppression ;
Myelosuppression ;; ;
tive serological studies as having seroconversions Encephalopathy ;
express surprise when told that they have experi- Polyradiculopathy ;
enced a virus infection. Addisonian
Occasionally, however, primary infections are ac- Rejection/graft-versus- ; ?
host disease
companied by the syndrome of infectious mono-
nucleosis. This is similar to the syndrome produced
by EBV except that lymphadenopathy is uncom-
tients present with virus dissemination to the retina
mon and the Paul—Bunnell test is invariably nega-
without other signs. Any part of the gut may be the
tive. The postperfusion syndrome, described earlier,
site of CMV replication, which may be asympto-
is essentially CMV mononucleosis acquired by
matic or may be associated with ulceration, which
blood transfusion. Sometimes, the hepatic compo-
can, in extreme circumstances, cause erosion of
nent of CMV mononucleosis is prominent, so a
neighbouring blood vessels with catastrophic
diagnosis of non-A, non-B hepatitis is considered
haemorrhage. Patients with AIDS may develop a
initially. Within a few days, however, the full clinical
low-grade encephalopathy or polyradiculopathy.
picture becomes clear, with persisting pyrexia and
At postmortem examination, AIDS patients often
atypical lymphocytosis.
have CMV adrenalitis. Finally, CMV may induce
CMV is such a common virus infection that pri-
an immunosuppressive syndrome where the patient
mary infection tends to occur by coincidence with a
becomes unable to deal with superinfecting bacteria
variety of medical conditions. In addition, since
or fungi. In this instance, and in some of the other
CMV is an opportunist, it will tend to reactivate
clinical presentations (pneumonia, hepatitis, gut ul-
when a patient becomes debilitated as a result of
ceration, septicaemia), the underlying nature of the
some underlying condition. If the underlying dis-
CMV infection is often not recognized by the clini-
ease is esoteric and has an unknown aetiology, a
cian, who is understandably distracted by the more
case report tends to appear describing the associ-
easily recognized superinfecting organisms.
ation. By scanning through Index Medicus, readers
The major clinical diseases associated with CMV
will find literally dozens of such spurious associ-
are summarized in Table 2C.6, according to the
ations, but they should be reassured: CMV is not
type of immunocompromised patient. Some dis-
the cause of all known diseases.
eases, such as gastrointestinal involvement, are
found in all patient groups. Some are found in all,
but predominate in one group; for example, 85% of
Immunocompromised Patients CMV disease presents as retinitis in AIDS patients,
compared to less than 5% in transplant patients.
Immunocompromised patients may respond to pri- While this observation remains unexplained, there
mary or recurrent CMV infections by remaining is extensive evidence to support the hypothesis that
entirely asymptomatic. More frequently, they will CMV pneumonitis in allograft patients is an
develop a spiking pyrexia which resolves after a few immunopathological condition caused by an ag-
days. Some may develop viraemia with fever and gressive cell-mediated response against a lung tar-
leucopenia, which is sometimes termed ‘CMV syn- get antigen coded by, or revealed by, CMV infection
drome’. This may progress to pneumonitis or this (Grundy et al., 1987). According to this hypothesis,
complication may supervene directly. In either case, AIDS patients are relatively spared from pneu-
once pneumonitis has become established, the monitis because, by the time they are sufficiently
prognosis is poor (mortality 80—90%). Some pa- immunocompromised to permit CMV dissemina-
CYTOMEGALOVIRUS 95
tion to the lung, they have insufficient T cell respon- without additives, since CMV is stable in urine.
siveness to mount the postulated immuno- Saliva should be allowed to soak on to a plain
pathological response. Likewise, the myelosup- cotton-tipped swab, which is then shaken in virus
pressive CMV disease results when CMV replicates transport medium and broken off.
in stromal supporting cells, releasing cytokines Heparinized blood samples should be collected
and producing a milieu unfavourable for haemato- with care since heparin inhibits CMV replication
poiesis. and the phenolic preservatives found in proprietary
These observations relating to different CMV- pathology bottles may be toxic to cell cultures.
associated diseases in distinct patient groups, com- Some 10 ml of peripheral blood should be mixed
bined with the increasing knowledge about the im- gently with 500 units of preservative-free heparin.
portance of CMV viraemia and viral load, lead to a For PCR, acid citrate-dextrose or EDTA is prefer-
potential explanation for the diversity of CMV dis- red to heparin; check with your local laboratory.
eases. CMV viraemia may be a prerequisite for Tissue biopsies should be placed into plain sterile
CMV disease at any target site. Increasing viral containers with no additives. Fluid and cells ob-
load may increase the chance that CMV may pen- tained by bronchoalveolar lavage should similarly
etrate tissues to cause disease. Other organ-specific be placed in a plain sterile container.
changes, distinct for each patient group, may then All specimens should be sent to the laboratory
dictate in which organ CMV localizes. For example, without delay. If delay of more than a few hours is
the marrow suppression is presumably found only anticipated, then all samples should be sent refrig-
after bone marrow transplant because these new erated, or on wet ice, but under no circumstances
cells are receptive to CMV replication. should any specimen be frozen at any temperature.
Several studies have associated CMV with allog-
raft rejection, or graft-versus-host disease in the
Selection of Sites for Examination
case of bone marrow transplant recipients. This
association could be causal or the immunosuppres- To diagnose congenital or perinatal infection, urine
sive therapy required for treatment of such episodes or saliva samples are usually collected. More invas-
could allow CMV to reactivate and so be found ive procedures such as lumbar puncture or liver
coincidentally in those with graft rejection. If con- biopsy are sometimes performed but identification
trolled studies of CMV vaccine or antiviral prophy- of CMV at these sites has not been shown to have
laxis were able to reduce graft rejection one could any prognostic value.
conclude that the association is causal; a situation If adults with mononucleosis or hepatitis are be-
which has been reported in a recent placebo-con- ing investigated, urine and blood are the best
trolled trial (Lowance et al., 1999). samples. It should be possible to detect CMV from
all urine samples collected within a few weeks of
onset, while viraemia is often detected in the first
DIAGNOSIS few days of illness. The identification of urinary
CMV excretion in a patient with such symptoms
As for all viruses infections, there are two potential might be coincidental but the detection of viraemia
strategies for providing a diagnosis: the detection of strongly supports the diagnosis of CMV mononuc-
virus or the demonstration of a specific immune leosis or hepatitis.
response. If pregnant women have symptoms, they should
be investigated for viraemia. Amniocentesis can be
considered after 21 weeks, with the amniotic fluid
Detection of Virus tested by PCR and culture. However, there is no
advantage in actively screening asymptomatic
women. Investigations involving the culture of
Collection of Specimens
urine, genital secretions, saliva and breast milk have
Urine must be fresh but otherwise can be obtained been carried out but the results are not predictive of
by midstream collection, by urine bags in neonates which women will have babies with congenital or
or by urinary catheter. Samples can be collected at perinatal infection. Since the ‘patient’ in these
any time of day and should be sent to the laboratory examples is the neonate not the woman herself,
96 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2C.9 Direct detection of cytomegalovirus in peripheral blood leucocytes. M : Monocyte; P : polymorphonuclear leucocyte
(Kindly provided by Professor H. Thé)
samples should not routinely be collected from any (van den Berg et al., 1991). These are reacted with
maternal site. monoclonal antibodies against an early protein of
Immunocompromised patients should be inves- M 65 000 (termed ppUL83) followed by im-
P
tigated by means of surveillance samples taken munoperoxidase staining (Figure 2C.9). Note that
weekly of blood, and possibly urine or saliva in the monoclonal antibody required for this assay
addition. This must be done as a routine on all does not recognize the immediate-early proteins as
patients rather than waiting for symptoms to devel- originally described. Presumably, the phagocytic
op. CMV excretion from urine and saliva is very activity of the leucocytes shown in Figure 2C.9 has
common in allograft recipients so the relative risk led them to ingest virus-infected material among
for future disease is typically 3. In contrast, PCR which ppUL83 is prominent. It is tempting to
viraemia is detected less frequently in allograft pa- speculate that this may derive from dense bodies,
tients but, when it does occur, it is indicative of a since ppUL83 is a major component of these aber-
relatively poor prognosis with a relative risk of rant forms.
about 10. However, even the detection of viraemia
does not guarantee that CMV is the cause of, say,
Histopathology
the patient’s hepatitis or pneumonitis. Ideally,
samples should be collected from these target or- Cytomegalovirus can be recognized in histological
gans whenever possible. preparations by its characteristic ‘owl’s eye’ inclu-
An alternative way of detecting viraemia is sions. These Cowdry type A intranuclear inclusions
through detection of antigenaemia in preparations have a surrounding halo and marginated
of peripheral blood mononuclear cells as targets chromatin. They can be found in kidney tubules,
CYTOMEGALOVIRUS 97
bile ducts, lung and liver parenchyma, gut, inner ear answer has stimulated research into more rapid
and salivary gland but are less prominent in brain ways of detecting CMV, which are discussed below.
tissue (see Figures 2C.8 and 2C.12 for examples).
Although histopathology provides a specific di-
Electron Microscopy
agnosis, it is known to be insensitive. One study has
shown that CMV can be cultured from tissues six Samples of urine from infants infected congenitally
times more frequently than typical inclusions can be or perinatally contain high titres (10—10 TCID
seen. ml\) of CMV. Using the pseudoreplica electron
microscopy technique, it has been possible to dem-
onstrate this viruria. Several authors have reported
Tissue Immunofluorescence
that approximately 80% of infected infants can be
Some biopsy samples (e.g. liver, lung) may contain detected, with the false-negative results being clear-
cells infected with CMV which can be visualized by ly attributable to low titre urine samples (
staining frozen sections with antisera to CMV. Al- 10TCID ml\). The viral specificity of the tech-
ternatively, the tissue can be disrupted and the cells nique has been reported at 100%, simply because it
fixed to glass slides before staining. Bronchoalveo- would be most unusual for any other herpesvirus to
lar lavage fluid contains a suspension of cells ex- be found at such high titre in urine from infants.
foliated from the respiratory tract, which can be Electron microscopy cannot be used in im-
centrifuged, washed to remove adherent mucus and munocompromised patients for several reasons.
then air-dried and fixed to microscope slides before First, the titre of CMV found in clinical samples
staining. from adults is generally lower than that found in
infants. Second, and more importantly, all human
herpesviruses frequently infect immunocom-
Cell Cultures
promised patients and cannot be distinguished
For conventional cell cultures, human fibroblasts from each other by electron microscopy. Since dif-
must be used, since they are the only cells which ferent antiviral therapy may be required for each
support CMV replication in vitro. Foreskins or em- herpesvirus, the results of electron microscopy
bryo lungs may be employed as a source of fibrob- would not be specific enough to influence patient
lasts and must be used only at low passage ( 25). management.
To detect wild strains of CMV reliably, the cul-
tures must be cared for obsessionally. The medium
Detection of Early Antigen Fluorescent Foci
must be drained, 0.2 ml of each clinical specimen
(DEAFF)
inoculated and incubated at 37°C for 1 hour to
permit virus adsorption. The cultures should then The technique of DEAFF was developed as a
be refed with maintenance medium to reduce the means of retaining the specificity and sensitivity of
toxic effects of the inoculum. This is especially im- cell culture without having to wait for the produc-
portant in the case of particulate samples such as tion of CPE as a diagnostic end-point (Griffiths et
blood and tissue homogenates. For the detection of al., 1984).
viraemia, buffy coat or unseparated heparinized Following inoculation on to cell cultures, CMV is
blood can be inoculated into cell cultures. If toxicity absorbed rapidly into the cell. CMV rapidly starts
is observed, denuded areas of the monolayer can be to produce - and -proteins but CMV DNA syn-
repaired by the addition of fresh fibroblasts. thesis is delayed and protracted until several days
All cultures should be observed at least twice after infection. This explains the long delay seen in
weekly for the typical focal cytopathic effect (CPE) conventional cell cultures, since CMV needs to rep-
of CMV (Figure 2C.10). Occasionally, urine licate, produce daughter virions and infect neigh-
samples from cases of congenital infection produce bouring cells in order to produce the cytopathic
widespread CPE within 24—28 hours which re- effect shown in Figure 2C.10.
sembles that of HSV. Usually, however, the CPE To speed up the diagnostic process, cells are in-
evolves only slowly, so the cultures must be main- oculated with clinical specimens and stained after
tained for a minimum of 21 days before being re- only 18 hours incubation using monoclonal antibo-
ported as negative. This delay in obtaining an dies directed against some of the - and -proteins
98 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2C.10 Local cytopathic effect typical of cytomegalovirus seen in monolayer cultures of human embryo lung fibroblasts.
(Photograph kindly prepared by Ms A. Grzywacz)
Figure 2C.11 Cytomegalovirus infection diagnosed by detection of early antigen fluorescent foci (DEAFF)
CYTOMEGALOVIRUS 99
of CMV (Figure 2C.11). This technique is termed in both transplant patients (Kidd et al., 1993) and
shell vial assay in the USA because of the vessels HIV-positive individuals (Bowen et al., 1997).
used for the sample processing (Gleaves et al., 1984). Clearly, ‘PCR’ is not a single procedure and it is
recommended that each laboratory should measure
the clinical significance of its results through formal
Enzyme Immunoassay (EIA) assessment. The availability of fully quantitative
The techniques of DEAFF and leucocyte antigen PCR assays for CMV (Fox et al., 1992) offers an-
detection provide results rapidly but still require other approach for further refining prognostic
subjective immunostaining and cell culture in the values, as well as for understanding pathogenesis, as
case of DEAFF. The technique of EIA could poten- discussed previously.
tially overcome both of these problems by detecting
soluble virus-specific proteins and by producing a Characteristics of the Various Assays
colour change, visible to the naked eye, whenever Described
CMV is present.
Some workers established EIA detection systems These are summarized in Table 2C.7. In previous
using polyclonal hyperimmune sera or monoclonal editions of this book, conventional cell culture was
antibodies. The results indicated that EIA could described as the ‘gold standard’ against which as-
detect small amounts of CMV complement-fixing says should be compared. In 1997, this process can
(CF) antigen or of virus when added artificially to be considered complete for PCR so that it can now
buffer systems. However, when clinical samples be recommended as the new ‘gold standard’ for
containing CMV were assayed, results of low sensi- providing diagnostic information in a timely man-
tivity and/or specificity were obtained. ner able to influence the management of individual
patients. Any PCR assay which has had a published
validation showing correlation with clinical end-
Detection of Viral DNA points could be employed (Kidd et al., 1993; Einsele
et al., 1995; Shinkai and Spector, 1997). Note that
Earlier reports of the detection of CMV by dot-blot
the samples required (e.g. whole blood or plasma),
methods have been completely superseded by PCR.
the sample extraction and assay-specific details all
Different parts of the CMV genome have been
differ among these validated assays. Colleagues
chosen as targets, but there is no obvious advantage
should therefore choose one method and follow the
in any particular one. Ideally, one would wish to
whole procedure exactly as described. Hopefully,
amplify a conserved region, but the degree of gen-
commercial assays will facilitate the widespread in-
etic variability found in clinical strains is only be-
troduction of PCR, although at present none has
ginning to be defined (Darlington et al., 1991). Po-
been formally evaluated against clinical end-points.
tentially, PCR methods could be so sensitive that
they detected low quantities, even latent virus,
which would not necessarily be clinically relevant. Advantages and Disadvantages of Virus
Indeed, some authors have concluded that their Detection
nested PCR procedures do not provide prognostic
information. A non-nested procedure was chosen in ∑ The anatomical site of the infection can be
the author’s laboratory to avoid this problem; PCR documented (e.g. lung involvement in a patient
was found to produce good prognostic information with pneumonitis).
Table 2C.7 CMV detection in body fluids
Proven
Method Sensitivity Specificity Reliability Rapidity prognostic value
Performance characteristics
Method Sensitivity Specificity Objectivity Rapidity
Neutralization ; ;; ; 9
CF ; ; ; ;
IFA-LA ;; 9 9 ;;
ACIF ;; ;; ; ;;
Latex agglutination ;; ;; ; ;;;
RIA ;;; ;; ;; ;;
EIA ;;; ;; ;; ;;
∑ The patient’s immune response is not required for Many CMV IgG assays have been described.
diagnosis, so all infected immunocompromised Those most frequently used are listed in Table 2C.8
patients, not just those able to mount an immune together with estimates of their specificity, sensitiv-
response, can be identified. ity, objectivity and rapidity. In patients with intact
∑ Rapid diagnostic methods have enabled infected immunity, the CF test is perfectly adequate, pro-
patients to be recruited into therapeutic trials of vided that a potent antigen is employed and that
potential antiviral agents so that patient manage- optimal incubation temperatures are used. Other
ment may be influenced. tests (marked ‘;;’ in Table 2C.8) are more sensi-
∑ PCR methods can quantify the amount of virus in tive in that they produce higher antibody titres but
a clinical samples. This should allow quantitative they do not detect substantially more seropositive
virological assessment of antiviral agents. individuals in a population.
∑ If resistant virus emerges during a course of treat- With immunocompromised patients, the CF test
ment, this may be detected by the assay and so often gives false-negative results by failing to detect
provide an opportunity for prescription of an low levels of IgG antibody. Thus, a more sensitive
alternative drug. technique is required and any of those marked
∑ A potential disadvantage is that the monoclonal ‘;;’ in Table 2C.8 are satisfactory. If an IF
antibodies or DNA probes used to detect CMV method is chosen, the IFA-LA assay should be
may be too specific and may identify only some avoided because all human sera bind to the IgG Fc
strains of the virus, although this has not as yet receptor induced by CMV and this perinuclear flu-
proved to be a problem. orescence can be difficult to distinguish from the
virus-specific nuclear fluorescence found with
seropositive samples. ACIF is the IF method prefer-
Detection of Immune Response red since it is unaffected by IgG binding to the Fc
receptor.
IgG Antibody as a Marker of Past Infection
IgG Antibody as a Marker of Acute
The detection of IgG antibodies against CMV is
Infection
indicative of infection sometime in the past. The
seropositive individual is liable to experience reac- Rising levels of IgG antibody were employed in the
tivations of latent infection. The presence of IgG past but this approach has been completely super-
antibodies against CMV is thus a marker of poten- seded by assays for detecting virus itself.
tial infectivity; although a seropositive individual is
‘immune’ in the immunological sense, this term
Detection of IgM Antibodies
should not be used to imply protection from en-
dogenous or exogenous infection. Attempts to detect CMV-specific IgM antibodies
CYTOMEGALOVIRUS 101
have been plagued by the use of methods of poor the presence of CMV in a child, say 9 months, is
sensitivity and specificity and by interference from taken as evidence of congenital infection, then such
rheumatoid factor. Testing for IgM antibodies may a diagnosis will be wrong in at least 90% of cases.
be helpful in cases of suspected CMV mononucleo- The clinicians may be happy to accept the diagnosis
sis. In all other cases of suspected active CMV when the clinical findings are reviewed but the child
replication, investigations using virus detection then has only a clinical diagnosis with compatible
methods are recommended. laboratory findings rather than a definitive virologi-
cally-proven diagnosis. Testing for specific IgM
antibodies is of no help in this situation since they
Problems Associated with Serological should be present following congenital infection
Diagnosis and will presumably be produced acutely in cases of
perinatal infection, although this remains to be in-
∑ Some immunocompromised patients fail to vestigated.
mount a typical immune response and die from If CMV is found in a child who develops symp-
disseminated CMV infection. If diagnoses are toms during infancy, it is worth bleeding the mother
made solely by serology, these cases will not be and contacting the laboratory servicing her antena-
identified. tal clinic to see if sera have been retained. Occa-
∑ The passive transfer of CMV IgG antibodies with sionally, a laboratory has kept a serum used to test
blood products may produce ‘seroconversions’ if for rubella status. If this serum has CMV IgG but
sensitive IgG assays are employed. not IgM antibodies, then it is unhelpful, since the
∑ The major objection to detecting CMV infection mother may have had recurrent infection during
serologically is that the diagnosis is delayed and pregnancy or, if she presented later than 16 weeks,
so can have little effect on the management of an primary infection early in pregnancy. If, however,
individual patient. the serum is IgM positive, it will confirm that she
had primary infection during pregnancy, which
makes it more likely that her child’s CMV was
acquired congenitally. Similarly, if the mother
MANAGEMENT seroconverts between early pregnancy and paediat-
ric presentation, this supports a congenital trans-
Congenital Infection mission. Finally, the mother may remain
seronegative, in which case the child cannot have
The most important part of the management of had intrauterine infection and so the diagnosis of
these cases is to make an unequivocal diagnosis. congenital infection can be excluded.
This is usually accomplished in the case of those It will be evident from this discussion that the
symptomatic at birth but is often impossible in management of these cases would be greatly facili-
those who are initially asymptomatic. tated if it were possible to screen all newborns for
Babies born with symptoms are usually inves- evidence of congenital infection, in a similar fashion
tigated using the appropriate tests; culture of urine to the established phenylketonuria screening pro-
or saliva for CMV. The presence of CMV in a gramme. At present, cell culture is too cumbersome
neonate aged less than 3 weeks is indicative of con- and expensive a technique to be used widely but it is
genital infection. hoped that one of the modern technologies de-
Those born without symptoms are unlikely to scribed earlier will be able to fulfil this role reliably
have cultures performed and typically present from and, of course, cheaply.
the age of 6 months onwards with sequelae such as Once the diagnosis of congenital infection has
hearing loss or mental retardation. These cases been established, an assessment should be made of
should be cultured for CMV but, if the virus is the prognosis for the child and for a future preg-
present, this does not guarantee that the infection nancy. An estimate of the child’s prognosis can be
was acquired congenitally rather than perinatally. given from the titre of neonatal viruria and the
Unfortunately, in each population which has been magnitude of the humoral immune reactivity pres-
investigated, perinatal infection is at least ten times ent in the cord serum, but this can only divide cases
more common than congenital infection so that, if crudely into high-, medium- and low risk categories.
102 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
More precise prognostic markers are required. The Perinatal Infection
prognosis for a future pregnancy is clear if the diag-
nosis has been definitely proven and the child was These cases produce few medical management diffi-
symptomatic at birth; since only one example of culties. Most remain asymptomatic and undiag-
cytomegalic inclusion disease has been reported in nosed. Cases of infantile pneumonitis should have
consecutive siblings, the risk of an identical recur- samples of urine, saliva and nasopharyngeal aspira-
rence must be very low. If, however, the child devel- te cultured for CMV but, apart from bronchoscopy
oped symptoms during infancy, then the position is and/or lung biopsy, there is no way of proving that
not so clear. No sibling cases have been reported the pneumonitis is due to CMV in an individual
but this might be because the correct diagnosis has case. The main medical importance of perinatal
not been made in the past. It remains possible, infection is the difficulty it produces for the diag-
therefore, that a women could have a future preg- nosis of congenital infection (see above) and the fear
nancy damaged in a similar way. To be scientifically of contagion that it stimulates.
and legally correct, the risk cannot be given as zero It is clear that children less than 18 months of age
but, on humanitarian grounds, the patient may be with perinatal infection can transmit CMV to their
reassured that the risk must be very low. Before a parents (Pass et al., 1986). This presumably results
definitive virological diagnosis is available, the dif- from close contact with infected saliva during nor-
ferential diagnosis often includes a recessive gene mal family life and so would be difficult to control
for presentations with hearing loss, which has a completely. Control should be considered, however,
recurrence risk of 25%. If the parents are prepared if the mother is contemplating pregnancy, although
to gamble with their genes, then the virologist must care must be taken not to induce feelings of social
not dissuade them from taking what must be a far isolation or rejection in the older sibling. There is no
lower risk with CMV. evidence that these children are contagious to
Having established the diagnosis and prognosis, adults outside the family but it seems prudent to
treatment must be considered, including remedial advise the parents that the infants should avoid
therapy to compensate for hearing, speech or devel- intimate contact with women who may be pregnant
opmental defects. No specific antiviral chemother- or with immunocompromised patients. Unfortu-
apy is recommended at present, although gancic- nately, this advice, when ultimately passed to
lovir has been studied in a dose-comparative trial schoolteachers, can lead to the children being
(Whitley et al., 1997). The results show that gancic- treated as lepers. The teaching staff may have to be
lovir was relatively well tolerated over the 6 weeks reassured strongly to ensure that the children are
duration of therapy with no excess toxicity from the not treated differently from any others. It usually
higher dose of 6 mg kg\ b.d. As a result, this dose is suffices to emphasize that 10—20% of the children in
now being compared against no treatment in a the classroom are asymptomatically excreting
randomized controlled trial. When the results of CMV as a result of perinatal infection, so that rou-
this trial are published, recommendations about tine hygienic precautions should be used by all staff
treatment can be made. Until such a time, this for all children. The same advice should be given
author concurs with opinions of the investigators when the child has congenital CMV infection, irre-
that ganciclovir should not be used outside the spective of whether or not the virus has induced
setting of a controlled clinical trial (Whitley et al., disease. It would be absurd to ostracize the occa-
1997). The toxicity profile of ganciclovir includes sional case of symptomatic congenital infection,
carcinogenesis in rodents and so possible thera- knowing that literally hundreds of other children of
peutic advantages must be balanced carefully the same age are excreting similar amounts of the
against potential adverse consequences. same virus.
Finally, the parents should be informed about the
nature of the child’s illness and advised on its infec-
tivity (see below).
Postnatal Infection
Figure 2C.12 Postmortem histological appearance of cytomegalovirus pneumonitis in a bone marrow allograft recipient.
(Reproduced with permission from Griffiths, P.D. (1984) Diagnostic techniques for cytomegalovirus infection. Clinics in Haematology,
13, 631—644)
to have congenital CMV infection. Virus isolates their serological status; it should be clear that pre-
were obtained in each instance from the index case, conceptional humoral immunity in these women
the mother and the aborted fetus. In both cases, the cannot be equated with a guarantee of protection
maternal and fetal isolates were shown to be indis- for the fetus.
tinguishable from each other by restriction enzyme
analysis but were quite different from their respect-
ive index case (Wilfert et al., 1982; Yow et al., 1982).
The conclusion is clear: primary CMV infection Immunocompromised Patients
occurs commonly during pregnancy and will be
found if women of childbearing age are investigated. The most important part of the management of
Typing of strains in the published cases showed that these patients is to make the diagnosis of CMV
the infections were not acquired from recognized infection rapidly. Once extensive damage to target
occupational exposures, a finding which has im- tissues has occurred, as is shown in Figure 2C.12,
portant medical implications. There is little evi- then no antiviral therapy can reasonably be ex-
dence that staff exposed professionally to infectious pected to have a successful outcome. To provide
cases have an increased risk of contracting CMV advance warning of CMV disease, blood should be
infection but it seems prudent to advise pregnant collected weekly from all allograft recipients and
staff to avoid such contacts if possible by practising processed by one of the rapid diagnostic methods,
good hygienic precautions such as hand-washing described in the previous section.
after patient contact. This cautious approach is ap- If CMV is found in an allograft recipient, then the
plied to female technical staff working with CMV in patient’s condition should be reviewed with the cli-
the laboratory, although we have never had a case nicians. Many are asymptomatic but some may
of seroconversion among our predominantly subsequently become unwell and the availability of
seronegative staff. It should be emphasized that the rapid virological results may allow the clinicians to
same advice is given to female staff irrespective of reduce immunosuppressive therapy before exten-
CYTOMEGALOVIRUS 105
sive dissemination of CMV has occurred. The im- Table 2C.10 Annual public health impact of congenital CMV
portance of rapid diagnosis in the clinical manage-
USA UK
ment cannot be overemphasized, since the differen-
tial diagnosis of some illnesses requires an increase Number of live births 4 000 000 700 000
in immunosuppressive therapy (e.g. renal allograft Proportion congenitally infected 1% 0·3%
rejection episodes). The strategies for the use of Number congenitally infected 40 000 2 100
antiviral drugs in asymptomatic patients found to Number with cytomegalic inclusion
have active infection with CMV will be discussed in disease (7%) 2 800 147
the next section. Number fatal (12%) 336 18
Number with sequelae (90%) 2 218 132
Number asymptomatic (93%) 37 200 1 953
Number with sequelae (15%) 5 580 293
PREVENTION
Total number damaged 8 134 443
be made. The number of volunteers required to in Safrin et al., 1997). Foscarnet is a low molecular
assess this formally is enormous. weight analogue of the inorganic pyrophosphate
Vaccines could also be employed for im- product of DNA polymerase activity and so acts at
munotherapy in patients receiving allografts. Clear- a different site and does not require prior anabol-
ly, there would be little point in attempting to pres- ism. All three drugs must be administered by intra-
ent T cell epitopes at this time because the patients venous infusion. Ganciclovir produces neutropenia,
would be receiving drugs such as cyclosporin in which may be dose limiting. In animal toxicology
order to suppress these responses. However, one studies, ganciclovir has a cytostatic effect on the
could aim to present B cell epitopes with the hope of testis; the clinical significance of this for humans is
boosting the humoral immune response to keep the unknown. Foscarnet is nephrotoxic, although this
CMV load below that required to cause disease. In effect can be reduced by large volumes of normal
the case of bone marrow transplantation, immuniz- saline. Foscarnet also affects ionized calcium levels
ation of the donor prior to marrow harvest may and produces a fixed drug eruption on the skin of
potentially lead to adoptive transfer of immunity to the genital area. Cidofovir is nephrotoxic and it is
the recipient. important that patients are hydrated and receive
Passive T cell immunotherapy has also been probenecid to decrease renal concentrations of
evaluated after bone marrow transplantation (re- drug.
viewed in Riddell and Greenberg, 1997). Donor
cells are expanded in vitro and passively adminis-
tered to recipients. The clinical results show that the
cells persist in recipients, and a controlled trial to
Strategies for Deploying Antiviral Drugs
evaluate protective efficacy is currently underway.
Based on the different stages of pathogenesis shown
in Figure 2C.6, four distinct treatment strategies can
be envisaged for CMV (Table 2C.11). While these
TREATMENT strategies have been evaluated formally in trans-
plant patients, it is disappointing that they have not
been applied to the design of studies in AIDS pa-
The drugs ganciclovir, foscarnet and cidofovir have
been licensed for serious or life-threatening CMV tients. Recent results have, however, indicated that
infections in immunocompromised patients. Gan- the same basic principles apply also to AIDS pa-
ciclovir is phosphorylated by the product of the tients, so I will discuss the findings under the appro-
UL97 gene of CMV (Sullivan et al., 1992) and then priate headings.
the triphosphate acts to inhibit virion DNA poly-
merase (UL54). Cidofovir is a phosphonate com-
Prophylaxis
pound which is structurally analogous to a nucleo-
side monophosphate. It does not require activation This approach is to give a drug active against CMV
by UL97 but is converted to the diphosphate (struc- from the time of transplant, and Table 2C.12 lists
turally analogous to a nucleoside triphosphate) by the double-blind, randomized, placebo-controlled
cellular enzymes, and then inhibits UL54 (reviewed trials which have been conducted to date of such
Table 2C.12 Double-blind, randomized, placebo-controlled trials of prophylaxis for CMV infection and disease after transplantation
BMT : bone marrow transplant; HT : heart transplant; LT : liver transplant; RT : renal transplant; ACV : acyclovir; GCV : ganciclovir; IFN : interferon-; Ig : immunoglobulin;
d : day; w : week; m : month; NG : not given.
? Third arm.
CYTOMEGALOVIRUS 109
prophylaxis. Ganciclovir, the most potent drug in presumably because the modest antiviral activity of
vitro, has been subjected to the rigours of several acyclovir is not offset by bone marrow toxicity, as is
such trials but the other two licensed drugs, foscar- the case with ganciclovir. A randomized compari-
net and cidofovir, have not. In addition, Table son of prophylaxis with acyclovir or ganciclovir
2C.12 shows that interferon-, acyclovir and im- after liver transplant (not included in Table 2C.12)
munoglobulin have also been studied, although shows ganciclovir to be superior in controlling
many physicians do not associate these therapies CMV disease (Winston et al., 1995), illustrating that
with anti-CMV activity. the toxicity of ganciclovir is less prominent if bone
The results summarized in Table 2C.12 show that marrow is not the organ being transplanted.
ganciclovir has consistently demonstrated activity
against CMV infection in all patient groups. It also
Early Treatment of Active Infection
reduced CMV disease in most groups, with some
discrepancies which require discussion. After bone Since allograft recipients are monitored closely to
marrow transplant, ganciclovir had a strong trend detect active CMV infection at the earliest possibil-
towards protection, which was significant in one ity, trials have been conducted to determine if early
study (Goodrich et al., 1993) but failed to reach intervention with antiviral drugs can provide clini-
statistical significance in a second (Winston et al., cal benefit. There are two main approaches: sup-
1993). After heart transplant, ganciclovir had a sig- pression, where the drug is given after CMV has
nificant effect in seropositive recipients (Merigan et been detected at a peripheral site such as in urine or
al., 1992) but not primary infections, while the op- in saliva; and pre-emptive therapy, where CMV is
posite was seen in a second study (Macdonald et al., detected systemically, either from blood or the lung
1995). Note that ganciclovir prophylaxis after bone sampled by bronchoalveolar lavage. The collection
marrow transplantation had no significant effect or of the latter sample is predicated on the im-
even a trend in favour of survival; presumably, the munopathological nature of CMV pneumonitis
drug-induced neutropenia facilitated bacterial and (Grundy et al., 1987), so that therapy is directed at
fungal infections to which the patients succumbed early virus replication before disease has become
(Goodrich et al., 1993; Winston et al., 1993). This established. Both approaches have produced clini-
illustrates the principle in Table 2C.11 that prophy- cal benefit (Goodrich et al., 1991; Schmidt et al.,
laxis exposes all patients to the risk of side-effects 1991). In particular, the study of ganciclovir in bone
and so should only be contemplated when a drug marrow transplants by Goodrich et al. (1991)
has no toxicity. In AIDS patients, a trial of oral showed this drug was literally life-saving when used
ganciclovir, 1 g t.d.s. versus placebo, was termed in pre-emptive mode, in contrast to its lack of effect
‘prophylaxis’ because patients did not have CMV on mortality when used prophylactically (Goodrich
disease at trial entry (Spector et al., 1996). Note that et al., 1993; Winston et al., 1993). Again, the prin-
this is not virological prophylaxis as defined in ciples summarized in Table 2C.11 are prescient,
Table 2C.11. Nevertheless, PCR data show that the showing that the toxicity of drugs for particular
greatest effect of ganciclovir was seen when it was organs is as relevant as their antiviral potency.
given prophylactically because the drug had less A ‘prophylaxis’ trial has been reported (Feinberg
effect in those who were already PCR-positive at et al., 1998) of valaciclovir versus doses of acyclovir,
trial entry (Spector et al., 1998). shown in two previous randomized, double-blind,
The results in Table 2C.12 also show that inter- placebo-controlled trials to be ineffective against
feron- and acyclovir each had anti-CMV effects in CMV disease. The PCR data at trial entry showed
vivo, whereas there was no evidence of anti-CMV that this drug worked best for pre-emptive therapy
activity of immunoglobulin. This suggests that, if (Griffiths et al., 1998). This interpretation has been
immunoglobulin does have clinical benefit against controversial but is now supported by recent natu-
CMV disease, this is provided by interference with ral history studies from three separate research
secondary phenomena rather than CMV itself. groups (Bowen et al., 1997; Dodt et al., 1997; Shin-
Note that the anti-CMV effect of acyclovir is clini- kai et al., 1997) showing that PCR viraemia identifi-
cally important; indeed, acyclovir is the only anti- es AIDS patients at high risk of imminent CMV
viral drug shown to improve survival when used for disease (Figure 2C.13).
prophylaxis after bone marrow transplantation, In AIDS patients, there is clearly a therapeutic
110 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
100 Table 2C.13 Current status of drugs for CMV pre-emptive
90 therapy in aids
80
Drug Comment
70
60 Valaciclovir 2 g q.d.s. poorly tolerated
%
No. of
Planned patients Significant markers of efficacy reported
duration
Patient Organ therapy Drug Drug Reduced Reduced Reduced Reduced Increased
group affected Drug 1 Drug 2 (days) 1 2 viraemia excretion disease dissemination survival Reference
BMT Upper GIT GCV 2.5 mg/kg Placebo 14 18 19 No Yes No No No Reed et al. (1990)
t.d.s.
AIDS Retina GCV 5 mg/kg Foscarnet 14 induction 127 107 ND ND No No Yes Anonymous (1992)
b.d. 14 d then o.d. 60 mg/kg t.d.s. then
14 d then maintenance
90 mg/kg o.d.
AIDS Lower GIT GCV 5 mg/kg b.d. Placebo 14 32 30 No Yes No? Yes No Dieterich et al. (1993)
14 d then o.d.
AIDS Retina GCV 5 mg/kg b.d. GCV plus Life 183 96 ND ND Yes ND No Anonymous (1996)
(relapsed or 14 d then 10 mg/kg foscarnet: continue
active retinitis o.d. or foscarnet existing
despite 90 mg/kg b.d. 14 d maintenance dose,
maintenance) then 120 mg/kg o.d. add induction dose
of second drug for
14 d. For
maintenance: GCV
5 mg/kg o.d.,
foscarnet 90 mg/kg
o.d.
BMT : bone marrow transplant; d : day; GCV : ganciclovir; GIT : gastrointestinal tract; ND : not determined.
? In intention-to-treat analysis. Yes for subsidiary analysis.
112 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 2C.15 CMV in AIDS patients during the era of highly active antiretroviral therapy (HAART): principles learned
from transplantation
Better : patients receiving HAART should have improved clinical outcome; worse : HAART may potentially exacerbate CMV disease.
the immunopathological response to target anti- laboratory support available. However, such pa-
gens in the lung. tients should clearly be managed by one of the
CMV strains resistant to ganciclovir, ganciclovir proactive strategies (prophylaxis, suppression, pre-
plus foscarnet, or to ganciclovir plus foscarnet plus emptive therapy), since it would be unethical to con-
cidofovir, have already been described in AIDS pa- tinue to allow the natural history of CMV disease in
tients (Chou et al., 1997). Genetic changes in UL97 these patients to proceed unchecked in the face of
usually confer low-level resistance to ganciclovir. overwhelming benefit demonstrated in controlled
Continued selective pressure may select for UL54 clinical trials. There should be transfer of this con-
mutants, some of which are cross-resistant to cept to AIDS patients so that CMV disease is pre-
cidofovir. Rare mutations in UL54 can confer resis- vented via pre-emptive therapy rather than treated.
tance to both ganciclovir and foscarnet (Chou et al., Indeed, I suggest we set ourselves the objective to
1997). regard CMV disease in AIDS patients as a failure of
Based on the principles of natural history and medical management, rather than the starting point
pathogenesis gleaned from study of transplant pa- of therapeutic intervention.
tients, highly active antiretroviral therapy
(HAART) should generally improve the long-term
control of CMV disease in AIDS patients (Table
2C.15). However, HAART might also alter the clini- REFERENCES
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the pathogenetic process includes an im- Anonymous (1992) Mortality in patients with the acquired im-
munodeficiency syndrome treated with either foscarnet or
munopathological component. Thus, physicians ganciclovir for cytomegalovirus retinitis. Studies of Ocular
should be aware of the possibilities of inducing Complications of AIDS Research Group, in collaboration
CMV pneumonitis in a patient with asymptomatic with the AIDS Clinical Trials Group. New England Journal of
CMV lung infection, or producing inflammatory Medicine, 326, 213—220.
Anonymous (1996) Combination foscarnet and ganciclovir ther-
responses to CMV in the liver, gastrointestinal tract apy vs monotherapy for the treatment of relapsed
or eye. These basic principles reinforce the objective cytomegalovirus retinitis in patients with AIDS. The
of preventing CMV disease through pre-emptive Cytomegalovirus Retreatment Trial. The Studies of Ocular
therapy, rather than trying to treat established dis- Complications of AIDS Research Group in Collaboration
eases. with the AIDS Clinical Trials Group. Archives of Ophthalmol-
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
2D
Epstein–Barr Virus
Dorothy H. Crawford
University of Edinburgh, Edinburgh, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
118 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2D.1 Electron micrographs of the thin section of cells form an EB virus-immortalized lymphoblastoid cell line (M-ABA).
Arrows indicate (a) immature virus particles and (b) enveloped virus particles. (; 39 000)
virinae, genus Lymphocryptovirus, showing a struc- outer, irregularly shaped, lipid-containing envel-
ture indistinguishable from other human herpes- ope, giving the mature particle a diameter of
viruses by electron microscopy. It is a large virus 150—200 nm. The envelope is derived from cellular
with a buoyant density in caesium chloride of membranes of infected cells and is acquired by bud-
1.2—1.3 and a molecular weight of 100 ; 10 Da. ding of the immature particles through the cell
The central nucleic acid of the virion is surrounded membrane. The envelope is essential for infectivity,
by an icosahedral capsid consisting of 162 triangu- and the sensitivity of the virus to ether and other
lar capsomeres and measuring 100 nm in diameter lipid solvents results from its destruction.
(Figure 2D.1). This, in turn, is surrounded by an
EPSTEIN–BARR VIRUS 119
LMP 2A, B EBNA LP EBNA 2 EBNA 3a EBNA 3b EBNA 3c EBNA 1 LMP 1 LMP 2A,B
N C W WW W WW WW WWWY H F Q U P O a M S L E Z R K B C D T V I A Nhet
h e g f cb x d
Bam HI
map
Figure 2D.2 Linear map of the EB virus genome, showing the open reading frames for the major latent proteins
Infection of B cell
virion contains double
stranded linear DNA
EBNA 2 + LP
Cell death CD23
release of HLA DR 12 h
virions
EBNA 1, 3a,b,c
VCA 24 h
MA
Blast
transformation
Inevitable
Genome
cell death
circularization
48 h
EA
LMP1
48 h
Phenotype of
latently infected
immortalized B cells
Figure 2D.3 EB virus gene expression and cellular events after infection of a resting B cell. EBNA : EB viral nuclear antigen;
LMP : latent membrane protein; EA : early antigens; MA : membrane antigens; VCA : viral capsid antigens
Figure 2D.4 A photomicrograph of cells from an EB virus-immortalized adherent lymphoblastoid cell line (M-ABA) stained for
EBNA. Bright nuclear fluorescence is seen in all cells. The cells are counterstained with Evans Blue. (; 70)
Latent membrane proteins (LMP). LMP 1 is The functions of these proteins are unknown, but
coded for by the BNLF 1 gene, and its expression is they appear to suppress reactivation from latency.
induced by EBNA 2. It is the most abundantly
transcribed region of the genome in latently infected
Lytic Cycle Proteins
cells. The protein is found in the viral particle as
well as in infected cells, where it has a membrane EBV lytic genes show extensive homology to those
location with six membrane spanning domains and of other herpesviruses, and their expression similar-
both the N-terminus and C-terminus in the cyto- ly follows an orderly cascade with the expression of
plasm. LMP 1 is essential for the immortalization of each set being activated by the previous, and in-
B cells and is a viral oncogene, inducing a tu- hibited by the following set. They are divided into
morigenic phenotype on transfection into NIH 3T3 immediate-early, early, and late, according to
cells. When transfected in B cell lines, LMP upregu- whether they are transcribed before (immedate-
lates expression of the cell adhesion molecules and early and early) or after (late) viral DNA synthesis.
CD21, CD23 and CD40 and induces B cell activa-
tion. These changes mimic those seen following
Immediate-early genes. EBV possesses two
CD40—mediated B cell activation, and, in both
genes that can be classified as immediate-early
cases, are mediated by tumour necrosis factor re-
genes, BZLF1 (Z) and BRLF1 (R), which together
ceptor-associated factor (TRAF)-signalling mole-
transactivate the early genes and thereby effect the
cules and the transcription factor NFkB. When
switch from latent to lytic infection in B cells.
transfected into squamous epithelial cell lines, LMP
induces the membrane receptor molecules CD40
and the epidermal growth factor (EGF) receptor Early genes. First identified by the staining pat-
and inhibits terminal differentiation processes. tern of sera containing antibodies to EBV when
LMP 2A and B (also called terminal proteins 1 applied to the Raji cell line which lacks expression
and 2) are formed by alternative splicing from an of the late genes, the early gene products (early
open reading frame which spans the terminal repeat antigens, EA) were characterized as diffuse (D) (nu-
sequences, and thus the intact gene is only formed clear and cytoplasmic staining) and restricted (R)
and transcribed after the genome has circularized. (nuclear staining) (Henle et al., 1971). It is now
122 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
known that each of these complexes consists of survival gene bcl2, which are thought to be import-
around 30 proteins, most of which have enzyme ant in vivo in immune evasion and viral persistence.
functions required for viral DNA replication.
Late genes—viral capsid proteins. This antigen Host Range and Growth In Vitro
complex (VCA) consists of the viral structural pro-
teins that have not yet been analysed in detail be- The host range of EBV is limited to humans, its
cause of the lack of a fully lytic system in vitro. The natural host, and some subhuman primates, includ-
major capsid protein p160 is coded for by the ing the gibbon, owl monkey, squirrel monkey and
BALF4 open reading frame. some species of tamarin, all of which can be infected
VCA is detected by indirect immunofluorescence experimentally.
in a minority of cells in a permissive cell line such as In humans, only B lymphocytes are regularly
P3HR1 using an EBV-positive human serum. infected by the virus and this restriction of infection
is probably accounted for by their expression of
CR2 (the receptor for the C3d component of com-
EBV glycoproteins. The EBV-coded glycop- plement, also called CD21), the cell surface receptor
roteins are involved in viral infectivity and spread. used by EBV. A similar molecule found on a sub-
Seven have been identified, most of which are in- population of squamous epithelial cells may result
serted into the membranes of the infected cell and in infection of these cells in the oropharyngeal cav-
three of these become components of the viral en- ity, although the extent and role of this infection in
velope (membrane antigens, MA). Two (gp340/220 normal individuals is a controversial issue. Ac-
and gp85) have been studied in detail because they tivated T cells and dendritic cells also express CR2
induce the production of neutralizing antibodies and may on rare occasions support EBV infection.
and are therefore potential vaccine candidates. In vitro, the outcome of infection of B cells and
Three show extensive homology to herpes simplex squamous epithelial cells differs markedly. In epi-
glycoproteins. thelial cells a lytic infection occurs with production
The major envelope glycoprotein, gp340/220, is of new viral progeny and cell death (Sixbey et al.,
coded for by the BLLF1 open reading frame. The 1984), whereas in vitro infection of B cells leads to
protein mediates virus attachment to the B cell immortalization (see below).
surface by binding to the EBV receptor CR2 (also The only method for demonstrating infectious
called CD21) (Nemerow et al., 1985). Thus both virus in patient samples is the lymphocyte ‘immor-
naturally occurring and experimentally induced talization’ test, in which lymphocytes from EBV-
antibodies to gp340/220 prevent infection by block- negative donors are exposed to the filtered sample
ing attachment. and then cultured. The presence of virus in the
Gp110 is coded for by the BALF4 open reading sample is indicated by the outgrowth of immortal-
frame and has homology with the herpes simplex ized B cells into a lymphoblastoid cell line (see
virus glycoprotein gB. It is localized to the nuclear below). The technique is very time consuming and
and cytopalsmic membranes of infected cells but is slow to give results and is therefore not recommen-
not detected in the viral envelope. ded for routine use. Polymerase chain reaction
Gp85 is coded for by the BXLF2 open reading (PCR) detection of EBV DNA is a quicker and
frame and has homology to the herpes simplex virus more sensitive test but does not necessarily denote
glycoprotein gH. The protein is localized to the infectivity.
plasma membrane and viral envelope, where it in-
duces fusion between the viral and cellular mem-
branes. EBV Immortalization
Association Disease
80
Causative agent Acute infectious mononucleosis
antibody (%)
Figure 2D.6 Basic pattern of serum antibodies to EB virus-associated antigens before, during and after primary infection.
HA : heterophile antibody; EA(D) : diffuse form of early antigen; other abbreviations as in Figure 2D.3
onset of clinical symptoms, IgM, IgA and IgG anti- Cellular Immunity
bodies to VCA are present in the serum, as are IgG
One of the most distinctive features of IM is the
antibodies to EA (D) and MA. Anti-MA antibodies
presence, at the time of onset of clinical symptoms,
are neutralizing and probably agglutinate virus par-
of a lymphocytosis and ‘atypical’ mononuclear cells
ticles, thus preventing further infection and spread
in the peripheral blood (Figure 2D.7, see Plate II).
of the virus. IgM and IgA antibodies to VCA and
The rise of lymphocyte count, which may be very
IgG anti-EA antibodies rise to a peak during the
marked, is due to a vast increase in absolute num-
acute disease and decline to undetectable levels dur-
bers of CD8 ; T-lymphocytes. This finding is ac-
ing convalescence. IgG antibodies to EBNA 1 are
companied by depression of T lymphocyte func-
not usually detectable in the serum until the conva-
tions, including those measured by in vivo delayed-
lescent period, although IgM antibodies to EBNA
type hypersensitivity testing and in vitro mitogen
have been detected transiently during acute IM.
stimulation. The CD8;T cells have cytotoxic ac-
Heterophile antibodies regularly appear in the
tivity against EBV-infected B cells which is mainly
serum early in IM but their relationship to the virus
MHC class I restricted. A recent study suggests that
and their role, if any, in controlling infection remain
this represents a response to an EBV-coded super-
unclear. A variety of autoantibodies may be found
antigen (Sutkowski et al., 1996). These T cells are
in IM, which include cold agglutinins (anti-i), rheu-
specific for latent and lytic EBV antigens and prob-
matoid factors, antinuclear antibodies and antibo-
ably have an essential role in the complete recovery
dies to platelets and to smooth muscle. These anti-
from IM. Acting together with the humoral el-
bodies, which may account for the raised total
ements of the immune response, they control and
serum IgM level found in IM, are thought to be the
confine the infection.
result of the polyclonal activation of B cells caused
by EBV infection. They are usually transient and
harmless.
EPSTEIN–BARR VIRUS 127
Histological Findings enough to cause extreme pain and difficulty with
swallowing, and occasionally gross tonsillar en-
Generally, tissue from IM patients is not available
largement may lead to pharyngeal obstruction. In
for study, but occasionally liver, bone marrow or
25% of cases secondary infection of the pharynx
lymph node biopsies are performed before the diag-
occurs with a -haemolytic streptococcus. Less
nosis is made. Also the histological findings on tis-
commonly, patients present with jaundice, cough,
sue from surgically removed tonsils or ruptured
myalgia or symptoms of one of the neurological
spleens, and postmortem specimens have been de-
complications of IM (see below).
scribed. In all cases the tissues are infiltrated with
mononuclear cells, which are immunoblastic in ap-
pearance. In the lymph node the pattern is generally
described as ‘reactive’, with the infiltrate found in
widely dilated sinuses and intersinusoidal cords ex- Physical Signs
tending into the interfollicular compartment, often
Lymphadenopathy is present in the majority of
obscuring the follicular centres. Occasional Hodg-
cases at some time during the acute illness. The
kin or Reed— Sternberg-like cells may be present. In
cervical nodes are most obviously involved, but
the liver the portal areas are infiltrated and in the
generalized lymphadenopathy occurs, with the
spleen the white pulp may be obscured by the exten-
nodes often remaining palpable for several weeks.
sive infiltrate which extends throughout the paren-
The lymph nodes are discrete and not severely ten-
chyma. Small aggregates of monocytoid cells and
der. The additional presence of rubbery small
immunoblasts can be seen in the bone marrow.
lymph nodes in the axilla and groin indicates IM
Immunological studies showed that the virus-infec-
rather than a throat infection with involvement of
ted B cells are localized in the paracortical region of
cervical nodes. Splenic enlargement and tenderness
the lymph node. The histological findings are not
occur in 50—60% of patients, and this is accom-
diagnostic of IM in themselves, and may be difficult
panied by mild hepatomegaly in 15—25% of pa-
to distinguish from other causes of immunoblastic
tients. Clinically apparent jaundice occurs in
proliferations, Hodgkin’s disease and non-Hodgkin
5—10% of cases. Fevers of 38—40°C are a regular
lymphomas.
feature during the first 1—2 weeks of IM, with the
highest temperature often occurring at midday and
being followed by drenching sweats.
Pharyngitis and palatal petechiae occur during
Clinical Features the first week of the illness. These may be accom-
panied by a grey-white membrane which, when as-
Incubation Period sociated with pharyngeal oedema and tonsillar en-
As the source of infection is generally not deter- largement, can result in obstruction to the pharynx
mined, the incubation period is difficult to calculate; of trachea. Periorbital oedema is common early in
however, a period of 30—50 days is usual before this disease and may lead to the mistaken diagnosis
symptoms occur. of nephritis.
Two types of skin rashes can occur: a faint mor-
billiform eruption which lasts 24—48 hours, or a
maculopapular rash which occurs in almost all pa-
Symptoms
tients receiving ampicillin. The cause of the latter is
Characteristically IM begins abruptly with a sore unknown, but may be due to the production of
throat and swelling of the neck, accompanied by antibodies specific for ampicillin and the deposition
non-specific symptoms such as malaise, fever, of immune complexes in the skin arterioles; its pres-
sweating, chills, headaches, stiff neck, anorexia and ence is regarded by some as diagnostic of IM.
vague abdominal discomfort. A prodromal period Other associated clinical conditions include en-
characterized by lassitude and slight fever is de- cephalitis, meningitis, delirium, coma, psychosis,
scribed by some patients. The sore throat, which transverse myelitis, polyneuritis, mononeuritis,
occurs in 80—90% of patients, is usually mild and pericarditis, myocarditis, interstitial pneumonia
clears after 7—14 days. It may, however, be severe and pleural effusions. None of these are common.
128 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
IM in Children and the Elderly perature may continue to rise at midday. The pa-
tient often feels exhaustion on the slightest exertion
When IM occurs outside the classic age range of
and complains of an inability to concentrate for
17—25 years it tends to present a less typical clinical
several weeks after apparent recovery. However, a
picture. In children the disease is usually mild and
study carried out on the achievement of students in
does not require medical attention. Sore throat and
their examinations after an attack of IM showed no
cervical lymph node enlargement are usually pres-
significant change from the expected performance.
ent but not invariably so. Occasionally children
Occasional patients, particularly those over 25
exhibit classic IM even as early as the age of 2 years.
years of age, may experience intermittent fatigue
The clinical onset of IM in the elderly is often
over the following 2 years. Any contribution they
insidious and occasionally bizarre. The disease can
make to the group described as having chronic
be severe, with hepatic, neurological and renal in-
fatigue syndrome (CFS) can only be ascertained
volvement.
when full documentation of serological tests during
the acute and subsequent phases are available (see
below). Other patients suffer ‘relapses’ during the 6
IM in Pregnancy
months to 1 year following IM, with return of fever,
Infectious mononucleosis during pregnancy is un- sore throat and lymphadenopathy accompanied by
common, but where it has occurred there has rarely a positive heterophil antibody test. The exact na-
been any deleterious effect on the fetus, and termi- ture of these relapses is unclear, since the serological
nation of pregnancy is not indicated. Lymphoma in markers of primary infection may remain positive
babies born to mothers with IM has occasionally for up to a year even in sublinical cases of serocon-
been reported but a direct association with EBV version.
infection has been difficult to establish. Rare cases
of fatal lymphoproliferative disease in women who
developed IM during pregnancy are better
documented, and suggest that the immunological Complications
changes associated with pregnancy may have in-
hibited the normal immune control of IM (see In the vast majority of cases IM is a benign and
above). self-limiting disease from which complete recovery
is the rule. However, certain morbid complications
have been described in the literature, which account
IM in the Immunosuppressed for around 30 deaths per year in the USA. The main
causes of death are neurological complications,
When primary EBV infection occurs in im- splenic rupture, hepatic failure and secondary infec-
munocompromised patients, particularly after or- tion. Approximately half the deaths are associated
gan transplantation, it is often asymptomatic due to with X-linked lymphoproliferative syndrome (see
the patient’s inability to mount an immune reac- also page 137).
tion; however, it may result in atypical disease, with
gastrointestinal symptoms and/or signs of renal
graft rejection and failure being reported. Antibo- Neurological Complications
dies to EB viral antigens may be slow to develop,
with neither IgM antibodies to VCA nor the hetero- These include meningitis, encephalitis and the
phile antibody test being invariably positive. Some Guillain—Barré syndrome. Each of these conditions
of these primary infections progress to lympho- may precede, accompany or postdate IM by several
proliferative disease and lymphoma (see below). weeks. Recovery is usual.
Hepatic Complications
Course and Convalescence
Although many IM patients have biochemical evi-
The illness may last for several weeks, and even dence of hepatocellular damage, overt jaundice is
when obvious symptoms have resolved the tem- uncommon (5—10%), and complete recovery is the
EPSTEIN–BARR VIRUS 129
rule. However, more severe cases have been re- rather than a specific aetiological association be-
ported, and these include massive hepatic necrosis tween EBV and CFS.
resulting in death.
Treatment
BURKITT’S LYMPHOMA
Figure 2D.9 Male child with a Burkit’s lymphoma of the jaw (a) before treatment and (b) after treatment with cyclophosphamide
(30 mg per kg body weight)
African BL is a tumour which occurs in children The diagnosis of BL in an endemic area is very often
aged 3—15 years, with a peak age incidence of 6—7 clear from the clinical features described above;
years. In those areas of Africa and Papua New however, hisotological evidence should be sought
Guinea where BL is endemic it occurs with an (Figure 2D.10). The tumour shows a characteristic
incidence of 15 per 100 000 children aged 5—10 histological picture of a diffuse monomorphic infil-
years, and is the commonest malignancy in this age trate of medium-sized blast cells with variable
range. It is more common in boys than girls and numbers of infiltrating histiocytes giving the classic
arises extranodally, typically in the area of the jaw, ‘starry sky’ appearance. BL is a monoclonal tumour
giving a chracteristic presentation (Figure 2D.9). of B cell origin, and in over 90% of cases the cells
The tumour is usually found to be multifocal at express surface IgM.
presentation, the other sites commonly involved
being the postorbital region, gastrointestinal tract,
thyroid, liver, kidney, skeleton, testicles and the
ovaries, and the breast in pubertal girls. Treatment
BL is a highly malignant tumour, with death
supervening within a few months of clinical onset in Burkitt lymphoma is very sensitive to chemother-
untreated cases. The tumour is, however, sensitive apy, one dose of cyclphosphamide often being
to chemotherapy. enough to cause complete regression of the tumour
mass. Relapses do occur, however, and are pro-
gressively less responsive to therapy. For this rea-
134 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2D.10 Histological section of Burkitt’s lymphoma, showing a uniform population of nucleolated lymphoid cells and scattered
vacuolated macrophages (H & E, ; 800.) (Courtesy of Professor P. Isaacson, University College Hospital)
son a full course of treatment should be given in- the rarer, the more differentiated forms, is contro-
itially, in which case the prognosis is good. versial.
Seroepidemiology
Prevention
The association between NPC and EBV was first
The prevention of BL may theoretically be achieved demonstrated serologically (Old et al., 1966) and
either by the eradication of malaria, which is prob- this study has been extended to show that 100% of
ably a necessary cofactor in the disease, or by the sera from cases of undifferentiated NPC have high-
prevention of EBV infection by vaccination (see titre antibodies to the EB viral antigen VCA, re-
page 138). gardless of their geographical location. As in BL,
Where the eradication of malaria has been the antibodies are present at a ten times higher
achieved in small areas, such as some of the islands geometric mean titre than in matched controls, and
of Papua New Guinea, and the incidence of BL has show a unique reaction pattern. Thus the anti-EA
fallen. (D) component is the most frequently seen and is
present at a higher titre than anti-EA (R). IgG and
IgA anti-EA (D) antibody titres rise as the disease
NASOPHARYNGEAL CARCINOMA progresses and fall in remissions. They can be unde-
tectable in long-term survivors. Similarly, IgA anti-
Nasopharyngeal carcinoma (NPC) is a malignant bodies to VCA are present in NPC sera and corre-
tumour of the squamous epithelium of the late with disease progression. These IgA antibodies
nasopharynx, which is very prevalent in southern are also found uniquely in the saliva of NPC pa-
China, where it is the commonest tumour in men tients.
and the second most common in women. In most
other areas of the world the tumour is rare, but
pockets of high incidence occur in North and Cen- Pathogenesis
tral Africa, Malaysia, Alaska and Iceland (Figure
Association with EBV
2D.8). The most undifferentiated form of the tu-
mour, which occurs most commonly, is always as- As with BL, it is difficult to distinguish an aetiologi-
sociated with EBV, whereas the association with cal association between EBV and NPC from a pure-
EPSTEIN–BARR VIRUS 135
ly passenger role for the virus in the tumour cells; The tumour most commonly arises on the posterior
however, the following evidence suggests that there wall of the nasopharynx in the fossa of Rosenmül-
is more than a casual association involved. Multiple ler, where it often remains silent, and rapidly meta-
clonal copies of the EB viral genome can be detec- stasizes to the draining lymph nodes. Thus the most
ted in the malignant epithelial cells of 100% of frequent presenting symptoms of NPC is bilateral
undifferentiated NPC biopsy specimens. All the enlargement of lymph glands in the neck, which are
malignant epithelial cells express the EBV-coded firm, non-tender and fixed. The upper cervical chain
antigen EBNA 1, and LMP is expressed in around of glands are most often involved in the initial
50% of tumours. Seroepidemiological data show spread. At this stage the primary tumour may be
that 100% of sera from undifferentiated NPC pa- very small and difficult to locate. Less frequently the
tients have high-titre antibodies to EB viral anti- presenting symptoms are associated with invasion
gens which correlate with clinical events (see above). by the primary tumour, and include nasal obstruc-
Finally, the malignant epithelial cells from NPC tion, postnasal discharge, epistaxis, partial deafness
produce EBV particles when explanted into tissue and cranial nerve palsies. If untreated, the disease is
culture following growth in nude mice to remove rapidly fatal, with death being most often due to
the non-malignant, infiltrating lymphocytes. This laryngeal and pharyngeal obstruction.
NPC-derived virus has been shown to be distin-
guishable to EBV derived from other sources.
Diagnosis
Cofactors in the Pathogenesis of NPC The diagnosis of NPC is often made on biopsy
NPC is a genetically restricted tumour, being most material from an enlarged cervical lymph node. The
common in the southern Chinese. It has an inter- cells are squamous epithelial in origin and three
mediate frequency in some Negroid and Mongoloid histologial types are described in the World Health
races and is rare in Caucasians. It has been noted Organization classification: (1) a well-differentiated
that the first-generation immigrants from southern squamous cell carcinoma with intercellular bridges
China to the USA retain the high frequency of the and/or keratinization; (2) a non-keratinizing car-
disease, although later generations show a declining cinoma; and (3) an undifferentiated carcinoma in
incidence, which may be due to intermarriage with which a heavy lymphocytic infiltration is often pres-
non-Chinese races. These data strongly suggest a ent which may be so extensive as to lead to the
genetic factor in the aetiology of the disease, and mistaken diagnosis of lymphoma (Figure 2D.11).
this is backed up by family clustering and the find- However, the lymphocytes, which are mainly T
ing that the HLA haplotype A BW36 confers a 4—6 cells, are non-malignant. The term ‘lympho-
epithelioma’ has been used to describe this third
times increased risk of disease. Environmental fac-
tors have also been suggested in the aetiology of type, which occurs most commonly in the high-risk
NPC, in particular dietary components, such as areas.
salted fish, and phorbol ester contamination of soil Serum antibody titres to EBV antigens can be
and food by common local plants (Ito, 1986). used to confirm the diagnosis of NPC and to moni-
tor the progress of the disease. Large-scale screen-
ing programmes have been undertaken in China,
and those individuals with persistent IgA antibo-
Clinical Features dies to VCA in serum samples have been followed
up. NPC has developed in some, and ‘precancerous’
NPC occurs at a rate of 98 per 100 000 of the lesions have been diagnosed in others. It is hoped
population in southern China and is more common that this type of project may lead to early treatment
in men than in women. The age of onset of NPC and a reduced mortality from NPC. IgA antibodies
varies with geographical distribution and histologi- to VCA and EA in the saliva are considered diag-
cal type, the undifferentiated type being more nostic of NPC.
common in high-risk areas in young patients,
whereas the more differentiated types occur in older
patients and constitute the bulk of sporadic cases.
136 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2D.11 Histological section of undifferentiated nasopharyngeal carcinoma, showing scattered malignant epithelial cells and a
heavy infiltrate of small lymphocytes. (H & E, ; 400)
This syndrome (first called Duncan’s syndrome) It is well known that there is an increased incidence
was recognized in 1974 by Purtilo and colleagues, of malignancy following organ transplantation and
who described a family in which six male kindred concomitant immunosuppression, and around 20%
died of acute IM and/or malignant lymphoma (Pur- of this is accounted for by malignant lymphoma.
tilo et al., 1974). Since then many such families have These are mainly classified as large cell lymphomas
been reported, with affected members having an which develop in 1—10% of patients following solid
apparent inability to mount an effective cell-me- organ transplantation. The tumours tend to occur
diated immune response to primary EBV infection. in the central nervous system, or in lymphoid tissue
The defective gene is located on the X chromosome in the gastrointestinal tract or the transplanted or-
and has recently been cloned (Coffey et al., 1998; gan. Over 90% of these tumours carry the EB viral
Sayos et al., 1998). It codes for a protein which genome and the tumour cells generally express all
regulates T cell activation. the latent viral genes. Examination of lympho-
Clinically the affected males are healthy until proliferative lesions from multiple sites for immuno-
primary EBV infection occurs. The course of the globulin gene rearrangement reveals that most are
disease is then either fulminating and rapidly fatal monoclonal in origin, although separate clones may
or it may progress to a chronic phase, often culmi- proliferate at each individual site. However, pro-
nating in a fatal, B cell lymphoproliferation. These gression from a polyclonal lymphoproliferative
tumours often occur in the central nervous system lesion to a monoclonal lymphoma has also been
or gastrointestinal tract. Hypogammaglobu- described. Serological studies in transplant recipi-
linaemia, agranulocytosis and aplastic anaemia ents show that, in around 50% of those developing
have also been reported following IM in some of EBV-associated lymphoma, there is evidence of a
these patients. recent primary infection.
Many abnormal laboratory findings have been The immunosuppressive therapy following trans-
reported in X-LPS, although none are consistently plantation reduces the T cell-mediated immunity to
found. These include defective natural killer cell EBV (Crawford et al., 1981), and it is therefore
activity and a reduction in EBV-specific T cell postulated that, in the absence of specific immune
cytotoxicity. The pattern of antibodies to EB viral mechanisms, EBV-infected B lymphocytes prolifer-
antigens may be abnormal, with high anti-VCA and ate in an uncontrolled manner. This postulate is
EA antibodies and low anti-EBNA antibodies. This strengthened by the finding of regression of some of
pattern is identical to that seen in many im- these lesions when the immunosuppressive therapy
munosuppressed states. Female carriers of the ab- is reduced or withdrawn. This is now the first line of
normal X chromosome may show a milder derange- treatment, and the antiviral drug acyclovir is some-
ment of antibody pattern and can in that case be times added to this regimen, but its role in tumour
recognized in a family where a fatal case of IM has regression is unclear. However, relapse and recur-
occurred. Carriers generally have normal EBV-spe- rence are common, and recently donor-derived
cific T cell killing. EBV-specific cytotoxic T cells grown in vitro have
138 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
been used to prevent or treat EBV-associated B early in life to prevent natural infection in the BL-
lymphoproliferative disease in bone marrow trans- and NPC-susceptible populations. An effective vac-
plant recipients (Rooney et al., 1995). cine preparation could also be useful in
seronegative organ transplant recipients, and per-
haps even those at risk of developing severe IM,
Acquired Immune Deficiency Syndrome such as male offspring of X-LPS carriers.
The antigen chosen for vaccine development is
(AIDS) the MA antigen gp340/220 (page 122), since it is this
antigen to which neutralizing antibodies are mainly
With the profound cellular immunodeficiency seen directed. Cotton top tamarins have been used as an
in patients infected with human immunodeficiency animal model for testing vaccine preparations, since
virus (HIV) it is not surprising that EBV-associated these animals develop EBV-associated tumours
lymphomas frequently occur, and these are in- after inoculation with EBV. Preinoculation of gp
cluded in the CDC diagnostic criteria for AIDS. 340/220, presented in immune-stimulating com-
However, whereas some of these tumours are of the
plexes, protects the tamarins from virus challenge
large cell histological type seen in the other im-
(Morgan et al., 1988). It remains to be seen whether
munodeficiency states, others are of the BL pheno- this vaccine will be effective in humans.
type. The latter tumours are monoclonal and
around 50% show the cellular and viral gene ex-
pression described for African BL (page 132). The
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
2E
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
142 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
(HCMV), HHV-6 and HHV-7 are not frequently
shed in urine (GautheretDejean et al., 1997).
Growth
Figure 2E.2 The cytopathic effect of HHV-6 on CD4 ; T lymphocytes. This shows uninfected cells (JJhan T leukaemia cell line) in (a),
and infected cells 5 days postinfection in (b). The cytopathic effect includes clumped, fused cells, cytomegalia (large cells, including
multinucleated) and ‘balloon’ cells (cells which appear to have grossly enlarged cytoplasm). Infection and cell fusion can result
subsequently in cell lysis
(Lusso et al., 1994). After infection the surface ex- kidney, as well as lymphoid or progenitor cell types
pression and transcriptional control are also down- such as in the bone marrow and lymph nodes (see
regulated (Furukawa et al., 1994; Secchiero et al., below). In vitro infection has been observed, but
1997b). In contrast to HHV-7, HHV-6 upregulates with limited replication, in NK cells, B lym-
CD4 expression and this can enhance infection of phocytes, astrocytes, megakaryocytes, bone mar-
these cells with HIV (Lusso et al., 1991a, 1995). row progenitor cells, monocytic/macrophage cells,
as well as hepatic, cervical and retinal epithelial
cells. HHV-6 has also been identified as a commen-
Salivary Gland Epithelium
sal of the brain on the basis of sensitive PCR DNA
In vivo antigen staining and in situ hybridization detection and limited antigen expression (Challoner
studies have shown that specialized cells within the et al., 1995). The exact target cell in the brain is not
salivary gland epithelium harbour persistent infec- clear, although astrocyte or oligodendrocyte-like
tions with HHV-6 and HHV-7 (Fox et al., 1990; cells have been suggested and the virus DNA can be
Sada et al., 1996). There is virus replication at these detected by PCR in cerebrospinal fluid of some
sites and both viruses are shed (see above). HHV-7 patient groups.
is easier to detect and isolate from saliva then HHV-
6 (DiLuca et al., 1995). This is likely to be the main
route of host-to-host transmission. Other routes
include iatrogenic transfer with blood products or Latency
organ transplants.
Like all herpesviruses, HHV-6 and HHV-7 can es-
tablish latent infections in specialized cell types.
Growth in Other Cell Types
There is evidence for latency within monocytic/
The viruses may be identified in vivo by polymerase macrophage cells as well as bone marrow progeni-
chain reaction (PCR) in a number of cell types, but tor cells (Kondo et al., 1991; Gompels et al.,
there is no evidence that these are sites of permissive 1993;1994; Kempf et al., 1997; Yasukawa et al.,
active replication. Since these are blood-borne in- 1997). HHV-7 can be detected by PCR in blood and
fections, PCR detection in tissue must be controlled saliva of asymptomatic adults in higher levels than
for blood contamination. In addition to T lym- HHV-6 (DiLuca et al., 1995; Kidd et al., 1996;
phocytes and salivary gland epithelium, there are GautheretDejean et al., 1997). On this basis it has
other related relevant sites in vivo with evidence for been suggested that HHV-7 may exist in a less
active replication, including regions with specializ- controlled state of latency than HHV-6. Evidence
ed epithelium such as in the retina, lung, liver and has been presented that HHV-7 can reactivate
144 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
HHV-6 (Katsafanas et al., 1996). Both HHV-6 and virus, with approximately two-thirds of the genes
HHV-7 have been detected in cells with monocytic encoding similar proteins (Figure 2E.3). However,
markers in Kaposi’s sarcoma (KS) biopsies (Kempf this relationship is distant and can only be deter-
et al., 1997). It has been suggested that these cells mined by encoded amino acid sequences. Further-
carry latent infections and that they have been re- more, HCMV is almost double the size, 230 kb,
cruited and reactivated through a chemotactic re- containing extended glycoprotein gene families ab-
sponse to chemoattractant cytokines in the KS sent from HHV-6 and HHV-7. HCMV is represen-
lesion. tative of beta-1 herpesviruses, and HHV-6/-7 of
beta-2 herpesviruses. There are no animal herpes-
viruses identified to date more related to HHV-6/-7
than HCMV, although murine CMV has properties
Molecular Biology similar to all these viruses and may be used as an
animal model for some features (Rawlinson et al.,
The derivation of complete genomic sequence for 1996). There are HHV-6— and HHV-7—specific
both HHV-6 and HHV-7 have been described, in- genes (Figure 2E.3). HHV-7 has fewer genes than
cluding overall reviews of their molecular biology HHV-6. HHV-6 encodes a Rep protein, U94, found
and known functions of encoded proteins (Gompels in adeno-associated viruses (AAV) and can comple-
et al., 1995; Nicholas, 1996). This information is ment this function in replication defective AAV
briefly summarized in the following section. (Thomson et al., 1994). The HHV-6 Rep gene poss-
The genomes of HHV-6 and HHV-7 are respect- ibly represents the first transfer of a gene between
ively 153 kb and 145 kb in size. They are both two DNA viruses, in that the amino acid sequences
bounded by terminal direct repeats of 8 and 6 kb are more closely related to the homologue in some
respectively, which themselves are bounded by re- AAV strains then they are to each other. This Rep
peated sequences similar to the telomeric repeats homologue is absent in HHV-7 and may indicate
(GGGTTA) at the end of human chromosomes. replication differences between these viruses. Both
Roles for these sequences in replication and latency viruses have conserved and unique glycoprotein
have been proposed (Gompels et al., 1995). The genes which mediate infection and are targets for
open reading frames (ORFs) in HHV-6 strain immunlogical recognition (Gompels et al., 1995).
U1102 are designated U1—U100, with those in the The virus infects and spreads by cell fusion and
direct repeats as DR1—DR7 (Gompels et al., 1995). candidate glycoproteins which mediate this process
In HHV-7 similar nomenclature is used, with have been identified in each virus, the conserved gH
homologous genes U2—U100 (Nicholas, 1996). A and gL complex, encoded by HHV-6 and HHV-7
few genes are lacking in HHV-7 compared to HHV- U48 and U82 (Liu et al., 1993a, 1993b; Gompels et
6 but there are also a few HHV-7 specific genes, al., 1995; Nicholas, 1996; Anderson and Gompels,
designated H1—H7 (Figure 2E.3) (Gompels et al., 1999). Conserved replication and structural genes
1995; Nicholas, 1996). have been identified. An origin of lytic replication
The genomes are most closely related to each has been localized and characterized, plus viral
other, with similarity between the encoded amino genes involved in replication, including enzyme tar-
acid sequences in over 90% of the ORFs. Similari- gets of established antiviral drugs, such as a viral
ties to other vertebrate herpesviruses can be identi- DNA polymerase, HHV-6 and HHV-7 U38, and
fied in only 20—30% of the ORFs, the ‘conserved phosphotransferase, HHV-6 and HHV-7 U69. Un-
herpesvirus genes’. The organization of these genes like HCMV, HHV-6 and HHV-7 encode an origin
can be grouped into seven gene blocks (Gompels et binding protein, OBP, homologue, HHV-6 and
al., 1995). The arrangement of these gene blocks is HHV-7 U73, which is found in alphaherpesviruses.
subgroup specific. HHV-6 and HHV-7 share with This suggests a difference in replication strategy in
HCMV a betaherpesvirus organization of these HCMV, although HHV-6 and HHV-7 have more
gene blocks (Figures 2E.3 and 2E.4). complicated origins then HSV and are more similar
Analysis of HHV-6 and HHV-7 coding sequences to other betaherpesviruses. A conserved protease
also places them within the betaherpesvirus sub- gene has been identified, HHV-6 and HHV-7 U53,
group of the Herpesviridae. They share the closest and the specificity of the HHV-6 enzyme character-
relation with HCMV, the prototype betaherpes- ized on peptide substrates (Tigue et al., 1996) and
Figure 2E.3 Genome organization of HHV-6 and HHV-7 in comparison to HCMV. The coding sequences and their organization are compared between these three human
betaherpesviruses. The boxes labelled I to VII indicate the seven gene blocks which encoded proteins conserved in all mammalian and bird herpesviruses studied (see text).
These include functions for infection, transcriptional control, replication and morphogenesis. There are also genes which encode these functions but are either betaherpesvirus
group specific or unique to each virus, reflecting biological properties characteristic of the group or the individual virus. Shaded genes indicate encoded proteins that are shared
between betaherpesviruses, which include genes outside the overall gene blocks. Black genes are those shared between HHV-6 and HHV-7. Some of these genes are part of a
gene family characteristic for betaherpesviruses and first described for HCMV, the US22 gene family, which include transcription factors (DR1, DR2, DR6, DR7, U2, U3, U7,
U8, U16, U25 and U95). Positional homologues are indicated in HCMV, but these are not always the closest sequence homologue. White genes are specific for each virus. Genes
are numbered according to their designation from respective genomic sequencing projects, and functions of selected genes are described further in the text. Regions underlined
in all three viruses encode immediate-early genes which are conserved in position, designated IE-A and -B for HHV-6 and HHV-7, MIE and IE for HCMV
146 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
RR1 pol gB dbp dU gH MCP Sp PT X O udg gL
0 * 159
0 37 145
27 38 39 41 42 46 48 53 56, 57 60 77 81, 82
HCMV beta I II III IV V VI VII
0
* 230
41 39,38 53 48 57,56 60 77 81, 82 46 42 27 37
Figure 2E.4 Genome organization of HHV-6 and HHV-7 is betaherpesvirus subgroup specific. The organization of the conserved
gene blocks shown in Figure 2E.3 are compared to prototypes of alpha-, beta- and gammaherpesviruses. HHV-6 and HHV-7 share a
betaherpesvirus subgroup arrangement of conserved genes. Rearrangement of gene blocks are shown for HSV-1 and EBV, representing
the alpha- and gammaherpesviruses, respectively. Thus the overall number of genes encoding similar products, the degree of their
similarity and their overall organization group HHV-6 and HHV-7 with HCMV in the betaherpesvirus subgroup (see also Figures 2E.1
and 2E.3). By these three criterion HHV-6 and HHV-7 are also more closely related to each other then to HCMV. Genes encoding
conserved functions in the gene blocks are indicated as follows: RR1 : ribonucleotide reductase subunit (u28); pol : DNA polymerase
(U38); gB : glycoprotein B (U39); dbp : DNA binding protein (U41); dU : dUTPase (U45); gH : glycoprotein H (U48); MCP :
major capsid protein (U57); Sp : late spliced DNA packaging gene (U60/66); PT : phosphotransferase (U69); XO : exonuclease
((U70); udg : uracil-DNA glycosylase (U81); gL : glycoprotein L (U82). Black boxes indicate repeat sequences, stars indicate
positions of origins for DNA replication
full length protein (C. Parry and U.A. Gompels, strain variants (Yamamoto et al., 1994). The IE
unpublished results). In all herpesviruses studied, region has a lower CG dinucleotide frequency then
this protein cleaves itself and a product encoded by expected and this feature is a mark of regions in the
an in-frame transcript. This cleavage is essential for host genome that are subject to DNA methylation
virus production and is a new target for antiviral (Gompels et al., 1995). Methylation can affect gene
therapy. regulation and it is interesting to note that the IE
Gene regulation appears to follow the same cas- regions of all betaherpesviruses have this signature
cade as in other herpesviruses. There are immedi- CG suppression indicative of methylation. It is
ate-early (IE) or genes, which include regulators of possible to speculate that this observation may be
virus gene expression, followed by early (E) genes, correlated to the observed similarities in the sites of
including enzymes for DNA replication, then late latency between the betaherpesviruses (i.e. mono-
(L) or genes, which include structural genes for the cytic/macrophage and bone marrow progenitor
virus particle. The IE genes are important in the cells), suggesting similarities in molecular mechan-
switch between lytic replication and latency. One of isms for latency.
the IE genes, HHV-6 or HHV-7 U86, encodes simi-
larity with the IE2 protein of HCMV. Another IE
gene, HHV-6 or HHV-7 U89, is unique to these
Unique and Cellular Genes
viruses, although it is a positional homologue of
IE1 in HCMV (Schiewe et al., 1994). In HHV-6 this About a third of HHV-6 and HHV-7 genes are
IE gene also shows different organization between specific to these two viruses and presumably reflect
Figure 2E.5 Comparison of strain variation in HHV-6. A summary of sequence comparisons for different strains from A and B variant groups are shown. Most sequences
within conserved regions (gene blocks I to VII) average sequence variation at 5% (white boxes), whereas at regions near or within repetitive sequences variation is higher (black
boxes). Sometimes this is confounded by rearrangement of repeat motifs, as in the U47 glycoprotein gene, leading to higher scores of overall variation due to misalignments
across these sequences. Using a region of U47 outside these repeats to analyse A and B strains from a population where both are common, there are gradual steps between 2 and
5% variation between A and B variant groups, with some strains in between these group distinctions (Kasolo et al., 1997). Less variation has been observed for HHV-7
analysing a similar region (2%) (N. Obel, F. Kasolo and U.A. Gompels, unpublished results). Conserved genes indicated as for Figure 2E.3. Repetitive sequences indicated by
boxes with diagonal shading
148 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
adaptations to their particular cellular tropism and Tanaka-Taya et al. (1996)). However, there is
(Figure 2E.3). Both HHV-6 and HHV-7 also encode evidence for geographic variation, as in Zambia
proteins with similarity to cellular products, variant A strains have been identified in febrile in-
chemokine receptors (U12 and U51) and members fants in greater prevalence, 40% (Kasolo et al.,
of the immunoglobulin superfamily, including an 1997). Antigenic differences between strains have
OX-2 homologue (HHV-6 and HHV-7 U85) (Gom- been described, consistent with results for other
pels et al., 1995; Nicholas, 1996). Distant similarities herpesvirus strains (CampadelliFiume et al., 1993;
are also identified within glycoproteins encoded by Pellett et al., 1993). In addition, some differences in
other betaherpesviruses and selected gammaherpes- growth properties in leukaemic cell lines (for
viruses. These proteins may play a role in the ability example Sup-T1 cells) have been noted (Ablashi et
of the viruses to persist and replicate within cells al., 1993). However, later studies show efficient
which are a normal part of cellular immunity. propagation of all strains of HHV-6 as well as
HHV-7 in Sup-T1 cells (Cermelli et al., 1997). These
observations may reflect properties of different sub-
clones of these leukaemic cell lines. Differences in
Strain Variation virulence are difficult to identify, since most com-
parisons to date, with the exception of the Zambian
At least two strain groups of HHV-6 have been study, have been made in countries where variant A
identified, and termed variant A and variant B by a strains are rare. It has been suggested that variant A
nomenclature agreement (Ablashi et al., 1993). Fur- strains are more virulent and have a relation to
ther subdivisions in both groups have also been HIV/AIDS on the basis of their initial identification
described (Gompels et al., 1993; Challoner et al., in AIDS patients from HIV endemic parts of Africa
1995). In conserved genes strain group differences in (Gompels et al., 1995; Kasolo et al., 1997), as well as
protein or nucleotide sequences averages 5%. data from HHV-6 variant A-related complications
Greater divergence is found at the ends of the in USA AIDS patients (see below).
genome which are near repetitive sequences that
may be mutagenic (Figure 2E.5) (Gompels et al.,
1995). Selected genes have higher rates of variation
CLINICAL CHARACTERISTICS
and correlate with regions of the genome which are
rearranged in other herpesviruses (Gompels et al.,
HHV-6 is the causal agent of a paediatric fever
1995). These genes also have higher rates of non-
which can present as exanthem subitum (also
synonymous substitutions resulting in coding
known as ‘roseola infantum’) and there can be seri-
changes, which may be indicative of selection taking
ous complications. In secondary reactivated infec-
place at these sites (Figure 2E.5). These regions have
tion in immunosuppressed AIDS or transplant pa-
been used as markers for variation (Gompels et al.,
tients, disseminated infections with associated
1993; Kasolo et al., 1997). Less variation has been
pathology have been observed. In bone marrow
observed for HHV-7, 2% for HHV-7 in comparison
transplant patients, HHV-6 reactivation is related
to 5% for HHV-6, even in related regions where
to growth suppression of some progenitor cells. The
HHV-6 variation has been observed (N. Obel, F.C.
virus is an immunosuppressive agent in certain con-
Kasolo and U.A. Gompels, unpublished results).
ditions. Further associations with pathologies are
Closely related genomic strains of HHV-7 have
under evaluation.
been reported, averaging less than 1% variation
(Megaw et al., 1998). Greater variation has been
observed for HCMV strains and Epstein—Barr virus
(EBV) strains, including large genomic deletions in Primary Infection in Childhood
laboratory strains (Gompels et al., 1995) (Chapters
2C and 2D). Variant B strains appear to be more Most causal relations have been established during
prevalent in studies emanating from Japan, USA, the primary infection during childhood. A de novo
Germany, Italy and Austria; primary infection with primary infection can be described by seroconver-
variant A strains is rare, less than 5% of popula- sion, virus isolation and high levels of virus DNA in
tions studied (see references in Gompels et al. (1995) the blood. Primary infection with HHV-6 usually
HUMAN HERPESVIRUSES 6 AND 7 149
occurs after maternal immunity abates at about 6 6 antibodies, with a mean age of infection at 7.3
months. Maternal antibodies are present at birth, months (Pruksananonda et al., 1992; Hall et al.,
then decrease to 6 months of age when the seroposi- 1994).
tive rate increases (Yoshikawa et al., 1989; Enders et In exanthem subitum (ES) patients diagnosed
al., 1990; Hall et al., 1994). With age the serological with primary HHV-6 infection, there are other
titre appears to gradually decrease, but persists, and symptoms aside from fever and rash which are typi-
the virus establishes lifelong infections characteris- cal for ES. These symptoms can also occur during
tic of other herpesviruses. Up to 100% of adult the more common HHV-6 febrile illness without ES
populations are seropositive for this virus rash (Hall et al., 1994). These appear to be related to
(Yoshikawa et al., 1989; Enders et al., 1990; Ward et the level of virus replication, and include diarrhoea,
al., 1993a). A similar pattern exists for HHV-7, al- respiratory symptoms, convulsions, cervical lymph
though infection appears to occur later during in- node swelling and anterior fontanelle bulging
fancy and early childhood, with seropositivity in- (Asano et al., 1991, 1994; Okada et al., 1993). During
creasing to adulthood (Yoshikawa et al., 1993; primary infections there is a transient leucopenia
Huang et al., 1997). which is consistent with virus replication and killing
of lymphocytes.
In studies among children admitted to hospital
Childhood Fever and Rashes (Exanthem
with acute febrile illnesses, in the USA and Italy,
Subitum, Roseola Infantum)
aged less than 2—3 years, 10—24% were due to pri-
Exanthem subitum was the first causal relationship mary HHV-6 infection (Hall et al., 1994; Portolani
to disease established for HHV-6 (Okada et al., et al., 1993). In the USA study, HHV-6 accounted
1993; and references therein). This presents as a for 20% of emergency ward visits from febrile illness
mild skin rash in infants and young children which for infants between 6 and 12 months (Hall et al.,
is accompanied by fever (Figure 2E.6, see Plate III). 1994). In the Italian study, primary infections with
Rare but serious complications have been reported. this virus accounted for 40% of childhood admis-
Recently, it has been established that HHV-7 can sion to hospital for virus infections, higher by ten-
also cause exanthem subitum; this finding is com- fold then any other identified agent (Portolani et al.,
plicated by the fact that HHV-7 usually infects later 1993).
than HHV-6, and that HHV-7 can reactivate There is also evidence for congenital and
HHV-6. Furthermore, HHV-7 antibodies can neonatal infections (Hall et al., 1994), although the
cross-react with HHV-6. However, HHV-7—specific majority of children appear to be infected during
exanthem subitum has been identified (Tanaka et infancy by horizontal transmission, probably via
al., 1994). salivary fluid. There is no evidence for transmission
More recent and extensive studies of childhood by breast milk. Studies of strains in families show
infections with HHV-6 in a USA paediatric popu- the strain type is conserved and that either parent or
lation of 3000 have shown that actually the pri- closely related adult/siblings may transmit HHV-6
mary characteristic of infant infections with HHV- or HHV-7. Evidence for sequential infection with
6 is a high fever for 3—4 days (mean 39.4—39.7°C in multiple strains of HHV-6 or HHV-7 have been
different studies). Only a small proportion of these recorded, either asymptomatically or associated
(6—10%) additionally develop the rash characteris- with complications (see below) (Van Loon et al.,
tic of exanthem subitum. These are measles- or 1995; Torigoe et al., 1996).
rubella-like macular or papular rashes on the face, In primary infections among British children, vir-
the trunk or both. Most diagnoses of exanthem al load in PBMC is high as detected by quantitative
subitum can be attributed to HHV-6 infections, DNA PCR, with 10 HHV-6 or 10 HHV-7 genome
although a proportion may be due to HHV-7 or equivalents per 10 PBMC (Clark et al., 1997). Simi-
dual infections. Some cases may be misdiagnosed lar or higher levels of HHV-6 have been detected in
as measles or rubella in some countries in which whole blood of febrile infants in Zambia (Kasolo et
such infections still occur commonly (see below). al., 1997). The virus DNA can be detected in plasma
Of those classed as infants with exanthem during childhood viraemia and adult reactivations
subitum, 80% had seroconversion or rise in HHV- but not in healthy adults (Secchiero et al., 1995).
150 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Complications, Febrile Seizures, Primary Infection in Adults
Encephalitis, Bone Marrow Suppression,
etc. Due to the frequency of primary infection during
If up to 100% of populations may be infected with childhood, primary HHV-6 and HHV-7 infections
HHV-6 or HHV-7 even minor complication rates, in adults are rare; indeed primary infection with
although rare are of importance because the poten- HHV-7 has not yet been documented among
tial numbers involved are large. Complications ob- adults. For HHV-6, primary adult infection has
served in primary childhood infections with HHV-6 been associated with fever and convulsions in a liver
include skin rash (6—10%), febrile seizures (10%), transplant patient (Ward et al., 1989). It has also
hepatitis, bone marrow suppression, recurrent en- been associated with an infectious mononucleosis-
cephalitis, gastrointestinal (10%) and respiratory like illness where patients are negative for HCMV
symptoms (sinusitis and pneumonitis, 10%). Some and EBV. Although HHV-6 and -7 are widespread,
of these complications may occasionally result in a it may be important to monitor transplant patients
fatal outcome. (Asano et al., 1990; Hall et al., 1994). for both rare primary infections as well as re-
Fatal cases associated with haemaphagocytic syn- activated infections. Furthermore, like other herpes-
drome have been described (Portolani et al., 1997). viruses, after primary infection there is evidence for
The appearance of primary infection can be mis- infections with other strains. Infection may be
diagnosed as measles or rubella infections and, con- asymptomatic or associated with disease (Cone et
versely, measles and rubella infections may be al., 1996; Iuliano et al., 1997).
superficially misdiagnosed as HHV-6 or HHV-7
(Black et al., 1996a; Tait et al., 1996; Kasolo et al.,
1997). Measles may reactive HHV-6 infections Secondary Reactivated Infections in
(Suga et al., 1992). Thus, HHV-6 and HHV-7 infec- Childhood
tions need to be taken into account in any measles/
rubella surveillance programme.
Reactivated infections in children have been re-
HHV-6 infection accounts for a third of all febrile
corded shortly after the primary episode as well
seizures under the age of 3 (Hall et al., 1994). It also
more distantly (Kondo et al., 1993; Hall et al., 1994).
is associated with recurrent seizures, meningoen-
Complications include fever, recurrent encephalitis,
cephalitis and other CNS complications which may
recurrent febrile seizures, bone marrow sup-
have a fatal outcome, including occasional observa-
pression, rash and disseminated infections in im-
tions of epilepsy (Asno et al., 1992; Yoshikawa et al.,
munosuppressed patients (see below) (Kondo et al.,
1992; Kondo et al., 1993; Suga et al., 1993; Vinters et
1993; Hall et al., 1994). A diagnosis and possible
al., 1993; Hall et al., 1994). It is not clear whether the
association with reactivated infection is difficult to
seizures are associated with the fever or with a
establish because of the presence of antibodies re-
direct interaction of the virus with the CNS. HHV-7
sulting from the primary infection. However, as
has also been associated with infant febrile convul-
vireamia can accompany reactivation, diagnosis by
sions during seroconversion (Clark et al., 1997), as
quantitative PCR is possible; preliminary evidence
well as CNS complications during exanthem subi-
supports this (Clark et al., 1997; Kasolo et al., 1997).
tum (Torigoe et al., 1996).
Antibody avidity tests have also been proposed to
The impact of primary HHV-6 infections in de-
distinguish IgM from the primary infection from
veloping countries has not yet been assessed where
higher affinity IgG raised during secondary infec-
there may be other complicating factors such as
tions (Ward et al., 1993b). Factors influencing virus
malnutrition, malaria, tuberculosis or HIV. Pre-
reactivations include immunosuppression and in-
liminary PCR-based studies in a Zambian infant
fection with other agents such as measles (Suga et
population suggest the virus is widespread and this
al., 1992), other herpesviruses (Linde et al., 1990) or
is supported by serological studies in other parts of
infections with different strains of HHV-6 or HHV-
Africa (Kasolo et al., 1997).
7 (Katsafanas et al., 1996). Some of these multiple
infections and virus reactivations have been asso-
ciated with fatalities. In a Beijing study, HHV-6
aetiology accounted for 2% of acute childhood en-
HUMAN HERPESVIRUSES 6 AND 7 151
cephalitis (age 7 months to 13 years), about the ciated pneumonia plus HHV-6 replication in the
same proportion as found for HSV. This accounted bone marrow (Gompels et al., 1994). In vitro studies
for 7% of all encephalitis linked to viral aetiology also support a role for HHV-6 in bone marrow
(Xu et al., 1996). suppression where the virus inhibits the outgrowth
of progenitor cells, including granulocyte/macro-
phage and erythroid lineages, while HHV-7 had no
effect (Isomura et al., 1997). In one study where both
Secondary Reactivated Infections in
HHV-6 and HHV-7 were assayed, only HHV-6
Adults reactivation was associated with bone marrow sup-
pression (Wang et al., 1996). However, in a separate
Since primary infection in children is widespread, study, HHV-7 was associated with a longer time for
there is considerable interest in the pathology asso- neutrophil engraftment and with symptomatic
ciated with reactivated infections in adults. Im- HCMV disease (Chan et al., 1997).
munosuppressed patient populations are primary
targets but reactivation has also been observed in
Other Transplant Patients
apparently ‘normal’ hosts. In asymptomatic adults,
0 to 10 HHV-6 genome equivalents per 10 PBMC HHV-6 infection, reactivation or reinfection has
have been observed, which is lower than in viraemic been observed in liver and kidney transplant pa-
primary infections during childhood (see above), tients. Clinical manifestations were observed in
whereas HHV-6 is not detected in cell-free plasma those with concomitant HCMV infection (Herbein
during latency. Both primary infections in children et al., 1996; Ratnamohan et al., 1998). In one liver
and virus reactivation in immunosuppressed adults transplant patient, a rare adult primary infection
are accompanied by viraemia, which can lead to with HHV-6 was accompanied by fever and fits
detection of virus DNA in the plasma (Secchiero et (Ward et al., 1989). In four other liver transplant
al., 1995). patients studied, HHV-6 reactivation was asso-
ciated with bone marrow suppression as well as, in
one case, with pneumonitis (Singh et al., 1997). Stu-
Bone Marrow Transplant Patients dies of the reactivation of HHV-6 together with
In transplant patients receiving immunosuppres- HCMV after liver transplantation have shown that
sive therapy, HHV-6 reactivation has been HHV-6 is a marker for symptomatic HCMV infec-
documented as one of the first human herpesviruses tion (Dockrell et al., 1997). Similar effects have been
to reactivate, before HCMV and EBV infections suggested for HHV-7 reactivation in relation to
(Wang et al., 1996). This can be asymptomatic or HCMV disease in renal allograft patients (Osman et
accompanied by rash and fever with general mal- al., 1996). It is not clear whether this is a result of
aise, sinusitis and association with HCMV infec- direct interactions, the immunosuppressive effects
tions (Kadakia et al., 1996). In bone marrow trans- of HHV-6 or HHV-7 favouring HCMV reactiva-
plant patients there is also evidence that virus tion, or other factors, including similar immu-
reactivation, either from the donor or the host, can nological conditions in patients with dual reactiva-
result in virus replication in the bone marrow, asso- tions.
ciated with suppression of outgrowth of particular
progenitor cells. In one study this was a general Pneumonitis
effect; in others delayed engraftment was observed HHV-6 infections have been associated with pneu-
for granulocytes, platelets or red cells (Drobyski et monia, particularly in immunosuppressed patient
al., 1993; Kadakia et al., 1996; Wang et al., 1996). An groups, but whether this is a direct effect is not clear
association with increased graft-versus-host disease (Carrigan et al., 1991; Cone et al., 1993a, 1996;
has been reported (Wilborn et al., 1994a; Appleton Gompels et al., 1994). In some cases this has led to
et al., 1995) but not confirmed by others (Kadakia et fatalities (Knox et al., 1995b).
al., 1996; Wang et al., 1996). The bone marrow
suppression observed in the transplant patients has
Associations with Neurological Conditions
also been observed in an apparently ‘normal’ adult
who also had both HHV-6 and legionella-asso- All human herpesviruses have been detected in the
152 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
CNS, but the specificity of this interaction differs; infection, why MS occurs in some groups and not
only the alphaherpesviruses, HSV and Varicella others needs to be explained. Perhaps the question
zoster virus (VZV), have a demonstrable site of of HHV-6 and its relationship to AIDS is similar.
latency within neuronal tissue. HHV-6 and HHV-7 Thus HHV-6 can be immunosuppressive and can
infections have been associated with neurological destroy helper T lymphocytes; why pathology in
conditions but the mechanism for this has not yet some patients and not in others? Is it local viral load
been established. HHV-6 DNA has been detected in from new infections or virus reactivations from
the CNS within brain autopsy tissue of normal and latency; are host factors involved or is it a combina-
patient groups and has been suggested as a com- tion of different factors?
mensal of the brain. Whether this is a lytic, latent or Abnormal expression of a viral antigen, pp41
carrier effect has not been established (Challoner et (U27), has been demonstrated in association with
al., 1995; Merelli et al., 1997). MS plaques, in oligodendrocyte-like cells (Chal-
The association of HHV-6 with neurological con- loner et al., 1995). In the same study, DNA was
ditions has been suggested, often on the basis of detected in CSF of some MS patients. Similarly, an
identification of virus DNA within the cerebro- earlier study detected HHV-6 DNA in the CSF of
spinal fluid (CSF) of different groups of patients. 3/21 MS patients but not in matched sera, while no
HHV-6 DNA has been detected in the CSF of pa- virus DNA was detected in other neurological dis-
tients with recurrent seizures/fits (Kondo et al., ease or control groups (Wilborn et al., 1994b). How-
1993). In patients with CNS disease HHV-6 DNA ever, this has not been confirmed in a wider study of
has also been identified in CSF (Wilborn et al., 1994; 115 MS patients where HHV-6 DNA was not detec-
Cinque et al., 1996). Acute encephalitis, meningoen- ted in the CSF or serum (Martin et al., 1997). These
cephalitis and demyelinating encephalitis have been recent results do not support a role for disseminated
attributed to HHV-6 in some cases via identifica- infection in MS, although they do not rule out the
tion of virus DNA in CSF, as well as DNA and important possibility of associations with local
antigen in local white matter lesions of autopsy lesions (as suggested in the Challoner study and the
samples (Moschettini et al., 1996; Kamei et al., 1997; encephalitis reports), and this requires further
Novoa et al., 1997). Further association with men- evaluation.
ingoencephalitis with basal ganglia infarcation was Increased antibody levels have been observed in
demonstrated by serology showing acute infection two studies of MS patients (Wilborn et al., 1994b;
(Webb et al., 1997). Fatal cases of HHV-6—asso- Soldan et al., 1997). Where MS patient groups have
ciated encephalitis have been shown in patients been further subdivided, an increased IgM response
with immune disorders, such as AIDS, bone mar- to HHV-6 pp41/38 antigen was shown in a sub-
row transplant and a multiple sclerosis (MS) patient group of patients, relapsing-remitting MS (RRMS),
(Asano et al., 1992; Drobyski et al., 1994; Merelli et versus those with chronic progressive MS (CPMS)
al., 1996), but also in an immunocompetent adult or other neurologic disease, autoimmune disease
(Novoa et al., 1997). HHV-7 has also been asso- and normal controls (Soldan et al., 1997). In this
ciated with neurological conditions but further ana- latter study, viral DNA was also detected in some of
lyses have not been done (Torigoe et al., 1996; Clark the patients’ sera and this was used as a marker for
et al., 1997). viraemia (Secchiero et al., 1995; Soldan et al., 1997).
HHV-6 has also been recently associated with However serum DNA has not been consistently
MS by antibody and CSF studies, but a clear rela- observed in other studies including both HHV-6
tionship remains to be established. Analyses are and HHV-7 (Wilborn et al., 1994b; Martin et al.,
complicated by the widespread nature of this virus 1997).
infection and frequent detection of virus DNA in
normal brain autopsy material. However, the pre-
liminary data does point to a possibly interesting
correlation, but further work is required to deter- Evaluation as an Immunosuppressive
mine whether this is a cause or effect of MS or Agent
merely a fortuitous association of HHV-6 latently
carried in an MS-associated cell type. If HHV-6 can There is evidence from in vitro studies and some in
cause MS, with such a widespread persistent virus vivo observations to suggest that both HHV-6 and
HUMAN HERPESVIRUSES 6 AND 7 153
HHV-7 can act as immunosuppressive agents. In kin’s disease may be an indirect effect or possibly
vitro both viruses infect and kill T lymphocytes by carrier effect, in that, although integrated HHV-6
both direct cell lysis mediated by cell fusion and by has been observed in cells derived from Hodgkin’s
apoptotic mechanisms. Other immune cells can be lymphoma, there is no evidence for HHV-6 DNA or
infected and compromised, as demonstrated by gene expression within the Reed—Sternberg cells
HHV-6 in vitro infections of B-lymphocytes, natural (Valente et al., 1996). There is no evidence to date
killer cells and monocytic/macrophage cells (see be- for cellular transformation by HHV-7 or associ-
low). HHV-7 infections appear to have a more re- ation with abnormal cellular proliferations, al-
stricted cellular tropism, probably due to their re- though a homologue encoded by the DR7 gene has
quirement for CD4 antigen as a receptor on the cell been identified (Nicholas, 1996).
surface (Lusso et al., 1994). In vivo infection is asso-
ciated with leucopenia during primary infection as
well as hypocellularity in lymph nodes in AIDS
patients. HHV-6 replication within the bone mar- Diagnosis
row has been observed and this has been associated
with bone marrow suppression. Unlike HIV, life- Infection with HHV-6 can be diagnosed by indirect
long immunity to these viruses is generated, which immunofluorescence, virus isolation, PCR, reverse
can be compromised in certain situations, leading transcriptase (RT)-PCR, ELISA capture assays and
to disease. Reactivation of HHV-6 and HHV-7 virus neutralization assays. By electron microscopy
could lead to further immunosuppression, which (EM) the virus has the classic herpesvirus nuc-
could underlie or enhance other infections such as leocapsid morphology, with irregular envelopment
HIV/AIDS or pneumonitis; there is some evidence including tegument proteins and a lipid bilayer
to support this. membrane containing surface projections com-
posed of glycoproteins. EM is not sufficient to diag-
nose HHV-6 or HHV-7 from other herpesviruses.
Indirect immunofluorescence using acetone-fixed
Evaluation as an Oncogenic Agent whole infected cells in reaction with human sera and
fluorescein isothiocyanate (FITC) conjugates is the
One region of HHV-6, ORF 1 in a restriction en- main diagnostic tool. This is generally a sensitive
zyme DNA fragment (‘Sal L’), has been shown to assay but will not distinguish between HHV-6
have the ability to morphologically transform 3T3 strains or primary versus reactivated infections. In
cells and that these cells injected into nude mice can addition, there is a large level of cross-reaction be-
develop tumours (Thompson et al., 1994). This gene tween HHV-6— and HHV-7—positive sera. Some
product of gene DR7 also has the ability to transac- assays incorporate cross-blocking steps for both
tivate the HIV promoter (see below), so appears to these viruses and claim specificity; others incorpor-
function normally in gene regulation. Recent stu- ate specific PCR-based assays (Black et al., 1996c;
dies have shown that it can bind the antioncogene Tanaka-Taya et al., 1996; Clark et al., 1997). There
P53, which may contribute to its in vitro properties are some antigens which can also cross-react with
of cellular transformation (Kashanchi et al., 1997). certain conserved proteins in HCMV, but these are
A tumour suppressor activity has been defined and rare.
cell lines established which express this property in HHV-6 and HHV-7 serologies are not sufficient
inhibiting H-Ras and BPV-1 transformation via to detect virus reactivation and associated pathol-
downregulation of their promoters for gene expres- ogy. Different assays have been used to distinguish
sion (Araujo et al., 1997). In vivo there is some primary versus reactivated infections, including (gG
evidence for associations with some lymphopro- avidity assays, levels of blood DNA, differential
liferative disorders, but these seem rare events. For detection of DNA in the blood versus saliva (Ward
example, an EBV-negative but HHV-6—positive et al., 1993b; Clark et al., 1997). Primers for sensitive
Burkitt lymphoma cell line has been described, as DNA PCR or cDNA RT-PCR have been described
well as isolated cases of HHV-6—associated rare and, when used in conjunction with quantitative
small T cell lymphoma (Braun et al., 1995; Bando- PCR, can be used to determine if an infection is
bashi et al., 1997). Early associations with Hodg- viraemic. These methods are under evaluation. In
154 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
latently infected adults the level of circulating cells tors for entry but chemokines which inhibit HIV
harbouring latent HHV-6 or HHV-7 DNA have entry do not inhibit HHV-6 entry (Cocchi et al.,
been estimated to be as little as 1 in 10 or 10, but 1995). Interestingly both HHV-6 and HHV-7 en-
during a viraemia levels can be between 10 and code proteins with features of G-protein-coupled
10 times higher (Cone et al., 1993b; Clark et al., receptors which have similarity to chemokine re-
1997; Kasolo et al., 1997). Consequently, HHV-6 ceptors, and infection with these viruses can up-
virus isolation from the blood is usually only ob- regulate expression of a chemokine receptor, CCR7
served if there are high levels of circulating, actively (Gompels et al., 1995; Nicholas, 1996; Yoshida et al.,
replicating virus. However, HHV-7 was first iso- 1997). Unlike HIV, both HHV-6 and HHV-7 can
lated from an apparently healthy individual (Fren- initially infect and persist in epithelial cells and
kel et al., 1990b). There appears to be higher levels specialized sites such as salivary gland epithelium.
of HHV-7 circulating then HHV-6 (Cone et al., Expression of one of the HHV-6 chemokine recep-
1993b; Kidd et al., 1996; Clark et al., 1997). In tor-like genes U51 in epithelial cells results in down-
exanthem subitum infants, DNA is detected in regulation and secretion of selected chemokines
acute and convalescent stages but not before onset which can inhibit HIV infection (R. Milne and U.A.
of HHV-6 or HHV-7 disease (Tanaka-Taya et al., Gompels, unpublished results).
1996).
Specific tests for antigens which correlate with
active infections or virus reactivation are being de-
Reactivated Infections in HIV/AIDS
veloped. In HHV-6, a 100 kDa phosphoprotein,
p100 or p101 encoded by U11, is present in the Patients
tegument and has been identified as one of the
immunodominant targets for antibody responses In AIDS patients human herpesviruses reactivate
(Neipel et al., 1992; Pellett et al., 1993). In HHV-7, and cause major viral opportunistic infections lead-
proteins with similar immunogenicity, but lower ing to both morbidity and mortality. The contribu-
molecular weight, 85—88 kDa have been described tions by HHV-6 and HHV-7 to these infections,
and specific antibody reagents defined. The target either singly or in combination with other herpes-
for the 85 kDa protein is distinct, the HHV-7 U14 viruses or other infections, are being evaluated.
tegument protein (Black et al., 1996b; Foa-Tomasi Both viruses can lead to disseminated infections
et al., 1996; Stefan et al., 1997). Antigenicity and and there is evidence for fatalities associated with
antibody reagents to late glycoproteins which are HHV-6 reactivations. The big question is whether
markers for infection have also been characterized, HHV-6 and HHV-7 also synergize with HIV in
glycoproteins gB and gH (U39 and U48), and these progression to AIDS.
are targets for protective humoral immunity (Cam-
padelliFiume et al., 1993; Ellinger et al., 1993; Liu et
Disseminated Infections
al., 1993, 1993b). None of these markers have 100%
specificity. Both childhood and adult AIDS patients show evi-
dence for disseminated HHV-6 infections. The virus
appears to be one of the first herpesviruses to reacti-
vate in HIV patients. By PCR the HHV-6 can be
RELATIONSHIPS TO HIV/AIDS detected at multiple sites with increased viral load
(Corbellino et al., 1993; Knox and Carrigan, 1994;
Both HHV-6 and HHV-7 share cellular targets with Clark et al., 1996). There may be a complicated
HIV. These viruses can infect and kill CD4 ; T relationship with HIV, because HHV-6 and HHV-7
lymphocytes. HHV-6, like HIV, can also infect and can reactivate each other (Katsafanas et al., 1996).
remain latent within monocytic/macrophage cells. Studies of viral load detected by DNA PCR in
This may also be demonstrated for HHV-7. Both saliva show increased secretion of HHV-7 in HIV/
HIV and HHV-7 use CD4 as a cellular receptor. AIDS patients (DiLuca et al., 1995). These infec-
HIV additionally uses as a coreceptor members of tions are difficult to monitor at the periphery in
the chemokine family of receptors. It is not known blood since the level of the target cell for both
whether HHV-6 or HHV-7 also use these corecep- HHV-6 and HHV-7, the CD4 ; cell, becomes very
HUMAN HERPESVIRUSES 6 AND 7 155
much reduced in patients with late stage AIDS, infections/reactivations with multiple herpes-
which also results in the depletion of the target cell viruses, but also with multiple strains. For example,
for HHV-6 and HHV-7. As detected by DNA PCR, the first isolate in USA of HHV-6 was in an AIDS
levels of HHV-6 and HHV-7 decrease in PBMC in patient and was one of the A variant strains which
late stage AIDS patients (Fairfax et al., 1994; Fabio are rare in that country. Multiple A and B strains
et al., 1997). In contrast, in disseminated infection have been found in HIV/AIDS patients (Iuliano et
the level increases in other tissues (Emery et al., al., 1997).
1999) and virus can be detected in plasma, which is Treatment for multiple herpesvirus infections in
a marker for viraemia and virus reactivation (Sec- AIDS patients has been put forward as a strategy
chiero et al., 1995). In a longitudinal study of HHV- for management of opportunistic infections which
6 reactivation in two HIV-1 infected patients, the may affect AIDS progression and survival (Whitley,
CD4 ; cell count decreased coincident with HHV- 1997).
6 reactivation and in both cases involved variant A;
one of the patients secreted both A and B variant
strains (Iuliano et al., 1997). Whether HHV-6 and Direct Interactions
HHV-7 directly contribute with HIV to the de-
pletion of the T lymphocytes in the blood is not There can be both direct and indirect interactions
known. between HHV-6 and HHV-7 with HIV. Indirect
interactions include those described above, in which
Retinitis the viruses may reactivate during periods of im-
munosuppression in AIDS patients. The viruses
HHV-6 has been detected in vivo in AIDS retinitis. may also have direct interactions with HIV by en-
Usually it is detected in coinfections with HCMV, hancing replication or interrupting latency of HIV.
but it has also been identified on its own. HHV-6 By these actions HHV-6 and HHV-7 could contri-
and HCMV coinfections may affect their patholo- bute to AIDS progression in those infected with
gies and interacting effects have been shown in vitro HIV.
(Fillet et al., 1996). HHV-6 has been shown in vitro
to infect retinal epithelium.
Relationship to CD4 Cell Count
Complications, Lymphadenopathy, Both HHV-6 and HHV-7 are markers for AIDS
Pneumonitis, Encephalitis progression. The levels of DNA quantitated in
Although the total contribution remains to be de- PBMC of AIDS patients are inversely correlated
termined, isolated accounts show that HHV-6 reac- with the CD4 ; T lymphocyte cell count (Fairfax
tivations in AIDS patients can be associated with et al., 1994; Fabio et al., 1997). Thus as the ‘CD4’
fatalities due to pneumonitis and encephalitis count decreases, the levels of detectable HHV-6 and
(Knox and Carrigan, 1994; Knox et al., 1995b). HHV-7 also decrease. However, this is also at a time
HHV-6 reactivations have been associated in HIV- where the whole body load of the virus increases
infected individuals with early phase lym- due to disseminated infections. The simple interpre-
phadenopathy syndrome but not with malignant tation of this data is that depletion of CD4 cells by
lymphoproliferations (Dolcetti et al., 1996). HHV-6 HIV infection results in fewer cells for reactivated
can be detected in brain biopsies from deceased HHV-6 and HHV-7 replication. The more complex
AIDS patients, where the CNS infections were simi- interpretation is that reactivation of all these viruses
lar to HHV-6—associated neuropathology in a bone have contributed to the CD4 depletion. Both HHV-
marrow transplant patient with fatal encephalitis. 6 and HHV-7 can contribute to this depletion by
In addition, fulminant HHV-6—associated encepha- direct cell lysis. HHV-7 can also contribute by
litis has been detected in an HIV-infected infant downregulation of the CD4 antigen.
(Knox and Carrigan, 1995; Knox et al., 1995a).
Transactivation of HIV Genes and
Multiple Herpesvirus Infections Reactivation HIV Latency
The picture is complex. Thus AIDS patients show HHV-6 encodes several genes which regulate gene
156 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
expression. Their role in the normal virus life cycle infection and cell types infected. Alternatively,
is to regulate the cascade of gene expression from HHV-6 can kill the CD4 ; T lymphocytes, reduce
immediate-early to early to late genes. They also the target cell population susceptible to HIV infec-
may have roles in regulating cellular gene expres- tion and contribute to selective pressure on HIV
sion to enhance virus production and in roles for towards wider host ranges. Other receptors may be
establishing, maintaining or reactivating latency. involved in these interactions, in that HHV-6 infec-
Some of these genes can also transactivate the HIV tion of progenitor cell lines renders these cells sus-
promoter. This has been shown in vitro using repor- ceptible to HIV infection (Furlini et al., 1996).
ter gene assays for HHV-6 proteins encoded by Since HHV-7 downregulates CD4 expression,
DR7, U16, U27, U89 and U94, as reviewed previ- this virus may interfere with HIV infections at an
ously (Gompels et al., 1995). It has also been shown early stage. Such inhibitory effects have been ob-
in vivo, whereby HHV-6 gene expression (DR7) has served in cell culture (Crowley et al., 1996). Multiple
been shown to reactivate a tat-deficient HIV levels of control of CD4 expression have been
provirus from latency (Gompels et al., 1995; shown which affect RNA and protein production as
Kashanchi et al., 1994, 1997). The DR7 homologue well as cell surface expression; these activities are
exists in HHV-7 but its function has not been com- independent of p56lck levels (Furukawa et al., 1994;
pared to HHV-6 DR7. Secchiero et al., 1997b).
2F
Kaposi’s Sarcoma-associated
Herpesvirus (Human herpesvirus 8)
Thomas F. Schulz
University of Liverpool, Liverpool, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
168 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2F.1 Electron micrograph of KSHV in lytically induced KS-1 cells (Said et al., 1996). The KSHV virion has the characteristic
features of a herpesvirus. (Courtesy of D. Ablashi, Advanced Biotechnologies)
lytic cycle by treatment with phorbol esters, is (Russo et al., 1996; Neipel et al., 1997) places KSHV
shown in Figure 2F.1. PEL-derived cell lines may with the subgroup of herpesvirus, the rhadino-
be infected latently with KSHV only, or may be viruses (Figure 2F.2). This group includes several
coinfected with EBV, if the original tumour also other animal herpesviruses such as HVS, Equine
contained EBV (Renne et al., 1996; Said et al., 1996; herpesvirus 2 (EHV-2), Murine herpesvirus 68
further references in Schulz, 1998). In cell lines (MHV-68), and three recently discovered
which are only infected with KSHV (e.g. BCP-1, rhadinoviruses of macaques, Macaca nemestrina
BCBL-1, KS-1) most cells only express latent (RFHVMm) and Macaca mulatta (RFHVMm and
KSHV genes (see below), but some will switch spon- RRV) (Desrosiers et al., 1997; Rose et al., 1997).
taneously into lytic replication. In contrast, the EBV, the only other known human herpesvirus, is
dually KSHV/EBV infected cell line HBL-6 (or BC- classified as a herpesvirus. At present, KSHV
1) is much more strictly latent. The lytic replication appears to be most closely related to the three
programme can be switched on in all these cell lines macaque rhadinoviruses (Figure 2F.2). This close
by treatment with either phorbol esters (Renne et relationship is supported by a very similar organisa-
al., 1996) or Na-butyrate (Miller et al., 1996), but tion of the RRV genome, including the presence in
some cell lines are better virus producers (e.g. KS-1) RRV of a homologue of IL-6 (Desrosiers et al.,
than others. KSHV virus preparations obtained in 1997), and other viral genes first found in KSHV
this manner are infectious in vitro and can, albeit (see below). The KSHV genome shares with two of
only inefficiently and transiently, be propagated on the macaque viruses (RFHVMm, RFHVMn) also a
the 293 embryonal kidney cell line (Foreman et al., higher GC content and CpG ratio than is found in
1997). It is also possible to isolate replicating KSHV other rhadinoviruses, in particular HVS, which
on 293 cells from KS biopsies (Foreman et al., 1997) could be indicative of latent persistence in resting
or saliva (Vieira et al., 1997), but detection of rep- rather than dividing cells (Rose et al., 1997). It is
licating KSHV requires the use of PCR and can therefore possible that the present group of Old
usually only be sustained for a few passages. World herpesviruses will be further subdivided in
future.
The genome structure of KSHV is similar to that
of other herpesviruses (Figure 2F.3). A long
Phylogeny and Genome Organization unique region (LUR) of 140.5 kb is flanked by
multiple 801 bp terminal repeats (TRS) of high
Phylogenetic analysis of its genomic sequence (85%) GC content, which are involved in the
KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (HUMAN HERPESVIRUS 8) 169
Figure 2F.2 Phylogenetic relationship between herpesviruses. The phylogenetic tree shown here was created by the neighbour-joining
method of the glycoprotein B sequence of all the herpesviruses shown. For the - and -herpesvirus group only human viruses are
shown. To illustrate the relationship between KSHV and other herpesviruses, the gB sequence of several nonhuman viruses were
included. Numbers at branch points denote bootstrap values to indicate the reliability of this analysis (values over 75% are generally
taken to indicate a robust assignment to a branch). The figure shows that KSHV belongs to the subgroup of herpesviruses
(‘rhadinoviruses’) and appears most closely related to a rhadinovirus of rhesus macacaques (RRV), shown here as a representative for a
group of three closely related macaque viruses (see text). These four viruses may define a further subgroup within the ‘rhadinoviruses’
(see text)
packaging of the viral genome. At present at least 83 gene blocks also contain genes which are found in
viral genes have been either predicted, or some other (but not all) rhadinoviruses. (Russo et
documented experimentally, within the LUR, and al., 1996; Neipel et al., 1997; Nicholas et al., 1997a).
others may be found in the future (Russo et al., 1996;
Neiper et al., 1997; Nicholas et al., 1997a).
Like other herpesvirus genomes, that of KSHV Function of Individual Viral Genes
contains genes encoding proteins which are re-
‘Conserved’ Viral Genes
quired for replication and assembly of new virus
progeny. These are grouped together and arranged Only a few studies have so far been carried out on
in six blocks (gene blocks I—IV, IV, V), as shown in viral proteins encoded within the ‘conserved’ gene
Figure 2F.3. The designation of these conserved blocks I—V, and it is likely that their function is
viral genes follows the nomenclature previously similar to that of their counterparts in other herpes-
adopted for HVS, the first herpesvirus to be fully viruses (Chapters 2 and 2A—E). Open reading frame
sequenced. Genes that are unique to KSHV have (ORF) 17 in gene block II (Figure 2F.3) has been
been designated with a ‘K’ prefix (for KSHV) and shown to encode the viral proteinase and assembly
numbered sequentially. These are located in six protein which is required for the proteloytic pro-
‘non-conserved’ gene blocks (Figure 2F.3). These cessing of capsid proteins. ORF 50 encodes the
170 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2F.3 A comparison of the genomes of the two human herpesviruses, EBV and KSHV. Open boxes with roman numbers
denote groups of structural genes which are conserved among herpesviruses and also many other herpesviruses. The solid line
represents the long unique (coding) region; open rectangles internal or terminal repeat regions. Solid circles in the EBV genome
represent ori-Lyt (L) and ori-Lyt(R), the open circle ori-P. Solid circles in the KSHV genome represent putative ori-(L) and ori-(R)
domains. Small hatched boxes represent GC-rich repeats and inverted palindromic areas. The position and transcriptional orientation
of viral genes discussed in the text is indicated by pointed boxes. In KSHV, dark shading indicates genes known to be expressed in
latently infected spindle cells and PEL cells (see text), chequered boxes represent genes which are weakly expressed in PEL cells, but
whose expression is inducible by phorbol esters or butyrate (see text). As far as this has been investigated, these are only expressed in
lytically infected KS spindle cells or in infiltrating mononuclear cells, but not in latently infected spindle cells. The viral IL6 is expressed
in PEL cells and B-cells of MCD lesions in vivo (see text). Open (pointed) boxes indicate ‘class III’ genes (see text). The arrow represents
the nut-1/T1.1 untranslated RNA expressed during lytic replication. Lines joining LANA and v-cyc represent the alternative splicing
pattern for these genes (see text). In EBV, dark shading indicates genes expressed during type I latency (EBNA-1, BARF0),
cross-hatched genes are in addition expressed during type II latency (LMP1, 2A/2B), and stippled genes also during type III latency
(EBNA-2, 3A, 3B, 3C, EBNA-LP). Lines joining the different genes represent the splicing pattern for latent genes (see page 173)
equivalent of the EBV R transactivator, upregulates membrane glycoprotein with a cysteine-rich ex-
several early genes, and may play a role in the tracellular domain, a single transmembrane do-
switch from latency to lytic replication (Sun et al., main, and a cytoplasmic domain containing several
1998). potential tyrosine phosphorylation sites. K1 has
transforming properties in T cells when recombined
into a herpesvirus saimiri vector, and in rodent fi-
‘Non-conserved’ Viral Genes broblasts (Lee et al., 1998a). Its cytoplasmic domain
More information is available presently on viral can activate intracellular signalling cascades involv-
proteins which are either unique to KSHV, or ing Syk, vav, PI3 kinase and induce Ca> influx
shared by KSHV and only some other herpes- (Lee et al., 1998b). K1 is not expressed in latently
viruses. infected PEL cell lines, and appears to be an im-
mediate early or early gene (Russo et al., 1996;
Lagunoff and Ganem, 1997; Neipel et al., 1997).
Transmembrane proteins. ORFK1, at the left Whether it is expressed in vivo in latently infected
end of the viral genome, is predicted to encode a KS spindle (endothelial tumour) cells or PEL re-
KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (HUMAN HERPESVIRUS 8) 171
mains to be investigated, and its role in the the cytoplasmic domain is located at the C-terminal
pathogenesis of KSHV-associated neoplasia there- end and contains possible binding sites for TRAFs,
fore requires further detailed study. There is con- known to be involved in NFkB activation induced
siderable sequence variability in the K1 coding re- by the TNF receptor and LMP 1 (Glenn et al.,
gion among different KSHV isolates. While the 1999). The ORF K15/LAMP transcript is weakly
biological significance of this sequence variability is expressed in PEL cell lines in vitro, but nothing is
still unknown, it can be used for phylogenetic stu- known at present about its expression in tumour
dies to define different KSHV lineages (see below). cells in vivo.
ORK K8.1 (Figure 2F.3) encodes, by means of
alternative splicing, two transmembrane glyco-
proteins. This feature, and the position of ORF Cytokines encoded by KSHV. A large block of
K8.1 in the viral genome, makes it a likely equival- ‘non-conserved’ viral genes contains several with
ent of EBV gp340/220, which is involved in the homologies to mammalian proteins and appears
binding of EBV to its receptor (Raab et al., 1998). more extensive than in other rhadinoviruses (Russo
The K8.1 glycoprotein, gp35/37, is immunogenic et al., 1996; Neipel et al., 1997; Nicholas et al., 1997a,
and recognized by sera from most KSHV-infected 1997b, 1998; Figure 2F.3). Several growth factor
individuals and is therefore a good diagnostic anti- homologues, including vIL-6 (ORF K2) two MIP-
gen (see below). I homologues (ORF K4/v—MIP-II; ORF K6/v-
Similar to HHV-6 and HHV-7, KSHV contains a MIP-I) and a MIP-I homologue (ORF K4.1;
homologue of an NCAM-like adhesion molecule, MIP-III) are encoded in this region (Russo et al.,
encoded by ORF K14 (Russo et al., 1996). Its func- 1996; Neipel et al., 1997; Nicholas et al., 1997a,
tion is still unknown. 1997b).
The ORF 74 gene encodes a member of the family The IL-6 homologue encoded by ORF K2 is
of seven transmembrane domain G-protein- capable of maintaining the proliferation of IL-6—de-
coupled receptors (GCRs). Members of this family pendent mouse and human myeloma cell lines
are also found in several herpesviruses, including (Moore et al., 1996; Neipel et al., 1997; Nicholas et
cytomegalovirus (CMV) and HVS. Transient ex- al., 1997b). Similar to human IL-6, v-IL-6 activates
pression studies of the KSHV v-GCR indicate that STAT1, STAT3 and Jak1 phosphorylation in HEp-
it binds to several CXC and CC chemokines and G2 hepatoma cells (Molden et al., 1997). In contrast
that it appears constitutively active (Arvanitakis et to human IL-6, v-IL-6 appears not to require the
al., 1997). The KSHV GCR enhances the prolifer- chain (gp80) of the IL-6 receptor complex and to use
ation of rat kidney fibroblast (NRK-49F) cells, exclusively its chain (gp130) (Molden et al., 1997;
transforms rodent fibroblasts to tumourigenicity, Nicholas et al., 1997b). v-IL-6 is secreted by PEL
and induces the secretion of vascular endothelial cells infected by KSHV, expressed by CD20 ; B
growth factor (VEGF) (Arvanitakis et al., 1997; Bais cells in lymphoid tissues from KSHV-infected pa-
et al., 1998). However, v-GCR appears not to be tients and in multicentric Castleman’s disease, but
expressed in latently infected PEL cells (Sarid et al., not in KS tissues, suggesting that v-IL-6 may con-
1998; Figure 2F.3) and it is still unclear whether it is tribute to the proliferation of hematopoietic but not
expressed in neoplastic cells in vivo. endothelial cells (Moore et al., 1996; detailed refer-
Another membrane protein, with multiple trans- ences in Schulz, 1998; Schulz et al., 1998).
membrane regions and a c-terminal cytoplasmic The KSHV genome also encodes three proteins
domain (ORF K15/LAMP), is encoded at the with significant sequence similarity to cellular
‘right’ end of the viral genome and has similarities chemokines (Moore et al., 1996; Russo et al., 1996;
to the two latent membrane proteins of EBV, LMP Boshoff et al., 1997; Kledal et al., 1997; Neipel et al.,
1 and LMP 2 (Poole et al., 1999; Glenn et al., 1999). 1997). The protein encoded by ORF K6 (MIP-I
Like LMP 2, which is thought to play a role in the homologue) has been shown to interact with the
control of EBV latency, ORF K15 has up to 10 CCR5 chemokine receptor and to block the entry of
transmembrane regions (alternative splicing results CCR5—dependent primary HIV-1 strains (Moore et
in smaller proteins with fewer transmembrane seg- al., 1996). The ORF K4 encoded chemokine (v-
ments) and a cytoplasmic domain with putative MIP-II) interacts preferentially with CCR3, inhibits
SH2—binding sites (Glenn et al., 1999). As in LMP 1, the entry of dual-tropic HIV-1 strains and is
172 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
chemotactic for eosinophils. Both v-MIP-I and v- given the name FLICE-inhibitor protein (v-FLIP)
MIP-II have angiogenic properties when tested on (Thome et al., 1997). The presence of DEDs in
the chorioallantoic membrane (Boshoff et al., 1997). KSHV K13 suggests a similar role. Whereas HVS
There is some basal expression of v-MIP-I and ORF 71 is expressed during lytic replication and,
v-MIP-II in PEL cell lines, which increases marked- like v-BCL-2, has been hypothesized to protect cells
ly after induction of the lytic cycle (Moore et al., infected lytically against apoptosis (Thome et al.,
1996). v-MIP-II appears to be expressed only in 1997), KSHV K13 is encoded on a bicistronic
cells infected lytically within KS lesions, but not in mRNA which also codes for ORF 72, the viral
the majority of (latently infected) spindle cells. Al- homologue of a D-type cyclin (see below), and
though not yet firmly established, it is conceivable which is expressed in latently infected PEL cell lines
that these viral chemokines, released by cells lyti- and KS spindle cells (Davies et al., 1997; Rainbow et
cally infected, may play a role in the development of al., 1997; Sarid et al., 1998; further references in
KS by promoting neoangiogenesis, eosinophil infil- Schulz, 1998) (Figure 2F.3). It is therefore conceiv-
tration, or the proliferation of spindle cells latently able that KSHV v-FLIP (K13) may play a role in
infected (see below), latently infected KS spindle and B lymphoma cells.
Orf K1 protein Transforms fibroblasts and T-cells Lagunoff and Ganem (1997)
Lee et al. (1998a,b)
Orf K15/‘LAMP’@ Similarities to the transforming Glenn et al. (1999)
LMP-1 of EBV Poole et al. (1999)
Others
LNA (ORF 73)? Nuclear protein, required for episome Rainbow et al. (1997)
maintenance Ballestas et al. (1999)
‘kaposin’ (ORF K12)? Putative hydrophobic protein Zhong et al. (1996)
Possible transforming properties Muralidhar et al. (1998)
strictly latent in KS tissue (see below). The expres- those genes which are expressed in PEL cell lines
sion pattern of individual viral genes has been and whose expression is not increased by agents
studied mainly in PEL cell lines, which are more which induce lytic viral replication (e.g. some phor-
amenable to experimentation than KS tumour tis- bol esters or sodium butyrate). At present there are
sue. Three different patterns of expression can be at least four such genes: ORF K13 (v-FLIP), ORF
distinguished (Sarid et al., 1998). ‘Class I’ KSHV 72 (v-cyclin), ORF 73 (LNA), and a 4.5 kb transcript
genes (Table 2F.1 and Figure 2F.3) are defined as of as yet unknown coding potential (Sarid et al.,
KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (HUMAN HERPESVIRUS 8) 175
1998). Three of these, ORFs K13/v-FLIP, 72/v-cyc, assess the efficacy of these drugs has yet been re-
73/LANA are encoded next to each other, and ported. If their efficacy against KS in vivo can be
translated from two alternatively spliced mRNAs substantiated, this would suggest that lytic KSHV
controlled by the same latent promoter (Figure replication, known to occur in a proportion of KS
2F.3; Dittmer et al., 1998; Rainbow et al., 1997; spindle cells and in some mononuclear cells infil-
Sarid et al., 1998). These three genes are also ex- trating KS lesions (Orenstein et al., 1997; Staskus et
pressed in most KS spindle (endothelial tumour) al., 1997), plays an important role in the pathogen-
cells and considered ‘latent’ genes. esis of KS. However, treatment of HIV with combi-
A second set of KSHV genes (‘class II’ genes) is nation antiretroviral therapy is also very effective in
also, in most cases only weakly, expressed in PEL some patients against AIDS KS, underlining the
cell lines, but, unlike ‘class I’ genes, their expression importance of HIV-1 replication as a cofactor in the
is enhanced after induction of the lytic replication pathogenesis of AIDS KS (Blum et al., 1997; Con-
cycle. This class of genes include some, such as ORF ant et al., 1997; and see below).
K12 (‘kaposin’), which are, like ‘class I’ genes, also
strongly expressed in latently infected PEL cells and
all KS spindle cells (Figure 2F.4; Plate III). The
ORF K12 transcript (T 0.7) is in fact among the Different subtypes of KSHV
most abundant in KS tissue (Zhong et al., 1996).
While this transcript is therefore also a marker of On the basis of two KSHV genomes, which have
latent infection, other ‘class II’ genes, such as the— been sequenced completely (Russo et al., 1996), or
also strongly expressed—untranslated nut-1/T1.1 nearly completely (Neipel et al., 1997), and several
RNA (Figure 2F.3) (Zhong et al., 1996) are only seen others which have been sequenced partially
in lytically infected cells in KS tissue by in situ (Lagunoff and Ganem, 1997; Nicholas et al., 1997a,
hybridization (Staskus et al., 1997). 1998), it appears that only the two ends of the LUR
Also among the ‘class II’ genes are the virus- exhibit significant sequence variability. An earlier
encoded cytokines (v-IL-6, v-MIP-I, v-MIP-II, v- study suggested the existence of three KSHV ‘vari-
BCK/MIP) and the interferon regulatory factor (v- ants’ or ‘subtypes’, termed A,B,C, by sequence
IRF/ORF K9), and the ORF K15/LAMP tran- analysis of the conserved ORF 26 and ORF 75
script (Sarid et al., 1998; Glenn et al., 1999) (Figure genes (Zong et al., 1997). More recent efforts, based
2F.3). As discussed above, some of these, e.g. v-IL-6, on the more variable K1 gene at the left end of the
are expressed in KSHV-infected B cells in vivo, but viral genome (Figure 2F.3), indicate the existence of
do not appear to be expressed in KS spindle cells. four main groupings (Cook et al., 1999; Zong et al.,
A third set of viral genes (‘class III’ genes) is 1999). Group B strains predominate in Africa,
expressed in PEL cell lines only after induction of whereas group A and C strains are found in all parts
the lytic replication cycle and comprises many con- of Europe and group D strains in Taiwan. The
served structural genes, but also some genes unique evolution of the K1 glycoprotein appears to be
to KSHV, such as ORFs K1 and K8, K8.1 (Sarid et driven by some still unidentified selective pressure
al., 1998) (Figure 2F.3). (Cook et al., 1999; Zong et al., 1999). At the right
end of the viral genome, the presently available
KSHV sequences appear to fall into two major
groups, with a highly divergent primary sequence to
ANTIVIRAL THERAPY the right of ORF 75 in the region of ORF K15/
LAMP (Figure 2F.3) (Glenn et al., 1999; Poole et
In vitro, the replicative cycle of KSHV in PEL cell al., 1999). In contrast to ORF K1 these two variants
lines can be inhibited with ganciclovir, cidofivir and of ORF K15LAMP appear not to have evolved
foscarnet, but not acyclovir (Kedes and Ganem, recently, and one of them may have originated from
1997; Medveczky et al., 1997). In vivo treatment a recombination event with a closely related virus.
with ganciclovir or foscarnet reduced the rate of KS At present there is no evidence for the existence of
lesions developing in treated individuals in retro- KSHV variants with a different pathogenic poten-
spective analyses (Glesby et al., 1996; Mocroft et al., tial. However, most of the longer KSHV sequences
1996). No controlled prospective clinical trials to examined so far have been obtained from tumour
176 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 2F.2 Serological assays for the detection of antibodies to KSHV/HHV-8
Latent antigen
LANA IFA 85—94 71—88 0—3 (UK/US) Gao et al. (1996b)
(latency-associated Kedes et al. (1996)
nuclear antigen) Simpson et al. (1996)
Lennette et al. (1996)
4—35 (Italy, Simpson et al. (1996)
Greece) Calabrò et al. (1998)
Whitby et al. (1998)
ORF 73 protein WB 100 80 0 (US) Gao et al. (1996a)
(226/234 kDa nuclear
protein; main
component of LANA)
Structural (lytic)
antigens
vp19 (ORF 65) ELISA/WB 91 75—84 1.7—5 (UK/US) Simpson et al. (1996)
9—22 (Italy, Calabrò et al. (1998)
40 kDa protein WB 67 Greece) Miller et al. (1996)
Gp35/37 ELISA/WB approx. 90 approx. 80 5 Raab et al. (1998)
Chandran et al. (1998)
Whole cell IFA 96—100 90—100 20—25 (US) Lennette et al. (1996)
ELISA/IFA 100 0—12 (US) Ablashi et al. (1997)
Chatlynne et al. (1998)
IFA 100 0 (US) Smith et al. (1997)
Antigens and assay formats used to detect antibodies to KSHV and approximate prevalence rates in patients with KS, and blood donors in
different geographic regions. The range of values shown for Italy are due to marked regional variation of KSHV seroprevalence within Italy
(Calabrò et al., 1998; Whitby et al., 1998), whereas the range of values reported by lytic IFA for US blood donors is likely due to differences in
technical protocol (see text).
tissue, rather than from infected, healthy individ- ward et al., 1997; Vieira et al., 1997; further refer-
uals, and only shorter variable regions such as ORF ences in Schulz, 1998; Schulz et al., 1998). To quanti-
K1 have been compared between KS patients and tate the amount of KSHV DNA in peripheral
infected healthy controls (Cook et al., 1999). The blood, a quantitative competitive PCR assay has
existence of more pathogenic variants can therefore been developed (Lock et al., 1997).
not yet be excluded.
Serological Assays
DIAGNOSTIC ASSAYS
Several serological assays have been developed of
Detection of KSHV DNA by PCR varying sensitivity and specificity (Table 2F.2). With
the help of these assays it has been possible to gain
By PCR, KSHV is easily detectable in fresh or some insight into the epidemiology of KSHV (see
frozen biopsies of KS, PEL or MCD, although the below). However, their use in clinical practice, es-
amount of KSHV DNA can be quite variable, in pecially to diagnose infection with KSHV in an
particular in KS biopsies. Detection in paraffin- asymptomatic individual from a non-endemic
embedded specimens may require the use of nested country, is still associated with some uncertainty.
PCR, as does the detection in peripheral blood, Sera from KSHV-infected individuals contain
saliva or semen, of KSHV-infected individuals with antibodies to proteins expressed in latently infected
or without KS. Different primer combinations have cells, as well as those produced during lytic viral
been used successfully for this purpose (Chang et al., replication. The main latent KSHV antigen, LNA,
1994; Boshoff et al., 1995; Whitby et al., 1995; Ho- (Gao et al., 1996a), is a 225—234 kDa nuclear protein
KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (HUMAN HERPESVIRUS 8) 177
encoded by ORF 73 (Rainbow et al., 1997). It is part virions as antigen has also been reported (Chat-
of, if not identical with, LANA, originally defined by lynne et al., 1998).
immunofluorescence (Gao et al., 1996b; Kedes et al., In an attempt to circumvent these difficulties,
1996). For routine testing of antibodies against this several groups have investigated the use of recom-
protein, an immunofluorescence assay (IFA) on binant structural proteins, concentrating either on
latently infected PEL cell lines, such as BCP-1 (Gao those with low homology to the corresponding
et al., 1996b) or BCBL-1 (Kedes et al., 1996) is EBV proteins, or on proteins without a counterpart
presently the most widely used technique, but as- in the EBV genome. At present, only two recom-
says based on recombinant proteins are under de- binant structural proteins appear to hold any
velopment. As shown in Table 2F.2, the LANA IFA promise. The minor capsid-related protein encoded
detects antibodies in about 80—90% of KS patients by ORF 65 (Simpson et al., 1996) gives a sensitivity
and therefore has high, but not perfect, sensitivity. of 80—90%, while reacting with a slightly higher
There is no homologue of ORF 73/LNA in EBV, a proportion of UK blood donors than detected by
ubiquitous and related herpesvirus (Russo et al., LANA immunofluorescence (Table 2F.2). It detects
1996). Furthermore, in countries where infection some KS sera that are missed by LANA IFA and
with KSHV appears to be rare, such as the UK, the two assays can therefore be used in combination
only 0—3% of blood donors react with this antigen. to increase the sensitivity (Simpson et al., 1996). The
This suggests that ORF 73/LNA is a highly specific glycoprotein encoded by ORF K8.1 does not have a
serological antigen. At present there is still con- counterpart in the EBV genome and is also recog-
siderable interlaboratory variation with LANA nized by the majority of KS sera, but only rarely by
IFAs (Rabkin et al. (1998); detailed references in US and northern European blood donor sera
Schulz, 1998; 1999). Although other latent viral pro- (Chandran et al., 1998; Raab et al., 1998).
teins exist, none has so far found a diagnostic appli- In contrast, recombinant proteins or peptides de-
cation. rived from a minor capsid protein encoded by ORF
As for other herpesviruses, many proteins ex- 26, or the major capsid protein (ORF 25), do not
pressed during the lytic cycle of viral replication are discriminate well enough between sera from KS
immunogenic and recognized by antibodies in pa- patients and blood donors to be used for routine
tient sera (Miller et al., 1996; Simpson et al., 1996; serology (Rabkin et al., 1998; and further references
Smith et al., 1997; Raab et al., 1998). The lytic in Schulz, 1998, 1999).
replication cycle of KSHV can be induced in latent-
ly infected PEL cell lines by treatment with phorbol
esters or butyrate, and such ‘induced’ cells have EPIDEMIOLOGY OF KSHV
been used for immunofluorescence using different
experimental conditions (Lennette et al., 1996; Ab- Geographical distribution
lashi et al., 1997; Smith et al., 1997; Figure 2F.5).
These lytic IFAs have been reported to detect anti- The distribution of KSHV among asymptomatic
bodies in a higher proportion of KS patients than individuals from different geographical regions has
LANA IFA. However, presumably as a result of been studied by PCR and the serological assays
using different experimental conditions, variable available at present. Both approaches suggest that
seroprevalence rates have been reported with lytic KSHV is more common in some Mediterranean
IFAs for US blood donors (Lennette et al., 1996; countries and in parts of Africa than in northern
Smith et al., 1997; Chatlynne et al., 1998; further Europe or the US, and that there may be marked
references in Schulz, 1998, 1999). Some of the struc- regional differences in KSHV prevalence even with-
tural proteins of KSHV have relatively high homol- in countries. Although the exact prevalence rates
ogy to the corresponding EBV proteins (Russo et are still uncertain, KSHV appears to be more
al., 1996), and serological cross-reactivity between common in those countries which were known to
the major capsid proteins of both viruses may occur have higher incidence rates for classical KS.
(references in Schulz et al., 1998). Therefore it will be By PCR, KSHV has been detected in peripheral
important to establish the best conditions for lytic blood mononuclear cells (PBMCs) or semen
IFAs to allow results to be reproducible in different samples from asymptomatic donors in northeastern
laboratories. An ELISA using purified KSHV Italy and Sicily, but not in northwestern Italy,
178 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2F.5 Immunofluorescence pattern for (a) latent and (b) lytic KSHV antigens in PEL cells. (a) shows the characteristic speckled
nuclear pattern for the ‘latency associated nuclear antigen’, LANA (Gao et al., 1996b: Kedes et al., 1996), which is encoded by ORF73
(Rainbow et al., 1997). (b) shows the diffuse nuclear and cytoplasmic fluorescence seen on TPA- or Na-butyrate-induced PEL cells with
patient sera. Only a few of the viral proteins contributing to this pattern have so far been identified (see text)
KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (HUMAN HERPESVIRUS 8) 179
France or the UK (detailed references in Black- investigated so far, e.g. Uganda, Tanzania, The
bourn and Levy, 1997; Schulz, 1998, 1999). KSHV Gambia, Cameroon, South Africa (Gao et al.,
has occasionally been found in PBMCs, but not 1996b; Lennette et al., 1996; Simpson et al., 1996;
reproducibly in semen samples, from healthy US Ariyoshi et al., 1998; Mayama et al., 1998; further
donors. In The Gambia, West Africa, KSHV has references in Schulz, 1998, 1999). By IFA, antibodies
been detected by PCR in the peripheral blood of to lytic KSHV antigens have been found in
10% of HIV-negative antenatal mothers. In Zam- 30—100% of sera from different parts of Africa (Len-
bia, it was found in the peripheral blood of 8% of nette et al., 1996). These high seroprevalence rates,
young febrile children, and in Uganda in 14% of obtained with assays which detect antibodies to
adult controls with tumours other than KS (detailed defined antigens, suggest that KSHV is widespread
references in Schulz, 1998, 1999). PCR carried out in Africa, and occurs not only in those regions (East
on PBMCs, underestimates the number of KSHV- and Central Africa) where KS was known to exist
infected individuals because of the low viral load in before the arrival of HIV-1 (endemic KS) (see be-
peripheral blood (see below). These results therefore low). This could suggest an involvement of addi-
indicate only the presence of KSHV in healthy indi- tional cofactors in the pathogenesis of endemic KS
viduals of these countries, but cannot be used to (see below).
infer the exact prevalence of KSHV infection. Although less frequent than in Africa, infection
The seroprevalence of KSHV in different geo- with KSHV also appears to be widespread in Italy
graphic regions has been studied using the LANA and Greece (Gao et al., 1996b; Simpson et al., 1996;
IFA, an ORF 65 ELISA and Western blot, as well Calabrò et al., 1998; Whitby et al., 1998; further
as lytic IFAs (Gao et al., 1996b; Lennette et al., 1996; references in Schulz, 1998, 1999). Unlike in the ‘non-
Simpson et al., 1996; Calabrò et al., 1998; Whitby et endemic’ countries discussed above, approximately
al., 1998; further references in Schulz, 1998, 1999) 13% of all Italian blood donors were found to have
(Figure 2F.5). Like PCR, serology also suggests that antibodies to both KSHV LANA and the ORF 65
KSHV is relatively rare in the UK, Denmark, protein, and 24% had antibodies to at least one of
France and The Netherlands, with less than 5% of these antigens (Calabrò et al., 1998) (Figure 2F.6). It
blood donors having antibodies to either KSHV is likely that the ‘true’ prevalence of KSHV is closer
LANA or the ORF 65 protein. In the US, reported to the latter figure. In Italy, infection with KSHV
prevalence rates in blood donors have varied from 0 appears to be more common among blood donors
to 3% for antibodies to LANA, 0 to 5% for antibo- from regions previously reported to have a higher
dies to the ORF 65 protein, and 0 to 25% for incidence of classic KS, such as Sicily, Sardinia,
antibodies measured by lytic IFA or an ELISA on than in central Italy or in prealpine regions (Cala-
purified virions (Gao et al., 1996b; Kedes et al., brò et al., 1998; Whitby et al., 1998; further refer-
1996; Lennette et al., 1996; Simpson et al., 1996; ences in Schulz, 1998, 1999). KSHV seroprevalence
Smith et al., 1997; Chandran et al., 1998; Chatlynne is increased markedly in blood donors aged over 50,
et al., 1998; further references in Schulz, 1998, 1999). i.e. the age group in which the incidence of classic
As there is considerable variability not only be- KS is highest (Calabrò et al., 1998). Whether this
tween assays, but also between laboratories under- reflects a higher rate of infection among individuals
taking the same assay with slightly different proto- born 50 years ago, or a reactivation of KSHV
cols (Rabkin et al., 1998), these figures only give a replication in older age, is unclear at present.
rough indication as to the exact prevalence rates in Thus, the increased prevalence of KSHV infec-
these ‘non-endemic’ countries. Of note, only very tion in some Mediterranean countries is in accord-
few donors in non-endemic countries have antibo- ance with their higher incidence of classical KS. In
dies which react in more than one assay, adding to Africa, KSHV appears at the present time to be
the current uncertainty about the exact KSHV highly prevalent in the East, West and South,
prevalence in these countries. whereas ‘endemic’ (HIV-negative African) KS is
In contrast, all studies reported so far concur that confined to East and Central Africa. The geo-
antibodies to KSHV are frequently found in several graphic distribution of KSHV in Africa therefore
parts of Africa. Antibodies to LANA and ORF 65 agrees partially with that expected for the ‘KS
are found in approximately 40—50% of adults and agent’, suggesting an involvement of cofactors in
adolescents in most parts of sub-Saharan Africa KS pathogenesis (see below).
180 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 2F.6 Seroprevalence of KSHV in different countries and HIV transmission groups Seroprevalence rates for KSHV, as
measured by antibodies to LANA (IFA) and to vp16/ORF65 (ELISA/WB) by Simpson et al. (1996) and Calabrò et al. (1998) are shown.
As discussed in the text, and illustrated in Table 2F.2, somewhat higher prevalence rates have been found in American blood donors
with an IFA on lytically infected cells by some (Lennette et al., 1996), but not others (Smith et al., 1997; Chandran et al., 1998; Chatlynne
et al., 1998). However, the overall rank order of seroprevalence, from low in the UK/USA, to intermediate in some Mediterranean
countries, to high in several parts of Africa, is supported by all studies
Additional Cofactors Required for the revalence in these two countries (Calabrò et al.,
Development of KS? 1998). In the Gambia, West Africa, AIDS KS is
markedly more common among HIV-1—infected
A number of epidemiological observations suggest
than among HIV-2—infected individuals, although
that additional cofactors may be required in addi-
the KSHV prevalence appears to be comparable in
tion to KSHV for the development of KS. KSHV
these two groups (Ariyoshi et al., 1998). These ob-
seroprevalence rates of 20—35% in Italy, and ap-
servations suggest that infection with HIV-1 repre-
proximately 20% in Greece (see above and Table
sents an important cofactor. Although KS is more
2F.2; Fig. 2F.6), when compared to the population
common in transplant recipients, and therefore re-
based incidence rates of 1—3/100 000 for ‘classic’ KS
lated to immunosuppression, the role of HIV-1 in-
in these regions, suggest that the development of KS
fection may go beyond that, since KS in HIV-2—in-
is a rare event in a KSHV-infected individual who is
fected individuals is comparatively rare (Ariyoshi et
not infected with HIV or otherwise immunosup-
al., 1998), and some AIDS KS lesions can respond
pressed. In contrast, AIDS-associated KS appears
rapidly to antiretroviral therapy (Blum et al., 1997;
to be relatively common among homosexual men
Conant et al., 1997). An angiogenic role for the
infected with HIV-1 and KSHV. Of note, AIDS-
HIV-1 Tat protein or inflammatory cytokines re-
associated KS is not more common among HIV-
leased during HIV infection has been suggested,
infected intravenous drug users and patients with
and an induction of KSHV replication by HIV-1
haemophilia from Italy compared to the UK, in
Tat protein claimed (detailed references in Biberfeld
spite of the marked difference in KSHV serop-
et al., 1998; Schulz, 1998), but such a link has not yet
KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (HUMAN HERPESVIRUS 8) 183
replication (see above).
The four latent genes expressed in KS spindle
cells (ORF K12/‘kaposin’; ORF K13/v-FLIP, ORF
72/v-cyc, ORF 73/LANA; see above) are also ex-
pressed in PEL cells, as is a fourth, so far unmapped,
transcript (Rainbow et al., 1997; Sarid et al., 1998).
In addition, PEL cell lines show some basal expres-
sion of v-IL-6, MIP-I, MIP-II and v-IRF (ORF K9)
and a non-translated nuclear RNA T1.1/nut-1
(Moore et al., 1996; Zhong et al., 1996). By im-
munohistochemistry, PEL cells and KSHV infected
B cells in lymphatic tissue, but not KS spindle cells,
express v-IL-6, an inducible gene (Moore et al.,
1996). There are thus some well-documented dif-
ferences in gene expression between KS spindle cells
and KSHV-infected B cells, and further differences
Figure 2F.7 Proportion of UK AIDS patients of different are likely to be found in the future.
transmission groups who develop KS. KS is common in homo-
sexual/bisexual men, but infrequent among intravenous drug
users, patients with haemophilia, or heterosexually infected
women. Detailed data and references in IARC Monographs on
Multicentric Castleman’s Disease
the Evaluation of Carcinogenic Risks to Humans, vol. 67: Human (MCD)
Immunodeficiency Viruses. IARC Press, 1996
Multicentric Castleman’s disease is an atypical lym-
been established clearly. phoproliferative disorder which occurs in two his-
tological variants, the hyaline-vascular variant and
the plasma cell variant. Most cases of MCD in
Primary Effusion Lymphoma (PEL) HIV-infected patients, in particular the plasma cell
variant, contain detectable KSHV. In contrast,
KSHV genomes are also found consistently in PEL, KSHV is much less frequently detected in MCD of
also termed ‘body-cavity associated lymphoma’ HIV-negative patients (Soulier et al., 1995; further
(BCBL) (for example, Cesarman et al., 1995; further references in Schulz, 1998; Schulz et al., 1998). The
references in Schulz, 1998), a rare form of AIDS- precise role of KSHV in the pathogenesis of MCD
related B cell lymphoma which is characterized by is thus not yet clear. However, v-IL-6 is expressed in
malignant effusions in the pleural or abdominal B cells in these lesions and, on the basis of its in vitro
cavity, immunoglobulin gene rearrangement, lack properties (see above), is likely to contribute to the
of most surface markers and, unlike Burkitt’s lym- proliferation of KSHV infected B cells in these
phoma, a lack of c-myc rearrangements (for a re- lesions.
view, see Jaffe, 1996). Many cases of PEL are dually
infected with EBV and KSHV (Cesarman et al.,
1995), but occasional cases of EBV-negative/ Other Lymphoproliferative Disease
KSHV-positive PEL have been reported (Renne et
al., 1996; Said et al., 1996). A few cases of PEL have KSHV has been detected in occasional cases of
also been described in HIV-negative patients (de- HIV-negative angioimmunoblastic lymphadeno-
tailed references in Schulz, 1998; Schulz et al., 1998). pathy and germinal centre hyperplasia (Luppi et al.,
PEL lymphoma cells, and the cell lines estab- 1996). Histologically, there may be prominent
lished from them (see above), harbour multiple (ap- plasma cell proliferation and angiogenic changes in
proximately 50—100) copies of KSHV as episomes these cases, suggesting that viral proteins like v-IL-
(Cesarman et al., 1995a; Renne et al., 1996; Said et 6 and v-MIP-I/II may have induced these transient
al., 1996), as is typical for latent herpes viral infec- changes.
tion. As in the case of KS spindle cells, a minority of KSHV and its IL-6 homologue have also been
cells in a PEL cell line culture can undergo lytic claimed to play a role in the pathogenesis of
184 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
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Royal Free and University College Medical School, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
188 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
some steatosis can be observed in chronic HCV
infection. A mononuclear cell infiltration, which is
particularly marked in the portal zones, is the char-
acteristic mesenchymal reaction. This is also ac-
companied by some proliferation of bile ductules.
Kupffer cells and endothelial cells proliferate and
the Kupffer cells often contain excess lipofuscin pig-
ment. In the icteric phase of typical acute hepatitis,
the walls of the hepatic vein tributaries may be
thickened and frequently are infiltrated, with prolif-
eration of the lining cells in the terminal hepatic
veins. Cholestasis may occur in the early stages of
viral hepatitis, and plugs of bile thrombi may be
found in the bile canaliculi; this is a more common
feature in hepatitis E. Figure 3.1 Section of a liver biopsy from a patient with chronic
hepatitis C seen at low magnification. There is a moderate lym-
Spotty or focal necrosis with the associated mes- phocytic infiltrate in the portal tracts, with piecemeal necrosis. A
enchymal reaction may also be found in anicteric scattered mild parenchymal inflammatory infiltrate is also pres-
hepatitis, but on the whole the lesions tend to be less ent. (H&E.) (Courtesy of Dr A.P. Dhillon, Department of His-
severe than in the icteric type of illness. At the other topathology, Royal Free Hospital and School of Medicine)
extreme, there is rapid massive necrosis of the liver
HCV RNA, for example following successful -in-
cells in fulminant hepatitis.
terferon treatment, is followed by histological im-
Repair of the liver lobules occurs by regeneration
provement. Typically, patients with chronic hepati-
of hepatocytes; frequent mitoses, polyploidy, atypi-
tis C have mild portal tract inflammation with
cal cells and binucleated cells are found. There is
lymphoid aggregates or follicles and mild periportal
gradual disappearance of the mononuclear cells
piecemeal necrosis. Parenchymal steatosis, apopto-
from the portal tracts, but elongated histiocytes and
sis and mild lobular inflammation are present. and
fibroblasts may remain. The outcome of acute viral
portal fibrosis or portal-central fibrosis may be
hepatitis may be complete resolution or fatal mass-
present in later stages of disease. Bridging necrosis
ive necrosis.
is not common. Rarely, granulomas can be ob-
served. Although many of the lymphoid follicles are
Chronic Hepatitis associated with bile ducts, ductopenia is not ob-
served. Advanced disease, with cirrhosis or hepa-
The pathological features of chronic hepatitis B
tocellular carcinoma is not generally associated
depend upon the stage of the disease, the host im-
with distinguishing features.
mune response and the degree of virus replication.
HCV antigens have been detected in scattered
In chronic hepatitis B with mild activity, only rare
groups of cells, with granular cytoplasmic staining.
piecemeal necrosis is seen. Characteristic hepa-
The periportal lymphocytes around lymphoid fol-
tocytes with eosinophilic ‘ground-glass’ cells are
licles are mixed, but contain relatively large numb-
relatively common in anti-HBe-positive patients
ers of CD4 lymphocytes. Figure 3.1 shows his-
with low levels of virus replication. Lobular hepati-
topathological changes typical of chronic hepatitis
tis is more common in patients with active virus
C. A characteristic histologic pattern of mild chro-
replication, and raised serum aminotransferases.
nic hepatitis with portal lymphoid follicles and
CD8 ; cells predominate in areas of piecemeal
varying degrees of lobular activity is found in many
necrosis. HBsAg and HBcAg can be detected by
patients with persistent hepatitis C infection.
immunoperoxidase staining in routinely fixed liver
biopsy sections. Patients with high levels of vir-
aemia may have minimal hepatitis.
The pathological features of HCV infection are Biochemical Tests of Liver Function
quite characteristic, albeit not pathognomonic. The
presence of HCV RNA in serum tends to correlate The serum levels of aspartic and alanine aminotran-
with some degree of hepatitis and disappearance of sferase are elevated in acute hepatitis, as are levels of
HEPATITIS VIRUSES 189
other enzymes released by the damaged liver cells. have palpable splenomegaly. Convalescence may be
Usually, the levels of alanine aminotransferase are prolonged, although complete recovery in adults
higher than those of aspartic aminotransferase, a usually takes place within a few months. In children,
difference particularly marked in hepatitis C. Elev- the prodromal features may be mild or even absent,
ation of these enzymes may be the only abnormality although anorexia, when present, tends to be severe.
to be found in individuals with asymptomatic and The icteric or posticteric phase in children is short.
anicteric infections who are tested because of There is no definite evidence of progression of hepa-
known exposure. Bilirubin is found in the urine and titis A to chronic liver disease, but it has been sug-
conjugated and total serum bilirubin levels are gested that autoimmune hepatitis may be triggered
raised in most symptomatic infections. The leuco- in genetically predisposed individuals. These obser-
cyte count usually is normal but some atypical lym- vations have not been confirmed. The prodromal
phocytes are found frequently. phase of hepatitis B and C is often prolonged and
A progressive decline in serum albumin concen- more insidious. Low grade fever, arthralgias and
trations and prolongation of the prothrombin time skin rashes, particularly in hepatitis B, are not un-
are characteristically observed after decompensated common. The clinical features of the icteric phase
cirrhosis has developed. The serum aspartate are similar in all types of acute viral hepatitis. The
aminotransferase may be higher than the serum mortality rate of acute hepatitis is low, approxi-
alanine aminotransferase concentrations in patients mately 0.1—3 deaths per 1000 cases.
with hepatic cirrhosis. A subset of patients with Fulminant hepatitis can occur following acute
chronic hepatitis C infection may test positive for hepatitis A—E; it is more common in hepatitis B.
autoantibodies, including LKM1 antibody. Hepatocellular failure develops rapidly; the patient
may be deeply jaundiced or encephalopathy may
occur before conspicuous jaundice is evident. Wide-
spread haemorrhage occurs. The prothrombin time
Clinical Manifestations is prolonged; an altered prothrombin time is a more
reliable indicator of prognosis, and of the need for
Differences between the clinical syndromes of acute liver transplantation, than the serum bilirubin or
hepatitis A, acute hepatitis B and other forms of serum aminotransferases.
viral hepatitis become apparent on analysis of large Fulminant hepatitis is unusual following hepati-
numbers of well-documented cases, but these dif- tis C infection, but has been reported, particularly
ferences are not sufficiently reliable for the diag- following chemotherapy. High mortality rates for
nosis of individual patients with icteric disease. Epi- hepatitis occurring during pregnancy have been re-
demiologic risk factors, for example travel, in- ported from India, the Middle East and North Afri-
jections or sexual risk, can indicate a possible aetiol- ca. The infection was often considered to be caused
ogy. Fever and headache are more frequent during by HAV, but is now known to be associated par-
the prodrome of acute hepatitis A. The late incuba- ticularly with HEV infection.
tion period—early clinical phase is frequently heral-
ded by a variety of non-specific symptoms such as
fatigue, anorexia, malaise and myalgias. A few days
later, anorexia, nausea, vomiting and right upper HEPATITIS A
quadrant abdominal pain can appear, followed by
passage of dark urine and clay-coloured stools and Hepatitis A is endemic in all parts of the world, but
the development of jaundice of the sclera and skin. the exact incidence is difficult to estimate because of
With the appearance of jaundice, there is usually a the high proportion of asymptomatic and anicteric
rapid subjective improvement in symptoms. The infections, differences in surveillance and differing
jaundice usually deepens during the first few days patterns of disease. The degree of under-reporting is
and persists for 1 or 2 weeks. The faeces then darken known to be very high. Serological surveys have
and the jaundice diminishes, at first rapidly and shown that infection with HAV is almost universal
then more slowly, over an additional period of 2 and, in developing countries, 80—90% of children
weeks or so. The liver may be palpable in acute have serological markers of past infection by the
severe hepatitis, but only a minority of patients age of 5. In industrialized countries (particularly in
190 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
northern Europe, North America and Australia) ters and often with only few identified cases. Hepati-
improvements in sanitation have resulted in a de- tis A is highly endemic in many tropical and sub-
crease in the incidence of hepatitis A. The preva- tropical areas, with the occasional occurrence of
lence of antibodies in young adults in such countries large epidemics. The infection frequently is acquired
is 5—20%. As a result, large epidemics have occur- by travellers from areas where the infection is of low
red, such as in Greece and Shanghai. prevalence to areas where hepatitis A is hyperen-
demic.
Incubation Period
Age Incidence
The incubation period of hepatitis A is between 3
and 5 weeks, with a mean of 28 days. Subclinical All age groups are susceptible to hepatitis A and
and anicteric infections are common, particularly in disease severity increases with age. As noted above,
children, and, although the disease has, in general, a most individuals in highly endemic areas are infec-
low mortality, adult patients may be incapacitated ted before 5 years of age but in many countries in
for many weeks. There is no evidence of persistence northern Europe and in North America most clini-
of the infection in the liver, and progression to cal cases occur in adults. In countries where there
chronic liver damage does not occur. The virus has been improvement in socioeconomic conditions
replicates in vivo in the liver but it seems likely that and sanitation, such as southern Europe and China,
the initial site of virus replication may be in the gut. there has been an increase in the mean age of infec-
This is not proven and the mechanism by which the tion. In many developed countries, the prevalence
virus reaches the liver is unknown, although a tran- of antibodies to hepatitis A has fallen to 5—10% of
sient viraemia has been postulated. young adults and there is thus a large susceptible
population. The diminishing incidence of hepatitis
A is now matched by an increasing incidence of
clinically apparent disease.
Mode of Spread This shift in age incidence is similar to that which
occurred with poliomyelitis during and after World
Hepatitis A virus is spread by the faecal—oral route, War II, reflecting improvement in socioeconomic
most commonly by person-to-person contact, and and hygienic conditions and a consequent shift in
infection is particularly common in conditions of herd immunity.
poor sanitation and overcrowding. Common
source outbreaks result most frequently from faecal
contamination of drinking water and food, but
waterborne transmission is not a major factor in the Seasonal Pattern
industrialized communities. On the other hand,
many food-borne outbreaks have been reported in In temperate zones, the characteristic seasonal
developed countries. This can be attributed to the trend is for an increase in incidence in the autumn
shedding of large amounts of virus in the faeces and early winter months, falling progressively to a
(Figure 3.2) during the incubation period of the minimum during the midsummer. However, more
illness in infected foodhandlers, and the source of recently the seasonal peak has been lost in some
the outbreak can often be traced to uncooked food countries. In many tropical countries the peak of
or food which has been handled after cooking. The the infection tends to occur during the rainy season,
consumption of raw or inadequately cooked shel- with a lower incidence during the dry periods.
lfish cultivated in polluted water is associated with a
high risk of hepatitis A infection. For example, an
epidemic with approximately 300 000 cases in Biology of HAV
Shanghai in 1988 was attributed to the ingestion of
raw clams (Halliday et al., 1991). However, al- The hepatitis A virion is a non-enveloped particle
though hepatitis A is common in the developed measuring 25—28 nm in diameter (Figure 3.3) and
countries, the infection occurs mainly in small clus- containing a linear genome of single-stranded
HEPATITIS VIRUSES 191
Figure 3.2 Electron micrograph showing hepatitis A virus in faecal extracts obtained during the early acute phase of illness. The
particles measure about 27 nm in diameter. (; 170 000) (From a series by Anthea Thornton and A.J.Zuckerman)
Figure 3.3 Electron micrograph showing a large aggregate of hepatitis A virus particles heavily coated with antibody, giving the
appearance of a ‘halo’. ‘Full’ and ‘empty’ particles are shown. (; 340 000) (From a series by Anthea Thornton and A.J. Zuckerman)
192 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
RNA, approximately 7500 nucleotides in length leotides and a shorter non-coding region and
and coding for three major structural polypeptides poly(A) tract at the 3 terminus. A small protein
with molecular weights of 33 000, 29 000 and 27 000 (VPg) is bound covalently at the 5 terminus. A
(VP1, VP2 and VP3). A fourth, truncated VP4 poly- single open reading frame (ORF) extends from nuc-
peptide of only l7 amino acids has been predicted leotide 710—750 to about 60 nucleotides in advance
from the nucleotide sequence of the virus but has of the 3 terminal poly(A) tract. This sequence can
not been detected experimentally. X-ray crystallo- encode a polyprotein with molecular weight of
graphic studies have not yet been reported but the about 250 000. The predicted amino acid sequence
basic structure of the capsid may be predicted from compared with analogous regions of other picor-
such studies of other picornaviruses; the antigenic naviruses suggests that the 5 region of the ORF
structure has been defined further by analysis of codes for the three major structural proteins of the
neutralization escape mutants selected by mon- virus along with the fourth, small VP4. The 3 re-
oclonal antibodies. These studies suggest that the gion encodes a protease which processes the poly-
immunodominant neutralization site is a conforma- protein and the polymerase and other functions
tional epitope comprising residues of VP1 and VP3 involved in genome replication. Dipeptide cleavage
(Ping and Lemon, 1992). It is believed that second- sites which have been identified in a number of
ary or higher orders of protein structure may play picornavirus polyproteins are not conserved in
essential roles in this antigenic site, since it has not HAV but attempts have been made to predict the
been possible to detect this predominant antigen in cleavage pattern and post-translational processing
virus preparations disrupted with detergent or fol- of the polyprotein (Figure 3.4). The cellular receptor
lowing expression of recombinant protein (al- for HAV has been identified recently (Kaplan et al.,
though expression of the entire HAV polyprotein 1996).
may enable assembly of antigenic virus-like par-
ticles in cell culture, as noted below).
HAV is exceptionally stable; it is ether resistant,
stable at pH 3.0 and relatively resistant to inactiva- Cell Culture
tion by heat. HAV retains its physical integrity and
biological activity at 60°C for 10 hours but is inac- The successful propagation of HAV in l979 in pri-
tivated after 5 minutes at 100°C. The virus also may mary monolayer and explant cell cultures and in
be inactivated by ultraviolet irradiation and by continuous cell lines of primate origin was a major
treatment with a 1:4000 concentration of formalde- advance and opened the way to the preparation of
hyde solution at 37°C for 72 hours. There is also hepatitis A vaccines. The viral capsid antigens are
evidence that HAV is inactivated by chlorine at a detectable by immunofluorescence and radioim-
concentration of 1 mg/l\ for 30 minutes. munoassay, and the viral RNA by an indirect,
quantitative autoradiographic plaque assay and by
complementary DNA—RNA hybridization and re-
verse transcriptase (RT)-PCR. HAV does not in-
Genetic Organization duce cytopathic changes in culture but tends to
establish persistent infections and remain largely
The organization of the genome of HAV is similar cell associated. However, primary isolation of wild-
to other picornaviruses and it was originally classi- type virus is difficult and several weeks elapse before
fied as enterovirus type 72 (Gust et al., 1983). How- antigen is detectable in the cytoplasm of infected
ever, there are substantial differences between HAV cells. Thus, virus isolation is not a practical diag-
and the established four genera of the picornavirus nostic technique in routine laboratories.
family, for example an unusually low GC content of Adaptation to growth in cultured cells occurs
38% and very limited sequence homology. Conse- with repeated passage, with more rapid production
quently, HAV has been reclassified in its own genus, of intracellular antigen and with higher final yields.
Hepatovirus, of the family Picornaviridae. Virus adapted to growth in cell culture may become
Cloning and sequencing data (Cohen et al., 1987) attenuated and the nucleotide sequences of wild-
indicate that the genome of HAV consists of 7478 type and attenuated strains have been compared.
nucleotides with a 5 non-coding region of 733 nuc- There is evidence that changes in the 5 non-coding
HEPATITIS VIRUSES 193
Figure 3.4 Organization of the hepatitis A virus genome. See text for details
region and the region encoding non-structural (2B/ HAV . These data support the hypothesis that liver
2C) polypeptides may be associated with attenu- cell injury in acute HAV infection is mediated by
ation and adaptation to cell culture. HAV-specific CD8 ; T lymphocytes, and is not
Only one serotype of hepatitis A has been identi- entirely due to an intrinsic cytopathic effect of the
fied in human volunteers infected experimentally, in virus itself. The molecular targets of these cells are
patients from different outbreaks of hepatitis A and unknown. Serum neutralizing antibodies protect
in naturally and experimentally infected chimpan- against HAV infection.
zees and monkeys. This has also been confirmed by
cross-neutralization tests and by the protective effi-
cacy of pooled human immunoglobulin obtained
from different geographical regions. However,
strain specific differences exist at least at the level of Laboratory Diagnosis
the nucleotide sequences of viral RNA from differ-
ent isolates of HAV. A pairwise comparison of the Specific diagnosis of hepatitis A can be established
nucleotide sequence of 168 bases from 152 strains of by demonstrating the virus in faeces by enzyme
HAV revealed that these can be classified into seven immunoassay and radioimmunoassay or by im-
unique genotypes, three of which were isolated from mune electron microscopy and RT-PCR. Isolation
Old World monkeys. The RNA genome of the vi- of the virus in cell cultures is not yet feasible nor
rus, like those of other picornaviruses, is subject to appropriate for routine diagnosis.
relatively high rates of spontaneous mutation. As stated above, the capsid antigen is highly con-
served and there is only a single serotype of HAV.
Specific serological tests for hepatitis A antigen
(HAAg) and antibodies include radioimmunoassay
Pathogenesis and enzyme-linked immunosorbent assay. Hepati-
tis A antibody (anti-HAV) is always demonstrable
The mechanisms underlying liver injury in hepatitis by such assays during the early phase of the illness
A are not understood. The initial non-cytopathic and titres increase rapidly (Figure 3.5). Since anti-
phase, during which virus replicates and is released, body develops very early in the course of the infec-
is followed by decreased virus multiplication and tion, serological diagnosis of recent infection can be
inflammatory cell infiltration, suggesting that im- established by titrations of serial samples of serum
mune mechanisms are involved in pathogenesis. or, more conveniently, by the demonstration of
Experimental evidence suggests that HLA-restric- hepatitis A antibody of the IgM class. This is the
ted, virus specific T cells play a significant role in simplest and most economical method of establish-
HAV-related hepatocellular injury. T cell clones ing the diagnosis. Hepatitis A IgM is detectable in
have been derived from patients with acute hepatitis serum for 45—60 days after the onset of symptoms.
A and analysed for their phenotype. CD8 ; clones Liver biopsy is not usually required in acute hepati-
isolated during the acute phase of the disease pre- tis A. Titres of anti-HAV in IgG rise with convales-
dominate over CD4 ; clones; these CD8 ; clones cence and the antibody usually persists for many
have cytotoxic activity and show specific cytotoxic- years. Recovery from infection is associated with
ity against autologous fibroblasts infected with lifelong immunity.
194 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Epidemic hepatitis, which resembles but is serologi- Virus-like particles have been detected in the
cally distinct from hepatitis A, has been reported stools of infected individuals by immune electron
from the Indian subcontinent (Khuroo, 1980), cen- microscopy using convalescent serum (Figure 3.6).
tral and southeast Asia, the Middle East, North However, such studies often have proved inconclus-
Africa and Central America. The disease also is a ive and a large proportion of the excreted virus may
common cause of acute sporadic hepatitis in these be degraded during passage through the gut. The
countries. Sporadic cases have been observed in particles are slightly larger than those of hepatitis A,
developed countries among migrant labourers and with a mean diameter of 32—34 nm. Cross-reaction
travellers returning from such areas. In contrast to studies between sera and virus in stools associated
prior epidemics of enterically transmitted non-A, with a variety of epidemics in several different coun-
non-B hepatitis, HEV was found to be a common tries suggest a single viral serotype (Bradley, 1992).
cause of acute hepatitis in a paediatric population
in Egypt. Seroprevalence studies in Hong Kong
suggest that hepatitis E accounts for a third of
non-A, non-B, non-C hepatitis, and that coinfection Biology of HEV
of hepatitis A and E can occur (Lok et al., 1992).
The average incubation period is slightly longer Research into the viral agent of enterically transmit-
than for hepatitis A, with a mean of 6 weeks. The ted, non-A, non-B hepatitis (ET-NANBH), now
infection is acute and self-limiting. Clinical disease known as Hepatitis E virus, advanced following the
occurs predominantly in young adults, and high development of animal models (Bradley et al., 1988).
mortality rates (up to 20%) have been reported in HEV was first transmitted to cynomolgous
the third trimester of pregnancy. The infection is macaques and subsequently a number of other spe-
spread by the ingestion of contaminated water and cies of monkeys and chimpanzees have been infec-
probably by food, but secondary clinical cases seem ted. The problem of degradation of HEV in the gut
to be uncommon. was circumvented when the gallbladder bile of in-
HEPATITIS VIRUSES 197
fected monkeys was found to be a rich source of
virus. This material enabled the molecular cloning
of DNA complementary to the HEV (RNA)
genome and the entire 7.2 kb sequence was deter-
mined (Tam et al., 1991). The organization of the
genome is distinct from the picornaviruses; the non-
structural polypeptides are encoded in the 5 region
and the structural polypeptides at the 3 end. HEV
resembles the caliciviruses in the size and organiz-
ation of its genome as well as the size and morphol- Figure 3.7 Organization of the hepatitis E virus genome. See
ogy of the virion. text for details
Recently, a virus which is closely related to HEV
has been discovered in several herds of swine in the
USA (Meng et al., 1997). Whether this virus also Pathogenesis
infects humans remains to be determined but the
detection of anti-HEV in around 2% of random After HEV inoculation macaque monkeys develop
blood donors in the USA (Dawson et al., 1992) may acute viral hepatitis associated with a rise in liver
be significant. enzymes, the presence of HEV specific viral par-
ticles in the stool and histological changes in the
liver from 21 to 45 days after infection. Ultrastruc-
tural changes in the livers of these experimental
Organization of the HEV Genome monkeys include infiltration of lymphocytes and
polymorphonuclear leucocytes around the necrotic
The HEV genome is a polyadenylated, positive- area, swelling of mitochondria, dilatation of smooth
sense RNA of around 7200 nt which contains three endoplasmic reticulum and presence of 27—34 nm
ORFs (Figure 3.7). The first, of approximately 5 kb, virus particles during the acute phase of the disease.
begins 28 nt from the 5 end of the genome and It is not known whether these changes reflect
encodes motifs associated with NTP-binding, heli- cytopathic liver injury or immune-mediated dam-
case and RNA-dependent RNA polymerase activ- age.
ities. A second ORF of around 2 kb begins 37 nt
downstream of the first, terminates 68 nt from the
poly(A) tail and is believed to encode the structural
polypeptides. The third ORF is very short (369 nt) Diagnosis
and overlaps the other two. Subgenomic RNAs
may be synthesized for translation of the second With the availability of recombinant antigens and
and third ORFs and the products of all three may synthetic peptides, serological assays were develop-
be subject to proteolytic processing and other post- ed to test for antibody to HEV (anti-HEV). These
translational modifications. are based on antigens that were initially identified
Sequencing of the HEV genome has enabled the by blind immunoscreening using convalescent sera
development of a number of specific diagnostic from infected individuals. Western blot assays are
tests. For example, HEV RNA was detected, using able to detect IgM anti-HEV. Specific IgM is detec-
the PCR, in stool samples obtained during an epi- ted infrequently at initial presentation and has dis-
demic in Kanpur (north India) and may also be appeared within 3 months of the onset of jaundice.
detected in infected liver. ELISA assays, which de- Specific IgG titres can be quite high, but tend to
tect IgG and IgM anti-HEV, have been developed disappear over time. The diagnosis can be con-
(Dawson et al., 1992) and used to detect antibodies firmed by detecting the virus genome in faecal ma-
in sporadic cases of ET-NANBH in children in terial from acutely infected patients using RT-PCR.
Egypt and elsewhere. One assay employs four rec- Bile from experimentally infected monkeys is also
ombinant HEV antigens expressed in Escherichia positive by this technique (Jameel et al., 1992). At-
coli as fusion proteins with glutathione-S-transfer- tack rates have been higher in males than females
ase, and another is based on synthetic peptides. and for adults rather than children. Evidence of
198 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
secondary intrafamilial spread is uncommon (Naik intravenously with HEV, rather than via the natu-
et al., 1992). ral (oral) route, and that macaques do not suffer
overt symptoms of hepatitis during infection—pro-
tection often is defined as the absence of elevated
transaminases despite evidence of virus replication
Clinical Features
(excretion in the faeces).
An early observation was the high mortality
(10—20%) in pregnant women due to fulminant
hepatitis. Fulminant hepatitis E has been reported HEPATITIS B
in the UK, but appears to be a relatively infrequent
cause elsewhere. In general, the disease is self-
Epidemiology
limited with no evidence of chronic infection. Liver
biopsies obtained during the acute illness show por-
The discovery in 1965 of Australia antigen (now
tal inflammation and cytoplasmic cholestasis. Stool
referred to as hepatitis B surface antigen, HBsAg)
specimens may reveal 27—32 nm virus-like particles
and the demonstration by Blumberg and his col-
when tested by immune electron microscopy (IEM)
leagues and others of its association with type B
but RT-PCR is a more reliable assay for the virus.
hepatitis (Blumberg et al., 1965) led to rapid and
unabated progress in the understanding of this
complex infection. Hepatitis B remains a globally
Immunization important disease. Low (less than 2% of the popula-
tion seropositive for HBsAg), intermediate (2—8%)
Although individuals who recover from hepatitis E and high prevalence (more than 8%) areas are rec-
mount an antibody response, including to putative ognized. Several epidemiological studies indicate
capsid antigens, it is not clear whether these antibo- that the reported rates of hepatitis B infection have
dies protect against subsequent infection or, if so, declined in western and northern Europe, and the
how long that immunity lasts. Adult populations in USA. Infection rates in children have declined in
endemic areas are susceptible to hepatitis E, with high prevalence areas where the universal immuniz-
high attack rates in epidemics. Preliminary findings ation of infants has been introduced.
indicate that hyperimmune rabbit antisera against In the past, hepatitis B was diagnosed on the
HEV antigens contain neutralizing activity. How- basis of infection occurring approximately 60—180
ever, the degree and longevity of protective immun- days after the injection of human blood or plasma
ity of macaque monkeys following recovery from fractions or the use of inadequately sterilized syr-
experimental infection or immunization with rec- inges and needles. The development of specific lab-
ombinant DNA or antigen preparations is contro- oratory tests for hepatitis B confirmed the import-
versial. Prospects for the development of hepatitis E ance of the parenteral routes of transmission, and
vaccines were reviewed recently (Panda and Nanda, infectivity appears to be especially related to blood.
1997). Purdy et al. (1993) demonstrated that immu- However, several factors have altered the epi-
nization with a bacterially-expressed fusion protein, demiological concept that hepatitis B is spread ex-
derived from the capsid region of the Burmese clusively by blood and blood products. These in-
strain, protected cynomolgus macaques from chal- clude the observations that under certain
lenge with the homologous strain of virus. How- circumstances the virus is infective by mouth, that it
ever, monkeys challenged with the Mexican strain is endemic in closed institutions and institutions for
excreted virus in their faeces, although there was no the mentally handicapped, that it is more prevalent
biochemical evidence (i.e. raised transaminases) of in adults in urban communities and in poor
liver disease. In another study, an immunogen de- socioeconomic conditions, that there is a huge res-
rived from the capsid region of the Pakistan strain ervoir of carriers of markers of HBV in the human
was found to protect against infection with the population, and that the carrier rate and age dis-
Mexican strain (Tsarev et al., 1994). Aside from the tribution of the surface antigen vary in different
duration of a protective antibody response, other regions.
caveats are that animals frequently are challenged There is much evidence for the transmission of
HEPATITIS VIRUSES 199
hepatitis B by intimate contact and by the sexual China and southeast Asia. There is also a substan-
route. The sexually promiscuous, particularly male tial risk of perinatal infection if the mother had
homosexuals, are at very high risk. HBsAg has been acute hepatitis B in the second or third trimester of
found in blood and in various body fluids, such as pregnancy or within 2 months of delivery. Although
saliva, menstrual and vaginal discharges, seminal hepatitis B virus can infect the fetus in utero, this
fluid, colostrum and breast milk, and serous ex- appears to be rare and is generally associated with
udates, and these have been implicated as vehicles antepartum haemorrhage and tears in the placenta.
of transmission of infection. The presence of the The mechanism of perinatal infection is uncertain,
antigen in urine, bile, faeces, sweat and tears has but it occurs probably during or shortly after birth
been reported occasionally, but has not been con- as a result of a leak of maternal blood into the
firmed. It is not surprising, therefore, that contact- baby’s circulation, or of its ingestion or inadvertent
associated hepatitis B is of major importance. inoculation.
Transmission of the infection may result from acci- However, mother-to-infant transmission does
dental inoculation of minute amounts of blood or not account for at least 50% of infections in
fluid contaminated with blood during medical, sur- children, and horizontal transmission, i.e. child-to-
gical and dental procedures; immunization with in- child transmission is equally important. The preva-
adequately sterilized syringes and needles; intra- lence in children is quite low at 1 year of age but
venous and percutaneous drug abuse; tattooing; ear increases rapidly thereafter, and in many endemic
and nose piercing; acupuncture; laboratory acci- regions the prevalence reaches a peak in children
dents; and accidental inoculation with razors and 7—14 years of age. Clustering of HBV also occurs
similar objects that have been contaminated with within family groups, but does not appear to be
blood. Additional factors may be important for the related to genetic factors and does not reflect ma-
transmission of hepatitis B infection in the tropics; ternal or venereal transmission. The probability of a
these include traditional tattooing and scarifica- childhood infection becoming persistent declines
tion, blood letting and ritual circumcision. Investi- with age, from around 90% in neonates to less than
gation of the role that biting insects may play in the 5% in adolescence.
spread of hepatitis B has yielded conflicting results.
HBsAg has been detected in several species of mos-
quitoes and in bed-bugs that were either trapped in
the wild or fed experimentally on infected blood, Chronic Hepatitis B
but no convincing evidence of replication of the
virus in insects has been obtained. Mechanical Chronic hepatitis B is defined as persistence of
transmission of the infection, however, is a possibil- HBsAg in the circulation for more than 6 months.
ity (Byrom et al., 1973). The ‘carrier state’ (the term may be inappropriate)
may be lifelong and may be associated with liver
damage varying from mild chronic hepatitis to se-
vere, active hepatitis, cirrhosis and primary liver
Perinatal Transmission cancer. Several risk factors have been identified in
relation to its development. It is more frequent in
Viraemic mothers, especially those who are males, more likely to follow infections acquired in
seropositive for HBeAg, almost invariably transmit childhood than those acquired in adult life, and
the infection to their infants at the time of, or shortly more likely to occur in patients with natural or
after, birth. Such perinatal infections lead to a high acquired immune deficiencies. Chronic hepatitis B
rate of chronicity, estimated at around 90%. It has infection occurs in 90% of neonates or young in-
been suggested that the soluble HBeAg may cross fants, but in only 1—5% of immunocompetent
the placenta and tolerize the fetus in utero. Individ- adults. In countries where hepatitis B infection is
uals infected at such an early age exhibit a high common, the highest prevalence of HBsAg is found
degree of immune tolerance and may remain vir- in young children, with steadily declining rates
aemic for decades. Perinatal transmission is an ex- among older age groups. HBeAg has been reported
tremely important factor in maintaining the reser- to be more common in young than in adult carriers
voir of the infection in some regions, particularly in of hepatitis B, whereas the prevalence of anti-HBe
200 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 3.2 Prevalence of hepatitis B
Eastern Europe
Northern Europe Mediterranean
Western Europe Former USSR Parts of China
Central Europe Southwest Asia Southeast Asia
North America Central America Sub-Saharan
Australia South America Africa
seems to increase with age. Heron hepatitis B virus (HHBV) has also been char-
Survival of HBV is ensured by the reservoir of acterized but reports of hepadnaviruses infecting
carriers, estimated to number over 300 million other species remain to be confirmed.
worldwide. The prevalence of carriers, particularly
among blood donors, in northern Europe, North
America and Australia is 0.1% or less; in central
and eastern Europe up to 5%; in southern Europe, Structure of HBV
the countries bordering the Mediterranean, and
parts of Central and South America the frequency is Electron microscopy of HBV-positive serum re-
even higher; and in some parts of Africa, Asia and veals three morphologically distinct forms of par-
the Pacific region as many as 20% of the apparently ticle (Figure 3.8). The small, 22 nm spherical par-
healthy population may be HBsAg positive (Table ticles and tubular forms of roughly the same
3.2). There is an urgent need to introduce methods diameter are composed of the virus surface protein
of interruption of transmission. The management of embedded in lipid and are synthesized in vast excess
choronic hepatitis B is complex, with personal, so- over the 42 nm, double-shelled virions. The latter
cial and economic implications. comprise a 27 nm, electron-dense core surrounded
by HBsAg that is distinct from the subviral particles
in that pre-S1 epitopes are present (see below). The
core or nucleocapsid consists of the genome sur-
Biology of HBV rounded by a second protein, hepatitis B core anti-
gen (HBcAg). A third antigen, ‘e’ antigen (HBeAg),
In addition to the human HBV, a number of similar is found in soluble form in virus-positive sera and is
viruses, which infect mammals and birds, have been related to the core antigen, as described below. The
described and the virus family has been named genome is DNA (Figure 3.9) and comprised of two
Hepadnaviridae (hepa-tropic DNA viruses). The vi- strands held in a circular configuration by base-
ruses have a similar genetic organization and mode pairing at the 5 ends (cohesive end region). One of
of replication and are characterized by a high de- the strands is incomplete (usually 50—80% full
gree of host specificity and tropism for the liver. Of length) and is associated with a polymerase which is
the mammalian viruses, the Woodchuck hepatitis able to fill in the single-stranded region when pro-
virus (WHV) and Beechey Ground squirrel hepatitis vided with suitable substrates.
virus (GSHV) have been well characterized, the for- The genomes of a variety of isolates of HBV have
mer being of interest as a model system for primary been cloned and the complete nucleotide sequences
liver cancer because there is a high probability of determined. Although there is some variation in
progression to tumour for WHV-infected wood- sequence (up to 12% of nucleotides) between these
chucks. Of the avian viruses, the Pekin Duck hepati- isolates, the genetic organization and other essen-
tis B virus (DHBV) has been well characterized and tial features are conserved. The genome is around
this animal model has been valuable in the elucida- 3200 bp in length and analysis of the protein coding
tion of the hepadnavirus replication process. A potential reveals four conserved ORFs, the prod-
HEPATITIS VIRUSES 201
Figure 3.8 Electron micrograph of serum containing hepatitis B virus after negative staining. The three morphological forms of the
antigen are shown: (1) small pleomorphic spherical particles 20—22 nm in diameter (hepatitis B surface antigen); (2) tubular structures (a
form of the surface antigen); (3) the 42 nm double-shelled virions, with a core surrounded by the surface antigen. ( ; 300 000) (From
Zuckerman, A.J. (1975) Human Viral Hepatitis. North Holland/American Elsevier, Amsterdam)
Figure 3.9 Structure and genetic organization of the HBV genome. The broad arrows surrounding the genome represent the four
large open reading frames of the L ( 9 ) strand transcript. The number of amino acids (aa) encoded are indicated in each case. The two
thin arrows surrounding the broad arrows represent the two major HBV mRNAs. The partial restriction map and the numbering of the
nucleotides indicated on the inner circle correspond to the ayw3 genome. DR1 and DR2 are the directly repeated sequences involved in
the replication of the genome. (From Tiollais P. et al. (1985) Nature, 317, 389—495. Macmillan, London)
gp36) with a 55 amino acid N-terminal extension, the common, group-specific antigen, a, which is
the pre-S2 domain. These pre-S2 proteins are minor believed to form a ‘double loop’ structure (amino
components of virions and subviral particles. acids 124—137 and 139—147) on the surfaces of the
Translation of the entire ORF (pre-S1 ; pre- virions and subviral particles. The formation of
S2 ; S) gives rise to the largest proteins (p39 and anti-a antibodies following vaccination seems to be
gp42) which seem to be found predominantly in sufficient to confer protective immunity. The major
virions and perhaps the tubular, 22 nm forms. A HBsAg protein also carries a pair of mutually ex-
domain within the pre-S1 region may be respon- clusive subdeterminants, d or y and w or r, which, in
sible for the attachment of the virus to the receptor each case, seem to correlate with variation at single
on the hepatocyte. Synthesis of the pre-S1 protein amino acid positions (122 and 160, respectively).
also may act as a signal for virion assembly in the Thus, four principal phenotypes of HBsAg are rec-
infected cell. Control of abundance of the major ognized—adw, adr, ayw and, more rarely, ayr—and
surface, pre-S2 and pre-S1 proteins seems to be at these show differing geographical distribution. For
the transcriptional and translational level. example, in northern Europe, the Americas and
The subviral particles and the virion surface are Australia subtype adw predominates, while ayr oc-
composed of HBsAg anchored in a lipid bilayer curs in a broad zone that includes northern and
derived from the host cell endoplasmic reticulum. western Africa, the eastern Mediterranean, eastern
The major antigenic determinant on the particles is Europe, northern and central Asia, and the Indian
HEPATITIS VIRUSES 203
subcontinent. Both adw and adr are found in precore region, between the two initiation codons,
Malaysia, Thailand, Indonesia and Papua New encodes a signal sequence which directs p25 to the
Guinea, whereas subtype adr predominates in other endoplasmic reticulum, where it is cleaved by a
parts of southeast Asia, including China, Japan and cellular signal peptidase. HBeAg is secreted follow-
the Pacific Islands. The subtypes provide useful ing further proteolysis which removes the C-ter-
epidemiologic markers of HBV. Unusual variants minal domain. HBeAg is also expressed on the sur-
which lack the group specific antigen, a, may be face of the infected hepatocyte and is a major target
selected by antibody in immunized infants infected for the cellular immune system. HBeAg is not an
perinatally and in persistently infected individuals essential protein of the virus but it may cross the
following treatment with hepatitis B immune placenta during pregnancy, tolerizing the fetus and
globulin or a natural antibody response. increasing the probability that a perinatal infection
Surface variants of HBV were first described in will progress to chronicity. Variants of HBV with
Italy infecting children and adults in the presence of mutations in the precore region (precore mutants)
specific hepatitis B surface antibodies (anti-HBs) and which are defective for the synthesis of HBeAg
several months after successful immunization with are discussed below.
two generally licensed hepatitis B vaccines given The P ORF, which overlaps the other three, en-
with and without hepatitis B immunoglobulin (Car- codes the viral polymerase. This enzyme has both
man et al., 1990). Epitope mapping of HBsAg in DNA- and RNA-dependent activities and the pre-
these patients revealed that monoclonal antibodies dicted amino acid sequence has been shown to have
which normally bind to the a determinant failed to homology with retroviral reverse transcriptases.
bind, suggesting that it was not present or that it The polymerase protein also acts as the primer for
was masked. Anticore antibody was found in these minus-strand DNA synthesis and has an ‘RNase H’
patients. Further work established that, in at least activity which degrades the RNA pregenome dur-
one case, the infection was caused by a variant with ing minus-strand synthesis. The fourth ORF has
a mutation leading to a substitution of arginine for been termed X because the function of its product
glycine at amino acid position 145 in the im- was originally obscure. It is now known that this
munodominant domain. This change, which seems protein acts as a transcriptional transactivator and
to have been selected by antibody, has since been may enhance the expression of the other viral pro-
observed independently in the USA (McMahon et teins. Experiments using the woodchuck model (see
al., 1992), Singapore (Harrison et al., 1991) Japan below) confirm that a functional X gene is required
and elsewhere. Other mutations have been de- for the establisment of infection in vivo.
scribed, leading to altered HBsAg which escapes
neutralization by anti-HBs, but the arginine for
glycine substitution at amino acid position 145
seems to be the most common (Oon et al., 1995). Replication of the Virus
Figure 3.10 The replication strategy of the HBV genome. (1) Priming of minus ( 9 ) strand synthesis by the terminal protein
(cross-hatched) on the bulge of the signal near the 5 end of 3.5 kb pregenomic RNA. (2a) Translocation of the primer to a position ()
near the 3 end of the RNA template. (2b) The template shown as a linear structure, solid boxes indicate the direct repeat (DR) sequences
and R the redundancy of the RNA. (3) Minus ( 9 ) strand DNA synthesis by reverse transcription with concommitant hydrolysis of the
RNA template (RNase H-like activity). (4) and (5) Translocation of the remaining 5 oligoribonucleotide from DR1 to DR2 and priming
of plus ( ; ) strand DNA synthesis. (6) Circularization of the genome through the redundancy in the minus ( 9 ) strand and
continuation of plus ( ; ) strand synthesis. (Modified from Lien et al. (1986) Journal of Virology, 57, 229—236. American Society of
Microbiology, Washington)
(approximately 3500 nucleotides) and, while some polymerase to secondary structure at the 5 end
act as mRNAs for the synthesis of HBeAg, HBcAg (epsilon signal, ) of the pregenome leads to packag-
and the viral polymerase, a subset are intermediates ing into immature viral cores in the cytoplasm (Fig-
in the synthesis of progeny genomes. Binding of the ure 3.10, step 1). The N-terminal domain of the viral
HEPATITIS VIRUSES 205
polymerase acts as the primer for minus-strand In the first, high levels of virus replication occur and
DNA synthesis and, following synthesis of a four the patient is seropositive for markers of the virion
nucleotide nascent strand, translocates to a comple- and for HBeAg. Although HBeAg correlates with
mentary four base sequence () near to the 3 end of the presence of the virus, in some cases when virus
the RNA template (Figure 3.10, steps 2a and 2b) replication declines to very low levels or when
This protein remains covalently attached to the 5 precore mutants are present, seroconversion to
end of that strand in the mature virion. Minus- anti-HBe may occur without clearance of the virus.
strand synthesis proceeds by ‘reverse transcription’ Therefore, direct tests for the virus are more reliable
of the pregenome by the viral polymerase with con- for establishing infectivity; for example, detection of
commitant degradation of the tempate (RNase H- viral DNA by hybridization or the PCR. During the
like activity, Figure 3.10, step 3). The remaining first phase of chronicity, replicative forms of the
oligoribonucleotide (which was the 5 end of the HBV genome may be detected in the liver by South-
pregenome) is at the position of the direct repeat, ern hybridization of DNA extracted from biopsy
DR1 (Figure 3.10, step 4), and is now believed to material (Figure 3.13, Lane A7). This replicative
translocate to the other copy of the direct repeat, phase may persist for years, and even lifelong in
DR2, on the minus strand and to prime synthesis of individuals infected perinatally and whose immune
the plus strand (Figure 3.10, step 5). The minus systems are tolerant. More usually, levels of virus
strand has a short terminal redundancy (approxi- replication decline gradually until this is eliminated
mately eight nucleotides) which permits the circu- with seroconversion to anti-HBe. Rarely, there will
larization of the genome as the plus strand is syn- be seroconversion also to anti-HBs, but in many
thesized (Figure 3.10, step 6). Completion of the cases, HBsAg will persist in the absence of virus
core presumably starves the polymerase of precur- replication. Examination of liver biopsies from pa-
sor nucleoside triphosphates leaving the plus strand tients in this second phase of chronicity often re-
incomplete. The cores are then coated with HBsAg veals that HBV DNA is now integrated chromo-
to form mature virus particles. somally in the hepatocytes (Figure 3.13, lane B) and
HBsAg seems to be produced following transcrip-
tion of this integrated DNA. In fact, integration of
virus DNA into the hepatocyte chromosomes
HBV Infection and Chronic Hepatitis B seems to take place throughout the period of virus
replication, and expansion of clones of such cells
Following HBV infection, the first marker to ap- may be a stage in progression to neoplasia (see
pear in the circulation is HBsAg, which becomes below). Integration of the viral genome is not be-
detectable 2—8 weeks prior to biochemical evidence lieved to be required for replication of the virus and
of liver damage or the onset of jaundice (Figure may, in fact, be the result of an abortive infection.
3.11). Next to appear are the markers of the virion, Although replicative and integrated HBV DNA
such as the virus-specific DNA polymerase activity may sometimes be observed in an individual biopsy
and the viral DNA, along with the soluble antigen, specimen (Figure 3.13, lane C) it is not clear that
HBeAg. Antibody to the core (anti-HBc) is detect- both may be present in the same cell.
able 2—4 weeks after the appearance of the surface
antigen, this persists throughout the infection and
after recovery. In acute infections, clearance of the
virus is marked by seroconversion with the disap- Occurrence of HBV in Extrahepatic
pearance of HBeAg and the appearance of antibo- Tissues
dies to it (anti-HBe). Later during convalescence,
HBsAg also disappears with the production of anti- As stated above, hepadnaviruses are essentially
surface antibody (anti-HBs). hepatotropic and it is not clear that they can repli-
In the case of 2—10% of infected adults, and a cate in other tissues. Sensitive hybridization tech-
much larger percentage of children infected niques have, however, enabled the detection of viral
perinatally, the immune system fails to clear virus DNA at other sites, particularly in peripheral leuco-
replication and the infection persists. Chronic hepa- cytes, the bone marrow and spleen. Viral DNA in
titis B may be divided into two phases (Figure 3.12). white blood cells usually occurs as monomeric or
206 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 3.13 Analysis of the state of HBV DNA in the hepatocyte using the Southern blot hybridization technique. Lane A1:
integration of HBV DNA in the PLC/PRF/5 cell line. Lane A6: HBV DNA replicative intermediates in the liver biopsy from an
HBeAg-positive patient. Lane B: integration of HBV DNA in an anti-HBe, HBsAg-positive patient without tumour. Lane C: biopsy
from an HBeAg-positive patient with both replicative forms and integrated HBV DNA. Lanes D: Biopsies from an anti-HBe,
HBsAg-positive patient with primary liver cancer. 1: Biopsy from the non-tumorous part of the liver. 2: Biopsy from the tumour
showing integration of HBV DNA at three sites. All DNA samples were digested with the restriction enzyme Hind III. The leftmost lane
shows radioactively labelled Hind III fragments of bacteriophage DNA as size markers—sizes are given in kilobase pairs alongside.
(Modified from Harrison et al. (1986) Journal of Hepatology, 2, 1—10. Elsevier Science Publishers BV, Amsterdam)
bDNA assay for HBV DNA. A new to appear in serum, followed by HBV DNA, HBeAg
chemoluminescent assay for HBV DNA based on and DNA polymerase, and anti-HBc (Krogsgaard
oligonucleotide amplification of a signal rather et al., 1985). Levels of HBV DNA usually reach
than target appears to be more sensitive than dot- 10—10 genome equivalents per millilitre with the
blot hybridization. The PCR for HBV DNA per- onset of symptoms, after which the levels decrease.
mits the detection, cloning and sequencing of HBV In contrast, in patients who develop chronic hepati-
genomes from such patients. It is at least 10 times tis B, levels of HBV DNA remain high. A positive
more sensitive than dot-blot hybridization assays IgM anti-HBc test typically distinguishes acute
for HBV DNA. from chronic hepatitis B. By the time the patient
consults a physician, HBV DNA and HBeAg are
often no longer detectable in serum. The loss of
Acute Hepatitis B
HBeAg is a sign that the patient will clear HBsAg.
A simplified guide to the interpretation of the test HBsAg may be present only transiently in serum.
results is shown in Table 3.3. Detection of HBsAg is The only evidence of infection may therefore be the
the frontline assay for infection and used widely for presence of IgM anti-HBc or subsequent develop-
screening, for example, in transfusion centres. ment of anti-HBc and anti-HBs. Anti-HBs may be
The diagnosis of acute hepatitis B generally rests quantified and the test is valuable in determining
upon the finding of HBsAg and IgM antibody to the outcome of immunization.
hepatitis B core antigen (anti-HBc) in the serum of a Serum alanine aminotransferase (ALT) concen-
patient with clinical and serum biochemical evi- trations typically rise in acute hepatitis B. Serum
dence of acute hepatitis. HBsAg is the first marker bilirubin concentrations increase in proportion to
208 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 3.3 Interpretation of results of serological tests for hepatitis B
Anti- HBc
HBsAg HBeAg Anti-HBe IgM IgG Anti-HBs Interpretation
; ; 9 9 9 9 Incubation period
; ; 9 ; ; 9 Acute hepatitis B or persistent carrier
state
; ; 9 9 ; 9 Persistent carrier state
; 9 ; < ; 9 Persistent carrier state
9 9 ; < ; ; Convalescence
9 9 9 9 ; 9 Recovery
9 9 9 ; 9 9 Infection with HBV without detectable
HBsAg
9 9 9 9 ; 9 Recovery with loss of detectable
anti-HBs
9 9 9 9 9 ; Immunization without infection.
Repeated exposure to antigen without
infection, or recovery from infection
with loss of anti-HBc
the severity of hepatic damage. During this phase, from serum, followed by loss of HBeAg. This may
IgM anti-HBc in serum correlates with active hepa- occur following a sudden, asymptomatic increase in
titis in patients. Antibodies to pre-S components serum aminotranferases. Once HBeAg is cleared,
appear early in the disease, and correlate with the the disease remits temporarily and serum amino-
disappearance of serological markers of HBV repli- transferases become normal.
cation, suggesting a role in immunological clear-
ance of HBV (Cupps et al., 1990). Immune com-
Natural History of Chronic Hepatitis B
plexes may be responsible for some of the
manifestations of the acute disease. A mild decrease Chronic hepatitis B generally lasts for many years,
in serum C3 and C4 concentrations occurs in acute but may not necessarily be lifelong. Some patients
hepatitis B and may reflect antigen—antibody com- with chronic hepatitis B may spontaneously
plex formation. Autoantibodies, including abnor- seroconvert from being HBeAg positive to anti-
malities in rheumatoid factor, and anti-nuclear and HBe positive and enter a phase of remission with an
anti-smooth muscle antibodies are also detectable improvement in serum aminotransferases, albeit
in acute hepatitis. In fulminant hepatitis extremely that they remain HBsAg positive. The term healthy
rapid clearance of HBeAg and HBsAg may occur. hepatitis B carriers has been used for these persons,
but the term is somewhat inappropriate, as they are
at risk of reactivation of active virus replication,
Chronic Hepatitis B
and, if cirrhosis has developed, they may ultimately
Many carriers are detected through routine screen- develop hepatocellular carcinoma, in the face of
ing for HBsAg or the presence of abnormal liver relatively low levels of viraemia. Recent retrospec-
function tests. Older patients may present for the tive studies have focused on survival in compen-
first time with complications of cirrhosis, or even sated cirrhosis due to hepatitis B. In a European
hepatocellular carcinoma. Typically the levels of multicentre longitudinal study to assess the survival
serum aminotransferases are elevated in patients of 366 HBsAg- positive compensated cirrhosis,
with HBeAg, HBV DNA-positive chronic hepatitis, death occurred in 23% of patients, mainly due to
but some patients may have normal values. The liver failure or hepatocellular carcinoma The cumu-
levels of aminotransferases fluctuate with time. A lative probability of survival in this cohort was 84%
spontaneous remission in disease activity may oc- and 68% at 5 and 10 years, respectively. The worst
cur in approximately 10—15% of HBeAg-positive survival was in HBeAg and HBV DNA-positive
carriers per year, characterized by disappearance of subjects (Fattovich et al., 1997).
detectable (by molecular hybridization) HBV DNA As stated above, a number of patients have ongo-
HEPATITIS VIRUSES 209
ing virus replication without detectable HBeAg, class II alleles (DRB*1302) with lack of persistent
and often in the presence of anti-HBe (Hadziyannis HBV infection has been reported (Thursz et al.,
et al., 1983). It is now known that such patients 1995). In fulminant hepatitis, extremely rapid clear-
frequently are infected with variants of HBV with ance of HBeAg and HBsAg may occur.
mutations in the precore region and which cannot The elimination of virus-infected hepatocytes is
synthesize HBeAg (Carman et al., 1989). The major- dependent on the recognition by cytotoxic T cells of
ity of these patients are anti-HBe positive and may viral determinants, in association with HLA pro-
have been infected initially with a mixture of wild- teins on the infected cells. Data derived from most
type and variant virus, with clearance of the wild experimental systems suggest that an acute, poly-
type on seroconversion to anti-HBe. Precore vari- clonal and vigorous cytolytic T cell response
ants may be selected during the process of serocon- usually occurs in acute symptomatic icteric hepati-
version to anti-HBe when hepatocytes expressing tis B and that patients with acute self-limiting hepa-
HBeAg are targeted for lysis by cytotoxic T cells. titis B develop a polyclonal HLA class I restricted,
Precore mutants can be detected in patients with cytotoxic T cell response against numerous epi-
fulminant hepatitis B (Carman et al., 1991) but it is topes in the HBV envelope, nucleocapsid and poly-
controversial whether infection with these variants merase proteins. Several HLA-A2 restricted
ab initio, in the absence of wild type virus, often cytotoxic T cell epitopes have been defined.
leads to acute liver failure. Not all patients with The failure to eradicate HBV reflects an inad-
fulminant hepatitis B are infected with precore mu- equate immune response to the virus, but the pre-
tants, and other mutations, such as in the core cise impairment of humoral and cellular immunity
promoter, as well as host factors, may be important that determines the development and outcome of
(Sterneck et al., 1996). hepatitis B has not been characterized. Persistent
infection is an unusual outcome in those patients
with acute icteric hepatitis B and the mechanisms
that lead to viral persistence are not well under-
Pathogenesis of Hepatitis B Infection stood. In neonates, specific suppression of the cell
mediated immune response may favour infection,
HBV is not cytopathic to hepatocytes under most perhaps because of intrauterine exposure to HBeAg
circumstances. Host immunity plays an important inducing tolerance to epitopes that are usually the
role in cellular injury and there is little correlation target of the cytotoxic T cell response at a time
between the severity of the illness and the level of when the immune system is ontogenically imma-
HBV replication. Patients with a poor T cell re- ture. Clonal deletion of HBV-specific T cells may
sponse may have high concentrations of virus in occur as a consequence of transplacental infection
liver and the mildest disease. The development of of the developing fetus or transplacental passage of
chronic infection is due to a failure of an adequate viral antigens. In contrast to the vigorous poly-
immune response, but the immune response is also clonal class I and class II-restricted T cell response
responsible for disease pathogenesis during chronic that can be identified in patients with acute icteric
infection. The subsequent expression of the disease hepatitis B, in chronic disease the response in pe-
involves a poorly understood interplay between vir- ripheral blood is relatively weak and focused, and
al and host factors. The nucleocapsid antigens insufficient to clear replicating virus.
(HBcAg and HBeAg) expressed on the cell mem- Other mechanisms may also operate to prevent
brane contain important targets of the immune re- clearance of HBV: mutations abrogating the recog-
sponse and cytolytic T cells in acute hepatitis B. nition of the wild-type hepatitis, including the natu-
In acute hepatitis B, intense lysis of hepatocytes ral variants of the HBcAg 18—27 core epitope that
occurs. Immune lysis is believed to eradicate hepati- interfere with the recognition of the wild type epi-
tis B. However, experimental studies in the trans- tope and act as T cell receptor antagonists (Be-
genic mouse model suggest that HBV replication rtoletti et al., 1994). There is some evidence of a
may also be reduced by a complex interplay of a failure of interferon production in patients with
cytotoxic T cell response, apoptosis and down- chronic hepatitis B.
regulation of hepatitis B gene expression by several Recent experiments indicate that, although many
inflammatory cytokines. An association of MHC patients will not have a discernible cytotoxic T cell
210 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
response, a proportion do mount a ‘secondary’ im- the chance of the baby developing the persistent
mune response, associated with seroconversion to carrier state is reduced by about 70%. More recent
anti-HBe, and at least a temporary remission in studies using combined passive and active immu-
disease. A proliferative T cell response is evident in nization indicate an efficacy approaching 90%. The
active disease. In addition, an HLA-restricted dose of HBIG recommended in the newborn is
cytotoxic T cell response can be detected around 1—2 ml (200 iu of anti-HBs per ml).
the time of anti-HBe seroconversion during sponta-
neous or interferon-induced HBeAg clearance, or
Active Immunization
when there has been an exacerbation of the disease
associated with an increase in HBV DNA. Failure to grow HBV in cell culture directed atten-
Cytokines also are released in acute and chronic tion to the use of other sources of antigen for active
hepatitis. Necrosis of hepatocytes may also be am- immunization. Since recovery from acute hepatitis
plified by antigen non-specific release of cytokines, B usually is associated with an anti-HBs response
rather than by a directed cytotoxic T cell response. and consequent immunity, an obvious approach
In transgenic mice, non-cytolytic mechanisms have was to use HBsAg as an immunogen in vaccine
been observed, including the suppression of viral formulations. First-generation vaccines, which are
gene expression and replication by a post-transcrip- still in use in some countries, are prepared from the
tional mechanism mediated by -interferon, tumour plasma of HBsAg-positive individuals by purifica-
necrosis factor (CTNF) and interleukin(IL)-12 tion and inactivation of 22 nm spherical surface
(Tsui et al., 1995; Cavanaugh et al., 1997). Experi- antigen particles. Trials of protective efficacy in
ments in mice have suggested that a predominance high-risk groups in the early 1980s demonstrated
of HBeAg-specific, T 2—type cells may contribute the value of the vaccines and their safety. These
&
to chronicity in HBV infection (Milich et al., 1995a). vaccines have been superseded in most countries by
preparations of HBsAg expressed in yeast or mam-
malian cells (recombinant vaccines). Local reac-
tions reported after immunization with these prep-
Prevention and Control of Hepatitis B arations have been minor, occurring in less than
20% of immunized individuals, and consisted of
slight swelling and reddening at the site of inocula-
Passive Immunization
tion. Temperature elevations of up to 38°C were
Hepatitis B immunoglobulin (HBIG) is prepared observed in only a few individuals.
from pools of plasma with high titres of hepatitis B HBV vaccines currently used in the UK and else-
surface antibody and may confer temporary passive where in the West are prepared from yeast (Sacchar-
immunity under certain defined conditions. The omyces cerevisiae). The region of the surface ORF
major indication for the administration of HBIG is specifying the 226 amino acids of the major surface
a single acute exposure to HBV, such as occurs protein, but without the pre-S sequences, was
when blood containing HBsAg is inoculated, inges- placed under the control of a yeast promoter in a
ted or splashed on to mucous membranes and the plasmid which also contained components necess-
conjunctiva. The optimal dose has not been estab- ary for replication and maintenance in yeast cells.
lished but doses in the range of 250—600 iu have The plasmid, which was constructed using E. coli,
been used effectively. HBIG should be administered was then used to transform yeast protoplasts. The
as early as possible after exposure and preferably transformed yeast, which synthesizes HBsAg, is
within 48 hours, usually 3 ml (containing 200 iu of grown from seed stocks in fermenters. The cells are
anti-HBs per ml) in adults. It should not be admin- harvested and disrupted mechanically and the sur-
istered 7 days or more after exposure. It is generally face antigen is purified by a combination of precipi-
recommended that two doses of HBIG should be tation steps, ultrafiltration, gel permeation and ion
given 30 days apart. exchange chromatography. The purified antigen is
Results following the use of HBIG for prophylax- sterilized by membrane filtration and adsorbed to
is in babies at risk of infection with HBV are en- buffered aluminium hydroxide.
couraging if the immunoglobulin is given as soon as The emergence of variants of HBV which are not
possible, and certainly within 12 hours of birth, and neutralized by vaccine-induced anti-HBs, as de-
HEPATITIS VIRUSES 211
scribed above, is of major concern. Horizontal Disease Control and Prevention, USA, recommen-
spread of these viruses has not yet been reported but ded that the arm be used as the site for hepatitis B
remains a possibility. If the number of potential vaccination in adults, as have the Departments of
variants is limited it may be possible to include Health in the UK.
these in future formulations of hepatitis B vaccine.
It seems that the mutation affecting amino acid
residue 145 (glycine to arginine) is the most Combined Immunoprophylaxis
common but variants with other changes are being Whenever immediate protection is required, as, for
investigated (Oon et al., 1995). example, for infants born to HBsAg-positive
There has been some interest in including pre-S mothers (see above) or following transfer of an indi-
sequences in recombinant hepatitis B vaccines to vidual into a ‘high-risk’ setting or after accidental
potentiate the immunogenicity of HBsAg and ad- inoculation, active immunization with the vaccine
dress the problem of non-responsiveness (Zucker- should be combined with simultaneous administra-
man, 1996). Enhancement of the immunogenicity of tion of HBIG at a different site. It has been shown
the pre-S region of HBsAg has been demonstrated that passive immunization with up to 3 ml (200 iu of
in mice, using chemically synthesized peptides anti-HBs per ml) of HBIG does not interfere with
(Milich et al., 1985). The immune response to the an active immune response. A single dose of HBIG
pre-S2 region was shown to be regulated by H-2 (usually 3 ml for adults; 1—2 ml for the newborn) is
linked genes which are distinct from those which sufficient for healthy individuals. If infection has
regulate the response to the S region. It was also already occurred at the time of the first immuniz-
demonstrated that immunization of a ‘non-respon- ation, virus multiplication is unlikely to be inhibited
der’ murine strain with particles which contain both completely, but severe illness and, most important-
S and pre-S2 can circumvent non-responsiveness. ly, the development of the carrier state of HBV may
Furthermore, HBV is believed to attach to the be prevented in many individuals, particularly in
hepatocyte receptor through a domain in the pre-S1 infants born to carrier mothers.
protein and antibodies to that domain are neu-
tralizing (Neurath et al., 1986). A variety of vaccines
containing pre-S sequences are now undergoing cli- Indications for Immunization
nical trial and there are some indications that the
The current indications for the use of hepatitis B
rate of non-responsiveness may be lower than with
vaccines in the UK are given below. Many coun-
vaccines containing S alone (Zuckerman et al.,
tries, including the USA and Italy, introduced uni-
1997). It is not clear whether the inclusion of extra
versal immunization for infants in 1992. The World
antigenic domains will circumvent the problem of
Health Organization recommended a decade ago
antibody escape variants.
that universal immunization should be in place in
areas with a prevalence of HBsAg greater than 8%
by 1994 and in all countries by 1997, with integra-
Site of Injection for Vaccination tion of hepatitis B vaccine into the Expanded Pro-
gramme of Immunization (EPI). These targets were
Hepatitis B vaccination should be given in the up-
not met and universal immunization of infants
per arm or the anterolateral aspect of the thigh and
worldwide remains a goal.
not the buttock (Zuckerman et al.,1992). There are
In 1996 the Department of Health and other
over 100 reports of unexpectedly low antibody
government offices in the UK recommended immu-
seroconversion rates after hepatitis B vaccination
nization for the following risk groups:
using injection into the buttock. In one centre, a low
antibody response was noted in 54% of healthy ∑ Babies born to mothers who are chronic carriers
adult health-care personnel. Many studies have of hepatitis B virus or to mothers who have had
since shown that the antibody response rate was acute hepatitis B during pregnancy. In addition,
significantly higher in centres using deltoid injec- babies born to mothers who are HBeAg positive,
tion than centres using the buttock. On the basis of who are HBsAg positive without e markers (or
antibody tests after vaccination, the advisory com- where HBeAg/anti-HBe status has not been de-
mittee on immunization practices of the Centers for termined), or who have had acute hepatitis B
212 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
during pregnancy should receive HBIG as well as B. These therapeutic preparations of type I inter-
active immunization. Currently, vaccine without ferons consist either of a mixture of interferon spe-
HBIG is recommended for babies born to cies derived from virus-stimulated Namalwa cells
mothers who are HBsAg positive but known to (Wellferon), or recombinant DNA-produced single-
be anti HBe positive. component -interferon species. All are adminis-
∑ Parenteral drug misusers. tered parenterally, either intramuscularly or subcu-
∑ Close family contacts of a case or carrier. Sexual taneously, but can be given intravenously for pa-
contacts of patients with acute hepatitis B should tients with bleeding disorders. All type 1 interferons
also receive HBIG. share a common pattern of biological effects that
∑ Families adopting children from countries with a begin with binding of the interferon to the cell sur-
high prevalence of hepatitis B, particularly some face receptor. Binding is followed by activation of
countries in eastern Europe, southeast Asia and tyrosine kinases, which leads to the production of
South America. several interferon-stimulated gene products. The in-
∑ Haemophiliacs (and others receiving regular terferon-stimulated gene products are responsible
blood transfusions or blood products, including for the pleiotropic biological effects of type-1 inter-
carers responsible for the administration of such ferons including antiviral, antiproliferative and im-
products). munomodulatory effects, cytokine induction and
∑ Patients with chronic renal failure. Higher doses HLA class I and class II regulation, macrophage
of vaccine may be required in those who are activation and regulation of natural killer and
immunocompromised. cytotoxic T cells. Examples of interferon-stimulated
∑ Health-care workers, including students and gene products include 2, 5-oligoadenylate syn-
trainees. thetase (2, 5-OAS), -microglobulin and the hu-
∑ Staff and students of residential accommodation man MXa homologue.
for those with severe learning disabilities. Alpha interferon is not indicated for acute icteric
∑ Other occupational risk groups, including certain hepatitis B. Although non-specific symptoms may
members of the emergency and prison services. be improved, there is no evidence that antiviral
∑ Inmates of custodial institutions. therapy accelerates healing or clearance of virus or
∑ Those travelling to areas of high prevalence. that early treatment with interferon prevents the
development of chronic disease. Low levels of viral
Despite the availability of a vaccine, infection
replication are found in patients with fulminant
persists worldwide. In populous regions where mass
hepatitis B, suggesting that the pathogenesis of the
immunization has not started, there has not been a
disease is due to an exaggerated immune response
significant decline in HBsAg carrier rates, and
to HBV, and -interferon has not been found to be
therefore the carrier pool has increased with the
beneficial in these severely ill patients.
increase in population. None the less, notable suc-
Treatment of chronic hepatitis B is targeted at
cesses through vaccination have emerged, in par-
patients with active disease and viral replication,
ticular in Taiwan and Alaska. In Taiwan, the long
preferably at a stage before signs and symptoms of
awaited impact of vaccination on the risk of hepa-
cirrhosis or significant injury have occurred
tocellular carcinoma has been discernible (Chang et
(Dusheiko et al., 1985). Eradication of the infection
al., 1997).
is possible in only a minority of patients. Permanent
loss of HBV DNA (undetectable by molecular hy-
bridization) and HBeAg, and normalization of the
Antiviral Therapy
serum aminotransferases are considered a response
Alpha interferon. Alpha interferons are naturally to antiviral treatment, as this result is associated
occurring intercellular signalling proteins used with an improvement in necro-inflammatory dam-
therapeutically for their properties of inducing an age and reduced infectivity. It is possible, but un-
antiviral state in cells, inhibiting cellular prolifer- proven, that the accompanying reduction in his-
ation and immunomodulation. Several -inter- tological chronic active hepatitis lessens the risk of
ferons, including HuIFN--N1 (Wellferon), rIFN- cirrhosis and hepatocellular carcinoma (Niederau
-2b (Intron A), and rIFN--2a, (Roferon-A) have et al.1996).
been licensed for the treatment of chronic hepatitis Approximately 35—40% of HBeAg-positive pa-
HEPATITIS VIRUSES 213
tients are treated effectively by -interferon, at a adults and usually accept treatment reasonably well
dose of 9—10 million iu three times weekly (5 million (Ruiz-Moreno et al., 1990). The appropriate dose of
daily in the USA) for 4—6 months. Response rates interferon in children is 3 mu m\ three times week-
tend to be higher in carriers with higher baseline ly for 4 months.
serum aminotransferase levels. The subclinical ex- Glomerulonephritis caused by chronic HBV has
acerbation of the hepatitis and increases in serum been treated with rIFN-2b (Lisker-Melmen et al.,
aminotransferases frequently seen in responders 1989). In patients in whom aminotransferases fall
suggest that interferon acts by augmenting the im- into the normal range and HBeAg disappears, urine
mune response to HBV, perhaps triggered by the protein excretion also decreases and resolution of
inhibition of viral replication, the effects of inter- the glomerulonephritis occurs (Lin, 1995).
feron on cytotoxic T cells, and the upregulation of Relative or absolute contraindications to -inter-
MHC class I display. Sustained loss of HBeAg gen- feron therapy include severe depression, Childs B/C
erally is associated with histological reduction in cirrhosis, cirrhosis and hypersplenism, autoimmune
inflammation. Longer term follow-up of responders hepatitis, hyperthyroidism, coronary artery disease,
has shown that up to 65% of those who lose HBeAg renal transplant, pregnancy, seizures, concomitant
become HBsAg negative (Korenman et al., 1991; drugs, including several herbal remedies, diabetes/
Lau et al., 1997). Although parameters that predict hypertension and retinopathy, thrombocytopenia,
responsive patients are difficult to characterize fully, leucopenia, anaemia, high titres autoantibodies and
those carriers with elevated pretreatment serum hyperthyroidism. The side-effects of -interferon are
aminotransferase levels, with chronic active hepati- relatively common, but are acceptable in most pa-
tis, lower serum HBV DNA, females, those negative tients. Toxicity can be predicted in patients with
for anti-HIV, those with a history of hepatitis, Cau- low baseline white cell counts or throm-
casians with more recent onset of chronic hepatitis bocytopenia, or pre-existing thyroid disease
B, and with IgM anti-HBc, may respond more fa- (Dusheiko, 1997). The risk of serious complications
vourably. Although these factors provide some pre- from -interferon is rare. However, serious idiosyn-
dictive information, none of these criteria are abso- cratic complications, such as immune disorders,
lute. pneumonitis, retinal disease, renal disease or deaf-
Alpha interferon also has been used to treat pa- ness, can occur and the drug always must be pre-
tients after induction and subsequent withdrawal of scribed with caution. Close monitoring is required
corticosteroids. Serum aminotransferases may in- throughout therapy. Monitoring requires regular
crease in a proportion of patients as corticosteroids clinical examinations, vital signs, urinalysis and
are withdrawn. The initial pretreatment may thus usually monthly measurement of serum chemistries,
‘prime’ patients, thus increasing the efficacy of - complete blood counts and thyroid function tests,
interferon. (Perillo et al., 1988; 1990). including thyroid stimulating hormone. A serum
Patients who are anti-HBe positive but who have pregnancy test should be done to exclude preg-
HBcAg in hepatocytes and histological chronic ac- nancy before starting treatment.
tive hepatitis due to infection with HBV ‘precore
mutants’, which do not synthesize HBeAg, may
have severe disease. In parts of Europe, and else- Nucleoside analogues. Antiretroviral therapies
where, disease caused by this variant of HBV is used in human immunodeficiency virus (HIV) infec-
more common than disease caused by the ‘wild tion have been attempted in chronic HBV infection.
type’ (HBeAg-positive chronic hepatitis). Approxi- Both these viruses utilize a reverse transcriptase, a
mately 10—25% of patients have long term re- potential target for antiviral inhibition. However,
sponses to treatment doses of 9—10 million thrice the usefulness of all the ‘first generation’ nucleoside
weekly for 6—12 months, as relapse rates tend to be analogues in patients is limited (Marcellin et al.,
high in these patients (Brunetto et al., 1989). 1989; Berk et al., 1992; Fried et al., 1992, Catterall et
Children who are infected perinatally, and have al., 1992).
mild disease activity, respond poorly to interferon Ganciclovir [9—[2—hydroxy-1—hydroxymethyl)-
treatment (Lok et al., 1989). Conversely, children ethoxymethyl]guanine] inhibits HBV DNA repli-
with active disease and high serum aminotran- cation but needs to be given intravenously. The
sferases respond to interferon therapy similarly to drug has been used to treat recurrence of hepatitis B
214 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
after transplantation, but replication re-ensues therapy of this disease in the USA. Lamivudine is
when treatment is stopped. Oral ganciclovir has active in vitro against human hepatitis B transfected
been used to treat HBeAg and anti-HBe-positive cell lines and in ducklings affected with DHBV, as
hepatitis B in a preliminary trial. well as in chimpanzees infected with HBV.
Famciclovir is the oral precursor of penciclovir, a Large phase II and III trials in patients with
purine nucleoside [9—(4—hydroxy-3—hydroxy- chronic hepatitis B are in progress. Doses above
methylbut-1—yl)guanine; BRL 39123]. Penciclovir 25 mg reproducibly decrease HBV DNA levels in
is poorly absorbed orally, but famciclovir is readily serum. Treatment has been extended to 1 year and
absorbed and rapidly converted into the active mol- more, in dose ranging studies with 5, 20, 100, 300 or
ecule penciclovir by enzyme hydrolysis; Phos- 600 mg per day. The results of a several relatively
phorylation leads to the triphosphate compound short randomized controlled trials have recently
and interference with synthesis of viral DNA; pen- been published. HBV DNA generally became unde-
ciclovir triphosphate reduces varicella zoster and tectable (by hybridization assay) in more than 90%
herpes simplex concentrations in cells, and oral of patients who received 25—300 mg per day. In
famciclovir is an effective treatment of shingles. most patients, HBV DNA reappears after therapy is
The duck model was first used to assess this new completed. Between 15 and 40% of patients treated
agent for hepatitis B. Plasma levels of DHBV DNA for a year have sustained suppression of HBV
were significantly reduced compared to levels in DNA; in an extended retreatment trial, approxi-
samples collected before treatment (Tsiquaye et al., mately 40% of patients have remained HBeAg
1994). Famciclovir has been tested in a phase II trial negative off therapy; in a large Asian trial, approxi-
in 333 HBeAg and HBV DNA patients with chronic mately 15% of patients became HBeAg negative
hepatitis B, using dosing schedules of 125, 250 and after 12 months of treatment, compared to 4% of
500 mg three times daily for 16 weeks. The trial has placebo recipients; treatment has been continued in
been completed but the results have only been pub- this and other cohorts. Histological improvement
lished in abstract form. A significant reduction in has been noted after 1 year of treatment. Patients
HBV DNA and ALT within the treatment period with chronic hepatitis B are currently being treated
was observed in the 500 mg group, and the HBeAg for 1 year with combinations of lamivudine and -
seroconversion rate was increased over placebo. interferon in controlled trials.
However, some patients do not appear sensitive to In these trials, only minor non-specific adverse
famciclovir, and resistant mutations have been re- events were seen that were not related to dose.
ported. Similar results have been observed after 6 months of
Lamivudine (2,3-dideoxy-3-thiacytidine (( < )- treatment; a degree of histological improvement has
SddC), 3TC (Epivir, or lamivudine) is a potent in- been noted.
hibitor of HBV as well as HIV. The drug acts by The drug appears to be well tolerated, and rela-
inhibiting DNA synthesis through chain termina- tively few serious side-effects have been reported.
tion. The ( 9 )-form (( 9 )-SddC), which is resistant Serious side-effects have been observed in about 5%
to deoxycytidine deaminase, is the more active anti- of patients; these include anaemia, neutropenia, an
viral stereoisomer than the ( ; )-form. The negative increase in liver enzymes, nausea and neuropathy.
enantiomer ( 9 )-SddC does not appear to affect Increased lipases may occur, but uncommonly, and
mitochondrial DNA synthesis. Metabolic studies serious lactic acidosis has not been observed. Severe
have shown that the drug is converted to the mono- exacerbations of hepatitis accompanied by jaundice
phosphate, diphosphate, and triphosphate form. have been reported in patients whose HBV DNA
The ( 9 )-form is phosphorylated to ( 9 )-SddCMP became positive after stopping treatment, or after
and is subsequently converted to ( 9 )-SddCDP the development of resistance (Honkoop et al.,
and ( 9 )-SddCTP (Chang et al., 1992). 1995). We have observed reactivation of hepatitis B
The drug is rapidly absorbed after oral adminis- in two immunosuppressed liver transplant patients
tration, with a bioavailability of 80%. The ma- who developed a methionine to valine or isoleucine
jority of drug is excreted unchanged in the urine. substitution in the highly conserved tyr-met-asp-
Since 1990, lamivudine has been used in trials of asp (YMDD) motif of the HBV polymerase (Ling et
treatment of HIV infection, and this compound has al., 1996). This motif is part of the active site of the
recently been licensed for combination antiviral polymerase, and this mutation parallels the M184
HEPATITIS VIRUSES 215
mutation seen in resistant HIV, where substitutions who received active drug had a greater than 90%
of valine and isoleucine for methionine also have decline in HBV DNA. Phase II and III studies
been found. designed to find the optimal dose and involving
After lamivudine is stopped, HBV replication larger numbers of patients are now in progress.
may reactivate if viral genomes have not been Preliminary in vitro evidence suggests that M552I,
cleared during treatment. In this case, recurrence of and M552V variants of HBV which are relatively
HBV replication can be associated with severe resistant to lamivudine are susceptible to inhibition
‘flares’ or exacerbation of hepatitis as HBV DNA by adefovir.
increases in serum. The pathogenesis of this injury is Phase I and II trials with several new nucleoside
not fully understood. Probably some of the injury is analogues are in progress. At this time the long-
related to an immune response, despite the increas- term efficacy and safety of lamivudine or other new
ing levels of viraemia. The emergence of resistance nucleoside analogues are unproven; chronic type B
could have a similar effect, as viral DNA increases. hepatitis disease will require relatively long courses
of treatment, often in asymptomatic carriers, per-
haps including children, and viral resistance may
Adefovir dipivoxil (bis-POM pMEA) 0-[2-[[bis- emerge. The end-points of treatment will require
[(pivaloyloxy)methyl]phosphinyl]methoxy]- careful evaluation. Combination treatment may be-
ethyl]adenine. This new agent is a phos- come necessary but may not be required for all
phonomethylether analogue. It is an oral nucleotide patients.
(nucleoside monophosphate) analogue and does
not require cellular nucleoside kinases for activa-
tion. The drug inhibits viral polymerases and ter- Biologic response modifiers. Interleukin 12 is a
minates the growing DNA chain by acting as a 75 kDa heterodimeric protein and a member of the
competitive inhibitor of dATP. Adefovir has an TNF receptor superfamily. This cytokine is a prod-
effect on HIV as well as on other retroviruses and uct of macrophages and dendritic cells (i.e. antigen
also suppresses DHBV and HBV replication. presenting cells). Interleukin 12 promotes T 1 and
&
In experimental studies, adefovir dipivoxil has suppresses T 2 cell development, suggesting that
&
been shown to inhibit viral DNA synthesis in HBV- IL-12 may be useful therapeutically to promote a
transformed hepatoma cell lines and in primary cellular immune response. The compound induces
duck hepatocytes from DHBV-positive ducklings. secretion of -interferon from T and natural killer
During treatment, viral antigen expression is reduc- (NK) cells, and increases the lytic activity of NK
ed in bile duct epithelial cells and pancreatic islets. cells and facilitates specific cytotoxic T lymphocyte
This agent also has some immunomodulatory ac- (CTL) responses. In experimental murine models of
tivity and stimulates natural killer activity. acute viral infections, IL-12 reduces morbidity and
Dose-dependent toxic effects observed in prevents reinfection with HSV-2 and cytomega-
multiple dose studies in rats and monkeys include lovirus (CMV). The effect of systemic IL-12 treat-
nephropathy, characterized by tubular cell necrosis, ment on autoantibody synthesis in vivo in HBeAg-
elevations in hepatic transaminases, elevations in expressing transgenic mice has been tested. Low-
creatinine kinases, and degenerative changes in dose IL-12 significantly inhibited autoantibody
small and large intestine. (anti-HBe) production by shifting the T 2—me-
&
A small pilot study has been concluded, in which diated response towards T 1 predominance.
&
20 patients (13 of whom were coinfected with HIV) Additionally, previous studies suggest that a pre-
were treated with adefovir dipivoxil 125 mg (n : 15) dominance of HBeAg-specific T 2—type cells may
&
or placebo (n : 5) for 28 days. The drug was discon- contribute to chronicity in HBV infection. There-
tinued in two recipients because of an ALT increase. fore, IL-12 may also prove beneficial in modulating
An ALT increase of more than three times the base- the antibody and cellular immune response in acute
line was observed in four patients on drug or after hepatitis B to improve clearance of hepatitis B
dosing. A significantly greater decline in HBV DNA (Milich et al., 1995b). It has been shown that sup-
was seen in drug recipients compared to placebo. pression of intrahepatic replication of HBV DNA in
The mean percentage change was 9 97 compared transgenic mice is mediated through cytokines such
to 9 7 in placebo recipients, and all 15 patients as -interferon and TNF. Fever, lethargy, anorexia,
216 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
anaemia, thrombocytopenia, stomatitis, pulmonary Recently, DNA-based immunization has been
oedema and increased serum ALT have been ob- tested using purified plasmid DNA containing pro-
served in experimental animals. Pilot studies have tein coding sequences and the regulatory elements
begun in HBV infected patients. IL-12 can also required for their expression. The DNA is introduc-
aggravate autoimmune processes, and deaths in pa- ed into tissues of the organism by means of a par-
tients with renal carcinoma have been observed. enteral injection and the number of cells transfected
Thymosin is an immune stimulant which is and the amount of protein produced is sufficient to
known to enhance suppressor T cell activity and B produce a broad-based immune response to a wide
cell synthesis of IgG in vitro. Peptide preparations variety of foreign proteins. A response to HBsAg
of thymosin have been evaluated in small controlled has been achieved using this form of antigen presen-
trials and HBV DNA has been noted to become tation. A CD8 ; CTL response has been induced
negative in some of these patients and in chimpan- in BALB/c mice, suggesting a pathway for
zees with chronic hepatitis B (Mutchnick et al., exogenous presentation of hepatitis B envelope pro-
1991). It is given parenterally by subcutaneous in- tein via MHC class I expression (Davis et al., 1995).
jection. This interesting agent’s possible therapeutic If the safety of DNA-mediated immunization can be
role was evaluated in a larger controlled trial, in assured, this form of vaccination may also have
which HBeAg seroconversion rates were not signifi- therapeutic potential (Whalen and Davis, 1995).
cantly different from recipients given placebo. The
place of this drug is still being appraised, as few side
effects are observed with thymosin injected subcu-
Hepatitis B Virus and Primary Liver
taneously.
Cancer
Immunotherapy Persistent infection with HBV is associated with a
There has been a resurgence of interest in the possi- high risk of developing hepatocellular carcinoma
bility of immunizing chronically infected, HBV- (HCC); however, the precise role played by the virus
positive patients to activate an immune response in causing this tumour remains to be elucidated.
and eradicate viraemia. In a recent pilot study, 32
patients with HBeAg positive, HBV DNA positive
Epidemiological Evidence
chronic hepatitis B were immunized with three
standard doses of GenHevac vaccine at monthly Hepatocellular carcinoma is one of the 10 most
intervals. Six months after the first injection, 37% common cancers in the world, with over 250 000
had undetectable DNA. Eight responders were new cases each year. In areas where the tumour is
given interferon to maintain virus inhibition (Pol particularly common—for example, some regions
et al., 1994, Pol, 1995). These results require expla- of sub-Saharan Africa, China and southeast Asia—
nation, as earlier trials of HBsAg vaccination had the age-adjusted incidence of HCC is over 30 new
no demonstrable efficacy for chronic disease. The cases per 100 000 population each year; whereas it is
effects may be the result of altered antigen presenta- less than 5 cases per 100 000 per year in western
tion. Larger controlled trial are in progress. Europe and North America. Primary liver cancer is
There is considerable interest in the possibility of more common in males than among females, and
provoking a cytotoxic immune response to HBV the incidence of the tumour increases with age,
core epitopes, as the CTL response contributes to reaching a peak in the 30—50 age group.
viral clearance in acute hepatitis B. A strong When specific tests for the serological markers of
cytotoxic T cell response has been observed in HLA HBV infection became available, it became clear
A2.1 individuals with acute hepatitis B against an that the geographical areas with a high incidence of
epitope (FLPSDFFPSV) that contains an HLA A2 primary liver cancer were coincident with those
binding motif located between residues 18—27 of the with a high prevalence of seropositivity for HBsAg.
hepatitis B nucleocapsid protein (Bertoletti et al., Furthermore, most patients from high risk areas
1993). This identification has led to development of presenting with primary liver cancer proved to be
a therapeutic vaccine (Theradigm HBV tm) which is HBsAg positive or to have high titres of anti-HBc.
now undergoing assessment. HBV infection in these areas generally occurs at a
HEPATITIS VIRUSES 217
young age: in China and southeast Asia the virus HBV-associated primary liver tumours have also
usually is acquired perinatally from a carrier been shown to contain integrated HBV DNA.
mother, while in Africa the infection tends to be Analysis by DNA:DNA hybridization reveals in-
acquired in early childhood through horizontal tegrated HBV DNA in approximately 80% of pri-
spread of the virus. Thus, there is often a consider- mary liver tumours from HBsAg carriers (Brechot
able interval between the initial virus infection and et al., 1980; Chen et al., 1986). An example is illus-
development of HCC, although the tumour does trated in Figure 3.13, lane D2. It is possible that
occur in younger age groups in high-risk popula- more sensitive techniques, such as the PCR, may
tions. enable the detection of viral DNA sequences in
The relative risk of an HBsAg carrier, compared tumours which test negative by hybridization. In
to a matched non-carrier, developing primary liver most cases, the tumours seem to be clonal with
cancer was estimated in an elegant prospective respect to the integrated viral DNA and may have
study carried out in Taiwan by Beasley and arisen from a single cell. There is, however, con-
coworkers. Over 22 000 men, including more than siderable variation between tumours in terms of the
3000 HBsAg carriers, were followed for 75 000 man- number of integrants and their location. Although
years. The HBsAg carriers proved more than HBV DNA is often found integrated into Alu or
200—fold more likely to develop HCC than mem- other satellite sequences, this is likely to reflect the
bers of the non-carrier group and more than 50% of size of such targets and it seems that the sites of
the deaths in the former group were due to the integration in the cellular chromosomes are ran-
tumour or to cirrhosis, another possible conse- dom.
quence of long-term HBV infection (Beasley and Nucleic acid sequencing of the junctions between
Hwang, 1991). viral and cellular DNA shows that the direct re-
peats in the virus genome (see the account of the
replication of the viral genome above) frequently
HBV DNA in Primary Liver Tumours
are located close to these junctions and may be ‘hot
If HBV does indeed play a causal role in the devel- spots’ for recombination with host DNA. Since syn-
opment of primary liver cancer then the tumours thesis of progeny viral DNA in infected cells is
might be expected to contain virus-specific nucleic cytoplasmic, it is likely that it is an intermediate(s)
acid and perhaps also to express viral proteins. The in the process of conversion of virion DNA, or
first cell line to be established from a primary liver progeny HBV DNA recycled from the cytoplasm to
tumour from an HBsAg carrier (PLC/PRF/5) pro- the nucleus, to covalently closed circular DNA
ved to secrete in culture HBsAg in the form of 22 nm which is involved in the recombination.
particles with a morphology similar to those found
in the plasma of persistently infected individuals.
Some other HCC-derived cell lines are HBsAg posi-
Animal Models
tive but many are negative. Similarly, immunohis-
tochemistry of HBV-associated tumours shows that Infection of eastern woodchucks (Marmota monax)
a minority produce HBsAg. Production of HBcAg with WHV, which is phylogenetically related to
by tumours seems to be rather less common. HBV, may progress to chronic hepatitis and pri-
Following the molecular cloning of the HBV mary liver cancer. WHV DNA may be found integ-
genome, which made available hybridization rated in the tumours and, in several cases, has been
probes of high specific activity, the PLC/PRF/5 cell shown to be involved with the rearrangement and
line was shown to contain HBV DNA integrated altered expression of endogenous myc oncogenes,
chromosomally at several sites (Figure 3.13, lane including a pseudogene which is not present in the
A1). Further analysis reveals considerable rearran- human genome (Wei et al., 1992). It is not clear what
gement of the viral DNA, although it is not clear role environmental cocarcinogens may play in the
whether these rearrangements were present in the development of primary liver tumours in patients
original tumour or occurred during the establish- (see below) but, by careful control of diet, it has been
ment of the cell line (the genotype of the cultured possible to demonstrate that WHV infection is suffi-
cells appears to be stable with respect to the integ- cient to produce tumours in woodchucks in the
rated HBV sequences). Other cell lines derived from absence of dietary cofactors.
218 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
tumours has not been forthcoming. Expression of
Mechanisms of Oncogenesis
the core antigen in tumours appears to be relatively
Because primary liver cancer most often develops in rare, and there are a number of possible explana-
a liver which is affected by chronic hepatitis or tions for this observation. Firstly, the beginning of
cirrhosis (or both), it has been suggested that the the core antigen gene is near to one of the direct
involvement of HBV is mediated through these repeats which appear to be involved in the integra-
pathological changes and subsequent regeneration. tion process, so these sequences may often be dis-
However, it is clear that HCC is much more likely rupted. Secondly, cellular control mechanisms such
to develop in an HBV-infected cirrhotic liver than as DNA methylation may prevent transcription of
in the cirrhotic liver of an uninfected patient and the core gene. Finally, expression of the core gene
that tumours often develop in the livers of HBV- may lead to presentation of HBeAg on the cell
infected patients without an intermediate cirrhotic surface and targeting of the cell for lysis by the
stage. The common finding of integrated viral DNA immune system. This may effectively select clones
in tumours implies a more direct role of viral on- with rearrangements of integrated DNA for sur-
cogenesis. However, the fact that up to 20% of vival. The tumour cells do not seem to support virus
tumours from HBsAg-carriers may be negative for replication (except perhaps in very early tumours)
viral DNA suggests that other mechanisms some- and this may also give a growth advantage in the
times may be involved. virus-infected liver.
Integrated viral DNA also may be detected in the One mechanism whereby viruses cause neoplas-
livers of some HBsAg carriers without tumour, both tic transformation of cells is via the expression of a
in patients with ongoing virus replication and in transforming gene introduced in the integrated viral
those who have cleared replicating virus. The hy- genome. The HBV genome does not seem to con-
bridization techniques used are able to detect these tain such a gene and it has not been possible to
integration events only if they occur at the same site transform cells in vitro using virus or viral DNA.
in many cells and, since sites of integration in the The long interval between virus infection and tu-
chromosomes appear to be random, this implies the mour development also argues against such a direct
clonal expansion of a cell with integrated viral mechanism. Nevertheless, the so-called X gene of
DNA. The establishment of such clones in the liver HBV has been shown to encode a transcriptional
may be the first step in a multistage process leading transactivator and the possibility that this protein
to carcinoma and there may be a role for other plays a role in disrupting the normal transcriptional
environmental factors (such as mycotoxins in the control of the cell cannot be ruled out. Transactiva-
diet) in such a process. The long interval often seen tion by the X protein seems to be through respon-
between the initial virus infection and tumour de- sive elements such as the transcription fector AP-1
velopment fits with this concept. Furthermore, (jun/fos), AP-2, NF
B and the CRE site. It has been
since integration appears to occur repeatedly shown that truncated pre-S proteins also may have
throughout the period of the virus replication, the transactivating properties. These may be produced
continuing accumulation of preneoplastic clones in tumour cells by expression of integrated HBV
within the liver would increase the probability of DNA with virus—host junctions in the surface ORF.
progression to tumour in patients with long-term A second mechanism of viral oncogenesis is
chronic infection. known as insertional mutagenesis. Here, integra-
Production of HBsAg in the livers of carriers who tion of the viral genome results in the introduction
have cleared virus replication, as well as by some of an enhancer sequence or promoter (as discussed
tumours, indicates that the surface mRNA may be above) or may physically disrupt a cellular gene.
actively transcribed from integrated viral DNA. Although this hypothesis is attractive for HBV,
The activity of this promoter makes attractive the screening of HBV-associated primary liver tumours
promoter insertion hypothesis that aberrant tran- with a number of oncogenes as probes has not
scription of cellular genes may result in loss of proved fruitful. In one tumour, the HBV genome
growth control. In fact, one such RNA transcript in has been found to be integrated into the gene for a
the PLC/PRF/5 cell line is a hybrid of viral and retinoic acid receptor (Dejean et al., 1986) and, in
cellular sequences. However, despite the attractions another, a cyclin A gene was involved (Wang et al.,
of this hypothesis, supportive data from analysis of 1990). However, these are isolated instances and it
HEPATITIS VIRUSES 219
seems that a common mechanism of oncogenesis tients who have received liver transplants. Hepa-
via insertional mutagenesis is extremely unlikely. tocytes in the graft become infected with HDV cir-
Despite this caveat, the evidence that HBV plays culating at the time of transplantation. In the ab-
a causative role in tumour development remains sence of HBsAg, there is no cell to cell spread of the
convincing. It is possible that further research will virus but HDV replication persists in isolated hepa-
uncover more of the mechanisms involved and tocytes.
some of these may have ramifications in the field of
human oncology in general. However, many re-
searchers are now focusing on the roles of the X
Epidemiology
gene product and other transactivators of transcrip-
tion and of concomitant changes in the gene for p53.
Limited serological studies indicate a worldwide
Meanwhile, mass vaccination campaigns in many
distribution of hepatitis D in association with HBV.
countries are aimed at preventing the spread of the
The infection is epidemiologically important in
virus to the next generation. Already, there is evi-
Italy, the Middle East (the Gulf States and Saudi
dence that the incidence of primary liver cancer in
Arabia), parts of Africa and South America (Riz-
children in Taiwan has fallen markedly (Chang et
zetto, 1996). It has been estimated that 5% of
al., 1997).
HBsAg carriers worldwide (approximately 15 mil-
lion people) are infected with HDV. In areas of low
prevalence of HBV, those at risk of hepatitis B,
particularly intravenous drug users, are also at risk
HEPATITIS D
of HDV infection. Epidemics with high mortality
have been described in South America in associ-
Delta hepatitis was first recognized following detec-
ation with severe hepatitis B. Delta infection is asso-
tion of a novel protein, delta antigen (HDAg), by
ciated with acute and chronic hepatitis, always in
immunofluorescent staining, in the nuclei of hepa-
the presence of hepatitis B, and superinfection in a
tocytes from patients with hepatitis B (Rizzetto et
carrier of HBV often leads to exacerbation of severe
al., 1977). HDV is now known to be defective and
hepatitis.
require a helper function from HBV for its trans-
The mode of transmission of hepatitis D is similar
mission. HDV is coated with HBsAg, which is
to parenteral spread of hepatitis B, so serological
needed for release from the host hepatocyte and for
evidence of infection is found most frequently in
entry in the next round of infection. The agent is
western Europe and North America in multiply
unique among human viruses and consists of a
transfused individuals, such as patients with hae-
particle measuring 35—37 nm in diameter, with an
mophilia, and in drug addicts.
internal nucleocapsid comprising the genome sur-
rounded by the delta antigen and enveloped by an
outer protein coat of HBsAg. The genome consists
of a single-stranded, circular RNA of around 1700 Structure and Replication of HDV
nucleotides, the delta antigen being encoded by
antigenomic RNA (reviewed by Taylor, 1997). The HDV particle is approximately 36 nm in diam-
Two major modes of delta hepatitis infection are eter and composed of an RNA genome associated
known (Hadziannis, 1997). In the first, a susceptible with HDAg, surrounded by an envelope of HBsAg.
individual is coinfected with HBV and HDV, often The virus reaches higher concentrations in the cir-
leading to a more severe form of acute hepatitis culation than HBV, up to 10 particles per mil-
caused by HBV. Vaccination against HBV prevents lilitre have been recorded. The HDV genome is a
such infections. In the second, an individual chroni- closed circular RNA molecule of 1679 nucleotides
cally infected with HBV becomes superinfected with with extensive sequence complementarity that per-
HDV. This may accelerate the course of the chronic mits pairing of approximately 70% of the bases to
liver disease and cause overt disease in asympto- form an unbranched rod structure. The genome
matic HBsAg carriers. HDV may be cytopathic, thus resembles those of the satellite viroids and
and HDAg directly cytotoxic. A less common type virusoids of plants and, similarly, seems to be rep-
of infection has been seen in HDAg-positive pa- licated by the host RNA polymerase II with
220 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
autocatalytic cleavage and circularization of the Pathogenesis
progeny genomes via trans-esterification reactions
(ribozyme activity) (Lai, 1995; Lazinski and Taylor, The pathogenesis of the disease is uncertain. It has
1995). Consensus sequences of viroids, which are long been held that HDV is pathogenic and that the
believed to be involved in these processes, also are liver injury in hepatitis D is related to HDV itself.
conserved in HDV. These data have recently been challenged with the
Unlike the plant viroids, HDV codes for a pro- observation that HDV reoccurs in liver trans-
tein, HDAg, in an ORF in the antigenomic RNA. planted patients soon after grafting but without
Around 600 copies of a polyadenylated mRNA, signs of HBV recurrence or evidence of liver dam-
approximatey 800 nt in length, may be detected in age. In these individuals, HDV may establish latent
the cytoplasm of infected hepatocytes. The antigen, infection that is not dependent upon HBV for repli-
which contains a nuclear localization signal, was cation and which is only associated with recrudes-
originally detected in the nuclei of infected hepa- cent liver injury after the acquisition of HBV. Hepa-
tocytes and may be detected in serum only after titis D virions cannot be released from the infected
stripping off the outer envelope of the virion with hepatocytes without an envelope supplied by HBV.
detergent. The delta antigen is detectable in two Fulminant hepatitis may occur in acute HDV
forms in the infected hepatocyte. The 195 aa (small) and HBV infection, and outbreaks of severe hepati-
form is required for HDV RNA replication and tis have been reported in Indians of the Amazon
binds to the rod-like structures of the genome and basin and in areas of Central Africa. Degenerative
genome complement. The larger (214 aa) form, changes were observed in these patients, character-
which is structural, and therefore required for virion ized by fine steatotic vacuolization of hepatocytes,
assembly, seems to be synthesized following RNA in keeping with a cytotoxic inflammatory lesion.
editing. This process converts the termination It is known that hepatitis D may result in interfer-
codon at the end to the ORF for the short form to a ence of HBV replication but the molecular mechan-
tryptophan codon, resulting in a 19 aa C-terminal ism has not been established. An increase in HDV
extension (Casey and Gerin, 1995). replication has been noted in concurrent HDV and
HIV replication, but this may not necessarily cause
more severe hepatitis (Buti et al., 1991). There is also
a large body of data to suggest that the pathogen-
Laboratory Diagnosis of HDV Infection esis of of HDV hepatitis is in part immunologically
mediated.
Specific serological tests are available to detect anti-
body to HDV; anti-HD IgM and anti-HD IgG, and
for HDV RNA and HDAg. Coinfection and
superinfection can be distinguished by correlation Clinical Features
of the results of these tests with those for markers of
HBV infection. Thus, in coinfection, HBsAg, Hepatitis D causes acute, fulminant and chronic
HBeAg and HBV DNA become detectable in serum hepatitis, either as a coinfection with hepatitis B or
along with HDAg and HDV RNA. Coexistence of as a superinfection in patients with chronic hepatitis
anti-HBc in IgM with markers of HDV infection is B. Clinically there is a spectrum of disease, and in
a reliable indication of coinfection; anti-HD IgM some coinfected or superinfected persons HDV ap-
becomes detectable, followed by anti-HD IgG. pears to be a pathogenic agent, and to aggravate the
Markers of virus replication usually become unde- underlying HBV infection. There is much interest in
tectable during convalescence. the study of pathogenicity caused by this agent.
Superinfection of HBV carriers with HDV results
frequently in persistent HDV infection. HD vir-
aemia is followed by an anti-HD IgM, and then Treatment
IgG, response. Markers of HBV replication may be
suppressed during acute HDV infection. Anti-HD A number of investigators have evaluated inter-
IgM persists with HDAg and HDV RNA in serum feron treatment of chronic type D hepatitis. HDV is
in chronic delta hepatitis. sensitive to interferon, and doses of 3—10 million iu
HEPATITIS VIRUSES 221
three times weekly for 6 months result in repression al., 1985; He et al., 1987). These studies made avail-
of hepatitis D and amelioration of biochemical able a pool of plasma known to contain a relatively
signs of hepatitis, with a decrease in serum amino- high titre of the agent. In order to clone the genome,
transferases (Rizzatto et al., 1986; Saracco et al., the virus was pelleted from the plasma. Because it
1989; Rosina et al., 1991). Coincident with this de- was not known whether the genome was DNA or
crease, HDV RNA disappears from serum in ap- RNA, a denaturation step was included prior to the
proximately 50% of cases. Unfortunately many pa- synthesis of complementary DNA so that either
tients relapse when treatment is stopped. DNA or RNA could serve as a template. The result-
ing cDNA was then inserted into a bacteriophage
expression vector and the libraries screened using
Vaccination serum from a patient with chronic non-A, non-B
hepatitis.
Vaccination against hepatitis B prevents HDV in- This approach led to the detection of a clone
fection. Active immunization with HDV synthetic (designated 5—1—1) which was found to bind to anti-
or expressed gene products may modulate the infec- bodies present in the sera of several individuals
tion in woodchuck models (Karayiannis et al., infected with non-A, non-B hepatitis (Choo et al.,
1993). 1989). This clone was used as a probe to detect
larger, overlapping clones in the same library. It
was possible to demonstrate that these sequences
hybridized to a positive-sense RNA molecule of
HEPATITIS C around 10 000 nt which was present in the livers of
infected chimpanzees but not in uninfected controls.
Hepatitis C Virus is Responsible for No homologous sequences could be detected in the
Almost All Cases of Parenterally chimpanzee or human genomes. By employing a
Transmitted Non-A, Non-B Hepatitis ‘walking’ technique, it was possible to use newly
detected overlapping clones as hybridization
The specific diagnosis of hepatitis types A, B and D probes in turn to select further virus-specific clones
revealed a previously unrecognized form of hepati- from the library. Thus, clones covering the entire
tis which was clearly unrelated to any of these three viral genome were assembled and the complete nuc-
types. Results obtained from several surveys of leotide sequence determined. The organization of
post-transfusion hepatitis in the USA and elsewhere the genome (Figure 3.14) closely resembles those of
provided strong epidemiological evidence of ‘guilt the enveloped RNA viruses, the pestiviruses and
by association’ of an infection of the liver referred to flaviviruses, as discussed below.
as non-A, non-B hepatitis (Alter et al., 1988). This
was the most common form of hepatitis occurring
after blood transfusion in some areas of the world
following the introduction of tests for HBsAg. Stu- Organization of the HCV Genome
dies also showed that this infection was common in
haemodialysis and other specialized units, that it The genome of HCV (Figure 3.14) resembles those
occurs in a sporadic form in the general population of the pestiviruses and flaviviruses. It comprises
and that it can be transmitted by therapeutic around 9400 nt of positive-sense RNA, lacks a 3
plasma components, and that a prolonged carrier poly(A) tract and has a similar gene organization
state in the blood may occur. There was also con- (Choo et al., 1991). It has been proposed that HCV
siderable evidence that the parenterally transmitted should be designated the prototype of a third genus
infection, like hepatitis B, may become persistent in the family Flaviviridae, hepacivirus. All of the
and progress to chronic liver disease, cirrhosis and viral genomes from this family contain a single large
hepatocellular carcinoma. ORF which is translated to yield a polyprotein (of
Transmission studies in chimpanzees helped es- around 3000 amino acids in the case of HCV) from
tablish that the main agent of parenterally acquired which the viral proteins are derived by post-transla-
non-A, non-B hepatitis was likely to be an envel- tional cleavage and other modifications.
oped virus with a diameter of 30—60 nm (Bradley et There is a short, untranslated region at the 5 end
222 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 3.14 Organization of the hepatitis C virus genome. See text for details.
of the genomic RNA and a further untranslated lates of the virus, and divergence seems to be driven
region at the 3 end, the large ORF accounting for by the generation of neutralizing antibodies which
over 95% of the sequence. The structural proteins are targeted to that domain of E2. This hypothesis
are located towards the 5 end and the non-struc- is supported by the observation that much less
tural proteins towards the 3 end. The first product variability occurs in agammaglobulinaemic indi-
of the polyprotein is the non-glycosylated capsid viduals. Thus, efforts to develop hepatitis C vac-
protein, C, which complexes with the genomic RNA cines are hampered by variability of the most obvi-
to form the nucleocapsid. As with the flaviviruses, a ous target molecule and the efficacy of candidate
hydrophobic domain may anchor the growing vaccines is likely to be hampered by the rapid evol-
polypeptide in the endoplasmic reticulum and fa- ution of antibody escape mutants.
cilitate cleavage by a cellular signalase, releasing a The non-structural region of the HCV genome is
nucleocapsid precursor (anchored C). The amino divided into regions NS2 to NS5 (Figure 3.14). In
acid sequence of the nucleocapsid protein seems to the flaviviruses, NS3 has two functional domains, a
be highly conserved among different isolates of protease which is involved in cleavage of the non-
HCV. structural region of the polyprotein and a helicase
The next domain in the polyprotein also has a which is assumed to be involved in RNA replica-
signal sequence at its C-terminus and may be pro- tion. Motifs within this region of the HCV genome
cessed in a similar fashion. The product is a glycop- have homology to the appropriate consensus se-
rotein, E1 or gp35, which is probably found in the quences, suggesting similar functions. The HCV
viral envelope. The third domain may be cleaved by protease, which uses NS4a as a cofactor, is a major
a protease within the viral polyprotein to yield a target of efforts to develop specific antiviral agents.
second surface glycoprotein, E2 or gp70. Alterna- Unlike the flaviviruses, HVC encodes a second pro-
tive cleavage sites at the C-terminus of E2 may tease activity: sequences at the NS2—NS3 junction
generate a small addition protein, p7. These glycop- are responsible for self-cleavage of that site. The
roteins have not been visualized in vivo and the HCV NS5, unlike that of the flaviviruses, is cleaved
molecular sizes are estimated from sequence data to yield NS5a and NS5b. NS5b contains the Gly-
and expression studies in vitro. These envelope pro- Asp-Asp (GDD) motif common to viral RNA-de-
teins are the focus of considerable interest as poten- pendent RNA polymerases and so is likely to be the
tial targets in tests for the direct detection of viral HCV replicase, but NS5a also may be involved in
proteins and for anti-HCV vaccines. genome replication.
As with many other RNA viruses, replication of
the HCV genome is an error-prone process and the
resulting mutations lead to the generation, within
an infected individual, of a population of viruses Diagnosis of HCV Infection
with closely related, but different, nucleotide se-
quences. This has been termed the ‘quasispecies Cloning of the HCV genome made possible the
effect’. The effect is particularly noticable in the development of specific diagnostic tests including
region of the genome (the hypervariable region, enzyme immunoassays (EIAs) for antibody and RT-
HVR-1) which encodes a domain at the N-terminus PCR for viraemia. Since the 5—1—1 antigen orig-
of E2. Sequences within this region are highly vari- inally was detected by antibodies in the serum of an
able within each individual, as well as between iso- infected patient, it was an obvious candidate for
HEPATITIS VIRUSES 223
development of an EIA to detect anti-HCV antibo- tection of the primary product by Southern hybrid-
dies. A larger clone, C100, was assembled from a ization is required. There is considerable variation
number of overlapping clones and expressed in in nucleotide sequences among different isolates of
yeast as a fusion protein using human superoxide HCV and the 5 non-coding region, which seems to
dismutase sequences to facilitate expression. This be highly conserved, is the preferred target for diag-
fusion protein formed the basis of first-generation nostic PCR (Garson et al., 1991). Hepatitis C viral
tests for HCV infection (Kuo et al., 1989). It is now load may be estimated by quantitative RT-PCR or
known that antibodies to C100 are detected rela- using a hybridization assay based on branched
tively late following an acute infection. Further- oligonucleotides (bDNA assay).
more, the first generation assays were associated
with a high rate of false positivity when applied to
low incidence populations, and there were further Interpretation of Serological Tests for HCV
problems with some retrospective studies on stored A negative EIA test is sufficient to rule out infection
sera. Data based on this test alone should, therefore, in individuals, such as blood donors, without risk
be interpreted with caution. factors for HCV infection. For those of low risk, a
Second- and later-generation tests include anti- positive EIA requires confirmation and a supple-
gens from the nucleocapsid and further non-struc- mentary assay for antibodies, such as RIBA, is valu-
tural regions of the genome. The former (C22) is able. Where the RIBA is negative, the EIA result is
particularly useful: the sequence of the HCV core likely to have been a false positive. If the RIBA is
protein is relatively highly conserved, compared to positive, the patient is likely to have (or to have had)
other HCV proteins, and antibodies appear rela- hepatitis C. RT-PCR may then be used to deter-
tively early during infection. Supplementary tests mine whether the patient is viraemic. Follow-up
involving several viral antigens bound to a solid RT-PCR also is indicated where the result of the
substrate, recombinant immunoblot assays (RIB- RIBA is indeterminate.
As), are available and give a more detailed evalu- Individuals with even slightly raised serum trans-
ation of the antibody profile of the patient. Anti- aminases should be tested for anti-HCV by EIA and
body tests based on synthetic peptides also are any positive results confirmed by RIBA or assay for
available. HCV RNA. In patients with biochemical or clinical
Routine testing of blood donations is now in evidence of liver disease, a positive EIA is sufficient
place in many countries and prevalence rates vary to diagnose hepatitis C, and testing for HCV RNA
from 0.2—0.5% in northern Europe to 1.2—1.5% in is valuable for confirmation. Quantitative assays for
southern Europe and Japan. Many of those who are HCV RNA are valuable in monitoring the efficacy
found to be antibody positive have a history of of antiviral therapy and may help predict long-term
parenteral risk, such as transfusion or administra- outcome.
tion of blood products or intravenous drug use.
There is minimal evidence for sexual or perinatal
transmission of HCV and it is not clear what are the
‘natural’ routes of transmission. Persistence of HCV Infection is
The availability of the nucleotide sequence of Common
HCV made possible the use of the PCR as a direct
test for the (genome of the) virus itself. The first step Current data suggest that at least 50%, and perhaps
is the synthesis of a complementary DNA copy of more than 80%, of infections with HCV progress to
the target region of the RNA genome using reverse chronicity. Thus, in contrast to HBV infection,
transcriptase (RT) primed by the antigenomic PCR where persistent infections of immunocompetent
primer or random hexadeoxyribonucleotides. The adults are rare, persistent HCV infection seems to
product of this reaction is a suitable target for am- be the norm. The morbidity of chronic hepatitis C is
plification (RT-PCR). The concentration of virus in affected by many interactive factors, including age
serum samples is often very low so that the mass of of acquisition, concomitant alcohol abuse, gender,
product from the PCR reaction is insufficient for coexisting viral disease and the host immune re-
visualization on a stained gel. Therefore, a second sponse.
round of amplification (with nested primers) or de- Histological examinations of liver biopsies from
224 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
‘healthy’ HCV carriers (blood donors) reveal that genotypes (1a, 1b, 2a, 2b, 3a, 4a, 5 and 6), so there is
none has normal histology and that up to 70% have little evidence so far for variants of HCV that are
chronic active hepatitis and/or cirrhosis (Esteban et completely non-pathogenic.
al., 1991). It is not clear whether pathological
changes result from direct cytopathology of the vi-
rus or are mediated by the immune response to Treatment of Hepatitis C
infection. HCV infection also is associated with pro-
gression to HCC. For example, in Japan, where the Alpha Interferon
incidence of HCC has been increasing despite a
decrease in the prevalence of HBsAg, HCV is the A considerable number of large, controlled thera-
major risk factor. There is no DNA intermediate in peutic trials of -interferon have been undertaken in
the replication of the HCV genome or integration of acute and chronic hepatitis C infection. Treatment
viral nucleic acid, and viral pathology may contrib- is indicated for acute HCV infection because of the
ute to oncogenesis through cirrhosis and regener- propensity of this virus to cause chronic disease;
ation of liver cells. HCV infection rarely seems to controlled trials indicate that treatment of the acute
cause acute liver failure. infection lessens the risk of chronic disease. It is not
always clear whether treatment has benefited those
patients who might have been convalescing sponta-
neously. However, if a diagnosis of acute hepatitis C
Genotypes can be made, and the patient does not appear to be
convalescing 2—4 months after the onset of the dis-
The tremendous variation in the sequence of the ease, i.e. HCV RNA remains detectable, -inter-
genomes of various isolates of HCV has led to their feron is recommended at a dose of 3—6 million iu
classification into types and subtypes (Simmonds et thrice weekly for 12 months, or -interferon and
al.,1994) and up to 11 major genotypes are recog- ribavirin for 6 months.
nized. There is some evidence of variation between
genotypes of viral pathogenicity and responsiveness
Chronic Hepatitis C
to treatment (see below) but data are incomplete for
most genotypes and other variables, such as the age There is evidence that alcohol and hepatitis C may
of the patient and the duration of infection, con- act synergistically in causing hepatic injury, and the
found interpretation. drinking of excess alcohol is discouraged because of
Infection with types 1b and 1a are relatively this evidence (Miyakawa et al., 1993; Sawada et al.,
common in Europe; infection with type 1b is fre- 1993). The patient should be advised not to donate
quent in southern Europe. Epidemiological dif- blood. Patients can be told that the parenteral route
ferences in age distribution of major types, and the is the most important route of transmission and
risk factors associated with particular genotypes, that the virus is not easily transmitted except by this
have become apparent. In Europe, types 3a and 1a route.
are relatively more common in young individuals Alpha interferon is indicated for the treatment of
with a history of intravenous drug use. Type 1b patients with chronic hepatitis C infection who are
accounts for most infections in those aged 50 or HCV RNA positive and have elevated serum
more. Type 4 infection is the most prevalent infec- aminotransferases with histologically moderate
tion in Egypt, and many parts of the Middle East chronic hepatitis or more advanced lesions (Anony-
and Africa. mous, 1997; Hoofnagle and Tralka, 1997). Thus a
Although an inherently greater pathogenicity of liver biopsy generally is required. There is currently
type 1 HCV has been implied, these studies have not no substitute for liver biopsy to ascertain the grade
always been based on prospective follow-up or ap- (severity and extent of hepatic inflammation) or
propriately controlled to account for influences of stage the disease (fibrosis).
several interdependent parameters and cofactors. Serum aminotransferases, bilirubin, alkaline
Moreover, several clinical investigations have phosphatase and prothrombin time should be
documented severe and progressive liver disease measured. In patients whose lifestyle or geographic
after infection with each of the well-characterized origin suggest that they are at risk of other viral
HEPATITIS VIRUSES 225
infections, HBsAg and HIV infection need also be spond, although long-term responsiveness is uncer-
considered. It is not easy to chart the prognosis for tain. This is of particular importance in liver trans-
patients seen at one point in time, however, as the plant patients.
disease progresses at variable rates in individual Within genotypes, response rates may be higher
patients. Treating relatively mild, early disease in for patients with lower virus concentrations and less
younger patients to prevent cirrhosis is a rational advanced disease. Younger patients and patients
objective. without fibrosis or cirrhosis are also more respon-
Four -interferons have been studied in large sive to therapy (Chemello et al., 1995). The mechan-
controlled trials of -interferon. Several, including isms by which different genotypes might differ in
HuIFN--N1 (Wellferon), rIFN--2b (Intron A) responsiveness to treatment remain obscure. Pa-
and rIFN--2a (Roferon-A) and consensus inter- tients with diverse circulating quasispecies may be
feron have been evaluated for the treatment of chro- less responsive to therapy than those with a single
nic hepatitis C. In the large placebo-controlled stu- major species. Unfortunately, the issue remains
dies approximately 50% of patients with chronic complex: there is not yet a standardized system or
hepatitis C have an initial response to 3 million iu nomenclature for quantitating concentrations of
three times a week (Davis et al., 1989; Di Bisceglie et HCV RNA in serum. Also, these factors may be
al., 1989). Serum ALT concentrations generally are interdependent.
used to assess response, and if these have not be- Patients with normal ALT prior to treatment
come normal in 3 months, then treatment is stop- may develop an exacerbation after failed interferon
ped, as most responsive patients will respond within therapy. The mechanism is unknown but could be a
this period. However, after stopping treatment after result of displacement of interferon sensitive clones.
6 months, approximately one half of the responsive Recent molecular evidence indicates that an inter-
patients will relapse. Between 15 and 25% of pa- feron sensitive region can be defined in the NS5a
tients have sustained virological responses and be- region in patients with genotype 1b infection. Pa-
come HCV RNA negative. Responsive patients tients with more amino acid substitutions in the
usually show histological improvement. It is rea- NS5A gene (region 2209—2248) were more likely to
sonable to infer that those patients who, a year after have a complete response than patients with wild
stopping interferon treatment, have normal serum type 1b (Enomoto et al., 1996).
ALT and are negative for HCV RNA by sensitive Because autoimmune hepatitis is treated differ-
PCR, have responded to therapy, but long term ently, and may be aggravated by interferon therapy,
studies to ascertain the effect on survival have not it is particularly advisable to exclude this diagnosis
been done. The majority of patients who relapse do by measuring the titres of anti-smooth muscle and
so within 6 months of stopping treatment. Serum anti-liver kidney microsomal antibodies, even in
aminotransferases usually increase in patients who those with a positive anti-HCV test, and to measure
are HCV RNA positive at the end of therapy, al- HCV RNA in anti-HCV-positive patients in whom
though in some cases the relapse may be delayed for -interferon is contemplated.
several months (Chayama et al., 1991). Prolonging
therapy for 1 year to 18 months is now recommen-
Ribavirin
ded. Higher (induction) doses may be beneficial, but
are associated with a greater incidence of side- Ribavirin is a synthetic guanosine nucleoside ana-
effects (Kakumu et al., 1990). logue, which possesses a broad spectrum of activity
Unfortunately, responsiveness to -interferon re- against both DNA and RNA viruses in vitro and in
mains somewhat unpredictable. Factors which pre- vivo (Fernandez et al., 1986). Efficacy has been dem-
dict a greater likelihood of response are now being onstrated in several viral diseases. The drug exerts
studied. Multivariate analyses of several pretreat- its action after intracellular phosphorylation to
ment parameters indicate that patients without cir- mono-, di- and triphosphate nucleotides. The pre-
rhosis, with genotype 2 and 3 infection and with cise mode of action probably includes pertubation
lower levels of viraemia are more likely to respond of intracellular nucleoside triphosphate pools, in-
(to 6 months treatment) than patients with geno- terference with the formation of the 5 cap structure
type 1 (and perhaps type 4) infection. Immunosup- of viral mRNA by competitive inhibition of both
presed patients and patients with HIV may re- guanyltransferase and methyltransferase capping
226 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
enzymes (although this may not be relevant for feron- Hepatocellular Carcinoma Study Group,
HCV), direct inhibition of the viral RNA—poly- 1998). The risk reduction was apparently greater for
merase complex and, possibly, enhancement of patients with chronic hepatitis C and no evidence of
macrophage inhibition of viral replication. infection with HBV.
The major side-effects of the drug that have been
reported include anaemia, a metallic taste, dry
mouth, flatulence, dyspepsia, nausea, headaches, ir-
ritability, emotional lability, fatigue, insomnia, skin Ribavirin and Interferon
rashes and myalgia. Mild reversible anaemia is Two large studies have been undertaken to assess
common. Modest increases in uric acid have been the effect of ribavirin and -interferon combined,
reported. Ribovirin is also teratogenic and contra- versus -interferon alone, in naive and relapsed pa-
indicated in pregnancy, patients should not con- tients. Current trials indicate that approximately
ceive for at least 6 months after stopping therapy.
45—50% of patients who have relapsed after -inter-
feron have sustained biochemical and virological
Studies of Ribavirin in Hepatitis C Infection responses to combined therapy. The mechanism of
apparent synergism is not yet understood (Lai et al.,
Small pilot studies indicated that ALT and aspar- 1996; Schalm et al., 1996, 1997; Bizollon et al., 1997;
tate aminotranferase (AST) declined in patients Lindsay 1997; Reichard et al., 1997). Approximately
treated with ribavirin, but both returned to pret- 35% of nave patients respond to a combination of
reatment levels when treatment was stopped. In an ribavirin and -interferon.
open label study in Sweden, in which ribavirin was
prescribed to 10 patients with chronic hepatitis C
(1000—1200 mg per day) for 12 weeks, the median
serum AST levels declined but rose to pretreatment
levels upon completion . A small study, using esca- Prevention of Hepatitis C
lating doses of ribavirin (600—1200 mg) showed a
somewhat slower fall in serum aminotransferases, There are no vaccines available to protect contacts
perhaps reflecting the lower starting dose of rib- of patients with hepatitis C. However, secondary
avirin (Di Bisceglie et al., 1992). There was a signifi- transmission should be relatively easy to prevent.
cant decrease in geometric mean titres of HCV The role of intrafamilial transmission requires clari-
RNA. Several placebo-controlled trials have in- fication, but is relatively infrequent. Sexual trans-
dicated that, despite the significant improvement in mission is possible and has been described, but this
serum ALT, a marked decline in serum HCV con- fortunately is a relatively inefficient and infrequent
centrations does not occur. Most patients relapse route. Young sexually active adults could be told of
when therapy is stopped (Dusheiko et al., 1996; the advisability of condom use in general for casual
Bodenheimer et al., 1997). sexual contact.
Mother-to-infant transmission has been ob-
served, but appears to be unusual. Differences in the
Interferon Treatment of Cirrhosis
rate of maternal—infant transmission in different
Alpha interferon treatment should be targeted to countries remain unexplained, and the importance
patients before the development cirrhosis, as pa- of this route in perpetuating the reservoir of human
tients are at risk of developing sequelae once cirrho- infection is unknown, but could be relevant. Ma-
sis has developed. Several small trials have sugges- ternal—infant transmission is more likely in mothers
ted that interferon therapy improves liver function with HCV RNA concentrations higher than 10
and reduces the incidence of HCC in patients with genomes per millilitre. Transmission from infected
cirrhosis due to hepatitis B or C (Nishiguchi et al., surgeons to their patients has now been
1995). A recent, retrospective analysis of data for documented and verified by molecular epi-
913 patients with chronic viral hepatitis and cirrho- demiological evidence (Esteban et al., 1996). It is
sis from Italy and Argentina showed that interferon unknown whether higher levels of viraemia and
treatment lowered the rate of progression to hepa- particular forms of surgery are more likely to trans-
tocellular carcinoma twofold (International Inter- mit infection.
HEPATITIS VIRUSES 227
GB AND ‘HEPATITIS G’ VIRUSES tempts were made to clone the genome of the puta-
tive viral agent using immunoscreening (gt11 ex-
The discovery of HCV and HEV and their roles in pression libraries) and representational difference
causing parenterally transmitted, epidemic and analysis (RDA). The genomes of two distinct, but
sporadic cases of non-A, non-B hepatitis does not related, viruses were present in the cloning source
rule out the possibility that there are other, uniden- and these viruses have been provisionally desig-
tified human viruses which may cause hepatitis. nated GB viruses A and B (GBV-A and GBV-B)
Incubation times varied following transmission of (Simons et al., 1995b). The genomes are of a size
non-A, non-B hepatitis to non-human primates, (around 9.5 kb) and organizational strategy typical
suggesting the involvement of more than one agent. of the Flaviviridae and, more specifically, resemble
The results of subsequent cross-challenge experi- those of the pestiviruses and HCV.
ments seemed to support this conclusion, although Given the passage history of the cloning source, it
retrospective analyses of some of these studies im- seems possible that one, or both, of these viruses
plicate reinfection with HCV. Some sporadic cases originated from the experimental animal(s); GBV-A
of acute liver failure may be associated with a virus seems to be a representative of a number of closely
infection, candidate hepatitis F (fulminant) virus. related viruses found in New World monkeys. The
Virus-like particles were detected by electron question of whether GBV-B is the virus which
microscopy (Fagan et al., 1992) but, although other caused hepatitis in the surgeon, or is a second mon-
viruses were excluded rigorously (Fagan and Har- key virus, remains unanswered; further isolates of
rison, 1994), a viral aetiology has not been con- this virus have not been discovered in humans or
firmed by experimental transmission to susceptible other animals.
animals. Other viruses may exist which are spread
by the faecal—oral route and have a tropism for the
liver. Discovery of GBV-C and HGV
Viruses which cause acute and persistent infec-
tions of the liver may be difficult to detect, particu- Cross-reactive antibodies were detected in around
larly if those infections are asymptomatic in most 1.5% in volunteer blood donors from the USA,
individuals. A candidate recently has been de- 14.0% of intravenous drug users and 19.9% of indi-
scribed by two groups working independently and viduals from West Africa, using enzyme immunoas-
given the provisional names GB virus C (GBV-C) says based on recombinant GBV-A and GBV-B
(Simons et al., 1995a) and hepatitis G virus (HGV) proteins. The presence of highly conserved motifs in
(Linnen et al., 1996). Formal classification awaits the non-structural helicase region (NS3) allowed
the decision of the International Committee on the the development of a PCR assay with degenerate
Taxonomy of Viruses. primers which enabled amplification of GBV-A,
GBV-B and HCV. The PCR product from one of
the antibody-positive, West African sera led to clon-
ing and sequencing of a further novel virus, GBV-C.
GB agents Working independently, another research group
cloned viral cDNA from the sera of a patient with
Hepatitis viruses are notoriously difficult to grow in chronic post-transfusion hepatitis and of a
cell culture, and experimental transmission to ani- phlebotomist with intermittent rises in ALT levels,
mals, especially primates, has played an essential and named this virus hepatitis G virus (HGV, Lin-
role in their discovery. In 1967, Deinhardt described nen et al., 1996). Sequence comparisons of GBV-C
attempts to transmit to monkeys viral hepatitis and HGV confirm 85% identity at the nucleotide,
from a number of human sources (Deinhardt et al., and 95% at the amino acid, level and that they are
1967). Administration of acute phase plasma from a separate isolates of the same virus.
surgeon (with the initials GB), who suffered from
hepatitis with 4 weeks of icterus, gave rise to abnor-
mal liver function tests in all of four tamarins tested. Biology of the GB Viruses and HGV
In more recent experiments, infection was transmit-
ted to Saguinus labiatus from stored sera, and at- The structure of the genomes of all of these new
228 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
viruses is typical of the Flaviviridae and, more spe- the multiply transfused and HCV-positive individ-
cifically, is similar to HCV. The genomes are posi- uals. Antibodies to the envelope (E2) glycoprotein
tive-sense, single-stranded RNA molecules with seem to be a marker of recovery from past infection
short, untranslated regions at either end. They en- but their longevity remains to be established. GBV-
code single, large polyproteins organized with the C/HGV viraemia has been detected in patients with
structural polypeptides at the N-terminal end and a variety of disorders, including chronic liver dis-
with conserved motifs in the non-structural region ease and acute liver failure, but a causative role
which include a serine protease (NS3), RNA heli- remains unproven. Many individuals who have
case (NS3) and an RNA-dependent RNA poly- been found to be infected are symptomless, or have
merase (NS5). All three novel viruses seem to en- only mild elevations of serum transaminases as the
code two putative envelope glycoproteins, E1 and sole evidence of liver involvement. Indeed, definitive
E2, as is the case for HCV. However, while the evidence that this virus infects the liver is lacking
nucleocapsid polypeptide of GBV-B (156 amino and the site(s) of replication remains to be deter-
acids) seems roughly equivalent to that of HCV (191 mined.
amino acids), clearly discernible nucleocapsid pro- Whether GBV-C/HGV causes significant disease
teins cannot be identified in the genomes of GBV-A in a minority of those infected remains to be estab-
and GBV-C/HGV. In up to 25% of isolates of the lished. Clearly, the virus may cause persistent infec-
human virus, the first available AUG codon for tions but it is not known whether some acute infec-
initiation of translation of the long ORF is at the tions are cleared over a relatively short time frame.
beginning of the envelope glycoprotein region. It is Preliminary studies suggest that individuals who
questionable whether viruses without a nucleocap- have cleared an infection with GBV-C/HGV may
sid protein are viable and one may speculate that outnumber those persistently infected severalfold
defective viruses, with deletions towards the 5 end, but chronic infections persisting for several years
are generated during infection. Translation of the have been documented and the average duration of
ORFs of GBV-A and GBV-C/HGV involves an such chronic infections is unknown. GBV-C/HGV
internal ribosome entry site (IRES) in the 5 untran- may share a common pattern of transmission with
slated region of the genome, as is the case for HCV. HCV. Infection is common in risk groups, such as
Sequence variation among isolates of GBV-C/ intravenous drug users and the recipients of blood
HGV, including in the region encoding the surface products, including haemophiliacs and patients
glycoproteins, seems to be rather less than for HCV. with combined variable immunodeficiency. Instan-
More diverse variants of this virus may await dis- ces of transmission through transfusion have been
covery. Meanwhile, application of the term ‘geno- documented. Given their common mode of trans-
types’ to isolates of GBV-C/HGV describes a level mission, it is not surprising that coinfections may
of genetic diversity roughly equivalent to subtypes occur with HCV and GBV-C/HGV.
(variation within a genotype) of HCV. Two recent articles question further the role of
GBV-C/HGV in the aetiology of liver disease.
Among 79 patients with post-transfusion hepatitis,
only three were infected with GBV-C/HGV alone—
Clinical Significance of GBV-C/HGV they had only mild hepatitis, and serum levels of
Infection ALT and GBV-C/HGV RNA were poorly corre-
lated (Alter et al., 1997b). Furthermore, there was
Despite much effort, it has not been possible to no evidence that coinfection with GBV-C/HGV
identify recombinant antigens which react consist- worsened the outcome of HCV infection. The other
ently in immunoassays with antibodies in the sera study included an investigation of 45 patients with a
of infected individuals. Detection of the genome by diagnosis of non-A—E hepatitis (Alter et al., 1997a).
RT-PCR remains the sole reliable method for diag- Although four were positive for GBV-C/HGV
nosing viraemia. Estimates of the prevalence of RNA, the authors concluded that there was no
GBV-C/HGV infection in healthy individuals va- evidence to implicate the virus as an aetiologic
ries from 0.8% (blood donors with normal ALT agent of non-A—E hepatitis, nor was there evidence
levels in the USA) to 5.7% (Vietnam) and are much that persistent infection with GBV-C/HGV leads to
higher for those with parenteral risk factors, such as chronic liver disease. Detection of GBV-C/HGV in
HEPATITIS VIRUSES 229
the absence of other known viruses does not rule such an association (Naoumov et al., 1998; Sim-
out possible coinfection with yet undiscovered, par- monds et al., 1998).
enterally transmitted viruses. Calls for the routine
screening of donated blood for GBV-C/HGV are
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
236 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 4.1 Electron micrographs of (a) rotavirus, (b) enteric adenovirus, (c) SRSV, (d) calicivirus, (e) astrovirus, (f) enterovirus and (g)
parvovirus. (Negative staining with 3% phosphotung-state, pH 6.3; bar : 100 nm) (Astroviruses: courtesy Mr T. Lee and Dr J. Kurtz,
Oxford Public Health Laboratory; all other viruses: courtesy Dr J. Gray, Clinical Microbiology and Public Health Laboratory,
Cambridge)
the method of reverse transcription (RT)—poly- infected food (e.g. shellfish) or water (Hedberg and
merase chain reaction (PCR) and serological tech- Osterholm, 1993).
niques using recombinant antigens have greatly In essence, treatment follows guidelines estab-
helped to estimate the true extent of human infec- lished in 1985, mainly using oral rehydration fluids
tions with SRSVs and astroviruses, which was containing electrolytes and sugar (reviewed by
found to be much higher than previously thought. Bhan et al., 1994; Desselberger, 1999). Bismuth sub-
Most viral gastroenteritis infections follow two salicylate has been found to be beneficial in children
distinct epidemiological patterns. In childhood, di- wirh acute watery diarrhoea (Figueroa-Quintanilla
arrhoea occurs as endemic disease, mainly caused et al., 1993). Agents such as diphenoxylate or
by rotaviruses of group A, adenoviruses of sub- loperamide against abdominal cramping should be
group F, astroviruses and classic human cal- avoided as they can have side effects. In severe cases
iciviruses. By the age of 5 years many children have rapid fluid replacement by parenteral administra-
been infected with these agents, often without ap- tion may be required. In developing countries
parent symptoms. The main mode of transmission where children are often malnourished as well,
seems to be orofaecal, possibly also by droplets and supplementary nutrition is an important compo-
close contact. By contrast, epidemics are mainly nent of the therapy. Public health measures to con-
caused by SRSVs and sometimes by astroviruses fine outbreaks include frequent handwashing,
and group B and C rotaviruses. All ages can be proper disinfection and removal of infected faeces
affected, and viruses are quite often transmitted by and contaminated food or water, and taking infec-
VIRUSES ASSOCIATED WITH ACUTE DIARRHOEAL DISEASE 237
Figure 4.2 RNA profile (10% SDS polyacrylamide gel, silver stained), protein products (gene protein assignment) and particle
structure (reconstruction from cryoelectron micrographs) of rotavirus. (From Mattion et al., 1994; with permission of authors and
publishers)
ted individuals out of work. Outbreaks in clinical been elucidated in a number of excellent studies by
wards may require temporary closure to new ad- the groups of Prasad and Chiu (Prasad et al., 1988,
missions and restrictions on placements of staff. 1990, 1996; Shaw et al., 1993; Lawton et al., 1997a,
Viruses regularly and irregularly causing acute 1997b; Prasad and Estes, 1997) and Yeager (Yeager
gastroenteritis will be briefly described below (for et al., 1990, 1994). According to their findings the
concise reviews see Hart and Cunliffe, 1997, Dessel- double-shelled capsid is ordered in 6 -, 5— and
berger, 1998b). According to the relative degree of 3—fold symmetry axes and is perforated by 132
importance of different viruses for clinical human aqueous channels (in 3 symmetry positions). Com-
disease, rotaviruses are presented more extensively plete gene protein assignments have been estab-
than the other viruses. lished for several strains; the protein function corre-
lations are only partially known. Details on sizes of
RNA segments and their products as well as on
post-translational modification and possible func-
ROTAVIRUSES tion(s) of the viral proteins are summarized in Table
4.1. Functions are reviewed in more detail below.
Structure, Genome and Gene Protein
Assignment
Classification
Rotaviruses are the major cause of infantile gastro-
enteritis worldwide and also of acute diarrhoea in
A classification scheme for rotaviruses has been
the young of many mammalian species (calves, pig-
derived from immunological reactivities of three of
lets, lambs, fowl, etc.). Rotaviruses possess a genome
its components as well as from genomic sequence
consisting of 11 segments of double-stranded (ds)
comparisons and has been established as follows
RNA encoding six structural proteins (VP1, VP2,
(Estes, 1996):
VP3, VP4, VP6 and VP7) and five non-structural
proteins (NSP1—NSP5). The structural proteins 1. According to the serological cross-reactivity of
constitute the core or inner layer (VP1—VP3), an the inner capsid protein VP6, five groups (A—E)
inner shell or intermediate layer (VP6), and an outer have been firmly established, and two more
shell/layer (VP4, VP7) of the double-shelled/triple- groups (F,G) are likely to exist. Within group A
layered particle (Figure 4.2). The wheel-like struc- rotaviruses, there are subgroups (I; II; I ; II;
ture of the particle (Latin rota : wheel) as seen by nonI,nonII) according to their exclusive reactivi-
electron microscopy is pathognomonic (Figure 4.1). ties with two VP6—specific monoclonal antibo-
Details of the three-dimensional structure have dies.
238 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 4.1 Genes, gene protein assignments and function(s) of proteins of group A rotaviruses
Protein product
Deduced
RNA Size molecular Post-translational
Segment (bp) Designation weight (kDa) modification Location and function
2. Both surface proteins, VP4 and VP7, elicit the As VP4 and VP7 are coded for by different RNA
production of antibodies which neutralize in segments (segment 4 and 7, 8 or 9 depending on
vitro and in vivo (Offit et al., 1986a, 1986b; Green strain, respectively; see Table 4.1) and as rotaviruses
et al., 1992) and are thought to be involved in were found to reassort readily in doubly-infected
protection. Therefore, a dual classification cells in vitro and in vivo (Garbarg-Chenon et al.,
scheme into types similar to that developed for 1986; Urasawa et al., 1986), various combinations of
influenza viruses has been established (so far VP4 and VP7 types in natural rotavirus isolates
only for group A rotaviruses): it differentiates G have been observed (Estes, 1996; Desselberger,
types (VP7—specific, G for glycoprotein) and P 1998a; Table 4.2).
types (VP4—specific, P for protease-sensitive pro-
tein). So far, 14 different G types and more than
20 P types have been detected, indicating exten-
sive genomic diversity within group A rota- Replication
viruses. Whereas the correlation of G serotypes
and their genotypes is practically complete, for Rotaviruses spread via the orofaecal route and in-
many P genotypes no serotype has yet been es- fect the small intestine after oral ingestion. Multipli-
tablished. Thus, it has been agreed to designate cation occurs in the mature epithelial cells at the
the P serotype and genotype separately but tips of the villi of the small intestine. Rotaviruses
jointly, the latter in square brackets: for example, grow well in secondary monkey kidney cells and in
the human Wa strain is classified as G1P1A[8], permanent monkey kidney cell lines (MA104) in the
the human DS-1 strain as G2 P1B[4], etc. (For a presence of trypsin, and therefore their replication
recent update of the nomenclature of human and in vitro could be studied in detail (Estes, 1996).
animal rotavirus strains see Estes, 1996.) Double-shelled particles attach to the host cells via
VIRUSES ASSOCIATED WITH ACUTE DIARRHOEAL DISEASE 239
Table 4.2 Rotavirus G types (VP7 specific) and P types (VP4 specific) in the combinations found in natural isolates of
animals (A), humans (H) or both animals and humans (B)
G type
P type 1 2 3 4 5 6 7 8 9 10 11 12 13 14 NK
1 A A
2 A
3 B
4 H A A
5 A A A A
6 H H A B H
7 A A A A
8 H H H H
9 H B A H H
10 H
11 A A H B A
12 A B A A A
13 B A H
14 B
15 A
16 A
17 A A A
18 A
19 A
NK B H B B A A A H H B H A A
NK : not known.
All G types are genotypes and serotypes, not all P types (genotypes) have yet been differentiated as serotypes (Estes, 1996).
[Modified from Desselberger, 1998a]
the outer capsid protein VP4 (Ludert et al., 1996). ever, shed before complete maturation). Double-
The cellular receptor is not fully characterized yet shelled infectious virions are released by cell lysis.
but has a sialic acid component. There may be The non-structural proteins have been implicated
coreceptors. Virus entry is by receptor-mediated as supporting various stages of morphogenesis, and
endocytosis or direct penetration. Replication is ex- NSP4 was proposed to act as intracellular receptor
clusively in the cytoplasm. After removal of the in the RER to attract single-shelled particles for
outer capsid, the viral RNA-dependent RNA poly- maturation to double-shelled particles. (For details
merase (coded for by RNA 1) is activated, and large of structure—function correlations and replication
numbers of positive-stranded RNAs are tran- see Estes, 1996; Prasad and Estes, 1997; Lawton et
scribed, which leave the single- shelled particle via al., 1997a, 1997b).) In immunodeficient hosts and
its aqueous channels. They act as mRNAs, and their under certain experimental conditions, rotaviruses
translation products accumulate in the cytoplasm. undergo genome rearrangements (Desselberger,
Single-shelled particles form, consisting of VP1, 1996).
VP2, VP3 and VP6 and containing one genome
equivalent of packaged single-stranded RNA
(which is then replicated to form dsRNA). They
accumulate and form pseudocrystalline aggregates Pathogenesis
termed ‘viroplasm’ (intracytoplasmic inclusion bo-
dies). By budding through the rough endoplasmic Increasing cellular necrosis of the gut epithelium
reticulum (RER), single-shelled particles incorpor- leads to villous atrophy, loss of digestive enzymes, a
ate VP7 and VP4 to form the outer capsid (with a reduction of absorption and increased osmotic
transient, RER-derived envelope, which is, how- pressure in the gut lumen, resulting in the onset of
240 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 4.3 Rotavirus pathogenesis. Development of damage to gut mucosa, diarrhoea and repair. (From Phillips, 1989; with
permission of author and publisher)
diarrhoea. This is followed by a reactive crypt cell Yuan et al., 1996). These might be directed towards
hyperplasia accompanied by increased fluid secre- the type-specifying antigens VP7 and VP4, but also
tion, which also contributes to the severity of diar- towards the inner capsid protein VP6 (Burns et al.,
rhoea. Local pathogenesis is shown diagrammati- 1996; Herrmann et al., 1996; Feng et al., 1997).
cally in Figure 4.3. There is also a rotavirus-specific cytotoxic T cell
Viral factors determining pathogenicity of response (Dharakul et al., 1990), but its exact role in
rotaviruses have been investigated in several animal overcoming the infection or in protection against
models (piglets, mice, rabbits; for review see Burke subsequent infections is not known (Offit, 1994;
and Desselberger, 1996). The protein product of Franco and Greenberg, 1995). Natural infection or
RNA segment 4, VP4, has been found to be a major appropriate vaccination (see below) seem to protect
determinant of pathogenicity in several systems, but from severe disease in subsequent infections (Coul-
products of other structural genes (VP3, VP7) and son et al., 1992; Velasquez et al., 1996), even if the
of non-structural genes (NSP1, NSP2, NSP4) have serotype of the challenging virus differs from that of
also been implicated (Broome et al., 1993; Hoshino the previous infections or in the vaccine.
et al., 1995). NSP4 has been described recently as a
viral enterotoxin (Tian et al. 1994, 1995; Ball et al.,
1996).
Illness, Diagnosis and Treatment
Replication
Clinical Symptoms and Treatment
All adenoviruses grow well in human epithelial
cells, with the exception of the enteric adenoviruses Clinically, adenovirus-associated diarrhoea does
types 40 and 41. Those can be grown in Graham 293 not differ from those caused by other viruses, al-
cells (a human embryonic kidney cell line trans- though the duration of symptoms may last slightly
formed by adenovirus type 5 DNA; Takiff et al., longer (3—11 days). The stools are watery and non
1981). Replication has been studied in detail, mainly bloody (in contrast to bacterial diarrhoeas). Fever
with adenoviruses types 2 and 5 of subgroup C. Cell and vomiting are common. Usually adenovirus
attachment is facilitated by the fibre protein, and gastroenteritis is a mild disease, and treatment is
uptake is via receptor-mediated endocytosis, fol- symptomatic.
lowed by uncoating, movement of viral DNA to the
nucleus and phased early and late gene expression.
The early protein E1A is a potent blocker for apo- Prevention and Control
ptosis and interferon (IFN)- and - expression.
Late gene expression is at the onset of viral DNA At present there is no vaccine candidate for enteric
replication and is accompanied by blockage of cel- adenoviruses. Control of hospital outbreaks is by
lular and RNA expression. Virus assembly is in the cohort nursing of patients, use of gloves, gowns and
cytoplasm. Virus particle release is after cell death, goggles, and disinfection with sodium hypochlorite.
mediated by disruption of the cytoskeleton. Some
adenovirus proteins seem to decrease expression of
MHC class I antigens on the surface of infected SMALL ROUND STRUCTURED
cells, thus hindering susceptibility to adenovirus- VIRUSES (SRSVs)
specific cytotoxic T lymphocytes, and also to
counteract tumour necrosis factor expression (for Structure and Genome
details see Shenk, 1996).
This group of viruses was first recognized from an
outbreak of gastroenteritis in an elementary school
Diagnosis in Norwalk, Ohio, USA in 1968, affecting half of the
students and teachers (for review see Cubitt, 1994;
Detection of enteric adenoviruses in faecal speci- Kapikian et al., 1996). The outbreak was not due to
mens is mainly by ELISAs using subgroup F-speci- a bacterial pathogen, and, finally, using IEM, the
fic monoclonal antibodies. Electron microscopy fol- causative agent Norwalk virus (NV) was visualized
lowed by immune electron microscopy (IEM) can as a 27—35 nm virus particle. Upon cloning and
also be used to identify these agents. Adenoviruses sequencing of the genome, NV has been classified
are detected in 4—15% of stools from children with unequivocally as a member of the Caliciviridae fam-
VIRUSES ASSOCIATED WITH ACUTE DIARRHOEAL DISEASE 243
Figure 4.4 Genomic organization of Norwalk virus (NV; genogroup I), caliciviruses of genogroups II and III, and of astrovirus
(serotype 2). For nomenclature of NV-like viruses see Table 4.3. ORF : open reading frame; aa : amino acids; (A) : poly(A) tail of
RNA. (From Estes et al. (1997) and Matsui and Greenberg (1996); slightly modified)
ily. The genome consists of single—stranded RNA of 7.7 kb (Figure 4.4). The genome composition of the
positive polarity and approximately 7.7 kb in size. so-called ‘classical’ caliciviruses bears a greater
Typically, the surface of the particle carries cup- similarity to that of several animal caliciviruses/
shaped depressions which have given the name to SRSVs than to human SRSVs (Matson et al., 1995;
this viral family (Latin calix : goblet, cup). Liu et al., 1995), justifying their classification as a
The morphology of SRSVs has been analysed in separate subgenus. All calicivirus genomes have
more detail using cryoelectron microscopy and im- three open reading frames (ORFs), ORF 1 encoding
age reconstruction, when it was possible to produce non structural proteins with helicase, protease and
recombinants and NV-like particles from insect RNA polymerase functions; ORF 2 the viral capsid
cells which were infected with a baculovirus recom- protein; and ORF 3 at the 3 end of the genome
binant expressing the capsid protein (Prasad et al., encoding a small protein of as yet unknown func-
1994a, 1994b). The capsid consists of 90 dimers of a tion (Figure 4.4). As present, human SRSVs cannot
single capsid protein of 58K molecular weight be grown in cell culture.
(monomer) which are arranged in such a way that
large hollows are seen at the fivefold and threefold
axes and represent what appears to be the cup-like
structures in typical caliciviruses. Classification
Full-length sequences of cDNAs from three dif-
ferent caliciviruses (NV, Southampton virus and Classification according to morphology differenti-
Manchester virus) have now been obtained (Jiang et ates between well-structured and featureless small
al., 1993; Lambden et al., 1993; Liu et al., 1995). The round viruses; the SRSVs comprise NV and Nor-
single-stranded RNAs of positive polarity are poly- walk-like viruses (diameter 27—35 nm; Hawaii,
adenylated and have a length of between 7.3 and Snow Mountain, Mexico, Lorsdale, Sapporo, etc.
244 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 4.3 Genogroups of human SRSVs et al., 1984; for further details see Estes et al., 1997).
Name derived Calicivirus
Virus from morphology genogroup
Clinical Course
Norwalk virus SRSV I
Southampton virus SRSV I
Desert Shield virus SRSV I The incubation period ranges from 10 to 50 hours,
Hawaii agent SRSV II and diarrhoea lasts 24—48 hours. Ileum biopsies
Snow Mountain agent SRSV II from ill volunteers showed that a symptomatic ill-
Mexico virus HuCV/MX II ness correlated with blunting of intestinal villi, crypt
Lorsdale virus HuCV/LV II
Hu CV UK1—UK4 HuCV/UK1—UK4 II and untyped hyperplasia, cytoplasmic vacuolization and lym-
Toronto virus Minireovirus II phocytic infiltration of the lamina propia. Small
Sapporo virus HuCV Sapporo III intestinal brush border enzymes are decreased, and
(HuCV Japan) malabsorption and diarrhoea with abdominal
Manchester virus HuCV/MV III
cramps, nausea and vomiting result.
Plymouth virus HuCV/PV III
viruses), so-called classical caliciviruses (diameter This is mainly by direct electron microscopy or
30—40 nm) and astroviruses (diameter 28—30 nm) IEM, by ELISA (Herrmann et al., 1995) or, more
(Estes et al., 1997). Among the featureless small recently, by RT-PCR assays (Ando et al., 1994,
round viruses (SRVs), picornaviruses (diameter 1995; Moe et al., 1994). For the latter to be success-
27 nm) and parvoviruses (diameter 18—20 nm) are ful, broadly reactive primers must be available in
subsumed. order to detect most of the human SRSVs. Initial
Using IEM and ELISAs it was discovered that at use of such primers looks promising (Green et al.,
least five serotypes of SRSVs/caliciviruses exist, rep- 1995). Alternatively, several sets of primers directed
resented by NV, Hawaii virus (HV), Snow Moun- against the same or different regions of distinct
tain agent (SMA), Taunton agent (TA) and Sapporo genogroups can be used. These techniques have also
virus (SV), respectively. Sequencing of correspond- been applied to detecting SRSVs in shellfish and
ing regions of the genomes of a number of SRSVs other foodstuffs (Lees et al., 1995; Atmar et al., 1996)
(e.g. Wang et al., 1994) has allowed three different from which human infection frequently originate.
genogroups to be distinguished. Within the geno-
group, similarity is 92—99% for the polymerase re-
gion and 78—98% for the capsid region (Estes et al., Immune Responses
1997; Table 4.3).
The immune responses against SRSV infections
have been examined in adult volunteer studies, us-
ing IEM and more recently ELISAs with recom-
Replication binant antigens. However, there is the problem that
most volunteers have been infected with such vi-
As there is no in vitro cell culture system available ruses some time before experimental exposure, and
for human SRSVs, their replication can at present proper neutralization assays are not available. In-
be deduced only from that of animal caliciviruses terestingly, more than 50% of adult volunteers ap-
which have a similar genome organization and can pear to be susceptible (Parrino et al., 1977; Johnson
be propagated in cell cultures. From this it appears et al., 1990; Graham et al., 1994; Gray et al., 1994).
that viruses interact with species-specific receptors. Volunteer studies have shown that pre-existing
Proteins deduced from ORF 1 may arise from ap- SRSV-reactive antibodies do not protect from rein-
propriate co- and post-translational cleavage of a fection in the longer run; on the contrary, higher
polyprotein precursor, in a way similar to the cleav- pre-existing antibody levels seem to condition for
age cascade identified for picornaviruses (Pallansch more severe illness upon reinfection (Parrino et al.,
VIRUSES ASSOCIATED WITH ACUTE DIARRHOEAL DISEASE 245
Figure 4.5 Age-related prevalence of antibodies to Norwalk virus in England. (From data of Gray et al., 1993.)
1977; Gray et al., 1994). On the other hand, some ly to be a large number of inapparent infections in
volunteers who fall ill after initial NV challenge early childhood as it has been shown that 50% of
remained immune when rechallenged 6—14 weeks 3—year-old children already have NV-specific anti-
later (Parrino et al., 1977). bodies, a number that increases to 80% in early
adulthood (Figure 4.5; Gray et al., 1993). In less-
developed countries, NV-specific antibodies are
produced after primary infection even earlier in life.
Clinical Course
In general, children appear to be infected with hu-
man SRSVs much more frequently than was recog-
The clinical symptoms were studied in 50 volun-
nized previously (Gray et al., 1993; Numata et al.,
teers and were as follows: 41 (82%) became infected,
1994).
of those 68% were symptomatic and 32% asympto-
Outbreaks of acute gastroenteritis which can be
matic. Most common symptoms were nausea, head-
related to food or waterborne sources, occur fre-
ache and abdominal cramps followed by diarrhoea
quently in recreational camps, hospitals, schools,
and vomiting. Severe symptoms lasted for only
cruise ships, nursing homes, etc. and are associated
12—48 hours (Graham et al., 1994). Persistent infec-
with ingestion of contaminated drinking or recre-
tion with SRSVs and other viruses and chronic
ational water, uncooked shellfish, eggs, salads and
diarrhoea has been found in children with severe
cold foods. Mixed infections with SRSVs of differ-
combined immunodeficiency (SCID) (Chrystie et
ent genogroups have been observed (Gray et al.,
al., 1982) and in patients with the acquired immune
1997). Worldwide outbreaks occur year round, and
deficiency syndrome (AIDS) (Grohmann et al.,
nosocomial infection with SRSVs seems to be quite
1993).
common. The viruses are highly infectious and
spread rapidly in volunteer studies. The primary
and secondary attack rates are high (above 50%).
Epidemiology Transmission is by the faecal-oral route and by
projectile vomiting producing an aerosol which
The availability of recombinant proteins expressed scatters these viruses in the environment (Caul,
from cloned cDNAs has allowed the study of age- 1994). Improvement of sanitary conditions and per-
dependent antibody prevalence in both developed sonal hygiene, as well as sometimes closure of facili-
and developing countries. Epidemic gastroenteritis ties (hospitals, nurseries, etc.), are necessary to con-
produced by SRSVs is relatively mild. There is like- fine the infections. Viral shedding does not
246 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
normally persist beyond 100 hours after the initial Table 4.4 Incidence of astrovirus serotypes in Oxford, UK,
infection, but can be prolonged for up to 2 weeks. 1976—1992, and prevalence of astrovirus type-specific
antibodies in Utrecht, the Netherlands, 1994
Shedding has also been observed in volunteers who
remained well. Astrovirus isolates Astrovirus antibody
Oxford, UK? Utrecht, The Netherlands@
Astrovirus (n : 291) (n : 260)
type (% type) (% type-specific antibody)
Prevention
1 64.9 91
The most efficient methods of prevention are 2 11.3 31
3 9.3 69
measures of good hygiene (handwashing, disinfec- 4 11.3 56
tion and disposal of contaminated faeces and ma- 5 2.1 36
terial, hygienic processing of food, withdrawal from 6 0.3 16
work of ill food handlers, etc.). A vaccine seems 7 0.7 10
possible, for example derived from VLPs (Jiang et
?From Lee and Kurtz (1994).
al., 1992) and possibly from transgenic plants (‘ed- @From Koopmans et al. (1997a).
ible vaccines’; Mason et al., 1996), but better under-
standing of the significance of antibody responses
for protective immunity is required. Prophylactic Classification
surveillance of key personnel in hospitals or food
production (including keeping people with vomit- So far, seven serotypes have been distinguished by
ing and loose stools off work) and of water sources solid phase IEM and have been confirmed recently
helps to reduce SRSV-induced outbreaks. as different genotypes (Noel et al., 1995). The inci-
dence of different serotypes was determined from a
collection of isolates in Oxford, UK (Lee and Kurtz,
ASTROVIRUSES 1994) and has been complemented recently by an
age-stratified study of the prevalence of neutralizing
antibodies in Utrecht, The Netherlands (Koopmans
Astroviruses are members of the newly defined fam-
et al. 1997; Table 4.4). According to this, serotype 1
ily Astroviridae (Monroe et al., 1993; Murphy et al.,
is most frequently found, followed by serotypes 2—4
1995). Virions possess a genome of single-stranded
at medium and serotypes 5—7 at the least frequency.
RNA of positive polarity and no envelope, like the
Interestingly, the seroprevalence figures suggest
Picornaviridae and Caliciviridae (Matsui and
that individuals can become infected by more than
Greenberg, 1996).
one serotype.
Parvoviruses
ACKNOWLEDGEMENTS
These viruses are well-known pathogens of diar-
rhoea in animals. However, in humans no firm asso- The author gratefully acknowledges fruitful dis-
ciation with diarrhoea has been made, although cussions with Dr J. Gray (Cambridge) and Dr J.
parvovirus-like particles are not infrequently ob- Kurtz (Oxford), and the secretarial support of Mrs
served as ‘small round viruses’ in human faeces. Isabel Harvey and Mrs Lynne Bastow.
Influenza
Chris W. Potter
University of Sheffield, Sheffield, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
254 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Studies on the structure of influenza viruses carried
out during the past years have brought knowledge
of this subject to a point far in advance of that
known for other viruses, with the exception of hu-
man immunodeficiency virus. Influenza viruses
grown in embryonated eggs and examined in the
electron microscope show particles approximately
spherical and with a diameter of 80—120 nm: after
serial passage in the laboratory, some strains pro-
duce filamentous particles, and pleomorphic forms
are not uncommon (Stuart-Harris et al., 1985). The
electron microscopic appearance of influenza virus
particles is shown in Figure 5.1a, and a diagram
showing the components that make up the particles
is shown in Figure 5.1b.
The chemical composition of the virus particles
has been determined. Each particle is composed of
approximately 1% RNA, 70—75% protein, 20—24%
lipid and 5—8% carbohydrate. Structurally, the vi-
rus consists of a single strand of RNA of molecular
weight 4—6 ; 10 segmented into eight fragments;
the molecular weight of the fragments ranges from
3 ; 10 to 1 ; 10. The RNA is closely associated
with the nucleoprotein (NP) to form a helical struc-
ture, the nucleocapsid; the NP has a molecular
weight of approximately 60K, and there are ap-
proximately 1000 molecules in each virus particle.
The NP is a type-specific antigen and occurs in one
of three antigenic forms, and these different forms
provide the basis for the classification of human
Figure 5.1 Structure of influenza virus particle: (a) electron influenza viruses into types A, B and C.
microscopic appearance; (b) diagram of structural components.
HA : haemagglutinin; L : lipid bilayer in which the HA and Surrounding the nucleocapsid is a second protein
NA subunits are inserted; M : matrix protein; NA : referred to as the matrix or membrane protein (M1);
neuraminidase; NC : nucleocapsid consisting of nucleoprotein this protein is the major protein of the virus particle,
(NP) subunits closely associated with the viral RNA. (Courtesy occupying 35—45% of the particle mass. The mol-
of Stuart-Harris and Schild, 1976) ecular weight of the M1 protein is 23K, and there
tions concerning influenza and influenza viruses are approximately 3000 molecules in each virus
remain unanswered, much of the scientific informa- particle. A second M protein, termed M2 and coded
tion necessary to achieve the above aims is avail- by the same gene segment, is important in virus
able; but an enormous number of resources are replication (see below); there are 14—68 molecules of
necessary for their implementation, and this must M2 present in each virus particle which are thought
be considered in competition with other priorities. to have a function in virus assembly. About the M1
protein the particles have a viral membrane; this is a
lipid bilayer which constitutes approximately
20—24% of the virus particle.
THE VIRUSES Two virus glycoproteins are inserted into the
membrane; these are rod-shaped structures radi-
Structure ating out from the virus particles to give a spiky
appearance to the surface. The first of these glyco-
The influenza viruses belong to the genera Influenza proteins is the haemagglutinin (HA), which is com-
virus A, B and C in the family Orthomyxoviridae. posed of two separate molecules, termed HA1 and
INFLUENZA 255
HA2, joined together by a disulphide bond (Skehel early after infection, while NS2 appears late and is
et al., 1984): the complete HA molecule is composed found in virus particles (Richardson, 1991). The
of three of these subunits, each of molecular weight function of these molecules is unknown, but mutant
75—80K, and 20% carbohydrate to give a total mol- viruses with defects in the RNA fragment coding for
ecular weight of approximately 225K. There are these proteins are unable to synthesize sufficient
approximately 1000 HA molecules on each virus viral RNA or M1 protein.
particle: each HA particle is 14—16 nm in length and Most studies on the structure of influenza virus
4 nm in diameter, and is attached to the lipid mem- particles have been carried out on influenza A vi-
brane by a hydrophobic tip; and the HA forms ruses; however, sufficient work has been done with
25—30% of the protein of the virus. The subtype influenza B viruses to suggest general similarity in
classification of influenza viruses is based princi- size, composition and structure. Some differences
pally on the different antigenic forms of the HA have been found, but the significance of these to
molecule. The function of the HA is the attachment virus behaviour and epidemiology is not known.
of the virus to receptors on the surface of host cells The influenza B viruses differ from influenza A vi-
during the initial stages of virus infection: the HA ruses by the presence of an antigenically distinct
also attaches to receptors on erythrocytes, causing NP, and this, together with the antigenic specificity
haemagglutination. The second glycoprotein radi- of other virus proteins and glycoproteins, charac-
ating from the surface of the virus particle is the terizes a virus type with marked differences in epi-
neuraminidase (NA). There are 100—200 NA mol- demiological behaviour. The size, composition and
ecules on the surface of each virus particle: they are structure of influenza C viruses are similar to those
10 nm long and 4 nm in diameter; and are composed of influenza A: the main difference is an antigeni-
of four identical subunits, each of molecular weight cally distinct NP, and the absence of the NA glyco-
approximately 60K and an unknown amount of protein. These differences again characterize a virus
carbohydrate. The NA molecules are attached to which has epidemiological properties distinct from
the viral membrane by a stalk terminated by a those of influenza viruses A and B.
hydrophobic tip; the complete structure has a
mushroom appearance (Laver et al., 1984), and a
total molecular weight of approximately 200—250K.
Again, the NA glycoprotein is antigenically vari- Replication
able, and these differences are used in the classifica-
tion of the viruses. The function of the NA is not Most studies on the replication of influenza viruses
fully understood: the enzyme hydrolyses sialic acid have been carried out using influenza A strains; and
residues from specific glycoproteins, and can thus the limited number of studies with influenza B vi-
affect or modify cell receptors of absorption; alter- ruses have not indicated major differences in the
natively, NA may mediate in the release of newly mechanism of replication of this virus type. When
formed virus particles from the surface of infected Influenza A virus is inoculated on to cell cultures,
cells. The NA does not appear to have a function in three possible consequences may result. First, the
virus entry, replication or assembly (Liu et al., virus may fail to initiate infection. Secondly, the
1995). virus may undergo an incomplete growth cycle,
Influenza virus particles also contain three fur- known as abortive infection; this occurs in a variety
ther non-glycosylated P-proteins; these are termed of cell lines, including HeLa cells, L cells and human
PB1, PB2 and PA: the molecular weights vary from diploid fibroblast cells. There is at present no accep-
80 000 to 90 000K; there are approximately 50 mol- ted explanation for abortive infection, but because
ecules of each species in each virus particle; and they of a block at some stage of the normal replication
are internal components of the viral particles, prob- cycle large numbers of virus particles are produced
ably associated with the nucleocapsid. The function which are deficient in RNA content and are non-
of the P-proteins is not fully understood; however, infective. Abortive infection is also seen when large
these molecules are involved in virus-specific RNA amounts of virus are inoculated on to permissive
synthesis associated with virus replication. Finally, cells, such as the cells of the amniotic membrane of
the virus RNA codes for two non-structural pro- the embryonated egg, where the effect is described
teins, termed NS1 and NS2: NS1 is synthesized as the Von Magnus phenomenon. Thirdly, infection
256 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 5.2 Model of replication of influenza virus. (Reproduced with permission from J.S. Oxford and the British Society of
Antimicrobial Chemotherapy; Journal of Antimicrobial Chemotherapy (1975), 1, 7)
may be permissive, and result in the production of tion of the virus coat by lysosomal protease and
infective virus; the various stages of replication are lipase enzymes, and requires a proton channel for-
shown in Figure 5.2. med by the M2 protein (Lamb et al., 1994). The
Permissive infection by influenza virus occurs in released virus genome (vRNA) together with the
human, baboon and monkey kidney cells and in the polymerase enzyme complex then migrate to the
cells lining the amniotic and allantoic cavities of the infected cell nucleus (Martin and Helenius, 1991).
embryonated hen’s egg, and is initiated by absorp- The events of viral replication in the cell nucleus
tion of virus to the cell surface. This phenomenon are complex, and remain to be fully understood.
requires the interaction of two molecules: these are Our information to date suggests that transcription
a virus receptor on the cell surface which is a of the messenger RNAs (mRNAs) from all eight
neuraminic acid-containing glycoprotein; and the fragments of vRNA takes place immediately after
virus HA, since antibody against the HA prevents entry into the cell nucleus. Initially, similar amounts
virus adsorption. Following adsorption, virus en- of each mRNA are produced; however, this is fol-
ters the cell: penetration is suggested by electron lowed by a regulatory phase in which the synthesis
microscopic studies to be a pinocytotic process re- of the mRNA coding for NP and NS1 predomi-
sulting in virus-containing endosomes appearing in nates: it is clear that mRNA synthesis is regulated at
the cell cytoplasm at approximately 20 minutes all stages, and that this regulation is controlled by
after virus infection. The subsequent stage of virus virus proteins such as P-proteins, NP and NS1
uncoating is not fully understood; however, it oc- (Krug, 1983). The synthesis of mRNA involves se-
curs as a result of fusion of virus particles with quentially cap recognition and binding, endonuc-
endosomal membranes, a fall in pH and the disrup- lease activity, initiation and then elongation to pro-
INFLUENZA 257
duced functional mRNA. First, the polymerase cells, and it is this structure which supplies these
complex binds to a methylated cap structure pres- virus proteins to the assembling particles; it is sug-
ent on heterologous, cellular RNA; this cap recogni- gested that this organization of assembly is control-
tion is a function of the PB2 protein. Secondly, a led by the M2 protein, which is present in high
nucleotide sequence of 10—13 bases is cleaved from concentration at the cell surface. The process of
the cap structure by endonuclease activity, termed virus release is by budding from the membrane of
cap snatching, and this structure forms the starter the infected cells; this is detectable at 5—6 hours after
molecule for mRNAs synthesis: it has been sugges- infection and is maximal at 7—8 hours after infec-
ted that the cleavage is one of the functions of the tion. The mechanism of virus release from the host
PB1 and PB2 proteins (Blok et al., 1996). The cap cell is not known; studies have suggested that it is a
sequence incorporates a G complementary to the function of the virus NA, since antibody to the NA
penultimate C of the vRNA to complete the initi- inhibits virus release. Alternatively, the NA might
ation step, and this is a second function of the PB1 reduce virus aggregation at the cell surface by re-
protein. Finally, elongation takes place in the nor- moving neuraminic acid from the viral envelope.
mal manner to produce complete mRNAs; this is a
function controlled by the PA protein. Cap snatch-
ing from the host RNA requires host RNA syn- Viral Variation
thesis, and virus replication is dependent on this
process; for this reason, inhibitors such as -
Historical Aspects
amanitin inhibit influenza virus replication. The
mRNAs leave the cell nucleus and bind to ribo- The explosive nature of epidemic influenza, to-
somes, and translation of virus proteins is initiated: gether with the large numbers of patients involved
this process again has not been fully elucidated, and and the specific clinical features of this disease, has
the exact role(s) of the P-proteins and the regulation given credibility to records of this infection from the
of the events are not clearly established. The syn- beginning of the eighteenth century. Improved and
thesis of cRNA, and subsequently vRNA, occurs more precise data, and the laboratory isolation of
after the time of peak production of mRNA and the influenza virus in 1933, have given accurate
protein synthesis. Full-length vRNAs are produced records for the past 100 years: the incidence of
and exist in approximately equimolar amounts of outbreaks and the numbers of patients involved
each throughout infection, and are all synthesized have not decreased during this period. History re-
in the infected cell nucleus. cords frequent, almost annual, epidemics of influ-
The time sequence for the appearance of the vari- enza; and, since 1700, fourteen pandemics. Accurate
ous virus proteins has been studied extensively: the documentation of the latter is first seen for the
presence of NP can be detected approximately 2 pandemic which spread from Russia into Europe
hours after virus infection, and the concentration and the USA in the years 1889—1892: the infection
rises to a maximum at 5—6 hours; the M1 protein presented as an acute upper respiratory tract infec-
appears at 5 hours after infection, and has been tion of sudden onset and short duration; the total
identified in both the cytoplasm and to a lesser number of fatal cases was high, particularly among
extent in the nucleus of the infected cells; and the infants and the elderly, but deaths were recorded in
HA and NA appear approximately 3 hours after only a relatively small percentage of the 25—30% of
infection. the world’s population infected.
Virus assembly occurs when vRNA migrates to The pandemic of 1918—1920 originated in China
the cell surface and virus proteins have also accu- or the USA, and is known as the Spanish influenza.
mulated at this site. Assembly requires the aggrega- The strain of virus which caused the second wave of
tion of one of each of the vRNA components of the this pandemic had unusual virulence since it
complete virus RNA; this is believed to be a random commonly caused a severe and unique form of
process which accounts for both the numbers of pneumonia which resulted in at least 20—25 million
defective particles, and the relative ease with which deaths, principally in young adults. The effects of
reassortant viruses can be produced during double the pandemic caused international panic and the
infections. The virus HA, NA and M1 proteins are number of deaths recorded may be only a fraction
present in the plasma membrane of the infected of the true number: many countries did not report
258 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 5.1 Antigenic shift in the haemagglutinin of influenza A viruses
NK : not known.
?Influenza A viruses grown in eggs were tested in HI tests with antisera from ferrets infected intranasally with
live virus and bled 3—4 weeks later. The serum HI antibody titre against 8 HA units of virus is listed.
figures; India was said to have lost a generation; one occur approximately every 10—40 years. Since 1933
and a half million deaths occurred in sub-Saharan the viruses which cause these two types of outbreak
Africa; the propaganda surrounding the world war have been isolated and compared in the laboratory.
of 1914—1918 undermined the accuracy of data from Viruses isolated from patients infected in the years
Europe; and the Bolshevik revolution in Russia 1933—1946, 1947—1956, 1957—1967 and from 1968
resulted in ignorance of the effects of the pandemic onwards show wide variations, and cross-haemag-
in that country (Crosby, 1976). glutination inhibition (HI) test using antisera from
The definition of a pandemic of influenza is an virus-infected ferrets show no serological cross-re-
outbreak of infection arising in a specific geographi- activity (Table 5.1). In addition, RNA sequences
cal region, spreading worldwide and infecting a recovered and amplified by the polymerase chain
high percentage of people; and caused by a strain of reaction (PCR) from postmortem lung tissue of a
influenza virus which could not have arisen by mu- patient who died of influenza in 1918 indicated that
tation from strains circulating previously. Thus, the the 1918—1920 pandemic was also caused by an
influenza outbreaks of 1932—1933 and 1947—1948 influenza A (H1N1) virus (Taubenberger et al.,
were caused by viruses related to each other, and to 1997): this virus and a virus now infecting pigs and
the virus thought to have caused the pandemic of the epidemic strains A/PR/8/34 and A/FM/1/47 all
1918—1920: by definition, the outbreaks of 1933 and belong to one subtype, although showing no cross-
1947 were not pandemics. However, a pandemic did reactions in the HI test. The absolute specificity of
occur in 1957 which originated in China, spread the HA of the different subtypes indicates that in the
through the world, infected 40—50% of people and years when pandemic infections are recorded, new
caused over one million deaths, mainly in the elder- influenza virus subtypes have emerged against
ly population. Pandemic infection occurred again which the population has no immunity: this is
in 1968, and the event was similar to that in 1957. known as antigenic shift. Since it is principally anti-
Since 1968 and in the years between the pandemic body to the virus HA which is equated with immun-
years, epidemics of influenza occurred in some ity (see later), infection by influenza viruses of a
countries each year; thus, the pattern is for frequent previous era does not induce HI antibody to the
epidemics interspersed with explosive pandemics newly emerged virus strain. The historical pattern
which occur every 10—20 years. Since the last pan- for pandemic influenza is for a new virus subtype to
demic was in 1968, it may be said that the next emerge and spread rapidly, causing explosive out-
pandemic is overdue at the time of writing (1999). breaks in many countries. As a result of infection,
immunity to the new subtype is built up over a
period of years, and further epidemics are more
Antigenic Shift
limited. However, after 10—40 years, when the popu-
The recorded patterns on influenza infection con- lation is largely immune, a new virus subtype
tain two phenomena: the first is the almost annual emerges with an HA which does not cross-react
epidemics which occur in one or more countries, with antibody to the HA of the previous virus sub-
and the second are the extensive pandemics which type. Consequently, the new virus initiates a new
INFLUENZA 259
cycle of influenza infection. These sudden changes the dual infection was by two subtypes of human
in antigenicity can also occur in the virus NA mol- influenza viruses. However, influenza A viruses in-
ecule. fect other species, including horses and in particular
Although the HA antigens of influenza A virus birds: dual infection with human and animal or bird
subtypes show no cross-reactivity in HI tests, some viruses in a species which could support the replica-
expression of relatedness is seen in the phenomenon tion of both could result in the production of a
known as original antigenic sin, first reported by reassortant virus with surface antigen(s) from the
Francis et al. (1953). This term describes the obser- animal virus and infectivity for humans. This theory
vation that infection by influenza virus induces anti- is scientifically tenable: reassortant viruses can be
body to the infecting virus and recalls antibody to produced in the laboratory from human and animal
the other virus subtypes that the individual has parents; reassortants can be identified following
experienced previously; indeed, the titre of antibody mixed infection of animals or birds; influenza vi-
to the earlier infecting subtypes can be severalfold ruses can cross species barriers; and antigenic
greater than that to the current infecting virus. This analysis of two human influenza virus subtypes has
is probably due to stimulation of B-memory cells revealed similarities between the HA of these vi-
which persist after infection being stimulated by the ruses and known avian viruses. In contrast, al-
new virus serotype, and suggests a relatedness for though animals have been infected with human in-
the HA antigens of different virus subtypes not fluenza virus following epidemic infection of
detected in HI and other serological tests. humans, no epidemiological evidence has been
The importance of antigenic shift in producing found to suggest that the HA of an animal or avian
new antigenic subtypes which spread to cause pan- influenza virus has subsequently occurred on a
demic infection is evident; but despite some 50 years novel human influenza virus subtype which then
of intensive research, the origin of these new influ- caused pandemic infection. Influenza B viruses do
enza viruses is unknown. The problem has been of not occur in animals and do not exhibit antigenic
great interest to a large number of virologists; and shift: this has been put forward as indirect evidence
experiments, epidemiological studies and imagin- for reassortment as the mechanism for the emerg-
ation have produced a number of theories. For ence of new influenza A virus subtypes.
example, new serotypes of virus may arise by spon- Finally, it has been suggested that there are a
taneous mutation; alternatively, it has been sugges- limited number of influenza A subtypes which are
ted that new viruses could have been introduced recycled in the human population. Evidence for this
from cosmic sources, since the years of some epi- theory comes from seroepidemiological studies of
demics due to newly emerged subtypes show a loose antibody to influenza viruses in sera taken at differ-
connection with the occurrence of astral phenom- ent times from subjects of different ages. In a numb-
ena. Although both these views are present in the er of such studies, antibody to a human influenza
literature, only a minority of scientists consider virus subtype was identified in serum samples from
them valid explanations, and convincing evidence elderly persons taken years before the appearance
to support either of these theories is lacking. Thus, of the same subtype as a cause of pandemic infec-
random mutations would be expected to produce tion. From these observations, it has been suggested
intermediate strains, and such strains have not been that all influenza virus subtypes exist cryptically in
identified; detailed analysis has shown that new nature, and emerge when the antibody status of the
virus subtypes have multiple sequence difference, population has fallen to levels which allow pan-
and it is difficult to imagine these all occurring demic infection: a cycle of approximately 70 years
simultaneously. and involving the subtypes which caused the pan-
The most widely held theory for the origin of the demics recorded since 1889 would satisfy this re-
new influenza virus is that new virus subtypes are quirement. The evidence supporting this theory is
reassortant viruses resulting from double infection. fragile. Although serological studies have detected
The appearance of a new influenza virus subtype is the presence of antibody to the influenza virus sub-
paralleled by the disappearance of the old subtype type (H3N2) which was first seen in 1968 in sera
(an exception has occurred in most recent times, from aged persons collected before the epidemic, no
when two virus subtypes have circulated concur- reservoir for the virus in humans or animals before
rently), and it would therefore seem unlikely that this epidemic has been found. Again, it can be ar-
260 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 5.2 Antigenic drift in the haemagglutinin of influenza A (H3N2)
Figure 5.3 Seasonal occurrence (a) and age and associated infection (b) in 367 cases of Reye’s syndrome. URI : upper respiratory
infection. (Data from Corey et al., 1976)
(Conover and Roessmann, 1990) and neural tube ties but small changes consistent with viral en-
defects (Lynberg et al., 1994) following influenza cephalitis have been shown, and virus has been
virus infection during pregnancy. Transplacental isolated at autopsy from the lungs of fatal cases of
passage of the virus has been demonstrated, but encephalitis. The pathogenesis of these neurological
prospective studies of congenital abnormalities fol- complications is unknown, since virus recovery
lowing epidemics of influenza have failed to estab- from the brain has been infrequently documented.
lish a relationship between influenza virus infection Epidemiological studies have associated some
and these abnormalities. No conclusions are justifi- subtypes of influenza A with sudden unexplained
able from our present knowledge; there may be a death and sudden infant death syndrome, some-
risk of anencephaly following influenza infection in times called cot death syndrome (Zink et al., 1987).
the first trimester of pregnancy, but this risk is Firm data associating acute influenza infection with
probably very small. sudden infant death syndrome have been sought by
many workers in the past, but have been difficult to
obtain; and the association is made on circumstan-
tial evidence and remains speculative. Influenza vi-
Other Complications rus infection, and the association of this infection
with secondary Staph. aureus infection (see above),
Although influenza in healthy adults is normally can result in toxic shock syndrome; this complica-
severe but of short duration, resolving in 3—5 days, tion of influenza infection has been reported by a
complications can occur, particularly in elderly pa- number of observers. Finally, reports of an associ-
tients and those with predisposing conditions, and ation of maternal influenza with schizophrenia in
these have been outlined above. In addition, virus offspring and Parkinson’s disease are in the litera-
infection can result in a number of other less well ture, but the evidence of association is both fragile
understood complications. Influenza can cause and contradictory (O’Callaghan, 1991).
ketoacidosis in diabetic patients, even in relatively
mild cases of virus infection. Infection has been
implicated in acute viral encephalitis and in
Guillain—Barré syndrome, and deaths have been DIAGNOSIS OF INFECTION
reported in both these groups. Histological examin-
ation of brain tissue has shown no gross abnormali- During epidemics of influenza, large numbers of
266 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
patients are seen with similar symptoms; if it has Virus Isolation and Identification
been established that influenza virus is circulating
in the community, an association of patients with Pathological Specimens
contacts diagnosed as having influenza may suggest
that the diagnosis is self-evident. However, influ- Influenza viruses replicate in the upper respiratory
enza A and B can coordinate, and mixed epidemics tract, and are present in these tissues and in respir-
of influenza and other viruses have been reported; atory secretions; virus is not found in other tissues,
under these circumstances presumptive diagnoses although occasional reports of virus isolation from
can be misleading. Again, the symptoms of influ- brain, heart tissue and blood are documented. Ser-
enza in any group of patients are clearly different ial specimens of respiratory secretions from patients
from those due to other virus infections; however, with influenza indicate that the maximum titre of
the symptoms and signs for any one patient may virus is present on days 2 and 3 after the onset of
vary, such that a diagnosis on clinical criteria can- symptoms, but virus is detectable from days 1 to 5.
not be confidently made. Theoretically, it is advis- Throat or nasopharyngeal swabs can be taken into
able that all diagnoses should be confirmed by lab- a suitable transport medium, or nasal washings can
oratory tests, but in practice this is not always be collected. Comparative studies have shown that
feasible. More importantly, isolated cases of suspec- virus can more commonly be isolated from nasal
ted influenza should be investigated, since these washings than from other specimens.
may represent the first case of an impending epi-
demic or infection by a new virus strain: diagnosis Culture in Embryonated Eggs
of these cases is not of benefit to the individual
patient but is an important signal of what may Influenza viruses A and B, present in pathological
happen subsequently in the community. The recog- specimens and collected into transport media, can
nition of index cases activates preventative be cultured by amniotic inoculation of 10—12 day
measures to protect subjects for whom influenza is a embryonated eggs. The virus is absorbed from the
life-threatening infection. fluid of the amniotic cavity on to the cells of the
The laboratory diagnosis of influenza is based amniotic membrane in which they multiply, releas-
upon the isolation and identification of virus from ing newly formed virus back into the amniotic
pathological specimens, and/or the demonstration fluids. After 2—3 days incubation, virus can be pres-
of a significant increase in specific antibody titre ent in high titre in the amniotic fluid and can be
between serum specimens collected at the onset of detected by adding aliquots of harvested amniotic
infection and 2—3 weeks later. The isolation of virus fluid to chick, turkey, guinea-pig or human eryth-
can only be achieved in a limited number of cases, rocytes and observing haemagglutination. Egg
since virus may have disappeared by the time the fluids negative for virus can be passed to further
specimens are taken, and the methods used for virus embryonated eggs and retested: experience has
isolation lack sensitivity; however, direct methods shown that further passage is unwarranted, and
of detecting viral nucleic acid by PCR, and viral specimens which do not reveal virus after two egg
protein by ELISA tests or with fluorescein-labelled passages are recorded as negative.
antibody are increasingly available, refined and
rapid. Serological tests provide the most sensitive
Tissue Culture
and practical alternative for diagnosis in the ab-
sence of virus isolation; but, since they require a Pathological specimens can be inoculated on to
convalescent serum specimen, the diagnosis is retro- tissue cultures of kidney cells from rhesus monkeys,
spective. However, a diagnosis may be made on a baboons, chicks or a variety of other species; again,
single serum sample by demonstrating the presence experience has shown that rhesus monkeys and ba-
of a virus-specific IgM response; such responses boon kidney cells are probably the most sensitive.
may be present for about 8 weeks, occasionally After absorption and incubation of virus-infected
longer, following influenza infection. cells, newly produced virus can be detected by a
number of methods. First, free virus released into
the maintenance medium of the tissue culture can
be detected by haemagglutination with eryth-
INFLUENZA 267
rocytes, as described for amniotic fluid (see above). virus to the same titre as against homologous virus;
Secondly, since virus is released slowly from the cell the indicator system in this test is an NA substrate,
surface of infected cells, erythrocytes will adhere such as fetuin.
directly to these infected cells; this phenomenon is
termed haemadsorption, and can be observed un-
der the microscope. Finally, and more rapidly, virus Rapid Diagnosis
can be detected in infected cells by fixation and
staining with specific, fluorescein-labelled antibody Immunoreactions
(Brumbeck and Wade, 1996).
Since influenza virus is very rarely isolated from
symptom-free persons, the isolation of virus from
Virus Recognition patients may be taken as proof of infection without
Influenza viruses isolated from embryonated eggs the need for diagnostic serology; however, using
or tissue culture can be identified by serological conventional egg and tissue culture techniques (see
methods. First, influenza viruses can be recognized above) this procedure takes at least 2—3 days, and in
as influenza A, B or C by complement fixation (CF) practice probably a week, to complete. Since clini-
tests using extracts of infected cells or embryonic cians may confidently expect antiviral agents for the
membranes, which contain high titres of NP anti- treatment of influenza to be available in the near
gen, and standard antisera against influenza A, B or future, more rapid methods of diagnosis are needed
C viruses. An NP antigen is found for all influenza if these compounds are to be used rationally. In
A, B or C virus types, and antibody against one type addition, more rapid diagnostic methods would
does not react with soluble antigen of another type: allow the earlier initiation for measures to limit the
thus, the influenza type can be unequivocally identi- spread of infection. One procedure for rapid diag-
fied in this manner. nosis has been investigated by many workers, and
The further classification of influenza isolates relies on the direct identification of virus and virus
into subtypes and strains is a highly specialized antigens in infected cells of the respiratory epi-
responsibility of WHO reference laboratories: these thelium, and these are constantly shed into the re-
determinations are carried out on virus isolates for- spiratory secretions: these cells can be removed
warded from laboratories to these centres. Each either in throat washings or scraped from the tissue
virus isolate is tested by HI tests against antisera surface with a metal spoon. In the laboratory, the
raised in experimental animals against a range of cells are placed on a glass slide, fixed and stained by
virus subtypes and strains: the titre of each sera an indirect immunofluorescence (IF) or an ELISA
against homologous virus is known from prior test- technique using antisera against influenza virus; the
ing. The submitted virus srains are standardized to IF staining can be accelerated by treatment with
contain a fixed amount of HA activity by titration microwave irradiation for a few seconds (Hite and
against chick erythrocytes, and then reacted against Huang, 1996). The procedure can be completed
a range of dilutions of each antisera. By observing within 1—2 hours of specimens arriving in the lab-
the patterns of HI against the various antisera, the oratory, and offers obvious advantages. Many
virus subtype and strain is identified. Should the workers have investigated this method; some are
virus isolate not be inhibited by any of the sera to convinced of the value of the technique, but others
the same titre as known for homologous virus then have been disappointed with the specificity of anti-
the strain may be a new subtype or strain: homo- sera available and the level of background reaction,
logous sera against this strain is then prepared in particularly fluorescence, which makes the test diffi-
animals, and more extensive cross-HI tests per- cult to interpret; however, better reagents are be-
formed. These tests require the constant addition of coming available and the method offers a consider-
new control sera to the battery of antisera, and able advance over existing methods for the future.
experience in interpreting the results.
In addition, to identify the HA of the virus isolate,
Molecular Biology
the NA is also typed. This is done by identifying
which antisera prepared against the various influ- Developments in molecular biology have provided
enza virus NAs will inhibit the NA of the unknown reagents and techniques for the diagnosis of numer-
268 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ous microorganisms, and these techniques are series of dilutions made: to each dilution is then
equally applicable to the detection of influenza vi- added a standard quantity of intact virus, and after
ruses. Thus, influenza virus RNA can be detected incubation chick erythrocytes are added. The pres-
using DNA probes by molecular hybridization; the ence of antibody is indicated by inhibition of haem-
reactions can be detected by using either isotope- or agglutination. A further technique for detecting
biotin-labelled probes (Uryvaev et al., 1990). Vi- serum antibody is the haemadsorption inhibition
ruses can be detected by PCR: virus RNA sequences test. In this test, mixtures of standard virus and
are transcribed into cDNA by reverse transcriptase, serum dilution are inoculated on to tissue cultures
and then amplified using specific DNA primers; the of monkey kidney cells and incubated: after 2—3
amplified sequence is then detected by polyac- days, the cultures are washed, guinea-pig eryth-
rylamide gel electrophoresis as a molecule of pre- rocytes added, and the cultures viewed under the
dicted molecular weight. The sensitivity of the PCR microscope. The presence of haemadsorption indi-
technique is unsurpassable, since theoretically one cates virus replication and the absence of antibody,
RNA sequence can be amplified to several million whilst no haemadsorption indicates neutralization
DNA sequences; thus, the technique offers exquisite by antibody in the serum. By testing each serum
sensitivity, but great care must be taken to avoid over a range of dilutions, the titres of antibody can
cross-contamination (Zhang and Evans, 1991). Al- be determined; again, a fourfold or greater rise in
though influenza virus is not isolated using conven- titre is diagnostic of infection.
tional techniques from normal persons (see above), Infection by influenza viruses results in a rise in
this conclusion must be independently tested if the serum antibody titre, but the requirements for an
PCR reaction is used. The method can be refined for equal or greater than fourfold rise in titre of HI or
the detection and identification of virus strains CF antibody reflects the inaccuracy of these tests
(Wright et al., 1995). for detecting increases in antibody. A more precise
method of measuring antibody is by the single
radial haemolysis technique. Here, influenza virus is
coated on to sheep red cells, and suspended in
Serology melted agar with complement: the agar is poured
into dishes or on to glass slides and, after setting,
Although isolation of virus from respiratory secre- wells are cut in the agar and inoculated with dilu-
tions is recommended to establish the diagnosis for tions of test sera. The presence of antibody in the
all suspected cases of influenza, virus cannot be serum is detected by lysis of the red cells as antibody
isolated from all cases of infection. More commonly combines with complement and antigen on the red
the diagnosis is made retrospectively by the demon- cell surface. This lysis can be seen with the naked
stration of a rise in serum antibody to the infecting eye, and the amount of antibody present is directly
virus. For this, blood samples are taken as early related to the area of the zone of haemolysis. The
after the onset of symptoms as possible (acute speci- procedure is more sensitive than CF or HI tests and
men), and 14—21 days later (convalescent specimen): has a greater degree of precision: a 50% increase in
these sera are titrated for virus antibody, and the zone area represents a rise in antibody and is evi-
demonstration of a fourfold or greater increase in dence of recent infection. Sera do not require pre-
antibody titre in the convalescent sera as compared treatment to remove the non-specific inhibitors
to the acute sera is diagnostic of infection. A which plague the HI test.
common method for measuring antibody titre is by
CF: soluble antigen is extracted from embryonic
membranes of infected eggs, and is reacted against a
range of dilutions of the acute and convalescent TREATMENT AND PREVENTION
sera. This test is often positive for patients from
whom virus could not be isolated. The clinical severity of influenza, with high tem-
A more specific test for antibody to influenza perature, respiratory symptoms, myalgia and severe
virus is the HI test. For this, paired sera from pa- prostration, requires most patients to seek bed rest
tients with suspected influenza virus infection are during the acute phase of illness; the exhaustion and
treated to remove non-specific inhibitors, and a depression which follow may require further rest
INFLUENZA 269
and convalescence. During epidemics symptoms has been made, and this can be summarized under
can affect tens of thousands of individuals, causing the separate headings of treatment and prevention.
disruption to industry, services and social life; caus-
ing a significant number of deaths; and resulting in
complications such as pneumonia and Reye’s syn-
drome which are life-threatening to both old and Treatment
young. From these observations, it is clearly desir-
able that adequate means of treatment and preven- At the present time treatment of influenza is usually
tion should be developed; but despite this need, symptomatic. Patients are advised to remain in bed
which has been recognized throughout this century, for 2—3 days until the acute symptoms have subsid-
progress has been relatively slow, and the impact of ed. The symptoms of headache and fever were
epidemic influenza has changed little since the virus commonly treated with salicylates, but in view of
was first isolated some 60 years ago. Some progress the evidence that a combination of salicylates and
270 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
acute influenza virus infection may underlie the indicated two features of antiviral activity. First, the
pathogenesis of Reye’s syndrome in children, para- compound acts at the level of virus uncoating by
cetamol should be used in place of salicylates. Co- lysosomal enzymes: this process requires the ion
deine linctus may relieve the cough; insomnia may channel formed by the M2 protein and a fall in pH
be treated with barbiturates or promethazine (an (see above) for optimal enzyme activity, but aman-
antihistamine with hypnotic side-effects); and anti- tadine blocks this channel and thus uncoating and
biotics are indicated where chest complications are subsequent viral replication are limited. Secondly,
present or suspected. The use of prophylactic anti- the action of amantadine on the M2 ion channel
biotics in patients with chronic chest disease who activity inhibits the transport of HA and other viral
thus have a higher risk of developing postinfluenzal subunits at the cell surface, which in turn limits
pneumonia is contentious. Some advocate this virus assembly (Lin et al., 1997).
practice, but the incidence of secondary bacterial Clinical studies with amantadine have shown
pneumonia is not reduced and infection may be by that the compound can occasionally induce mild
antibiotic-resistant organisms. neurological symptoms; these include insomnia,
Since the earliest conception of antiviral chemo- loss of concentration and mental disorientation.
therapy, influenza has been one of the target dis- The symptoms are quickly shown by susceptible
eases against which suitable antiviral compounds individuals, and cease when treatment is stopped. If
should be developed. The search for such com- these symptoms do not occur within 24 hours of
pounds has used two methods. One is the rational initiation of amantadine treatment they are unlikely
approach, which predicts potential valuable sub- to occur at all. With these reservations in mind,
stances that would interfere with virus replication; amantadine has been subjected to clinical trials, and
this requires an exact knowledge of the molecular satisfies the basic requirements of non-toxicity.
events of virus infection and multiplication, and Both prophylatic and therapeutic studies have been
such information is not complete. The other is the carried out. In the most convincing prophylactic
serendipity approach, which requires the random trials, index cases of influenza were identified in
screening of chemical compounds in the hope that closed groups of subjects in schools or universities,
an active compound will be found by chance. To and unaffected volunteers from among the contacts
date both strategies have had similar limited suc- were given either amantadine or a placebo: these
cess: no entirely satisfactory agent has been found, studies indicated an approximate 70% protection
but compounds with antiviral activity against influ- for those taking amantadine prophylactically
enza have been identified. (Dolin et al., 1982). In a therapeutic study carried
out among patients attending general practitioners,
volunteers with symptoms of influenza were treated
Amantadine, Rimantadine
with either amantadine or placebo in a double-
Amantadine and rimantadine are synthetic, water- blind study. The diagnosis of influenza was con-
soluble primary amines with a symmetricl structure; firmed by demonstrating a significant rise in serum
basically, they consist of a stable base with an active HI antibody, and only patients in whom the diag-
amino group (Figure 5.4). In experimental studies, nosis was confirmed were included in the subse-
the compounds have been shown to inhibit the quent analysis. Amantadine significantly reduced
growth of influenza viruses in cell culture; to limit the duration of the fever (51 hours as opposed to 74
virus replication in mice, ferrets and other experi- hours in the placebo group) and illness (2.5 days in
mental animals; to reduce tissue damage by influ- bed as opposed to 3.5 days). The study inadvertent-
enza virus in infant rats; and to protect hamsters ly included patients shown subsequently to be suf-
from infection from virus-infected animals placed in fering from influenza B infection; this provided an
the same cage (Potter and Oxford, 1977). On the added control to the study, since no therapeutic
other hand, the compounds are not equally active effect was demonstrated against this infection, and
against all influenza virus strains; resistant strains amantadine is known to have no antiviral activity
can arise during treatment; naturally occurring re- against Influenza B virus. It is suggested that riman-
sistant strains have been isolated; and the com- tadine is the preferred compound, since, although
pounds are not active against influenza B viruses. not as effective as amantadine, it is less toxic (Ar-
Studies on the mode of action of amantadine have ruda and Hayden, 1996).
INFLUENZA 271
Ribavirin and other compounds may show synergy and re-
duce the chance of resistant strains arising: these
The structure of the synthetic nucleoside analogue
studies lie in the future.
1—--ribofuranosyl-1, 2,4—triazole-carboxymide
(ribavirin) is shown in Figure 5.4. The compound
has been shown to inhibit the replication of a wide
range of RNA and DNA viruses in vitro, including Prevention
both influenza A and influenza B, and to limit influ-
enza virus replication in mice and ferrets. The com- It is clear from knowledge of the epidemics of influ-
pound probably acts by inhibition of virus nucleic enza that individuals, groups and communities
acid synthesis. Ribavirin is well tolerated at concen- would benefit from the development of effective
trations 200—fold greater than those necessary to vaccines. To this end various forms of vaccines have
inhibit virus replication; however, the compounds been developed during the past 50 years: despite
has been reported to have immunosuppressive ef- this, the efficacy of influenza vaccines is still ques-
fects. Clinical studies have demonstrated a signifi- tioned, and the ability of vaccines to limit epidemic
cant therapeutic effect on symptoms of both influ- infection has not been proven.
enza A and B infection (Stein et al., 1987); and the
compound is very effective in animal studies when
given by aerosol combined with rimantadine or Immunity to Influenza
amantadine (Hayden, 1996).
The antigens of the influenza virus particle which
stimulate immunity to subsequent infections have
been identified. The virus proteins have been purifi-
4–Guanidine-Neu5Ac 2en (GG167)
ed and separately inoculated into groups of experi-
The successful development of the few antiviral mental animals; the results of challenge studies have
compounds available has given enormous impetus indicated that immunity is induced by the host
to the development of further such compounds. responses to the virus HA and, to a lesser extent, to
Thus, compounds are being designed, tested and the NA. Some evidence suggests that the immune
developed at the present time, and are both ana- response to the M2 and the NP proteins may con-
logues of known antiviral compounds and novel tribute to immunity, but there is little evidence to
structures (Arruda and Hayden, 1996). These initi- date to suggest that these are major factors.
atives are particularly important since viral strains Studies to determine which immune responses
resistant to amantadine are easily generated. One correlate with protection against infection have in-
such compound to come from these initiatives is dicated that the serum antibody titre against the
4—guanidine-neu5Ac 2en (GG167); the structure of HA is the most important; thus, susceptibility to
this compound is shown in Figure 5.4. Since anti- influenza virus infection is inversely related to the
body against NA glycoprotein of influenza virus titre of serum HI antibody. This is true for both
provides partial protection against infection, and experimental challenge with attenuated virus and
the crystal structure of NA is known (Laver et al., natural infection with virulent viruses; and a serum
1984), compounds which specifically block NA ac- HI titre of approximately 30—40 represents 50%
tivity can be designed and synthesized: GG167 is protective level of antibody against infection by
such a compound, acting on a conserved pocket of homologous virus (Potter and Oxford, 1979). In
the NA of both influenza A and B viruses. The addition, the degree of cross-immunity for strains of
antiviral activity of GG167 can be shown in vitro, in virus of the same subtype is directly related to the
animal tests and in volunteer studies (Hayden et al., degree of cross-reacivity of the HA antigens: immu-
1996), and it is anticipated that the compound will nization with influenza virus confers no protection
be licensed for use in the near future. Virus strains against challenge infection with virus of a different
resistant to GG167 can occur; these have a reduced subtype, since there is no cross-reactivity of the HAs
dependence on NA activity and evolve slowly (Blick of the two viruses. In addition to conferring relative
et al., 1995), but the observation indicates the need protection against infection, serum HI antibody is
for further compounds in the future. Alternatively, reported both to reduce the severity of infection and
treatment strategies using a combination of GG167 decrease virus spreading from infected persons.
272 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 5.4 Response of volunteers to immunization with whole or ether-split influenza
virus vaccine and results of subsequent challenge infection
?Vaccine given subcutaneously in 0.5 ml volume, and containing equivalent concentration of virus HA;
control group given 0.5 ml saline only.
@10
egg infectious dose (EID) of live virus 1 month after immunization.
Similar studies have shown that serum neura- tent for subcutaneous inoculation. The final in-
minidase-inhibiting (NI) antibody also contributes oculum is a mixture of vaccines of all the prevalent
to protection against influenza virus infection. This virus strains of the year. Clinical studies with vac-
has been shown in studies with experimental ani- cine prepared in this manner have recorded some
mals and in observations of natural infections in local pain in approximately 20—30% of vaccinees,
humans. A generally held view is that the serum and systemic reactions such as fever, headache and
antibody is more important in determining immun- muscle pain in about 5% of persons; however, these
ity than the serum NI antibody. It is less clear responses are usually mild and ephemeral. The vac-
whether other immune responses to the virus HA cine induces serum HI and NI antibody responses
and NA antigens are important; local IgA antibody which confer protection on 60—90% of volunteers
against virus antigens probably contributes to im- against challenge virus infection (an example is
munity, but the importance of cellular immune re- shown in Table 5.4) and significant prevention of
sponses claimed by a number of authorities remains hospitalization of the elderly in subsequent epi-
to be established (McMichael et al. 1983). demics. The vaccine can be given annually, each
From the above, it is clear that an influenza vac- year the strains being changed to correspond to the
cine must contain surface HA and NA antigens of currently circulating virus; to young and elderly;
the virus in a form which will stimulate serum HI and in higher doses to the elderly who give relative-
and NI antibody, local IgA antibody and possibly ly poor immune responses to conventional vaccine
cellular immunity. It is essential that the vaccine (Keitel et al., 1996). The antibody response persists
contains the antigens of recently isolated virus at a protective level for 1—5 years, depending on the
strains: although some cross-reactivity and corre- vaccine virus strain and the age of the vaccinees;
sponding cross-immunity are seen between viruses however, subsequent infecting virus strains may
of the same subtype, the most solid immunity is show antigenic drift, and the vaccine-induced anti-
found following immunization with virus homo- body will be less effective in protecting against these
logous to the infecting strain. Thus, whatever the new strains. In contrast, the antibody response to a
type of vaccine used, the virus content should be vaccine containing virus of a new subtype against
annually reviewed, and changed when new variants which the vaccinees have had no past experience is
occur. relatively short lived, and 60—80% of the antibody
titre may have disappeared by 12 months after im-
munization.
Whole Virus Vaccines
Whole, inactivated virus vaccines are prepared by
Split Virus Vaccines
inoculating the currently circulating strain of influ-
enza virus into embryonated eggs. The allantoic Because of the relatively high incidence of reactions
fluids are harvested after 2—3 days incubation, cen- seen in vaccinees given whole, inactivated virus vac-
trifuged by zonal centrifugation to concentrate and cine, attempts have been made to produce a prod-
purify the virus particles, inactivated with formalin uct which is less reactogenic while preserving the
or -propriolactone and standardized by HA con- ability to induce satisfactory titres of serum anti-
INFLUENZA 273
Figure 5.5 Reactions of volunteers to immunization with influenza virus vaccines. (See also Table 5.4)
body. For this, virus pools grown, purified and sponse to whole, subunit-adsorbed or aqueous
inactivated as described in the previous section are subunit vaccines is similar; and since aqueous
treated with detergents to disrupt the virus par- vacines are less reactogenic, these are to be prefer-
ticles: inoculation of virus manufactured in this red (Table 5.5). However, when the population has
manner induces fewer reactions in volunteers than not been primed by previous exposure to viruses of
whole virus vaccine (Figure 5.5), and the serum the same subtype, the serum HI antibody response
antibody responses and protection afforded against to whole virus is significantly greater than for
subsequent challenge are similar (Table 5.4). For subunit vaccine (Table 5.5). But at these times, two
these reasons, many prefer split vaccines to whole doses of whole virus vaccine are needed: since satis-
virus vaccines. factory levels of antibody can be induced with two
doses of either whole or subunit vaccines, and the
latter are less reactogenic, these are again preferred.
Subunit Virus Vaccines
It must be mentioned that attempts to increase
Since only virus HA and NA antigens are required the immunogenicity of virus vaccine for humans by
to induce immunity, vaccines containing purified incorporating adjuvants have not to date identified
surface antigens and free of virus RNA and core a safe and suitable carrier. The best vaccines avail-
proteins have been investigated; these are used in able at the moment are aqueous subunit vaccines;
aqueous suspension, or may be absorbed to carriers but many authorities hold that these are not suffi-
such as cholera B toxin, ISCOMs, or with immune ciently potent for completely successful immuniz-
modulators, such as IL-2. These adjuvant subunit ation, and further developments are needed (Potter,
vaccines confer protection when given early to ex- 1982).
perimental animals (Scheefers and Becht, 1994;
Katz et al., 1997), but this has not been tested in
Live Virus Vaccines
humans: a review of adjuvants for human vaccine
by Gupta and Siber (1995) extends this subject. There is evidence that immunization with live, at-
Volunteers given aqueous subunit vaccines intra- tenuated influenza virus vaccines induces a more
muscularly experience fewer reactions than those solid immunity than inactivated vaccines. These
given whole virus vaccines or absorbed subunit vac- findings, the known shortcomings of inactivated
cine (Figure 5.6). In years of antigenic drift when the vaccines, the resistance of general practitioners and
population is primed, the serum HI antibody re- the public to immunization against influenza by
274 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
overcome. The development of a suitable influenza
virus vaccine initially involves the production and
purification of a suitable reassortant. Then, under
strictly controlled conditions, the virus must be
shown to be attenuated for humans and to be gen-
etically stable, since some vaccine virus strains in
the past have been found to revert to virulent vari-
ants when inoculated into volunteers. In a series of
developmental tests the vaccine virus must be
shown to be attenuated for various groups of volun-
teers of different ages and susceptibility, to induce
antibody responses, and to protect against chal-
lenge virus infection before it can be licensed for
general use. At the present time these studies are
estimated to take approximately 2 years to com-
plete: this makes their development impractical,
since by the time the vaccine can be made available,
the epidemic strain against which it has been pre-
Figure 5.6 Reactions of volunteers to immunization with influ- pared will probably have disappeared, to be re-
enza virus vaccines
placed by new strains requiring a new vaccine. Des-
injection and the reactogenicity to inactivated virus pite these setbacks, studies are continuing to find
vaccines have continued to encourage the develop- methods for the more rapid development of live
ment of live vaccines against this infection. Influ- vaccine; should a method be found that reduces the
enza viruses can be attenuated by serial passage in development period to 6—9 months, live influenza
embryonated eggs, by chemical mutation or by lab- virus vaccines, with all the attendant advantages,
oratory passage at reduced temperatures; using will become practical. Alternatively, if the long his-
these techniques virus can be produced which in- tory of efficacy and safety of reassortant vaccine
fects volunteers without producing appreciable cli- virus based on A/Ann Arbor/6/60 (H2N2) can be
nical illness. Unfortunately, these methods require a accepted, many of the protracted studies on safety
long and unpredicatable time to complete—prob- listed above may be considered unnecessary and the
ably too long for vaccine to be available for immu- development of these vaccines accelerated. No live
nization against current influenza variants. To cir- influenza virus vaccines are avilable for general use
cumvent this problem, attenuated strains produced in Western countries at the present time, and vac-
by one of the above methods have been mixed with cines are limited to one of the forms of inactivated
wild-type virus to produce reassortants which con- vaccines discussed above.
tain the RNA fragments coding for wild-type HA
and NA, and all the other genetic material from the
Other Approaches to Vaccine Development
attenuated strain. These recombinants can be pro-
duced relatively quickly in the laboratory and, In addition to the development of recombinant in-
when inoculated intranasally into volunteers, pro- fluenza virus as vaccines, several other approaches
duce few and mild symptoms, induce both serum are possible, and are being researched at the present
and local HI and NI antibody against the wild-type time. The gene coding for the HA glycoprotein can
virus and immunity to challenge virus infection be cloned into a variety of vehicles, such as
(Beare, 1982). One attenuated strain, A/Ann Arbor/ baculovirus, by genetic engineering, and can be in-
6/60 (H2N2) has been used to produce attenuated duced to express virus HA; large quantities of pure
vaccine reassortants for over two decades (Maassab virus HA can be produced for immunization by
and DeBorde, 1985); and in all studies these have these techniques. Recent studies have shown that a
been shown to be safe and effective, easily adminis- DNA copy of the virus RNA gene coding to virus
tered and suitable for all ages (Potter, 1994). HA can be cloned into Vaccinia virus; immuniz-
Some problems in the development of live at- ation with this material induced serum HI antibody
tenuated influenza virus vaccines have not been and cellular immunity to the virus HA. Fears that
INFLUENZA 275
Table 5.5 Response of volunteers to immunization with inactivated influenza vaccines
Postimmunization
Previous Nature of influenza A
experience? (H1M1) vaccine given Preimmunization 1 dose 2 doses
?Older subjects who had been exposed to influenza A (H1N1) viruses in 1933—1957 (primed) and
subjects born after 1957 (unprimed).
Number of subjects in each group varied from 12 to 30.
Vaccinia virus is too dangerous a vector for immu- few reactions, and such reactions are usually mild
nization of humans against influenza are based on and of relatively short duration; they produce
reactions of vaccinees to this virus when used in the serum antibody in the majority of subjects, and
past for immunization against smallpox; however, it immunity to infection in 60—90% of vaccinees. Vac-
is suggested that the above virus construct is less cine is recommended for elderly and ‘at risk’ per-
pathogenic than native Vaccinia virus. Again, sons for whom influenza is a threat to life. In addi-
cDNA derived from virus RNA can be selectively tion, key personnel of industry and social services
mutated and then rescued from transfected cells should be offered vaccine, particularly when new
with helper virus (Parkin et al., 1996): these deletion subtypes emerge. For the remainder of the popula-
mutants cannot revert to virulent virus and are tion, immunization against influenza is a debatable
therefore safe, attenuated vaccines. Vaccines can be subject: some authorities hold that vaccines should
constructed as DNA plasmids containing the DNA be available for all and used according to the wishes
copy of the virus RNA coding for the virus HA of doctors and patients, while others protest that
(Ulmer et al., 1996): these species induce serum anti- the widespread use of vaccine is not justified.
body and cellular immunity and protection in ani-
mal studies, and are of great current interest. Final-
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Parainfluenza Viruses
Donald M. McLean
University of British Columbia, Vancouver, Canada
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
280 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
envelope proteins. After replication within the
cytoplasm, mature virus particles are released by
budding.
Inhibitors of protein synthesis block viral
genome replication but not transcription, and
carbohydrate analogues interfere with glycosyla-
tion of virion proteins. Inhibitors of DNA replica-
tion or transcription have no direct effects on para-
influenza virus replication.
Biological Properties
Toronto MelbourneC
NewcastleA WashingtonB SeattleD Chapel HillE
Serotype 1960—63? 1964—67@ 1969—70 1957—61 ’81—’82 ’85—’86 1966—71 1966—75
*Represents 189 strains of parainfluenza types 1, 2 and 3; proportions of each were not stated.
?McLean et al. (1963); @McLean et al. (1967); AGardner et al. (1971); BParrott et al. (1962); Fairfield Hospital (1982, 1986); DFoy et al. (1973); EChapman et al.
(1981).
Table 6.2 Parainfluenza virus isolates from children with acute lower respiratory infections
isolated have shown rising antibody titres against firmed among otherwise healthy children aged un-
the homologous serotype during convalescence, der 5 years in Nashville, Tennessee, between 1974
which confirms that virus infection occurred at the and 1993 (Reed et al., 1997). Conversely, respiratory
time of illness. syncytial virus, which is a major aetiological agent
A strong association between parainfluenza vi- in bronchiolitis and pneumonia, is found in a rela-
ruses and croup, but infrequent isolations of parain- tively small proportion (1.0—10.6%) of croupy pa-
fluenza viruses from other lower respiratory tract tients.
infections, is demonstrated clearly in hospitalized Highest age-specific attack rates for croup were
patients at Newcastle upon Tyne and Washington observed regularly in children aged less than 3
DC, and in ambulatory patients in Seattle and years, both in the above centres and elsewhere.
Chapel Hill (Table 6.2). This association was con- Highest parainfluenza virus isolation rates were ob-
284 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
served in that age group. For example, in Toronto
between November 1962 and March 1963, parain-
fluenza viruses were isolated from 92 of 224 croupy
children and influenza A from an additional three
children (total rate 42%). Of the 166 children aged
less than 3 years, 46% yielded viruses, and, of the 58
children older than 3 years, 33% yielded viruses
(McLean et al., 1963).
Parainfluenza 1 and parainfluenza 3 viruses have
been isolated virtually in every month of the year,
when routine surveillance is pursued for three or
more years. In Toronto, these serotypes were iso-
lated during every month except June and July, and
similar observations have been recorded from
centres including Washington DC, Seattle and Figure 6.3 Acquisition of parainfluenza neutralizing antibo-
dies by age. (Adapted from Parrott, R.H. et al. (1962) American
Nashville. However, in each location, abrupt in- Journal of Public Health, 52, 907—917)
creases in monthly virus isolation rates, accom-
panied by substantially increased clinical attack demonstrated for infants in Houston, Texas, with
rates of croup, occur either early or late in the cord blood antibody titres 9 256 (Glezen et al.,
winter season, between October and February in 1984).
the northern hemisphere and the corresponding Family studies at Tecumseh, Michigan, between
winter months April through August in the south- 1976 and 1981 (Monto et al., 1986) have shown a
ern hemisphere. Frequently, parainfluenza infec- sixfold higher isolation rate of parainfluenza viruses
tions associated with croup precede, by several among children aged : 5 years than in older sub-
weeks, an increase in incidence of respiratory syn- jects and 50—80% of virus excreters of all ages ex-
cytial virus infections inducing bronchiolitis and perienced upper respiratory infections, but about
pneumonia. However, in some localities during par- twice as many infected children aged : 5 years
ticular years, both categories of paramyxovirus may experienced fever than older subjects. Peak occur-
circulate simultaneously. rence of parainfluenza 1 infections was noted during
Parainfluenza 1 neutralizing antibodies have September and October; parainfluenza 3 infections
been found in sera from 48% of infants aged 0—5 were more evenly distributed throughout winter.
months at a Washington DC shelter, which de- Surveillance studies at Houston, Texas, between
clined to 4% at 6—12 months (Parrott et al., 1962). 1975 and 1980 revealed that a higher proportion of
Antibody rates increased steadily with advancing parainfluenza-associated respiratory infections
age to 74% among children aged 9 4 years (Figure among children aged : 5 years were due to parain-
6.3). Comparable trends were observed for neu- fluenza 3 virus (66%) than parainfluenza 1 virus
tralizing antibodies to parainfluenza 2 and parain- (25%) (Glezen et al., 1984) and this age group com-
fluenza 3 viruses, but in each age category the high- prised 69% of all parainfluenza virus-positive cases.
est antibody acquisition rates were against para- Although parainfluenza 3 virus infections remained
influenza 3. Antibodies during the initial 5 months endemic during all months of 1975 and 1976, they
of life were acquired transplacentally from mothers, showed peaks of prevalence during March (spring)
and during subsequent months by active virus infec- of each year 1977—1980, in contrast to parainfluenza
tion. Parainfluenza 3 neutralizing antibody at low 1 infections which peaked in January (winter) of
titres 8—32 conferred about a twofold reduction of each year. Family studies showed that parain-
the rates of development of febrile illness and isola- fluenza 3 virus induced about 67 infections per 100
tion of virus following a subsequent infection with child-years in infants aged : 2 years, and this rate
the same serotype, in contrast to rates for decreased to 16 per 100 child-years by age 5 years.
seronegative infants with titres : 8; high titres However, only 11% of infants aged : 1 year devel-
64—1024 were associated with a further twofold re- oped lower respiratory disease such as croup or
duction of rates for illness and virus isolation. Simi- bronchiolitis, but 21% aged 1—2 years developed
lar protective effects of maternal antibody were these complications. Second or subsequent infec-
PARAINFLUENZA VIRUSES 285
Table 6.3 Laboratory diagnosis of respiratory tract infections usually associated with parainfluenza viruses
Virus identification
CF : complement fixation; ELISA : enzyme-linked immunosorbent assay; HI : haemagglutination inhibition; n/p : nasopharyngeal secretions
obtaied by suction; NT : neutralization in tissue culture; throat : throat gargling or swab.
?Also detects influenza virus (haemadsorption), enterovirus and rhinovirus (cytopathic effect).
@If respiratory syncytial virus is suspected by immunofluorescence, use diploid human cells also.
AIf parainfluenza or influenza viruses are suspected by immunofluorescence, use monkey kidney cells also.
tions with parainfluenza 3 virus were observed tion of parainfluenza 1 (77 isolates) and parain-
twice as commonly among infants aged 1—2 years fluenza 2 (33 isolates) was confined to April through
without detectable neutralizing antibody (titre : 8) December, with peak isolation rates in autumn dur-
than in those with titres elevated to 128, and this ing October. Age-specific infection rates for parain-
difference persisted through age 2—3 years. fluenza 3 virus were highest in children aged 0—3
Parainfluenza 3 virus excretion in throats of years, while parainfluenza 1 and parainfluenza 2
Houston children with acute respiratory infections viruses showed no significant age-specificity. Rein-
persisted longer than other respiratory viruses. fections with parainfluenza 3 virus were observed in
Parainfluenza 3 virus was detected in one or more 19 children after intervals of 6 months to 3 years
specimens each day among nine of 26 (35%) throat (average 1.36 years); no reinfections were noted after
secretions collected up to 6 days before onset of infection with parainfluenza 1 or parainfluenza 2
illness and on each day subsequent among 60 of 125 viruses.
(48%) secretions obtained 0—10 days after onset
(Frank et al., 1981). After 10 days, parainfluenza 3
Parainfluenza Virus Infections in Adults
virus was excreted intermittently until 19 days. In
four children whose throat secretions yielded para- Respiratory tract illnesses affecting adults at
influenza 3 virus 4 days before to 5 days after onset McMurdo Station, Antarctica, between 15 October
of respiratory illness, virus was again detected as and 15 November 1975, were attributed principally
late as days 29—32. Excretion of influenza A and to parainfluenza virus infections (Parkinson et al.,
respiratory syncytial virus, however, was confined 1979). Of 39 personnel with respiratory illnesses,
almost exclusively to the initial 7 days after onset of five viral isolates (from four subjects) were parain-
respiratory illness. fluenza 1 and three isolates were parainfluenza 3.
Longitudinal studies at Nashville, Tennessee be- The only additional isolates were strains of
tween 1974 and 1993 revealed 286 (5.6%) parain- rhinovirus. Respiratory infections began shortly
fluenza virus isolates among 5099 otherwise healthy after the arrival of successive groups of personnel
children aged 0—5 years who developed acute respir- who had been transported directly by air from the
atory infections. These included 66% with croup, USA, commencing during September 1975. Parain-
22% with lower respiratory illness and 18% with fluenza infections were prevalent in USA at that
upper respiratory illness (Reed et al., 1997). Parain- time. Between April and August 1975, when Antarc-
fluenza 3 virus (157 isolates) were achieved during tic personnel received no visitors or supplies from
every month of the year, with peak isolation rates in the outside world, no respiratory tract illnesses were
spring from March through May. However, detec- recorded. These observations demonstrated: (1)
286 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
that parainfluenza viruses were important aeti- pharyngeal secretions from children aged less than
ological agents of respiratory infections among 5 years by the Auger suction technique (McLean
Antarctic personnel; (2) the necessity of continuing and Wong, 1984). Briefly, a fine-bore catheter is
to introduce parainfluenza viruses into remote passed through a nostril into the nasopharynx. Se-
communities in order to maintain their trans- cretions are aspirated using suction by a 20 ml or
mission between personnel. 50 ml capacity syringe attached to the external end
of the catheter. For older children and adults, pa-
tients gargle 5—10 ml of 0.15 mol L\ saline for 1
DIAGNOSIS minute, and expectorate the fluid into suitable
screw-capped or snap-capped containers. Throat
Principles swabs provide less satisfactory specimens for vi-
rological diagnosis, and they should be collected
Laboratory diagnosis depends upon the selection only when it is not feasible to obtain garglings or
and application of a combination of virological nasopharyngeal suctions.
tests appropriate to the infective agent inducing the
syndrome. Although croup is a well-defined, easily Preparation for Tests
recognized clinical entity, and despite the 80% or
better likelihood that the causative virus is parain- For rapid non-cultural tests, microdrops of secre-
fluenza in most communities, the capability of any tions or garglings are placed directly on carbon-
test system must be sufficiently broad to include coated electron microscope grids (300 mesh) or
additional agents such as influenza and respiratory swabs are smeared directly on grids, which are dried
syncytial viruses. Conversely, although bronchioli- in air and stained for 30 seconds with 2% phos-
tis in children is regularly caused by respiratory photungstic acid. Cellular deposits after centrifu-
syncytial viruses, a few cases may be induced by gation of secretions or garglings are applied to each
parainfluenza virus (Table 6.2). cup of 12—cup Teflon-coated slides, or throat swabs
Virological diagnosis involves three principal are smeared directly over cups (Figure 6.4). Slides
categories of test (Table 6.3): (1) rapid non-cultural are dried on a warming plate at 40°C, and fixed for
(morphological) tests employ optimally a combina- 10 minutes in ice-cold acetone. If the laboratory is
tion of electron microscopy plus immunofluores- remote from the patients, these samples may be
cence (McLean and Wong, 1984), or immuno- stored at ambient temperatures for several days, or
fluorescence alone (Gardner and McQuillin, 1980); shipped unrefrigerated by postal services or
these provide a serotype-specific diagnosis of the common carriers.
infecting virus within 3 hours after receipt of For virus isolation tests, secretions or swabs
nasopharyngeal secretions or throat swabs in the should be suspended immediately in 2 ml tissue cul-
laboratory; (2) virus isolation tests, using tissue cul- ture maintenance medium containing 10% inac-
ture techniques and subsequent serotyping of iso- tivated fetal bovine serum or 0.75% bovalbumin
lates, require 3—10 days for completion (McLean, and held at 4°C until inoculated into tissue cultures
1982); (3) serological tests on paired sera are com- within the ensuing 24 hours. If longer transit or
pleted within 2 hours to 7 days after receipt of holding times are likely, the suspensions should be
convalescent-phase sera, collection of which must placed in screw-capped or snap-capped vials and
be delayed until 1—3 weeks after onset (McLean, held in refrigerated cabinets at 9 60°C until tested.
1982). These principles are consistent with recent Serological tests require the collection of two
statements by an Expert Committee of the World blood samples. The early (acute phase) sample is
Health Organization (1981). collected aseptically by venepuncture as soon as
possible after initial examination of the patient,
which is usually 1—2 days after onset of the illness.
Specimens The later (convalescent phase) sample is collected
1—3 weeks subsequently, when fever and other
symptoms have resolved completely. Sera should be
Collection
separated from clots within a few hours after collec-
Every attempt should be made to collect naso- tion of blood samples, and stored at 9 20°C to
PARAINFLUENZA VIRUSES 287
from virus-positive patients. Figure 6.1 shows an
enveloped virion of 140 nm total diameter with an
outer coat comprised of spikes of haemagglutinin
and neuraminidase 12—15 nm high present in the
nasopharyngeal secretions of a patient with bron-
chiolitis.
Immunofluorescence
Antisera to each seroptype of parainfluenza and
influenza virus prepared in rabbits, and to RS virus
prepared in guinea-pigs, are diluted appropriately
in phosphate-buffered saline (PBS) pH 7.0 (usually 1
in 10), and microdrops are added to each pair of
wells in Teflon-coated acetone-fixed slides contain-
ing the patient’s specimens. Slides are incubated at
37°C for 30 minutes in a humidified atmosphere
and washed three times with PBS; each cup is over-
laid with a microdrop of the appropriate fluor-
escein-labelled antirabbit or antiguinea-pig anti-
serum diluted 1 in 20. Slides are incubated for an
additional 30 minutes, washed three times with
PBS, counterstained for 1 minute with 0.01% naph-
thalene black in PBS, then washed three times in
PBS and examined by incident light fluorescence
microscopy. Immunofluorescent foci in the cyto-
plasm of cells treated with rabbit anti-parainfluenza
1 serum followed by antirabbit serum, but not in
cells which received other antiviral serum, indicate
that the patient was infected with parainfluenza 1
virus. This result, which is serotype specific, is ob-
tained within 3 hours of receipt of specimens, thus
Figure 6.4 Immunofluorescence for detection of virus in throat
secretions. (Reproduced with permission from McLean, D.M.
providing same-day confirmation of preliminary re-
Virology in Health Care, Figure 3.5, p 38, © 1980 Williams & sults obtained by electron microscopy.
Wilkins, Baltimore) When an accurate virological diagnosis is
achieved by same-day non-cultural techniques it
may be possible to discharge the patient one or
await testing. Provided that transit time does not
more days earlier than those without demonstrable
exceed 2—3 days, sera may be shipped between insti- viral aetiology if management of altered respiration
tutions unrefrigerated. is not required. Studies at the University of British
Columbia show the benefit—cost ratio to be 12:1,
even if only a single day of hospitalization is saved.
Rapid Non-cultural Tests
Electron Microscopy
Virus Isolation
Electron microscopic examination of grids is per-
formed immediately after negative staining with Nasopharyngeal or throat specimens are suspended
phosphotungstic acid. Within 5—15 minutes, virions in 2 ml of tissue culture maintenance medium con-
morphologically typical of the Paramyxoviridae are taining 10% fetal bovine serum, centrifuged at
observed in throat or nasopharyngeal secretions 2000g for 10 minutes to deposit bacteria and de-
288 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
squamated cells, and supernatants are inoculated restricts severely the clinical usefulness of virus iso-
into tissue cultures of primary monkey kidney and lation in the management of patients. However,
continuous diploid human fibroblasts. It is advis- when viral concentration in nasopharyngeal secre-
able to incubate duplicate tissue cultures at both tions is too low for detection by electron micro-
33°C and 37°C, because some influenza and parain- scopy or immunofluorescence, virus isolation tech-
fluenza viruses replicate optimally at the lower tem- niques are essential to identify the virus causing the
perature. Haemadsorption is performed after ap- patient’s infection.
proximately 3, 7 and 10 days of incubation by the Experience in Arizona between 1983 and 1985
addition of 0.1% suspensions of guinea-pig and (Ray and Minnich, 1987) has shown that immuno-
avian erythrocytes in 0.15 mol L\ sterile saline to fluorescence detected parainfluenza 1 in only 63%
each tissue culture cup from which the supernatant of throat specimens from which this serotype was
fluid has been removed aseptically by pipetting. isolated in tissue cultures, and parainfluenza 3 in
When haemadsorption is observed, the haemag- 31% virus culture-positive throat secretions.
glutinin titre of virus in the supernatant fluid is Although libraries of cDNA clones of mRNAs for
determined by serial twofold dilutions of 0.025 ml the nucleocapsid protein, nucleocapsid phospho-
volumes in 96—cup plastic microplates using 0.5% protein, matrix protein, haemagglutinin-neura-
suspensions in 0.15 mol L\ saline of guinea-pig or minidase glycoprotein, fusion glycoprotein and
avian erythrocytes. sundry non-structural proteins of parainfluenza 3
Serotyping of the viral isolate is performed by virus (Spriggs and Collins, 1986) have been con-
mixing of antisera to each parainfluenza and influ- structed through recent developments in molecular
enza serotype, diluted to contain 10 antibody units virology, selection and adaptation of appropriate
per 0.025 ml, with the fresh viral isolate diluted to cDNA base sequences for use as probes for detec-
contain four haemagglutinating doses. Typing anti- tion of parainfluenza 3 virus in throat secretions
sera should be treated shortly beforehand with heat from patients must await future technical refine-
and periodate or receptor-destroying enzyme to re- ments.
move inhibitors. Serum—virus mixtures are held at
bench temperature (22°C) for 10 minutes before the
addition of 0.5% suspensions of erythrocytes. Inhi-
bition of haemagglutination after mixture with anti- Serology
parainfluenza 1 serum, but not with antisera to
other serotypes, indicates that the new isolate is Paired sera should always be examined simulta-
parainfluenza 1 virus. This serotyping procedure neously against currently circulating parainfluenza
can be completed within about 2 hours. viruses, most conveniently by HI tests. Acute-phase
Alternatively, the serotype of the fresh isolate sera should be collected 1—2 days after the onset of
may be determined by immunofluorescence. After croup or other signs of acute respiratory infection;
aspiration of fluid from tissue culture cups showing convalescent-phase sera are collected 1—3 weeks
positive haemadsorption reactions, tissue culture subsequently. After treatment with heat and per-
cells are removed by forcible pipetting and deposi- iodate or receptor-destroying enzyme, serial two-
ted in wells of Teflon-coated immuno-fluorescence fold dilutions of patients’ sera are examined for their
slides. After drying and acetone fixation, antisera to ability to inhibit haemagglutination by four haema-
each paramyxovirus serotype, followed by fluor- gglutinating doses of each parainfluenza serotype.
escein-labelled anti-species antibody, are applied. Antibody conversions from negative to positive, or
Examination by incident fluorescence microscopy at least a fourfold increase of antibody titre, indicate
is performed, as for throat secretions (page 287). current virus infection. Although the performance
This serotyping procedure is completed within 3 time of this test can be as short as 2 hours, the
hours. clinical usefulness of this test is limited due to the
Although virus isolation and serotyping are de- obligatory delay in collection of the convalescent-
sirable to confirm the virological identification al- phase serum. Furthermore, the appearance of anti-
ready obtained within 3 hours by rapid non-cul- body in sera of young infants following initial para-
tural techniques, the prolonged incubation time influenza virus infections is somewhat erratic; anti-
(3—10 days) to detect the presence of virus growth body may be detected only after repeated infections.
PARAINFLUENZA VIRUSES 289
Neutralizing antibodies may be detected in pa- in this example, the benefit—cost ratio for the result
tients’ sera by absence of haemadsorption in tissue of a rapid non-cultural technique in reducing by
cultures after 3 days incubation following inocula- one day the duration of hospitalization of a patient
tion of mixtures of serial twofold dilutions of pa- exceeds 12:1.
tients’ sera with 100 tissue culture-infective doses of
prototype parainfluenza strains. Appearance and
persistence of neutralizing antibodies coincide with
MANAGEMENT
that for HI antibodies.
Complement-fixing antibody titres to parain-
Appropriate clinical management of patients de-
fluenza viruses usually appear in patients’ sera
pends upon their rapid and accurate clinical assess-
somewhat later than HI and neutralizing antibo-
ment, combined with presumptive or serotypic
dies. Although this technique is useful in epidemiol-
identification of the causative virus using same-day
ogy (Parrott et al., 1962), its relevance to the diag-
diagnostic tests. Most parainfluenza virus infec-
nosis of acutely ill patients is minimal.
tions of infants and children manifest clinically as
Serological investigation of a community for evi-
acute laryngotracheobronchitis (croup), but some
dence of parainfluenza virus infection has been
patients may develop tracheobronchitis, bron-
undertaken by radioimmunoassay (RIA) (Parkin-
chiolitis or pneumonia. Young children are usually
son et al., 1979) with results comparable to those
nursed in plastic tents supplied with cool,
obtained by the more convenient, rapid and inex-
moistened oxygen (croupette), where they are held
pensive HI technique. The enzyme-linked im-
for 1—2 days until their respiration becomes quiet
munosorbent assay (ELISA) technique also pro-
and unlaboured. Severe respiratory obstruction
vides results comparable to HI tests, but the ELISA
may require endotracheal intubation followed by
test requires a spectrophotometer for reading re-
tracheotomy to establish a satisfactory airway. Ac-
sults; moreover, 4—6 hours are required for its per-
cumulation of excessive tracheobronchial secre-
formance, in contrast to 2 hours for HI tests which
tions in severe bronchiolitis and pneumonia may
require no sophisticated instrumentation.
necessitate their removal by emergency broncho-
scopic aspiration. Antibiotics are not normally pre-
scribed in croup, tracheobronchitis or bronchiolitis,
but appropriate antibiotics may be required if bac-
Economic Factors
teriological investigations of these conditions or
pneumonia reveal concomitant infections with hu-
Fees for the performance of virus diagnostic tests
man bacterial pathogens.
were established most recently on 1 April 1991 by
the Medical Services Plan of the Province of British
Columbia, Canada, and provide an example of the
benefits of laboratory diagnosis of parainfluenza PREVENTION
virus infection. They are based on detailed cost-
accounting of supplies, equipment, technical and Spread of parainfluenza virus infections within hos-
professional components of each test. Virus identifi- pitals is well documented. In Newcastle upon Tyne
cation by rapid non-cultural techniques costs between November 1970 and October 1972, parain-
$44.50, virus isolation in tissue culture including fluenza virus infections were identified in 19
serotyping of the isolates costs $81.00 and serologi- children who were contacts on paediatric wards of
cal tests on a pair of sera cost $42.20. These fees 134 patients who were admitted to hospital with
contrast with the current per diem charge of $620.00 respiratory infections due to parainfluenza viruses
for acute care at Vancouver Hospital: University of (Gardner et al., 1973). In one episode, clinically
British Columbia Site in Vancouver. When an accu- manifest and virologically confirmed parainfluenza
rate virological diagnosis is achieved by same-day 1 virus infections developed in three paediatric
non-cultural techniques, patients may be dis- ward contacts of the index case, and also the attend-
charged from hospital one or more days earlier than ing house physician who subsequently carried the
those without demonstrable viral aetiology, pro- infection to a surgical patient in a different ward.
vided that a croupette is no longer required. Thus, However, in Newcastle, and also in Rochester, New
290 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
York (Hall, 1981), respiratory synctial and influenza REFERENCES
viruses continue to cause a higher proportion of
virus-induced nosocomial respiratory infections Banatvala, J.E., Anderson, T.B. and Reiss, B.R. (1964) Parain-
than parainfluenza viruses. fluenza infections in the community. BMJ, 1, 537—540.
Dissemination of parainfluenza 1 virus by air- Chanock, R.M., Parrott, R.H., Vargosko, A.J. et al. (1962) Acute
borne droplets was demonstrated on a croup ward respiratory diseases of viral aetiology IV. Respiratory synctial
virus. American Journal of Public Health, 52, 918—925.
in Toronto (McLean et al., 1967). Parainfluenza 1 Chapman, R.S., Henderson, F.W., Clyde, W.A. Jr et al. (1981)
virus (3 TCD ) was isolated from 150 litres of air The epidemiology of tracheobronchitis in pediatric practice.
sampled from a croupette which housed a croupy American Journal of Epidemiology, 114, 786—797.
child who excreted 300 TCD ml\ in his naso- Coelingh, K.L.V., Winter, C.C., Tierney, E.L. et al. (1988) Attenu-
ation of bovine parainfluenza virus type 3 in non-human
pharyngeal secretions in January 1967; 10 TCD
primates and its ability to confer immunity to human parain-
parainfluenza 1 virus was recovered from 150 litres fluenza virus type 3 challenge. Journal of Infectious Diseases,
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10 000 TCD parainfluenza 1 per ml nasopharyn- Dick, E.C., Mogabgab, W.J. and Holmes, B. (1961) Characteris-
tics of parainfluenza 1 (HA-2) virus. I. Incidence of infections
geal secretions during February 1967.
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In the absence of effective antiviral agents and 73, 262—272.
vaccines against parainfluenza virus infections, Fairfield Hospital, Melbourne, Australia, Annual Report, 1982,
limitation of nosocomial spread of these viruses 1986.
should be attempted along the following lines Foy, H.M., Cooney, M.K., Maletzky, A.J. et al. (1973) Incidence
(McLean et al., 1967; Gardner et al., 1973). Paediat- and etiology of pneumonia, croup and bronchiolitis in pre-
school children belonging to a prepaid medical care group
ric patients who are admitted to hospital with croup over a four-year period. American Journal of Epidemiology, 97,
should be nursed in wards, preferably with cubicles 80—92.
or barriers, which are reserved for that syndrome Francki, R.I.B., Fauquet, C.M., Knudson, D.L. et al. (1991) Clas-
alone. Patients with other acute respiratory infec- sification and nomenclature of viruses: fifth report of the Inter-
tions such as bronchiolitis should also be nursed in national Committee on Taxonomy of Viruses. Archives of
Virology, Suppl. 2.
separately designated areas. Avoid, if possible, ad- Frank, A.L., Taber, L.H., Wells, C.R. et al. (1981) Patterns of
missions to wards containing paediatric patients shedding of myxoviruses and paramyxoviruses in children.
with respiratory infections those patients with con- Journal of Infectious Diseases, 144, 433—441.
ditions which predispose to parainfluenza infec- Gardner, P.S. and McQuillin, J. (1980) Rapid Virus Diagnosis:
Application of Immunofluorescence, 2nd edn. Butterworth,
tions such as cystic fibrosis and immunosuppressive
London.
states. Promote the dilution of virus below infective Gardner, P.S., McQuillin, J., McGuckin, R. et al. (1971) Observa-
limits, in expired air adjacent to patients with re- tions on clinical and immunofluorescent diagnosis of parain-
spiratory infections, through more frequent ex- fluenza virus infections. BMJ, 2, 7—12.
changes of air by more adequate ventilation, with Gardner, P.S., Court, S.D.M., Brocklebank, J.T. et al. (1973)
Virus cross-infection in paediatric wards. BMJ, 2, 571—575.
discharge of room air to the exterior. Glezen, W.P., Frank, A.L., Taber, L.H. et al. (1984) Parainfluenza
Prophylaxis against parainfluenza virus infec- virus type 3: seasonality and risk of infection and reinfection in
tions in humans through vaccines must await future young children. Journal of Infectious Diseases, 150, 851—857.
developments. Bovine parainfluenza 3 virus evoked Hall, C.B. (1981) Nosocomial viral respiratory infections: peren-
serotype specific antibody responses and protection nial weeds on pediatric wards. In Nosocomial Infections (ed.
R.E. Dixon), pp 227—233. Yorke Medical Books, New York.
against subsequent challenge with human parain- Heilman, C.A. (1990) Respiratory syncytial and parainfluenza
fluenza 3 virus in non-human primates (Coelingh et viruses. Journal of Infectious Diseases, 161, 402—406.
al., 1988). Envelope glycoproteins of parainfluenza McKinney, R.W., England, B.L. and Froede, S. (1959) Studies
2 virus solubilized selectively with actylglucoside with haemadsorption virus type 1. I. Recovery from two cases
have induced high-titre serotype-specific antibody of influenza-like disease in military personnel and related in-
vestigations. American Journal of Hygiene, 70, 280—296.
responses in serum and respiratory secretions of McLean, D.M. (1982) Immunological Investigation of Human
hamsters after intranasal immunization and pro- Virus Diseases. Vol. 5, Practical Methods in Clinical Immu-
tected completely against challenge with live para- nology (series ed. R.C. Nairn). Churchill Livingstone, Edin-
influenza 2 virus (Ray et al., 1990). Comparable burgh.
serotype-specific responses were observed with McLean, D.M. (1988) Virological Infections. Thomas, Springfield
IL.
parainfluenza 3 envelope glycoproteins (Ray et al., McLean, D.M. and Wong, K.K. (1984) Same-day Diagnosis of
1988).
PARAINFLUENZA VIRUSES 291
Human Virus Infections. CRC Press, Boca Raton FL. Ray, R., Glaze, B.J., Moldoveanu, Z. et al. (1988) Intranasal
McLean, D.M., Bach, R.D., Larke, R.P.B. et al. (1963) immunization of hamsters with envelope glycoproteins of hu-
Myxoviruses associated with acute laryngotracheobronchitis man parainfluenza virus type 3. Journal of Infectious Diseases,
in Toronto, 1962—63. Canadian Medical Association Journal, 157, 648—654.
89, 1257—1259. Ray, R., Matsuoka, Y., Burnett, T.L. et al. (1990) Human parain-
McLean, D.M., Bannatyne, R.M. and Givan, K.F. (1967) fluenza virus induces a type-specific protective immune re-
Myxovirus dissemination by air. Canadian Medical Associ- sponse. Journal of Infectious Diseases, 162, 746—749.
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Monto, A.S., Koopman, J.S. and Bryan, E.R. (1986) The Tecum- and clinical impact of parainfluenza virus in otherwise healthy
seh study of illness. XIV. Occurrence of respiratory viruses, infants and young children : 5 years old. Journal of Infectious
1976—1981. American Journal of Epidemiology, 124, 359—367. Diseases, 175, 807—813.
Parkinson, A.J., Muchmore, H.G., Scott, L.V. et al. (1979) Para- Rendtorff, R.C., Walker, L.C. and Roberts, A.N. (1963) A parain-
influenza virus upper respiratory tract illnesses in partially fluenza-3 outbreak in an orphanage nursery. American Jour-
immune adult human subjects. A study at an Antarctic station. nal of Hygiene, 77, 82—97.
American Journal of Epidemiology, 110, 753—763. Spriggs, M.K. and Collins, P.L. (1986) Human parainfluenza
Parrott, R.H., Vargosko, A.J., Kim, H.W. et al. (1962) Respir- virus type 3: messenger RNAs, polypeptide coding assign-
atory diseases of viral etiology. III. Myxoviruses parain- ments, intergenic sequences and genetic map. Journal of Virol-
fluenza. American Journal of Public Health, 15, 907—917. ogy, 59, 646—654.
Ray, C.G. and Minnich, L. (1987) Efficiency of immunofluores- World Health Organization (1981) Rapid laboratory techniques
cence for rapid detection of common respiratory viruses. Jour- for the diagnosis of viral infection. Report of a WHO scientific
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
294 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
between G proteins of the two strain groups is
: 55%, and the antigenic relatedness is only 3—7%.
The F protein, on the other hand, is relatively con-
served, with prototype A and B strains exhibiting
greater than 90% amino acid homology.
In tissue culture RSV produces a characteristic
syncytial appearance with eosinophilic cytoplasmic
inclusions. RSV has a growth cycle which, after
inoculation, consists of a period of adsorption of 2
hours, followed by an eclipse period of 12 hours.
The subsequent log phase of replication of the new
virus lasts for approximately 10 hours. At the time
when maximal quantities of virus are obtained,
about half of the virus remains cell-associated on
the surface of the cell. Under certain conditions,
such as repeated high passage, persistent infection
Figure 7.1 Negative contrast electron micrograph of RSV. The may develop which is associated with a diminished
fringed envelope is variable in shape. The surface projections are amount of cell-free virus and a loss of the character-
about 15 nm long and the envelope contains a helical nucleocap- istic cytopathic effect.
sid which may occasionally be visible. (Negative contrast 3%
potassium phosphotungstate, pH 7.0; ; 200 000; courtesy of
Professor C.R. Madeley)
Figure 7.3 Pathological findings of an infant dying with RSV bronchiolitis. The wall of the bronchiole appears inflamed and
oedematous, and sloughed, necrotic material fills the lumen in the small airway
invariably and often abundantly present, have en- the lower respiratory tract disease of infancy has
gendered speculations that immune mechanisms also been postulated. The inactivated RSV vaccine
may actually contribute to the pathogenesis of the produced not only serum antibody, but also an
disease. An immune complex reaction between ma- aggravated systemic cell-mediated immunity re-
ternally acquired IgG antibody and viral antigen sponse. Stimulation of T cell responses by RSV or
has been hypothesized as occurring in the lungs of by vaccinia virus recombinants carrying RSV genes
severely affected infants. Such a mechanism has also in experimental animals has produced both the
been suggested by the experience with the inac- benefit of enhanced viral clearance and the detri-
tivated RSV vaccine developed in the 1960s. This mental result of pathologic lung inflammation
vaccine evoked high levels of circulating antibody, (Connors et al., 1994).
but when the vaccinees were subsequently exposed The third type of immune reaction which has
to the wild virus they developed more severe disease been suggested is one involving IgE antibody. In
than did those children who had not received the this theory bronchiolitis results from an allergic
RSV vaccine. This theory, however, does not ex- response, mediated by specific IgE generated by
plain the observation that severe disease may occur previous sensitization to the virus. Specific IgE anti-
in infants who no longer possess detectable anti- body has been found on epithelial cells in the nasal
body. Furthermore, a number of studies have sug- secretions of infants with RSV infection. In some
gested that maternal antibody may actually be pro- studies infants with wheezing tended to have higher
tective. Also, complement levels do not decline levels of IgE antibody and histamine in their secre-
during acute infection, as would be expected during tions (Welliver and Duffy, 1993). However, in other
an immune-complex process. However, antibody studies only low concentrations of RSV IgE anti-
may contribute to disease by other mechanisms, body were found in the acute and convalescent
such as by suppressing the infant’s own immune phases of infants with RSV infection, and these
response to integral proteins, or by augmenting levels did not differ from those found in control
viral replication (Toms, 1995). In vitro infection of infants without viral infection (Toms et al., 1996).
macrophages has been enhanced by antisera to the The phenomenon observed following the use of
F and G proteins (Krilov et al., 1989). the inactivated RSV vaccine has been studied by the
A cell-mediated immune reaction occurring in use of animal models (Vaux-Peretz et al., 1992; Con-
298 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
nors et al., 1994). Rodents challenged with live RSV ripheral airways is much larger than in older
after immunization with formalin-activated vaccine children.
develop an enhanced pulmonary pathology similar The normal immune response to RSV infection
to that observed in the children who received the has recently been better delineated. Primary infec-
inactivated vaccine and subsequently acquired tion in infants is associated with a specific serum
natural RSV infection. High levels of antibody to IgM antibody response which is transitory. In
the F and G proteins, measured by enzyme-linked about the second week after infection IgG antibody
immunosorbent assay, but low levels of neutralizing may be detected, but the amounts diminish after
antibody developed both in the experimental model 1—2 months. An IgA serum antibody response in
and in the children who received the inactivated young infants may not be detectable. After repeated
vaccine, suggesting a detrimental effect of formalin infection an anamnestic response generally occurs
on the neutralizing epitopes of the surface glyco- in all three immunoglobulin classes.
proteins (Murphy et al., 1986). The importance of the specificity and type of
Recent studies have offered an explanation for antibody in protection and recovery from RSV is as
this abnormal response in the recipients of the inac- yet unclear. Young infants may have a poor neu-
tivated vaccine by delineating the divergent cellular tralizing antibody response and diminished res-
responses elicited by live RSV infection compared ponses to the F and G proteins. These proteins are
to that from inactivated or killed RSV (Graham et glycosylated, especially the G protein, and thus are
al., 1991, 1993; Bright et al., 1995; Toms, 1995). Live poor immunogens, and maternal antibody may in-
RSV, as with other natural live viral infections, terfere with the antibody response in infants.
characteristically evokes a T 1 helper cell response, Specific secretory antibody has also been identifi-
&
in which T helper cells are associated with IL-2 and ed in the secretions of infected infants. IgA, IgG and
-interferon production, as well as IgG2a neutraliz- IgM antibodies bound to epithelial cells and free in
ing antibody and CD8 ; cytotoxic T cells (CTLs). nasal secretions have been identified during the
Inactivated RSV and other non-replicating anti- course of infection.
gens on the other hand usually elicit a Th2 cytokine Litle is known about systemic and, especially,
pattern (IL-4, IL-5, IL-6) and IgG antibody with- local cell-mediated immune responses in infants
out CTL production. The formalin inactivated vac- with RSV infection. However, the importance of
cine, therefore, may have altered the usual immune cellular immunity in protection against RSV dis-
response evoked by natural RSV infection to an ease is supported not only by the animal model
abnormal one, producing primarily a T 2—type re- studies mentioned above, but also by the observa-
&
sponse and affecting multiple components of the tion that children, as well as adults, who have defi-
immune system. In vitro studies have suggested fur- cient cell-mediated responses tend to have pro-
ther that after prolonged (6 days or more) rechal- longed, severe and sometimes fatal infection with
lenge with RSV antigen the T response in cells RSV (Hall et al., 1986). Specific lymphocyte trans-
&
primed by live RSV switches from T 1 to T 2, while formation is detectable in infants within 1 month of
& &
in cells primed by the F protein, the response re- infection, but it has not been correlated clearly with
mains T 1 (Bright et al., 1995). In individuals with the severity of the infant’s illness nor with recovery
&
previous natural RSV infection a T 1 response and and protection. Of interest also is the observation
&
specific CTL production have been demonstrated in that RSV infection in both infants and adults is
vitro (Bangham et al., 1986; Isaacs and McMichael, associated with little or no detectable interferon in
1987; Anderson et al., 1994). the nasopharyngeal secretions.
Others have suggested that it is not necessary to
postulate an abnormal immune response to explain
the severity of lower respiratory tract disease in the
first months of life. Anatomical considerations and CLINICAL FEATURES
the relative immunological immaturity of the young
baby may explain much of the severity of RSV Infection in Infants and Young Children
infection during the early months of life. The in-
fant’s airway is small, and the proportion of the RSV is such an important cause of lower respir-
total pulmonary flow resistance from the small pe- atory tract disease in the young that at the periods
RESPIRATORY SYNCYTIAL VIRUS 299
of peak occurrence of pneumonia and bronchiolitis Evaluation of the severity of the lower respir-
in young children the presence of RSV in the com- atory tract disease in these young infants is often
munity may be assumed. RSV has been reported in problematic. Fever and the extent of the wheezing
various studies as causing 5—40% of the pneu- or crackles generally do not correlate with the se-
monias and bronchitis in young children, as well as verity of the illness, and the degree of hypoxaemia
50—90% of the cases of bronchiolitis. Its contribu- may not be clinically evident. Cyanosis is present in
tion to cases of croup is relatively smaller, with less only a minority of infants, despite the observation
than 10% being associated with RSV infection. that most infants hospitalized with RSV lower re-
The severity of RSV infection appears to be af- spiratory tract infection are hypoxaemic. Hy-
fected not only by age, but also by gender and poxaemia of a moderate to severe degree may be
socioeconomic factors. Despite an equal attack present in an infant who does not appear cyanotic.
rate, boys require hospitalization more often than An increasing respiratory rate has been noted to be
girls, and a higher proportion of infants from one of the better clinical indicators of hypoxaemia.
crowded and industrialized environments tend to The hypoxaemia results from the characteristi-
be hospitalized. In non-urban, middle to upper class cally diffuse involvement of the lung parenchyma,
areas, one of 1000 infants infected in the first year of which may not be evident on chest radiography.
life may require hospitalization, whereas in indus- This produces an abnormally low ratio of ventila-
trialized, poorer economic environs the rate has tion to perfusion. Alveolar hypoventilation and
been reported to be as high as one in 50 infants. progressive hypercarbia may develop, but are rare
Primary infections are almost always sympto- in infants given good oxygenation and supportive
matic but may be as mild as a common cold or as care.
severe as a life-threatening lower respiratory tract The radiological picture in infants with lower
infection. The initial manifestations of RSV infec- respiratory tract disease due to RSV may vary from
tion in infants are usually those of a febrile upper a virtually normal appearance to one which mimics
respiratory tract infection. Lower respiratory tract bacterial pneumonia. However, the severity of the
involvement commonly becomes manifest within infant’s illness is not generally mirrored by the
several days. Although fever is common during the radiological changes. Infants with the typical find-
early phase of the illness, the infant may be afebrile ings of bronchiolitis, hyperinflation and minimal
by the time the lower respiratory tract disease be- peribronchial increased markings may be severely
comes prominent and hospitalization is under- ill and hypoxaemic. The most characteristic find-
taken. ings on the chest radiograph with RSV lower respir-
The harbinger of the lower respiratory tract dis- atory tract disease are hyperinflation, infiltrates—
ease is often a worsening cough. As the disease which are often perihilar involving more than one
progresses, tachypnoea and, often, dyspnoea devel- lobe—and atelectasis, especially of the right middle
op, overtly marked by retractions of the chest wall. or right upper lobes. In the Newcastle upon Tyne
In bronchiolitis the respiratory rate may be strik- studies of infants hospitalized with lower respir-
ingly elevated, and wheezing and hyperinflation are atory tract disease due to RSV, hyperinflation was
hallmarks of the disease. However, crackles may present in over half the children and increased
also be present. In pneumonia the crackles may be peribronchial markings in 39% (Simpson et al.,
localized or diffuse and may be accompanied inter- 1974). Hyperlucency, which occurred as the sole
mittently by wheezes. Indeed, bronchiolitis and abnormality in 15% of the cases, was particularly
pneumonia often appear to represent a continuum associated with RSV infection. Consolidation,
and may be difficult to differentiate clinically. The which sometimes appeared similar to that found
variability in auscultatory findings and in the res- with bacterial infection, was present in approxi-
piratory rate within short periods of time is frequent mately one-fourth of the cases and was usually sub-
enough in infants with lower respiratory tract infec- segmental in distribution. The radiological abnor-
tion due to RSV to be considered characteristic. malities, as well as the hypoxaemia, tend to persist
The course of the illness similarly may be variable, beyond the time of clinical improvement.
lasting from one to several weeks, but most infants RSV infection in newborns had been thought to
will show clinical improvement within 3—4 days of occur rarely, but is now recognized as being rela-
the onset of the lower respiratory tract disease. tively common. The manifestations of the disease in
300 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
the newly-born, however, may be atypical and mis- Certain groups of children appear to be at risk of
sed clinically (Hall et al., 1979). Neonates, especially developing complicated, severe or even fatal RSV
in the first 2—3 weeks of life, infrequently have the infection. Infants hospitalized in the first few
typical clinical findings of lower respiratory tract months of life with congenital heart disease appear
disease. More commonly RSV is manifest by such to be at particular peril. Although all the factors or
non-specific signs as lethargy and poor feeding. Up- conditions associated with a poor prognosis for
per respiratory tract signs may be present in only those infants with cardiac lesions and RSV infection
about half. Premature infants appear to be more have not been defined, pulmonary hypertension ac-
susceptible to infection, and the mortality may be companying the congenital heart disease appears to
high in neonatal intensive care units. increase the risk appreciably. Recent surgical and
Primary infections may also be milder, especially technical advances and early correction currently
in older infants, manifest only as tracheobronchitis have reduced appreciably the mortality and mor-
or upper respiratory tract disease. Such upper res- bidity in infants with congenital heart disease, al-
piratory tract infections, however, do tend to be though their risk for fatal RSV disease still appears
more prolonged and severe than the usual cold. to be more than three or fourfold greater than that
Otitis media is a frequent accompaniment of RSV estimated for other infants hospitalized with RSV
infection in the first few years of life, particularly in infection (Navas et al., 1992).
the first year. RSV has been recovered from middle Infants with underlying pulmonary disease, es-
ear aspirates of such infants, both as the sole patho- pecially bronchopulmonary dysplasia, also appear
gen and in conjunction with bacterial agents. to be at risk of developing prolonged and compli-
cated infection with RSV, even in their second year
of life. Another group of children who are prone to
extensive and severe RSV infection are those who
Complications are immunosuppressed or have a congenital im-
munodeficiency disease (Hall et al., 1986; Pohl et al.,
Apnoea is a frequent complication of RSV infection 1992). These children may develop lower respir-
in young infants, occurring in approximately 20% atory tract disease at any age and tend to have
of hospitalized cases. Apnoea may be the initial sign extended shedding of the virus. RSV infection has
of RSV infection, preceding overt respiratory signs. also been shown to be associated with exacerba-
The apnoea is most likely to occur in premature tions and complications in children with nephrotic
infants with a gestation of 32 weeks or less and in syndrome and cystic fibrosis.
those of young postnatal age, less than 44 weeks Perhaps the most frequent complication from
postconceptional age (Church et al., 1984). A his- RSV lower respiratory tract disease in early infancy
tory of apnoea of prematurity has also been identifi- is prolonged alterations in pulmonary function
ed as a significant risk factor for the development of which may predispose to chronic lung disease in
apnoea with RSV infection. The apnoea associated later life. A number of studies have associated bron-
with RSV infection is non-obstructive and tends to chiolitis in infancy with surprisingly high rates of
develop at the onset or within the first few days of recurrent wheezing and lower respiratory tract dis-
the illness. Although the prognosis for such infants ease. In some children these clinical manifestations
has not been defined accurately, it does not appear improve with age, and in others pulmonary func-
to place the infant at increased risk of subsequent tion abnormalities may persist but be clinically si-
apnoea (Church et al., 1984). lent. Whether such children who go on to have
Despite the often prolonged and severe course of recurrent wheezing and lower respiratory tract dis-
lower respiratory tract disease in young children, ease are those who are genetically predisposed to
secondary bacterial infection is an unusual compli- hyperreactive lungs, have smaller airways or an
cation. In developing countries, however, concur- atopic diathesis, or whether the bronchiolitis aug-
rent bacterial infection is common and may be a mented or engendered a lasting hyperreactivity of
major factor leading to the high mortality rate in the airways, is unclear (Landau, 1994). Studies in
such infants. Furthermore, antibiotic therapy has which the initial infection was identified as being
been shown not to improve the rate of recovery of caused by RSV and in which the children were
infants with RSV lower respiratory tract disease. prospectively followed have indicated that atopy
RESPIRATORY SYNCYTIAL VIRUS 301
does not appear to be the major factor in predicting ation of proper infection control procedures and for
which children will develop such long-term pul- antiviral chemotherapy.
monary abnormalities. In a subgroup of children, Both direct and indirect immunofluorescent re-
however, atopy does appear to be a major factor in agents, utilizing either polyclonal or monoclonal
increasing their risk of more severe RSV disease and antibodies, are available, possessing a high degree
subsequent complictions such as recurrent wheez- of specificity and sensitivity. The fluorescent pattern
ing (Welliver and Duffy, 1993). observed will depend on the antibody used. Mono-
clonal antibody to the nucleocapsid protein or the
phosphoprotein will produce specific large and
small cytoplasmic inclusions in RSV-positive cells,
Infection in Older Children and Adults
while monoclonal antibody to the fusion (F) protein
shows diffuse cytoplasmic staining. Polyclonal anti-
Repeated RSV infections occur throughout life, and
serum produces both the specific inclusions and
the interval between infections may be only weeks
diffuse staining in the cytoplasm (Figure 7.4).
or months (Hall et al., 1991). In older children and
A variety of rapid assays for detecting RSV anti-
adults these repeated infections are usually manifest
gen have become available recently. Most are based
as upper respiratory tract infections or sometimes
on an enzyme immunosorbent assay (EIA). A
as tracheobronchitis. In a minority of adults,
number of commercial kits, which rely on a quali-
usually less than 15%, the infection may be asymp-
tative colour change, are simple and suitable for
tomatic. Even in young healthy adults, RSV infec-
outpatient clinics and offices. However, the sensitiv-
tion may be associated with pulmonary function
ity and specificity of these kits may be variable and
abnormalities, mostly a hyperreactivity of the air-
results are likely to be considerably less accurate in
way to cholinergic stimulus, which may last for a
a busy clinic than those reported in published stu-
couple of months.
dies from evaluating laboratories.
RSV infection in the elderly may be more severe
The sensitivity of the EIA assays against isolation
and have some similarities to infection at the other
of the virus in cell culture has ranged from about 50
end of the age spectrum (Falsey et al., 1995). In the
to 95% and the specificity from about 70 to 100%.
elderly, RSV has been associated with exacerba-
A major factor in the reported sensitivity of these
tions of chronic bronchitis and can produce a flu-
assays is the quality and sensitivity of the viral
like syndrome indistinguishable from that of influ-
isolation in tissue culture to which it is being com-
enza. Outbreaks of RSV infection may occur more
pared. The success of viral isolation has varied
frequently than is generally recognized in institu-
greatly in different laboratories, partly because of
tions for the elderly and can result in a high inci-
the lability of RSV, the quality and transport of the
dence of pneumonia. The relative role of the virus
specimen, and, particularly, the sensitivity of vari-
and of the usual bacterial pathogens in pneumonia
ous cell lines.
in this elderly population is unclear.
Nasal washes or tracheal secretions are generally
On acute medical wards RSV infections, both
the best specimens for isolation. The specimen
nosocomially and community acquired, in adult
should be inoculated on to tissue culture promptly
patients and staff are being increasingly recognized
and without subjecting it to major temperature
(Takimoto et al., 1991; Falsey et al., 1995). Those
changes. RSV is a relatively labile virus and with-
with the most severe clinical manifestations gen-
stands freezing and thawing poorly. At 37°C ap-
erally are those with chronic or immunosuppressive
proximately 10% of infectivity of the virus in tissue
underlying conditions.
culture medium remains after 24 hours. However, at
4°C in certain media, such as veal infusion broth,
little loss in virus titre may occur for 2—3 days.
DIAGNOSIS Control of the pH of the media is important in
preventing loss of infectivity, the optimum pH being
RSV infection may be diagnosed by identification of 7.5.
the viral antigen by a rapid diagnostic technique or Human heteroploid cells, such as HEp-2 and
by isolation of the virus in tissue culture. Rapid HeLa cells, generally provide the best tissue culture
diagnosis is important in management for the initi- for the isolation of RSV. The sensitivity of such cell
302 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
have greater specificity and are more suitable for
testing smaller numbers of daily specimens. The
disadvantages are the requirements for a technician
trained in reading the fluorescent patterns and for a
specimen that contains adequate nasopharyngeal
cells. Ideally a diagnostic laboratory should use a
combination of techniques, preferably a rapid diag-
nostic assay in combination with viral isolation.
Serological diagnosis by detecting antibody rises
in acute and convalescent sera is unlikely to be of
help in the management of the patient because of
the time required. Furthermore, the serological re-
sponse in young infants may be poor and not de-
tectable by some antibody assays. Seroconversion
usually does not occur for at least 2 weeks and may
require 4—6 weeks. Several serological tests are
available, mostly in research laboratories. Neutral-
ization and EIA most frequently are used and offer
Figure 7.4 Positive indirect immunofluorescent antibody test the possibility of detecting individual antibody class
on nasal secretions from an infant with lower respiratory tract and specificity.
disease due to RSV
Figure 7.5 Stained preparations of uninfected HeLa cells (left) and cells infected with RSV (right). Syncytial formation is well
illustrated by the infected monolayer
ministered by small-particle aerosol into an oxygen ary function the results have been conflicting, with
tent, oxygen hood or via a ventilator, usually for 12 some demonstrating no change in pulmonary resis-
or more hours per day until clinical improvement, tance and some even showing a paradoxical
which is usually in 2—5 days (Hall et al., 1983). For worsening. Some experts would advise that bron-
which infants ribavirin should be used is controver- chodilator therapy, usually aerosolized, be con-
sial. Controlled double-blind studies have shown sidered for the more severely affected infants, par-
ribavirin to have a beneficial effect on the clinical ticularly those above 6 months of age, and that it be
course of the RSV bronchiolitis and pneumonia in carefully monitoried initially. Systemic therapy
infants who were mostly mildly to moderately ill, as with corticosteroids has not been shown to be of
measured by the rate of improvement in illness benefit in bronchiolitis or in RSV pneumonia
severity and on the measured levels of the infants’ (Springer et al., 1990).
arterial oxygen saturation, as well as the duration of
mechanical ventilation and supplemental oxygena-
tion (Smith et al., 1991); however, the degree of
benefit, especially relative to the cost, has been ques- Prevention
tioned (American Academy of Pediatrics, 1997).
Ribavirin should be considered on an individual For more than a decade research has been directed
basis, especially for those infants who have underly- towards the development of a safe and effective
ing conditions which put them most at risk for vaccine for RSV. A vaccine for this virus, however,
severe RSV disease. poses particular problems. It would have to be ad-
No toxicity has been associated with ribavirin ministered at a very young age, within the first few
therapy and development of resistance to ribavirin weeks of life, and should be able to produce an
by RSV strains or any other virus has not been immunity more durable than that often seen after
observed, even with prolonged treatment. natural infection.
Aerosolized bronchodilators have also been em- The first vaccine developed—the alum-precipi-
ployed intermittently for some patients with bron- tated, formalin-inactivated vaccine tested in the
chiolitis. Their use is controversial, as most studies 1960s—produced high levels of serum antibody but
have failed to show benefit in infants under 18 then resulted in augmented disease following infec-
months of age. In those studies evaluating pulmon- tion with the wild virus (Toms, 1995). Subsequently,
304 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
candidates for an attenuated live vaccine have been Of the infection control procedures employed to
sought. Vaccine developed from both cold-adapted limit the nosocomial spread of RSV, hand-washing
strains and from temperature-sensitive (ts) mutants is the most important. When compliance with con-
appeared promising in initials trials, but were later sistent and careful hand-washing is less than opti-
shown to be too reactive, genetically unstable or mal, the use of gloves may be of benefit. The routine
overly attenuated. Renewed interest in the use of an use of gowns and masks has not been shown to be of
attenuated live viral vaccine has been engendered additional benefit. The use of gowns, however, may
recently by the promising advances in the develop- be advisable during periods of close contact in
ment of genetically stable mutant viruses (Chanock which an infant’s secretions are apt to contaminate
et al., 1992; Toms, 1995; Crowe et al., 1996). the clothing. Since RSV primarily infects via the
New techniques are revealing the molecular soul eyes and nose, masks are of limited value, but
of RSV. It can be genetically engineered, thus bring- eye—nose goggles have been shown to be beneficial.
ing new hopes for immunoprophylaxis against RSV Other procedures of potential benefit include iso-
infection. Research has focused on the two major lation or cohorting of infected infants and assigning
surface glycoproteins, the F and G proteins, as nursing personnel to care for either infected infants
possible immunizing agents (Oien et al., 1993; Para- or uninfected infants, but not both simultaneously.
diso et al., 1994; Toms, 1995). These proteins have During epidemic periods the number of patient
appeared promising in experimental animals as contacts and visitors should be limited. Recognition
subunit vaccines and as carried by vaccinia and and cleaning of objects and surfaces contaminated
adenorivus recombinants. However, the recom- with infant secretions are also advisable. Elective
binant vaccines have appeared to be less im- admissions of infants with high-risk conditions
munogenic and protective in chimpanzees than in should be avoided during the epidemic periods of
rodents. Delineation of specific B and T cell epi- RSV. For those infants who must be admitted, par-
topes on these surface glycoproteins which elicit ticular care must be employed to prevent such
protective immunity is also in progress (Toms, 1990; cross-infections.
Hsu et al., 1994).
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Takimoto, C.H., Cram, D.L. and Root, R.K. (1991) Respiratory with glycoprotein subunits of respiratory syncytial virus to
syncytial virus infections on an adult medical ward. Archives of protect cotton rats against viral infection. Journal of Infectious
Internal Medicine, 151, 706—708. Diseases, 155, 1198—1204.
Toms, G.L. (1990) Respiratory syncitial virus: virology, diag- Welliver, R.C. and Duffy, L. (1993) The relationship of RSV-
nosis, and vaccination. Lung, 1685, 388—395. specific immunoglobulin E antibody responses in infancy, re-
Toms, G.L. (1995) Respiratory syncytial virus—how soon will current wheezing, and pulmonary function at age 7—8 years.
we have a vaccine? Archives of Disease in Childhood, 72, 1—3. Pediatric Pulmonology, 15, 19—27.
Toms, G.L., Quinn, R. and Robinson, J.W. (1996) Undetectable West, W.H., Lounsbach, G.R., Bourgeois, C. et al. (1994) Biologi-
IgE responses after respiratory syncytial virus infection. Ar- cal activity, binding site and affinity of monoclonal antibodies
chives of Disease in Childhood, 74, 126—130. to the fusion protein of respiratory syncytial virus. Journal of
Vaux-Peretz, F., Chapsal, J.M. and Meignier, B. (1992) Compari- General Virology, 75, 2813—2819.
son of the ability of formalin-inactivated respiratory syncytial
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Adenoviruses
Göran Wadell
University of Umea , Umea , Sweden
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
308 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 8.1 Human adenovirus serotypes
Prototype
capsomer is formed by five copies of polypeptide to various subgenera. Each fibre is a trimer of poly-
III. peptide IV.
The fibres are glycoproteins and display a char- The polypeptides IIIa, VI, VIII and IX connect
acteristic varying length in adenoviruses belonging hexons and vertex capsomers into a dense capsid.
ADENOVIRUSES 309
particles are taken into endosomes by absorptive
endocytosis. An ATP-driven protein pump lowers
the pH within the endosome, resulting in a hydro-
phobic alteration to the surface of the adenovirus
capsid.
Penetration of the lipid bilayer by the virion ap-
pears to cause disruption of the endosome, allowing
the capsid to be transported to the cell nucleus
(Varga et al., 1991). The viral cysteine protease is
activated by the reducing environment of the host
cell and degrades protein VI which anchors the
viral DNA to the capsid (Gerber et al., 1996). The
viral capsid is transported to a nuclear pore via
microtubules. The remaining capsid is lost during
the deposition of the virus core into the cell nucleus.
Transcription and DNA synthesis of adenovirus
occur in the nucleus.
Six different early transcription regions have
been identified. Transcription is initiated by early
region E1A which contains enhancer-like struc-
tures. Both regions E1A and E1B are needed for the
expression of other viral genes and are also required
Figure 8.1 Electron micrograph of Human adenovirus 4
for adenovirus transformation. Regions E2A and
E2B encode several proteins which are required for
This capsid contains a nucleoprotein complex viral DNA replication. The E3 region can be re-
which is composed of polypeptides V and VII and a garded as a cassette containing immunomodulating
linear double-stranded DNA molecule containing peptides, e.g. the 19 kDa glycoprotein, which binds
33—45 kb pairs. The ends of linear DNA molecules the heavy chain of MHCI, thus impairing its
are covalently bound to a protein with a molecular glycosylation and transport to the cell surface. Sev-
mass of 55K. The adenovirus particle is stable to eral of the open reading frames (ORFs) in the E3
low pH, bile and proteolytic enzymes. For these region have a structure compatible with membrane
reasons, adenoviruses can replicate to high titres in proteins and are translated into peptides that have
the intestinal tract. immune modulating functions. The E3 14.7 kDa
and the E3 10.4/14.5 kDa protein inhibit tumour
necrosis factor (TNF)-induced apoptosis and TNF-
induced release of arachidonic acid.
Replication Several other early virus-specific proteins are for-
med before viral DNA synthesis can be initiated.
Replication of DNA, transcription and maturation This occurs 7 hours after infection. Five hours later
of adenovirus take place in the cell nucleus. The the synthesis of virus structural proteins can be
spliced mRNA is translated in the cytoplasm. initiated. The synthesis of the cellular proteins then
Infection by Ad2 and Ad5 is initiated by attach- gradually ceases.
ment of the fibre to CAR, a glycoprotein of the Newly formed virus structural polypeptides are
immunoglobulin superfamily (Bergelson et al., transported within a few minutes into the nucleus,
1997). The penton base then binds to the second where the assembly of virus particles starts 7 hours
receptor, the fibronectin binding integrin v3 or after the initiation of DNA synthesis. Up to 10
v5 (Wickham et al., 1994). This initial step of the virus particles are assembled in each cell within 30
infection cycle may account for the distinctly differ- hours after infection with the most rapidly growing
ent tropisms of adenoviruses of different subgenera. types of human adenoviruses. The proportion of
Penetration of virions occurs by an incompletely infectious particles among these varies with differ-
understood mechanism. Cell-associated adenovirus ent serotypes from 1 in 10 to 1 in 10000. The transla-
310 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
tion of viral mRNA coding for structural proteins In general, the critical classification of viruses
continues uninterrupted for about 40 hours. This should be based on nucleotide sequence differences
results in an accumulation of a tenfold excess of between genomes from different viruses. DNA
viral polypeptides. Efficient release of virions re- homology of adenoviruses has been studied by filter
quires expression of E3 11.6 kDa adenovirus death hybridization and liquid hybridization (Green et al.,
protein (ADP) (Tollefson et al., 1996). 1979). The classification of adenoviruses by this
means is in complete agreement with the data ob-
tained by classification of human adenoviruses
based on the molecular weight of the internal struc-
Classification of the 49 Human tural polypeptides of the virion. Human adeno-
Adenoviruses into Six Subgenera viruses display characteristic differences in the G-C
content in their DNA, subgenus A (47—49%), sub-
Human adenoviruses were originally classified by genus B (49—52%), subgenus F (52%). This differ-
Rosen (1960) into four groups, I—IV, based on differ- ence can be exploited by using SmaI DNA restric-
ent haemagglutination properties with rat and rhe- tion endonuclease that cleaves at 5CCC GGG
sus monkey erythrocytes. Although the portion of (Wadell et al., 1980a).
the genome coding for the receptor-binding sites of SmaI has been used to obtain DNA restriction
the adenovirus fibre represents only a fraction of the patterns of 41 human adenovirus serotypes (Figure
total adenovirus genome, this property is apparent- 8.2). It should be noted that a limited range of the
ly critical and a large number of other biological number of SmaI fragments of the adenovirus
properties are shared by adenoviruses grouped to- genomes is characteristic for each subgenus. Fur-
gether according to Rosen. The importance of genes thermore, the restriction patterns of the adenovirus
coding for fibre proteins in the classification of ad- types belonging to the same subgenus display a high
enoviruses is also illustrated by the fact that aden- proportion of comigrating DNA restriction frag-
oviruses belonging to different subgenera display ments. In the case of the heterogeneous subgenera
characteristic differences in the length of their fibres. A, B and F, clusters of DNA restriction patterns are
The charge, i.e. the isoelectric point of the fibre revealed. In general, there is comigration of more
knobs, varies from 4.5 (Ad35) to 9.5 (Ad37). than 50% of DNA restriction fragments of aden-
It was suggested in 1967 that human aden- oviruses belonging to the same subgenus or the
oviruses could be divided into subgenera on the same cluster, whereas pairwise comparison of
basis of their oncogenicity in newborn hamsters. genomes classified into different subgenera reveals
Subgenus A comprises adenovirus serotypes which that less than 10% of the DNA restriction frag-
are highly oncogenic, i.e. causing tumours in every ments comigrate.
animal within 2 months. Subgenus B adenoviruses It is evident that the access to DNA restriction
are weakly oncogenic, meaning that few of the ani- patterns of adenovirus prototypes provides efficient
mals developed tumours after an observation per- means of identification and classification of un-
iod of 1 year. The non-oncogenic adenoviruses can known strains and can reveal the existence of gen-
transform rodent cells in vitro and are divided into etic variability within one serotype (Adrian et al.,
subgenera C and D on the basis of differences in the 1986).
antigenicity of the early T antigen. Genetic variability has been demonstrated within
The polypeptides of the virion represent a major each adenovirus serotype studied. Four genome
portion of the products of the adenovirus genome. types of Ad3 and 12 of Ad7 have been detected
Internal polypeptides would be expected to be more among more than 700 wild-type strains recovered
conserved and offer a means of classifying human from different parts of the world by using only two
adenoviruses. The 49 human adenovirus serotypes restriction enzymes, SmaI and BamHI (Figure 8.3).
could be divided into six subgenera by SDS-polyac- This technique allows the introduction of molecular
rylamide gel electrophoresis of virion polypeptides epidemiology into the study of adenovirus infec-
(Wadell et al., 1980a). This method was compatible tions and is a tool which can be used with others in
with the previous methods, and extended these as attempting to distinguish between virulent and less
regards Ad4 (subgenus E) and the newly identified virulent adenovirus strains. Phylogenetic studies
enteric Ad40 and Ad41 (subgenus F) (Table 8.2). based on the ITR and VA-RNA genes showed that
Table 8.2 Properties of human adenovirus serotypes of subgenera A to F
DNA
Sub Serotype Intrageneric Intergeneric G ; C Number of V Vl Vll Haemag Length of Oncogenicity in Tropism symptoms
genus (%) Small? glutination fibres (nm) newborn hamsters
fragments pattern@
A 12,18,31 48—69 8—20 48 4—5 51.0—51.5 25.5— 18 IV 28—31 High (tumours in most Cryptic enteric
46.5—48.5A 26.0 animals in 4 months) infection
B:1 3,7,16,21, 89—94 9—20 51 8—10 53.5—54.5 24 18 I 9—11 Weak (tumours in few Respiratory disease
B:2 14, B11, 34, 35 animals in 14—18 Persistent infections of
months) the kidney
C 1, 2, 5, 6 99—100 10—16 58 10—12 48.5 24 18.5 III 23—31 Nil Respiratory disease
persists in Iymphoid
tissue
D 8—10, 13,15, 17, 94—99 4—17 58 14—18 50—50.5D 23.2 18.2 II 12—13 Nil Keratoconjunctivitis
19, 20, 22—30, 32,
33, 36, 37, 38, 39,
42—47C
E 4 4—23 58 16—19 48 24.5 18 III 17 Nil Conjunctivitis
Respiratory disease
F 40, 41 62—69 15—22 52 9—12 46.0—48.5 25.5 17.5 IV 28—33 Nil Infantile diarrhoea
?The restricted DNA fragments were analysed on 0.8—1.2% agarose slab gels. DNA fragments smaller than 400 bp were not resolved.
@I, Complete agglutination of monkey erythrocytes; II, complete agglutination of rat erythrocytes; III, parallel agglutination of rat erythrocytes (fewer receptors); IV, agglutination of rat erythrocytes discernible only
after addition of heterotypic antisera.
APolypeptide V of Ad31 was a single band of 48 kDa.
BMembers of subgenus B are divided into two clusters of DNA homology based on pronounced differences in DNA restriction sites.
COnly polypeptide analysis and/or DNA restriction analysis has been performed with Ad32—Ad39 and Ad42—Ad47.
DPolypeptides V and Vl of Ad8 showed apparent molecular mass of 45 and 22 kDa, respectively. Polypeptide V of Ad30 showed an apparent molecular mass of 48.5 kDa.
Modified from Wadell (1984).
312 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
A B C D E F
12 18 31 3 7 16 21 11 14 34 35 1 2 5 6 8 37 4 40 41
10
0.5
0.4
Figure 8.2 Schematic presentation of Smal DNA restriction patterns obtained after cleavage of DNA from adenovirus prototypes
belonging to subgenera A, B, C, D, E and F
simian adenoviruses were closely related to mem- used only when the two parent genomes have been
bers of the different subgenera of human aden- identified. An evolutionary variant is one in which
oviruses (Bailey and Mautner, 1994; Kidd et al., genetic alteration was generated via insertion or
1995). intragenomic recombination in progeny of the same
strain. A genomic cluster is a group of genome types
that are significantly more closely related to each
other than to any other genome type. The genome
Taxonomic Terminology type denotes a distinct viral entity within a genomic
cluster identified by DNA restriction analyses.
It is clear from the above that the classification of Strain corresponds to the progeny of each wild-type
adenoviruses has evolved from schemes based on isolate.
biological properties to a reproducible and dis- As more DNA variants have been discovered and
criminating system based on genomic differences. It studied in greater detail, their classification has be-
is advisable at this stage to list some definitions. come more complex and their relatedness with each
Members belonging to the genus of adenoviruses other better understood. There has been some con-
share common epitopes on the hexons. Subgenus is fusion due to different working groups using the
defined by the DNA homology of more than 50% same letters and numbers for quite different genome
between members within a subgenus and less than types (Wigand and Adrian, 1991). The adoption of a
20% between members of different subgenera. The unified system will require the renaming of some
serotype is defined by quantitative neutralization genome types.
with hyperimmune sera. A ratio of homologous to
heterologous neutralization titre must be greater
than 16. The designation recombinant should be
ADENOVIRUSES 313
bases can cause cell detachment of monolayer cell
cultures after 2 hours incubation. This activity is
particularly strong in preparations from aden-
oviruses of subgenera B and C. In addition, the
adenovirus capsid and/or pentons exert a direct
effect on the lipid bilayer of the endosome, allowing
the release of endosome content into the cytosol.
However, the gene products of the adenovirus
genome responsible for the adenovirus-induced
symptoms in the host are, on the whole, not defined.
Ad1, Ad2 and Ad5, members of subgenus C, per-
sist in tonsils for several years, through a low-grade
replication. Whether only a subset of lymphocytes
is infected is not known. Shedding of infectious
virus in stools for at least 2 years has also been
documented (Fox and Hall, 1980). The source may
be Peyer’s patches of lymphoid tissue in the gut but
this has not been demonstrated.
The mimicry such as the immunological cross-
reactivity between gliadin and the Ad12, E1B, 54K
protein has yet to be critically evaluated in relation
to sequelae of adenovirus infections of the gut.
Members of subgenus B:2, Ad11, Ad34 and Ad35
cause persistent infections of the kidney. Silent
shedding of Ad11 for 6 months into the urine of a
Figure 8.3 Schematic presentation of BamHI DNA restriction
healthy pregnant woman has been documented by
patterns obtained after cleavage of DNA from Ad3 and Ad7 Gardner (personal communication). Ad11 has also
genome types been demonstrated to be transmitted via the kidney,
giving a haemorrhagic cystitis in a transplant recipi-
ent. These serotypes are also activated in AIDS
patients and bone marrow transplant recipients.
PATHOGENESIS Animal models for human adenovirus infections
are available, such as hepatitis induced by Human
The propensity of adenoviruses to shut off expres- adenovirus 5 in the mouse and pneumonia induced
sion of the host mRNA, and the unregulated excess by Human adenovirus 5 in cotton rats.
synthesis of adenovirus structural proteins, results
in accumulation of viral proteins as intranuclear
inclusion bodies which are incompatible with nor-
mal cell function (Figure 8.4). This will give rise to EPIDEMIOLOGY
the characteristic histopathological intranuclear in-
clusions in the alveolar cells in a severe adenovirus General
pneumonia. In the upper respiratory tract cells, cili-
ary abnormalities and microtubular aberrations Information on the role of adenoviruses in viral
lead to a defective mucociliary clearance (Carson et respiratory diseases has been gathered in series of
al., 1985). reports to the World Health Organization (WHO).
Differences in target cell specificity between the Reports of adenovirus isolation to the WHO regis-
adenovirus fibres can contribute to the distinctly ter have been evaluated over a 10—year period
different tropisms of members of the six different (1967—1976) (Schmitz et al., 1983). During this
adenovirus subgenera. Furthermore, a toxin-like 10—year period adenoviruses accounted for 13% of
activity has been associated with vertex cap- a total of 135 702 reported isolations. Adenovirus
somers—the base of the penton. Isolated penton infections were second only to influenza A, which
314 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 8.4 Intranuclear inclusion of an Ad5—infected KB cell 48 hours after infection. (Reproduced with permission from R. Marusyk
et al. (1972) Journal of General Virology, 14, 261—270)
represented 28% of the reported isolates. is valid to present the epidemic distribution accord-
A stratification of the reported adenovirus isola- ing to subgenus.
tions by age revealed the following: 22% ( 1 year);
42% (1—4 years); 18% (5—14 years); 10% (15—24
years); 7% (25—29 years); 1% ( 30 years). Between
47 and 55% of the infections resulted in an illness so Subgenus A
slight that it escaped attention. Fox and Hall (1980)
have concluded on the basis of seroconversion that Ad12, Ad18 and Ad31 represent only 0.5% of the
the true contribution of adenoviruses to illness is reported typed virus isolates. Members of subgenus
double that usually estimated on the basis of virus A share several properties with the enteric aden-
isolation alone. oviruses and are otherwise distinguished from all
Members of different adenovirus subgenera show other adenovirus serotypes by three characteristics:
distinctly different organ tropisms. Consequently, it (1) the majority of isolates have been obtained from
ADENOVIRUSES 315
0—12-month-old infants; (2) 91% of the reported adult sera. However, in Japan only 2% of the aden-
isolates were recovered from stools; and (3) 60% of ovirus strains were typed as Ad7, whereas 52% of
the children had a gastrointestinal disease. In the the isolates were typed as Ad3. Recently an out-
absence of information on the serological response break of Ad7h infectious has emerged in Japan.
of children shedding adenoviruses of subgenus A, it The situation in Europe is different; thus in the
is difficult to evaluate their role as a cause of diar- former West Germany Ad3 and Ad7 accounted for
rhoea. 11% and 25%, respectively, of typed adenoviruses.
Infections with subgenus A members are relative- The Japanese strains were genome typed as the Ad7
ly common. An extensive study of the prevalence of prototype, which appears to be of low virulence.
neutralizing antibody in children in Rome demon- Isolation frequency may be influenced by the sever-
strated that Ad31 and Ad18 were second only to ity of virus-associated diseases and perhaps also by
members of subgenus C and Ad3. The use of the the tendency of virus strains to cause persistent
SmaI DNA restriction patterns (Figure 8.2) is of infections with shedding of infectious virus over
value in the identification of Ad12, Ad18 and Ad31, extended periods.
as they cross-react in both neutralization and hae- DNA restriction analysis of adenovirus strains
magglutination-inhibition assays. can reveal the genetic variability within each sero-
type (Wadell, 1984). This is best exemplified by the
analysis of Ad7. Twelve distinct genome types of
adenovirus 7 — Ad7p, Ad7a, Ad7b, Ad7c, Ad7d,
Subgenus B Ad7e, Ad7f, Ad7g, Ad7h, Ad7i, Ad7j, Ad7k—were
identified by using the restriction endonucleases
DNA restriction analysis has revealed that this sub- BamHI and SmaI (Azar et al., 1998). The worldwide
genus should be divided into two clusters of DNA distribution of the different Ad7 genome types is
homology. shown in Figure 8.5. In Africa, type Ad7c predomi-
nated. In Australia, the distribution of the Ad7
genome types was similar to that in Europe, the
DNA Homology Cluster B:1
difference being that a shift from Ad7c to Ad7b took
The members of this cluster are associated with place in 1975. In Brazil, Ad7e was the only genome
respiratory infections. Ad3 and Ad7 account for type detected and this genome type was unique to
13% and 19.7% of all adenovirus isolates typed and Brazil. In China, severe outbreaks of Ad7—asso-
reported to WHO. Both Ad3 and Ad7 show an ciated respiratory disease with mortality among in-
epidemic appearance with 4—5—year intervals. Both fants have been reported. Three strains recovered
serotypes are most frequently isolated from children from autopsy cases in 1958 were genotyped as
below the age of 4 years. Ad7a. A11 strains which have been genotyped dur-
Three different epidemic patterns of Ad7 infec- ing the last years were identified as Ad7d, this
tion have been identified. The first pattern consists genome type being unique to China. In Europe
of outbreaks mainly in the winter season among more than 90% of all isolates from patients were
infants. These infections may be severe, and fatal genotyped as Ad7b and Ad7c. These two appear to
among institutionalized children. The second pat- be mutually exclusive, a shift from Ad7c to Ad7b
tern consists of epidemic outbreaks of respiratory occurring in 1969. In the USA, Ad7b strains have
disease among schoolchildren. Severe infections predominated among the Ad7 isolates collected on
with fever for 7 days are usually encountered but a the west coast since 1962. Ad7p and Ad7a strains
fatal outcome is rare. The third pattern is seen as the were detected among Ad7 isolates recovered
outbreaks among newly enlisted military recruits. through the Virus Watch Program in Seattle,
It is evident from the WHO register that pro- Washington. During an outbreak of respiratory dis-
nounced differences prevail in the frequency of iso- ease in the South Cone of South America
lation of different adenovirus serotypes from differ- (1984—1994), 537 adenovirus strains from hospital-
ent regions of the world. The frequency of reported ized children with ALRI were analysed: 63% repre-
Ad7 isolates in Japan and the antibody prevalence sented subgenus B; 281/338 strains were typed as
were highly discordant. Ad7—specific antibodies the newly emerging Ad7h genome type associated
were detected in 30% of the children and in 50% of with 32/33 fatalies. Kajon et al. (1996). In Ad7h the
316 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 8.5 Worldwide distribution of different adenovirus 7 genome types. (Reproduced with permission from G. Wadell et al. (1985).
Journal of Clinical Microbiology, 21, 403—408)
Ad3 fibre had recombined into the Ad7 genome and has been reported to cause outbreaks of respir-
(Kajon and Wadell, 1996). atory disease among military recruits. Ad11, Ad34
Seventeen genome types of Ad3 have been identi- and Ad35 are closely related and can cause persist-
fied (Li and Wadell, 1988). The Ad3 prototype pre- ent infections of the kidneys, and possibly also of
dominated in Europe, Brazil, Africa and Australia, other parts of the urinary tract. Ad11 has been
whereas strains genome typed as Ad3a were found demonstrated to cause haemorrhagic cystitis. The
in China, Japan, USA and recently also in Europe. persistent nature of these adenoviruses is also well
No mutually exclusive shift between genome types illustrated by the fact that they are the most
could be detected among Ad3 isolates. common adenoviruses to be isolated from patients
Of the remaining members of DNA homology with the acquired immune deficiency syndrome
cluster B:1, Ad16 can be associated with conjuncti- (AIDS) or from bone marrow transplant recipients.
vitis, whereas Ad21 can cause severe respiratory
disease. Ad21 has been isolated from military re-
cruits at all major basic training centres in the US
Army, which has prompted the introduction of a Subgenus C
live Ad21 vaccine.
The subgenus C members account for 50% of all
adenovirus strains reported to WHO. The relative
DNA Homology Cluster B:2
frequencies are 25.4, 20.4, 11 and 2.4% for Ad2,
Ad11, Ad14, Ad34 and Ad35 account for less than Ad1, Ad5 and Ad6, respectively. They are most
1% of reported adenovirus isolates. The prevalence frequently isolated in children under the age of 4
of antibodies against Ad11 among Italian children years. Distinct epidemics of genome types of these
is about 2%. Ad14 differs from the other members adenoviruses occur. Among children below the age
ADENOVIRUSES 317
of 5, 50—70% have antibodies against the most Subgenus E
commonly detected Ad1 and Ad2 (Fox and Hall,
1980). Adenovirus 4 is the only member. Ad4 is rarely
The subgenus C adenoviruses have been desig- isolated from children in Europe or the USA, being
nated endemic adenoviruses, due to their propen- detected in only four children out of 1800 with
sity to cause persistent infections and shedding for proven adenovirus disease. Ad4 accounts for 2.4%
years in stools. The infection can be perpetuated of the adenovirus isolates reported to WHO. Most
within families, being spread to the newly born child isolates were obtained from adults, and the fre-
of the family. Because these adenoviruses are har- quency of Ad4 from adults was superseded only by
boured in the tonsils and shed from the gut, superin- the serotypes of subgenus D. The prevalence of
fections occur frequently and a vast number of Ad4—specific antibodies among adults is 30, 50 and
genome types of the four serotypes have been dem- 60% in the USA, Japan and Taiwan, respectively.
onstrated. Adenovirus 5 is of particular interest, as Two strikingly dissimilar genome types of aden-
this virus can cause severe infections in im- ovirus 4, the Ad4 prototype and Ad4a, have been
munocompromised hosts. demonstrated by DNA restriction analysis. The
Ad4 prototype has been isolated from military re-
cruits and identified as a cause of the characteristic
epidemic outbreaks of respiratory disease. The
Ad4a genome type is commonly isolated in Japan
Subgenus D as a cause of conjunctivitis. Ad4a was identified in
outbreaks of eye disease causing acute haemor-
Subgenus D contains 31 serotypes, i.e. more than rhagic conjunctivitis with subconjunctival haemor-
half of all recognized human adenoviruses. How- rhage in Rome in 1974. In 1977, a 5—year-old boy
ever, only 4.1% of all typed adenovirus strains re- died with disseminated Ad4 infection after treat-
ported to WHO belong to subgenus D. It should be ment at an intensive care unit in Buffalo, New York.
emphasized that the global situation for these aden- A nosocomial outbreak of pharyngoconjunctival
oviruses is not well mirrored by the WHO register. fever affected 35 of his attendants at the hospital. All
Infections caused by members of subgenus D are the Ad4 strains were genome typed as Ad4a. In
more common in Asia and Africa. Ad8 is the classic Japan, Ad4 is second only to Ad8 as a cause of
cause of epidemic keratoconjunctivitis (Jawetz, adenovirus-associated eye disease. During the per-
1959). iod 1967—1976 only seven Ad4 strains associated
Ad19 was isolated in Saudi Arabia in 1955 from a with eye disease were reported to the WHO register.
child with trachoma. This type was not reported However, during recent years several reports on
again until 1975 when several epidemic outbreaks Ad4—associated conjunctivitis have appeared, 12
of keratoconjunctivitis caused by Ad19 appeared in genome subtypes being isolated from outbreaks oc-
both Europe and the USA. These outbreaks were curring between 1985 and 1989 in Sapporo, Japan
demonstrated to be caused by a new genome type, (Itakura et al., 1991).
Ad19a, which was distinctly different from the orig-
inal Ad19 prototype. In 1976 a new adenovirus type
Ad37, related to Ad19a, appeared. Ad37 is now an
important cause of keratoconjunctivitis. This type Subgenus F
may also appear as sporadic infections. Further-
more, Ad37 may be sexually transmitted, as Ad37 The enteric adenoviruses Ad40 and Ad41 are the
isolates from penile lesions and cervicitis are re- only members of subgenus F. They are shed in large
ported. The fibre genes of Ad19a and Ad37 are amounts (10 virus particles per gram of faeces)
identical (Arnberg et al., 1997). These three sero- from children with diarrhoea. In contrast to
types are the predominant pathogens of aden- rotaviruses, enteric adenoviruses cause diarrhoea in
ovirus-associated keratoconjunctivitis in Western children throughout the year. Outbreaks have been
countries. In Japan, Ad3, Ad4, Ad8, Ad19 and Ad37 reported from hospitals and boarding schools.
have been demonstrated to cause 39% of the virus Ad40 and Ad41 do not share the property of aden-
infections of patients treated at an eye hospital. oviruses of subgenus C which can be shed for long
318 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 8.3 Illnesses associated with adenovirus infections
periods into the stools. Enteric adenoviruses cannot pitalized respiratory infections in this age group.
be found in stools from healthy controls. In a pros- Characteristic symptoms are pharyngitis with an
pective study in Uppsala in 1981, Ad40 and Ad41 exudative tonsillitis and frequently conjunctivitis,
accounted for 8% of the children seeking hospital together with nasal congestion and cough. In some
care for diarrhoea. Seroconversion could be dem- children the diseases can be difficult to distinguish
onstrated in 70% of the patients (Uhnoo et al., from group A streptococcal tonsillitis. Children
1984). usually have elevated temperature, myalgia and
Ad40 and/or Ad41 have been isolated in stool headache.
specimens from Africa, Asia, Europe, Latin America Adenoviruses can also cause laryngotracheob-
and North America. About 50% of 6- to 8-year-old ronchitis, but the pneumonias which occur in young
children in Africa, Asia and Europe have neutraliz- children are the most serious clinical manifesta-
ing antibodies against Ad41. In Europe during the tions. These may occur as a consequence of infec-
1980s, the relative incidence of Ad41 compared with tion with the endemic Ad2 and Ad5, among which
Ad40 infections increased from 30% to 90—95%. certain strains (genome types) can be more aggres-
During this same period Ad41/M3 superseded sive than others. In particular, Ad3 and Ad7, which
Ad41/Ml and Ad41/M2 as the major genome type appear in epidemic outbreaks due to the low herd
isolated (De Jong et al., 1993). immunity, may also result in severe infections. Ad-
enoviruses have been reported to account for 10%
of pneumonias in childhood. The clinical symptoms
are characterized by fever, cough, dyspnoea and
CLINICAL SYNDROMES
respiratory wheezing. Severity of the infections is
often related to overcrowding, and infections occur
Table 8.3 lists the clinical syndromes associated
preferentially among institutionalized children. In
with adenovirus infection and the principal sero-
the winter of 1959, 3398 cases of adenovirus pneu-
types involved.
monia with a fatality rate of 15.5% were seen at a
Peking children’s hospital. Fatal cases among
children below the age of 2 have also been reported
Respiratory Disease in Children from France, Germany, the Netherlands, Finland,
Canada and the USA (review by Wadell et al.,
Adenoviruses are responsible for 5% of acute res- 1980b). Eight studies performed between 1958 and
piratory infections in children under the age of 4 1988 on patients in China, the majority of whom
years, whereas they account for about 10% of hos- had adenovirus-associated pneumonia, revealed
ADENOVIRUSES 319
that 82% of the 1878 strains were typed as Ad3 or Acute Respiratory Disease (ARD) in
Ad7 (reviewed in Li et al., 1996). Military Recruits
Among the survivors of severe respiratory infec-
tions, in particular among Inuits in Canada, In- ARD is usually caused by Ad4, Ad7 and Ad21.
dians in the USA and Polynesians in New Zealand, Ad14 has been reported from Holland. Ad3 is much
residual lung damage thought to be due to second- more rarely isolated, probably because of the more
ary obliterative bronchiolitis has been reported. extensive herd immunity against this virus. In gen-
Furthermore, a so-called hyperlucent lung disease eral, outbreaks do not involve seasoned troops but
has been traced back to adenovirus infections in cause a high morbidity among newly enlisted re-
reports from Canada. The most common serotypes cruits. Adenovirus infections among healthy civil-
involved in this condition are Ad3, Ad4, Ad7 and ian adults are much less common. Crowding of
Ad21 representatives of subgenera B and E. people from widely different parts of the country,
In a prospective 10 year follow-up study from allowing repeated exposure to highly infectious
Finland, Simila et al. (1981) reported sequelae after doses, and the strenuous physical exercise may ac-
Ad7 pneumonia: 22 children were examined 10.7 count for the unusually high degree of severe infec-
years after the pneumonia; 12 had abnormal chest tions among recruits, some of which may have a
X-ray (six with bronchiectasis) and in 16 the results fatal outcome. ARD usually appears during the
of pulmonary function tests were abnonnal. This is third week in training. Characteristic symptoms in-
in agreement with the sequelae reported from clude fever for at least 4 days, malaise, headache,
North American Indians or Inuits. In all these stu- nasal congestion, sore throat, hoarseness and
dies children below the age of 2 were more prone to cough. About 10% develop patchy pulmonary infil-
develop both severe primary infections, occa- trates.
sionally with a fatal outcome, and long-term se- Cases of adenovirus pneumonia display charac-
quelae. teristic histopathological features, with bronchioli-
Adenoviruses can also be a severe threat to tis and retracted intranuclear inclusion bodies in
children in developing countries who are exposed to bronchiolar epithelium and alveolar cells. Preven-
measles infections. In a series of autopsies on tion of this disease is discussed later.
children dying from respiratory disease after
measles in South Africa, adenovirus pneumonias
were considered to be the cause of death in one-
quarter. Pertussis Syndrome
Adenovirus 7 sometimes appears in clear-cut out-
breaks which may involve children of school age. In Although whooping cough is caused by Bordetella
such cases a high fever with a mean duration of 7 pertussis, adenoviruses may be isolated very fre-
days, bronchopneumonia, gastroenteritis and men- quently (39%) from patients infected with B. pertus-
ingism may occur, but the outcome is generally sis. The contribution of reactivated adenoviruses to
favourable. the clinical features of pertussis is not known but
serotype Ad5 may result in fatal adenovirus pneu-
monia complicating pertussis infection.
Pharyngoconjunctival Fever
Infections of the Eye
This disease, first reported among those using
swimming pools, is characterized by conjunctivitis, Pharyngoconjunctival fever is a characteristic ad-
fever, pharyngitis and adenoidal enlargements. Ad3 enovirus syndrome. The acute follicular conjuncti-
and Ad7 are most frequently associated with this vitis which is part of this syndrome may also appear
syndrome, whereas Ad4 is common in Asia but as a separate entity. After an incubation period of
unusual among children in Western countries. Ade- about 8 days, engorgement of both the bulbar and
quate levels of chlorine are usually sufficient to the palpebral conjunctiva is seen, with involvement
inhibit outbreaks connected with the use of swim- of the cornea in 5—10% of patients. In addition, a
ming pools. characteristic preauricular lymphadenopathy is
320 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 8.4 Clinical characteristics of 55 children with adenovirus gastroenteritis
Fever
Ad40 14 14 (100) 8.6 11 (79) 7 (50) 1 (7) 8 (57) 1 (7) 2 (14) 3 (21)
Ad41 19 18 (95) 12.2 15 (79) 7 (37) 1 (5) 8 (42) 7 (37) 3 (16) 4 (21)
Established
adenovirus 14 14 (100) 6.2 7 (50̂ 5 (36) 9 (64) 14 (100) 5 (36) 2 (14) 11 (79)
Untyped
adenovirus 8 8 (100) 4.5 6 (75) 2 (25) 3 (38) 5 (63) 4 (50) 1 (13) 2 (25)
Figure 8.6 Cytopathic effect of A-549 cells 48 hours after infection with adenovirus 7b
Antigens
specimens are centrifuged gently on to the cell cul- (Turner et al., 1993; Towbin et al., 1994). Allard and
tures prior to incubation (Espy et al., 1987). coworkers have also developed an enteric aden-
Generally, ELISA is less sensitive than FA when ovirus-specific PCR using primers located in the
performed on respiratory specimens and is most early region E1 gene. Sequencing of serotype speci-
suited for the detection of adenovirus in faecal ma- fic regions can be used to type adenovirus isolates
terial where antigen is not intracellular. (Li et al., 1999).
DNA restriction enzyme analyses and DNA hy-
bridization techniques, in particular dot-blot as-
says, are also useful for identifying adenoviruses,
especially those which are difficult to grow in tissue Typing of Isolates
culture (Allard et al., 1985). The use of intracellular
viral DNA is recommended, as this enables analysis An adenovirus serotype is defined by the capacity of
of numerous isolates. Efficient extraction methods a hyperimmune reference serum to neutralize its
have been elaborated by Shinagawa et al. (1983) and infectivity. A serotype is regarded as new if the ratio
Fife et al. (1985). For diagnostic purposes confirma- of homologous to heterologous neutralization titre
tion of an adenovirus CPE in tissue culture and is equal to or greater than 16. The dominating
information on the subgenus of an isolate can be neutralizing epitopes are localized on the external
obtained by a general polymerase chain reaction portion of the hexons (Table 8.5). The hexon gene is
(PCR) based on VA RNA (Kidd et al., 1996). Fur- localized between map units 50 and 62 of the aden-
ther information on typing can be obtained by re- ovirus genome. Haemagglutination inhibition
striction fragment length polymorphism (RFLP) measures the interaction between IgG and type-
analysis after digestions of a hexon or a VA gene specific epitopes on the tip of the fibre of the an-
specific amplimer by restriction endonucleases (Al- tenna-like projection at each vertex. The fibre gene
lard et al., 1994). is localized at map units 86—91. For most isolates
PCR is beginning to prove a useful tool for the there is concordance between the results of neutral-
identification of adenoviruses. Hexon-coded ization and haemagglutination inhibition. Haema-
primers have been used in group-specific assays to gglutination-inhibition is a simple and rapid tech-
detect adenovirus DNA in stool samples (Allard et nique, provided that rat and monkey erythrocytes
al., 1990, 1992) and in post-mortem specimens are available.
ADENOVIRUSES 325
Typing of adenoviruses can also be achieved by erythrocytes in a complete pattern. The agglutinat-
ELISA assays using monoclonal antibodies pro- ion patterns of the other adenoviruses are less well
vided that the epitopes are commonly expressed discernible. A heterotypic rabbit antiadenovirus
and correlate with the appearance of epitopes in- serum (antiadenovirus type 6 is frequently used)
ducing neutralizing antibodies. may be added in order to convert the excess of
Solid-phase immune electron microscopy monovalent haemagglutinins (i.e. fibres and pen-
(SPIEM) is a versatile technique which can be used tons) into divalent, complete haemagglutinins.
for serological typing of viruses in general (Sven- Serum neutralization assays are cumbersome but
sson and von Bonsdorff, 1982). the most efficient means of detecting specific antibo-
One strain may carry neutralization and haema- dies against each adenovirus serotype. All the
gglutination inhibition epitopes of two different above-mentioned techniques have been adapted to
serotypes provided they are members of the same microtitre assays.
subgenus. These strains are designated intermediate
strains or haemagglutination variants.
PREVENTION OF ADENOVIRUS
INFECTIONS
Serological Diagnosis
Treatment of keratoconjunctivitis by adenoviruses
The worldwide distribution of adenoviruses can be during the acute phase with adenine arabinoside
assessed by analyses of prevalence of antibodies and iododeoxyuridine has been demonstrated to be
and/or frequency of isolation of adenovirus strains. unsuccessful. Adenovirus-associated respiratory
Screening of adenovirus-specific antibodies gives disease is difficult to distinguish from respiratory
reliable information on the prevalence of aden- disease from other causes. However, in the USA the
ovirus infection in various populations. Mam- clinical problem has been considered of such im-
malian adenoviruses share group-specific antigen portance that vaccines have been developed. The
epitopes which can be detected by CF or ELISA. early adenovirus vaccines were grown in primary
CF for adenovirus-specific antibodies is ideally per- monkey kidney cells which have been demonstrated
formed with a pool of antigen representing a sero- to contain SV40 virus. SV40 virus can integrate into
type of each subgenus. If this is difficult to accom- the E3 region of the adenovirus genome and this
plish, representative serotypes of subgenus B (e.g. approach was therefore abandoned. A new prin-
Ad7) and subgenus C (e.g. Ad2) should be used. ciple for vaccination against respiratory viruses has
Sonicated, clarified, infected cells provide a good been introduced in the form of live, oral, enteric-
source of antigen as a tenfold excess of structural coated adenovirus vaccines. These strains were cul-
proteins (predominantly hexons, pentons and tivated in human embryo fibroblasts. Initially there
fibres) carrying cross-reactive epitopes is produced. was some concern about the possibility of the ap-
A fourfold or greater rise in CF antibody titre is a pearance of additional adenovirus strains in place
sign of current adenovirus infection. of the Ad4 and Ad7 which were eliminated by the
CF tests have frequently been used to ascertain vaccination introduced 1971. Adenovirus 21 did
the prevalence of antibodies against adenoviruses. appear as a cause of outbreaks of respiratory dis-
However, CF antibodies usually need to be boosted ease. However, since 1976 Ad21 has been incorpor-
if they are to persist for longer periods. Further- ated into the enteric-coated adenovirus vaccine and
more, the CF assay is highly inefficient in detecting adenovirus-associated pneumonia has ceased to be
an immune response to adenoviruses in infants. The a major problem in the US Army (Takafuji et al.,
ELISA assay is therefore preferred. 1979). It has been estimated that at least 15 million
Serotype-specific assays must be applied to ob- recruits have been vaccinated. The vaccines are,
tain information on the response to each aden- however, not licensed for administration to
ovirus serotype. Haemagglutination-inhibition as- children. For logistic reasons the production of the
says using erythrocytes of monkeys (subgenus B) or adenovirus vaccine was terminated in 1995, and by
rats (subgenera A, C, D, E or F.) can be used. 1999 the supplies of the vaccine were depleted. Ad-
Adenoviruses of subgenus B and D agglutinate enovirus infections among conscripts are now re-
326 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
emerging. Arnberg, N., Mei, Y-F. and Wadell, G (1997) Fiber genes of
Ad14 has been reported to cause outbreaks of adenoviruses with tropism for the eye and the genital tract.
Virology, 277, 239—244.
respiratory disease in the Netherlands but not the Azar, R., Varsano, N., Milequir, F. et al. (1998) Molecular epi-
USA. Adenovirus 3 is known to cause infections demiology of adenovirus type 7 in Israel: identification of two
among children. The herd immunity appears to be new genome types, Ad7k and Ad7d . Journal of Medical Virol-
sufficient to prevent outbreaks among military re- ogy, 54, 291—299.
Bailey, A. and Mautner, V. (1994) Phylogenetic relationships
cruits. Children living in crowded conditions may among adenovirus serotypes. Virology, 205, 438—452.
experience severe respiratory disease with Ad3 and Bergelson, J.M., Cunningham, J.A., Droguett, G. et al. (1997)
Ad7. It is possible that vaccination should be con- Isolation of a common receptor for coxsackie B viruses and
sidered in children less than 2 years old since this is adenoviruses 2 and 5. Science, 275, 1320—1323.
the group principally at risk. However, vaccination Carson, J.L., Collier, A.M. and Hu, S.-C.S. (1985) Acquired cili-
ary defects in nasal epithelium of children with acute viral
of children with live vaccines cannot be contem- upper respiratory infections. New England Journal of Medi-
plated until the virulence of different adenovirus cine, 312, 463—468.
genome types has been evaluated. De Jong, J.C., Bijlsma, K., Wermenbol, A.G. et al. (1993) Detec-
Adenoviruses are used as vectors for vaccination tion, typing, and subtyping of enteric adenoviruses 40 and 41
and gene therapy. They have been deleted in E1, E3 from fecal samples and observation of changing incidences of
infections with these types and subtypes. Journal of Clinical
and E4 regions in order to provide for the transgene Microbiology, 31, 1562—1569.
and ascertain that the vector will remain non-rep- Espy, M.J., Hierholzer, J.C. and Smith, T.F. (1987)The effect of
licative. The advantages are that they can be pro- centrifugation on the rapid detection of adenovirus in shell
duced in large amounts and transduce both rep- vials. American Journal of Clinical Pathology, 88, 358—360.
licating and nonreplicating cells. They are not Fife, K.H., Ashley, R., Shields, A.F. et al. (1985) Comparison of
neutralization and DNA restriction enzyme methods for typ-
integrating into the host chromosome and are non- ing clinical isolates of human adenovirus. Journal of Clinical
oncogenic. The expression is usually transient. Microbiology, 23, 95—100.
However, as vectors there are disadvantages be- Flomenberg, P., Babbit, J., Drobyski, W.R. et al. (1994) Increas-
cause they do not interact with the host chromo- ing incidence of adenovirus disease in bone marrow tranplant
recipients. Journal of Infectious Diseases, 169, 775—781.
some and expression is often transient, as they are
Fox, J.P. and Hall, C.E. (1980) Viruses in Families. PSG, Lit-
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
Rhinoviruses
Nikolaos G. Papadopoulos
and
Sebastian L. Johnston
University of Southampton, Southampton, UK
INTRODUCTION TAXONOMY
Rhinoviruses (Figure 9.1) are the major cause of the Rhinoviruses are small RNA viruses belonging to
common cold. Although the majority of infections the picornavirus (pico : small ; RNA) family.
produce only mild disease, their impact on overall Picornaviridae also include the enteroviruses, such
morbidity and their economic cost worldwide are as polio, coxsackie and echo viruses, cardioviruses
considerable. More recently, their role in acute ex- such as the rodent Encephalomyocarditis virus and
acerbations of asthma and other airway disease has aphthoviruses or foot-and-mouth disease viruses.
been demonstrated, revealing a less benevolent na- Rhinoviruses are more closely related to en-
ture than previously thought. teroviruses than the other genera. They are the most
While the recorded history of the common cold is numerous of the Picornaviridae, with 100 serotypes
over 2000 years old (Hippocrates, c. 400 ), identified and numbered by specific antisera in a
rhinoviruses where first propagated in 1953 in tis- collaborative programme supported by the World
sue cultures whose supernatants were able to pro- Health Organization (Hamparian et al., 1987)
duce common cold symptoms in volunteers. The (Table 9.1). New virus types are certainly emerging
first rhinovirus was initially isolated from patients by a process of random mutation and immune se-
with colds and named in 1957, while 3 years later lection (Patterson and Hamparian, 1997).
cytopathogenic viruses were recovered using hu- Rhinoviruses are divided into major (90%) and mi-
man embryo kidney cell cultures (Tyrrell and Par- nor (10%) groups, according to their cellular recep-
sons, 1960). Other susceptible cell lines were subse- tor usage (Uncapher et al., 1991). An alternative
quently discovered and used to identify additional categorization, dividing the viruses into groups A
serotypes and to study rhinoviral biology and role and B based on sensitivity to antiviral compounds
in disease. Recently, the genomes of several sero- and correlating with sequence similarities and
types have been sequenced and three-dimensional pathogenicity, has also been proposed (Andries et
structures have been revealed in atomic resolution. al., 1990).
Molecular technologies are rapidly progressing in
the development of novel diagnostic tools and will
hopefully aid the so far unsuccessful search for an
effective treatment.
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
330 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 9.1 Three-dimensional computer enhanced electron Figure 9.2 Rhinoviral icosahedral symmetry
micrography of Human rhinovirus 14. The canyon can be seen
surrounding the axis. (Courtesy of Dr J.Y. Sgro)
axis of the capsid there is a 2.5 nm deep depression
shaped by the five VP1 units, forming a ‘canyon’
STRUCTURE (Figure 9.1). In most rhinoviruses, VP1 also con-
tains a hydrophobic ‘pocket’ under the canyon,
The Capsid containing an incompletely characterized fatty-acid
‘pocket factor’. This pocket is thought to be in-
Rhinoviruses are among the simplest infectious volved in virus uncoating and is the major target of
agents. Their virion consists of a non-enveloped antiviral compounds (Hadfield et al., 1997).
capsid surrounding a single-stranded positive-sense The characteristic conformation of the canyon,
genomic RNA. In 1985, X-ray crystallography re- being physically unreachable by the immunog-
vealed the structure of Human rhinovirus 14 lobulin Fabs, was hypothesized to be the receptor
(HRV14) and Poliovirus 1 at atomic resolution binding site. Evidence in favour of this hypothesis
(Hogle et al., 1985; Rossman et al., 1985). These and has been produced (Colonno et al., 1988), demon-
subsequent studies showed considerable structural strating that the amino acid sequences at the base of
similarities amongst picornaviruses, such as promi- the canyon are very well conserved, while the edges
nent -sheets forming a -barrel, even though the of the canyon and the most external portions of the
sequence of each virus differs considerably from virus capsid are hypervariable and correspond to
others in the group. The rhinoviral capsid is com- neutralizing immunogenic sites (Rossmann and
posed of 60 identical subunits arranged as 12 pen- Palmenberg, 1988).
tamers in an icosahedron (Figure 9.2). Each subunit
consists of all four structural proteins of the virus,
named VP1—VP4, with molecular masses of 32, 29,
26 and 7 kDa respectively. VP1, 2 and 3 are surface Antigenicity
proteins interacting with antibody and correspond-
ing to the part of the genome with the highest Four such neutralizing immunogenic (NIm) sites,
variability. VP4 is confined to the interior of the localized in different areas of the capsid of RV14,
capsid and is closely associated with the viral RNA. along the edge of the canyon, were characterized by
The arrangement of these structural proteins can be RNA sequencing of mutants that escaped neutraliz-
seen in Figure 9.3. Around the fivefold symmetry ation by specific antisera (Sherry et al., 1986). NIm-
IA and NIm-IB on VP1 are positioned above the
RHINOVIRUSES 331
Table 1 Rhinovirus serotypes
1A Echo-28 51 F01—4081
1B B632 52 F01—3772
2 HGP 53 F01—3928
3 FEB 54 F01—3774
4 16/60 55 WIS 315E
5 Norman 56 CH82
6 Thompson 57 CH47
7 68—CV 11 58 21—CV 20
8 MRH-CV 12 59 611—CV 35
9 211—CV 13 60 2268—CV 37
10 204—CV 14 61 6669—CV 39
11 1—CV 15 62 1963—CV 40
12 181—CV 6 63 6360—CV 40
13 353 64 6258—CV 44
14 1059 65 425—CV 47
15 1734 66 1983—CV 48
16 11757 67 1857—CV 51
17 33342 68 F02—2317—Wood
18 5986—CV 17 69 F02—2513—Mitchinson
19 6072—CV 18 70 F02—2547—Treganza
20 15—CV 19 71 SF365
21 47—CV 21 72 K2207
22 127—CV 22 73 107E
23 5124—CV 24 74 328A
24 5146—CV 25 75 328F
25 5426—CV 12 76 H00062
26 5660—CV 27 77 130—63
27 5870—CV 28 78 2030—65
28 6101—CV 29 79 101—1
29 5582—CV 30 80 277G
30 106F 81 483F2
31 140F 82 03647
32 363 83 Baylor 7
33 1200 84 432D
34 137—3 85 50—525—CV-54
35 164A 86 121564—Johnson
36 342H 87 F02—3607—Corn
37 151—1 88 CVD-01—0165—Dambrauskas
38 CH79 89 41467—Gallo
39 209 90 K2305
40 1794 91 JM1
41 56110 92 SF-1662
42 56822 93 SF-1492
43 58750 94 SF-1803
44 71560 95 SF-998
45 Baylor 1 96 SF-1426
46 Baylor 2 97 SF-1372
47 Baylor 3 98 SF-4006
48 1505 99 604
49 8213 100 K6579
50 A2 No. 58
canyon, while NIm-II on VP2 and NIm-III on VP3, High variability of these regions is implied by the
are positioned below. RV2 has a slightly different large number of distinguishable serotypes and has
pattern; nevertheless, the antigenic sites are still been, to a certain extent, molecularly characterized
located on the most protrusive regions of the virion. (Horsnell et al., 1995).
332 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
mune response and upregulating the viral receptor
itself (Papi et al., 1998), while there is evidence that
at the same time rhinovirus—ICAM-1 binding indu-
ces conformational changes promoting viral un-
coating and cell entry (Casanovas and Springer,
1994).
The minor group receptors have been recently
identified as the low density lipoprotein receptor
(LDLR) and - -macroglobulin receptor/LDLR-
related protein ( MR/LRP) (Hofer et al., 1994).
Limited information is currently available on their
interactions with the virus.
Replication
Figure 9.3 Arrangement of the three main structural Rhinoviral RNA has a molecular weight of approxi-
rhinovirus proteins. The fourth structural protein, VP4, is con- mately 2 MDa, consisting of some 7200 nucleotides
fined in the interior, hence it is not visualized (Duechler et al., 1985). A 5 non-coding region of
around 620 bases in length and a 3 non-coding
Receptors region of 50 bases before a polyadenylated tail are
characteristic of all currently sequenced serotypes
An important function of the viral capsid is to de- (Figure 9.4). The long 5 untranslated region is high-
liver the genomic material intact into a cell follow- ly conserved and contains several AUG codons
ing attachment to a cell surface receptor. Initially, it upstream of that used to initiate translation. These
has been shown that almost 90% of rhinoviruses sequences are within a complex structure, termed
(major group) bind to one receptor on HeLa cells, the internal ribosome entry site or IRES, which can
while the remaining (HRVs 1a, 1b, 2, 29, 30, 31, 44, bind ribosomal subunits directly, without the re-
47, 49, 62) use another (Colonno et al., 1986). The quirement for a free 5 end or a cap binding protein
major group receptor was independently reported (Jackson et al., 1990). The organization of the
by three different groups in 1989 (Greve et al., 1989; genome is shown in Figure 9.4. The structural pro-
Staunton et al., 1989; Tomassini et al., 1989) as the teins VP1—4 derive from the P1 region, while P2 and
intercellular adhesion molecule 1, (ICAM-1). P3 code for the proteins required for virus replica-
ICAM-1 is a 95 kDa glycoprotein member of the tion. The non-structural proteins, seven in total,
immunoglobulin superfamily, physiologically act- include two proteases with specific viral cleavage
ing as a receptor for the lymphocyte-function asso- sites (2A, 3C), an RNA-dependent RNA-poly-
ciated antigen-1 (LFA-1, CD11a/CD18) and Mac-1 merase (3D) and a small protein Vpg (3B) which is
(CD11b/cd18) found on leucocytes. It facilitates the covalently bound to the 5 end of the RNA and
interaction of lymphocytes with antigen-presenting possibly acts as a primer for RNA synthesis (Row-
cells as well as their migration to inflammatory sites lands, 1985). The remaining non-structural proteins
(Montefort and Holgate, 1991). The binding sites of (2B, 2C, 3A) are associated with RNA replication.
LFA-1 and major rhinoviruses on the ICAM mol- The single stranded, positive-sense RNA can act
ecule are distinct but significantly overlapping as messenger RNA and is infectious on its own.
(Staunton et al., 1990). Furthermore, cryoelectron Similarly to the other picornaviruses, the genome is
microscopy proved that the rhinovirus binding site translated into a polyprotein from a single long
is positioned in the canyon, verifying the ‘canyon open reading frame. This polyprotein is processed
hypothesis’ (Rossman et al., 1994). Binding of into the mature proteins by several cleavage steps
rhinoviruses to ICAM-1 blocks LFA-1—ICAM-1 performed by virus-specific proteases. With the
interaction, possibly downregulating the local im- polymerase, a negative-sense copy of the genome is
produced, from which further positive strands are
RHINOVIRUSES 333
made by viral RNA polymerase, and these can act ability to resist heat inactivation at 50—56°C, with
either as messenger RNA, being subsequently trans- some serotypes completely inactivated at lower
lated, or can be incorporated as genomic RNA in temperatures and others quite resistant. Optimal
the progeny virus. Rhinoviruses are capable of com- culture temperature ranges from 33 to 35°C (Kil-
pleting numerous replicative cycles within 6—8 lington et al., 1977).
hours, with a yield of up to 100 000 viruses per cell
(Belsham and Sonenberg, 1996).
Figure 9.5 Pathogenetic mechanisms of rhinoviral infection. Rhinovirus entry and replication in the nasopharynx induces immune
mediators and neurogenic pathways. Viruses are eradicated, but the concurrent vascular leakage and glandular secretion result in the
well-known symptomatology
The mechanisms of exacerbations of asthma in- IgG and IgA serum antibodies remain low for the
duced by viral infections in susceptible individuals first week after inoculation and subsequently begin
is another issue in rhinovirus pathophysiology cur- to increase, to reach their peak approximately a
rently under investigation. Rhinovirus infection month later. IgG antibodies stay at high levels for at
seems to potentiate allergic responses (Calhoun et least a year, while IgA declines slowly but remains
al., 1994), however, the precise mechanisms have yet detectable during the same period. Nasal IgA is also
to be determined. Inflammatory mediators such as produced, becoming detectable 2 weeks after inocu-
kinins, and proinflammatory cytokines induced by lation, reaching its peak 1 week later and slowly
rhinovirus infection (IL-1, IL-6, IL-8, GM-CSF, declining to its original levels by 1 year (Barclay et
IFN- and IFN-), altered cell-mediated immunity, al., 1989). The late rise in antibody titres indicates
virus-specific IgE and other mechanisms have been that humoral immunity is not essential for recovery
proposed, but the relative importance of each of from viral illness. On the other hand, existing anti-
these currently remains unclear (Johnston, 1995). bodies, especially in high titres, may protect against
reinfection with the same serotype (Barclay et al.,
1989). Possible ways for antibody-mediated virus
inactivation include virus aggregation, activation of
IMMUNITY the complement cascade, prevention of binding to
receptor and inhibition of uncoating (Rowlands,
Both cellular and humoral immunity are activated 1995).
in response to rhinovirus infection. Virus-specific An important role in virus eradication has been
336 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
attributed to cellular immunity; however, the mech- density, but most importantly family structure,
anisms involved are not yet understood in detail. In strongly influence the incidence of rhinovirus infec-
contrast to the high specificity of humoral immun- tions. An infection is usually introduced by a child
ity, rhinovirus-specific T cells can recognize both to other siblings and parents at home. Mothers are
serotype-restricted and shared viral epitopes (Gern more susceptible than fathers, possibly because of
et al., 1997b). Peripheral blood lymphocyte counts increased exposure. School and day care trans-
are decreased during infection, followed by recovery mission is also very high, due to overcrowding, low
or even leucocytosis (Skoner et al., 1993). An in- immunity and children’s unhygienic habits (Levan-
creased production of IL-2 and IFN- after mitogen dowski, 1992).
stimulation, as well as increased natural killer A seasonal pattern has been documented in tem-
cytotoxicity, has been found in peripheral blood perate climates, with two peaks occurring—one in
mononuclear cells after experimental rhinovirus in- autumn, coinciding with the opening of schools,
fection (Hsia et al., 1990). Lymphocytes can be ac- and another one in late spring. During winter the
tivated both specifically and non-specifically occurrence of rhinovirus infections is reduced (Fox
through a monocyte-dependent mechanism (Gern et al., 1975), although more recent studies have
et al., 1996a). Rhinoviruses enter but do not repli- suggested that school attendance is the major factor
cate inside monocytes and airway macrophages, in determining seasonal patterns, and that infec-
indicating a potentially direct effect of these cells in tions occur throughout the winter months, peaking
antiviral immunity (Gern et al., 1996b). Locally pro- in the first 2—3 weeks after children return to school
duced soluble factors by activated immune cells, (Johnston et al., 1996).
such as interferons or tumour necrosis factors, have All populations are affected. The prevalent sero-
potent antiviral activities (Wong and Goddel, 1986). types vary from year to year. It is possible that a
small number of serotypes may cause most of the
illnesses (Monto et al., 1987); however, virus isola-
tion and serotyping techniques are still cumber-
EPIDEMIOLOGY some and inadequate for detailed evaluation.
There is a well-documented epidemiological rela-
The common cold is probably the illness most fre- tionship between psychological stress and the sus-
quently afflicting humans, and is certainly the most ceptibility to rhinovirus infection (Cohen, 1995), the
common cause for primary care consultations and mechanisms of which are still speculative.
absenteeism from work or school. The average
number of yearly infections in adults is estimated to
be between two and five, while this number in-
creases up to 12 in children. A simple calculation CLINICAL FEATURES
would reveal that a normal individual spends be-
tween 1 and 2 years of life suffering from colds! The symptoms most easily described are those that
Rhinoviruses cause about 35—60% of common everybody has experienced (Hippocrates, c. 400 ).
colds. Interestingly, in contrast to popular belief, Rhinoviruses produce the symptoms of the
early studies were unable to demonstrate any in- common cold, including rhinorrhoea, sneezing, na-
crease in susceptibility to rhinoviral infections after sal obstruction, sore throat and cough. General
exposure to cold temperatures (Douglas et al., malaise and headache may occur, while fever is less
1968); however, several other epidemiological fac- common. The course of the disease correlates with
tors are involved. Age is certainly important. Infec- virus titres. Symptoms appear after a 24—48 hour
tions increase significantly from the second year of incubation period, reach their peak 2—3 days later
life and throughout school age, decreasing subse- and last for 5—7 days in total, persisting occasionally
quently, probably due to neutralizing antibodies for as long as 2—4 weeks. Symptom severity is highly
induced by previous exposures (Monto, 1995). In- variable; on many occasions the disease may be
creased morbidity and complications reappear in hardly noticed.
the elderly (Nicholson et al., 1997). Apart from the On the other hand, rhinovirus infections may
age-related susceptibility to the virus, socio- cause significant morbidity in specific patient
economic factors such as nutrition and population groups. Infants with bronchopulmonary dysplasia
RHINOVIRUSES 337
Figure 9.6 Chart of diary card recordings from a 10—year-old child with intermittent wheeze during community aqcquired rhinovirus
infections (indicated by arrows; major group rhinovirus infections indicated by boxes). Episodes of falls in peak expiratory flow (PEF)
associated with severe lower respiratory symptoms (wheeze and cough, chest score) and upper respiratory symptoms (cold score) are
clearly seen
may develop serious respiratory illness, necessitat- the virus in as many as 50% of episodes of commu-
ing admission to an intensive care unit and occa- nity-acquired sinusitis (Pitkaranta et al., 1997).
sionally mechanical ventilation (Chikedel et al., Moreover, rhinoviruses account for approximately
1997). Pulmonary function abnormalities, disease 40% of viral acute otitis media (Buchman et al.,
progression and secondary bacterial infections can 1994). In both instances, the viruses may also facili-
result in children with cystic fibrosis (Collinson et tate or contribute to concurrent bacterial infec-
al., 1996). Senior persons, especially residents of tions.
nursing homes, are also prone to severe disease that The role of rhinoviral infection in exacerbations
can exceptionally prove fatal (Wald et al., 1995). of asthma has been well documented in recent stu-
Furthermore, atopic individuals seem to be predis- dies. Upper respiratory tract infections are asso-
posed to more severe rhinoviral colds than normal ciated with up to 80% of asthma episodes in school-
subjects (Bardin et al., 1994). children, with rhinovirus being the most commonly
isolated agent (Johnston et al., 1995) (Figure 9.6). In
adults, the figures are smaller (Beasley et al., 1988)
although studies employing methodology as sensi-
Complications tive as that used in children have yet to be reported.
Time trend analysis suggests that viral infections
Acute sinusitis and acute otitis media have been are indeed important in exacerbations in adults as
associated with rhinoviral infections. As previously well as children (Johnston et al., 1996).
mentioned, sinus involvement is detected by CT in Rhinoviruses are also implicated in acute and
a considerable percentage of rhinovirus infections, chronic bronchitis but this subject has yet to be
while there is evidence suggesting a causal role of adequately studied.
338 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 9.7 Rhinovirus infected Ohio-HeLa cell culture with the characteristic cytopathic effect (right), in comparison to control cells
(left)
Figure 9.8 Agarose gel electrophoresis of PCR products reveals a band at 380 bp. Both genomic and replicative strands (using OL27
primer and OL26 primer on the reverse transcriptase, respectively) are shown for rhinoviruses 2, 9, 14 and 16 (left half). Dilutions of
RV9 can be detected up to 10\
10
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
346 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 10.1 Electronmicrographs of (a, b) human enteric torovirus showing torus (arrow), crescent (arrow head) and rod (double
arrow head), and (c) a human enteric coronavirus. The latter has spikes which are more prominent and readily discernible than those of
the torovirus. The shorter, 10 nm, projections on the torovirus particles may be the haemagglutinin-esterase protein (HE).
(Bar : 100 nm) (Reprinted from Duckmanton et al. (1997) by permission of Academic Press)
CORONAVIRUSES AND TOROVIRUSES 347
name Feline infectious peritonitis virus (FIPV).
Murine hepatitis virus (MHV, murine coronavirus)
strains, in addition to infecting one or more of
respiratory, enteric and hepatic tissues, replicate in
cells of the central nervous system, causing en-
cephalitis and demyelination (Dales and Anderson,
1995; Houtman and Fleming, 1996). Porcine hae-
magglutinating encephalomyelitis virus (HEV) also
selectively infect neuronal tissue. Coronaviruses
have been assigned to one of groups 1, 2 and 3
(Table 10.1) on the basis of amino acid sequences,
supported to some extent by antigenic analyses.
Structure
Growth In Vitro
Antigenic Structure
Previously OC43 and serologically related strains
Virus-neutralizing (VN) antibodies are induced by
of HCV were considerably more difficult to propa-
both the S and HE proteins of coronaviruses (re- gate in cell culture than 229E-related strains and so
viewed by Cavanagh, 1995), and some antibodies organ cultures were used for OC43, hence the letters
against the M protein neutralize virus in the pres- ‘OC’. Now, however, cell lines are available for the
ence of complement. Monoclonal antibodies have propagation of both of the laboratory-adapted
shown that neutralizing and haemagglutination- HCV types (consult HCV sequence papers referred
inhibiting antibodies are induced by the torovirus S to above), although primary isolation remains diffi-
protein. cult (Myint, 1994, 1995). Cultures of fetal and adult
Epitope mapping of coronaviruses has been done astrocytes were infected by both types of HCV but
mostly for S and has shown that VN epitopes are infectious virus was only released from the fetal cells
formed largely by the amino-terminal S1 half of the (Bonavia et al., 1997). Human torovirus has not
protein, which correlates with the much greater
been grown in vitro.
amino acid sequence variation in S1 than in S2
(Cavanagh, 1995). Removal of glycans from S of
IBV greatly diminished the binding of VN mono-
clonal antibodies. The S protein has been shown to CELL RECEPTORS FOR HCVs
be a major inducer of protective immunity, al-
though the other structural proteins, particularly Attachment of Coronaviruses to Cells
the nucleocapsid protein, contribute to the induc-
tion of protection. In the case of FIPV it is the The attachment of a virus to host cells is a crucial
immune response to S which exacerbates the dis- early step in infection, and specificity of attachment
ease. It is thought that FIPV attached to anti-S can be a major factor in determining host cell range
antibody remains infectious and is more readily and hence pathogenesis. This has been elegantly
taken up by macrophages (antibody-dependent en- demonstrated by the transfer of coronavirus recep-
hancement), in which FIPV replicates, than virus tors from susceptible cells to other cells, rendering
without antibody (de Groot and Horzinek, 1995). the latter susceptible to infection. It is the S protein
Coronaviruses have been placed into one of three that serves to attach the virus to cells. The extent to
groups (Table 10.1). Species have higher amino acid which the HE protein, when present, is also respon-
identities with viruses within their group than with sible for cell attachment is not clear. This topic has
those in the other groups. Previously the groups been reviewed by Holmes and Compton (1995) and
had been referred to as serological groups. This, Lai and Cavanagh (1997).
however, was misleading, as some members e.g. Human aminopeptidase N (APN) has been
HCV-229E and PEDV, had only low antigenic identified as a receptor for HCV-229E (Yeager et al.,
cross-reactivity with other members of group 1. 1992). This protein is a metalloprotease located on
Until recently TCV had been in group 2 but recent the surface of epithelial cells, including those of the
sequence and antigenic analyses have indicated that intestine, lung and kidney. Porcine APN is the re-
it should be assigned to group 3 (Stephenson et al., ceptor for TGEV (Delmas et al., 1992). TGEV repli-
1999). cates mostly in enterocytes of the small intestine
350 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
and produces its greatest tissue damage at this site Involvement of Glycans
but it also replicates to a lesser degree within the
respiratory tract. This correlates with the incidence Both the S and HE proteins of BCV bind to cell
of APN, which is greater in the small intestine than surface components, a vital component of which is
in lung tissue. the glycan component N-acetyl-9—O-acetylneura-
The cellular receptor for MHV is a member of the minic acid (Neu5,9Ac ). This residue acts as a recep-
carcinoembryonic antigen (CEA) family of glyco-
tor not only on erythrocytes but also on susceptible
proteins in the immunoglobulin superfamily (re- tissue culture cells (Schultze and Herrler, 1992). The
viewed by Lai and Cavanagh, 1997). This CEA S protein binds more efficiently than HE to
protein is a 424 amino acid glycoprotein with four Neu5,9Ac and it has been proposed that the BCV
immunoglobulin-like domains. Human and ham-
S protein is responsible for the primary attachment
ster cells, refractory to infection by MHV, became to cells (Schultze and Herrler, 1992). The HEF
susceptible to MHV when this receptor was ex- glycoprotein of influenza C virus also binds to
pressed following transfection with the murine CEA Neu5,9Ac , while influenza A and B viruses do not
gene (Dveksler et al., 1991). Chen et al. transfected
(Vlasak et al., 1988).
COS-7 cells, which lack a functional receptor for It has been proposed that the attachment of co-
MHV, with genes of human CEA and human bili- ronaviruses might be a two-step process. Primary
ary glycoprotein; the cells were then susceptible to attachment might be mediated by a first receptor,
MHV (Chen et al., 1997). Experiments with chim- e.g. Neu5,9Ac , for some coronaviruses, a second
eras of human and murine CEAs revealed that the
receptor, e.g. APN or CEA proteins, bringing the
immunoglobulin loop of human CEA conferred vi- virus and cell membranes closer together. The latter
rus-binding specificity. Different isoforms of murine step might be necessary for the fusion of the two
CEAs exist. These have extensive differences in the membranes for the release of the virus genome.
amino-terminal immunoglobulin-like domain to Some receptors might fulfil both functions for some
which MHV binds, and bind MHV to different coronaviruses.
extents. Analysis of chimeras indicated that amino IBV (and TGEV) attaches to 2,3—linked
acids 38—43 were key elements for binding MHV neuraminic acid on erythrocytes and, provided that
and virus-induced membrane fusion (Rao et al., the virions are first treated with neuraminidase
1997). (from Vibrio cholerae or Newcastle disease virus),
Human cells which were not susceptible to CCV can cause haemagglutination (Schultze et al., 1992).
or FCV became susceptible when transfected with a This removes 2,3—linked neuraminic acid, which is
human/canine chimera of APN (Benbacer et al., present on the S protein of the virus and interferes
1997). The critical, carboxy-terminal domain was with the attachment of the S protein to neuraminic
formed by amino acids 643—841 of the canine APN. acid on the erythrocyte surface. In view of the find-
When amino acids 255—348 of porcine APN were ing that TGEV attaches to porcine enterocytes, as
replaced by amino acids 260—353 of human APN, opposed to erythrocytes, via APN, it may be that
the resulting chimeric protein was able to function either 2,3—linked neuraminic acid does not play a
as a receptor for HCV-229E (Kolb et al., 1996). role in the attachment to these susceptible cells or
Thus different parts of APN molecules may func- that it may function as a primary receptor, insuffi-
tion as receptors for different coronaviruses within cient for efficient infection, part of APN forming a
viruses of group 2 (Table 10.1). Expression of feline secondary receptor essential for efficient infection.
APN in rodent cells rendered the cells susceptible The role played by the HE protein where present,
not only to FCV but also to HCV-229E, CCV and e.g. in HCV-OC43, is not clear. Although it can
TGEV (Tresnan et al., 1996). Previous work had mediate binding to erythrocytes, the main function
shown, however, that human APN would bind of HE might be to remove neuraminic acid from the
HCV-229E but not TGEV, while porcine APN virus and cell surface. The esterase activity of the
would bind TGEV but not HCV. HE, and HEF protein of influenza C, specifically
Less work has been done on the receptor for cleaves Neu5,9Ac and does not hydrolyse the re-
HCV-OC43. Collins (1995) has demonstrated that
ceptors for influenza A and B. Following haemag-
OC43 binds to major histocompatibility complex glutination at 4°C, subsequent incubation at 37°C
class I molecules.
CORONAVIRUSES AND TOROVIRUSES 351
results in the destruction of the neuraminic acid and Lower Respiratory Tract Disease
subsequent elution of the virus and reversal of the
haemagglutination. There have been some reports indicating a more
serious lower tract involvement in young children
and old people (Isaacs et al., 1983). It is not clear
that HCVs infect the lower respiratory tract but the
occurrence of HCV upper respiratory tract infec-
CLINICAL FEATURES tions coupled with other factors may cause more
serious disease. Up to 30% of acute wheezing epi-
Diseases associated with coronaviruses in humans sodes in asthmatic children may be due to co-
have been reviewed by Myint (1994). ronavirus infection.
A study in a neonatal intensive care unit revealed
that all premature infants infected with co-
ronaviruses had symptoms of bradycardia, apnea,
hypoxaemia, fever or abdominal distention. Chest
Upper Respiratory Tract Disease X-ray revealed diffuse infiltrates in two cases (Sizun
et al., 1995).
Most information on the clinical features of HCV
infections in humans has been obtained from volun-
teers experimentally infected with HCVs, and epi-
demiological studies using paired sera collected Molecular Bases of Tropisms
from individuals during and after naturally ac-
quired HCV infections. HCVs are believed to be Little is known about the molecular basis of the
second only to rhinoviruses with respect to the tropism of human coronaviruses and toroviruses.
percentage of colds that they cause. Observations made with MHV are instructive in
HCVs generally cause mild upper respiratory this regard. Variation in the sequence of S would
tract infections in humans, and up to 30% of appear to have marked effects on tissue tropism and
common colds are caused by HCVs. The incubation pathogenicity. Mutations in S of MHV were asso-
period of HCV infections is relatively short, with a ciated with the rate of spread of the virus in neurons
range of 2—5 days to the development of symptoms. and whether the infection resulted in demyelination
Illness—nasal discharge, mild sore throat, sneezing, or encephalitis. Persistent infection of mouse glial
general malaise, perhaps with headache (Tyrrell et cells by MHV resulted in the production of mutants
al., 1993)—then lasts for an average of 6—7 days, with uncleaved spike protein. These did not cause
sometimes lasting for 18 days. Fever and coughing cell—cell fusion and consequently spread slowly
may be exhibited in 10% and 20% of cases, respect- (Gombold et al., 1993). These and other changes in
ively. No difference is observed between 229E and tropism and pathogenicity probably did result from
OC43 strains in the pathology of infection. Gen- changes in the S gene, although the possibility that
erally symptoms are indistinguishable from those of mutations had occurred elsewhere in the genome
colds caused by rhinoviruses. Virus excretion cannot be excluded (Cavanagh, 1995). Gallagher
reaches a detectable level at the time symptoms (1997) has shown that a tissue culture-adapted mu-
begin and lasts for 1—4 days. Subclinical or very tant of MHV JHM, the JHMX strain, did not elute
mild infections are common and can occur through- from its receptor at 37°C, whereas the parental
out the year. JHM strain did do so. This difference in interaction
An electron microscopic examination of the nasal with the receptor was associated with a large dele-
epithelium of a person infected by HCV revealed tion from the S protein of JHMX.
virus particles within and outside the ciliated cells
but neither within or outside the goblet cells or
other cells of the nasal mucosa (Afzelius, 1994). The Epidemiology
ciliated cells seemed not to be destroyed, although
in many cases the cilia were withdrawn. This loss of Generally HCV epidemics occur during the winter
cilia was considered likely to cause rhinorrhoea. and early spring but the peak period may vary by
352 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
several months. However, other periods of high Coronaviruses Associated with the
infection rates have been observed and it appears Human Enteric Tract
that major peaks may occur at any time of the year.
All age groups are infected with HCVs and infection There has been an increasing number of reports
rates have been shown to be relatively uniform for describing coronavirus-like viruses isolated from
all age groups (Monto and Lim, 1974). This is differ- faecal specimens from humans (Macnaughton and
ent from the situation with some other respiratory Davies, 1981; Myint, 1994; Duckmanton et al.,
viruses, such as respiratory syncytial virus, where 1997). Some of these viruses were isolated from
there is a distinct decrease in infection rates with infants with necrotizing enterocolitis, patients with
increase in age. non-bacterial gastroenteritis and from homosexual
The incidence of OC43 and 229E types of HCV men with diarrhoea who were symptomatic and
has been studied in depth among approximately seropositive for human immunodeficiency virus.
1000 people in Tecumseh, USA, over a period of 4 Some isolates were shown to be serologically re-
years. Serology revealed that 17% of individuals lated to OC43. The discovery that a protein found
had a cold caused by OC43 in any one year (Monto in enterocytes functions as a receptor for HCV-
and Lim, 1974). Infections by 229E in the same 229E strengthens the possibility that coronaviruses
community averaged 8% each year, while double might replicate in the human alimentary tract.
this rate was detected elsewhere in another study. In
some years the incidence of disease was much
greater than the average. In a study in the Rhone
Toroviruses Associated with the Human
Alpes region of France, virus was isolated from
approximately one-third of nasal swabs, 18% of Enteric Tract
these yielding HCV (Lina et al., 1996).
There is a periodicity of infections caused by Duckmanton and colleagues (1997) have isolated
229E and OC43 group viruses which follows a com- and purified toroviruses from faeces of paediatric
plex pattern, although they usually cause peaks of patients with gastroenteritis, extending previous
infection with intervals of 2 or 3 years. In general, work (Beards et al., 1984; Koopmans and Horzinek,
high infection rates in any particular year are 1994). That the viruses detected were indeed
caused by either 229E or OC43 group viruses, with toroviruses was demonstrated by nucleotide se-
only the occasional sporadic HCV infection belong- quencing. More studies of these little-known viruses
ing to the other group. HCV infections have been may not be expected.
shown to occur throughout the world with the same
cyclic pattern.
Certain individuals are more prone than others Coronaviruses in the Central Nervous
to HCV infections. Reinfection of individuals with System
the same HCV serotype often occurs within 4
months of the first infection, suggesting that Multiple sclerosis (MS) is a chronic disease of the
homologous HCV antibodies are protective for central nervous system involving multifocal regions
about 4 months. In addition, there are indications of inflammation and myelin destruction. A number
that antibodies to one HCV group may not be of environmental factors have been proposed for
protective against infection with viruses from the triggering MS, foremost among them being infec-
other HCV group. tious agents (Kurtze, 1993). Many viruses have been
The serological response to both naturally ac- linked with MS, including coronaviruses. Co-
quired and experimentally induced infections is ex- ronavirus-like particles were observed in the brain
tremely variable and depends on a number of fac- of an MS patient and titres of antibodies to HCVs
tors, including the infecting HCV strain and the were greater in cerebrospinal fluid from MS pa-
serological test employed. Many individuals have tients compared to control subjects. Two putative
high antibody levels after infection, and reinfection coronaviruses (CV-SD and CV-SK) were isolated
with the same or related strains is common. after passage in mice of brain material from two MS
patients. Doubt has been cast on whether the SD
and SK isolates are of human, rather than murine,
CORONAVIRUSES AND TOROVIRUSES 353
origin. However, analysis of brain tissue from MS Almost one-third of T cell lines established from
and non-MS patients by in situ hybridization using MS patients, but 2% from normal control sub-
DNA probes complementary to CV-SD and MHV jects, showed an HLA-DR-restricted cross-reactive
RNA revealed the presence of nucleic acid to which pattern of antigen activation after in vitro selection
the probes bound (Murray et al., 1992a). The inci- with either MBA or HCV-229E antigens (Talbot et
dence of this RNA was fivefold greater in the MS al., 1996). This further supports the possibility that
patients. Stewart et al. (1992) analysed RNA from molecular mimicry is involved in MS, HCV being
brain tissue of MS patients using polymerase chain one of several pathogens that have exhibited this
reactions (PCRs) specific for HCVs OC43 and phenomenon with MBA (reviewed by Talbot et al.,
229E. RNA of the latter type was detected four of 11 1996).
MS patients and in none of six neurological and five
normal controls. No OC43 RNA was detected.
Although these observations do not prove that
DIAGNOSIS
coronaviruses cause MS in humans, it should be
remembered that strains of MHV cause both acute
Diagnosis of HCV infections is not routinely done
and chronic infections, including demyelination, of
in most diagnostic virus laboratories. This is mainly
the central nervous system of mice and rats (Dales
due to their fastidious growth requirements in cell
and Anderson, 1995; Houtman and Fleming, 1996).
culture and to their inadequate immunological
Moreover, intracerebral inoculation of Owl mon-
characterization, which makes serological diag-
keys and African green monkeys with either MHV
nosis difficult. In addition, the infections caused by
or the CV-SD isolate resulted in acute to subacute
HCVs have so far proved to be largely of minor
panencephalitis and/or demyelination. Replication
clinical significance and therefore have not merited
of these viruses was demonstrated by detection of
extensive study. However, the application of nucleic
viral RNA and proteins (Murray et al., 1992b). Ex-
acid technology, described below, promises to make
periments with MHV and rodents have shown that
detection of human coronaviruses, and toroviruses,
the virus was able to enter the central nervous sys-
much easier.
tem from peripheral trigeminal or olfactory nerves
Diagnostic procedures which have been used
after intranasal inoculation (see references in Mur-
comprise virus isolation in cell and/or organ cul-
ray et al., 1992b).
tures, and serological diagnosis on paired sera from
Infection by OC43 in primary cultures of fetal
infected individuals (Myint, 1994, 1995).
astrocytes and of adult microglia and astrocytes
was demonstrated by fluorescence (Bonavia et al.,
1997). This technique failed to detect 229E but RNA
replication of this type of HCV was detected by Virus Isolation
PCR and probing of the resultant DNA. Interest-
ingly, only the fetal astrocytes released infectious Virus isolation can be done from nasal and throat
virus. Clearly HCVs have the capacity to replicate swabs, and nasopharyngeal aspirates taken from
in some cells of the CNS of primates. infected individuals.
One feature of MS is an increased frequency of 229E and related strains can be isolated in roller
myelin-reactive T cells, a characteristic also of ro- culture monolayers of human embryonic lung fi-
dents infected with neurotropic strains of MHV. broblasts, such as W138 and MRC5 cells. In virus-
Transfer of these rodent T cells to naive animals positive cultures a gradual cytopathic effect consist-
triggered experimental allergic encephalomyelitis ing of small, granular, round cells appears through-
(EAE), an autoimmune disease that is used as a out the monolayers, although especially around the
model for MS, triggered by injection of myelin basic periphery of the monolayers. However, cell sheets
protein (MBP) (Watanabe et al., 1983). Epitopes are rarely destroyed completely on initial isolation.
common to MBP and MHV—molecular Isolates are generally confirmed as being 229E-re-
mimicry—have been invoked as the basis of this lated by standard serum neutralization tests. Other
autoimmunity. HCV-229E has a non-structural cell types that have been used are described by
protein which has a short amino acid sequence Myint (1995).
identical to one in MBP (Jouvenne et al., 1992). OC43—related strains usually cannot be grown in
354 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
cell cultures, at least on initial isolation, and for HCV in nasal washings from volunteers infected
these strains isolation has to be performed in organ with 229E. This techniques was of comparable sen-
cultures of human embryonic tissues. Small pieces sitivity to standard culture. However, when a non-
of tracheal or nasal epithelium with the ciliated radioactive probe was used, preferable in a diagnos-
surface uppermost are inoculated with the respir- tic laboratory, there was loss of sensitivity (Myint,
atory specimens and the culture examined for cili- 1995).
ary activity daily for up to 2 weeks. The medium A much more sensitive method of detection is the
from these preparations is then harvested and PCR or, strictly speaking, the reverse transcriptase
examined after negative staining by direct electron PCR, as the RNA of HCV must first be copied into
microscopy or by immune electron microscopy us- DNA by the enzyme reverse transcriptase (RT).
ing convalescent-phase serum from the infected pa- Stewart et al. (1995) have described a RT-PCR,
tient. Immobilization or reduction in ciliary activity followed by Southern blotting, for the detection of
of infected organ cultures in comparison to that in HCV-229E and OC43 in cerebral autopsy material.
controls has also been used to identify HCV infec- Cristallo et al. (1996) have also described a RT-PCR
tion, but this is not a reliable measure of infection. and probing method for detecting HCV-OC43.
Myint et al. (1994) have used a nested RT-PCR to
detect HCVs on nasal swabs. This approach, which
Serological Diagnosis does not require probing to detect the PCR prod-
uct, was much more sensitive than cell culture or
229E- and OC43—type infections can be identified probe methods. Of all the methods used to detect
using paired serum samples taken from acute and HCV, the nested PCR would seem to be the one
convalescent stages of infection; tests used include most likely to be used in future. PCR technology is
virus neutralization, complement fixation and hae- commonly used in major diagnostic laboratories
magglutination inhibition (for OC43—related for many purposes. The specific reagents required
strains). A more sensitive method for detecting sig- for HCV detection—oligonucleotides correspond-
nificant changes in HCV antibody in paired human ing to an HCV gene—are readily and inexpensively
sera is ELISA. made as the nucleotide sequences of both 229E and
OC43 are in the public domain. This approach can
also be used to detect coronaviruses in faeces. It is
possible, however, that some human coronaviruses
Detection of HCV Proteins present in faeces might have nucleotide sequences
significantly different from those of 229E and/or
HCV particles can be detected in epithelial cells OC43. Degenerate oligonucleotides, possibly based
shed from the nasopharynx of individuals with co- on sequences in the more conserved polymerase
ronavirus infections. Indirect immunofluorescence gene, might be useful in this regard (Stephenson et
assays have shown that coronavirus fluorescence al., 1999).
can be visualized in nasopharyngeal cells from vol- The sequencing of part of the genome of
unteers inoculated with HCVs. An antigen-capture toroviruses present in human faeces is a great ad-
ELISA has been used to detect HCV antigens in vance for the study of these little known viruses,
nasal and throat swabs, and nasopharyngeal aspi- making possible the application of RT-PCR anal-
rates, taken from children suffering from acute re- ogous to that described for HCV (Duckmanton et
spiratory infections. Nasal swabs were the best al., 1997).
specimens for obtaining suitable quantities of anti-
gen for the assay.
TREATMENT
Detection of HCV RNA
As yet there are no treatments which prevent the
Cloning of the N gene of HCV-229E made possible development of HCV infections. When a human
the production of a radiolabelled nucleic acid probe cerebral cortical neuron cell line, in which HCV-
which was used in a slot-blot technique to detect OC43 replicated very poorly, was treated with in-
CORONAVIRUSES AND TOROVIRUSES 355
terferon , the susceptibility to OC43 was increased diarrhea coronavirus (NCDCV). New Microbiologica, 19,
100—fold. This was due to membrane expression of 251—256.
Dales, S. and Anderson, R. (1995) Pathogenesis and diseases of
HLA class I which acts as a receptor for OC43 the central nervous system caused by murine coronaviruses. In
(Collins, 1995). Current studies of the various enzy- The Coronaviridae (ed. S.G. Siddell), pp 257—292. Plenum
matic activities within the large polymerase protein Press, New York.
of HCV may lead to the development of drugs that de Groot, R.J. and Horzinek, M.C. (1995) Feline infectious per-
itonitis. In The Coronaviridae (ed. S.G. Siddell), pp 293—315.
would inhibit HCV but have no adverse effect on Plenum Press, New York.
human cells. de Vries, A.A.F., Horzinek, M.C., Rottier, P.J.M. et al. (1997) The
genome organisation of the Nidovirales: similarities and dif-
ferences between arteri-, toro- and coronaviruses. Seminars in
ACKNOWLEDGEMENTS Virology, 8, 33—47.
Delmas, B., Gelfi, J., L’Haridon, R. et al. (1992) Aminopeptidase
N is a major receptor for the enteropathogenic coronavirus
The author acknowledges financial support from TGEV. Nature, 357, 417—420.
the Ministry of Agriculture, Fisheries and Food, Duckmanton, L., Luan, B., Devenish, J. et al. (1997) Characteriz-
and the Biotechnology and Biological Sciences Re- ation of torovirus from human faecal specimens. Virology, 239,
158—168.
search Council, UK. Dveksler, G.S., Pensiero, M.N., Cardellichio, C.B. et al. (1991)
Cloning of the mouse hepatitis virus (MHV) receptor: expres-
sion in human and hamster cell lines confers susceptibility to
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
11
Measles
Sibylle Schneider-Schaulies and Volker ter Meulen
Julius-Maximilians University, Würzburg, Germany
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
358 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
was only with the isolation of the virus by Enders brane) and appear on its inside surface (Fraser and
and Peebles in 1954 that experimentation became Martin 1978). It is the aminoterminus of the H
possible. The development of tissue culture systems, protein that protrudes through the cytoplasmic and
the availability of monoclonal antibodies and mol- viral membranes (type II glycoprotein), while the F
ecular biological approaches have then permitted a protein is anchored near the C-terminus (type I
greater dissection of the virus structure and replica- glycoprotein). One or both of the cytoplasmic do-
tion strategy. Experimental approaches to define mains are believed to interact with the matrix (M)
MV protein functions in detail have long been protein which, in turn, links the envelope to the
limited due to the lack of a system that allows a RNP core structure. The viral genomic RNA is fully
reverse genetic approach. Only recently has a plas- encapsidated by N (nucleocapsid) protein to form
mid-based system to rescue infectious MV in tissue the RNP core structure that resists RNAse degra-
culture been developed, making possible the intro- dation. As the viral genome cannot serve as mRNA,
duction of stable alterations into the viral genome the viral polymerase complex consisting of the P
and leading to an understanding of the contribution (phospho-) and the L (large) proteins is part of the
of any single viral gene product in the MV life cycle RNP core complex. Their location within this com-
and precisely how its functions are exerted plex has not yet been resolved. The same holds true
(Radecke et al., 1995). for cellular actin which is also known to be
MV is a member of the newly introduced order packaged into the virion structure.
Mononegavirales which includes the Rhabdoviridae, The virions are highly pleomorphic with an aver-
Filoviridae and Paramyxoviridae. As a para- age size of 120—250 nm and both filamentous and
myxovirus, MV reveals structural and biochemical irregular forms are known. The virion shown in an
features associated with this group; however, it electron micrograph is bounded by a lipid envelope
lacks a detectable virion-associated neuraminidase which bears a fringe of spike-like projections (pep-
activity. It has therefore been classified in a separate lomers) 5—8 nm long (Figure 11.2a). The membrane
genus, Morbillivirus, of which it is the type species. below the spikes is 10—20 nm in thickness and en-
Other members of this group include: Peste de petit closes the helical viral RNP core which has a diam-
ruminants virus (PPRV) which infects sheep and eter of 17 nm and a regular pitch of 5 nm. Immedi-
goats; Rinderpest virus (RPV), which infects cattle; ately below the membrane, M proteins appear as a
Canine distemper virus (CDV), which infects dogs; shell of electron dense material.
Phocine distemper virus (PDV), which infects seals
and sea-lions; Dolphin morbillivirus (DMV); and
Porpoise morbillivirus (PMV). More recently, a
morbillivirus has been isolated in Australia which Genome Structure
infects horses, and accidentally humans. All these
viruses exhibit antigenic similarities, and all pro- The viral genome is a non-segmented RNA mol-
duce similar diseases in their host species. Both MV ecule of negative polarity that is about 16 kb in
and CDV can persist in the central nervous system length. The genome, completely sequenced for the
(CNS) and produce chronic neurological diseases. MV Edmonston (ED) strain, encodes six structural
genes for which the reading frames are arranged
linearly and without overlap in the following order:
3 nucleoprotein (N, 60 kDa), phosphoprotein (P,
Morphology 70 kDa), matrix protein (M, 37 kDa), fusion protein
(F, disulphide-linked 41 kDa F and 22 kDa F pro-
MV particles consist of a lipid envelope surround- teins, cleavage products of a 60 kDa precursor F
ing the viral RNP complex which is composed of protein), haemagglutinin protein (H, 80 kDa, exist-
genomic RNA associated with proteins (Figure ing as disulphide-linked homodimer) and the large
11.1a). Both viral transmembrane proteins (fusion protein (L, 220 kDa) encoded on its 5 end (Figure
(F) and hemagglutinin (H) proteins) are present on 11.1b) (Rima et al., 1986).
the envelope surface and appear as projections from The genome is flanked by non-coding 3 leader
the particle. Portions of both the F and H proteins and 5 trailer sequences that are thought to contain
extend through the virion envelope (transmem- specific encapsidation signals and the viral promo-
Figure 11.1 (a) Measles virus particle. (b) MV mRNAs are sequentially transcribed from the genome with decreasing efficiency and
encode the structural proteins. In addition, the second gene (the P gene) encodes three non-structural viral proteins C, R and V. (c) At
the gene boundaries, MV genes are separated by conserved intergenic regions where the polymerase complex stutters to polyadenylate
the proximal mRNA to subsequently reinitiate transcription of the downstream gene. Alternatively, these signals are neglected by the
polymerase when bicistronic or polycistronic mRNAs are transcribed, as well as during genome replication
360 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ters used for viral transcription and/or replication.
The genomic RNA molecule is entirely complexed
with N protein with one N molecule covering six
nucleotides. This is thought to be the reason why
only viral genomes whose number of nucleotides is
a multimer of six are efficiently replicated by the
viral polymerase. Within the 3 leader sequence,
transcription of the viral monocistronic mRNAs
from the encapsidated genome is initiated. The
coding regions of the viral genome are separated by
conserved intergenic regions which consist of a
polyadenylation signal at the 3 end of each gene, a
conserved trinucleotide (CUU, except for CGU at
the H/L gene boundary) and a reinitiation signal for
the distal gene (Figure 11.1c). From the P gene,
three non-structural proteins, C (20 kDa), V
(46 kDa) and R (46 kDa) are expressed. Whereas the
genetic information for the C protein is encoded by
a separate reading frame (Bellini et al., 1985), V
protein can only be translated from edited P
mRNAs where a G residue not encoded by the viral
genome has been cotranscriptionally inserted at
particular sites. It thus shares a common N-ter-
minal domain with P, while a zinc finger-like do-
main is present on the C-terminus of V. About 50%
(to yield V) and 1.5% (to yield R) of the P mRNAs
are edited, and it has been established that editing is
an intrinsic activity of the MV polymerase. This is
because P mRNAs, once synthesized, are not edited,
and editing of P gene transripts is not performed by
heterologous polymerases such as that of Vaccinia
virus. At the M—F gene boundary a GC-rich region
of about 1 kb in length spans the 3 end of the M
gene and the 5 end of the F gene. Several open
reading frames have been predicted for this region
which could only be accessed by translation from a
rare bicistronic transcript by ribosomal reinitiation.
None of the putatively encoded proteins has yet
been detected in infected cells.
MV Protein Functions
Figure 11.2 Electron micrographs of (a) the measles virion Viral RNP and Non-structural Proteins
(bar : 50 nm) and (b) purified MV nucleocapsids (bar : 50 nm).
Both are negatively stained with phosphotungstic acid. (Repro- The fully encapsidated MV genomic RNA serves as
duced with permission from Meadeley, C.R. (1973) Virus Mor- a unique target for the viral polymerase to initiate
phology. Churchill Livingstone, Edinburgh)
transcription and replication. The N protein is
phosphorylated and acts to condense the viral
genomic and antigenomic RNAs into a smaller,
more stable and more readily packaged form. This
MEASLES 361
gives the nucleocapsid its helical form and its her- delivering the RNP core into the cytoplasm. Both H
ring-bone appearance in the electron micrograph and F are glycoproteins and, after infection, it is to
(EM) (Figure 11.2b). When expressed in the absence these polypeptides that neutralizing antibodies are
of viral RNA, N proteins aggregate into nuclear and raised. These proteins are protease-sensitive, as
cytoplasmic nucleocapsid-like structures which are virions appear smooth under the EM following
thought to contain encapsidated cellular RNAs. It protease treatment. Both types of spikes can be
is the formation of high-affinity protein complexes isolated following gentle detergent lysis of the
with the phosphorylated P protein by which self- virion, although F tends to remain strongly asso-
aggregation of N proteins as well as encapsidation ciated with cellular actin. Both spikes have the ten-
of cellular RNAs are prevented during replication dency to aggregate; this is presumably mediated by
of the viral genome. Both C-terminal and N-ter- the hydrophobic tail of each molecule which nor-
minal domains of P protein are involved in N—P mally serves to anchor the spike in the lipid bilayer.
complex formation, while another domain within The H protein can be isolated as a tetrameric
the P protein has been shown to be essential for its complex from the cell membrane and its ability to
dimerization. Equally important for both transcrip- agglutinate red blood cells from sheep and mon-
tion and replication, stable complexes between P keys, but not humans, has long been recognized.
and L have to be formed, and apparently L protein Glycosylation sites are bunched within a region of
is stabilized by this interaction. Moreover, P is 70 amino acids, and glycosylation has been shown
thought to act as a transactivator regulating L pro- to be essential for haemadsorption, probably by
tein functions. The L protein is a multifunctional stabilizing the highly complex tertiary structure of
RNP-specific RNA polymerase producing mRNAs, the protein. The strict structural constraints of this
replicative intermediates and progeny viral protein have so far prevented the identification of
genomic RNAs. Capping, methylation, editing and domains essential for interaction with the identified
polyadenylation are thought to be mediated by the MV receptor complex, which consists of CD46
polymerase protein, in addition to initiating, elon- physically associated with moesin. Both proteins
gating and terminating ribonucleotide polymeriz- reveal a wide tissue distribution in vivo, and it is of
ation. Active sites within the protein have not yet note that CD46 is only expressed on monkey but
been determined, however, conserved motifs have not on human erythrocytes. H protein most prob-
been identified which suggest a linear arrangement ably serves a dual function: (1) in mediating attach-
of the functional domains. The non-structural pro- ment to CD46; and (2) with certain MV strains, in
teins C, V and R are expressed in the cytoplasm of subsequent downregulation of CD46 from the cell
infected cells with no association with the virion surface. Amino acids essentially involved in the lat-
structure. Their role in MV replication has not been ter biological property have recently been identifi-
defined. With the availability of the recombinant ed. In addition, as revealed by transfection experi-
MV cDNA, viruses have been constructed that are ments, H exerts a helper function in F-mediated
defective in both V and C protein functions. Dele- membrane fusion, probably by directing the fusion
tion of either of these gene products apparently did domain into the optimal distance to the target cell
not affect the capacity of the recombinant viruses to membrane and stabilizing the interaction.
replicate in tissue culture (Radecke and Billeter Synthesized as a precursor protein (F ), F protein
1996; Schneider et al., 1997). is cleaved in the Golgi compartment by subtilisin-
like proteases to yield two disulphide-linked
subunits, F and F , a structure common to many
Envelope Proteins
virus proteins with membrane fusion activity.
The RNP core structure is enclosed by a lipid envel- Glycosylation of the F precursor is an essential
ope which contains two proteins on its external prerequisite for cleavage, and it is only the F
surface, the haemagglutinin (H) and the fusion (F) subunit that contains all the potential N-glycosyla-
protein that are organized as functional complexes tion sites. The N-terminal domain of the F subunit,
and constitute the spikes observed in the EM. The also called the fusion domain, is highly conserved
H protein mediates binding of the virus to receptors amongst the paramyxoviruses and is highly
on the surface of the target cells, while the F protein fusogenic in vitro. There are still only theoretical
causes the virus envelope to fuse with the cell, thus explanations as to why the fusogenic N-terminus of
362 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
the F subunit fails to induce membrane fusion
during intracellular transport. Most likely, the
fusogenic domain is masked during this process by
intramolecular folding to a distal domain within the
F protein. The fully processed F protein is incor-
porated into the cell membrane as an oligomer. In
the intact virion the active site of each protein is
presumably carried at the tip of the spike and orien-
tated outwards, away from the hydrophobic tail
and towards any possible target cell.
The M protein is thought to interact with the
viral RNP and with the plasma membrane, in which
the glycoproteins are inserted to stabilize the virion
structure. Recombinant MVs carrying deletions of
major parts of the M gene were found to bud highly
inefficiently, thus supporting the initial suggestions
that this protein might be essential in this process. A
physical interaction between MV M and other viral Figure 11.3 CD46 (MCP, membrane cofactor protein), the
structural proteins has not yet been demonstrated. major protein receptor for MV strain Edmonston. MV-binding
The generation of a recombinant MV defective for sites are located within the short consensus repeat (SCR) do-
the expression of the MV glycoproteins (and ex- mains I and II, whereas complement components C3b/C4b bind
to SCR III and IV, respectively. Proximal to the transmembrane
pressing instead the glycoprotein of vesicular stom- domain, oligosaccharide-rich serin/threonine/proline (STP) do-
atitis virus) revealed that the presence of the MV mains are located.
glycoproteins was required for packaging the M
protein into mature budding virions, thus indica-
ting that either F or H, or both, would at least
transiently have to interact with M protein. isoforms of CD46 (due to alternative splicing of a
precursor mRNA) are expressed in a tissue-specific
manner and all of them can support MV uptake.
Replication Cycle CD46 contains four repetitive conserved domains,
of which the two most distal from the cell mem-
brane have been found to be essential for binding
MV Receptor Usage and Tropism
MV or MV-H protein. The molecule’s physiologi-
One of the most important parameters determining cal ligand(s), complement components C3b/C4b,
viral tropism is the availability of specific receptors bind to other domains located proximal to the
on the surface of susceptible target cells that allow membrane (Figure 11.3). As a member of the RCA
viral attachment and penetration. MV is highly gene family, CD46 is essentially involved in protect-
species specific in that it does not naturally replicate ing uninfected cells from lysis by activated comple-
in non-primate hosts. In vivo it reveals a pro- ment by recruiting the C3b/C4b components, ren-
nounced tropism for cells of the haematopoetic lin- dering them accessible to degradation by serum
eage, but, at later stages can replicate productively proteases and thus interfering with the formation of
in a variety of cell types, as it does in tissue culture. membrane attack complexes. It is considered of
Thus, the receptor would be expected to be ex- pathogenic importance that CD46 is down-
pressed by most human cells both in vivo and in regulated from the surface of infected cells or fol-
vitro. This is in fact the case for the two major lowing interaction with MV-H protein, as these
components of the MV receptor complex so far cells are significantly less protected against comple-
identified (Griffin and Bellini, 1996): CD46 (MCP; ment mediated lysis in vitro (Schneider-Schaulies et
membrane cofactor protein), a member of the ‘regu- al., 1995). Most strikingly, however, the property to
lators of complement’ (RCA) gene family, and downregulate CD46 is MV strain dependent since
moesin (membrane organizing external spike pro- MV strains, predominantly those which have been
tein), which is tightly associated with CD46. Several exclusively isolated and passaged on lymphocytes,
MEASLES 363
Rough
ER
g
Golgi
H, F
L, N, P
Nucleus
do not reveal this phenotype. It is possible that, hibitory effect on the biosynthesis of the host cell.
when depleted of CD46—mediated protection, MV- The origin and activation stage of the host cells also
infected cells may be eliminated earlier in vivo. As all influence the efficiency of MV replication. It has, for
of the MV vaccine strains downregulate CD46, this instance, been shown that productive MV infection
particular biological property may be considered to in primary lymphocytes only occurs if the cells are
be an attenuation marker. activated, and does not generally occur in most
Expression of CD46 is not the only determinant rodent cells even after host cell penetration. In-
of MV tropism. Stable expression of human CD46 tracellular factors determining MV susceptibility
does not confer susceptibility to MV infection in have not yet been defined.
many mouse cells and in CD46 transgenic animals. MV replication is confined to the cytoplasmic
Rodent brain cells support MV replication both in compartment. Following delivery of the viral RNP
vivo, after experimental infection, and in tissue cul- complex into the cytoplasm of a susceptible host
ture, although they do not express CD46. Thus, cell, viral transcription is initiated after specific at-
additional or alternative receptors, as well as so far tachment of the polymerase complex to the promo-
uncharacterized intracellular factors, also essential- ter located within the 3 end of the genome and
ly determine MV tropism. progresses to the 5 end by transcribing mono- and
bicistronic mRNAs (Figure 11.4). At each gene
boundary, the polymerase complex resumes tran-
Intracellular Replication
scription of the distal gene or, controlled by un-
The time taken for MV replication in a suitable host known factors, leaves the template to reinitiate at
cell is highly variable and becomes shorter as the the promoter region. As a consequence, a polar
virus adapts to growth in vitro. For instance, the gradient is established for the frequency of viral
Edmonston strain replicates well in Vero cells, a mRNAs, with the N-specific mRNA being the most
permanent cell line derived from the kidney of green abundant and the L-specific mRNA the least repre-
monkies. Growth is complete within 6—8 hours and sented (Figure 11.1b). At the 3 end of each gene,
is accompanied by effective inhibition of host cell poly(A) tracts are added to the mRNA transcripts,
macromolecular synthesis. However, other strains, most probably by a polymerase stuttering mechan-
particularly freshly derived isolates, grow more ism at the termination signals (Figure 11.1c). This
slowly and replication times of 7—15 days are not stuttering again reflects the RNA editing activity of
uncommon. Such viruses often have very little in- the viral polymerase and the introduction of non-
364 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
templated nucleotides in primary transcripts. In ad- protein and possibly of glycoproteins is considered
dition, bi- and polycistronic polyadenylated tran- to be crucial in the pathogenesis of subacute scleros-
scripts spanning two or more adjacent genes are ing panencephalitis (SSPE).
produced. In the replication mode, the polymerase During the replication process the large amount
complex has to read through the intergenic bound- of glycoprotein inserted into the cell membrane
aries to yield a positive copy of the entire viral causes it to develop the capacity to haemadsorb,
genome, the replicative intermediate, which is about while the F protein promotes fusion with adjacent
100—fold less abundant than that of negative polar- cells. Multinucleate giant cells are thus formed
ity. It is not known how the polymerase complex which are pathognomonic for measles infection.
decides whether to interrupt transcription at the This fusion kills the cells more rapidly than the
gene boundaries and polyadenylate the nascent virus, and if it is prevented the cells survive longer
transcripts or to read through and continue tran- and yields of virus are increased.
scription to full genomic length. Transcripts of posi-
tive polarity containing the encapsidation signal at
their 5 end joined to the N gene sequence are BIOLOGICAL PROPERTIES OF MV
thought to be indicative of replication of the anti-
genome. Replication of, but not primary transcrip-
tion from, viral genomic RNA is dependent on pro-
Stability
tein synthesis, and it is thought that the switch to
the replication mode is determined by the accumu- The structure of the virion explains much of the
lation of levels of N protein that has to encapsidate early data concerning the stability of the virus. The
the nascent genome and may act as an antiterminat- particle is dependent upon the integrity of the envel-
ing protein. ope for infectivity and is inactivated by any pro-
The viral mRNAs direct the synthesis of viral cedure which disrupts this structure. Hence the vi-
proteins by host ribosomes. Those encoding the rus is sensitive to detergents or other lipid solvents
glycoproteins F and H become membrane bound in such as acetone or ether. Particles are acid labile
and inactivated below pH 4.5, although they remain
the rough endoplasmic reticulum. Protein products
infective in the range pH 5—9. The virus is also
are translocated and modified through the Golgi
apparatus (acquisition of N-linked glycosylation thermolabile. It may remain infective for 2 weeks at
and proteolytic cleavage of the F protein) and 4°C, but it is completely inactivated after 30 mi-
nutes at 56°C. At 37°C it has a half-life of 2 hours.
finally inserted into the plasma membrane. Progeny
RNA interacts with N protein to form the nuc- Thermolability is probably due to an effect on the
leocapsid, and P and L proteins bind to these struc- internal structure of the particle, since haemag-
tures in the perinuclear area. Late in infection, nuc- glutinin is relatively temperature resistant. Virus
leocapsids may also enter the cellular nucleus. M can be stored for prolonged periods at 9 70°C and
protein combines with some of the cytoplasmic nuc- also freeze-dries well. These properties have import-
leocapsids but also complexes with the plasma ant consequences for the transport and storage of
membrane and draws together the dispersed virus vaccine.
glycoproteins. It is quite possible that M protein
also interacts with cytoskeletal components during
this process as well as during intracellular transport Haemagglutinin
of viral RNPs. Progeny nucleocapsid structures line
up beneath these modified areas of membrane and Unlike other members of the morbilliviruses, MV
are pinched off in the budding process. displays haemagglutination activity. This is easily
The mechanism of budding is unclear, but the demonstrated using monkey erythrocytes from the
ability of M to aggregate in a crystalline array could rhesus, patas and vervet monkey and the baboon.
confer upon it the capacity to distort the membrane Human erythrocytes are not agglutinated. The vi-
into an outward-facing bulge, and ultimately to bud rus H protein acts as a means of attachment to
off the nucleocapsid inside a small vesicular struc- susceptible cells. Consequently, the ability to cross-
ture—the new virion (Figure 11.4). M is thought to link erythrocytes, which do not support virus repli-
act as a trigger in this process, and lack of this cation, represents an unnatural process in virus
MEASLES 365
multiplication. Thus the inability to agglutinate Haemolysis
erythrocytes from the primary host, which is based
on the lack of the major MV receptor component The ability to lyse red blood cells once the virus has
CD46 on these cells, is not surprising. Morbil- bound is mediated by the viral F protein. This
liviruses are not thought to reveal neuraminidase ability is also artificial in the same sense as haemag-
activity and there is no evidence that they attach to glutination, since the F protein is not normally
receptors containing sialic acid. Consequently, once called upon to lyse a target cell before productive
attached to a red blood cell, MV does not re-elute infection is accomplished. Nevertheless, haemolysis
rapidly. provides a convenient measure of F protein activity
H protein inserted into any membranous struc- which is more sensitive to both pH and temperature
ture is active in the haemagglutination test (HA). than haemagglutination. Optimum temperature for
Virus particles separated by isopycnic centrifu- haemolysis is 37°C and optimum pH is 7.4. The
gation have a buoyant density of 1.23 g ml\, and ability of the paramyxoviruses to fuse at neutral pH
HA activity is detected in this area of the gradient. A accounts for the characteristic cytopathic effect
large amount of haemagglutinating material is also (CPE) induced by these viruses—the formation of
found in the upper regions of the gradient (termed giant cells.
‘light haemagglutinin’), which probably represents Proteolytic activation of the F protein is vital for
H protein inserted into empty membranous frag- its fusion activity; although uncleaved molecules
ments of the infected cell, or defective virus par- can be inserted into mature virus particles, these
ticles. Haemagglutinating activity in this fraction have lost the ability to fuse with target cells and are
can exceed that associated with the intact particles. therefore not infectious. The mechanism of F pro-
It is of note, however, that some recent MV wild- tein activity is not understood. Most likely, interac-
type strains have very low haemagglutination activ- tion of H protein with its receptor triggers a struc-
ity, which may be caused by the presence of an tural rearrangement of the F protein that allows
additional N-linked glycosylation site within their insertion of its fusion domain into the cell mem-
H proteins. Alternatively, the recent finding that brane. This is also thought to have a destabilizing
some wild-type strains only bind poorly to CD46 effect on the local structure of the envelope. The
suggests the use of an alternative MV receptor. importance of the fusion domain in this process is
H is the major immunogen of the virus, and underlined by the fact that synthetic peptides with
antibodies directed against this polypeptide have similarity to this region efficiently impair cell fusion.
both haemagglutination-inhibiting (HI) and virus- The requirement for F protein cleavage could be
neutralizing (NT) activities. This is presumably ac- interpreted as necessary to permit the free move-
complished by blocking the attachment of virus to ment of the two chains during the conformational
target cells. However, these antibodies cannot pre- change. Antibodies directed against the F protein
vent the progressive cell-to-cell spread of the virus are required for effective containment of virus infec-
mediated by the F protein. The function of H as the tion because local infection can be maintained by
major viral attachment protein to host cell recep- cell to cell fusion.
tor(s) has already been outlined (see above), as has
the property of H proteins from certain MV strains
to modulate the expression of CD46. This latter
modulation has been shown to occur after mere EPIDEMIOLOGY AND RELATEDNESS
surface interaction between MV H protein and OF DIFFERENT VIRUS ISOLATES
CD46, and is thus also observed independent of
infection. Although not defined so far, this process is The efficient spread of the virus is mediated by
thought to involve membrane signalling, as it has aerosol droplets and respiratory secretions, which
also been found that cross-linking of CD46 by vari- can remain infectious for several hours. The disease
ous compounds including MV particles leads to an incidence in the northern hemisphere tends to rise
inhibition of interleukin (IL)-12 synthesis in mono- in winter and spring when lowered relative humid-
cyte/macrophages in tissue culture. ity would favour this form of transmission. In equa-
torial regions epidemics of measles are less marked
but can occur in the hot, dry seasons. Acquisition of
366 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
the infection is via the upper respiratory tract, the pathogenic potential (lymphotropism and neuro-
nose and, possibly, the conjunctivae. Virus is also virulence, the latter more likely to cause SSPE), or
shed in the urine but this is unlikely to be an import- to monitor potential antigenic drift in wild-type
ant means of transmission. MV strains that may impair the protective effect of
The spread of measles has been used as a conveni- the current vaccine. Moreover, in populations
ent example to illustrate the principles of epidemiol- where mass vaccination campaigns have been
ogy, and it has been calculated that any community undertaken it is important to define whether any
of less than 500 000 is unlikely to have a high single measles case would be due to an imported
enough birth rate to supply the number of suscep- virus or represent a still inadequate vaccine cover-
tible children required for the continuous mainte- age or vaccine failure. Thus, in 1995, 60% of the 309
nance of the virus in the population. In fact, the cases of measles reported in the United States were
complete elimination of measles from isolated either directly imported or were found to be directly
groups has been documented. Such communities linked to an imported case by routine investigation
remain free of the disease until it is reintroduced or molecular epidemiology methods (Rota et al.,
from outside, and susceptible individuals are once 1992).
more at risk. Measles often leads to a more serious Sequence analysis of vaccine and wild-type MV
disease in such communities experiencing the illness strains as well as SSPE isolates have enabled these
for the first time because all age groups are suscep- various MVs to be classified into lineage groups
tible to the infection. In general, measles mortality is (Table 11.1).
highest in children under 2 years of age and in The sequence of the C-terminal 151 amino acids
adults. Death from uncomplicated measles is rare in of the N protein has now been analysed in 46 strains
the developed world, but the introduction of the of MV. It shows that there is up to 7.2% divergence
virus to the Fiji Islands in 1875 resulted in an epi- in the coding sequence and 10.6% divergence in the
demic with a fatality rate of 20—25%, and introduc- amino acid sequence between the most unrelated
tion into Greenland in 1951 produced an epidemic strains in this region. MV strains fall into six or
which infected 100% of the susceptible population seven different genotypes some of which are extinct
and resulted in a death rate of 18 per 1000. (i.e. have not been isolated recently) or others which
MV isolates have been obtained from many dif- are still cocirculating in the human population. In
ferent locations and from patients with different this respect, MV does not differ from other para-
clinical conditions. Much effort has been invested in myxoviruses such as Human respiratory syncytial
attempts to distinguish between these viruses and, virus, Human parainfluenza virus type 3 and Mumps
in particular, to identify any strains which might be virus. The vaccine strains widely differ from the
predisposed toward the production of encephalitis wild-type isolates, and SSPE-derived sequences
or SSPE. Conventional serological techniques ap- were much more similar to those seen in wild-type
plying polyclonal antibodies have, so far, failed to viruses. Based on these sequence similarities, it was
demonstrate any significant differences. Thus infec- even possible to identify wild-type MVs that had
tion by any one MV confers immunity to them all. circulated in a given population as likely infectious
MV is monotypic in nature, i.e. only a single agents found later in SSPE brain material. These
serotype of the virus has been described. Antigenic findings resulted in two important conclusions:
differences as observed with monoclonal antibodies firstly, SSPE develops after infection with a wild-
between vaccine and wild-type viruses need further type MV and not following vaccination, and sec-
study, as it is not clear how much this affects the ondly, circulating wild-type viruses and not particu-
ability of wild-type strains to replicate and be trans- lar neurotropic strains initially infect the CNS. So
mitted from persons with waning titres of immunity far, evidence indicates that measles is an antigeni-
generated by vaccination with a different genotype. cally stable virus and that the development of com-
The monotypic nature of MV in serological terms plications is not determined solely by the virus.
has masked the existence of a set of genotypes which Susceptibility of the host, age and immune status at
accumulate mutations continuously. During the re- the time of infection, and possibly other factors, are
cent past, molecular epidemiology of MV has been almost certainly more significant than the invading
intensively studied. This has been undertaken to virus. The biological importance of the fact that all
reveal whether there are MV strains with different vaccine strains have a genotype substantially differ-
MEASLES 367
Table 11.1 Distribution of MV strains in lineage groups
Year of Country of
Strain isolation isolation Origin
Based on molecular epidemiologic analyses, MV strains group into six lineages (data from Rima et al.
in ter Meulen and Billeter (1995)). SSPE, subacute sclerosing panencephalitis; MIBE, measles inclusion
body encephalitis; the TT strain has accidentally been isolated from a case with Kawasaki disease.
0 2 4 6 8 10 12 14 16 18 20
Figure 11.5 The course of clinical measles. The events occurring in the spread of the virus within the body are shown in lower case
lettering. As the virus spreads by primary and secondary viraemia (vma) from the lymph node to the entire reticuloendothelial system
(r.e.s.) and finally to all body surfaces, epithelial cell necrosis occurs and disease is produced. The characteristics of the disease are given
in upper case lettering
body through the upper respiratory tract or con- ment cells. Virus is present in blood and secretions,
junctiva. Replication is assumed to occur at the site and the patient is highly infectious. During this
of entry. The first sign of infection is normally virus period Koplik’s spots, the pathognomonic enan-
replication in the draining lymph nodes and de- them of measles, appear on the buccal and lower
struction of lymphoid tissue. The virus then spreads labial mucosa opposite the lower molars. These
to the rest of the reticuloendothelial system and raised spots with white centers are characteristic of
respiratory tract through the blood (primary vir- measles and begin to fade some 2—4 days after the
aemia). Giant cells containing inclusion bodies onset of the prodromal phase as the rash develops.
(Warthin—Finkeldy cells) are formed in lymphoid The distinctive maculopapular rash appears
tissue and also on the epithelial surfaces of the about 14 days after exposure and starts behind the
trachea and bronchi. About 5 days after the initial ears and on the forehead. From there the exanthem
infection the virus overflows from the compart- spreads within 3 days and involves the face, neck,
ments in which it has previously been replicating, to trunk, and upper and lower extremities. Once the
infect the skin and viscera, kidney and bladder (sec- entire body is covered the rash fades on the 3rd or
ondary viraemia). Giant cells are formed in all infec- 4th day and a brownish discoloration occurs, some-
ted tissues. Unlike other viruses, measles infection is times accompanied by a fine desquamation. His-
characterized by lymphoid hyperplasia and inflam- tologically, the rash is characterized by vascular
matory mononuclear cell infiltrates in all infected congestion, oedema, epithelial necrosis and round
organs. cell infiltrates. Once the exanthem has reached its
After 10—11 days incubation the patient enters height, the fever usually falls and the conjunctivitis
the prodromal phase which lasts from 2 to 4 days. as well as the respiratory symptoms begin to sub-
The initial symptoms consist of fever, malaise, side. Antibody titres rise and virus shedding de-
sneezing, rhinitis, congestion, conjunctivitis and creases from this point. Normally, the patient shows
cough. These symptoms increase over the next days a rapid improvement. Continuation of clinical
and are quite troublesome. At the beginning of the symptoms of the respiratory tract or fever suggests
prodromal stage, a transient rash may develop, complications.
which has an urticarial or macular appearance, but
disappears prior to the onset of the typical exan-
them. At this time giant cells are present in the
sputum, nasopharygeal secretions and urinary sedi-
MEASLES 369
Figure 11.6 (a) In the course of and following measles, both delayed-type hypersensitivity reactions (DTH), as measured by tuberculin
test, and in vitro proliferative repsonses of lymphocytes to mitogen stimulation are suppressed (Data from Tamashiro et al. (1987) and
Hirsch et al. (1984)) (b) Illustration of current models to explain MV-induced immunosuppression. Responder cells (RC) are uninfected
lymphocytes
tion of T cells in vitro once they express viral glycop- lymph nodes. Thus, these MV-infected cells confer a
roteins on their surface (Schnorr et al., 1997). De- negative rather than a positive signal to lym-
ndritic cells with functional characteristics of epi- phocytes in the T cell areas of the lymph node and
dermal Langerhans’ cells form a continuous could play a central role in the induction of a wide-
network within the epithelial lining of the conduc- spread immune suppression. Interestingly, MV-in-
tive airways and are the most potent type of anti- fected follicular dendritic cells have been found in
gen-presenting cells for activation of naive and the B cell areas of lymph nodes of experimentally
memory T cells once they have homed to the local infected rhesus macaques, and generation of sec-
374 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ondary follicles was largely impaired in areas where by intrathecal antibody snythesis, and the un-
infected follicular dendritic cells were observed. protected cells develop massive inclusion bodies,
consisting of virus nucleocapsids, in both nucleus
and cytoplasm. The condition can develop follow-
Acute Measles Postinfectious ing exposure to measles or develop later. Infectious
virus has not been isolated by conventional
Encephalitis methods from brain tissue, suggesting defects in
replication. This assumption has been supported by
It is likely that CNS involvement, even in uncom- immunohistological and molecular biological stu-
plicated measles, is common. Transient abnormal- dies on brain tissue of a case of MIBE. Of the five
ity of the EEG is detected in about 50% of patients; major structural proteins of MV, only N and P
headache is common and CSF pleocytosis is also proteins were consistently detected in infected brain
observed. The question of whether and how MV cells, whereas the envelope proteins were missing.
may reach the CNS in the course of the acute infec- In contrast, the mRNAs specific for the five viral
tion is still a matter of controversy. MV is highly
proteins were detectable in brain-derived total
lymphotropic and could be carried into the CNS
RNA samples by Northern blot analyses, although
even in cases where encephalitis has not been recog- the mRNAs for the envelope proteins were under-
nized. However, only exceptionally can MV be iso- represented in comparison with lytically infected
lated from the brain tissue of AMPE patients. In the cells. In vitro, N and P proteins were efficiently
majority of cases studied, neither MV antigen nor synthesized from their corresponding mRNAs, in-
RNA have been found in the CNS. Therefore, cur- dicating a restriction of the expression of the MV
rent theories favour an autoimmune reaction as the envelope proteins in MIBE (Baczko et al., 1988).
possible cause of CNS damage, since AMPE pa- Partially, this restriction has been explained by se-
tients may exhibit a proliferative T lymphocyte re- quence analyses that revealed a high rate of muta-
sponse to basic myelin protein (MBP). In addition, tions distributed over the entire MV genome. For
in CSF specimens of such patients MBP was detec- the MV M gene, mutations have eliminated the
ted as a consequence of myelin breakdown. Such
initiation codon, which explains the failure of MV
MBP-specific lymphoproliferative responses have
M protein synthesis in infected MIBE brain tissue
not only been seen after measles but also in patients (Cattaneo et al., 1988). Defects in MV mRNA tran-
with postinfectious encephalomyelitis following ru- scription and envelope protein synthesis apparently
bella or varicella or after rabies immunization do not largely affect the activity of the RNP com-
(Johnson and Griffin, 1986). This last disorder is plex which spreads to different areas of the patient’s
probably the human equivalent of experimental al- brain. As infectious virus particles may never be
lergic encephalitis (EAE), as such patients received formed due to the restriction of the envelope pro-
rabies vaccine prepared in brain tissue. Since teins required for assembly and budding, and giant
AMPE is characterized by demyelinating lesions in cell formation has never been observed, the spread
association with blood vessels as in EAE, it is not is thought to occur by microfusion events.
surprising that the finding of an MBP-specific lym-
phoproliferative response in measles infection is
considered to be of pathogenetic importance. How
MV leads to a T cell-mediated autoimmune re- Subacute Sclerosing Panencephalitis
sponse is still unknown. At present, the possibilities
of molecular mimicry or a deregulation of autoreac- MV was first implicated in the aetiology of this
tive cells occurring secondary to viral infections of disease by immune fluorescence in 1967, and this
lymphocytes are being considered. has since been confirmed by electron microscopy,
immunoelectron microscopic methods, and finally
by the successful rescue of virus by cocultivation
Measles Inclusion Body Encephalitis techniques (ter Meulen and Hall, 1978; ter Meulen
and Carter, 1984). Despite this, the manner in which
As this condition arises in patients with underlying the persistent infection is first established in the
immunodeficiencies, it is not usually accompanied brain and exactly how this leads to the production
MEASLES 375
of disease are still largely unknown. Recent PCR- by immunohistochemistry. Molecular biological
based studies even suggested that MV may in fact studies on SSPE brain tissue revealed extensive
reach the brain and establish lifelong persistent in- transcriptional and translational alterations affect-
fections that remain asymptomatic and do not lead ing mainly MV M, F and H genes (Carter et al.,
to the development of SSPE (Katayama et al., 1995; 1983; Baczko et al., 1986; Liebert et al., 1986; Cat-
Nakayama et al., 1995). The virus is thought to gain taneo et al., 1986; Sheppard et al., 1986). As in
entry to the CNS during viraemia in acute measles, MIBE, the envelope protein-specific mRNAs were
or by infected lymphocytes, but, once there, replica- only detected at low copy numbers (Cattaneo et al.,
tion proceeds only slowly and a widespread en- 1987) and were highly impaired in directing the
cephalitis is not established. It is also not known to synthesis of the corresponding gene products in
what extent virus replication per se is responsible vitro (Figure 11.7). Sequence analyses revealed a
for the development of lesions, nor what part is high rate of mutations located all over the MV
played by the immune system. genome, although different genes were affected at
different levels. The highest number of alterations
were found in the M gene, followed by F, H, P and
Virological Aspects
N genes, which were mutated to about the same
Important clues have come from the study of MV extent, whereas the L gene was most conserved.
replication. The virus normally replicates with the Mutations introduced were either point mutations,
production of giant cells and release of infectious most probably accumulating due to the infidelity of
progeny. In SSPE, free infectious virus has never the viral polymerase, or appeared as clustered tran-
been isolated, either from the brain or from CSF, sitions which are thought to result from the activity
and histopathological examinations have consist- of a cellular enzyme complex that actively modifies
ently failed to reveal the morphological changes viral genetic information. As a result of either of
associated with virus maturation (ter Meulen et al., these events, translation of viral mRNAs was com-
1983; Schneider-Schaulies and ter Meulen, 1992). pletely abolished or led to the synthesis of truncated
As with MIBE, giant cells and thickening of the or unstable MV proteins. These molecular biologi-
plasma membrane at points of budding have never cal data explain the absence of infectious MV par-
been observed, suggesting the absence of viral ticles and the lack of a budding and a cell fusion
glycoproteins. Viral nucleocapsids present in the process in infected SSPE brain tissue, since for these
cytoplasm are randomly scattered and show no sign events biologically active MV envelope proteins are
of regular alignment beneath the plasma mem- required. Although infectious virus is not present in
brane. As budding viral particles and infectious vi- the CNS, virus can occasionally be rescued from
rus are not detected, the infection may spread slow- brain tissue obtained post mortem by cocultivation
ly, strictly in a cell-associated manner. MV-specific (Wechsler and Meissner, 1982). So-called SSPE iso-
antibodies produced in the CNS are oligoclonal, as lates can be of two different types: cytolytic budding
opposed to the polyclonal response observed in the or cell-associated viruses, the latter spreading
serum (Dörries et al., 1988). This suggests that anti- through the culture with a gradually enlarging area
body in the CSF is made locally by a much smaller of CPE. In the second type of isolate mRNAs for the
population of lymphocytes which have invaded this MV, envelope proteins are detectable in infected
compartment in response to antigens present in the cells, although their function is impaired since these
CNS. This is further supported by the finding that proteins are not synthesized in infected cells or in in
only MV-specific antibody titres are tremendously vitro translation experiments. As revealed by recent
elevated, titres against other viruses being normal. sequence analyses, some of the SSPE isolates are
In serum specimens, antibodies with specificity to likely contaminations and are in fact ordinary lab-
all MV proteins are present, although recognition oratory MV strains. It is still unclear whether those
of the M protein is low or sometimes absent, where- which are true SSPE isolates may represent a small
as antibodies in CSF samples generally fail com- subpopulation of replication/maturation compet-
pletely to detect M protein. ent viruses or revertants that have been selected by
In SSPE brain sections, only the expression of the the isolation procedure and may not be representa-
MV N and P proteins and not that of the envelope tive of the dominant virus population in the infected
proteins was consistently detected in infected cells brain.
376 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 11.7 Restrictions of MV gene expression found in SSPE brain tissue. MV mRNAs encoding the viral envelope proteins are
expressed at low frequency (most probably due to host cell control mechanisms) and, in addition, harbour mutations that prevent the
synthesis of functional translation products
These virological and molecular biological find- preventing a rapid host cell destruction. Whether
ings in SSPE help to explain the absence of MV these control mechanisms efficiently control the
particles in brain tissue and the failure of the hu- maintenance of the persistent infection or other
moral immune response to eliminate infected brain factors, such as the introduction of mutations into
cells. Factors involved in the establishment of per- the viral genome, are required has not been resol-
sistence by a non-defective MV in brain tissue are ved.
largely unknown, since these studies have been car-
ried out on autopsy material. In tissue culture ex-
Immunological Aspects
periments with cells of neural origin, and in brain
material of experimentally infected animals evi- One of the immunological hallmarks in SSPE is the
dence has been provided to show that intracellular hyperimmune response to MV antigens, which in-
factors intimately control the efficiency of MV repli- cludes neutralizing antibodies in serum and CSF.
cation (Schneider-Schaulies et al., 1990). This ap- Yet this immune response fails to control virus in-
plied particularly to attenuation of viral transcrip- fection. This phenomenon has led to the proposal
tion, as well as to translational control exerted that measles antibodies may support persistence
predominantly on MV-specific and not on cellular rather than interfere with it. In tissue culture, MV-
mRNAs in brain cells in vitro. In addition, the cellu- specific antibodies act to cross-link viral proteins
lar enzyme activity actively modifying the primary expressed on the surface of infected cells which sub-
sequence of viral RNAs has been demonstrated in sequently aggregate at the pole of the cell to form so
these cells. Thus, it is a likely assumption that in- called ‘cap-structures’. These caps are internalized
tracellular factors present in brain cells act to slow and thus removed from the cell surface. During this
down viral replication after primary infection, thus process, M protein cocaps with the viral glyco-
MEASLES 377
proteins. Since complement-mediated lysis is of low that a virus laboratory is seldom called upon to
efficiency in the brain as a result of low complement make a diagnosis. As the vaccination programme
concentration in this compartment, it is likely that takes effect, physicians may become less familiar
the major effect of antibody in brain tissue would be with the disease, and vaccination itself has led to the
to promote clearance of antigen from the brain cell emergence of atypical forms of measles. Diagnosis
surfaces rather than lysis of the infected cells. This of these manifestations may require laboratory
process might explain the lack of membrane glyco- work. Furthermore, as more patients are placed on
proteins on the surface of brain cells but cannot immunosuppressive regimens, the need for diag-
explain the lack of intracellular envelope proteins. nosis of MV may well increase. Several procedures
Yet some evidence has been obtained in tissue cul- are available and are described below.
ture experiments which suggests that the capping
process could interfere with viral protein synthesis.
It has been shown that antibody directed against Microscopy
the haemagglutinin of influenza viruses can exert an
inhibitory effect on the activity of the viral poly- Direct Examination
merase. Similar observations have been made in cell Production of multinucleate giant cells with inclu-
lines persistently infected with MV or in MV-infec- sion bodies is pathognomonic for measles during
ted rats. In the latter, passive transfer of neutralizing the prodromal phase. Such cells are detectable in
monoclonal antibodies directed against the MV H the nasopharyngeal secretions (NPS). Clear identifi-
protein led to the development of a subacute MV cation of giant cells is facilitated if the smear is first
encephalitis by preventing an acute CNS infection fixed in formalin and then stained with haema-
(Liebert et al., 1990). The molecular biological toxylin or eosin. The Cowdry inclusion bodies are
analysis revealed a transcriptional restriction of vir- then easily discerned.
al mRNAs. Thus in SSPE the host immune re-
sponse could contribute to the production of this
serious fatal disease. Fluorescence Microscopy
It is well known that cell-mediated immunity Both direct and indirect immunofluorescence (IF)
(CMI) is far more significant in the control of MV have been widely used to stain cells shed in nasal
infection than the humoral immune response. This secretions, although it may be necessary to remove
has led to considerable interest in the CMI response antibodies which already coat virus antigens with a
mounted by SSPE patients. In general, no evidence low pH buffer. Stained cells include macrophages
for a general impairment of CMI in these patients and ciliated cells as well as giant cells. Urinary
was obtained. T cells are present in normal sediment cells have also been examined with a high
amounts, and lymphoproliferation and interleukin degree of success. IF-positive cells may be shed in
synthesis in response to a variety of antigens are the urine from 2 days before to up to 5 days after the
normal. Similarly, skin grafts are rejected in a nor- appearance of the rash. This method may therefore
mal fashion. It is possible that the response to MV be more applicable in later stages than examination
antigens is impaired, since anamnestic skin tests of NPS specimens. Such cells may also be present in
with measles antigens are often negative. A dispar- the urine 4—16 days after vaccination with the live
ity of results was obtained by using MV antigens in vaccine. IF is useful for the diagnosis of measles in
assays for CMI. Depending on the test system em- the pre-eruptive phase or in children vaccinated
ployed and on the potency and purity of the virus with killed vaccine, where rash development is
antigens used, a minor inhibition of CMI or normal atypical. Immunoperoxidase histochemical stains
reactions in comparison to controls can be ob- have also been used, and the use of monoclonal
served in SSPE patients. antibodies has improved sensitivity and reliability
of virus detection.
The symptoms of acute measles are so distinctive In common with other infections, diagnosis of
378 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
measles may be made if antibody titres rise by more biotics. Urinary sediment cells are collected by low-
than fourfold between the acute and the convales- speed sedimentation and treated similarly. MV can
cent phases or if measles-specific IgM is found. Sev- be isolated directly from blood but efficiency is
eral methods are available for this determination increased if lymphocytes are first separated on a
and are also useful for the assessment of immune ficoll gradient and stimulated with mitogen before
status. The tests most commonly used are haemag- use. Samples are then inoculated in triplicate on
glutination inhibition (HI), neutralization (NT), susceptible tissue cultures, such as primary human
hemolysin inhibition (HLI), complement fixation embryo kidney (HEK) cells and primary monkey
(CF) and enzyme-linked immunoassay (ELISA). Of kidney cells. In the latter case, the monkeys should
them all, the NT is the most sensitive and specific have been serologically tested, since some animals
but it is not very practical and therefore rarely used. harbour monkey intranuclear inclusion agent
HI, HLI, ELISA and CF are more useful in practice, (MINIA) which is similar to measles. Continuous
although the decreased sensitivity of the CF test cells lines derived from green monkey kidney cells
renders it not useful for testing immune status. (Vero or CV-1) are also suitable. These are less
ELISA tests are usually applied for measles-specific likely to be successful than primary cell culture.
IgG and IgM. Other tests such as gel precipitation Recently, Epstein—Barr virus-transformed B lym-
are rarely used. In general, titres of 8 or higher are phoblastoid cell lines (B95a, BJAB) were used for
indicative of immunity. In the case of SSPE, it is MV isolation (Kobune et al., 1990). It was shown
important that CSF is also tested. that these cells revealed a significantly higher sus-
ceptibility to MV present in clinical specimens than
Vero cells. Moreover, MV isolated in B95a cells
differed in some biological properties from those
Virus Isolation and Detection of Viral adapted to Vero cells, suggesting the MV is subject
RNA to host cell-mediated selection. It is therefore rec-
ommended to include these cell lines in MV isola-
Both virus isolation and RT-PCR-based detection tion attempts. CPE develops usually between 48
of viral RNA should be applied only in specific hours and 15 days, and consists of either a broad
instances, such as suspected infection of the im- syncytium or a stellate form. Both types reveal in-
munosuppressed in the absence of rash when only clusion bodies, which may be present in both nu-
limited antiviral immumoglobulins might be ex- cleus and cytoplasm. It is not known what governs
pected, developing pneumonia without a rash or the different forms of CPE but the availability of
unexplained encephalitis. Finally, both techniques cellular nutrients as well as the type of virus have an
can be attempted as a means of retrospective diag- effect. Uninoculated identical cultures should also
nosis using tissue obtained post mortem. be maintained as controls. If CPE is slow or not
RT-PCR analyses using MV N- and/or F-gene clear, it is possible to test whether the cells have
specific primers are mostly performed on serum, acquired the ability to haemadsorb monkey eryth-
nasopharygeal aspirates and urine sediment cells; rocytes or contain MV antigens or RNA.
however, they can also be applied to tissue samples
such as brain material. This technique requires an
RNA extraction step prior to reverse transcription
and subsequent PCR amplification. Diagnosis of SSPE
In acute measles, virus isolation is difficult and
the success rate may be low. Isolation is most likely The diagnosis of SSPE presents certain problems.
to be achieved from material (such as throat or Formerly, brain biopsy was performed routinely
conjunctival washings, sputum, urinary sediment and tissue thus obtained was examined for inclusion
cells and lymphocytes) taken during the prodromal bodies and virus antigen by immunofluorescence.
phase and is unlikely to succeed once antibody After recognition of the characteristic intrathecal
titres have started to rise. Isolation is therefore only antibody synthesis, determination of measles anti-
usually attempted in unusual cases. For virus isola- body titres in the CSF may be sufficient with, if
tion, washings and swabs are collected and mixed necessary, demonstration of MV-specific hetero-
with buffered salt solution (pH 7.2) containing anti- geneity by isolelectric focusing in combination with
MEASLES 379
an immunoblot technique (Dörries and ter Meulen, is essential. Seizure control requires anticonvulsive
1984). Virus isolation from SSPE brain tissue is drugs. Application of steroids has not been shown
complicated. Expression of virus must be restored to be beneficial. No specific therapy is known for
by growing cells derived from brain tissue with cells subacute measles encephalitis.
susceptible to MV infection, or fusing them together A variety of approaches to the treatment of SSPE
directly. It is therefore necessary that samples from have been attempted. As yet no convincing effects
SSPE brain contain viable cells. As MV replication have been demonstrated and almost all cases have
does only occur within brain cells, and viral par- proved fatal. Evaluation of the effectiveness of treat-
ticles are not released, MV-specific sequences can ment is extremely difficult. First, the course of SSPE
generally not be amplified by RT-PCR in CSF is highly variable and spontaneous remissions are
samples. common. Secondly, SSPE is a rare disease, and
clinical trials are therefore inevitably based on a
very small number of patients and interpretation is
MANAGEMENT difficult.
The aim of any therapy must be to prevent the
Measles spread of virus within the CNS and to aid the
body’s immune system to bring about the ultimate
Measles is an acute self-limiting disease which, in elimination of the virus from the body. The disease
the absence of complications, will run its course is extremely difficult to treat because no antiviral
without the need for specific intervention. Infection drugs which are effective against MV yet exist. Fur-
of the undernourished, the immunocompromised thermore, access is limited because of the anatomi-
or children suffering from chronic debilitating dis- cal and physiological pecularities of the brain.
eases is more serious. Such patients, as well as Therefore most attempts at treatment have aimed
children of less than 1 year of age and pregnant towards the activation of potentiation of the im-
women, can be protected after exposure by the ad- mune defense in the brain. A possible deficiency in
ministration of human antimeasles gammaglobulin CMI to MV has already been discussed, and the
(0.25—0.5 ml kg\). If this is given within the first 3 following regimens have been used with no or
days of exposure, it is usually effective. The effective- doubtful effects: removal of blocking factors by
ness is diminished if the globulin is given 4—6 days complete CSF exchange; potentiation of helper T
after exposure, and abolished if more than 6 days cell activity; isoprinosine (inosiplex); levamisole (l-
have elapsed. tetramisole) and interferon treatment. In the case of
isoprinosine, it seems likely that treatment may
lengthen survival time if given early in the disease.
This effect becomes less pronounced if the drug is
Measles Pneumonia administered later.
Respiratory tract infections cause considerable
damage to the ciliated epithelium and this may lead
to superimposed bacterial infection. In the case of a PREVENTION
pneumonia it is sometimes difficult to distinguish
between primary viral infections and the superim- Since MV has no animal reservoir, it is an obvious
posed bacterial infection. In any case, treatment target for a controlled campaign aimed at the eradi-
with antibiotics is required. cation of the virus (Mitchell and Balfour, 1985). In
the United States and Canada, where vaccination of
all children is required at or before commencing
Encephalitis school, the results have been quite startling. Case
reports have fallen by over 99% but eradication has
Treatment of acute measles postinfectious encepha- not yet been achieved. In Germany and the UK,
litis is only symptomatic and supportive, and does where vaccination is not mandatory, distrust of vac-
not differ from any other postinfectious encephali- cination has led to a lower acceptance rate and less
tis. Careful attention to fluid and electrolyte balance dramatic results have been achieved. In developing
380 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
countries, where the consequences of MV infection not without risk and adverse reactions to the live
are most severe, considerable effort has been exp- vaccine may be seen in as many as 50% of the
ended with comparatively little return. Any vaccine revaccinees. A similar phenomenon occurs in the
used must be safe, as measles is not normally a case of live and killed vaccinia virus vaccines where
severe illness, and cheap enough for mass adminis- the killed virus lacks an important antigen found
tration. Its effectiveness should also be long lasting. only on mature released virus.
Natural immunity is known to last for at least 65
years in the total absence of restimulation from
virus in the environment. In 1781 measles disap-
peared from the Faroe Islands following an epi- Live Vaccine
demic. It was not reintroduced until 1846. Individ-
uals old enough to have experienced the disease 65 The first attenuated vaccine strain was the Edmon-
years previously were still protected. This unusual ston B strain produced by serial passage of the virus
persistence of immunity even in the absence of re- in human kidney cells, human amnion cells, chick
exposure from the environment has suggested that chorioallantoic membrane and, finally, duck em-
MV may normally persist inside the body, possibly bryo cells. This vaccine was administered intramus-
in lymphocytes, and thus restimulate immunity cularly or subcutaneously 12—18 months after the
from within. disappearance of maternal antibodies. It was effec-
tive and achieved seroconversion in 95% of recipi-
ents, but side-effects of mild measles were common
(5—10%). In order to oppose this, gammaglobulin
Inactivated Vaccine was administered. The amount of gammaglobulin
was crucial: too little and side-effects could occur;
This vaccine was intended for use in young children too much could promote vaccine failure. In 1966 a
less than 1 years of age, who are most prone to Medical Research Council (MRC) trial conducted
serious complications. It was therefore considered in the UK followed 36 000 children. Measles inci-
desirable to avoid the use of a live vaccine. With dence fell 84% in the first 9 months, and decreased
formalin-killed virus preparation it was found that by 14% in the next 2 years. A follow-up study
at least three vaccinations were necessary to elicit a showed that the vaccine remained effective for at
suitable antibody response. Titres were lower than least 12 years.
those induced by natural measles and soon waned. The side-effects and necessity of providing gam-
This left vaccinees open to virus attack, but the maglobulin led to the development of the further
nature of their partial immunity led to serious hy- attenuated and less reactogenic Schwartz and
persensitivity reactions to infection and the disease Moraten (Enders) strains. These were derived from
course was modified (see Atypical measles). Most the Edmonston B vaccine by further passage in the
likely, an important epitope was destroyed by the chick embryo at lowered temperature. Further
inactivation of the live virus and thus the vaccine MRC trials demonstrated that these were indeed
could only induce imcomplete immunity. Antibody less reactogenic and the incidence of postvaccina-
induced by natural measles or live vaccine is protec- tion febrile convulsions was reduced from 7.7 to 1.9
tive at much lower levels than that produced in per 1000 recipients. Both vaccines produced a 95%
response to the killed vaccine. This suggested that seroconversion rate, altough antibody titres in-
some qualitative difference existed between the two duced by the Schwartz strain declined more rapidly
types of antibody. It was shown that killed vaccine than those produced in response to the Moraten
failed to induce effective haemolysin-inhibiting strain but remained at protective levels. In recent
antibodies, which in vivo prevent virus spread from years, the Edmonston strain was further attenuated
cell to cell. The problems could perhaps be over- by passing it in human diploid cells. This vaccine,
come, but, in view of the rapid decline in induced referred to as Edmonston Zagreb strain, has been
antibody, live vaccination is now recommended, shown to produce higher seroconversion rates than
and individuals previously immunized with the the Schwartz vaccine when administered at the
killed vaccine should be reimmunized with the live same age. Similar observations have been made
attenuated strain of virus. Even this procedure is with the AIK-C vaccine, produced in Japan. Titres
MEASLES 381
of protective antibodies were seen to decline in iso- occurred annually in the United States and by the
lated communities but were still detectable 14 years age of 15 years 95% of the population was serocon-
later. Children living in an open environment verted. Following the rigorous implementation of
showed serological evidence of subclinical re-expo- the MV vaccination programme, case reports have
sure which acted to boost their immunity. The at- fallen dramatically from 500 000 annually to 26 000
tenuated strains now in use are so reduced in viru- in 1978 and 1500 in 1983; between 1984 and 1988
lence that encephalitis has only been noted in about only 3700 cases were registered (Figure 11.8). As a
1 in 1 million vaccine recipients, as compared to 1 in result of measles vaccination, mortality and AMPE
1000—5000 children with natural measles. The have also declined and the available experience in-
measles vaccine is administered subcutaneously, dicates that SSPE can also be prevented by measles
usually between the ages of 9 and 20 months, for vaccination. However, in 1989 and 1990 a dramatic
primary vaccination. Humoral immune responses, increase in acute measles cases was observed in the
as defined by HI, NT and ELISA, are good and United States, rising in 1989 to 18 193 and in 1990
protective as long as the vaccine is given after wan- to 27 786 cases (Figure 11.8) (Atkinson and Oren-
ing of the maternal antibodies. As with acute stein, 1992). Measles cases typically occur in two
measles, the major isotype of MV-specific antibo- distinct cohorts: preschoolchildren less than 5 years
dies is IgG1. Levels of antibodies induced are gen- of age, and schoolchildren aged 5—19. In the former
erally lower than after measles and may decay more group, failure to obtain recommended vaccination
rapidly but are still measurable in most individuals is prominent, while in the latter, the majority of
15 years after immunization in the absence of boost- patients are infected despite previous vaccination.
ing infections. Activation of CMI is generally This indicates that lifelong immunity may not be
thought to be similar to that of acute measles, in induced by the application of live measles vaccine.
that both MV-specific CD4 and CD8 T cells are The reasons for vaccine failure may be administra-
stimulated, although cytotoxic T lymphocyte re- tion of the vaccine in the presence of maternal anti-
sponses after restimulation in vitro are considerably bodies or an inadequate response to vaccination
lower than after natural infection. In common with (primary vaccine failure) or loss of immunity in time
acute measles, administration of live measles vac- (secondary vaccine failure). Molecular biological
cine is associated with transient lymphopenia, loss studies of contemporary MV strains revealed se-
of delayed-type hypersensitivity skin test responses quence changes to past strains which may be asso-
to recall antigen and decreased in vitro proliferative ciated with different biological properties, although
responses to mitogens. Both leucopenia and atypi- vaccine-induced MV antibodies still neutralize MV
cal lymphocytosis have also been found after revac- isolates from current epidemics. As a result of these
cination. Cytokine production after MV vaccina- measles epidemics, the American Academy of
tion includes a predominant synthesis of IL-4, Pediatrics (AAP) and the Immunization Practices
indicating that T 1 responses may be inefficiently Advisory Committee of the USA (ACIP) recom-
&
generated. In general, however, the immunosup- mended a change from a one-dose to a two-dose
pression observed after vaccination is less pro- schedule for measles vaccination which is expected
nounced than that of acute measles, and is usually to be optimal for measles control and eventual elim-
not associated with complications. Recent reports ination. The first dose of measles vaccination
that both measles (contracted early in childhood) should be administered at 15 months of age, as
and measles vaccine would predispose for the later delayed primary measles vaccination (at 15 months
developement of intestinal bowel diseases such as of age or later) significantly reduces measles risk at
Morbus Crohn or ulcerative colitis have not been later ages. Initial vaccination at 12 months of age is
substantiated (Feeney et al., 1997). recommended for children living in high-risk areas
(areas with a large inner-city urban population
where more than five cases among preschool-aged
children occurred during each of the last 5 years or
Effectiveness of Vaccination in the with recent outbreaks among unvaccinated pre-
Control of Measles school-aged children). Of course, initial vaccination
of infants 12—14 months of age is also recommended
In the prevaccine era, an estimated 4—5 million cases before travel to areas in which measles is endemic or
382 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 11.8 Reported number of measles cases in the United States by year, 1950—1990. (Data from the Centers for Disease Control
(1991), reproduced with permission from the Journal of the American Medical Association, 1991, 266, 1522—1547. Copyright 1991,
American Medical Association)
epidemic. A second dose is recommended at 4—6 ures following vaccinations do not increase the
years of age by the ACIP (the AAP recommends probability of subsequent epilepsy or neurological
revaccination at the age of 11—12 years), which is disorders. Most convulsions following measles vac-
expected to provide protection to most persons who cination are simple febrile seizures, and they affect
do not respond to their initial vaccination. Persons children without known risk factors. Nevertheless,
who received inactivated vaccine and are therefore parents of children who have a personal or family
at risk of developing severe atypical measles after history of seizures should be advised of the small
exposure to natural measles should receive two increased risk of seizures following measles vaccina-
doses of live vaccine separated by not less than 1 tion; this is, however, far outweighed by the benefits
month. It is of note that up to 55% of these vac- of the protective effects. CNS conditions such as
cinees may reveal reactions to the live vaccine, such encephalitis and encephalopathy have been re-
as local swelling and low-grade fever. ported with a frequency of less than 1 per million
doses administered, an incidence which is lower
than that of encephalitis of unknown aetiology.
This finding suggests that the reported severe
Side-effects of Live Vaccination, neurological disorders temporally associated with
Adverse Reactions, Precautions and measles vaccination were not caused by the vaccine.
Contraindications Live measles vaccine should not be administered
to women who are pregnant or who are considering
The vaccine has an excellent record of safety (Na- becoming pregnant within the next 3 months be-
tional Vaccine Advisory Committee, 1991). Al- cause of the theoretical risk of fetal infection. The
though on rare instances a transient rash or low- decision to administer or delay vaccination because
grade fever may be observed after 5—12 days in some of a current febrile illness depends on the cause of
vaccinees, they remain otherwise asymptomatic as the illness and the severity of symptoms. Hypersen-
for the vast majority of recipients. As with the ad- sitivity reactions following the administration of
ministration of any agent that can induce fever, live measles vaccine are rare and usually occur at
some children may have a febrile seizure. Although the injection site. Persons with a history of
children with a history of seizures are at increased anaphylactic reactions following egg ingestion
risk for developing idiopathic epilepsy, febrile seiz- should, however, be vaccinated with extreme cau-
MEASLES 383
tion, and individuals who have experienced during the acute phase and the following 9 months.
anaphylactic reactions to neomycin should not be Malnutrition aggravates measles infection, and ma-
given the vaccine. Unlike with natural measles, ex- jor complications are pneumonia and diarrhoea
acerbation of tuberculosis has not been observed which lead to an overall two- or fourfold increase in
after measles vaccination. mortality. Administration of vitamin A has been
shown to reduce measles morbidity and mortality.
Vaccination in these areas has so far failed to
Vaccination of Immunocompromised yield dramatic results. This is largely due to the
epidemiology of measles in these areas. Interrupting
and HIV-infected Individuals transmission through vaccination would require
high vaccination coverage rates (990%), and even
Replication of vaccine viruses can be enhanced in with extremely high vaccination coverage rates out-
persons with immune deficiency diseases and in breaks can be expected to occur. Measles is particu-
those with immunosuppression, as occurs with leu- larly severe in infants. Peak incidence of measles
kaemia, lymphoma, generalized malignancy, or
occurs in the very young, under 2 years of age, and
therapy with alkylating agents, antimetabolites,
97% of cases occur below the age of 5. Vaccination
radiation or large doses of corticosteroids. Thus, should therefore be performed on younger children
patients with such conditions or therapies (except than is the case in industrialized countries and fre-
for human immunodeficiency virus (HIV) infection) quent revisits to the same area are essential in order
should not be given live measles vaccine. Short- to vaccinate the large number of susceptible infants
term corticosteroid therapy does not contraindicate that may be born in a single year. The time of
live measles vaccination. The increasing number of waning of maternal antibodies, the incidence of
infants and preschoolers with HIV infection in measles infection in early life and the efficiency of
certain countries has directed special attention to measles vaccination are determinants of the earliest
the appropriate immunization of these children. possible age for vaccination against measles. Vacci-
Asymptomatic children in need of live measles vac- nation is generally performed in industrialized
cination should receive it without prior evaluation
countries at 12 months of age. This is to some extent
of HIV infection state. Moreover, vaccination
a compromise between vaccinating at 15 months,
should be considered for all symptomatic HIV in- when seroconversion would be efficient (95%) but a
fected children, including those with the acquired large number of children would already have con-
immune deficiency syndrome (AIDS), since measles tracted measles, and vaccination earlier, before in-
in these children can be severe. Limited data on fection, when the success rate of vaccination is lower
measles vaccination among both asymptomatic (50—75% in 6—month-old children) and the risk of
and symptomatic HIV-infected children indicate side-effects is greater. There is still an ongoing de-
that the vaccine has not been associated with severe bate over the optimum age at which vaccination
or unusual adverse events, although antibody re- should be performed, in particular since recent
sponses have been unpredictable. Exposed sympto- trials with a high-titre vaccine at the age of 5—6
matic HIV-infected (as well as other immunocom- months suggested an increase in child mortality
promised persons) should receive high doses of
over control groups (Garenne et al., 1991).
measles Ig regardless of their previous vaccination
Future studies will undoubtedly lead to further
status. elucidation of the regulatory mechanisms involved
in the normal replication of MV. This should in
turn lead to a greater understanding of the events
Vaccination in Developing Countries preceding measles invasion of the CNS, and poss-
ible new avenues of therapy. Moreover, progress
In developing countries 1—1.5 million measles-re- made in the molecular biology of MV could lead to
lated deaths are reported per year. Measles seems the development of genetically engineered measles
more severe in Africa than on other continents, with vaccines which should be free of side-effects and
a number of countries reporting case fatality ratios possibly suitable for the immunization of individ-
higher than in most areas. Infants are at special risk uals currently at greatest risk, such as young infants
for measles and case fatality ratios are high, both or those suffering from chronic debilitating diseases.
384 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
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MEASLES 385
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vitro. Proceedings of the National Academy of Sciences of the tive study of the magnitude and duration of changes in tuber-
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Restricted expression of measles virus in primary rat astroglial and disease. Progress in Medical Virology, 30, 44—61.
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(1995) Receptor usage and differential downregulation of considerations. Journal of General Virology, 41, 1—25.
CD46 by measles virus wildtype and vaccine strains. Proceed- ter Meulen, V., Stephenson, J.R. and Kreth, H.W. (1983) Sub-
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
12
Rubella
Jennifer M. Best and Jangu E. Banatvala
Guy’s, King’s and St Thomas’ School of Medicine, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
388 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 12.1 Main historical events
? MMR1 contained Measles virus (Moraten strain), Mumps virus (Jeryl Lynn) and RV (HPV77.DE5).
@ MMR vaccines licensed in the UK contain Measles virus (Schwartz strain), Mumps virus (Jeryl Lynn) and RV (RA27/3 strain).
1938. Between 1949 and 1953, further experimental groups of workers were exchanged and found to be
studies using human volunteers showed that the identical.
incubation period of rubella ranged from 13 to 20 It was subsequently shown that RV induced
days. Subclinical infection was demonstrated, since interference in continuous lines of monkey kidney
secondary cases of rubella occurred in contacts of and that viruses other than EV11 (e.g. Cox-
volunteers inoculated with serum who had not sackievirus A9, Bovine enterovirus M-6 and Vesicu-
themselves developed a rash. lar stomatitis virus) could be used to challenge
Rubella virus (RV) was not isolated in cell cultures rubella-infected cell cultures. Many other primary
until 1962, when two groups of workers published and continuous cultures were subsequently shown
simultaneously methods for the isolation of RV. to support the growth of RV.
Parkman and his colleagues (1962) inoculated pri- Rubella-neutralizing antibodies were detected by
mary vervet monkey kidney (VMK) cultures with Parkman and his colleagues (1962), who demon-
throat washings obtained from military recruits strated that convalescent sera neutralised the inter-
when rash was present. Although no cytopathic ference effect produced by a standard inoculum of
effect (CPE) was observed, when cultures were RV. The presence of neutralising antibodies was
challenged 7—14 days after inoculation with 10 shown to be associated with protection against
TCID Echovirus 11 (EV11), the distinct CPE reinfection. This technique was used extensively for
produced by this virus was not observed, suggesting diagnostic purposes and serological surveys. Stu-
the presence of an interfering agent. The interfering dies conducted in different countries showed that
agent was neutralized by rabbit antiserum raised the acquisition of rubella antibodies was related to
against one of the isolates. Weller and Neva (1962) age, social class and geographical location, and that
also isolated the virus from blood and urine taken approximately 80% of women of childbearing age
from typical cases of rubella in primary human living in urban areas in Western countries were
amnion cultures. After specimens were passed once immune. Initial attempts to develop a haema-
or twice in these cell cultures, a characteristic CPE gglutination inhibition (HI) test were unsuccessful,
with cytoplasmic inclusions was observed. These since it was not appreciated that inhibitors of
effects were neutralized by serum obtained from haemagglutination, now known to be serum lipop-
patients convalescent from rubella. The agents roteins, were present both in the serum incorporated
isolated in the different cell cultures by these two in culture medium and in the test sera. Details of a
RUBELLA 389
successful HI test were published in 1967 (Stewart et somers has been described. The lipoprotein envel-
al., 1967). ope bears surface spikes of 5—8 nm composed of the
Clinical and virological investigations carried out two glycoproteins. The non-rigid delicate character
during the extensive rubella epidemic in the USA of the envelope results in the virus particle being
during the winter and spring of 1963—1964 led to a pleomorphic; elliptical and oblong virus particles
greater understanding of the pathogenesis of con- and particles bearing finger-like protrusions have
genitally-acquired rubella (CAR), as well as a further been described.
appreciation of its clinical features and sequelae. Rubella virus contains a single positive-strand
During this epidemic many pregnant women were polyadenylated 40S RNA of 9762 nucleotides ex-
inevitably infected and this resulted in the delivery cluding the poly(A) tail, and is capped at the 5 end
of about 30 000 rubella-damaged babies (Cooper, (Frey, 1994; Pugachev et al., 1997). The RNA is
1975). Studies at the time revealed that multisystem infectious when extracted under appropriate condi-
involvement was common and the range of abnor- tions. The base composition of the genome is G
malities much wider than previously reported. 30.8%, C 38.7%, A 14.9% and U 15.4%. The
When maternal rubella occurred in early preg- genome consists of two non-overlapping open read-
nancy, a generalized infection developed in the fetus ing frames (ORFs) separated by an untranslated
which persisted during the remainder of gestation region of 123 nucleotides. The 5 proximal ORF is
and into infancy, despite the presence of rubella 6348 nucleotides in length and codes for a poly-
antibodies. Many infected infants excreted virus and protein precursor of the non-structural (NS) pro-
transmitted virus to susceptible contacts. This epi- teins. The 3 proximal ORF is 3189 nucleotides in
demic highlighted the importance of developing a length and codes for the polyprotein precursor of
vaccine rapidly. the structural proteins (Figure 12.3). The complete
genomes of three strains of RV have been sequenced:
M33 and Therien, which are wild-type strains
PROPERTIES OF THE VIRUS isolated in the USA in the early 1960s, and the
RA27/3 vaccine strain. Sequencing has been difficult
due to the high GC content of the genome (69%)
Classification and several mistakes appeared in early sequences
(Frey, 1994; Pugachev et al., 1997). RA27/3 differs
Rubella virus is classified as a non-arthropod-borne from M33 and Therien at 36 nucleotides, 18 of
togavirus and is the only member of the genus which are silent with respect to amino acid coding.
Rubivirus. The overall structure and strategy of As sequence differences are maintained in virus
gene expression of RV is similar to that of the recovered from vaccinees, they can be used to
alphaviruses of the Togaviridae, such as Semliki- identify this attenuated strain in clinical specimens.
Forest and Sindbis (Frey, 1994). By HI, however, no Other sequencing studies, which have concentrated
antigenic relationship has been shown between ru- on the E1 ORF, have demonstrated that the genome
bella and more than 200 alphaviruses and sequence is stable and there is no evidence of
flaviviruses. Humans are the only known hosts for antigenic drift, even in highly vaccinated popula-
RV.
tions (Best et al., 1993; Frey et al., 1998). However,
two genotypes have been described, with some
isolates from China and India differing from isolates
Structure of the Virus from Europe, North America and Japan by 8—10%
of nucleotides, but by only 1—3% at the amino acid
The virus particle is 58 < 7 nm in diameter, while level (Bosma et al., 1996; Frey et al., 1998). There is
the nucleocapsid is 33 < 1 nm in diameter and no evidence that reinfection is due to an antigenic
surrounds the RNA genome (Figures 12.1 and 12.2). variant, but two viruses isolated from joints ex-
The virion consists of two membrane-bound hibited changes in antigenic epitopes in E1 (Bosma
glycoproteins E1 (58 kDa) and E2 (42—47 kDa) and et al., 1996; Frey et al., 1998). Thus, the level of
C, a non glycosylated capsid protein. The symmetry diversity is low when compared with some alpha-
of the nucleocapsid was difficult to establish due to viruses and other RNA viruses, such as human
its instability, but an icosahedron with 32 cap- immunodeficiency virus (HIV) and poliovirus.
390 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 12.1 Negatively stained preparation of rubella virus. Inset: an enlarged particle, showing spikes. (Bar : 50 nm.) (Kindly
provided by Dr I. Chrystie; reproduced with permission from Banatvala and Best, Topley and Wilson, 9th edition, Vol. 1)
Figure 12.2 Vero cell culture 4 days after infection with Rubella virus. Virions are budding (arrow) from cell surface membrane.
Virions are labelled with antirubella antibody (polyclonal) and colloidal gold probe (GAR G10; ; 103 200.) (Courtesy of Dr S. Pathak.)
the three structural proteins C, E2 and E1 (Figure replication and are similar to structures found in
12.3). All three structural proteins are transported to alphavirus-infected cells.
the Golgi complex. E1 and E2 form heterodimeric The structure and replication of RV have been
complexes, while the C protein forms non-covalent- reviewed in more detail by Frey (1994) and
ly bonded dimers, which are stabilized by disulphide Wolinsky (1996).
bond formation shortly before virus release.
In cell cultures RV is released by budding from
intracellular membranes (e.g. Golgi, endoplasmic Physical and Chemical Properties of the
reticulum) and from the plasma membrane. The
RNA-containing nucleocapsid cores bud from the
Virus
cellular membranes, where they acquire an envelope
consisting of the E1 and E2 glycoproteins and host Physical properties of RV have been reviewed by
cell lipids, to form mature virus particles. The ability Horzinek (1981) and Banatvala and Best (1998). The
to bud from intracellular vacuoles allows the virus stability of the virus is enhanced by the addition of
proteins to the suspending medium. MgSO ap-
to evade the host’s immune response, which may
enable the virus to establish persistent infections pears to improve the thermostability of the virus.
Rubella virus is inactivated by detergents and
(Wolinsky, 1996).
organic solvents, since the viral envelope contains
In vertebrate cell cultures the replication of RV is
slow and less efficient than that of the alphaviruses. lipid. The effects of these and other chemicals have
In Vero cells virus production reaches a peak at 48 been extensively reviewed elsewhere (Norrby, 1969;
hours after infection at high multiplicities of infec- Herrmann, 1979; Frey, 1994).
tion (Hemphill et al., 1988) and infection has no
effect on total cell RNA synthesis. Defective interfer-
ing RNAs have been detected. Membrane alter- Antigenic Characteristics
ations and vacuolation are observed in infected cells
by electron microscopy. Membrane-bound cyto- Early work on RV identified haemagglutinating
plasmic vacuoles are replication complexes recently (HA), complement fixing, platelet aggregating and
shown to be virus-modified lysosomes (Magliano et haemolytic activity. Methods for producing HA and
al., 1998). These structures are the site of virus complement fixing antigens have been reviewed
Figure 12.3 Map of the rubella virus genome and strategy for the expression of rubella virus structural proteins. Translation of rubella
RNA (i) results in production of non-structural proteins (ii), which initiate the synthesis of a full-lenth negative strand of RNA (iii),
which acts as template for the synthesis of both full-length positive strand for new viral progeny (iv) and 24S subgenomic RNA (v).
Translation of this 24S RNA results in the production of p110, a polyprotein (vi) which is proteolitially cleaved to produce the three
structural proteins C, E2 and E1 (vii). Within the non-structural ORF the location of global amino acid motifs indicative of replicase
(R), helicase (H) and cysteine protease (P) activity are indicated. X indicates a region of homology between rubella and alphaviruses.
hydrophilic domain of C, putatively interacts with virion RNA; hydrophobic signal sequences that precede the N terminal of E2 and
E1; the transmembrane sequences of E2 and E1. (Reproduced with permission from Banatvala and Best, Topley and Wilson, 9th
edition, Vol. 1)
RUBELLA 393
elsewhere (Banatvala and Best, 1998). continuous cell cultures (Herrmann, 1979). It indu-
Humoral and cell-mediated responses are pro- ces a CPE only in such continuous cell cultures as
duced against all three structural proteins, although RK13 (rabbit kidney), SIRC (rabbit cornea) and
E1 appears to be immunodominant (Cusi et al., Vero (VMK), providing conditions are controlled
1989; Chaye et al., 1992). Information on im- carefully. These cell cultures, together with primary
munoreactive regions within the structural proteins VMK cell cultures, have been used most frequently
has been obtained using monoclonal antibodies to for virus isolation. Virus is identified in primary
map epitopes and by measuring antibody reactivity VMK by interference. Vero cells are useful for virus
and T cell proliferative responses to synthetic isolation because they do not produce interferon,
peptides and recombinant proteins. E1 has been and virus can therefore replicate more rapidly to
shown to have at least six independent linear high titre. Since RV does not produce CPE in all
epitopes, including two or three neutralizing epi- sublines of Vero cells, passage into another cell line
topes, within the amino acid region 214—285 (Terry has been used for virus identification. Although RV
et al., 1988; Wolinsky et al., 1991; Chaye et al., 1992; produced a characteristic CPE in RK13 and SIRC
Mitchell et al., 1992). However, conformation-de- cells, the virus may be identified more reliably in
pendent epitopes may also play an important role in both these and Vero cell cultures by immuno-
the induction of immune responses, and these have fluorescence (IF) or polymerase chain reaction
not been identified. The E2 glycoprotein in the (PCR) (p 407).
mature virion appears to be relatively inaccessible BHK-21 and Vero cell cultures are used exten-
to the immune response, but a number of B cell sively for producing high titres of RV, which are
epitopes have been identified (Green and Dorsett, required for use as antigens in serological tests
1986; Wolinsky et al., 1991; Mitchell et al., 1993). (p 406).
Strain specific antigens have been identified on E1 Rubella-induced interference is probably asso-
and E2 using Western blotting (Dorsett et al., 1985; ciated with the interferon pathway, although inter-
Cusi et al., 1989). Human sera react with synthetic feron is not always detected in infected cell cultures.
peptides comprising residues 214—285 of the E1 RV also induces intrinsic interference—that is, re-
protein, and a recombinant protein containing these sistance to superinfection by high multiplicities of
epitopes was recognized by most rubella antibody- Newcastle disease virus in human fibroblasts, in-
positive sera (Starkey et al., 1995). At least five T cell duced by the viral genome.
epitopes have been identified on E1. There appear to The growth of RV in cell cultures and organ
be no generally recognized T helper cell epitopes on cultures has been reviewed by Banatvala and Best
E1, although the region E1 202—283 appears to be (1990).
recognised by both B and T cells from some
individuals. Responses detected using T cell lines are
considered to be more specific. However, most
investigators have used peripheral blood mononuc- Pathogenicity for Animals
lear cells (PBMCs). Marttila et al. (1996) were
unable to detect proliferative responses to synthetic Rubella virus infects rhesus (Cercopithicus aethiops),
peptides when PBMCs were used, but when T cell vervet (Macaca mulatta) and Erythrocebus patas
lines from 14 seropositive individuals were em- monkeys, marmosets (Sanguinus species), chimpan-
ployed, three minimal T helper cell epitopes were zees, baboons, suckling mice, hamsters, ferrets and
identified on E1. At least four T and two B cell rabbits (reviewed by Herrmann, 1979; Banatvala
epitopes have been identified on the C protein and Best, 1990). Monkeys usually develop a sub-
(Wolinsky et al., 1991; Ou et al., 1992; Mitchell et al., clinical infection with viraemia, virus excretion and
1994). an immune response, similar to that in humans
(p 397). A viraemia and humoral and cell-mediated
immune responses have been detected in 6—8-week-
old BALB/c mice (Wolinsky, 1996). Persistent infec-
Growth in Cell Cultures tion has been established in suckling mice, adult
hamsters, ferrets and rabbits. Vaccinia-expressed E1
Rubella virus grows in a wide range of primary and and E2 proteins have induced autoantibodies to
394 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 12.4 Serologically confirmed laboratory reports of rubella, England and Wales: 1984 to July 1996 (Miller et al., 1997)
(Reproduced with permission of the PHLS Communicable Disease Surveillance Centre © PHLS)
pituitary cells and autoimmune lymphocytic hy- inoculation, nutrition and the effects of increased
pophysitis in Syrian hamsters (Yoon et al., 1992). handling of the animals.
Attempts to reproduce the teratogenic effects of
RV in an animal model have produced inconsistent
results. The monkey is the only animal whose
reproductive process is similar to that of humans, POSTNATALLY ACQUIRED
but even in these animals results have not been INFECTION
reproducible. Thus, some workers isolated rubella
from conceptuses but no malformations were ob- Epidemiology
served. An increased rate of spontaneous abortion
and lenticular changes were observed in some Rubella has a worldwide distribution. Before the
studies, while others failed to isolate virus or to find introduction of vaccination, outbreaks usually oc-
evidence of malformation, although they showed curred in the spring and early summer in temperate
that an immune response developed in utero. Con- climates. Infection is uncommon in preschool
genital infection has been reported in rabbits, ferrets children, but outbreaks involving schoolchildren
and rats, but these reports could not be confirmed, and young adults living in institutional population
the occurrence of malformations being inconsistent. groups are common. Women of childbearing age
Insufficient attention was given in these studies to are often infected as a result of exposure to children
the use of adequate controls and such factors as within their household or as a result of occupational
virus passage history, species adaptation, route of exposure. Unlike measles, rubella does not exhibit
RUBELLA 395
characteristic periodicity. Occasionally, extensive more than 25% were reported from only 12 of the 45
worldwide pandemics occur; for example, in the developing countries surveyed. Particularly high
early 1940s and again between 1963 and 1965, when susceptibility rates occurred in island populations,
a high incidence was reported in the USA, the UK e.g. Trinidad and Tobago and Jamaica, although
and Australia. More commonly, rubella exhibits an many islands are now adopting vaccination pro-
increased incidence every 3—4 years, although with- grammes.
in a particular country epidemics may be localized Although outbreaks of rubella may not always be
to certain areas only. Thus, in the UK, extensive recognized in developing countries, or rubella-in-
outbreaks of rubella occurred in 1978—1979, duced rashes misdiagnosed, the incidence of con-
1982—1983, 1993 and more recently in 1996 (Figure genital rubella has recently been reported to have
12.4). The augmentation of the rubella vaccination been considerably higher (range 0.6—2.2 per 1000
programme in 1988, in which MMR was offered to live births) than during the early years of the rubella
preschool children of both sexes (p 414), resulted in a vaccination programme in Britain (0.14 per 1000
marked reduction in the incidence of rubella, with during epidemics; at other times, 0.08 per 1000).
the lowest ever number of cases reported in 1992 Mathematical modelling from five WHO regions,
(Miller et al., 1993). The 1996 outbreak was largely excluding Europe, during 1998 estimated that there
confined to 17—24-year-old males who had never were approximately 236 000 cases of CAR in devel-
been offered rubella vaccine; about 16% of males in oping countries during non-epidemic years; follow-
this age group were susceptible. Fortunately, trans- ing epidemics the number might show as much as a
mission of rubella to pregnant women was limited, tenfold increase (Salisbury and Savinykh, 1991).
since 98—99% were immune, either as a result of
naturally acquired infection or vaccination as
schoolgirls or post partum.
Since rubella is not a notifiable disease in many Clinical and Virological Features of
countries, and the clinical diagnosis is unreliable, Primary Infection
seroepidemiological studies provide a more accu-
rate assessment of the incidence of rubella in Rubella is spread mostly by droplet via the respir-
different age groups and in different geographical atory route. The epithelium of the buccal mucosa
areas. Despite serological tests being carried out by and the lymphoid tissue of the nasopharynx and
different techniques in different laboratories, sur- upper respiratory tract probably represent the site
veys have produced remarkably consistent results. of initial virus replication, following which rubella
Thus, in temperate climates, the proportion of spreads to the lymphatic system and establishes a
seropositive persons increases progressively with systemic infection. It is likely that mononuclear cells
age. In general, about 50% of 9—11-year-old are involved in dissemination of virus to different
children have rubella antibodies. In the prevaccina- parts of the body, although extracellular virus may
tion era, about 80—85% of women of childbearing be detected in serum.
age were found to be immune (Dowdle et al., 1970). Rubella has an incubation period of 13—20 days,
Although CAR is now a rare disease in those following which the characteristic features of rash
developed countries which have adopted rubella and lymphadenopathy may appear. Among
vaccination programmes, the burden induced by children, onset is abrupt, with the appearance of
congenitally acquired infection imposes a consider- rash and constitutional symptoms usually mild or
able strain on scarce health and educational re- absent. The rash is at first discrete, and is in the form
sources in many developing countries. Unfortunate- of pinpoint macular lesions. It appears first on the
ly, this is insufficiently appreciated. The proportion face and spreads rapidly to the trunk and then to the
of susceptible women in many developing countries, limbs. Lesions may coalesce, but the rash seldom
although exhibiting considerable variation even lasts for more than 3 days; in many cases it is
within different parts of a country, shows that the fleeting. Adults may experience a prodromal phase
proportion of women susceptible to rubella with such constitutional features as malaise or fever
(15—20%) is little different from that in industrial- for a day or two before rash develops. At this time an
ized countries in the prevaccine era (Cutts et al., erythematous pinpoint enanthem may be visible on
1997; Robertson et al., 1997); susceptibility rates of the soft palate. The mechanism by which RV
396 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
induces rash has not been established. However, complications, although a postinfectious encepha-
virus has been isolated from skin biopsy specimens lopathy may develop in about 1 in 5000—10 000
taken from areas with and without rash, as well as cases within a week of onset of rash. In contrast with
from the skin of patients with subclinical infections, measles, the prognosis is usually good. The cereb-
suggesting an immunopathological mechanism. rospinal fluid (CSF) usually contains cells, mostly
Lymphadenopathy, which may be tender, may be lymphocytes, but CSF protein levels are normal.
present for up to a week before onset of rash and The encephalopathy, which may be immune me-
may persist for 10—14 days after it has disappeared. diated, since infectious virus or its nucleic acid by
Lymphadenopathy may be generalized, but the PCR has been detected only rarely (Wolinsky,
suboccipital, postauricular and cervical lymph 1996), is not associated with demyelination or
nodes are affected most frequently. inflammatory damage, which is present in other
Joint involvement represents the commonest postinfectious encephalitides.
complication of naturally acquired rubella as well as Thrombocytopenia is also a rare complication of
rubella vaccination, usually developing as the rash rubella in which a purpuric rash, epistaxis, haema-
subsides. Although relatively uncommon among turia and gastrointestinal bleeding have been re-
prepubertal females and males, it may occur in up to ported.
50% of postpubertal females. Symptoms may vary Rubella may present atypically with minimal
in severity from a transient stiffness of the joints to lymphadenopathy and an evanescent rash. In up to
an arthritis with pain, swelling and limitation of 25% of cases infection is subclinical. Conversely,
movement. This usually persists for 3—4 days, but typical rubelliform rashes may result from infection
may persist occasionally for up to a month. The by other viruses (e.g. enteroviruses). Infections by
finger joints, wrists, knees and ankles are most such viruses as the human parvovirus (B19) and
frequently affected. Arthralgia may be the result of some arboviruses (e.g. Chikungunya virus and Ross
direct viral invasion of the synovium, since virus has River virus) may cause both rubelliform rashes and
been isolated from joint aspirates of patients with arthralgia. Because clinical diagnosis is unreliable, it
naturally acquired rubella and vaccinees with vac- is essential that all women who have been exposed
cine-induced arthritis. In vitro studies have shown to, or develop rubella-like illnesses in pregnancy, be
that wild-type and attenuated strains of rubella will assessed virologically (p 406); a past history of
replicate in human synovial membrane cell cultures. rubella without laboratory confirmation of the
Immune mechanisms are more likely to be involved, diagnosis must never be accepted as indicative of
however, for in addition to virus, high levels of previous infection and consequent immunity.
rubella-specific IgG have been detected in joint The relationship between the clinical and vi-
aspirates, which suggests that joint symptoms may rological features of infection is shown in Figure
be mediated by immune complexes; indeed, the 12.5. Patients are potentially infectious over a
presence of immune complexes in the sera of prolonged period; pharyngeal excretion may be
vaccinees has been associated with a high incidence present for up to a week before the onset of rash, and
of joint symptoms. Hormonal factors may also be for 7—10 days thereafter. Although virus may be
involved, since in addition to being commonest in recovered from the stools and urine, excretion from
postpubertal females, joint symptoms are most these sites is more transient. Such specimens are
likely to develop within 7 days of the onset of the therefore less suitable for virus isolation and do not
menstrual cycle in vaccinees. Although there have play an important role in the transmission of virus.
been studies which have reported that RV or its Viraemia is present for about a week before the
antigens have been detected in the synovial fluid or onset of rash, but, as rubella antibodies develop,
synovium of patients with rheumatoid arthritis and viraemia ceases. The first antibodies to appear are
seronegative arthritis (rheumatoid factor negative), those detected by HI and neutralization. IgG anti-
more recent studies have failed to confirm these bodies detected by enzyme immunoassay (EIA),
findings, although RV has very occasionally been complement fixation (CF) and single radial
detected in both the synovial fluid and cells of haemolysis (SRH) usually develop a few days later.
patients with chronic joint disease, including some Differing results have been obtained by the various
who were immunosuppressed (Bosma et al., 1998). workers who have examined IgG subclasses.
Rubella is associated very rarely with other Thomas and Morgan-Capner (1988) compared the
RUBELLA 397
Figure 12.5 Relation between clinical and virological features of postnatally acquired rubella. (Reproduced with permission from
Banatvala and Best, Topley and Wilson, 9th edition, Vol. 1)
use of different sources of rubella antigens in EIAs or if sera taken prior to contact (e.g. for screening
and reported IgG1 in all sera containing rubella purposes) are not available (p 407).
antibodies detectable by SRH or latex agglutination Initial reports suggested that asymptomatic rein-
(LA), and IgG3 in most sera from cases of recent fection in early pregnancy was unlikely to be
naturally acquired rubella. IgG4 has been detected associated with fetal infection, even when a specific
in sera from occasional cases of remote rubella. IgM response was detected. However, several well-
documented cases have now been reported where
RV was isolated from the products of conception or
children with congenital rubella were born (Best et
Reinfection al., 1989). Morgan-Capner et al. (1991) have cal-
culated that the risk of fetal infection is approxi-
Natural infection is followed by a very high order of mately 8% following reinfection in the first 16 weeks
protection from reinfection. However, evidence of of pregnancy, but fetal malformations are rare.
reinfection, which is generally asymptomatic, may Rubella reinfection is not associated with a lack of
be obtained by demonstrating a significant increase neutralizing antibodies or a specific defect in rubella
in antibody concentration following natural and specific lymphoproliferative responses (O’Shea et
experimental exposure to rubella. Reinfection al., 1994); but a failure to produce epitope-specific
would provide a hazard to the fetus if it is associated antibodies is possible. RV strains isolated from cases
with viraemia; this has only rarely been of reinfection do not appear to have sequence
documented. It may be difficult to distinguish changes in the E1 ORF (Bosma et al., 1996; Frey et
between primary infection and reinfection, particu- al., 1998). Reinfection following rubella vaccination
larly if blood was not obtained shortly after contact is discussed on page 411.
398 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
*
Reproduced with permission from Banatvala and Best, Topley and Wilson, 9th edition, vol. 1.
RUBELLA 399
Figure 12.6 The frequency of virus excretion with age of infants with congenitally acquired rubella. (Reproduced with permission from
Cooper and Krugman, 1967), Arch. Opthal., 77, 434. © 1967 American Medical Association)
larly during the first few weeks after birth, those with insulin-dependent diabetes mellitus (IDDM) has
severe disease may excrete high concentrations of not been established. However, an experimental
virus and readily transmit infection to rubella- study employing human fetal islet cells showed that
susceptible contacts. RV may persist in infants with RV induced a depression of immunoreactive se-
congenitally acquired disease in secluded sites for creted insulin without being cytolytic (Numazaki et
even longer. Thus, RV has been recovered from a al., 1990). A further study suggested that autoim-
cataract removed from a 3-year-old child (Menser et mune phenomena might be involved since it was
al., 1967b) and detected by PCR in lens aspirates shown that immunoreactive epitopes in the RV
from children with CAR up to the age of 12 months capsid shared antigenicity (molecular mimicry) with
(Bosma et al., 1995a). RV has also been detected in islet cell protein (Karounos et al., 1993).
the CSF of children with central nervous system The mechanism by which RV persists throughout
(CNS) involvement up to the age of 18 months gestation and for a limited period during the first
(Desmond et al., 1967). By IF, rubella antigen was year of life has not been clearly established. Possible
detected in the thyroid from a 5-year-old child with mechanisms include defects in cell-mediated im-
Hashimoto’s disease (Ziring et al., 1977), and by munity, poor interferon synthesis and the possibility
cocultivation techniques RV was recovered from the that a limited number of infected fetal cells give rise
brain of a child who developed rubella panen- to infected clones which persist for a limited period
cephalitis at the age of 12 years (Cremer et al., 1975; (reviewed by Banatvala, 1977). It has also been
Weil et al., 1975). Experimental studies have shown suggested that selective immune tolerance to the RV
that, within the CNS, the astrocyte is the main cell E1 protein may play a role (Mauracher et al., 1993).
type in which virus replicates, high levels of virus Studies in vitro show that RV replicates in T
being expressed. Intrauterine infection involving lymphocytes and macrophages and can also persist
these cells may perhaps induce focal areas of in B lymphocytes, causing inhibition of host-cell
necrosis resulting in the pattern of neurological protein synthesis (Chantler and Tingle, 1980; van
deficit observed in congenitally acquired disease der Logt et al., 1980). Infection of macrophages may
(Chantler et al., 1995). interfere with their interactions with T cells. Pos-
The mechanism by which CAR may result in tnatally acquired rubella causes a transient reduc-
400 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
well beyond the time when RV can be recovered
from accessible sites.
sponses may limit or terminate infection, such Britain indicate that preconceptual rubella does not
infants rarely have severe or multiple anomalies. result in transmission of RV to the fetus. Thus, there
The results of four studies conducted in different was no serological or clinical evidence of in-
countries are shown in Table 12.2. Most surveys trauterine infection in 38 infants whose mothers’
have shown that deafness and retinopathy, which rash appeared before or within 11 days after their
per se does not affect vision, are likely to be the only last menstrual period (LMP). However, an interval
anomalies commonly associated with post-first of 12 days between LMP and rash resulted in fetal
trimester rubella (reviewed by Banatvala and Best, infection and all of 10 mothers who developed rash
1998). Figure 12.7 shows that deafness is usually the 3—6 weeks after their LMP transmitted infection to
sole clinical manifestation of fetal infection occur- their fetuses (Enders et al., 1988).
ring between 13 and 16 weeks, and is relatively
common, but deafness or any other defect is only
rarely encountered after this time. These findings
emphasize the importance of conducting careful Clinical Features
follow-up studies on infants with serological evi-
dence of intrauterine infection as a result of ma- The frequency and importance of congenital defects
ternal infection occurring between 13 and 16 weeks, involving the heart, eyes and ears were emphasised
particular importance being directed towards the in the early retrospective as well as in most prospec-
recognition of hearing defects. tive studies. However, following the extensive
1963—1964 epidemic in the USA, as well as in
subsequent epidemics in other parts of the world, a
Risks of Preconceptual Rubella
much broader range of rubella-induced congenital
If conception occurs shortly after the appearance of anomalies was observed. This phenomenon is more
rash there should be little risk of the conceptus being likely to be due to more careful and prolonged
infected, because the development of the rash co- observation than to any change in the biological
incides with the immune response and the termina- behaviour or teratogenic potential of the virus.
tion of viraemia. However, because RV may persist Thus, careful examination of the case notes of
after the onset of rash elsewhere, for example in the infants with congenitally acquired disease who were
genital tract, infection of the conceptus via this route born before this outbreak showed that such
is a possibility. Although six case reports were found anomalies as thrombocytopenic purpura and ostei-
prior to 1984 in which maternal infection occurring tis occurred fairly frequently, even though not
as long as 21 days before conception apparently recorded in the literature. In addition, careful and
resulted in intrauterine infection, it is encouraging prolonged follow-up studies showed that CAR was
that more recent studies conducted in Germany and not a static disease, since some features of in-
402 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 12.3 Clinical features associated with congenitally acquired rubella
Common
Low birthweight Sensorineural deafness Sensorineural deafness
Thrombocytopenic purpura Peripheral pulmonary stenosis Peripheral pulmonary stenosis
Hepatosplenomegaly Mental retardation Pulmonary valvular stenosis
Bone lesions Central language defects Patent ductus arteriosus
Meningoencephalitis Diabetes mellitus Ventricular septal defect
Retinopathy
Cataract
Microphthalmia
Psychomotor retardation
Microcephaly
Cryptorchidism
Inguinal hernia
Diabetes mellitus
Uncommon
Cloudy cornea Severe myopia Severe myopia
Hepatitis Thyroiditis Thyroid disorders
Generalized lymphadenopathy Hypothyroidism Dermatoglyptic abnormalities
Haemolytic anaemia Growth hormone deficiency Glaucoma
Penumonitis ‘Late onset disease’ Myocardial abnormalities
Adapted with permission from Banatvala and Best, Topley and Wilson, 9th Edition, Vol. 1.
trauterine infection might not be apparent for the number of megakaryocytes in the bone marrow,
months or indeed years (e.g. perceptive deafness, although they are morphologically normal. If se-
IDDM). verely affected infants survive, their platelet count
Clinical features of the congenital rubella syn- rises spontaneously during the first few weeks of life;
drome (CRS) have been reviewed by Cooper (1975), rarely, infants may die from such complications of
who categorized them into transient, developmental thrombocytopenia as intracranial haemorrhage.
or permanent (Table 12.3). Bony lesions may be present in about 20% of
congenitally infected infants. The metaphyseal por-
tions of the long bones are usually involved and
Transient Anomalies
radiologically appear as areas of translucency.
Transient anomalies usually present during the first These lesions, which are due to a disturbance in
few weeks of life, do not recur and are not associated bone growth rather than an inflammatory response,
with permanent sequelae. Their pathogenesis has usually resolve without residual sequelae within the
not been established, but such features as in- first 1—2 months of age.
trauterine growth retardation (small-for-dates About 25% of infants who present at birth with
babies), thrombocytopenic purpura (Figure 12.8), clinical manifestations of CAR also have CNS
hepatosplenomegaly and haemolytic anaemia are involvement, this usually being in the form of
common. About 60% of infected infants fall below meningoencephalitis. At birth, these infants may be
the 10th, and 90% below the 50th, growth percen- either irritable or lethargic with a full fontanelle and
tile. The above features, which often result from CSF changes consistent with a meningoencephali-
infection acquired at an early gestational age, tis. Although about 25% of infants presenting at
seldom occur without such other manifestations of birth with a severe neonatal encephalitis may, by the
congenital disease as heart and eye defects, and age of 18 months, be severely retarded and have
reflect extensive infection which may result in high communication problems, ataxia or spastic di-
perinatal mortality rates. Infants with throm- plegia, some infants progress well neurologically
bocytopenic purpura have platelet counts ranging despite poor development during their first few
from 3 to 100 ; 10 1\ (normal : 310 < months of life. Although RV has been recovered
68 ; 10 1\). This is associated with a decrease in from the brain and from patients’ lymphocytes, it is
RUBELLA 403
being insufficiently appreciated since there may
have been no history of maternal rubella. Deafness
may be the only anomaly present, particularly if
maternal infection occurred after the first trimester.
Thus, in a study on a large number of children aged
6 months to 4 years who attended the Nuffield
Hearing and Speech Centre in London, it was
estimated that about 15% of all cases of sen-
sorineural deafness were the result of CAR. IDDM
(juvenile-onset; type 1) is the most frequent endoc-
rine disorder among children with congenital ru-
bella (reviewed in Burke et al., 1985). It was thought
originally to be a rare complication of congenital
rubella, but it is now recognised that clinical
manifestations may be delayed until adolescence or
adult life. A follow-up study of patients in New
South Wales who were born with CAR in the 1940s
showed that 20% had developed eventually IDDM.
Follow-up studies in New York on children, most of
whom were infected in utero during the 1963—1964
epidemic, have shown that 30 of 242 (12.4%) had
developed IDDM. The mean age of children devel-
Figure 12.8 Purpuric rash in newborn infant with congenitally
oping IDDM in this US study was 9 years, while all
acquired rubella, who was subsequently found to have congeni-
tal heart disease and cataract. (Reproduced with permission from of the Australian patients were in their 20s (reviewed
Banatvala and Best, Topley and Wilson, 9th edition, Vol. 1) by Banatvala and Best, 1990). Lymphocytic infiltra-
tion of the pancreas of an infant with CAR but
without IDDM may suggest that RV can initiate the
possible that this manifestation of congenital ru- train of events which subsequently results in IDDM
bella may be autoimmune in nature, triggered in later life. However, it seems probable that autoim-
possibly by molecular mimicry between viral and mune mechanisms are involved in the pathogenesis
host epitopes. since the HLA types in these patients are typical of
those with autoimmune disease, there being a
significant increase in prevalence of HLA-DR2. In
Developmental Defects
addition, islet cell antibodies have been detected in
Developmental defects may take months or years 20% of these patients. These antibodies have a
before becoming apparent, but persist indefinitely. cytotoxic effect on cultured islet cells and predict the
Failure to recognise them may not merely be the diabetic state. Although autoimmune responses
result of difficulty in their detection, for there is may play an important role, the mechanism by
evidence to show that such defects as perceptive which RV may trigger them remains to be estab-
deafness, some CNS anomalies and some ocular lished. Possible mechanisms have been reviewed by
defects may actually develop or become progress- Banatvala (1987).
ively more severe. Thus, audiological examination
has shown that some infants, although having
Central Nervous System
apparently normal hearing at 9—12 months of age,
are found to have severe sensorineural deafness a The progressive nature of congenital acquired ru-
few months later. Rubella-induced deafness may be bella is emphasised by reports that occasionally
unilateral with no characteristic audiometric pat- children with previously clinically stable congeni-
tern. Prior to the introduction of vaccination pro- tally acquired disease may develop a widespread
grammes, congenitally acquired rubella probably subacute panencephalitis. Ten cases have been
represented the commonest cause of congenital reported in patients with congenital rubella, and
deafness in most developed countries, its impact two following postnatally acquired infection. All but
404 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
one of the patients were male and clinical features monary and renal arteries.
developed between the ages of 8 and 19. Clinical and
laboratory features are analogous to measles-in-
Ocular Defects
duced subacute sclerosing panencephalitis, since, in
addition to intellectual deterioration, spasticity and Most of the classical ocular defects which occur in
ataxis occur. Histological studies revealed a panen- CAR were described by Gregg (1941), who drew
cephalitis with a perivascular inflammatory re- particular attention to the pigmented retinopathy
sponse as well as a vasculitis. RV has been isolated and cataract. Although usually present at birth,
from the brains of such patients and has also been cataracts may not be visible until several weeks
recovered from their lymphocytes. Rubella antigens later. They are unilateral in about 50% of patients,
have not been detected by immunofluorescence in and may be subtotal, consisting of a dense pearly-
sections of brain. High levels of rubella-specific IgG white central opacity (Figure 12.9), or total, with a
and, occasionally, IgM may be present in the serum, more uniform density throughout the lens. Micro-
while in the CSF there are elevated levels of protein phthalmia may be present in eyes with cataract.
and immunoglobulin, and oligoclonal bands are Glaucoma occurs much less frequently than cata-
found. There is also a high CSF:serum rubella ract, but glaucoma and cataract do not seem to be
antibody titre ratio. It has been postulated that present in the same eye. Retinopathy is present in
post-rubella panencephalitis may be a disease me- about 50% of congenitally infected infants and its
diated by immune complexes or by RV-mediated presence may provide a useful clinical diagnostic
autoreactivity to brain antigens. marker. Retinopathy is not the result of an inflam-
matory response but is due to a defect in pigmenta-
tion and usually involves the macular areas. Hyper-
Late Onset Disease
pigmented and hypopigmented areas give the retina
Between the ages of about 3 and 12 months, some a ‘salt and pepper’ appearance. Chronic uveitis,
congenitally infected infants may develop a chronic choroidal neovascularization, corneal hydrops and
rubella-like rash, persistent diarrhoea and pneu- keratoconus have also been described. The mechan-
monitis, which is referred to as ‘late onset disease’. isms involved may include damage induced by viral
Mortality is high, but some infants improve dra- persistence causing reduced cell growth rate and
matically if treated with corticosteroids. Circulating lifespan, virally induced vascular damage or
immune complexes are probably responsible for autoimmune phenomena (reviewed by Arnold et al.,
inducing this syndrome (reviewed by Hanshaw et 1994).
al., 1985).
Outlook for Children with Congenital
Permanent Defects Rubella
Among the permanent defects and in addition to As a result of the extent and severity of rubella-
damage to the organ of Corti which may be present induced congenital malformations, any surviving
at birth, cardiovascular and ocular anomalies pro- children require continuous and specialized medi-
vide the greatest problems. cal care, rehabilitation and education. However,
assessment of 25-year-old patients whose mothers
acquired rubella during the extensive epidemic in
Cardiac Defects
Australia in the early 1940s showed that many had
Cardiac defects are responsible for much of the high developed far better than had been anticipated in
perinatal mortality associated with CAR, the early childhood, despite the presence of hearing and
commonest lesions being the persistence of patent eye defects. Most were of average intelligence, em-
ductus arteriosus, proximal (valvular) or peripheral ployed, and some had married and had normal
pulmonary artery stenosis or a ventricular septal children (Menser et al., 1967a). In contrast, studies
defect. In association with such anomalies, a on children with CAR resulting from the 1963—1964
neonatal myocarditis may occasionally occur. RV US epidemic showed that they had fared less fa-
may also damage the intimal lining of the arteries vourably (Cooper, 1975). This difference is probably
and this may cause obstructive lesions of the pul- due to the survival of children with some of the
RUBELLA 405
in which case it may be necessary to test additional
blood samples at intervals of 3—4 days. Although HI
antibodies may be present during the acute phase of
the illness, antibodies detected by SRH, and the IgG
response detectable by EIA, may be delayed (Figure
12.5). The absence of rubella antibody by such tests
but their presence by HI may be suggestive of re-
cently acquired infection. Tests for rubella-specific
IgM should also be carried out. This investigation is
also of value when patients present in the postacute
phase of their illness, at which time antibody levels
may already have reached their maximum levels. It
is important to stress that, regardless of technique
employed, no particular titre of antibody can be
regarded as indicative of recent or current infection.
Although the presence of rubella-specific IgM used
Figure 12.9 Congenital rubella cataract in a 9-month-old in- to be considered indicative of a recent primary in-
fant. Cataract was also present in the left eye but was removed fection, it is now recognised that, if sensitive tech-
surgically. (Reproduced with permission from Banatvala and niques are employed, low and transient concentra-
Best, Topley and Wilson, 7th edition, Vol. 4) tions of rubella-specific IgM may be detected in
reinfection, and low levels may persist for periods
more severe manifestations of congenital infection, ranging from a few months to up to 4 years follow-
which reflects more modern, vigorous and sophisti- ing rubella vaccination (reviewed in Burke et al.,
cated methods of treatment which were not avail- 1985). Such findings emphasize the importance of
able previously. assessing clinical and serological data carefully be-
fore making a diagnosis of primary rubella infec-
tion, the consequences of which may result in termi-
LABORATORY TECHNIQUES AND nation of a precious pregnancy.
Women who give a history of exposure to a ru-
DIAGNOSIS bella-like illness are much more likely to acquire
infection if exposure is close and prolonged; for
Serological Assessment of Women example, within their own household, or, in the case
Exposed to or Developing Rubella-like of a school teacher, to children in class. Although
Illnesses in Pregnancy rubella contacts may give a history of rubella vacci-
nation or have been found to have rubella antibo-
It is essential that, before attempting to interpret dies when screened during a previous pregnancy, it
serological results on sera from women who have is still advisable to investigate such persons
been exposed to or who have developed rubella-like serologically if they have been exposed recently to
illness in pregnancy, accurate information is ob- rubella-like illness. Thus, some vaccinees may fail to
tained relating to date and duration of exposure, develop an immune response following rubella vac-
date of onset of illness, including presence and dis- cination (p 411) and very occasionally the result of a
tribution of rash, lymphadenopathy and arthralgia. screening test may be recorded incorrectly. In addi-
A history of rubella vaccination as well as results of tion, rubella screening, if carried out by HI, may be
previous rubella antibody screening tests, together unreliable, giving rise to false-positive results
with the techniques by which they were carried out, (p 406). Blood should be obtained from pregnant
are also relevant. women for rubella antibody screening when they
Blood should be collected from pregnant women first book in at antenatal clinics. It is advisable to
with features of a rubella-like illness as soon as store these specimens until, say, 2—3 months after
possible after onset of symptoms. A significant rise delivery. Thus, if a patient subsequently gives a
in antibody titre can often be detected within 7 days, history of exposure to rubella, this pre-exposure
although occasionally the response may be delayed, specimen can be tested in parallel with postex-
406 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
posure serum samples. In addition, should an infant sera (PHLS Working Party on the Laboratory
be delivered with clinical features compatible with Diagnosis of Rubella, 1988).
an intrauterine infection, serum obtained in early Many tests for rubella antibody screening are
pregnancy can be tested in parallel with that ob- available commercially. EIAs are now used widely
tained after delivery and the infant’s cord or because they are readily automated and can be used
neonatal blood. Tests may be carried out for evi- in automated antenatal screening. LA has the
dence of congenital infection, not only by rubella advantage of providing a result within a few minutes
but also by such organisms as cytomegalovirus and and may be used to confirm negative results. SRH is
Toxo-plasma gondii. still used in some UK laboratories, commercially
If a history of exposure to a rubella-like illness is available reagents being used to prepare plates in
given prior to attendance at the first antenatal cli- the laboratory. These techniques have been de-
nic, or if no earlier blood sample is available, it is scribed in detail by Best and O’Shea (1995). Vacci-
important that blood be obtained as soon as poss- nation should be offered to women with antibody
ible after contact. The presence of antibodies in such concentrations 10—12 iu ml\ (Skendzel, 1996)
a sample suggests that any antibody present is lon- and this is the cut-off in most commercial assays.
gstanding, resulting from infection in the past, pro- Negative results should be confirmed using a differ-
vided that the sample was obtained well within the ent assay in order to identify sampling errors and
incubation period of rubella (e.g. within 10—14 days avoid the occasional false negative results obtained
of last exposure). Nevertheless, it is advisable to in automated assays. Some women fail to produce
collect more blood in 7—10 days, to ensure that there antibody levels 15 iu ml\ by SRH even after
is no change in rubella antibody concentration. Pa- several vaccinations. Many laboratories therefore
tients who present for virological investigations consider women with a well-documented history of
some time after exposure to possible rubella are more than one vaccination to be immune, if antibo-
more difficult to assess, since it cannot be deter- dies are detectable by two reliable assays.
mined whether antibody is longstanding or the re- Rubella-specific IgG antibodies have been detec-
sult of recently acquired infection. This can be es- ted in saliva by IgG capture radioimmunoassay
tablished, however, by testing sera for rubella- (GACRIA) (Perry et al., 1993). Thus, saliva may be
specific IgM. Women who are seronegative should used instead of serum for rubella antibody screening
be followed up for 1 month after the date of the last and seroepidemiological studies, especially those
contact to ensure that seroconversion does not oc- involving children and in developing countries
cur. Many of these women experience considerable (Eckstein et al., 1996).
anxiety during this period, which may be allayed by
conducting serological investigations on the index
case to determine whether or not the infection was Serological Techniques Used for
indeed rubella. Patients who have been followed up Diagnosis
but who remain seronegative should be offered ru-
bella vaccination post partum.
Detection of Rubella-specific IgM
Serological methods are used for the diagnosis of
rubella acquired postnatally, since RV is slow to
Serological Techniques Used for grow and difficult to identify in cell cultures. A
Rubella Antibody Screening diagnosis is usually made by detection of rubella-
specific IgM, but in the case of a pregnant woman it
Extensive rubella antibody screening of adult is advisable to confirm that diagnosis by demon-
women is carried out in order to identify suscep- strating a rise in specific IgG concentration or by
tibles who require vaccination. EIA, SRH and LA detecting specific IgM in a second serum. Rubella-
are the techniques used most frequently for screen- specific IgM may be detected by commercial EIAs.
ing purposes. HI is not recommended since it is time The M-antibody capture format is generally prefer-
consuming and labour intensive, and false-positive red to indirect assays, for which serum pretreatment
results may occur, this being due to failure to is required owing to the possibility of false-positive
remove all serum lipoprotein inhibitors from test results due to IgM antiglobulins, such as rheuma-
RUBELLA 407
toid factor. Care should be taken to ensure that the higher avidity (Thomas and Morgan-Capner, 1990).
test employed has a high level of sensitivity and
specificity. The performance of commercial assays
may differ, but it is possible usually to detect specific Detection of Rubella virus
IgM antibodies within 4 days of onset of rash and
for 4—8 weeks thereafter. Rubella virus may be detected in clinical samples by
isolation in cell culture or by reverse transcription
Detection of a Significant Rise in Antibody nested PCR (RT-nPCR), but these techniques are
Titres only available in specialized laboratories. RT-
nPCR is of value for postnatal and prenatal diag-
A significant rise in antibody titre can be detected by nosis of congenital rubella (see below) and has been
a quantitative EIA, HI or LA titration. Seroconver- described in detail by Bosma et al. (1995a, 1995b)
sion can be detected by SRH. Although HI antibo- and Revello et al. (1997).
dies may develop 1—2 days after onset of symptoms, Rubella virus can be identified by cytopathic effect
IgG antibodies detected by EIA, LA or SRH may be in RK13 or certain sublines of Vero cells (reviewed
delayed until 7—8 days. by Best and O’Shea, 1995; Banatvala and Best,
1998). In our experience, the technique of choice is
Use of Saliva two passages in Vero cells and a third passage in
RK13 cells in which RV can be detected by indirect
Rubella-specific IgG and IgM antibodies may be immunofluorescence (Figure 12.10). Alternatively,
detected in saliva using antibody capture im- RT-nPCR can be used to identify virus after two
munoassays and results correlate well with serum passages in Vero cells.
antibodies (Perry et al., 1993). The optimum time for
detecting specific IgM is 1—5 weeks after onset of
illness. Using saliva, it has been possible to demon- Virological Diagnosis of Congenitally
strate that rubella-like illnesses in children under 1 Acquired Infection
year of age are due to other viruses, such as Human
herpesvirus 6 (see Chapter 2E).
Postnatal Diagnosis
The diagnosis of CAR may be made by:
Diagnosis of Reinfection
1. Detection of rubella-specific IgM in cord blood
Rubella reinfection may be diagnosed by a signifi-
or serum samples taken in infancy;
cant rise in rubella IgG concentrations, sometimes
2. Detection of a persistent rubella antibody re-
to very high levels, or detection of specific IgM in a
sponse in the infant (i.e. presence of rubella
patient with pre-existing antibodies. If serum
antibody at a time beyond which maternal
samples obtained before reinfection are not avail-
antibody would normally have disappeared, this
able for retesting, evidence of pre-existing antibody
being approximately 6 months of age);
may be accepted if there are at least two laboratory
and/or
reports of antibodies 10 iu ml\ obtained by a
3. Detection of RV in samples (such as throat
reliable technique (not HI). A documented history of
swabs) from infected infants during the first few
rubella vaccination followed by at least one test for
months of life. Virus may be detected by isolation
rubella antibodies 10 iu ml\ is also acceptable
in cell culture, or more rapidly by RT-nPCR.
(Best et al., 1989). The rubella-specific IgM response
Specimens can be sent on dry ice or in formal
is usually lower and more transient than following
saline for RT-nPCR in a distant laboratory. This
primary infection.
has been used in our laboratory to test lens
It is often possible to distinguish reinfection from
aspirates and to confirm the diagnosis of con-
primary infection by examining the antigen-binding
genital rubella in children in India (Bosma et al.,
avidity of specific IgG. Sera taken from cases of
1995a, 1995b).
recent primary rubella reinfection have low IgG
avidity, while sera taken from persons with distant The National Congenital Surveillance Pro-
infection, including cases of rubella reinfection, have gramme in the UK classifies suspected cases of
408 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 12.10 Rubella virus detected in RK13 cell cultures by immunofluorescence. Note cytoplasmic and perinuclear fluorescence.
(Reproduced with permission from Banatvala and Best, Topley and Wilson, 7th edition, Vol. 4)
congenital rubella according to the criteria shown in Table 12.4 Congenital rubella: case classification criteria
Table 12.4.
Congenital rubella infection
Maternally derived rubella-specific IgG antibo- No rubella defects but congenital infection confirmed by
dies as well as specific IgG and IgM antibodies isolation of virus, or detection of specific IgM or persistent IgG
synthesized by the fetus in utero are present at birth. in infant
The detection of rubella-specific IgM in cord, Congenital rubella syndrome
neonatal or infant sera by an M-antibody capture Confirmed Typical rubella defect(s) plus virus-specific IgM
assay is the method of choice for the diagnosis of or persistent IgG in infant; or two or more
CAR. If other IgM assays are used, rubella-specific rubella defects plus confirmed maternal
infection in pregnancy
IgM may not always be detected at birth and further Compatible Two or more rubella defects with inconclusive
serum samples should be tested if indicated. Specific laboratory data; or single rubella defect plus
IgM has been detected by M-antibody capture RIA confirmed maternal infection in pregnancy
in all confirmed cases to the age of 3 months, 86% of Possible Compatible clinical findings with inconclusive
infants aged 3—6 months, 62% of infants aged laboratory data, e.g. single defect plus probable
maternal infection in pregnancy
between 6 months and 1 year, 42% children aged Unclassified Insufficient information to confirm or exclude
between 1 year and 18 months, but rarely in children
over 18 months of age (Chantler et al., 1982). The Reproduced from Miller et al. (1994) with permission of the PHLS
absence of specific IgM by M-antibody capture Communicable Disease Surveillance Centre ©PHLS, and from Banatvala
and Best, Topley and Wilson, 9th edition, Vol. 1.
assays in the neonatal period virtually excludes
symptomatic congenital rubella. If a low or equivo-
cal result is obtained by any assay, a further The detection of specific IgG may be of value for
specimen of serum should be examined. If the IgM the diagnosis of congenital infection when tests for
result is negative or equivocal where there is a specific IgM have not been conducted in early
history of rubella in pregnancy and the baby is infancy. Since rubella is uncommon under the age of
asymptomatic, a specimen of serum can be taken at 2 years, specific IgG detected between the ages of 1
approximately 9—12 months of age to examine for and 2 may be suggestive of congenital rubella.
persistence of specific IgG. However, each case must be assessed individually,
RUBELLA 409
taking into account such factors as age, maternal Prenatal Diagnosis
history, presence of clinical findings suggestive of, or
Prenatal diagnosis of suspected congenital infection
compatible with, congenital rubella, and rubelli-
is of value when the mother is reluctant to have
form illnesses since birth. Detection of low avidity
therapeutic abortion following rubella in the first
IgG1 may also assist the diagnosis in children up to
trimester, when maternal infection has occurred
the age of 3 years, as the avidity matures more
after the first trimester, in cases of possible maternal
slowly in children with congenital rubella than
reinfection and where equivocal serological results
following postnatal infection (Thomas et al., 1993).
were obtained, such as when the mother presents
Rubella-specific lymphoproliferative assays may
some time after a rubella-like illness.
also help to provide a retrospective diagnosis of
The method of choice for prenatal diagnosis is the
CAR in children between the ages of 1 and 3 (O’Shea
detection of rubella RNA in amniotic fluid by
et al., 1992). Although rubella-specific IgG detected
RT-nPCR, which has a sensitivity of 87—100%
by SRH and EIA may persist indefinitely, studies on
(Revello et al., 1997; G. Enders and D. Dirmeier,
223 children with congenital rubella following the
1998, personal communication). Amniotic fluid
1963—1964 US epidemic showed that the HAI
should be taken 8 weeks after onset of maternal
antibodies declined more rapidly among congeni-
rubella infection; however, as false-negative results
tally-infected children than among their mothers; by
may be obtained occasionally if the sample is taken
the age of 5, 20% of infants with congenital rubella
too early, it may sometimes be necessary to test a
no longer had HI antibodies. Nevertheless,
second sample at 22—23 weeks gestation. Viral RNA
seronegative children failed to develop an HI re-
may also be detected in fetal blood. Detection of
sponse or excrete virus when challenged with an
virus in chorionic villus biopsies should be inter-
attenuated vaccine. More recent studies suggest a
preted with caution, as the detection of RV in the
decreased production of antibodies to the epitopes
placenta may not always reflect fetal infection
on the E1 glycoprotein, which induce HI antibodies,
(Bosma et al., 1995a). When RT-nPCR is used a
in some children with congenital rubella. In order to
result can be available within 48 hours.
determine the immune status of such children in
A prenatal diagnosis may also be made by testing
later life it may be necessary to test sera by EIA, LA
fetal blood obtained by cordocentesis for rubella-
or immunoblotting.
specific IgM. Although rubella-specific IgM may be
As discussed on page 398 and shown in Figure
detected in fetal blood following infection in early
12.6, RV may be recovered from throat swabs from
pregnancy, the fetus may not produce sufficient IgM
most neonates with severe CAR which results from
before the 22nd week of gestation. Even at 23 weeks
maternal infection in the first trimester, but by the
gestation false negative results may be obtained, as
age of about 3 months the proportion excreting
concentrations of fetal rubella-specific IgM may be
virus has declined to 50—60%. Those with severe
too low to be detected by available techniques; thus,
disease, particularly during the first few weeks after
it is advised to test fetal blood for rubella-specific
birth, often excrete high titres of virus and may
IgM by more than one assay. This approach has the
readily transmit infection to rubella-susceptible
disadvantage that patients may have to wait for
persons. Women of childbearing age, some of whom
what might be an unacceptably long time for a
may be in early stages of pregnancy, must be
diagnosis to be established, with little time for
dissuaded from visiting such infants unless they
termination to be considered.
have had rubella vaccine or serological tests confirm
that they are immune. It is important that midwives
and nursing staff who may have to care for such
infants are also shown to be immune to rubella; it PREVENTION—RUBELLA
must be appreciated that midwives and nurses who VACCINATION
originate from certain tropical countries may be
more likely to be susceptible to rubella, as they may Development and Use of Attenuated
not have been offered rubella vaccination (p 395). Vaccines
Figure 12.11 Incidence rates of rubella and congenital rubella syndrome (CRS) in the USA, 1966—1993. Cases of rubella reported to
the National Notifiable Disease Surveillance System per 100 000 population; cases of CRS reported to the National CRS Registry per
100 000 live births. (From Centers for Disease Control, 1994)
13
Mumps
Pauli Leinikki
National Public Health Institute, Helsinki, Finland
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
420 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
14
Enteroviruses
Philip D. Minor, Peter Morgan-Capner and Peter Muir
NIBSC, Potters Bar, Hertfordshire, UK
Public Health Laboratory, Preston, UK
Guy’s, King’s and St Thomas’ School of Medicine, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
428 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 14.1 Electron micrograph of poliovirus, negatively stained with 4% sodium silicotungstate: (a) whole virus particles (D
antigen) approximately 30 nm in diameter; (b) empty capsids, showing penetration of stain; (c) shadowed virus (print reversed to give
black shadows) producing pointed shadows; (d) shadowed virus (print reversed to give black shadows) producing blunt-ended shadows.
Physical Characteristics of the Virus ined electron microscopically (Figure 14.1a). Non-
Particle infectious empty capsids, unlike virions, are pen-
etrated by stain, revealing that the virion shell is
The virion is a largely featureless, symmetrical par- approximately 2.5 nm thickness (Figure 14.1b). The
ticle, approximately 27 nm in diameter when exam- essentially spherical appearance of the particle is
consistent with a virus with icosahedral symmetry,
ENTEROVIRUSES 429
and this has been broadly confirmed by particle precursor protein, VP0, as the last stage in the
shadowing which produces pointed and blunt maturation of a virus particle, probably in an
shadows (Figure 14.1c,d). The sedimentation coeffi- autocatalytic process resulting from the encapsida-
cient of the intact virus on centrifugation is tion of the nucleic acid genome.
155—160S, while that of the empty capsid is 70—80S. The defined structure of the virus particle result-
The buoyant density of the virion in caesium chlor- ing from the absence of a lipid layer has made it
ide gradients is 1.34 g ml\. A particle with the possible to obtain crystals suitable for X-ray diffrac-
chemical composition of an enterovirus virion tion studies, and the molecular structures of po-
would be expected to have a density of 1.47 g ml\ if liovirus (Hogle et al., 1985), rhinovirus type 14
freely permeable to caesium ions; the low density of (Rossman et al., 1985), and CVB3 (Muckelbauer et
the enteroviruses implies that the virion is essential- al., 1995) have been resolved such that the interac-
ly impermeable consistent with the failure of elec- tions involved in maintaining the structural features
tron microscopic stains to penetrate infectious par- of the infectious virion are now clear. The three
ticles (Figure 14.1a). In the past, the behaviour of largest virion proteins VP1, VP2 and VP3 have a
the viruses on isopycnic centrifugation has been very similar core structure, in which the peptide
used as a criterion for differentiating enteroviruses backbone of the protein loops back on itself to form
from rhinoviruses and aphthoviruses, both of which a barrel of eight strands held together by hydrogen
have a higher buoyant density in caesium chloride. bonds (the barrel). This core forms a wedge shape,
The enteroviruses are stable to acid pH, also unlike and the amino acid sequences between the se-
the rhinovirus and aphthoviruses, which are inac- quences forming the barrel, and the sequences at
tivated by brief exposure to pH 2. This has also been the N- and C-terminal portions of the protein con-
used as a criterion for the identification of a picor- tain elaborations which include the main antigenic
navirus as an enterovirus. sites involved in the neutralization of viral infection.
The proteins are arranged with icosahedral sym-
metry, with VP1 molecules at the pentameric apices
of the icosahedron oriented such that the pointed
Biochemical Structure of the Virus end of the wedge-shaped protein points towards the
Particle apex. The other two proteins, VP2 and VP3, alter-
nate about the centre of the triangular face of the
The infectious particle consists of a shell of 60 co- icosahedron (the threefold axis of symmetry). There
pies, each of four proteins, VP1, VP2, VP3 and VP4, are extensive interactions between the three large
arranged with icosahedral symmetry about a proteins, and also with the fourth protein, VP4,
genome composed of a single strand of RNA of which is internal. In particular, the N terminus of
messenger sense. Like many eukaryotic messenger VP1 lies under VP3, and the N terminus of VP3
RNA molecules, the genomic RNA terminates in a under VP1. The apical structure formed by VP1 is
sequence of 40—100 adenylic acid residues at the 3 separated from the plateau formed by VP2 and VP3
end (the poly(A) tract.) At the 5 end, however, it by a cleft or canyon; the peak of the VP1 structure is
bears a small protein (VPg) approximately 22 16.5 nm from the virion centre whilst the plateau
amino acids long, covalently linked to the RNA by made up of VP2 and VP3 is approximately 15 nm
the hydroxyl group of a tyrosine residue. The com- from the centre. The base of the cleft is of the order
plete genomic sequences of several enteroviruses are of 11 nm from the centre, and it has been postulated
now known. The virion is believed to be entirely that this cleft may contain the regions of the virus to
devoid of carbohydrate, and lacks a lipid mem- which the cellular receptor attaches. The interac-
brane, although as described below lipid molecules tions between the component proteins are such as
form an entegral part of the structure. The particle to suggest that the most stable unit of the structure
has a relative molecular weight (M ) of 8 ; 10, of is a pentamer made up of five copies each of VP1,
which the RNA provides 32% and the protein 68% VP2, VP3 and VP4; it seems likely that this is the
by weight. The four principal structural proteins of basic unit from which the virus is assembled. A
the virion have molecular weights of approximately diagrammatic representation of the three-dimen-
30 000 (VPI), 27 000 (VP2), 24 000 (VP3) and 7000 sional structure of the capsid proteins of poliovirus
(VP4). VP2 and VP4 are formed by the cleavage of a type 1 is shown in Figure 14.2.
430 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 14.2 Three-dimensional structure of the capsid proteins of poliovirus (Hogle et al., 1985). The numbers indicate positions of
amino acid residues: (a) core structure common to capsid proteins; (b) VP1; (c) VP2; (d) VP3
In addition to the protein components of the feature of enteroviruses this lipid molecule is. How-
virus, lipid molecules are also found in the structure. ever, certain antiviral compounds insert into the
Myristic acid is found covalently attached to the N corresponding region of rhinovirus 14, and in so
terminus of the smallest capsid protein VP4 (Chow doing prevent upcoating. It is therefore possible
et al., 1987). The aliphatic chain penetrates the apex that the lipid plays a part in uncoating or assembly
of the icosahedron, and it is possible that this forms of enteroviruses.
a structural support for the virion. Myristic acid has
been found to be attached to virion proteins in
other viruses including retroviruses and rotaviruses.
The other lipid moiety has not yet been identified, Antigenic Structure of the Virus
but has been shown to be present in the structures
of poliovirus type 1 and type 3. It has a 16-carbon The antigenic properties of the enteroviruses pro-
aliphatic chain which lies in the pocket formed by vide one of the main methods of differentiating
the beta barrel of VP1. The other end of the mol- them. In most cases specific antiviral antisera neu-
ecule has not been observed. Rhinovirus 14 has no tralize only homologous virus, but this is not always
such insert, and it is not yet clear how general a so. For every picornavirus studied in detail, it has
ENTEROVIRUSES 431
been shown that empty capsids prepared under ap- ing amino acids 142, 166 and 253; (2) a complex site
propriate conditions present major antigenic deter- involving residues 220—222 of VP1 and 160—170 of
minants different from those of the infectious virus. VP2 and the C terminus of VP2; (3) a complex site
Moreover, the infectious virus can be readily de- involving residues 286—290 of VP1 and 58—60 of
natured by relatively mild conditions, such as treat- VP3 (Minor, 1990). The fourth site involves residues
ment with ultraviolet light, heating to 56°C for 10 in different pentamers and is therefore only found in
minutes or attachment to a plastic surface in en- the intact virion, while the other sites are also found
zyme-linked immunosorbent assay (ELISA) tests. in some viral subunits. For poliovirus type 1, the
The infectious particle is said to express D or N site located at residues 89—100 of VP1 has been
antigenic character, and the empty capsid or de- implicated in virus neutralization, although to a far
natured virus C or H antigenic character. C antigen lesser extent than for type 3 where the homologous
is less specific to a particular virus than is D antigen. region is the principal target for neutralizing anti-
Thus there is evidence for cross-reaction of C anti- bodies in sera from immunized animals. The site
gen-specific monoclonal antibodies with poliovirus involving VP3 is the next most common target of
of type 1, type 2 and type 3, whereas cross-reaction antibodies, including most of the antibodies show-
by neutralizing monoclonal antibodies or those re- ing extreme strain specificity for poliovirus, whilst
acting with D antigen is rare. However, monoclonal the site involving VP2 is the least common.
antibodies able to neutralize both type 1 and type 2 The antigenic structure of type 2 virus has been
poliovirus have been reported (Uhlig et al., 1990) less well characterized. The site to which cross-
and priming for a secondary response to type 1 or reactive antibodies bind appears to involve se-
type 3 by previous injection with type 2 is quences around 200—210 of VP3 and 239—245 of
documented (Katrak et al., 1991). Similarly there is VP2, and may be linked to the strain-specific site
evidence for cross-reaction of echoviruses 11, 16 involving VP1 and VP3 (Uhlig et al., 1990).
and 22 with poliovirus with appropriate C-specific
antibodies. The difference between particles ex-
pressing D and C antigen is due to the conforma-
tion of the proteins, rather than to the loss of a Cellular Receptor Sites
protein. These antigenic characteristics suggest that
methods based on virus neutralization by antibody Enteroviruses attach to and enter cells by specific
may give different results concerning the identity of receptor sites. This was first shown for poliovirus by
enterovirus strains to methods based on im- the observation that purified genomic RNA was
munochemical techniques such as ELISA or im- infectious for rabbit cells whereas the virus itself was
munoblot methods. not. Competition experiments between different vi-
The antigenic structures involved in the neutral- ruses have shown that, for example, all three po-
ization of picornavirus infectivity have been the liovirus serotypes utilize the same cellular receptor,
subject of much study and have been clarified by the which is distinct from that for the Coxsackie B
resolution of the molecular structures of poliovirus viruses. This phenomenon has been confirmed in a
and rhinovirus outlined above. The similarities in number of cases by the isolation of monoclonal
the structures which have been found to be import- antibodies, which will block specifically the attach-
ant in neutralization of these two types of virus ment of certain types of virus by reacting with the
suggest that homologous features may be involved host cell rather than with the virus (Minor et al.,
in all picornaviruses. Sites have been identified by 1984).
the isolation of antigenic variants which are resis- The enteroviruses and the picornaviruses in gen-
tant to monoclonal antibodies able to neutralize a eral are responsible for a range of diseases with
parental strain, and then characterized by se- different target organs. These include the heart
quencing the genomic RNA. This has revealed four (Coxsackie viruses), nerve tissue (polioviruses), liver
major sites to which neutralizing antibodies bind. (HAV) and others, as well as the intestinal tract. It is
For type 3 poliovirus they include: (a) a sequence of possible that the expression of the receptor sites in
VP1 about one-third of the way in from the N specific tissues plays a part in the tropism of the
terminus involving residues 89—100, together with virus, although it is unlikely to be the only factor.
sequences from three other loops from VP1, includ- This in turn may lead to a novel chemotherapeutic
432 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
approach to viral diseases; for example, by the use the second half encodes proteins necessary for virus
of chemically synthesized receptor analogues. replication, such as the RNA polymerase. The
The gene encoding the cellular receptor for po- fourth portion of the genome is a second short
liovirus has been identified (Mendelsohn et al., sequence with no known coding function. This ar-
1989) and is a previously unknown three-domain rangement is summarized in Figure 14.3. The non-
protein of the immunoglobulin superfamily. The coding regions are well conserved between related
terminal domain is required for activity. Transgenic viruses, and there is thus reason to believe that they
mice carrying the gene for the human receptor for are of importance in virus replication.
poliovirus have been prepared by two groups (Ren The 5 non-coding region of eukaryotic messen-
et al., 1990; Koike et al., 1991) and are susceptible to ger RNA usually terminates in a methylated gua-
poliovirus infection with a pathology similar to that nosine cap, which is important in binding ribo-
shown by primates. Mouse cells are not normally somes and thus translation of the RNA into protein.
susceptible to infection with poliovirus or most Thus most eukaryotic messenger RNA molecules
other human enteric viruses. When stably transfec- require a free 5 terminus, and initiation of transla-
ted with the human gene for the poliovirus receptor, tion internally is not possible. However, it has been
however, they are rendered sensitive, and can thus shown that the 5 non-coding regions of poliovirus
be used to identity polioviruses in the presence of and other picornaviruses act as internal initiation
other viruses. This is of value in clinical studies sites so that ribosomes may bind in the absence of a
related to the programme to eradicate poliomyeli- free 5 capped terminal structure (Pelletier and Son-
tis, as described later. nenberg, 1988). In fact, infection with poliovirus
The receptor for Echovirus 1 has been isolated results in the cessation of cap-dependent transla-
and identified as the integrin VLA-2, a known sur- tion. The mechanism of internal initiation is be-
face molecule (Bergelson et al., 1992). DAF (decay lieved to involve binding of as yet ill-defined factors
accelerating factor) a cell surface protein involved in to complex secondary and tertiary structures for-
the complement pathway has been shown to be a med by the 5 non-coding region. Both the 5 and 3
receptor site for many echoviruses (Bergelson et al., non-coding regions must also be involved in the
1994; Ward et al., 1994) some coxsackie B viruses attachment of the RNA polymerase during RNA
(Shafren et al., 1995), and enterovirus 70 (Kar- replication.
nauchow et al., 1996). Coxsackie virus B1—6 sero- The viral proteins are synthesized as one large
types also use a common receptor, also used by protein from a single open reading frame. The pre-
some adenoviruses, identified as a probable trans- cursor protein includes two known sequences
membrane protein of unknown function which con- which act as proteolytic enzymes and digest the
tains two immunoglobulin-like domains, and is ex- protein at specific positions as it, and they, are in the
pressed preferentially in brain, heart and pancreas process of being synthesized. The first (P2A) acts to
(Bergelson et al., 1997). Coxsackie virus A10, 15, 18 cleave the structural from the non-structural pro-
and 21 employ ICAM-1, as does the major teins, whilst the second completes all the other pro-
rhinovirus group. Coxsackie virus B3 also uses nuc- cessing of the protein, except the final maturational
leolin, and Coxsackie virus A9 v3 integrin. cleavage of VP0 to VP2 and VP4. Apart from these
two functions the non-structural proteins include a
polymerase and the small genome-linked protein
VPg, and a number of proteins of unknown but
Strategy of Replication important function. Protein P2C is known to be
virtually identical in all the polioviruses, for
The genome of the virus is a single positive-strand example, implying that it cannot be altered readily
RNA molecule, and thus acts as a messenger RNA without affecting virus viability.
for the synthesis of the proteins found in infected The synthesis of RNA is less well understood
cells. The genome can be divided into four regions, than that of protein. The positive-sense genomic
comprising a sequence of about 750 bases with no RNA acts as a template for the synthesis of a nega-
known coding function, followed by a large open tive-sense copy, which in turn acts as a template for
reading frame of which the first half codes for the more positive-sense strands. The positive-sense
structural protein found in the virus particle, while RNA is used both for translation and for packaging
ENTEROVIRUSES 433
Figure 14.3 Organization of the genome of poliovirus, a typical enterovirus showing the two non-coding regions and the location of
the regions coding for the various virus proteins. In the formal nomenclature (Rueckert and Wimmer, 1984), the P1 region encodes the
structural capsid proteins, and the P2 and P3 regions encode non-structural proteins found only in infected cells
into virus and is the major species made after the the different viruses, and reveals a surprisingly close
early stages of infection. All nascent strands carry a relationship between the enteroviruses and
5 terminal molecule of VPg. The polymerase activ- rhinoviruses where the genomes show a high degree
ity indicated in Figure 14.3 is able to elongate of homology. In contrast hepatitis A has no signifi-
nascent RNA in vitro but is unable to initiate RNA cant homology with the archetypal enteroviruses
synthesis on its own. The mechanism of initiation is Poliovirus type 1 at the nucleic acid level. Echovirus
not yet fully known. 22 is also unique in sequence, showing no homology
The assembly of the virus particle involves the with other enteroviruses.
synthesis of VP0, VP1 and VP3 as a single unit, The human enteroviruses can be further divided
which is then assembled into pentamers, followed into two groups on the basis of sequence compari-
by the association of 12 pentamers into the virion, a sons of the 5 non-coding region (Pöyry et al., 1996;
process which involves host factors. The X-ray crys- Hyypiä et al., 1997). One includes polioviruses,
tallographic structure of the virus implies that the Coxsackie virus A21 and A24, and enterovirus 70,
final maturation cleavage of VP0 to VP2 and VP4 is while Coxsackie B, Coxsackie virus A9, A16, en-
autocatalytic. Thus a serine residue is found close to terovirus 71, Echovirus 11, Echovirus 12 and all
the N-terminus of VP2. A number of serine pro- partially sequenced enteroviruses form the second.
teases are known, and it may be concluded that as Similar sequence comparisons of the coding region
soon as the proton donor, such as the genomic and 3 non-coding region result in four subdivisions
RNA, is brought into the vicinity of the serine resi- of the human enteroviruses. the first group com-
due the final cleavage will take place. The virus is in prises the polioviruses and Coxsackie virus A1, A11,
general released from the cell by lysis. A13, A17, A18, A21 and A24; the second group
includes only enterovirus types 68 and 70 so far; the
third includes Coxsackie virus B1—6, A9, all en-
Sequence Comparisons of Enteroviruses
teroviruses except types 22 and 23, and enterovirus
Sequences of the genomes of a number of en- 69. Coxsackie virus A2, A3, A5, A7, A8, A14, A16
teroviruses and other picornaviruses have been de- and enteroviruses 71 form the final group.
termined (Stanway, 1990). Comparison of such se- Sequenced animal enteroviruses, such as bovine
quences indicates the evolutionary relationship of enteroviruses, form a group distinct from the hu-
434 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
man enteroviruses when either the 5 non-coding orphan) viruses and recently characterized human
region or the remainder of the genome is consider- enteroviruses. The virus types comprising each
ed. Different regions of the genome vary in their group are summarized in Table 14.1, together with
homology between enteroviruses and other picor- the major diseases with which they are associated
naviruses. Certain sections in the 5 non-coding re- and their cultural characteristics.
gion of the genome are identical in all enteroviruses The classification of the enteroviruses has fol-
examined, except for Echovirus 22, implying some lowed closely the history of the development of
definite functional constraint; this identity may knowledge on the aetiology of the diseases they
carry over into the rhinoviruses, but it is possible to cause. Poliovirus was first identified in 1909 by
identify sequences which are apparently enterovirus inoculation of monkeys with specimens from cases
specific. It is further possible to identify regions such of paralytic poliomyelitis. Following the discovery
as the 3 non-coding region which are strongly con- by Enders et al. (1949) that poliovirus could be
served for a particular species, such as a poliovirus. grown in cell cultures, the importance of the en-
Finally, certain regions including areas coding for teroviruses as a cause of human disease came to be
the coat protein VP1, but also some sequences in appreciated. In 1948 Dalldorf and Sickles (1948)
the 5 non-coding region, are extremely strain speci- recovered a new group of agents by inoculation into
fic. In principle, as described later, it is possible to newborn mice of faecal extracts from two children
exploit these various types of region to devise with paralytic disease. These agents were named
genomic probes or polymerase chain reaction Coxsackie viruses after the town in New York State
(PCR) primers which are specific for enteroviruses where the isolations were made. Coxsackie virus
in general, for a particular species of enterovirus, or types A and B were identified on the basis of the
for a particular strain of a particular species. Such histopathological changes they produced in new-
probes are valuable in clinical and epidemiological born mice and their capacity to grow in cell cul-
studies. They also imply a major function for re- tures. Later a third group, the echoviruses, was
gions of the genome such as the 5 and 3 non- identified, which was also associated from time to
coding regions where they are strongly conserved, time with human diseases. Echoviruses were found
although such functions have not yet been fully to be non-pathogenic for subhuman primates and
resolved. newborn mice, but produced cytopathic changes in
cell cultures. Within a few years it was found that all
three groups of virus were antigenically distinct and
consisted of several and, in some cases, numerous
PATHOBIOLOGICAL AND CLINICAL serological subtypes.
ASPECTS OF HUMAN Since 1970, new enterovirus types have been allo-
ENTEROVIRUSES cated sequential numbers (68—71) following those
allocated previously to enteroviruses. The en-
The enterovirus serotypes of humans are distin- teroviruses have a worldwide distribution and col-
guished on the basis of their homotypic seroneutral- lectively cause a considerable impact in morbidity
ization, and exhibit a wide range of biological and and morality. The extraordinarily wide range of
pathogenic properties which are often characteristic diseases included in their clinical ‘repertoire’ con-
of individual enterovirus groups. An extensive lit- tributes to their attraction as subjects for study by
erature of up-to-date and authoritative reviews on the molecular virologist, pathologist, clinician and
the clinical, pathological and epidemiological prop- epidemiologist alike. The diverse clinical properties
erties of individual groups of human enteroviruses of the viruses may be a reflection of the specificity of
is available (Christie, 1987; Bendinelli and Fried- different viruses for different cell receptor sites,
man, 1988; Tracy et al., 1991; Muir, 1992; Rotbart, which in turn may determine the tissue tropism of
1995a; Melnick, 1996; Muir and van Loon, 1997), to the virus.
which the reader is referred for further detailed Enteroviruses are also found in lower mammals
information. and simians, and in some cases serological relation-
The viruses are divided into five groups: po- ships between human and animal viruses occur. For
lioviruses, Coxsackie virus group A, Coxsackie vi- example, swine vesicular disease virus is closely re-
rus group B, echo (enteric human cytopathogenic lated serologically to Coxsackie virus B5.
ENTEROVIRUSES 435
Table 14.1 Types and characters of human enteroviruses
Cytopathic effects
in cell cultures
Among the human enteroviruses it is the po- kidney and human origin and their pathogenesis for
lioviruses for which details of antigenic and molecu- suckling mice. A feature of Coxsackie viruses not
lar structure are best understood, as described shared by other enteroviruses is their pathogenesis
above. For most of the other viruses information is in suckling mice. Coxsackie viruses A unlike Cox-
sparse. However, it is becoming clear that, despite sackie B strains, replicate poorly if at all in monkey
their generally unique patterns of neutralization by kidney cell cultures. Poliovirus, unlike other en-
antibody (on which virus typing is based), there are teroviruses, causes flaccid paralysis in monkeys and
frequent and major antigenic cross-reactions be- chimpanzees when administered into the brain or
tween types which can be identified by other spinal cord. Group A Coxsackie viruses produce
serological methods. For the human enteroviruses widespread myositis in the skeletal muscles of new-
in general there is little documented evidence of born mice, resulting in flaccid paralysis without
progressive antigenic variation. Antigenically un- other obvious lesions. Group B viruses also pro-
usual strains can be isolated, however (Magrath et duce myositis but it is generally of more focal dis-
al., 1986). For example, type 3 polioviruses isolated tribution than that caused by group A viruses. In
from patients with paralytic poliomyelitis in Fin- addition they cause necrotizing lesions of the brown
land in 1984 were antigenically clearly distinguish- fat pads (intrascapular cervical and cephalic pads)
able from classic poliovirus type 3 strains isolated in and, in the case of certain strains, encephalitis with
the 1950s. It may be significant that the cases of spastic paralysis, pancreatitis, myocarditis, en-
poliomyelitis from which the viruses were isolated docarditis and hepatitis in both infant and adult
occurred in persons who were ‘immunized’ with mice.
inactivated vaccine. Other evidence of antigenic The common portal of entry for enteroviruses is
heterogeneity among viruses of a single serotype is generally thought to be the alimentary tract via the
the finding the Coxsackie virus B5 strains isolated in mouth. Replication of virus in the cells lining the
1973 in the USA are clearly distinguishable from alimentary tract may be preceded by, or accom-
the prototype 1952 strains (Melnick, 1990). pany, oropharyngeal replication.
The host range of the enteroviruses varies be- For several enterovirus types (e.g. poliovirus) a
tween groups and types. Table 14.1 summarizes the viraemic phase is followed by involvement of the
ability of the viruses to replicate and produce target organs (e.g. spinal cord and brain, meninges,
cytopathogenic effects in cell cultures of monkey myocardium or skin). Incubation periods before
436 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
onset of disease vary widely from 2 to 30—40 days with stiffness and pain in the back and neck. The
for different enteroviruses. The pathogenesis of po- disease may last for up to 10 days and recovery is
liovirus has been more fully studied (Minor, 1996) complete. Poliovirus is only one of several viruses
than that of any other enterovirus, but even in this which may cause such meningeal symptoms. In a
case it is not fully understood. According to Bodian small number of cases, this picture progresses to
the virus, having entered via the alimentary tract, paralytic poliomyelitis.
multiplies locally at the initial sites of virus coloniz- Most cases of paralytic poliomyelitis occur with-
ation (tonsils, Peyer’s patches) and associated out evidence of an earlier phase of illness. The most
lymph nodes. At this stage virus may be isolated predominant sign is flaccid paralysis resulting from
from both throat and faeces. Secondary spread of lower motor neuron damage. Painful spasms and
virus apparently occurs via the blood to other sus- incoordination of non-paralysed muscles may also
ceptible tissues (e.g. other lymph nodes, brown fat occur. Death may be due to respiratory paralysis. In
pads and nervous tissue). On the other hand Sabin those who survive, some recovery of muscular func-
held the view that the virus replicates chiefly in the tion is common but may take 6 months or longer. In
mucosal layers of the intestine or oropharynx, seed- epidemics in developed countries in the early 1950s,
ing the associated lymphoid tissues where it does typically 5% of cases were fatal, 10% showed full
not necessarily replicate. After a low-level, possibly recovery with no sequelae, while the remainder
undetectable viraemia, replication at distant un- showed permanent paralysis to some degree.
identified sites leads to a secondary viraemia which Many years after maximal recovery from the
may result in infection of either the peripheral or original attack of poliomyelitis, some patients de-
central nervous system. For further details see Mi- velop further muscle weakness, the postpolio syn-
nor (1996). drome. There have been suggestions based on the
Within the central nervous system the virus ap- detection of intrathecal poliovirus antibody, and
pears to spread along nerve fibres. If local multipli- the presence of poliovirus RNA in cerebrospinal
cation is extensive, motor neurons are destroyed fluid (Leparc-Goffart et al., 1996), that in some cases
and paralysis results. The anterior horn cells of the this may be due to persistent polio-virus infection of
spinal cord are most prominently involved but in neural cells, although there are alternative hypothe-
severe poliomyelitis the posterior horn and dorsal ses such as ageing (Munsat, 1991). Persistence of
root ganglia are affected. Shedding of virus occurs poliovirus or other enterovirus infection of the cen-
from the throat and in faeces and thus transmission tral nervous system has also been implicated in the
of infection occurs independently of invasion of the aetiology of motor neuron disease, and the presence
nervous system. of enterovirus RNA in cerebrospinal fluid or neural
The major clinical aspects of enterovirus infec- tissue has been reported in small numbers of pa-
tion are summarized below. tients (Woodall et al., 1994; Leparc-Goffart et al.,
1996). However, one other study found that, while
enterovirus RNA can be detected in postmortem
Poliomyelitis brain and spinal cord tissue of patients with post-
polio syndrome and motor neuron disease, viral
The clinical features and epidemiology of this dis- RNA was also detectable in patients with other
ease have been reviewed in detail by Christie (1987) neurological or non-neurological diseases (Muir et
and Minor (1996). Progress towards its eradication al., 1996), and was therefore unlikely to be related to
is described later. chronic neurological disease.
Although infection with poliovirus of type 1, 2 or Poliomyelitis, when epidemic, occurs primarily in
3 is inapparent commonly, or results in a mild the summer months in temperate zones. Non-polio
febrile illness, aseptic meningitis or paralytic polio- enteroviruses, particularly Coxsackie virus A7 and
myelitis may occur. However, only about 1% of enterovirus 71, occasionally cause polio-like illness,
infections result in illness with neurological involve- and in some regions where wild polioviruses have
ment. Cases of abortive poliomyelitis, often accom- been eradicated, enterovirus 71 is emerging as an
panied by fever, malaise, headache, nausea or important cause of acute flaccid paralysis (da Silva
vomiting, are followed by uneventful recovery. et al., 1996).
However, a few patients develop aseptic meningitis
ENTEROVIRUSES 437
15
Poxviruses
Derrick Baxby
University of Liverpool, Liverpool, UK
This chapter deals only with those poxviruses which The poxviruses of vertebrates are separated into
cause human infection. The last case of smallpox eight genera, and species within each genus are very
occurred over 20 years ago, and the remaining hu- closely related. Some poxviruses have yet to be
man poxvirus infections are relatively unimportant, assigned to genera, and the relationships of some
although occasional severe infection and even death viruses within genera needs further clarification (Es-
may occur. With the exception of molluscum con- posito et al., 1995; Baxby, 1998).
tagiosum, human poxvirus infections are acquired Genetic hybridization can occur within a genus,
from animals. This, and the fact that some are re- and serological relationship between species is very
stricted geographically, is of value when possible close. Traditionally virus isolates have been identifi-
cases are being investigated (Table 15.1). ed on the results of biological tests. These methods
Despite the eradication of smallpox there is still are still used for presumptive identification, but
considerable interest in poxviruses. Such interest increasing use is being made of genome analysis.
includes: studies on gene expression including the Fortunately the validity of species established on
expression of foreign genes, and in particular gene biological criteria has in general been endorsed by
products which may help the virus to evade the genome analysis. The human pathogens are reason-
host’s defence mechanisms; the use of poxviruses as ably well-characterized species and are distributed
vaccine vectors; the natural evolution of diseases among four genera (Table 15.1) (Esposito et al.,
such as myxomatosis; the maintenance of diseases 1995).
such as monkeypox, cowpox and possibly buf-
falopox in wildlife populations; the possible effects
of such viruses on their wildlife reservoirs; evalu-
ation of the importance of human monkeypox; the Structure and Replication
development of improved vaccines for animal pox-
virus infections (Moss, 1996; Baxby, 1998). Al- Morphology
though perhaps less important than foot and mouth In general a poxvirus may be recognized as such by
and rinderpest, diseases of domestic animals such as its large size and brick-shaped morphology. Most
sheeppox, camelpox and avianpox can cause con- work has been done on vaccinia, which can be taken
siderable problems for communities dependent on as representative of orthopoxviruses, molluscum
them (Baxby 1988, 1998). and tanapox viruses. Virions are 200—250 ;
250—300 nm, basically brick-shaped but somewhat
pleomorphic; parapoxviruses are slightly smaller
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
452 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 15.1 Poxviruses pathogenic for humans
Animals
Reservoir naturally Geographical
Genus Virus host infected distributed Comment
Parapoxvirus Orf Sheep Sheep Worldwide Common trivial zoonoses.
Pseudocowpox Goats Goats Occupational hazards
Cattle Cattle
Molluscipoxvirus Molluscum Humans — Worldwide Trivial infection. Often
sexually transmitted
Yatapoxvirus Tanapox Monkeys Monkeys Kenya, Zaire Rare trivial zoonosis
(Congo Republic)
and somewhat narrower (c. 160 nm) (Figure 15.1a, Chemical Structure
b). Virions released naturally from cells (‘extracellu-
The genome of poxviruses is one long piece of
lar enveloped virus’, EEV) have an outer envelope
double-stranded DNA which varies in size from
which is soon lost on manipulation and not found
130 kb (parapoxvirus) to 260 kb (fowlpox virus).
on virions released artifically (‘intracellular naked
The molecule has cross-linked inverted terminal
virus’, INV) (Figures 15.1a,b and 15.2b). The extra
segments and on denaturation forms a closed circle
envelope has antigens not present in INV, and EEV
of single-stranded DNA (Moss, 1996). The central
plays an important role in pathogenesis (Smith et
portion is highly conserved but differences in the
al., 1997). The appearance of both EEV and INV in
terminal regions of different species provide a
the electron microscope is affected by stain penetra-
means for separating them (Esposito and Knight,
tion. The majority of virions have short surface
1985). The complete sequences of some strains of
tubules c. 10 nm in diameter and are referred to as
vaccinia and smallpox virus and considerable se-
M (‘mulberry’ forms) (Figure 15.1b); a minority,
quences of other poxviruses have been determined,
slightly larger and electron dense, appear to have a
and the location and function of the genes is usually
thick (c. 20—25 nm) membrane or capsule (hence ‘C’
related to the Hind III restriction map of the
forms) (Figure 15.1c). The M form of parapoxvirus
Copenhagen strain of vaccinia virus (Figure 15.3)
is covered by one long tubule which winds round
(Johnson et al., 1993). The DNA is not infectious per
the virion (Figure 15.1a). This gives the characteris-
se. The virion contains a number of virus-coded
tic criss-cross effect due to superimposition of the
enzymes, in particular a DNA-dependent RNA
images of the top and bottom surfaces of the virion
polymerase, which transcribe the viral genome
when seen in the electron microscope. All the forms
(Moss, 1996).
described (EEV-M, EEV-C. INV-M, INV-C) are
The virions contain many polypeptides; over 100
infectious.
have been detected by two-dimensional polyac-
Thin sections show a central dumbell-shaped
rylamide electrophoresis, and there is coding poten-
core or nucleoid, flanked by two ‘lateral bodies’.
tial for c. 200. Some are glycolysated and some of
Inside the cell, virions are often bounded by double
these are found in the envelope of EEV (Moss,
or multiple membranes (Figure 15.2a). When re-
1996).
leased naturally, the outer membranes fuse with the
cell or Golgi membranes and the virion is extruded
Antigenic Structure
from the cell, invested by the envelope described
above (Figure 15.2b). Poxviruses are antigenically complex. Extracts of
POXVIRUSES 453
Figure 15.1 (a) Mulberry (M) form of non-enveloped parapox virion; (b) naturally-released (extracellular) M form of Vaccinia virus; (c)
capsule (C) form of Vaccinia virus. (Negative stain; bar : 150 nm)
infected tissue contain a number of precipitating virion-coded enzymes are essential for the final
antigens, the precise number of which is unknown. stages.
Such extracts will fix complement and react in Transcription and translation are under close
ELISA and RIA tests, but it is not known how control. Poxvirus genes cannot be controlled by
many antigens take part in these reactions. Virus mammalian promoters and there are differences be-
neutralization is a complicated process: analysis tween early and late poxvirus-specific promoters.
with monoclonal antibodies has detected nine neu- Some genes, e.g. DNA polymerase, are transcribed
tralizing epitopes distributed among the INV of the from inoculum DNA and switched off by postrep-
different species of orthopoxviruses (Czerny et al., licative gene products. Others, e.g. the haemag-
1994). Extra antigens are present on the outer envel- glutinin, are produced throughout the replication
ope of EEV, although for technical reasons progress cycle, but via different promoters. Many gene prod-
here has been less rapid (Vanderplasschen et al., ucts are subject to post-transcriptional modifica-
1997; Baxby, 1998). Orthopoxviruses produce a tion, for example by glycosylation and proteolytic
haemagglutinin antigen which reacts with erythro- cleavage.
cytes from certain fowls. This antigen is present on Replication takes place in cytoplasmic factories
the envelope of EEV and can also be detected in referred to as B-type inclusions, in which virions at
virus-free supernatants. various stages of assembly are seen. Cells infected
with some poxviruses (e.g. cowpox, avian pox-
viruses) also contain electron-dense A-type inclu-
Replication and Cultivation
sions, usually containing mature virions; A-type in-
Only the briefest summary can be given here but an clusions are easily seen by light microscopy (Figure
authoritative and extensively referenced account 15.4).
has been published recently (Moss, 1996). Molluscum contagiosum virus (MCV) has never
Virions are taken into the cell by pinocytosis been cultivated in vitro, although some early anti-
and/or phagocytosis, and there is increasing evi- gens and a non-transmissable cytopathic effect may
dence that INV and EEV attach to different recep- be detected in cell culture (Birthistle and Carrin-
tors (Vanderplasschen and Smith, 1997). Constitut- gton, 1997). The remaining human pathogens can
ive cellular enzymes initiate virus uncoating, but usually be cultivated in easily obtained cell cultures,
454 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ent that virulence was a complex multifactorial sys-
tem. Analysis of the genome has identified genes
whose expression is not necessary for replication,
and strains which do or do not express some of
these genes have been compared in vivo and in vitro.
The nucleotide and amino acid sequences and the
observed or predicted properties of these gene prod-
ucts provide evidence for their demonstrated or
predicted role in pathogenesis. Some examples are
given in Table 15.2, and further details are provided
elsewhere (Smith et al., 1997). Gene products identi-
fied include some which help the virus to evade the
immune system by binding components of, for
example, complement (gene C3L), interferon (B8R)
or interleukins (B16R), or which inhibit inflamma-
tion (B13/14R). Other products determine host
range and control apoptosis (gene CHO) or promo-
te cell proliferation (C11L). The febrile response to
infection is mediated by interleukin 1 and is in-
hibited by a virus-coded gene product (B16R) which
binds this cytokine. The dissemination of naturally-
released virions (EEV) is an important aspect of
pathogenesis and genes have been identified (e.g.
B5R) which coded for proteins necessary for the
proper development of EEV and expression of viru-
lence.
Figure 15.2 Thin sections of Vaccinia virus. (a) Virion within SMALLPOX
cell, invested with double membrane; (b) extracellular virion
showing outer envelope, partly detached. D : double mem-
In 1980, the WHO General Assembly accepted the
brane; E : outer envelope; I : inner membrane; L : lateral
body; N : nucleoid or core. (Bar : 100 nm) conclusion of an independent commission that
smallpox had been eradicated; destruction of the
remaining virus stocks, scheduled for 1999, has been
and orthopoxviruses will produce pocks on the
delayed. However, the importance of smallpox
chorioallantoic membrane (CAM) of 12-day-old
should not be forgotten. It had a measurable impact
chick embryos. Methods for isolation and identifi-
on the development of civilization, was the first
cation of individual virus species have recently been
virus disease for which vaccination and chemo-
reviewed (Eposito and Massung, 1995; Meyer et al.,
prophylaxis became available, and the first to be
1997, 1998).
eradicated. It is mentioned here only to emphasize
the importance of surveillance—containment rather
than mass vaccination in the eradication campaign.
Poxvirus Gene Products and Authoritative information on all aspects of small-
pox is available in the comprehensive and eminent-
Pathogenesis
ly readable account by Fenner et al. (1988).
There has always been interest in the ‘virulence’ (i.e.
safety) of Vaccinia virus, particularly given its close
relationship to Smallpox virus. Although some em- MONKEYPOX
pirical studies showed that, for example, TK\ mu-
tants were attenuated for some hosts, it was appar- Monkeypox virus was first isolated in 1958 from
POXVIRUSES 455
Figure 15.3 Representation of the genome of Copenhagen vaccinia showing HindIII restriction fragments labelled A—P according to
decreasing size. Inverted terminal repeats (boxes) and two large non-essential regions (horizontal bars) are indicated. (Redrawn from
Baxby (1998). Reproduced by permission of Edward Arnold (Publishers) Ltd)
Diagnosis
Epidemiology and Control (Douglas and Dumbell, 1992) and the results of
studies on strains isolated during 1996—1997 are
Management of individual cases will be supportive, awaited with interest.
with case-to-case spread reduced by isolation and, if Laboratory workers studying Monkeypox virus
available, the use of smallpox vaccine for contacts. should be vaccinated with vaccinia and handle the
virus in safety cabinets. However, the risk of infec-
tion other than by accidental inoculation is prob-
A Misnamed African Disease of Squirrels ably very low.
Monkeypox virus was so named because it was first
detected in captive Asiatic monkeys. However the
virus has only been found naturally in Àfrica and
evidence points to squirrels (Funisciurus spp., VACCINIA
Heliosciurus spp.) as important reservoir hosts. Sur-
veys in Zaire detected monkeypox-specific antibo- Vaccination
dies in 85 of 347 (25%) squirrels sampled but from
none of 233 terrestrial rodents. Monkeypox-specific
Smallpox vaccination, although an efficient pro-
antibody has been detected in very few monkeys,
phylactic against smallpox, was not without well-
which, like humans are probably only occasional
documented risks. These ranged from rare but se-
hosts (Khodakevich et al., 1988).
vere complications, such as generalized vaccinia
which occurred in about 200 per million primary
Control vaccinees, to relatively mild but still troublesome
satellite lesions or nondescript rashes which occur-
Human cases occur in villages in the rain forests red in about 8% of vaccinees (Baxby, 1993). Routine
where a variety of animals are captured for food. use of vaccine, except perhaps for certain military
Infection of children might be explained by their personnel, is now discontinued. It is necessary for
playing with carcases. The results of the compre- those working with Monkeypox virus, but policies
hensive surveys carried out in the 1980s indicated about its use for those working with Cowpox virus
that those infected were principally unvaccinated vary (Baxby, 1993).
children and that case-to-case spread was unusual.
Control measures are based in interposing a buffer
zone of cleared land between the arboreal reservoir
and cultivated land, the development of animal hus-
bandry as a source of meat, and on education in the Buffalopox
handling of wildlife, with emphasis on any trapping
done by those previously vaccinated; continued Attention has been drawn to ‘buffalopox’ in buf-
vaccination was not thought necessary (Khodakev- faloes and their handlers, particularly in India.
ish et al., 1988). Some outbreaks have certainly been caused by typi-
Only occasional human cases were reported cal strains of Vaccinia virus. However studies on
thereafter but there was a resurgence in 1996—1997 isolates obtained after the cessation of routine vac-
which has not yet been fully explained. Increased cination suggest that cases continue to be caused by
political unrest would lead to population displace- a virus different from vaccinia ((Dumbell and
ment and breakdown of routine control measures, Richardson, 1993). These isolates, unlike vaccinia,
and the levels of vaccine-induced immunity will do not produce pocks on the CAM above 39°C.
decline with time. A potentially serious finding Their DNA is sufficiently-similar to that of vaccinia
which requires clarification is the observation that virus to regard them as variants or subspecies of it
case-to-case transmission occurred more frequently (Dumbell and Richardson, 1993; Esposito et al.,
during 1996—1997 than earlier (Heymann et al., 1995). However, at present it is not known whether
1998). Comparison of the genomes of smallpox vi- buffaloes are the reservoir host or whether wildlife
rus and monkeypox virus strains isolated up to reservoirs are involved as in the case of cowpox (see
1986 suggested they have evolved separately below).
458 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 15.6 Human cowpox. (a) Primary and secondary lesions; the former at the early eschar stage, the latter at the early vesicular
stage; (b) at the late vesicular—early pustular stage showing a haemorrhagic lesion with marked oedema and erythema. (a) Reproduced
from Baxby et al. (1994) by permission of Blackwell Science Ltd. (b) From Baxby, D. (1982) The natural history of cowpox. Bristol
Medico-Chirurgical Journal, 97, 12; reproduced by permission of the Editor)
‘Foreign’ genes have been inserted into poxviruses, Most information is available about cowpox in the
particularly vaccinia. The resultant recombinant domestic cat, an accidental host in which a relative-
strains retain their infectivity and, in general, the ly mild generalized disease occurs following pri-
inserted genes are expressed properly. Thus, the use mary infection via a bite or scratch (Bennett et al.,
of such recombinants as vaccines has been ad- 1990).
vocated. Most work has been done on vaccinia as a Human infection, similarly acquired, usually re-
vector, and vaccinia recombinant vaccines have mains localized and is characterized by a marked
been used to control wildlife rabies in Europe (Pas- inflammatory and erythematous response (Baxby et
toret and Brochier, 1996). However, there is still al., 1994). The bulk of the lesion is caused by hyper-
concern about the use of vaccinia virus as a vector, trophy and proliferation of the basal cell layer of the
despite the development of attenuated strains epidermis, together with massive inflammatory in-
(Smith et al., 1997). Other poxviruses are being filtration. Infection usually spreads into follicles and
considered as vaccine vectors; in particular the use typical A-type inclusions are usually seen (Figure
of avian poxviruses as vectors for mammalian vac- 15.4). By analogy with smallpox vaccination a tran-
cines. These are of interest because avian poxviruses sient viraemia might be expected.
induce a good immune response to the foreign gene
product without initiating productive infection in
mammals (Limbach and Paoletti, 1996).
Clinical Features
Diagnosis
Clinical
The main drawback to clinical diagnosis of cowpox
is its rarity and the failure to appreciate important
epidemiological information (see below). Cowpox,
which is restricted geographically (Table 15.1),
should be considered in anyone, including young
children, presenting with a painful haemorrhagic
lesion or black eschar; with or without erythema
and oedema; accompanied by lymphadenopathy
and a systemic ‘flu-like’ illness. This applies particu-
larly to patients seen in July—October and/or who
have had contact with cats (see below). Attempts to
establish contact with cattle are usually counter-
productive. Differential diagnoses include parapox-
virus (see below), herpes and anthrax. Colour illus-
trations of all these lesions are available (Baxby et
al., 1994), which, together with a properly taken
history, should help clinical diagnosis. Unfortu-
nately the rarity of human cowpox means that gen-
eral practitioners or even consultants seldom have
an opportunity to investigate a second case.
Laboratory
Figure 15.7 Parapoxvirus infection showing lesions in different Electron microscopy of vesicle fluid or extracts of
patients. (a) At the ‘target’ stage; (b) an ulcerated granulomatous crusts is particularly valuable because it will differ-
lesion which could be misdiagnosed as a malignancy; (c) crusting entiate between parapox, herpes and presumptive
lesion. ((a) From Baxby, D. (1982) The natural history of cowpox.
cowpox infections. Of 24 cases of cowpox where
Bristol Medico-Chirurgical Journal, 97, 12; reproduced by per-
mission of the Editor. (b) Kindly supplied by Dr M.S. Lewis- adequate material was available, electron micro-
Jones. (c) Reproduced from Baxby, D. et al. (1994) by permission scopy was successful in 23.
of Blackwell Science Ltd) If necessary, virus may be isolated on the CAM,
where the production of intensely haemorrhagic
pocks is diagnostic. Cytopathic effect occurs in
black crust (Figure 15.6a). The lesion is usually very many cell lines (Vero, MRC-5, RK13) and detection
painful and erythema and oedema are common at of A-type inclusions is diagnostic, as it would be if
the late vesicular and pustular stages (Figure 15.6b). found in biopsy material. Not all strains of cowpox
There is usually lymphadenitis, fever and general are identical and genome analysis may show dif-
malaise, often referred to as ‘flu-like’. These features ferences of epidemiological value.
460 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Laboratory
Pathogenesis
Virions with the characteristic morphology of para-
poxviruses are usually easily seen in lesion extracts, After a variable, sometimes lengthy, incubation per-
and this provides a rapid, certain diagnosis. The iod, papules develop, formed by epidermal hyper-
virus can be grown in cell culture but this is not trophy. This produces a nodule and also extends the
attempted routinely. dermis downwards, but the basement membrane
usually remains intact. Characteristic inclusions
(Henderson—Patterson bodies) are formed in the
Epidemiology prickle cell layer and gradually enlarge as the cells
age and migrate to the surface. These cells are re-
Human infection is an occupational hazard of farm- placed by hyperplasia of the basal cell layer. The
workers, abattoir workers, veterinary surgeons and inclusion is a well-defined sac packed with virions
students, etc. It is most common in the lambing and (Shelly and Burmeister, 1986). The lesion is circum-
calving seasons, and more commonly reported in scribed by a connective tissue capsule and the der-
sheepworkers than cattleworkers; this probably re- mis, apart from distortion, remains essentially nor-
flects differences in animal husbandry. Of 191 cases mal. Occasionally an inflammatory infiltration of
with a known source surveyed during 1978—1995, the dermis may occur (Brown et al., 1981).
462 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Epidemiology
Diagnosis TANAPOX
The appearance of lesions in normal cases is gen- Human infection with Tanapox virus was first rec-
erally sufficiently characteristic to permit clinical ognized in the Lake Tana area of Kenya in 1957,
diagnosis. Virions can usually be seen in large and particular attention was paid to it during
numbers if material expressed from the lesion is posteradication smallpox surveillance. An account
examined by electron microscopy. The lack of a of 264 labortory-confirmed cases from Zaire
marked inflammatory response and failure to iso- (Congo Republic), with colour illustrations, is avail-
late an agent in cell culture or CAM should elimin- able (Jezek et al., 1985), as is information on the
ate other poxvirus infections. virus itself (Knight et al., 1989).
POXVIRUSES 463
usually erythema and oedema and lym-
phadenopathy is common. The lesions generally
disappear within 6 weeks. Most (78%) patients have
only one lesion, and very few have more than two.
They may occur on any exposed area but the head
tends to be spared.
Diagnosis
16
Alphaviruses
Nicola S. Brink
Royal Free and University College Medical School, London, UK
and
Graham Lloyd
Centre for Applied Microbiology and Research, Porton Down, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
468 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ture alphavirus particles. In addition, a small
glycoprotein termed E3 has been demonstrated in
Semliki Forest virus (Garoff et al., 1974). A further
small 6K polypeptide is also encoded, but has not
been demonstrated in any alphavirus particles.
REPLICATION
The nucleic acid of the alphaviruses consists of a The alphaviruses have a group-reactive nucleo-
single-stranded (ss) positive-sense RNA, almost protein. Complex and type-specific reactivity is de-
12 000 nucleotides in length, which is capped at the termined by the envelope glycoproteins. This anti-
5 end and polyadenylated at the 3 end (Figure genic comparison of alphaviruses has been conduc-
16.2). The naked genome is infectious (Strauss and ted using haemagglutination-inhibition (HI) and
Strauss, 1986). The sedimentation coefficient has complement fixation tests (CF) as well as cross-
protection tests in mice and neutralization tests in
been reported to be 42—49. The genome is divided
cell culture (Calisher et al., 1980). The E1 glyco9
into two regions. The 5 two-thirds of the viral
genome codes for the non-structural proteins and protein of Sindbis contains the antigen responsible
the 3 third encodes the structural proteins (Straus for haemagglutination (Chanas et al., 1982). It is
et al., 1984). Both the structural and non-structural assumed that this is also the case for other members
proteins are translated as polyprotein precursors. of the genus Alphavirus. The E2 glycoprotein indu-
The non-structural polyprotein precursor is cleaved ces virus-specific neutralizing antibody. On the
into four non-structural proteins, NSP1, NSP2, basis of this, alphaviruses have been divided into six
NSP3 and NSP4. These function as the replicase/ antigenic complexes: Western equine encephalitis
transcriptase of the virus (Strauss and Strauss, (WEE), Venezuelan equine encephalitis (VEE),
1986). The structural polyprotein precursor is Eastern equine encephalitis (EEE), Semliki Forest
cleaved to form three major polypeptides. The cap- (SF), Middleburg and Ndumu viruses (Peters and
Dalrymple, 1990). A further virus Barmah Forest
sid protein C (30—40 kDa) and the envelope glyco-
virus has been shown to be biochemically typical of
proteins E1 and E2 (45—59 kDa) are found in ma-
the genus, but is not related serologically to mem-
ALPHAVIRUSES 469
Figure 16.2 Organization of the alpha-
virus genome. The 5 two-thirds of the viral
genome encode the non-structural pro-
teins, NSP1, NSP2, NSP3 and NSP4. The
3 third encodes the structural proteins.
The capsid and envelop glycoproteins (E1
and E2) are present in mature alphavirus
particles. A third glycoprotein, E3, has
been demonstrated in purified SFV. The
6K polypeptide is not known to be a struc-
tural protein
bers of the six antigenic complexes (Table 16.1). ogy is to be expected as WEE is thought to be a
Each complex consists of either a single virus spe- recombinant virus which derives its non-structural
cies having no known close relatives, for example genes from an EEE virus ancestor (Pfeffer et al.,
EEE, VEE, Middleburg and Ndumu viruses, or 1997).
several species, subtypes and varieties that are more
closely related to each other than the other mem-
bers of the genus, for example Semliki Forest virus
and WEE virus. These variants can often only be SPECTRUM OF DISEASE CAUSED BY
distinguished by plaque reduction or kinetic HI ALPHAVIRUSES
tests. Kinetic HI tests have also been used to differ-
entiate between geographical variants of EEE virus Alphaviruses cause a wide range of disease in ani-
(Casals, 1964). Studies utilizing monoclonal antibo- mals and humans, ranging from a clinically inap-
dies have been able to define the alphavirus anti- parent infection to a severe disease that may result
genic cross-reactivities more precisely and map the in death. The main target organs are muscle, brain,
antigenic determinants. reticuloendothelial system and the joints. This
A recent study sequenced one strain of each al- group of viruses may be clinically divided into those
phavirus species and this allowed for the classifica- that are associated with fever, rash and polyarthritis
tion of alphaviruses into six genotypes (as com- or those that are associated primarily with encepha-
pared to seven antigenic complexes): Sindbis, litis (Table 16.2). Middleburg and Ndumu viruses are
Ndumu, VEE, WEE, Barmah Forest and Semliki not known to cause disease in humans. Some alpha-
Forest viruses. The authors included EEE and viruses are also responsible for disease in animals,
WEE in the same genotypic complex; this homol- particularly Equidae.
470 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 16.2 Range of human illness caused by alphaviruses to virus isolation in the diagnosis of an acute infec-
tion (Pfeffer et al., 1997).
Clinical features Virus
Detection of an appropriate specific antibody re-
Fever, rash Sindbis, Ockelbo sponse is frequently used to diagnose alphavirus
Fever, rash, Ross River, Barmah Forest infections. Class-specific IgM assays have been de-
polyarthritis veloped to diagnose an acute or recent infection
Fever, rash, Chikungunya, O’nyong-nyong, Mayaro, with a particular alphavirus. However, care must be
myalgia, Sindbis, Ockelbo Ross River, Barmah Forest, taken in the interpretation of the result as cross-
arthralgia Igo Ora reactions may occur with other members of the
Fever, Eastern equine encephalitis same alphavirus antigenic complex (Table 16.1)
encephalitis Western equine encephalitis (Calisher et al., 1986). A serological diagnosis of a
Venezuelan equine encephalitis recent alphavirus infection may also be made by
Everglades, Semliki Forest
demonstrating a rise in specific antibodies when
acute and convalescent serum samples are tested in
parallel. This is usually done using HI, CF or neu-
DIAGNOSIS OF ALPHAVIRUS tralization tests.
INFECTIONS
The clinical features in a patient living or having
recently travelled to an appropriate geographical MANAGEMENT AND PREVENTION
region provide an important diagnostic clue. How- Management of alphavirus infections is usually
ever, differentiation from other viral infections may supportive and directed at symptomatic relief, for
be difficult. For example, it is sometimes difficult to example the symptomatic relief of joint symptoms
differentiate infection with Ross River virus from with non-steroidal anti-inflammatory agents and
infections with rubella, parvovirus B19 or en- aspirin. Prevention of alphavirus infections has fo-
terovirus infections. Laboratory confirmation of in- cused on vector control and the protection of the
fection may therefore be required. individual against mosquito bites. A detailed
Laboratory diagnosis includes viral culture, the knowledge of the vector and vertebrate host range
detection of a specific antibody response and, more is therefore essential to allow for a coordinated
recently, the use of molecular techniques to detect strategy to prevent and control alphavirus infec-
viral genome. In general, virus isolation is often tions. Vaccines are also available against some of
only successful when acute-phase antibody-nega- the individual alphaviruses infections (see below).
tive serum samples are used, particularly when the
serum sample is taken in the first 48 hours of illness.
Thereafter the amount of virus in serum drops
rapidly and recovery becomes increasingly difficult. ALPHAVIRUSES ASSOCIATED WITH
Virus identification is facilitated by the use of FEVERS AND POLYARTHRITIS
murine monoclonal alphaviruses-specific antibo-
Sindbis Virus
dies. Virus may be isolated by intracerebral inocula-
tion of baby mice as well as in a variety of cell
culture systems, including monkey kidney (Vero) Sindbis virus is the prototype of the alphaviruses. It
and mosquito (C6/36) cell lines. More recently was originally isolated from Culex mosquitoes col-
methods have been developed to diagnose alpha- lected in the Egyptian village of Sindbis (Taylor et
virus infections using genomic detection methods. al., 1955). In Sweden symptomatic disease resulting
As these are RNA viruses any polymerase chain from infection with a Sindbis-like agent has been
reaction (PCR)-based assay must first transcribe the termed Ockelbo disease. A Sindbis-related virus has
RNA into DNA. A reverse transcription-poly- also been described as the cause of Pogosta disease
merase chain reaction (RT-PCR) has been develop- in Finland and as Karelian fever in the Karelian
ed for the genus-specific detection of alphaviruses. region of the former USSR. Babanki virus is a Sind-
This utilizes degenerate primers localized within a bis-like agent that occurs in West and Central Afri-
ca; the clinical significance of infection with this
conserved region of the non-structural protein 1
virus remains to be defined (Peters and Dalrymple,
and is likely to be a sensitive and rapid alternative
1990).
ALPHAVIRUSES 471
Epidemiology and Host Range ment less and have suggested that the involvement
is of tendon and periarticular tissue, rather than a
Sindbis virus occurs in many parts of the world,
true arthritis (McIntosh et al., 1964).
including Europe, Asia, Africa and Australia. The
virus is known to infect humans, domestic animals
and birds, with birds forming the principal reser- Pathogenesis
voir. Humans are not considered to be essential to
the survival of the virus in nature. Sindbis virus is It is not clear whether the skin lesions are the result
transmitted among birds by the Culex mosquitoes. of a direct viral cytopathic effect or are im-
Infection in birds does not appear to result in dis- munopathological. Evidence to support the former
ease. Where humans and birds exist in close prox- hypothesis comes from Malherbe and Strickland-
imity, for example in the Nile Valley, transmission Cholmley (1963) who demonstrated the isolation of
to humans may occur (Peters and Dalrymple, 1990). Sindbis virus from a vesicle in the absence of a serum
Studies from South Africa have shown that the viraemia. This was associated with a subsequent
virus is distributed widely throughout the country. rise in specific antibody. Other workers have, how-
Transmission to the avian population and probably ever, failed to isolate Sindbis virus from skin lesions
to humans occurs annually. Extensive human dis- (McIntosh et al., 1964). The pathogenesis of the
ease only occurs, however, during years of abun- joint complications, and whether they are articular
dant rainfall and flooding (McIntosh et al., 1964; or periarticular, is also unclear.
Jupp et al., 1986). In Europe, disease caused by
Sindbis virus occurs after excursions into forested Diagnosis
areas by, for example, lumberjacks or berry pickers.
Here the virus has been isolated from Culiseta, Isolation of Sindbis virus from human sera has been
Aedes and Culex mosquitoes. reported, but this is frequently not successful, even
using sera taken early on in the illness. This is
because the viraemia associated with Sindbis virus
Clinical Features infection is often of low level and transient. In addi-
tion, medical attention is frequently sought relative-
Sindbis virus is among the least virulent of the al- ly late in the course of the illness. As mentioned
phaviruses. Serological surveys suggest that infec- above, Sindbis virus has also been isolated from a
tion with Sindbis virus is relatively common, but skin lesion. When inoculated into 3—5-day-old mice,
clinically apparent disease is unusual. When symp- either intracerebrally or intraperitoneally, Sindbis
tomatic, the spectrum of disease in humans varies virus causes a fatal infection which is preceded by a
from a mild illness to one consisting of a rash and short period of paralysis. The virus has also been
arthralgia. The illness is usually characterized by a isolated in vitro in fibroblastic cell lines. Antibodies
sudden onset of the rash, but occasionally prod- appear 7—10 days after the onset of illness and may
romal symptoms may be present. The rash is be detected by HI, CF and immunofluorescence
usually macular or papular but may become vesicu- (IF) (McIntosh et al., 1964; Doherty et al., 1969;
lar or haemorrhagic. Malaise, fatigue and headache Espmark and Niklasson, 1984). Although the detec-
are frequently present (McIntosh et al., 1964; Jupp tion of Sindbis-specific IgM may be of use in diag-
et al., 1986). The initial joint involvement is usually nosis an acute infection, this class of antibody may
migratory, followed by a gradual resolution of remain detectable 3—4 years after the initial infec-
symptoms. tion. A positive IgM result must therefore be inter-
Ockelbo disease was first described in the central preted together with the available clinical informa-
part of Sweden in the 1960s in clusters of patients tion.
with fever, arthralgia and a rash (Niklasson and
Vene, 1996). Some patients with Ockelbo disease
have reported joint symptoms persisting for more Ross River Virus
than a year and reccurrent joint problems were
documented in one third of a group of Swedish Infection with Ross River virus (RRV), also known
patients interviewed 2 or more years post-infection as epidemic polyarthritis, is a major public health
(Niklasson et al., 1988). By contrast reports from problem in Australia. It is the most common ar-
South Africa have emphasized the joint involve- boviral infection of humans in Australia. In the late
472 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 16.3 Geographical distribution of the Semliki Forest that humans are the most important vertebrate host
virus complex in this region. A study from the Cook Islands
Species Subtype Geographical distribution showed that the incubation period could be as short
as 3 days and the explosive nature of this epidemic
Semliki Forest Semliki ForestAfrica led to speculation that mechanical transmission of
Chikungunya Chikungunya Tropical Africa, South & RRV may occur. This theory has been supported by
South-East Asia,
Philippines laboratory evidence of mechanical transmission of
Igbo Ora West & Central Africa RRV from viraemic donors to uninfected recipient
O’nyong-nyong East & West Africa, suckling mice. In addition, it has also been sugges-
Zimbabwe ted that transovarial transmission in mosquitoes is
Mayaro Mayaro South America a potential survival mechanism for this virus (Kay,
Una South and Central America 1982).
Getah Getah Malaysia, Japan, Australia,
Cambodia, South Pacific
Ross River Australia, South Pacific
Bebaru Malaysia Clinical Features
Sagiyama Japan, Okinawa
Infection with RRV does not necessarily result in
disease in humans and it has been estimated that 50
1970s and early 1980s RRV also became endemic in subclinical infections occur for each clinically rec-
the Pacific Islands (Table 16.3). In recent years it has ognized case. Adults are more likely to be sympto-
also been identified in travellers returning to matic than children. The onset of clinical symptoms
Europe. is characterized by joint pains (epidemic polyarthri-
tis) accompanied by a rash and fever. In a recent
postal survey of general practitioners in south Aus-
Epidemiology and Host Range
tralia the complaint of ‘pain in the joints’ was the
Until 1979 the distribution of RRV infections was most important symptom for suspecting an infec-
thought to be limited to Australia, New Guinea and tion with RRV, with joint effusion, rash and pyrexia
the Solomon Islands, all in the western half of the being important signs (Stocks et al., 1997). The rash
Pacific basin. In Australia both epidemic and spor- may occur simultaneously with the joint pains or
adic transmission of RRV occurs. Epidemics are may follow 1—2 days later. Although the rash is
characteristically preceded by heavy rainfall and usually macular, maculopapular or papular, ves-
are located in river valleys and irrigated lands in iculation and petechiae have been described. The
coastal areas. However, other factors such as tem- rash and joint pains are commonly accompanied by
perature, are important—a sudden period of cold some degree of constitutional upset, including
can delay the build-up of the vector and so abort an myalgia, headache, anorexia and nausea. The joint
epidemic (Hawkes et al., 1985). The virus was first involvement is usually asymmetrical and migratory
isolated from Aedes vigilax, which is accepted as and most often affects the small joints of the hands
being the major vector in the coastal regions of and feet together with the knees. In addition, there
Australia. Other mosquito species, including Aedes may also be periarticular swelling and a tenosynovi-
comptorhynchus and Culex annulirostris, are also tis (Clarke et al., 1973; Hawkes et al., 1985). The
thought to be important vectors (Peters and Dal- arthralgia and arthritis tends to run a relapsing
rymple, 1990). Besides humans, other possible ver- course but with an overall gradual improvement.
tebrate hosts include marsupials, domestic animals The symptoms of epidemic polyarthritis may last
and rodents (Doherty, 1977). for 30—40 weeks, with some patients having symp-
In 1979 an explosive epidemic of RRV infection toms for more than a year. While joint pain, rash
occured in Fiji (Aaskov et al., 1981). The virus and fever are the most common clinical features,
spread to American Samoa and then to the Wallis glomerulonephritis has also been reported in the
and Furuna Islands and the Cook Islands. The acute phase of RRV disease (Fraser et al., 1988).
mosquito vector in this region is unknown although This case report describes a patient presenting with
the virus has been isolated from Aedes polynesiensis, haematuria and proteinuria coincident with an
a mosquito that is found in many parts of the East- acute RRV infection; the frequency of this compli-
ern Pacific (Rosen et al., 1981). Evidence suggests cation has yet to be defined.
ALPHAVIRUSES 473
Pathogenesis used to detect RRV-specific IgM antibodies, includ-
ing indirect IF and an IgM-capture ELISA. IgM
The pathogenesis of disease is not completely
antibody cross-reacts with Mayaro, Chikungunya
understood. While virus has been isolated from the
and Semliki Forest viruses in patients with RRV
blood, it has not been isolated from either the skin
infections (Table 16.3). However, ratios of
or joints. However, RRV antigen has been detected
homologous to heterologous IgM titres are usually
by immunofluorescence at both of these sites. His-
high and so, although this cross-reaction somewhat
tological examination of the skin lesions shows a
limits the diagnostic usefulness of this test, it can
mononuclear cell infiltration (Fraser et al., 1983). It
provide a reasonably specific and rapid diagnosis in
has also been shown that RRV is capable of infect-
an epidemic situation (Calisher et al., 1986). A fur-
ing human synovial cells (Cunningham and Fraser,
ther complicating factor is the persistence of the
1985). Serum complement is normal and circulating
IgM response; it has been shown that the IgM
immune complexes do not usually exceed normal
reactivity may persist for 1—2 years after the acute
levels. It has therefore been suggested that RRV
infection (Carter et al., 1985). The presence of RRV-
plays a direct role at these sites of inflammation.
specific IgA or low avidity IgG may help in the
diagnosis of a recent infection (Carter et al., 1987).
Diagnosis
Isolation of RRV is only successful using acute- Vaccine
phase antibody-negative serum samples. Both mos-
quitoes and mosquito cell lines have been used. The An inactivated RRV vaccine is currently being de-
current method of choice for RRV isolation is the veloped. Preliminary studies have shown that this
inoculation of the mosquito cell line, C6/36. As this candidate vaccine neutralized all strains of RRV
virus does not display a cytopathic effect in C6/36 tested, but to different degrees (Aaskov et al., 1997).
cells, viral antigen is detected using IF (Rosen et al., Further studies are awaited with interest as, while
1981). RRV may also be isolated in Vero (E6) cells. RRV does not usually cause a severe disease, it may
More recently methods have been developed to result in considerable morbidity with an associated
detect RRV RNA using RT-PCR. This methodol- loss of productivity.
ogy has potential application to virus surveillance
programmes in mosquito populations (Sellner et al.,
1994). Barmah Forest Virus
While virus may be isolated in an appropriate cell
culture system, an acute infection is frequently con- This alphavirus has been recently reported to cause
firmed serologically. This may be done using the both clinical and subclinical infections in Australia.
classical serological methods and HI and neutraliz- Clinical disease resulting from BFV infection was
ation. The development of HI antibodies differs in first reported in 1986. The first epidemic of human
patients from Australia when compared to those disease occurred in 1992 in the Northern Territory
from the Pacific Islands. In the Australian cases (Mackenzie et al., 1994). To date, Australia is the
described most patients have detectable HI antibo- only country in which this virus has been detected
dies in the first week of illness (Clarke et al., 1973). In (Hills, 1996).
contrast, HI antibodies appear to become detect- Barmah Forest virus (BFV) is the sole member of
able later in patients from the Pacific Islands, the seventh alphavirus serocomplex (Table 16.1).
usually only after the first week of symptoms (Rosen The nucleotide sequence of the BFV genome has
et al., 1981). The reason for this difference is not recently been described and, from amino acid se-
known. CF antibodies appear later and a serocon- quence comparisons with sequenced alphaviruses,
version may be demonstrated using acute and con- BFV has been shown to be most closely related to
valescent serum samples. This is not, however, the RRV and Semliki Forest virus (Lee et al., 1997). In
method of choice as cross-reactivity has been ob- recent years there has been an apparent increase in
served with antibodies produced during other al- incidence of polyarthritis caused by BFV. This
phavirus infections. A more precise diagnosis can be prompted a study on the molecular epidemiology of
made using neutralization tests (Kanamitsu et al., this alphavirus which showed a high degree of se-
1979). More recently a variety of methods have been quence homology between BFV isolates with no
474 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
evidence of geographical or temporal divergence serological response to infection with this virus in
(Poidinger et al., 1997). humans remains to be defined precisely. However,
in contrast to RRV infections, where the antibody
response may be delayed for 7 days, a recent study
Epidemiology and Host Range
demonstrated the presence of both HI and neu-
Barmah Forest virus has been isolated from the two tralizing antibody titres in two patients with BFV
most common vectors of RRV—Culex annulirostris infection at the onset of symptoms (Phillips et al.,
and Aedes vigilax. Occupational and recreational 1990). This could either be due to a different
exposure to these vectors is therefore an important serological response to these two viruses or, alterna-
risk factor for infection with this virus. A higher tively, BFV-associated disease may have a less
incidence of antibodies to BFV in RRV antibody- clearly defined onset than that caused by RRV in-
positive blood donors compared to RRV antibody- fections.
negative blood donors suggests a common ecology.
Further information is required on the duration of
viraemia in humans to determine whether other Chikungunya Virus
vertebrate hosts are of importance in viral mainte-
nance. This virus was first isolated from patients and mos-
quitoes in the Newala district of Tanzania in
1952—1953. The name is derived from a local term
Clinical Features for the illness and means ‘that which bends up’,
As mentioned above, both clinical and subclinical referring to the crippling arthralgia and arthritis
infections occur. The most common symptoms associated with infection with this virus. Chikun-
noted in a study from Queensland were arthritis/ gunya virus is found in the savannahs and forests of
arthralgia, myalgia and fever, which were present in tropical Africa as well as in many parts of Asia,
approximately 75% of patients. In addition, half the including Thailand, Cambodia, Vietnam, Burma,
patients in this study had a rash (Phillips et al., Sri Lanka and India (Table 16.3). The chikungunya
1990). A more recent study from New South Wales strains from Africa and Asia have been shown to be
reported lethargy (89%), joint pain (82%) and rash closely related, using a panel of monoclonal antibo-
(68%) as the most common symptoms. In the latter dies prepared against strains from Africa and Asia
study just over half of the patients reported an (Blackburn et al., 1995).
illness lasting for more than 6 months. These
authors also reported a possible association be-
Epidemiology and Host Range
tween the presence of a rash and an improved prog-
nosis (Beard et al., 1997). In general, the symptoms The epidemiology of chikungunya virus infections
are similar to those associated with RRV infection; differs in Africa and Asia. In Africa this virus is
laboratory confirmation is therefore necessary to transmitted by Aedes mosquitoes. The most im-
achieve a precise diagnosis. The wider availability portant vertebrate in the cycle of infection is the
of serological testing for BFV infections has also non-human primate, for example baboons and Cer-
enabled their more unusual features to be described. copithecus monkeys. Bushbabies and certain species
Recently the first case of glomerulonephritis after of bats may also be infected in nature, but their role
BFV infection was diagnosed. The authors suggest in viral maintenance is likely to be of secondary
that BFV infection should be considered as a poss- importance. Humans may be infected in African
ible aetiological agent for glomerulonephritis (Katz villages and rural areas, particularly where Aedes
et al., 1997). aegypti is present in large numbers. In contrast to
the situation in Africa, transmission in Asia is pri-
marily from human to human by Aedes aegypti. It
Diagnosis
has been suggested that infection with this virus
Clinical differentiation from RRV infection in pa- may be common in certain parts of Africa. For
tients living in endemic areas is difficult. Laboratory example, 47% of sera collected from 132 adults
confirmation is therefore necessary for a precise living in the Karamoja district of Uganda had de-
diagnosis to be made. BFV has been isolated from a tectable antibodies to Chikungunya virus by HI
patient in the mosquito cell line, C6/36. The (Rodhain et al., 1989).
ALPHAVIRUSES 475
Although the most important vector for chikun- Pathogenesis
gunya virus transmission to humans is the Aedes
Chikungunya virus may be isolated from the blood
mosquito, transmission has also been described by
early in the course of disease. As the disappearance
Mansonia africana. The active replication of alpha-
of the viraemia correlates with the apperance of HI
viruses in the mosquito is essential for perpetuation
and neutralizing antibodies, it would appear that
in nature, but the explosive nature of the chikun-
recovery from infection correlates with the develop-
gunya epidemics has led to speculation that mech-
ment of viral-specific antibody rather than cell-me-
anical transmission of the virus may also occur from
diated immunity. This is further substantiated by
a viraemic host by an uninfected mosquito (Peters
the effect of serum antibody in mouse protection
and Dalrymple, 1990).
tests (Carey et al., 1969). The haemostatic abnor-
mality seen in both children and adult patients is
Clinical Features possibly the result of an acquired platelet defect as it
has been shown that the association of Chikungunya
The incubation period varies but is usually 2—4
virus with human platelets in vitro promotes platelet
days. In adults there is an abrupt onset of fever,
clumping (Chernesky and Larke, 1977).
headache and severe joint pains without prodromal
symptoms. The joint pains are the dominant com-
plaint and affect mainly the small joints of the
Diagnosis
hands, wrist and feet. This may be associated with
an erythematous flush over the face and upper chest The clinical features described above in a patient
in approximately 80% of patients. A maculopapu- who has recently returned from, or is living in,
lar rash together with a generalized lympha- sub-Saharan Africa or Asia enables a presumptive
denopathy appears 3—4 days later. Although the clinical diagnosis to be made. In addition, a minor-
arthralgia may resolve within a few weeks, pain, ity of patients will have a leucopenia with a relative
swelling and morning stiffness may continue for lymphocytosis. In most, however, the white cell
months and even years after infection. Petechiae count will be normal. The platelet count may be
and bleeding from the gums does occur, but there decreased, but this is usually not significant.
are no significant haemorrhagic manifestations. Chikungunya virus can be isolated by the in-
In contrast to the clinical presentation in adults, a tracerebral inoculation of suckling mice or by viral
study from Bangkok concentrating on a paediatric culture in either mosquito or mammalian cell cul-
outpatient department showed that the most fre- ture systems (Peters and Dalrymple, 1990). Virus is
quent presenting symptom of chikungunya virus best isolated from serum samples taken early on in
infection in children was vomiting, which was pres- the illness, particularly in the first 48 hours. There-
ent in 35% of patients. In addition, 18% had ab- after the amount of virus in serum drops rapidly
dominal pain or anorexia. None of the patients in and recovery becomes increasingly difficult. Both
this study were noted to have joint symptoms; ar- HI and neutralizing antibodies appear on day 5—7
thritis and arthralgia therefore appear to be less of the illness. The appearance of these antibodies is
prominent features in children. On clinical examin- associated with a decrease in viraemia (Carey et al.,
ation of this paediatric population the most fre- 1969). HI antibodies rise rapidly and peak in the
quent sign was a pharyngitis, which was present in second week. In contrast, the CF antibody titres rise
71% of patients, followed by facial flushing (24% of more slowly. A class-specific IgM assay to diagnose
patients). None of these children had a rash. This a recent or acute chikungunya virus infection has
study illustrates very clearly that the clinical presen- been described. However, cross-reaction occurs
tation of chikungunya infections in adults and with other members of the same alphavirus anti-
children differs (Halstead et al., 1969). genic complex, so the results obtained must be in-
Haemorrhagic forms of chikungunya virus infec- terpreted with caution (Table 16.1) (Calisher et al.,
tion have been described in India and southeast 1986). The use of a specific IgM assay may be es-
Asia, but these are seldom as severe as dengue pecially relevant in an epidemic situation. This was
haemorrhagic fever. In a hospital-based study in illustrated in an outbreak in Yangon in Burma.
Bangkok about 8% of children with suspected Utilization of a simple indirect ELISA was helpful
haemorrhagic fever had chikungunya virus infec- in achieving an acute-phase diagnosis and results
tion (Nimmannitya et al., 1969). showed a strong concordance with HI (90% agree-
476 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ment), although ELISA was able to identify twice as Clinical Features
many patients at initial hospital admission (Thein et
The clinical features are similar to those of chikun-
al., 1992). The differential diagnosis within the lab-
gunya virus infections, with a sudden onset of joint
oratory should include dengue, Sindbis and West
pains followed by the development of a rash.
Nile virus infections.
Diagnosis
Vaccine
Virus may be isolated in one day old mice from
A formalin-inactivated chikungunya virus vaccine serum samples taken during the acute stage of the
prepared in monkey kidney tissue culture has been infection. However, mouse passage or subinocula-
described. Despite being apparently safe and im- tion into chick fibroblasts may be necessary. The
munogenic, its use has been limited to initial safety mice demonstrate alopecia and runting. The virus
studies and laboratory workers (Harrison et al., may then be further identified using plaque inhibi-
1971). More recently a live-attenuated vaccine has tion, cross-neutralization assays or HI (Williams
been produced. and Woodall, 1961.) Antibodies to O’nyong-nyong
virus may be detected by CF, HI, ELISA, IF and
neutralization assays. Interpretation may be diffi-
O’Nyong-Nyong Virus cult due to cross-reaction with Chikungunya virus.
In general, however, the antibody titre to the
O’nyong-nyong literally means ‘weakening of the homologous virus will be higher than that to the
joints’. It was first described as an epidemic viral heterologous virus (Peters and Dalrymple, 1990).
disease in East Africa (Haddow et al., 1960). Anti-
genically O’nyong-nyong virus is a subtype of the
species Chikungunya (Table 16.3). Igbo Ora Virus
same antigenic complex does not pose a diagnostic ant vertebrate host. Endemic transmission results in
problem (Calisher et al., 1986). a few human cases. In addition, major equine epi-
demics may result in a significant number of human
cases. As with other arboviruses, climatic factors
Prevention and Control exert a major influence on the epidemiology and
Protection against mosquito bites and control of distribution of WEE virus. In the eastern USA
the vector is important. An effective equine vaccine WEE virus is replaced by Highlands J virus, whose
is licensed in the USA and is recommended for primary vector is Culiseta melanura, which is also
livestock in areas where EEE virus transmission is the primary vector of EEE virus. The strictly or-
known to occur. Specific control measures to pre- nithophilic nature of this vector explains the ab-
vent human disease include a formalin-inactivated sence of significant disease outbreaks in the eastern
vaccine. However, as human disease is rare, even USA. WEE virus and Highlands J virus are closely
during an equine epizootic, this is only available to related both serologically and at a molecular level.
those considered to be at risk of exposure. Prompt
notification of suspected cases of arboviral en- Clinical Features
cephalitis to the local public health authority is also
essential. Asymptomatic infection in adults is common, with
an estimated case:infection ratio of approximately
1:1000. This decreases to approximately 1:60 in
children and 1:1 in babies (Peters and Dalrymple,
Western Equine Encephalitis Virus 1990). Most cases of encephalitis occur in children,
frequently under the age of 4 years, in whom the
Western equine encephalitis virus causes more hu- onset is marked by convulsions. This may result in
man infections than the EEE virus, but the illness is permanent neurological damage. Drowsiness,
less severe and mortality seldom exceeds 10%. Its headache and mental confusion, sometimes leading
distribution covers the Pacific coast of the USA, but to coma, may be seen in adults. In utero infection
also includes the great plains of the USA and Can- followed by an acute encephalitis has been
ada and extends into Central America and the documented (Shinefield and Townsend, 1953). Re-
northern parts of South America. The WEE anti- covery from the acute illness may be slow and
genic complex consists of six species (Table 16.4). It symptoms such as fatigability, irritability and head-
has been suggested that WEE virus arose through ache may persist for up to 2 years (Earnest et al.,
recombination between a EEE virus-like virus and 1971).
a Sindbis-like virus (Hahn et al., 1988).
mononuclear and polymorphonuclear infiltrate to- tivities may also protect from vector-borne diseases
gether with parenchymal necrosis. and are complementary to mosquito control pro-
grammes (Gahlinger et al., 1986). An inactivated
vaccine is available to those at risk.
Diagnosis
During the acute phase of the illness viral isolation
may be attempted from blood, throat swabs or a Venezuelan Equine Encephalitis Virus
cerebrospinal fluid samples using suckling mice, but
this is frequently not successful. Virus may usually As the name suggests, Venezuelan equine encephali-
be isolated from brain biopsy material or postmor- tis virus was first isolated in Venezuela, where it
tem brain tissue taken early on in the illness. How- causes important epizootics in horses, but also in-
ever, serology, together with the appropriate clini- fects humans. The geographical distribution in-
cal manifestations in a patient living in or who has cludes Central and South America. VEE virus
recently travelled to an endemic area, forms the caused epidemics/epizootics among people and
most important method of diagnosing of disease horses in Latin America from the 1920s to the
associated with infection with WEE virus. Classical 1970s. It reached the USA in 1971, but no further
serological techniques, for example HI, CF or neu- activity was reported until 1992—1993, when a small
tralization can be used to demonstrate a rise in outbreak in Venezuela confirmed the re-emergence
antibody titre between acute and convalescent of VEE from its enzootic habitat to humans. This
serum samples. In addition, the detection of WEE was followed by a major outbreak occurring in
virus-specific IgM can provide a rapid acute-phase 1995, involving an estimated 75 000—100 000 people
diagnosis. However, cross-reaction may occur with (Rico-Hesse et al., 1995; Weaver et al., 1996). A
other viruses within the complex (Table 16.1). variant of the virus, known as Everglades virus, is
Knowledge of the geographical distribution of the endemic in Florida and another variant, Mucambo
viruses within the complex allows interpretation of virus, is found in Brazil, Trinidad and Surinam
a positive IgM result together with the appropriate (Table 16.5).
patient details (Calisher et al., 1986).
Epidemiology and Host Range
Prevention and Control
Both epizootic and enzootic strains of VEE virus
Vector control is an important aspect of the control occur. Serotypes IAB and IC are equine virulent,
of all mosquito-borne encephalitides. Outbreaks in while ID, IE and II, III and IV are equine avirulent
humans and horses are associated with unusually (Table 16.5) (Weaver et al., 1996). The principal
high densities of mosquitoes. There is therefore a vertebrate host for enzootic transmission is the ro-
correlation between Culex tarsalis density and hu- dent, although birds and other species may play a
man cases of WEE virus infection. Besides vector less important role. Horses may be infected by sev-
control, changing behaviour patterns, for example, eral subtypes or variants of VEE (Table 16.5), but
air-conditioning houses and promoting in-door ac- they do not play an important role in enzootic
ALPHAVIRUSES 481
transmission which occurs primarily between small et al., 1996). A further prospective study which in-
mammals and the Culex mosquitoes of the sub- cluded a total of 13 patients showed that six pa-
genus Melanoconion (Cupp et al., 1986). In contrast, tients had a severe incapacitating febrile illness last-
during equine epizootics horses do play an import- ing 3—5 days, two others had a ‘flu-like’ illness, and
ant role in viral spread. It has been suggested that the remaining five (38%) were asymptomatic (Mar-
epizootic strains of VEE are maintained in other tin et al., 1972). The overall mortality is thought to
mammalian hosts and birds until the conditions are be less than 1%. Fetal loss may occur in pregnant
favourable for the development of an equine epi- women with VEE.
zootic. A number of mosquito species have been
implicated as likely vectors during epizootics. Hu-
Pathogenesis and Pathology
man disease usually follows equine disease, but hu-
mans are not thought to be important in the main- The disease caused by VEE virus in humans is
tenance of the epidemic due to low-titre viraemia. relatively mild compared to that caused by either
The major outbreak of VEE that occurred in WEE or EEE viruses. In horses, as well as other
Venezuela and Columbia in 1995 was remarkably species, there is a clear difference in the pathogenic-
similar to an outbreak occurring in 1962—1964. ity of the epizootic and the enzootic strains of VEE.
Both outbreaks followed unusually heavy rains Epizootic strains are generally more virulent, with
which led to an increase in the mosquito vector, and infection in horses resulting in viral replication in
both occurred in the same geographic region. the lymphoid tissue and bone marrow. This is asso-
Phylogenetic analysis showed that isolates from the ciated with lymphoid necrosis and a lymphopenia
1995 epidemic of VEE were closely related to the and is accompanied by a high-titre viraemia. Spread
serotype IC viruses isolated during the 1962—1964 to the central nervous system probably occurs in
epidemic. As a similar virus was identified in mos- the bloodstream, resulting in a fatal encephalitis in
quitoes in Venezuela in 1983, the authors raise the horses, as well as rodents and some primates (Peters
possibility of a serotype IC enzootic transmission and Dalrymple, 1990).
cycle in northern Venezuela (Weaver et al., 1996).
Diagnosis
Clinical Features
The diagnosis of VEE-associated disease should be
In horses, enzootic strains of VEE virus usually suspected in patients presenting with a ‘flu-like’ ill-
produce a fever and mild leucopenia. In contrast to ness in an appropriate geographical region when
this, horses infected with epizootic strains exhibit there is a concurrent equine epizootic. Material
high fevers, severe leucopenia and signs of encepha- obtained from sick horses confirming an epizootic
litis. Infections in humans result in a range of symp- of VEE may therefore be an important indicator of
toms. These may be subclinical or result in clinical human disease. Isolation of virus may be attempted
symptoms of varying severity. Symptoms and signs from acute-phase serum and throat swabs as well as
in patients seeking medical care include fever, chills, from brain tissue obtained from aborted and still-
severe headache, myalgia, vomiting and diarrhoea. born fetuses (Weaver et al., 1996). Culture of VEE
Most patients will have an acute and self-limiting virus has been achieved by inoculation into Vero or
febrile illness; however, convulsions, disorientation mosquito cells or suckling mice (Dietz et al., 1979).
and drowsiness may also be present (Weaver et al., Intracerebral inoculation of suckling mice may re-
1996). The proportion of cases that develop neuro- sult in death within 72 hours. Isolates identified in
logical sequelae appears to vary from outbreak to cell culture may be characterized further using VEE
outbreak. A review of the medical records of pa- serotype-specific polyclonal sera. Alternatively,
tients with convulsions in the 1995 epidemic genetic characterization may be done using RT-
showed that, although the age varied greatly (from 2 PCR with sequencing of the products generated
months to 48 years), most were children; however, from the E3 and E4 genes (Weaver et al., 1996).
only approximately 20% of these were under 3 A number of serological assays (IFA, ELISA, HI)
years of age. Of interest is that about half of these have been described. Acute and convalescent serum
patients had their first seizure between the 5th and samples may be tested in parallel by HI. This may
10th day after the onset of their illness and when provide diagnostically useful information. More re-
their temperature had returned to normal (Weaver cently attention has been focused on the use of an
482 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
IgG ELISA based on an antigen prepared from the versions have been described. However, the clinical
attenuated VEE vaccine strain of virus. In experi- features of infection with SFV have not been well
mentally infected guinea-pigs, VEE-specific IgG defined. One scientist working with SFV developed
could be detected at 6 days postinoculation, com- an encephalitis—SFV was isolated from both
pared with 10 days for HI antibodies. However cerebrospinal fluid and postmortem brain tissue
RRV, EEE and WEE virus-specific IgG exhibit a (Peters and Dalrymple, 1990). This highlights the
weak cross-reaction on the IgG ELISA, so results necessity for caution when working with any infec-
must be interpreted with caution. The appearance tious agents of unknown pathogenic potential.
of IgG antibodies detectable by ELISA coincides Getah virus is closely related to RRV and has
with the development of antibodies detectable by been linked to disease in horses (fever, rash and limb
plaque reduction neutralization assay (PRN). While oedema). Àlthough it has not been implicated in
less sensitive than the IgG ELISA, PRN is more human disease, serological studies have demon-
specific and therefore eliminates some of the prob- strated antibodies to Getah virus in sera taken from
lems of cross-reaction discussed above. It may individuals from the Pacific Basin, Japan and
therefore be used as a confirmatory test. VEE-speci- Hainan Island in China (Johnson and Peters, 1996).
fic IgM antibody has been detected as early as 4 More recently, a newly recognised alphavirus has
days postinoculation in experimentally infected been isolated from mosquitoes from the Me Tri
guinea-pigs (at a time at which infectious virus was village in Vietnam; it appears to be most closely
present in the serum). In summary, suspected cases related to SFV and has been associated with central
of VEE virus infection may investigated serologi- nervous system illnesses in children. It has been
cally using a VEE-specific IgM and IgG ELISA, proposed that this virus is called ‘Me Tri virus’. The
together with the more specific PRN as a confirma- contribution of this virus to human disease in
tory test (Coates et al., 1992). southeast Asia remains to be defined (Ha et al.,
1995).
17
Flaviviruses
Barry D. Schoub and Nigel K. Blackburn
University of Witwatersrand, Sandringham, South Africa
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
486 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
The clinical manifestations of the majority of secretions, as well as vertically. The pestiviruses are
flavivirus infections are relatively non-specific. In not able to infect humans.
endemic areas, the diagnosis of infection is often
made on clinical suspicion, especially if the phys-
ician is alerted by epidemic activity in the region.
Hepacivirus
Occasionally outbreaks of flavivirus infection may
follow climatic events, for example, heavy rains
The third, and newest genus of the family
after a period of drought, which result in the forma-
Flaviviridae, is Hepacivirus, which is described in
tion of numerous stagnant pools facilitating the
detail in Chapter 3. The biochemical and biophysi-
breeding of mosquito vectors and also attracting
cal characteristics of the virus have been determined
bird life. Outbreaks may follow uncontrolled ur-
and have been shown to be characteristic of the
banization and the breakdown of vector control
family Flaviviridae. Hepatitis C virus (HCV) is not
measures, as has occurred in recent yellow fever
an arbovirus and is not known to infect any non-
outbreaks in West Africa. In some situations human
human host in nature. Experimentally the only ani-
cases may follow an extensive zoonosis in the verte-
mal able to be infected is the chimpanzee. Infection
brate host, especially where birds or livestock are
is transmitted by blood transfusion, penetrating in-
involved. Surveillance and epidemiological
juries with blood-contaminated needles and instru-
monitoring which involve, amongst other things,
ments, sexually, and from mother to child during
the continual and sustained sampling of arthropod
birth. Since the implementation of widespread
vectors and vertebrate hosts for arbovirus activity,
screening for Hepatitis B virus (HBV) in blood,
is of critical importance in the prevention and man-
HCV has become the major cause of post-trans-
agement of outbreaks of flavivirus infections. Re-
fusion hepatitis. Current screening programmes for
cently calls have been made for mobile units to be
hepatitis C have, in turn, significantly reduced the
established and sent into the field to conduct clini-
incidence of post-transfusion hepatitis due to this
cal diagnosis and therapeutic research as well as for
virus.
epidemiological surveillance in endemic areas.
The most recent member of the family
The diagnosis of infection due to flaviviruses be-
Flaviviridae is Hepatitis G virus (HGV). The virus
comes particularly difficult in individuals who have
was first detected in 1995 in laboratory tamarins
travelled from an endemic area and present them-
experimentally inoculated with blood from a sur-
selves for medical management many thousands of
geon with clinical hepatitis. Three distinct agents
miles away from their source of infection. In the
were defined and called GBV-A, GBV-B and GBV-
modern era of jet travel, a history of travel and a
C although it appeared that only GBV-C was a
knowledge of the prevalent infections in different
human virus (GBV-A and GBV-B were probably
parts of the world are now an important component
tamarin agents). HGV was described at approxi-
of infectious diseases medicine. In addition, travel-
mately the same time by a different laboratory but
lers should be educated to alert their attending
subsequent analysis of HGV and GBV-C has sug-
physician should they become ill on returning
gested that they are identical viruses. Both are, how-
home.
ever, distinct from HCV, having only 29% amino
acid homology with it. The pathological role, if any,
of GBV-C or HGV has not yet been established.
The virus is discussed more fully in Chapter 3.
Pestivirus
Figure 17.1 Electron micrograph of negatively stained spherical West Nile virus particles. The envelope is tightly applied to the
nucleocapsid with peplomers clearly visible in many particles (arrows). ( ; 117 000.) (Professor G. Lecatsas, Department of Virology,
Medunsa University, South Africa)
488 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 17.2 Organization of the genes of Flavivirus, Pestivirus (tentative) and Hepatitis C virus (tentative)
Morphology and Morphogenesis (Figure 1.22—1.24 g ml\ and 1.15—1.20 g ml\ in sucrose;
17.1) that of pestivirus is 1.12—1.13 g\ in sucrose and
that of hepaciviruses is 1.09—1.11 g ml\ in sucrose.
Virus particles are spherical with a diameter of
some 40—50 nm. The envelope is tightly applied to a Flaviviruses
25—30 nm spherical core and surface peplomers are
often visible. Replication of virus takes place in the The nucleic acid of flaviviruses consists of a single
cytoplasm of the cell in association with the rough molecule of positive-sense single-stranded (ss)
and smooth endoplasmic reticulum. Accumulation RNA. A single open reading frame (ORF) on the
of viral particles are seen within lamellae and ves- genomic RNA is translated directly into a poly-
icles and replication is characteristically associated protein which is further processed into the three
with the proliferation of intracellular membranes. structural proteins. These are, in order from the N
Hepatitis C particles produced in MT-2 (trans- terminal, the internal RNA associated C protein
formed T cell line) have been visualized by electron and then the two envelope proteins, pre-M and E.
microscopy and appear as particles of 60—70 nm size The pre-M protein is a glycosylated precursor pro-
with an envelope embedded in which are prominent tein which is cleaved during or shortly after release
glycoprotein spikes 6—8 nm in length. from the cell into the non-glycosylated M mem-
brane protein with no disulphide bridges (molecular
weight 7—9 kDa). The E membrane protein is
usually glycosylated, and is considerably larger,
Biophysical and Biochemical Properties with a molecular weight of 51—59 kDa and possess-
ing six disulphide bridges formed by 12 conserved
The S W of flaviviruses is 170—210, that of pes- cysteine residues. The core protein C is rich in ar-
tiviruses is 140 and that of hepaciviruses is P 150. ginine and lysine with a molecular weight of
The buoyant density of the flaviviruses in CsCl is 14—16 kDa.
FLAVIVIRUSES 489
Following the translation of the three structural itiated at the internal ribosomal entry site (IRES), as
proteins, seven non-structural proteins are pro- has been demonstrated with the pestiviruses and
duced - the glycoproteins NS1, NS2A, NS2B, NS3, also in the picornaviruses. The polyprotein is then
NS4A, NS4B and NS5. Two of these proteins, NS3 processed by both host and viral proteinases into at
and NS5, are probably components of the RNA least 10 distinct viral proteins. The structural pro-
replicase. The gene order is thus 5 — C — pre-M — E — teins occupy the amino-terminal third and the rep-
NS1 — NS2A — NS2B — NS3 — NS4A — NS4B — NS5 licated enzymes the carboxy-terminal two-thirds.
— 3 (Figure 17.2). The structural proteins are comprised of the highly
The lipid content of the flavivirus envelope, basic core protein (C) and two glycoproteins E1 and
which comprises some 17% of the viral weight, is E2. The core protein is probably the capsid of the
derived from the host cell membrane. The E and M virus. Two main core proteins have been detected in
proteins are inserted into the envelope. The glyco- vivo and in vitro, p21 the full length protein and p19
protein E is rich in mannose and complex glycans. a secondary cleavage product. The heavily
glycosylated E1 (gp35) and E2 (gp70) are the major
structural proteins forming a complex which is pre-
Pestiviruses
dominantly non-covalently associated. E2 is the
Considerably less is known about the molecular most variable region of the genome and antibodies
biology of the genus Pestivirus. It does, however, directed against it have been demonstrated to me-
follow the same general molecular features and rep- diate protection in chimpanzee studies. Multiple
lication strategies as in the case of Flavivirus. There species of E2 are found, caused by variable exten-
is, similarly, a large ORF which directly translates a sions at its carboxy-terminus with a smaller protein,
single polyprotein of approximately 4000 amino p7, which is situated adjacent to it on the genome.
acids. The definition of the structural proteins of the The NS2 protein appears next; its function is as yet
virus has not, as yet, been established. Three viral unclear. The 70 kDa NS3 protein functions as a
glycoproteins are produced (molecular weight is viral protease and is responsible for the proteolytic
53—57, 44—48 and 23—31 kDa, respectively). These processing of the downstream remainder of the viral
are probably envelope proteins and antibodies to polyprotein (the structural protein portion of the
the 55—57 glycoprotein neutralize virus infectivity. polyprotein is cleaved by host enzymes). In addi-
In addition, there is a core protein, which is prob- tion, NS3 may also have NTPase and RNA helicase
ably the first polypeptide (from the amino terminal) enzymatic functions and may also have a role in the
of the polyprotein (molecular weight 20—31 kDa). pathogenesis of some diseases, for example it has
The lipid composition of the virus has not been been shown to transform cells in vitro. The small
reported. The tentative gene order established by NS4A (8 kDa) protein serves a variety of functions
sequence specific antibody reactivities is 5 — p20 — such as anchorage of replication complexes and is a
gp48 — gp45 — gp55 — p125 — (p54/p80) — p10 — X cofactor for the NS3 protease; the function of the
(not yet identified) — p133 (p58/p75) — 3 (Figure NS4B is not yet known. The NS5A (p56) and NS5B
17.2). (p65) proteins probably play a role in the replica-
tion cycle and, in addition, the highly phos-
phorylated NS5A may be a crucial determinant of
Hepaciviruses
the susceptibility of the virus to interferon.
Studies of nucleotide sequences of cloned HCV Molecular studies of HCV isolates have revealed
have revealed a genome of somewhat smaller size significant differences in genomic and polypeptide
(molecular weight 3.5 ; 10) to the other genera of length and a considerable diversity of about 30% in
the Flaviviridae family. Comparative sequence stu- nucleotide sequences, suggesting a quasispecies dis-
dies have shown a close relationship with both pes- tribution of virus isolates. At least six genotypes and
tiviruses and flaviviruses. The single ORF directly over 30 subtypes have been identified throughout
translates a polyprotein of between 3008 and 3037 the world. Genotypic identification is not only of
amino acids depending on the genotype of the virus. significance for charting the molecular epidemiol-
Flanking the ORF are long untranslated regions ogy of the virus but is also of clinical diagnostic
(UTRs) on the 5 and 3 ends of the genome. Cap- value as certain genotypes appear to be virulent or
independent translation of the polyprotein is in- may display different sensitivities to antiviral treat-
490 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ment, especially interferon (Smith et al., 1997). each genus are antigenically related to each other,
but there is little cross-reactivity between the three
genera.
ANTIGENIC PROPERTIES
YELLOW FEVER
The antigenic properties of the genus Flavivirus are
defined by serological tests such as the highly cross-
The Yellow fever virus is the type species of the
reactive pH-dependent haemagglutination-inhibi-
genus Flavivirus and also the source of its name
tion (HI) test, the less cross-reactive complement
(flavus is Latin for yellow). Yellow fever has been
fixation (CF) test and the much more specific neu-
one of the classic diseases of antiquity, instilling
tralization (NT) or plaque reduction neutralization
dread and terror in the Americas, Europe and Afri-
(PRNT) test. Other techniques mainly used for di-
ca from the seventeenth to the twentieth century.
agnostic serology are IgG and IgM detection by
The disease has been romanticized in the classic
enzyme-linked immunoassay (ELISA) and im-
works of Samuel Coleridge Taylor’s Rime of the
munofluorescent (IF) assays. The antigenic features
Ancient Mariner and the Wagnerian opera The Fly-
of the virus are characterized by reactivity with
ing Dutchman. At the beginning of the twentieth
antigenic domains and epitopes on the membrane E
century the disease almost put paid to the construc-
protein of the virus.
tion of the Panama Canal, and in West Africa yel-
The genus Flavivirus is further subdivided into
low fever more than any other disease was respon-
subgroups on the basis of cross-neutralization tests
sible for the appellation of ‘the white man’s grave’.
using a polyclonal hyperimmune mouse ascitic fluid
As late as 1988, an estimated 44 000 cases with
prepared against each member virus. The members
25 000 deaths were estimated to have occurred in
of each subgroup give significant cross-neutraliz-
Nigeria in the 3 years 1986—1988.
ation results against each other, whereas the ‘unas-
signed’ subgroup contains a miscellany of
flaviviruses which will display only limited cross-
reactivity with each other or with at least one other History
virus in any of the established subgroups. The anti-
genic classification of members of the genus The disease entity of yellow fever was first described
Flavivirus of medical importance, together with in 1667 in Barbados, and in the following two cen-
their respective vectors, disease manifestations, geo- turies devastating epidemics raged on the conti-
graphic distribution, vaccine availability and fre- nents of America and Africa and also, to some
quency of reported cases, is given in Table 17.1. The extent, in Europe. The panic and chaos was match-
flaviviruses which either cause no human disease, or ed by the extravagance of the speculations as to the
have only rarely been reported in association with cause. The confusion was further aggravated by the
illness, will not be dealt with further. The genus difficulty in distinguishing it from the other tropical
Pestivirus, which does not cause human disease, will plagues of malaria and dengue. In 1848, Nott pro-
also not be discussed further. HCV and HGV are posed that yellow fever was transmitted by the bite
dealt with fully in Chapter 3. of a mosquito, a suggestion supported by Beauper-
The genus Pestivirus does not show haema- thuy in 1854 and Carlos Finlay in 1881. In 1900 the
gglutinating activity but cytopathic strains can be US Army Yellow Fever Commission, under Major
identified by NT or PRNT techniques. Many of the Walter Reed, demonstrated in historical experi-
virus isolates belonging to this genus propagate in ments on human volunteers that the infection was
host species cells but do not cause cytopathic effect. indeed transmitted by mosquitoes. The following
These may be typed using IF techniques. Serosur- year, Walter Reed demonstrated that yellow fever
veys or diagnostic serology can be carried out using was due to a filterable agent, the first human disease
ELISA tests or NT with a cytopathic strain as the shown to be due to a filterable agent. The discovery
indicator virus. Serological tests for HCV are con- of the aetiological agent of yellow fever, however,
fined to immunoassays, either enzyme-linked or had to wait a further 26 years when workers of the
radioimmunoassays. The member viruses within Rockefeller Foundation’s West Africa Yellow Fever
FLAVIVIRUSES 491
Table 17.1 Antigenic classification of flaviviruses of medical importance and their clinical and epidemiological features in
humans
? These viruses are of very little importance to clinical medicine and will not be discussed further.
@ Numerous : 9 1000 cases reported, usually many thousands to millions; uncommon : 10—1000 cases reported; rare : : 10 cases reported.
M : Mosquito; T : Tick; HF : Haemorrhagic fever; TBE : Tick-borne encephalitis; CEE : Central European encephalitis; RSSE : Russian
spring—summer encephalitis.
492 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
The features of jungle yellow fever differ in East and
West Africa. In East Africa, endemic yellow fever
activity is relatively quiet, with few notified cases.
However, two vast epidemics took place in 1940 in
the Nuba mountains of Sudan, causing 40 000 infec-
tions with over 15 000 clinical cases and 1 500
deaths. The second epidemic in 1960—1962, the lar-
gest epidemic of yellow fever ever recorded, took
place in southwest Ethiopia, causing 30 000 deaths
in 100 000 clinical cases in a rural population of
some 1 million. In contrast to this, in West Africa
Figure 17.3 Geographic distribution of Yellow fever virus frequent outbreaks have occurred, usually on a con-
siderably smaller scale to the two East African epi-
Commission demonstrated the transmission of in- demics, with the exception of the Nigerian epidemic
fection from a Ghanaian patient called Asibi, to a of 1986—1988, when an estimated 44 000 cases and
rhesus monkey, and the subsequent passaging be- 25 000 deaths may have occurred (WHO, 1991).
tween monkeys. The Asibi strain was later to be the Over the last decade significant epidemics have
parent strain of the 17D vaccine developed by No- been reported from several West African countries,
bel laureate Max Theiler at the Rockefeller Foun- both preceding the Nigerian epidemic, in the Ivory
dation in New York in 1937. The 17D vaccine strain Coast (1982) and Burkina Faso (1983), as well as
was developed by serial passage through mouse after it, in Mali (1987), Angola (1988), Cameroon
embryo culture to chick embryo culture and then (1990) and Niger (1990). Yellow fever has expanded
through minced chick embryo devoid of nervous into Gabon, Liberia and Kenya, countries which
tissue. The final vaccine virus propagated through reported their first cases since 1950 between 1992
fertilized chicken eggs is remarkably attenuated yet and 1995 (World Health Organization, 1996a).
immunogenic, and the vaccine has proved to be The transmission cycles of yellow fever and the
very safe and effective, probably producing lifelong ecological interrelationships of its vectors and reser-
immunity after a single injection. voir hosts are complex. Essentially three cycles of
transmission are recognized:
1. The enzootic forest cycle represents the predomi-
nant maintenance of infection in its major verte-
Epidemiology
brate host, the monkey. In South America,
Alouatta, Cebus and Ateles monkeys are the ma-
Yellow fever occurs today in the tropics on both
jor primate reservoir hosts and they are infected
sides of the Atlantic (Figure 17.3). The endemic zone
by tree-hole breeding Haemagogus mosquitoes
in the Americas stretches between the latitudes of
in the forest canopy. The monkey is, however,
10°N and 40°S and in Africa from 16°N to 10°S.
only a transient host because of the short-lived
In the Americas, effective control of Ae. aegypti
viraemia. The major amplification host is the
has resulted in the virtual disappearance of urban
mosquito, which remains infected for life and is
yellow fever from the western hemisphere. The last
also able to pass infection on transovarially. Oc-
such epidemic of urban yellow fever occurred in
casionally a single or a few human cases may
Trinidad in 1954. However, jungle fever remains
occur when humans ventures into the forests.
endemic in Bolivia, Brazil, Columbia, Ecuador,
The cycle in Africa is similar and involves Cer-
Peru, Panama, Venezuela and the Guyanas. The
copithecus and Colobus monkeys and the Ae.
annual incidence by notification is 100 cases, al-
africanus mosquito as the principal vector.
though this is probably a substantially under-
2. The jungle yellow fever cycle represents the most
estimated number. Remarkably, the infection has
important epidemiological form of yellow fever
not extended to the heavy concentrations of Ae.
with respect to human infection. Epizootic up-
aegypti which have built up in urban centres.
surges of yellow fever are frequent, both on the
In Africa, recent epidemics of yellow fever have
fringes of the rain forests as well as in the sur-
been far more extensive than those in the Americas.
FLAVIVIRUSES 493
rounding riverine gallery forests. Human infec- With more intensive and careful physical examin-
tion occurs when forest mosquitoes invade ad- ation of patients, signs and symptoms more sugges-
jacent plantations, clearings and villages. Once tive of yellow fever may be revealed, such as flushing
infection has been introduced into the human of the head and neck, conjunctival injection, straw-
host, human-to-human transmission sustains berry tongue and a relative bradycardia. Probably
the epidemics, resulting in the dramatic out- the majority of clinically manifest yellow fever infec-
breaks reported in recent years. In South Amer- tions are aborted at this phase of the illness, ac-
ica, Haemagogus mosquitoes and possibly other counting for the underestimating of the true numb-
mosquito species may establish epidemics in hu- ers of cases by up to 500-fold (WHO, 1991).
mans. In East and Central Africa east of In cases of severe yellow fever, the early acute
Cameroon, Ae. simpsoni will readily bite mon- illness is followed by a brief period of remission
keys and humans. In the East African epidemics before the onset of the haemorrhagic, hepatic and
in Sudan and Ethiopia, the anthropophilic Ae. renal disease. The latter is heralded by a return of
Aegypti was the most frequently responsible fever, vomiting, abdominal pain, dehydration and
mosquito, as well as a number of ‘wild’ mos- prostration. The onset of the haemorrhagic dia-
quitoes. In West Africa and Central Africa west thesis is usually marked by coffee-ground haema-
of Cameroon, Ae. simpsoni does not bite humans temesis, the classical ‘black vomit’, and bleeding
and Ae. furcifer is the major vector. In the Niger- from puncture sites where injections or drip needles
ian epidemic the major vector was Ae. africanus. have been inserted. This is accompanied by jaun-
3. The urban yellow fever cycle is maintained by Ae. dice, albuminuria and oliguria. Deepening jaundice,
aegypti and was the dominant form of yellow massive haematemesis or haemoptysis or intra-ab-
fever before extensive mosquito control elimin- dominal bleeding, progressive renal failure, hypo-
ated the disease from South American towns. tension and shock may occur, followed by stupor,
Periodic reinfestations with Ae. aegypti have coma and death by the 7th to 10th day. Occa-
been observed in many South American coun- sionally the illness may run a rapid fulminant
tries and have revived fears of a resurgence of course with death within a few days of onset. Mor-
urban yellow fever. In West Africa Ae. aegypti- tality has been estimated to be of the order of
transmitted yellow fever is still responsible for 20—50% of cases entering the second phase of ill-
outbreaks in towns and rural villages. ness, although case fatality rates of up to 83% have
been reported (WHO, 1990). These figures probably
represent a marked overestimate of the fatality rate
in the more severe cases which are likely to come to
Clinical Features the attention of the health authorities. The differen-
tial diagnosis of severe yellow fever is usually that of
The clinical presentation of yellow fever follows the the causes of viral haemorrhagic fever: Congo
general pattern of arbovirus disease — a short incu- Crimean haemorrhagic fever, Rift Valley fever,
bation period of 3—6 days followed by an acute meningococcal septicaemia, Marburg and Ebola,
biphasic illness. It is the severity and extent of the generalized herpes simplex as well as HBV infec-
second phase of the acute illness which has im- tion, leptospirosis and toxic hepatitis. During the
parted to this infection its classical awesomness. severe second phase of illness, virus is usually absent
The initial phase of illness is characterized by a from the blood and antibodies are present, suggest-
viraemia which renders the patient infectious to ing that autoimmunity may well play a major role
biting mosquitoes and is also responsible for the in the pathogenesis of severe yellow fever.
acute constitutional symptoms. These symptoms Patients who survive generally recover complete-
last about 3 days and are generally those of a non- ly, although a chronic phase of illness lasting weeks
specific febrile illness: headache, malaise, nausea, or sometimes even months may occur in some indi-
lassitude and widespread muscle pain, especially in viduals. This is characterized by prolonged jaundice
the back. The differential diagnosis is wide and and disturbances of liver function, as well as pro-
includes malaria, other arbovirus infections includ- longed renal failure. Occasionally sudden death
ing dengue, typhoid, rickettsial infections, influenza, may occur in the chronic phase as the result of
enterovirus infections, acute HIV infection, etc. myocardial damage or cardiac arrythmias.
494 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
A number of host factors may affect the clinical demonstrated before a definitive diagnosis could be
expression and severity of yellow fever virus infec- made. Specific diagnosis still may be difficult, par-
tion. Age has played a significant role in South ticularly if the patient has had previous experience
American epidemics, the majority of infections oc- of flavivirus infection. In these instances higher
curring in young adults, especially between the ages titres may appear against heterologous viruses, in
of 20—25. Age distribution has not, however, been a keeping with the doctrine of original antigenic sin.
significant characteristic of African epidemics. In Isolation of virus may be performed in specializ-
some epidemics gender has played a significant role ed reference laboratories, either by intracerebral or
in the distribution of cases. For example, in the intraperitoneal inoculation of suckling mice or by
1972—1973 Brazil epidemic there was a marked pre- the intrathoracic inoculation of male Ae. aegypti or
dominance (90%) of males affected, whereas in Toxorhynchites mosquitoes. These techniques are
others there was only a slight male preponderance particularly sensitive and are essential for epi-
(e.g. 53% males in the 1986 Nigerian epidemic). demiological monitoring and research, but unfortu-
More controversial has been the purported associ- nately take up to 3 weeks to provide an answer.
ation of race and susceptibility to yellow fever, es- More rapid viral isolation techniques using mos-
pecially in the classical literature, which frequently quito cell lines such as the lines from Ae. albopictus
made reference to the mildness of the disease in the (C6-36) and Ae. pseudoscutellaris (MOS 61), which
indigenous inhabitants of the African jungle. There are sensitive to infection, combined with the IF test
is, however, no evidence confirming any difference using monoclonal antibodies may give a diagnosis
in susceptibility between races. within 3—4 days. It is recommended that early anti-
There are no known viral factors affecting either body is dissociated from viral antigen using
transmissibility or virulence. Although genomic dithiothreitol prior to isolation attempts in mos-
heterogeneity has been demonstrated by finger- quito cell cultures. An antigen capture ELISA tech-
printing and sequencing studies, and some anti- nique has been developed which is slightly less sen-
genic heterogeneity has been observed between iso- sitive than virus isolation but is able to produce a
lates, there has been no demonstrable clinical or specific result with the use of monoclonal antibo-
epidemiological differences between American and dies in less than 24 hours.
African isolates. Liver biopsy is contraindicated in acute yellow
fever. However, liver tissue from postmortem exam-
inations may provide useful histopathological in-
formation. The classical liver pathology is that of a
Diagnosis coagulative midzonal necrosis. The inclusion bo-
dies which have been held to be pathognomonic of
The development of reliable rapid tests for the ur- yellow fever are those in the cytoplasm due to
gent diagnosis of yellow fever has become a major eosinophilic degeneration (Councilman bodies) and
public health priority in the management of the intranuclear eosinophilic inclusion bodies (Torres
viral haemorrhagic fevers. The IgM antibody cap- bodies). Many of these features are, however, also
ture ELISA has become the test of choice for rapid found in fatal cases of viral haemorrhagic fever due
serological diagnosis of yellow fever virus infection. to other viruses, and the histopathology is now no
Cross-reactions may occur but IgM antibody levels longer regarded as being diagnostic of yellow fever.
will normally be higher against yellow fever. Im-
munofluorescence tests using infected cells spotted
on to microscope slides and then acetone fixed can
be used for detecting IgG and IgM in patients’ sera. Control
In the IF test, however, the rheumatoid factor may
be a much greater problem than in the IgM anti- The worldwide control of yellow fever has been
body capture ELISA and pretreatment to remove achieved by immunization and by effective vector
IgG prior to IgM testing is necessary. The classical control, which have largely eliminated the urban
techniques such as HI, CF and NT still have a place yellow fever cycle due to Ae. aegypti. However, in
in yellow fever surveillance and diagnosis but a recent times, poverty, war and competing health
fourfold or greater rise in titre would have to be priorities have led to a reduction in immunization
FLAVIVIRUSES 495
and surveillance efforts, resulting in a resurgence of any rate, never be given to infants less than 4
disease, especially in the endemic zone of Africa. In months of age. The effect of immunization in preg-
addition, reinfestation of towns and villages adjac- nancy has not been determined, however, being a
ent to forests with Ae. aegypti, aggravated by the live attenuated vaccine, it should not routinely be
massive uncontrolled urbanization and population given to pregnant women, but if travel to an area
migrations to the towns of developing countries, with ongoing yellow fever cannot be avoided, the
also exacerbated particularly by the severe drought, danger of infection would far outweigh the theoreti-
has renewed the spectre of urban yellow fever in cal risk to a pregnant mother and her fetus. The
Africa. vaccine should also be avoided in persons with a
It is over 50 years since the development of the history of egg allergy or in immunosuppressed indi-
first yellow fever vaccine by Theiler, and the original viduals, for example, due to HIV infection, malig-
method for the production of chick embryo-pas- nancy or individuals on immunosuppressive treat-
saged 17D vaccine has undergone little modifica- ment. However, as in the case of pregnancy, the
tion over the years. The French neurotropic vaccine relative risks of travel to an endemic area versus the
developed by passage in mouse brain is now no slight or even theoretical risk of the vaccine would
longer used because of the prohibitive danger of need to be evaluated on an individual basis.
encephalitic complications. The vaccine is a live Immunization policies with regard to yellow fe-
attenuated purified product produced by growing ver immunization involve three aspects of the con-
vaccine virus in chick embryos and is supplied as a trol of infection:-
lyophilized preparation, which should be stored
1. International travellers going to yellow fever en-
frozen or at least kept at temperatures of not more
demic areas require prophylactic immunization
than 5°C. After reconstitution it should be used
as a condition of entry to these countries, or
immediately and any remnants discarded within an
exiting from endemic countries. There were two
hour. A single dose of 0.5 ml given subcutaneously
reports in 1996—1997 of travellers to the Amazon
provides excellent, long-lasting immunity to 99% of
region of Brazil who died from yellow fever after
vaccinees. Although international health regula-
returning to their respective countries. Neither
tions demand booster doses every 10 years, neu-
had been vaccinated.
tralizing antibodies have been shown to persist for
2. Within endemic zones, two kinds of immuniz-
over 30 years and immunity is probably lifelong.
ation policy have been practised. These are the
Side-effects to immunization occur in less than
so-called ‘fire-fighting’ policy, where there is a
5% of recipients and are generally mild, low-grade
response to an outbreak, or alternately proactive
fever, myalgia and headache. Occasionally hyper-
routine immunization to prevent outbreaks oc-
sensitivity reactions have been reported, especially
curring. ‘Fire-fighting’ mass immunization is
in individuals allergic to egg protein. The most seri-
usually put into operation in the early phases of
ous side reaction, encephalitis, has been reported in
an outbreak or in advance of an imminent epi-
18 cases out of more than 35 million doses of vac-
demic, if effective surveillance is able to predict it.
cine which have been administered to date. Only
Although this strategy may be cheaper than rou-
two cases have occurred in children over the age of 7
tine immunization, it can often only be imple-
months, although the only fatality was in a 3-year-
mented after a considerable number of individ-
old child. Regarding the latter case, it was recently
uals have already been infected, and the
postulated that a change identified in amino acid
protective effect will be further delayed by an-
position 303 of the isolate P-16065 as compared to
other 7 days while antibodies develop. A far
the parent vaccine virus 17D-204 USA may have
more effective way of controlling yellow fever is
affected the virulence of the vaccine virus (Jennings
proactive, routine and sustained administration
et al., 1994).
of vaccine. In Africa, 33 countries have been
The contraindications to immunization are those
identified by the World Health Organization as
determined by age, pregnancy, history of egg allergy
endemic high-risk regions where yellow fever
and immunosuppression (Centers for Disease Con-
vaccine should be incorporated into the Ex-
trol, 1990). The vaccine should not be given to
panded Programme on Immunization (EPI)
children under the age of 9 months unless travel to
schedule. However, only one of these countries,
an endemic area cannot be avoided, but should, at
496 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Gambia, has exceeded the target vaccine cover- isolated in 1975 from the brain of a fatal case of
age of 80% (87% in 1994). In other countries encephalitis diagnosed during an unusual epidemic
coverage has ranged from 1% in Nigeria to 55% of encephalitis which lasted from 1975 to 1976. No
in Mauritania (World Health Organization, further cases have occurred since 1980, although
1996b). Yellow fever vaccine should preferably two children from the Ribeira valley tested positive
be given together with measles vaccine at 9 for IgM antibodies in 1989. A total of 821 cases were
months of age and can be administered simulta- diagnosed between 1975 and 1978, twice as many in
neously with measles vaccine with no reduction males and usually between 15 and 30 years of age.
in efficacy of either vaccine. It can also be given Workers in contact with the forests and rivers were
simultaneously with other viral vaccines and at highest risk. The virus has been isolated from
also BCG, with no increase in reactivity or de- pools of mosquitoes such as Psorophora ferox and
crease in efficacy. (However, yellow fever and also from wild birds. The transmission cycle of the
cholera vaccines should preferably be separated virus has, however, not been established.
by an interval of 3 weeks.) Clinical and epidemiological features of the dis-
ease in 821 cases between 1975 and 1978 have been
The current worldwide production of yellow fever
reviewed (Iversson, 1980). The disease commences
vaccine is estimated to be of the order of 15 million
with non-specific signs of pyrexia, headache and
doses per annum. There are concerted efforts to
vomiting. This may then be followed by disturban-
improve on techniques for the production of yellow
ces of consciousness and signs of encephalitis, in-
fever vaccine, for example by the use of tissue cul-
cluding localizing signs. Death may follow a pro-
ture techniques and also the development of recom-
longed coma or there may be a sudden fulminant
binant technology with reference to yellow fever
course. Serious neurological sequelae occur in some
immunization (Shiu et al., 1991).
20% of clinical cases and the overall case fatality is
Vector control strategies, such as aerial spraying,
found to be 10%. The IgM capture ELISA has been
domiciliary spraying and the enforcement of public
used to diagnose Rocio virus infection in children
health regulations to reduce collections of stagnant
and is preferable to the HI test for identifying recent
water and other breeding sites, have had successes
infection.
in the past in eliminating Ae. aegypti and controll-
ing urban yellow fever. Present-day socioeconomic
difficulties have, however, hampered recent efforts
to again control the reinfestation of villages and
Wesselsbron
towns in Africa. Control of vectors responsible for
the jungle yellow fever cycle is impractical.
Wesselsbron virus was first isolated from a lamb in
There still remain many gaps in our knowledge of
the village of Wesselsbron in South Africa in 1955.
the epidemiology of the infection and the perma-
Virus has been isolated from 23 cases of infection,
nent control of the disease will probably need not
11 of them resulting from laboratory infection or
only resources to implement what is already known
infection of laboratory field workers. The vertebrate
but also the elucidation of the remaining enigmas
host is chiefly livestock, especially sheep, and isola-
regarding the infection.
tions have been made throughout sub-Saharan Af-
rica, especially South Africa. It has also been iso-
lated from pools of wild-caught Aedes mosquitoes
OTHER MEMBERS OF THE such as Ae. (Neo) lineatopennis. The major vector
‘UNASSIGNED’ SUBGROUP OF in sheep is Ae. caballus-juppi. Human are infected by
FLAVIVIRUSES mosquito bite or by direct contact in handling car-
casses or tissues of animals that have died of the
Rocio disease.
Human infection is characterized by a sudden
Rocio virus is an arboviral cause of encephalitis onset of pyrexia, severe headache and retro-orbital
localized to the Ribeira valley and Santista low- pain associated with photophobia, and hyperaes-
lands in the southern coastal region of Sao Paulo thesia of the skin with an evanescent skin rash is
state in southeastern Brazil. The virus was first frequently present. Muscle and joint pains are also
FLAVIVIRUSES 497
commonly seen. In severe cases signs of encephalitis
such as blurred vision and some mental impairment
may occur. Patients recover after a few days to a
week and no permanent sequelae have been re-
ported.
Diagnosis of infection may be achieved by isola-
tion of the virus from blood or serological tests. The
HI test is only useful in individuals with no previous
flavivirus infection history because of the extensive
cross-reaction between Wesselsbron and other
flaviviruses. The capture IgM assay is the best
choice for diagnosis of recent Wesselsbron infec- Figure 17.4 Geographic distribution of Dengue virus
tion.
sible for severe and often incapacitating muscle
pain, it was seldom lethal. However, the gravity of
the dengue pandemic was really confirmed by the
DENGUE
recognition in the 1950s of the severe complications
of infection—namely DHF/DSS affecting mainly
Dengue is, at present, the most important arboviral
children in the endemic areas. Epidemics of DHF/
cause of death and disease in humans (Gubler and
DSS ravaged southeast Asia and in the following 30
Costa-Valez, 1991). The infection has spread widely
years was responsible for over 700 000 children be-
from southeast Asia to the Americas, the Pacific
ing hospitalized and over 20 000 fatalities (Halstead,
and Africa, now involving several million people
1984).
annually (Figure 17.4). In all major tropical areas of
In the western hemisphere, the first major epi-
the world the incidence of dengue fever (DF) and
demic of DHF/DSS struck Cuba in 1981 and was
dengue haemorrhagic fever (DHF)/dengue shock
responsible for 24 000 cases of DHF and 10 000 of
syndrome (DSS) has increased dramatically over
DSS (Gubler and Costa-Valez, 1991). (Sporadic
the past few years, with an ever-increasing fre-
cases of suspected DHF had been reported from
quency and extent of epidemics and a greater sever-
Central America since 1968.) Only the energetic
ity of cases. The spectre of the introduction of infec-
response of the Cuban health authorities, with in-
tion to Ae. aegypti populations in non-endemic
tensive education and mass hospitalization, kept
countries is of great international concern.
the mortality down to just 158. Following on this
epidemic, confirmed or suspected cases of DHF
have been reported in the Americas almost every
History year, with the most severe outbreaks of DF occur-
ring in Peru in 1990 involving over 76 000 cases
Outbreaks of illnesses clinically resembling DF (Centers for Disease Control, 1991a). Dengue re-
have been recorded since 1779 and 1780 in Java turned to Cuba in 1997. A summary of the present
(Indonesia), in Cairo and in Philadelphia. Similar status of dengue worldwide is that there are 2.5
epidemics of dengue-like illnesses occurred at 10—30 billion persons at risk, more than 20 million cases
year intervals throughout tropical and subtropical per year and 30 000 deaths. Imported cases of
regions of the world. Although the precise aetiology dengue in travellers returning to temperate coun-
could not be established and infections such as tries are reported in the United States annually and
chikungunya are clinically and epidemiologically DHF was reported in two cases in the UK in 1991
very similar, the majority of these epidemics were (Jacobs et al., 1991).
probably dengue. The spread of the disease has
been markedly accelerated by the advent of wide-
spread air travel over the last three decades. Dengue
fever was generally regarded as a relatively benign Virus Properties and Host Range
illness, affecting predominantly colonial expatriots
living in tropical countries and, although respon- The dengue viruses form a subgroup of the genus
498 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Flavivirus and, although extensive cross-reactivity mates and involving forest species of Aedes; a rural
is seen with serological tests such as HI, which or semirural cycle in humans with the peridomestic
cannot reliably distinguish dengue from many other Aedes species being the vectors; and an urban cycle
flaviviruses, neutralization tests are able to define in humans involving domesticated Aedes species.
dengue virus as a distinct antigenic subgroup. There By far the most important of these three for both
are four serotypes of dengue based on neutraliz- endemic and epidemic human dengue is the urban
ation tests. However, with all serological tests there cycle. It is, in fact, doubtful whether the forest cycle
is extensive cross-reactivity between the four sero- plays any significant direct role in human dengue.
types, although they are distinguishable with the The two major mosquito vectors of urban dengue
highly specific plaque reduction neutralization test. are Ae. aegypti and Ae. albopictus. Although Ae.
Following natural infection, protective immunity is albopictus is considered more sensitive to oral infec-
homotypic. Dengue serotypes 1, 3 and 4 show a tion with dengue viruses, Ae. aegypti is a more
closer antigenic and genetic relationship to each important vector for human disease and is especial-
other than dengue 2. However, within each of the ly responsible for explosive epidemics. The an-
serotypes, considerable heterogeneity and strain thropophilic Ae. aegypti usually preferentially feeds
variation is demonstrable on nucleic acid sequence on humans and occasionally also on domestic ani-
analysis and DNA/RNA hybridization studies, as mals, while the purely domestic Ae. albopictus ran-
well as by antigen signature analysis using mon- domly feeds on humans, domestic animals and feral
oclonal antibodies. The importance of these strain animals and birds. To establish infection, Ae.
variations with respect to the epidemiology and aegypti would need to feed on individuals with high
virulence of the virus remains to be determined. levels of viraemia and this may select for viral
Molecular analysis of isolates from the South Paci- strains of higher virulence which are more likely to
fic suggested that the recent epidemics (1988—1989) produce the severe epidemics associated with Ae.
were due to a new genotype rather than a previous- aegypti. In addition, the biting habits of Ae. aegypti,
ly circulating strain. which characteristically takes interrupted blood
The only vertebrate hosts of Dengue virus in na- meals and will thus feed on a number of individuals
ture are humans and several species of Asian and before becoming engorged, enhances its ability to
African subhuman primates. Other vertebrates can spread the virus. It is thus not unusual for a single
be experimentally infected only with difficulty, in- mosquito to spread infection amongst several indi-
cluding baby mice which usually require several viduals at a single feeding. In both mosquitoes,
blind passages of patient material to obtain an iso- biting activities are maximal soon after daybreak
late. The invertebrate hosts of dengue are members and in late afternoon, and are related to human
of the genus Aedes, especially the subgenus activities and movements. Outbreaks associated
stegomyia. The most important mosquito hosts as with both vectors are climatically influenced by
far as human infection is concerned are Ae. aegypti, heavy rainfall and high temperature.
Ae. albopictus and Ae. polynesiensis. Other Aedes The recent upsurge of dengue in existing endemic
species, including Ae. africanus, Ae. leuteocephalus zones and the spread to new areas has been at-
and the Ae. furcifer group, are involved in the main- tributed to modern phenomena of human and so-
tenance of the forest cycle of dengue in Africa, cial behaviour. Infestations of Ae. aegypti in tropi-
whereas in Asia the mosquitoes concerned belong cal towns and villages, which were controlled by
to the Ae. niveus complex. extensive spraying and other vector control
measures in the 1950s and 1960s, have now resurged
due to uncontrolled urbanization and the mass mi-
gration of rural populations into informal housing
Epidemiology settlements and squatter camps on the peripheries
of cities and towns. These population movements
Dengue displays many epidemiological similarities have been accentuated by famine, poverty and war.
to yellow fever and chikungunya viruses and there Stagnant water pools and lack of reticulated water
is considerable overlap in the ecologies of these supplies have provided ample opportunities for
three virus infections. Essentially there are three mosquito breeding, coupled with the breakdown of
transmission cycles of dengue: a forest cycle in pri- vector control in many tropical countries. Over-
FLAVIVIRUSES 499
crowding and grossly inadequate housing, which is pared with 2.0% and 1.1%, respectively, of DF due
so characteristic of the sprawling slums of the tropi- to secondary infection (Halstead, 1981). It is also
cal world, greatly facilitate the spread of vector- rare in individuals over the age of 15 years. DHF/
borne diseases and are especially conducive to DSS resembles yellow fever in its biphasic presenta-
dengue transmission resulting from the interrupted tion. The first phase is not unlike uncomplicated
feeding habits of Ae. aegypti. The spread of infection DF. This is followed by a brief remission of symp-
has also been enhanced by the modern era of jet toms when the fever subsides to normal or close to
travel, which facilitates the transportation of infec- normal. There is then a sudden deterioration in the
ted individuals to non-infected areas, creating the patient’s condition, marked by profound prostra-
threat of the introduction of infection if infestations tion, hypotension, circulatory collapse and manifes-
of Ae. aegypti are sufficiently high. Modern air tations of bleeding and shock. Bleeding is
travel has also been responsible for increasing the commonly seen as petechiae in the skin and mucous
number of imported cases of dengue into countries membranes, especially in the oral cavity, ec-
where physicians are, in the main, ignorant of the chymoses, bleeding at injection and skin puncture
infection, resulting in perplexing diagnostic difficul- sites, gastrointestinal bleeding and haemorrhagic
ties until a history of travel to endemic zones is pneumonia.
elicited. More recently, the influence of interna- The cause of the haemorrhagic diathesis in DHF
tional trade in the spread of dengue has been ob- is complex. There is evidence of vascular injury with
served. Motor vehicle tyres and casings from south- increased permeability and extravasation of fluid
east Asia, containing remnant pools of water where from the vascular into the interstitial fluid compart-
infected Ae. albopictus mosquito larvae have been ment, producing hypotension and DSS. Bleeding
found, have been shipped to various non-endemic due to vascular damage is suggested by the presence
areas of the world, posing a serious threat of intro- of petechiae and a positive tourniquet test. There is,
ducing both the virus as well as its vector. in addition, a marked thrombocytopenia, although
it is not yet clear what the relative contributions are
of impaired platelet formation due to direct sup-
pression of megakaryocyte production in the bone
Clinical Features marrow or excess destruction due to endothelial
damage. Evidence exists for both. Thirdly, there is
The majority of infections with Dengue virus, based haematological evidence of a consumptive co-
on the extent of population seropositivity, are agulopathy with a moderate increase in fibrin
asymptomatic. Clinical manifestations of dengue degradation products which, however, rarely
occur in two forms: classical DF and DHF/DSS. reaches the stage of disseminated intravascular co-
Classical DF is an acute disease characterized by a agulation.
sudden onset of fever, severe headache which is The definition of DHF is controversial. The
typically frontal in distribution, together with retro- WHO criteria for DHF are an acute onset of fever,
orbital pain, nausea and vomiting. Severe muscle haemorrhagic manifestations which include at least
and bone pain and arthralgia are characteristic of a positive tourniquet test, a thrombocytopenia of
dengue and usually more pronounced in the back, 100 000 ml\ or less and a haemoconcentration
which has led to it being terms ‘break bone fever’ by with a haematocrit increase of 20% or more. Mani-
Dr Benjamin Rush in 1778. He also aptly described festations of haemorrhagic fever, however, do differ
the associated severe depression as ‘break heart from country to country and amendments to the
fever’. There is frequently a diffuse, discrete criteria have been proposed to include, amongst
maculopapular rash which usually heralds the re- others, references to the age of the patient (less than
covery phase of the illness. In spite of the severity of 16 years) (Halstead, 1989). The severity of DHF/
symptoms which may be incapacitating, the disease DSS has also been graded by the WHO from I to IV
is temporary and full recovery takes place. (Anonymous, 1980):
The grave complication of DHF/DSS is govern-
Grade I Fever with non-specific, constitutional
ed by two factors: prior infection and age. Thus,
symptoms and the only haemorrhagic
DHF and DSS occur in approximately 0.18% and
manifestations being a positive tourni-
0.007%, respectively, of cases of primary DF com-
500 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
quet test. proteins of dengue virus could be demonstrated,
Grade II As for grade I, but with specific haem- some of which could also be neutralizing and others
orrhagic manifestations. not (Kurane et al., 1991).
Grade III Signs of circulatory failure or hypoten- The receptor on the monocyte/macrophage cells
sion. which form the third component of the enhancing
Grade IV Profound shock with pulse and blood complex is thought to be the FcRI molecule which
pressure undetectable. binds the Fc portion of IgG with great avidity, and
also, but probably to a lesser extent, the FcRII
molecule. The effect of the attachment of the vi-
rus—antibody complex to the FcR receptor is both
Pathogenesis of DHF/DSS to enhance the binding of virus to its target cell and
also to induce a signal to the cell facilitating inter-
The pathogenesis of DHF/DSS has been intensively nalization of the complex. There remain, however,
studied for a number of decades. Nevertheless, the many unanswered questions with the antibody-me-
pathways to the development of severe dengue have diated enhancement hypothesis. Firstly, DHF/DSS
not, as yet, been definitively established. Essentially may well occur in the absence of pre-existing anti-
there are two hypotheses involving immunological bodies, even though it is far more common for them
or virological mechanisms. to be there. It is also not clear whether binding of
The immunological theory of DHF/DSS is based virus to its specific receptor is essential for infectiv-
on the phenomenon of antibody-mediated enhan- ity. The mechanism by which enhanced infection of
cement of infection. Investigations of antibody-me- cells produces the components of a DHF/DSS syn-
diated enhancement in dengue have been the hall- drome is not known.
mark studies of this phenomenon, which have since The alternative hypothesis of Rosen and col-
been shown to be important facets in the pathogen- leagues (Rosen, 1986) holds that the severe compli-
esis of a number of viral diseases, from rabies to cations of DHF/DSS are the direct results of prop-
HIV (Kurane et al., 1991). The major cell infected by erties of the virus, that is, the consequence of
Dengue virus in vivo is the monocyte/macrophage infection with unusually virulent strains of dengue
cell (even though a number of cell lines from a circulating in a particular area and giving rise to
variety of tissues may be infected in vitro). Three outbreaks of DHF/DSS. However, as mentioned
elements partake in the process of antibody me- above, although nucleic acid sequencing techniques
diated enhancement: antibody, virus and the recep- and antigen signature analysis have demonstrated
tor for the Fc portion of IgG. strain variations within serotypes, no consistent re-
Evidence for the involvement of pre-existing anti- lationship between strain variation and either viru-
bodies comes from epidemiological observations lence or heightened infectivity has been reliably
that DHF/DSS is much more common in individ- demonstrated. There has also been no consistent
uals with pre-existing antibodies. These pre-existing association with serotype and DHF/DSS. Earlier
antibodies may be derived from passively acquired observations both in Thailand, as well as in the
maternal antibodies in infants less than 1 year of 1981 Cuban epidemic, suggested that dengue type 2
age, or from previous infection in children over 1 may be more frequently associated with DHF/DSS.
year of age. This was particularly evident in the Subsequent observations have now shown that all
Cuban epidemic of 1981 when DHF/DSS due to four serotypes may be responsible (Table 17.2).
type 2 virus was usually observed in individuals Neither of these two hypotheses is able to eluci-
infected some 4 years previously with type 1 virus. date why the development of DHF/DSS is so de-
Experimental work by Halstead and colleagues pendent on age or why DHF/DSS is more likely to
demonstrated enhancement of infection in experi- follow when dengue infection occurs in some areas
mental dengue in monkeys using human serum as but not in others.
well as sera from hyperimmunized animals. It was
later demonstrated that only IgG antibody and not
IgM, which has potent neutralizing activity, was Diagnosis
able to produce enhancement. Using monoclonal
antibodies, enhancing epitopes on the E and pre-M The clinical diagnosis of dengue, both the uncom-
FLAVIVIRUSES 501
Table 17.2 Dengue virus serotypes in relation to major outbreaks of Dengue fever and DHF
DHF : dengue haemorrhagic fever; DSS : dengue shock syndrome; N.S. : data not supplied.
plicated DF form as well as DHF/DSS, is often a number of blind passages are usually required.
unreliable. Dengue fever may clinically resemble a Intracerebral inoculation of adult or larval Toxor-
variety of acute febrile illnesses, although the severe hynchites species is a sensitive and rapid method for
muscle and bone pain is suggestive of dengue. Simi- the isolation of Dengue virus, giving results in 2—3
larly DHF may resemble other causes of haemor- days. Intrathoracic inoculation of mosquitoes is
rhagic fever, although thrombocytopenia with easier and just as sensitive but head squashes can-
haemoconcentration and signs of a moderate con- not be made or tested for specific dengue antigen for
sumptive coagulopathy is suggestive of dengue. at least 7 days postinoculation. The most
The most widely used serological test is the HI commonly used system of virus isolation is the in-
test, detecting antibodies as early as 4 days post oculation of mosquito cell lines from Ae. albopictus
onset. A specific diagnosis of dengue can be made (C6-36), Ae. pseudoscutellaris (AP-61) and Toxor-
early in primary infections, but cross-reactions with hynchites ambionensis (TRA-248), which are almost
other flaviviruses occur in late primary or second- as sensitive as mosquito inoculation, which allows
ary infections. The IgM antibody capture ELISA specific results to be obtained within 2—3 days using
(MAC ELISA) is being used to an increasing extent fluorescent-labelled monoclonal antibodies.
during outbreaks of dengue, and there are now
rapid assays available for detection of dengue IgG
and IgM, although cross-reaction may occur with
the IgG assay. The IgM antibodies may persist for Control
over 3 months, so the test is also useful for retro-
spective studies, but this persistence may cause di- There is, at present, no licensed dengue vaccine.
agnostic problems in areas where dengue is en- Current strategy is to develop a vaccine against all
demic. Immunofluorescent assays have been used four serotypes. Successful trials of monovalent vac-
successfully to detect dengue IgG and IgM antibo- cines have been reported, but, as yet, not for experi-
dies. The CF test is more specific than the HI test, mental tetravalent vaccines. Because of the lack of
but the antibodies detected by this assay appear availability of a vaccine, control of dengue depends
later and disappear earlier. The NT and PRNT tests on: (1) surveillance to obtain early warning of epi-
are the most specific and sensitive but are difficult to demics or preferably to be able to predict impend-
perform and thus tend to be used only for specific ing epidemics; and (2) effective vector control.
purposes. Epidemiological surveillance may be of two
Virus isolation, the only definitive way of being types. Firstly, proactive surveillance in in-
able to type isolates, is difficult, and if mice are used terepidemic periods in endemic areas or in countries
502 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
which are not yet infected but are vulnerable to the
introduction of new infections because of high infes-
tations of Ae. aegypti. Secondly, reactive surveil-
lance, where monitoring is instituted once suspected
or confirmed cases of dengue have already occur-
red. This form of surveillance is very insensitive, as
numerous cases may occur which are not suspected
to be dengue, before authorities are alerted to the
outbreak. In the Comoros islands, dengue type 1
was diagnosed in 62 out of 116 clinical cases in 1993.
However, an investigation to determine the preva-
lence of dengue infection in those over 5 years old,
using a dengue IgM assay, estimated that at least Figure 17.5 Geographic distribution of Japanese encephalitis
60 000 recent cases had occurred at that time. virus
A number of instruments of surveillance may be
used for proactive monitoring. Disease surveillance car tyres, or travellers or migrants from endemic
requires intensive educational efforts, especially at areas.
peripheral primary healthcare level, to alert heal-
thcare workers to the possibility of dengue. It is,
however, very difficult to sustain interest, especially
JAPANESE ENCEPHALITIS
if no cases materialize. Viral and serological surveil-
lance involves the active recruitment of specimens,
Japanese encephalitis (JE) virus is the major ar-
as, for example, carried out in Puerto Rico, where
boviral cause of encephalitis worldwide. First de-
blood samples are collected by collaborating phys-
scribed as a clinical entity in Japan in 1870, it is now
icians on a regular basis and sent for analysis (Gub-
thought to be responsible for about 50 000 cases
ler and Costa-Valez, 1991). Interest and cooper-
annually, half of whom are left with permanent
ation are also difficult to sustain. The use of blood
neurological or psychological handicap, and in a
samples sent to laboratories for testing for acute
further quarter of whom it is rapidly lethal. Infec-
febrile illnesses or ‘viral syndromes’ may be an
tion has been demonstrated in 16 countries in
easier way of recruiting specimens for serological
southeast Asia, stretching to India in the west and
testing. Surveillance may also utilize the routine
to southern Russia in the north—a population of
investigation of all viral haemorrhagic fever cases,
over 2 billion people (Figure 17.5).
which should include tests for dengue. Vector sur-
veillance may be of benefit to demonstrate low in-
festations (house index of : 1%) or to look out for
the introduction of exotic mosquito species, for Viral Features and Host Range
example, Ae. albopictus.
Vector control aims at the elimination of the JE virus shows some cross-reactivity with St Louis
main mosquito vector, Ae. aegypti. While this is encephalitis virus, West Nile virus and Murray Val-
technically feasible with both adulticide campaigns ley encephalitis virus, and together they form the
using ultralow-volume spraying with malathion mosquito-borne encephalitis complex. The virus is
and larvicide treatment of stagnant water, the costs antigenically relatively homogeneous and recovery
are high and the effects are temporary. In many from infection results in solid protection. Nucleic
tropical countries where Ae. aegypti eradication acid analysis, however, has revealed some signifi-
had been achieved, reinfestations have taken place cant heterogeneity. Using primer extension se-
to levels equalling, or even exceeding, those in pre- quencing, isolates of JE virus could be separated
control times. However, long-term community- into three distinct genotypic groups, each group
based programmes need to be implemented in en- found in separate geographic divisions (Chen et al.,
demic countries. In non-endemic countries there 1990). The epidemiological significance of this re-
should be vigilant monitoring for the possible im- mains to be established.
portation of dengue, for example, through motor The vertebrate hosts of JE virus are humans and
FLAVIVIRUSES 503
their domestic animals and birds; no wild animals 1984) to 1 in 600—800 persons infected (Halstead,
and birds are known to be infected to any signifi- 1981).
cant extent. The major mosquito vector of the virus The major target cells for JE virus are the T
is Culex tritaeniorhynchus, although a number of lymphocyte and the peripheral blood mononuclear
other species of Culex, Mansonia, Aedes and Anoph- cells. In fatal cases of encephalitis, viral antigen is
eles genera have yielded isolates of JE virus. also demonstrable in the neurons. Factors deter-
mining the neuroinvasiveness of JE virus involve
both viral and host factors. Nucleotide sequencing
of non-neurovirulent mutants of JE virus have dem-
Epidemiology
onstrated single base changes in the coding region
for E protein (Cecilia and Gould, 1991). Age is an
Nestling birds, particularly of the heron family, play
important host factor determining neurovirulence,
an important epidemiological role in the dissemina-
encephalitis being more common and more severe
tion of JE virus, with a second cycle involving do-
in the young as well as in elderly individuals. Ex-
mestic animals, particularly the pig. In serop-
perimental work in rats has shown a relationship
revalence studies high NT antibodies were found in
between neurotropism and neuronal maturity, in
several other animal species, e.g. cattle, horses, dogs,
that the virus selectively infects immature neurons
monkeys and bats. As with the wild bird popula-
(Ogata et al., 1991). The reason for greater neuroin-
tion, the high turnover of the domestic pig popula-
vasiveness in the elderly is unknown, but is consist-
tion, resulting in a continuous supply of susceptible
ent with the features of the related St Louis encepha-
animals, is a contributing factor to the pig being a
litis virus and West Nile virus, which similarly
major amplifying host for JE virus. JE rarely causes
display greater neuroinvasiveness in the elderly.
disease in domestic animals, although fatal en-
The typical case of JE commences after an incu-
cephalitis in horses and abortions in sows have been
bation period of 1—2 weeks, with fever and head-
recorded. Studies on birds have implicated other
ache, followed rapidly by depression of the level of
species whose main habitat is the rice paddies, as
consciousness, progressing from stupor to coma.
vertebrate hosts of JE virus; these water birds in-
Localizing cranial nerve and other neurological
clude water hens and bitterns. In India ardeid birds
signs occur in about 30% of cases and in children
such as cattle egrets are implicated.
generalized seizures are common. A quarter of cases
JE virus has been isolated from many species of
of clinical encephalitis will recover with no perma-
mosquito but the principal vector in many areas is
nent sequelae and a quarter will die rapidly. The
C. tritaeniorhynchus. Other mosquitoes, mainly the
remaining half will recover with varying degrees of
Culex species, are considered to be important in
permanent neuropsychiatric sequelae. In addition,
specific regions, e.g. C. gelidus in southeast Asia, for
especially in children, the virus may persist in lym-
the transmission of JE virus.
phocytes and reactivate to give recurrent disease
Humans are dead-end hosts and play little role in
after recovery (Sharma et al., 1991).
the amplification of the virus. JE is thus predomi-
Grave prognostic signs include a short prod-
nantly a rural problem, with disease closely related
romal period, deep coma, decerebrate posture,
to rainfall and irrigation.
breathing abnormalities and the ability to isolate
virus from the cerebrospinal fluid (CSF). Recently a
reduction of serum iron levels has been demon-
Clinical and Pathological Features strated in patients due to the sequestering of iron in
the spleen, and a direct relationship between low
Infection with JE virus is considerably more wide- serum iron and prognosis has been shown (Bharad-
spread than the incidence of encephalitis would waj et al., 1991).
indicate. Infection may present non-specifically as a The pathology of the brain in fatal cases has
mild febrile illness or as an aseptic meningitis or, revealed microfoci of necrosis scattered throughout
rarely, as a variety of inflammatory manifestations the central nervous system, but especially involving
in the viscera. The typical disease manifestations of the thalamus, the basal ganglia and the deep cer-
acute meningomyeloencephalitis have been widely ebral nuclei.
estimated to occur at between 1 in 20 (Rodrigues,
504 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Clinical Features
Figure 17.6 Geographic distribution of St Louis encephalitis
virus The majority (over 90%) of infections with SLE
virus are asymptomatic. In clinically apparent
that this has any epidemiological implications. By
cases, the disease is characterized by an abrupt
means of T1 restriction mapping, a number of geno-
onset of a febrile illness accompanied by constitu-
types of SLE have been defined and have been used
tional symptoms of malaise, nausea, vomiting and
as epidemiological markers.
headache. Occasionally there may be a more slow
The major vertebrate hosts of SLE are birds,
insidious onset. Central nervous system involve-
especially domesticated sparrows (Passer domes-
ment may be in the form of aseptic meningitis or
ticus), which act as the main amplifying host (Cen-
focal encephalitis. Encephalitis signs may be mani-
ters for Disease Control, 1991b). In addition, small
fest as neck stiffness, dizziness, ataxia, mental con-
mammals such as racoons, opossums and rodents
fusion and disorientation. Cranial nerve palsies
are also infected, as well as a variety of domestic
may occur in about 20% of cases, but the absence of
animals. However, other than birds, there is no
focal findings or seizures may be useful differential
evidence that animals play any role as maintenance
features to distinguish SLE from cases of focal en-
or amplifying hosts.
cephalitis such as herpes simplex. In more severe
Invertebrate vectors of SLE are various species of
cases there may be midbrain involvement and pro-
Culex mosquitoes, depending on location. In the
gressively severe coma. Overall case fatality is ap-
rural west it is C. tarsalis, in the northern and
proximately 7% of symptomatic cases, although in
southern regions of the central United States it is
outbreaks it may reach 20%. The likelihood of
mainly C. pipiens and C. quinquefasciatus, and in
encephalitis, and also its severity, is directly related
Florida C. nigripalpus.
to age, with case fatalities in the elderly during
outbreaks reaching 30% (Monath, 1980).
Epidemiology
Diagnosis
In all parts of the United States the transmission
cycle of SLE involves birds and mosquitoes. Hu- For rapid serological diagnosis of SLE infection the
506 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
IgM antibody capture ELISA is the method of
choice, using either serum or CSF. Cross-reactions
may occur if other flaviviruses are active in the area,
but this is not a major factor in North America. The
IF test on SLE-infected cells is a useful alternative
to the IgM ELISA for testing sera and the HI test
may be used for surveys or diagnosis if acute and
convalescent sera are available. In regions where
flavivirus activity is confined mainly to one virus,
the HI test would be a useful screening test, fol-
lowed by the SLE IgM antibody capture ELISA
test to determine recent SLE infection.
Epidemiology
WEST NILE VIRUS
The transmission cycle of WN virus consists mainly
West Nile (WN) virus infection is one of the most of wild birds as the vertebrate host and or-
ubiquitous of human arbovirus infections, being nithophilic Culex mosquitoes as the maintenance
found throughout Africa, in Asia and in parts of vector. The major Culex mosquito vector in Africa
Europe (Figure 17.7). The virus was first isolated in and the Middle East is C. univittatus, in southeast
the West Nile province of Uganda in 1937. Asia C. tritaeniorhynchus, and in France C. modest-
us.
Major epidemics of WN disease have been re-
Viral Features and Host Range ported in Israel during the 1950s, in France in 1962
and the largest epidemic ever recorded took place in
WN virus is related by neutralization tests to the South Africa in 1974, which involved tens of thou-
other members of the JE subgroup of flaviviruses, sands of individuals. Epidemics have characteristi-
particularly Kunjin virus, which is believed to have cally occurred in relatively localized areas, for
evolved from, or is a variant of WN virus. example, close to Tel Aviv in the Israeli epidemic, in
The major vertebrate hosts of WN virus are the Rhone delta in France and the semidesert
birds, although in addition the virus is able to infect Karoo region and in and around major cities of the
a variety of domesticated and wild animals as well highveld of South Africa. The distribution of WN
as humans and subhuman primates. The mainte- virus in South Africa is predominantly in the inland
nance vectors of WN virus consist of a variety of plateau region where it shares the same geographi-
mosquitoes, especially of the genus Culex. cal distribution and ecology as Sindbis virus, which
On the basis of antigenic studies of isolated vi- also produces a disease which is usually clinically
FLAVIVIRUSES 507
indistinguishable from WN infection. recently, however, a growing number of cases in
Outbreaks of WN virus, as with many other ar- children have been reported from various parts of
boviruses, have been governed by climatic condi- the world.
tions such as heavy rainfall, particularly in early Rarely cases of visceral involvement including
summer, and high summer temperatures. During severe hepatitis, occasionally with a haemorrhagic
outbreaks high attack rates in humans have been presentation, have been reported.
observed, for example, in some of the worst affected
towns in South Africa, 50—80% of the human popu-
lation were infected due to the high rate of feeding Diagnosis
by C. univittatus. This mosquito is probably also
responsible for sporadic cases in interepidemic per- The HI, CF or NT tests can be used for serological
iods. Because viraemia is low in humans, epidemic diagnosis of WN infection. The HI test, which de-
activity is directly due to infection of mosquitoes tects antibodies within a few days after onset, is
from viraemic birds, and human outbreaks are probably the method of choice. Cross-reactivity is
merely the spill-over of extensive epizootic activity not a problem in patients with no previous expo-
in birds—an important factor which facilitates epi- sure to flavivirus infection or vaccination. An IgM
demiological surveillance. Furthermore, no human- antibody capture ELISA is increasingly used as a
to-human transmission occurs and, as in the case of diagnostic test for WN virus infection, normally in
JE, human population immunity has no bearing on conjunction with the HI, which is used as a screen-
the suppression of epidemic activity itself and sus- ing test.
ceptible individuals would remain vulnerable irre- Virus isolation may be readily achieved from
spective of the proportion of immune individuals in blood from infected individuals, despite lower levels
the population. of viraemia. Suckling mice are particularly sensitive
to intracerebral inoculation, and cell culture of
mammalian origin as well as insect cell lines are
commonly used for virus isolation.
Clinical Features
Clinical Features
TICK-BORNE ENCEPHALITIS
Viral Features and Host Range The tick-borne encephalitides are a closely related
subgroup of viruses within the genus Flavivirus.
The virus displays a close relationship with Kunjin Although serological tests such as HI and CF give
virus and also JE virus. Two distinct strains can be considerable cross-reactivity with members of the
demonstrated, a New Guinea and an Australian flavivirus group, the tick-borne encephalitis (TBE)
variant. Individual isolations within the two groups subgroup of flavivirus are far more closely antigeni-
are, however, remarkably conserved. cally related to each other. In contrast to many of
their mosquito-borne arbovirus counterparts, the
tick-borne encephalitis subgroup is found almost
exclusively (with some exceptions such as louping ill
in the UK and Powassan in North America) in Asia
Epidemiology and eastern and central Europe. Within the sub-
group is the entity of TBE, which consists of two
The major maintenance vector is C. annulirostris subtypes: TBE—Central European subtype or Cen-
and a number of vertebrate hosts, chiefly wild birds, tral European encephalitis (CEE) virus; and TBE—
are involved in the transmission cycle. Domestic Far Eastern subtype or Russian spring—summer en-
animals are infected, but they probably do not play cephalitis (RSSE) virus. Others within the subgroup
any significant role as amplifying hosts. Outbreaks of the TBE are Omsk haemorrhagic fever virus,
have characteristically occurred in the summer Kyasanur Forest disease virus and Powassan virus,
months. which will be considered separately.
FLAVIVIRUSES 509
pathogenicity and plaque size. The two viruses dis-
play clearly distinguishable biological differences
from each other. The distribution of the two infec-
tions closely follows that of their arthropod vectors,
Ixodes ricinus in the case of CEE and Ixodes persul-
catus in the case of RSSE. The geographical dis-
tribution of CEE stretches from Russia in the east
through Finland and Sweden in the north, through
Germany to France in the west and down to Italy,
Greece and Yugoslavia in the south, encompassing
the central European countries in between (Figure
17.9). RSSE is found predominantly in the taiga (the
coniferous forest belt on the edge of the steppes and
the tundra region of Siberia) and in western Siberia.
Their pathogenic potentials are also different, CEE
causing a very much milder disease in experimen-
tally infected sheep and monkeys as compared to
RSSE, and this is reflected also in the clinical ex-
pression of their respective infections in humans.
Domestic animals, such as sheep, goats and cows,
infected with CEE excrete virus in their milk. The
virus is also relatively stable to low pH and, experi-
mentally, animals can rarely be infected by oral
inoculation. In humans, milk-borne transmission of
CEE through ingestion of goat, sheep or cow’s milk,
or dairy products made from them, such as cheese,
is an important route of acquisition of infection.
Epidemiology
Epidemiology
Figure 17.10 Geographic distribution of Omsk haemorrhagic
Infection has been limited to the Omsk region in the fever virus
forest-steppe landscape of western Siberia adjacent
to TBE endemic zones (Figure 17.10). The majority
of cases were recorded between 1945 and 1949. as well as laboratory safety measures. Because of
Between 1945 and 1958, a total of 1488 cases were extensive cross-reactivity, CEE vaccine would
recorded, with no typically transmitted cases occur- probably impart good protection.
ring since then. Occasional laboratory acquired in-
fections have been reported (Jelinkova-Skalova et
al., 1974). KYASANUR FOREST DISEASE
The virus can be readily isolated from patients’ Anderson, S.G. (1954) Murray Valley encephalitis and Austra-
lian disease. Journal of Hygiene, 52, 447.
blood using mice or cell culture, and antibodies can Anonymous (1980) Guide for Diagnosis, Treatment and Control of
be detected by HI, CF, NT or ELISA tests. Vector Dengue Haemorrhagic Fever, 2 ed. Technical Advisory Com-
control programmes have been carried out in the mittee on DHF for the Southeast Asian and Western Pacific
forest, especially spraying in the vicinity of dead Regions, World Health Organization, Geneva.
monkeys when these are encountered. A formalin- Besselaar, T.G. and Blackburn, N.K. (1988). Antigenic analysis
of West Nile virus strains using monoclonal antibodies. Ar-
inactivated vaccine has been prepared in India and chives of Virology, 99, 75—88.
immunization programmes in villages in the af- Bharadwaj, M., Prakash, V., Mathur, A. et al. (1991) Prognostic
fected areas have been carried out. significance of serum iron levels in cases of Japanese encephali-
FLAVIVIRUSES 513
tis. Postgraduate Medical Journal, 67, 247—249. 169, 512—518.
Cecilia, D. and Gould, E.A. (1991) Nucleotide changes respon- Konishi, E., Pincus, S., Fonseca, B.A.L. et al. (1991) Comparison
sible for loss of neuroinvasive-ness in Japanese encephalitis of protective immunity elicited by recombinant vaccinia vi-
virus neutralization-resistant mutants. Virology, 181, 70—77. ruses that synthesize E or NS1 of Japanese encephalitis virus.
Centers for Disease Control (1990). Yellow fever vaccine. Recom- Virology, 185, 401—410.
mendations of the Immunization Practices Advisory Commit- Kurane, I., Mady, B.J. and Ennis, F.A. (1991) Antibody-depend-
tee (ACIP). Morbidity and Mortality Weekly Report, 39, 1—6. ent enhancement of dengue virus infection. Reviews in Medical
Centers for Disease Control (1991a). Dengue epidemic—Peru, Virology, 1, 211—221.
1990. Morbidity and Mortality Weekly Report, 40, 145—147. Monath, T.P. (ed.) (1980) Epidemiology in St Louis Encephalitis,
Centers for Disease Control (1991b). St Louis encephalitis out- chap. 6. American Public Health Association, Washington
break—Arkansas, 1991. Morbidity and Mortality Weekly Re- DC.
port, 40, 605—607. Nothdurft, H,D., Jelinek, T., Marschang, A. et al. (1996) Adverse
Chen, W.R., Tesh, R.B. and Rico-Hesse, R. (1990) Genetic vari- reactions to Japanese encephalitis vaccine in travellers. Jour-
ation of Japanese encephalitis virus in nature. Journal of Gen- nal of Infection, 32, 119—122.
eral Virology, 71, 2915—2922. Ogata, A., Nagashima, K., Hall, W.W. et al. (1991) Japanese
Gresikova, M, Thiel, W., Batikova, M. et al. (1973) Haemag- encephalitis virus neurotropism is dependent on the degree of
glutination-inhibition antibodies against arboviruses in hu- neuronal maturity. Journal of Virology, 65, 880—886.
man sera from different regions in Steiermark (Austria). Zen- Pavri, K. (1989) Clinical, clinicopathologic and hematologic fea-
tralblatt für Bakteriologie, Parasitenkunde, Infektionskrank- tures of Kyasanur forest disease. Reviews of Infectious Dis-
heiten und Hygiene; Erste Abteilung: Originale, 224, 298. eases, 11, S854—S859.
Gubler, D.J. and Costa-Valez, A. (1991). A program for preven- Rodrigues, R.M. (1984) Epidemiology of Japanese encephalitis in
tion and control of epidemic dengue and dengue hemorrhagic India. In National Conference on Japanese Encephalitis, 1982.
fever in Puerto Rico and the US Virgin Islands. Bulletin of Indian Council of Medical Research Publication.
PAHO, 25, 237—247. Rosen, L. (1986) The pathogenesis of dengue haemorrhagic fever.
Halstead, S.B. (1981) Arboviruses of the Pacific and South-East South African Medical Journal (suppl.), 40—42.
Asia. In Textbook of Paediatric Infectious Diseases (eds R.D. Ruff, T.A., Eisen, D. and Fuller, A. (1991) Adverse reactions to
Feigin and J.D. Cherry), p. 1132. W.B. Saunders, Philadelphia. Japanese encephalitis vaccine. Lancet, 338, 881—882.
Halstead, S.B. (1984) Selective primary health care: strategies for Sharma, S., Mathur, A., Prakash, V. et al. (1991) Japanese en-
control of disease in the developing world. XI. Dengue. Re- cephalitis virus latency in peripheral blood lymphocytes and
views of Infectious Diseases, 6, 251. recurrence of infection in children. Clinical and Experimental
Halstead, S.B. (1989) Antibody, macrophages, dengue virus in- Immunology, 85, 85—89.
fection, shock, and hemorrhage: a pathogenic cascade. Re- Shiu, S.Y.W., Morikawa, S., Buckley, A. et al. (1991) 17D yellow
views of Infectious Diseases, 11 (suppl. 4), S830—S839. fever vaccine virus envelope protein expressed by recombinant
Iversson, L.B. (1980) Aspects of the encephalitis epidemic caused baculovirus is antigenically indistinguishable from authentic
by arbovirus in the Ribeira Valley, Sao Paulo, Brazil, during viral protein. Journal of General Virology, 72, 1451—1454.
1975—1978. Revista de Saude Publica, 14, 9—35. Smith, D.B., Pathirana, S., Davidson, F. et al. (1997) Journal of
Jacobs, M.G., Brook, M.G., Weir, W.R.C. et al. (1991) Dengue General Virology, 78, 321—328.
haemorrhagic fever: a risk of returning home. BMJ, 302, Stephenson, J.R. (1988) Flavivirus vaccines. Vaccine, 6, 471—482.
828—829. World Health Organization (1990) Yellow fever in 1988. Weekly
Jelinkova-Skalova, E., Tesarova, J., Buresova, V. et al. (1974) Epidemiological Record, 65, 213—220.
Laboratory infection with virus of Omsk haemorrhagic fever World Health Organization (1991) Yellow fever—epidemic in
with neurological and psychiatric symptomatology. Ceskos- Cameroon, 1990. Weekly Epidemiological Record, 66, 76—77.
lovenska Epidemiologie, Mikrobiologie, Immunologie World Health Organization (1996a) Inclusion of yellow fever
(PRAHA), 23, 290—293. vaccine in the EPI. Weekly Epidemiological Record, 71,
Jennings, A.D., Gibson, C.A., Miller, B.R. et al. (1994) Analysis of 181—185.
a yellow fever virus isolated from a fatal case of vaccine- World Health Organization (1996b) Yellow fever in 1994 and
associated human encephalitis. Journal of Infectious Diseases, 1995. Weekly Epidemiological Record, 71, 313—318.
MMMM
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
18
Bunyaviridae
Robert Swanepoel
University of Witwatersrand, Sandringham, South Africa
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
516 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 18.1 Negatively stained Rift Valley Fever virus particles from a patient’s serum. Note the surface spikes around the equator of
these virions, the diameter of which is 90—100 nm. (;300 000)
G1 G2 N L M S
Figure 18.2 Rift Valley fever viruses maturing within host cell vacuoles (;37 500)
of diversity in the family, but experimental evidence marily by budding through endoplasmic reticulum
indicates that there are genetic restraints and that into cytoplasmic vesicles which are presumed to
reassortment occurs with facility only between fuse with the plasma membrane to release virus, but
closely related members of bunyavirus serogroups it appears that virus can also bud directly from the
or different strains of an individual phlebovirus plasma membrane (Figure 18.2) (Anderson and
(Peters and LeDuc, 1991). Smith, 1987). Most of the viruses have been isolated
The L RNA segment of the genome codes for the and propagated by intracerebral inoculation of
viral transcriptase, and the M segment for the G suckling mice, but the hantaviruses produce chro-
proteins, as well as a non-structural protein NS in nic and inapparent infection of laboratory rodents.
the Bunyavirus and Phlebovirus genera. The S seg- Members of the family can be grown in a variety of
ment RNA codes for the N protein, as well as a cell cultures (Vero cells have been used most
non-structural protein NS in the bunyaviruses and commonly), but some of the viruses are non-
phleboviruses. Non-structural proteins have not as cytolytic so that their presence has to be demon-
yet been demonstrated in the nairoviruses or han- strated by immunofluorescence or similar means.
taviruses. The viral glycoproteins are responsible Hantaviruses are difficult to grow in vitro, and sev-
for recognition of receptor sites on susceptible cells, eral of the more recently discovered members of the
manifestation of viral haemagglutinating ability genus have not yet been adapted successfully to cell
and for inducing protective immune response in the cultures (Monroe et al., 1999).
host. The N protein induces production of and An abridged classification of the family, showing
reacts with complement-fixing antibody. selected members known to cause infection of hu-
mans and livestock in relation to their vectors and
distribution, is presented in Tables 18.2—18.6
(Karabatsos, 1985; Calisher and Karabatsos, 1989;
Biological Characteristics Peters and LeDuc, 1991; Calisher, 1991). In addi-
tion, high prevalences of antibody to many other
Viruses which attach to receptors on susceptible viruses have been found in particular human popu-
cells are internalized by endocytosis, and replica- lations, but conclusive evidence of infection or
tion occurs in the cytoplasm. Virions mature pri- disease association is lacking: antigenic cross-
518 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 18.2 Abridged classification of the genus Bunyavirus showing members known to cause infection of humans and
domestic animals. Information derived from sources cited in the text
Serogroup
ANTIGENIC COMPLEX
Virus (Synonym) Human infection
Subtype Putative vectors Livestock
Variety vectors Natural Laboratory Distribution
Serogroup
ANTIGENIC COMPLEX
Virus (Synonym) Human infection
Subtype Putative vectors Livestock
Variety vectors Natural Laboratory Distribution
GUAROA (1)
Guaroa Mosquitoes ; C. and S. America
One other complex (1)
Capim: five complexes (10)
Gamboa: two complexes (8)
Guama
GUAMA (4)
Guama Mosquitoes ; C. and S. America
CATU (1)
Catu Mosquitoes ; ; S. America
Three other complexes (7)
Koongol: one complex (2)
Minatitlan: one complex (2)
Nyando
NYANDO (2)
Nyando Mosquitoes ; Africa
Olifantsvlei: two complexes (5)
Patois: two complexes (7)
Simbu
AKABANE (2)
Akabane Ceratopogonids ; Asia, Africa,
MANZANILLA (12) Australasia
Oropouche Ceratopogonids ; ; S. America
Tinaroo Ceratopogonids ; Australasia
SHUNI (3)
Shuni Ceratopogonids ; ; Africa
Aino Ceratopogonids ; Asia, Autralasia
Four other complexes (8)
Tete: one complex (6)
Turlock: two complexes (5)
Ungrouped (4)
?Figures in parentheses indicate the total numbers of recognized members of the relevant taxon.
reactivity between viruses can complicate the inter- viruses appear to be transmitted by culicine mos-
pretation of survey findings or render it difficult to quitoes including aedines, but some are transmitted
arrive at a serological diagnosis in individual cases by anopheline mosquitoes. Simbu serogroup vi-
of disease. ruses are associated particularly with ce-
There is a broad tendency for antigenic grouping ratopogonid midges (Culicoides spp.), while the
to correlate with geographic distribution and with sandfly fever serogroup of phleboviruses (apart
the type of vector involved in transmission (Tables from Rift Valley fever and a few other mosquito-
18.2-18.4). Although a greater variety of arthropod- borne viruses), are associated with phlebotomids
borne members occurs in tropical and subtropical (sandflies). The Tete serogroup of bunyaviruses,
countries of Latin America and Africa, many vi- Uukuniemi serogroup of phleboviruses and the
ruses, including several important pathogens, occur nairoviruses are associated with ixodid and argasid
in temperate countries and the distribution of the ticks. Some viruses have been isolated from more
family extends to the Arctic region. Moreover, there than one type of vector.
are many instances on record of residents of temper-
ate countries which lack indigenous disease, acquir-
ing infection during travels abroad. Most of the
520 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Serogroup Bunyamwera
Serology
Bunyamwera, Ilesha, Germiston and
Serological diagnosis of infections is beset with the
Shokwe viruses
same problems of cross-reactivity which apply to
antigenic identification of isolates, and the difficul- Bunyamwera virus is widely distributed in Africa
BUNYAVIRIDAE 523
and has been isolated from aedines and other culi- virus, which had previously been isolated from culi-
cine mosquitoes, and/or human blood in Uganda, cine mosquitoes in Malaysia in 1955. Furthermore,
South Africa, Kenya, Nigeria, Central African Re- the name Olyka was provisionally used for Batai
public, Cameroon and Senegal. Antibody has been virus isolated from mosquitoes in the Ukraine, and
found in humans and/or domestic animals, rodents, Chittoor virus isolated from anophelines in India is
bats and subhuman primates in the same countries also considered to be closely related or identical to
as well as in Mozambique, Tanzania, Angola, Batai virus. Altogether, the cluster of viruses has
Congo, Egypt and Tunisia. However, some of the been isolated from anopheline and culicine mos-
antibody reactions recorded in surveys may have quitoes in Malaysia, Thailand, Cambodia, India,
been due to infection with related viruses. Despite Yugoslavia, Austria and the former USSR and
the widespread occurrence of antibody, human dis- Czechoslovakia. Antibody has been found in the
ease has seldom been recognized and the few cases same countries, as well as in Sri Lanka, Roumania,
which have been described include several labora- Hungary, Germany, Portugal and Finland in the
tory infections. Clinical findings included fever, sera of humans and/or birds, rodents, domestic ru-
maculopapular rash, arthralgia, neck stiffness, ver- minants and deer. The findings in seroprevalence
tigo and temporary loss of visual acuity. Severe studies suggest that human infection is seldom ac-
encephalitis occurred in experimental infection of a companied by overt disease, but febrile illness with
tumour patient. Infection was confirmed in patients malaise, myalgia, anorexia, and sometimes abdomi-
by isolation of virus from blood or demonstration nal pain, tonsillitis, cough and dyspnoea (associated
of an immune response. It seems likely that disease with lung infiltration), has been reported from
may be more common than at present realized. Czechoslovakia and Malaysia on the basis of
Ilesha virus has been isolated from the blood of serological diagnoses.
febrile humans in Nigeria, Uganda, Cameroon, and
the Central African Republic, and from anopheline
Cache Valley, Maguari, Fort Sherman,
mosquitoes in the latter country. In addition there
Tensaw and Wyeomyia viruses
is serological evidence that the virus occurs in
Senegal and Ghana. Few cases of disease have been Cache Valley virus has been isolated from culicine
reported, and these consisted of undifferentiated and anopheline mosquitoes from widely separated
febrile illness with a rash. Shokwe virus has been locations in the USA and from Jamaica, and anti-
isolated from mosquitoes, mainly aedines but also body has been found in the sera of humans and/or
from other culicines in South Africa, Senegal, Ivory horses, sheep, cattle, wild rodents, raccoons, deer
Coast and Kenya, and from rodents and the blood and monkeys in the USA, Canada, Trinidad and
of a febrile human in Ivory Coast. Little is known of Guyana. Despite the occurrence of high antibody
the pathogenic potential of the virus. Germiston prevalence rates, human disease has not been re-
virus has been isolated in South Africa, Zimbabwe, ported. Recently, however, the results of serological
Mozambique, Kenya and Uganda from Culex studies and pathogenicity trials, and the isolation of
rubinotus, a mosquito which selectively feeds on virus from a sentinel sheep, incriminated Cache Val-
rodents, and from myomorph rodents (rats and ley virus as the causative agent of an outbreak of
mice) in Uganda. Antibody has been found in the congenital abnormalities (hydranencephaly-ar-
sera of humans and/or cattle and rodents in South throgryposis syndrome) among sheep in Texas
Africa, Botswana, Namibia and Angola. Two lab- (Chung et al., 1990a; 1990b). Maguari, a subtype of
oratory infections have been reported; one with Cache Valley virus, has been isolated from mos-
undifferentiated febrile illness with rash, and the quitoes, mainly aedines, in Brazil, Argentina,
other with signs of mild encephalitis. Virus was French Guiana, Colombia and Trinidad, and from
isolated from the blood of the patients. horse blood in Guyana and Colombia. Antibody
has been found in the same countries as well as in
Peru, Surinam and Venezuela in the sera of humans
Batai virus
and/or horses, cattle, sheep, water buffalo and birds.
Calovo virus, first isolated from anopheline mos- Human disease has not been reported, but the virus
quitoes in 1960 in what was then Czechoslovakia, is is suspected of causing disease in horses. Fort Sher-
believed to be closely related or identical to Batai man, yet another subtype of Cache Valley virus, was
524 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
isolated from the blood of a patient with fever, represented 25% of arbovirus infections diagnosed
malaise, myalgia and sore throat in Panama, but no over a 13 year period. The patients suffered pros-
further information on the virus is available. Ten- trating illness with fever, headache, conjunctivitis,
saw virus has been isolated from several species of rash, epigastric pain and myalgia, and many had
anophelines in south-eastern USA, where antibody meningeal signs.
has been found in humans, dogs, cattle and rac- There have been numerous isolations of Pongola
coons. A single case of encephalitis was reported in virus from mosquitoes, mainly aedines and other
1973. Wyeomyia virus has been isolated from a culicines, in South Africa, Mozambique, Kenya,
range of culicine moquitoes in Colombia, Panama, Uganda, Ethiopia, Central African Republic and
French Guiana and Trinidad, and antibody has Ivory Coast. There has been one isolation of the
been found in human sera in Panama and Trinidad. virus from a febrile patient with headache and
The virus has been isolated once from the blood of a myalgia in Uganda (Kalunda et al., 1985). Neu-
patient with febrile illness in Panama. tralizing antibody has been found in humans in
South Africa, Mozambique, Botswana, Namibia
and Angola, and in cattle, sheep, goats and donkeys
Serogroup Anopheles A in South Africa, but interpretation of the findings is
complicated by the fact that there is unidirectional
cross-neutralization of Pongola virus by antibody to
Tacaiuma virus
its close relative, Bwamba virus. Moreover, the fact
Tacaiuma virus was isolated from the blood of a that most human isolates have reacted as Bwamba
sentinel monkey and from forest mosquitoes in the serotype while most mosquito isolates have reacted
Amazon region of Brazil, as well as from mos- as Pongola serotype merits further investigation,
quitoes in Argentina. Antibody has been found in particularly in view of a report that passage of a
humans in Brazil and French Guiana, and in Bwamba isolate in mosquitoes led to selection of
horses, rodents, bats and birds in Brazil. The virus virus reacting as Pongola serotype (Johnson et al.,
has been isolated once from the blood of a patient 1978).
with febrile illness in Brazil.
Serogroup C
Serogroup Bwamba
Apeu, Caraparu, Ossa, Madrid, Marituba,
Bwamba and Pongola viruses Murutucu, Restan, Nepuyo, Itaqui and
Oriboca viruses
Bwamba virus was originally isolated from blood
samples from nine road workers with febrile illness Apart from one virus which occurs in Florida, USA,
in Uganda, and subsequently from eight febrile pa- all of the known members of serogroup C occur in
tients in Nigeria, three in the Central African Re- Central and South America and tend to be asso-
public and one in Kenya, and from anopheline mos- ciated with tropical forests. The viruses named
quitoes in Uganda, Nigeria and Senegal. Antibody above have all been isolated from the blood of
was found in human sera in Uganda, Tanzania, febrile humans, and variously from sentinel mon-
Mozambique, South Africa, Botswana, Angola, keys, rodents, occasional marsupials and fruit bats,
Congo, Nigeria and Guinea; generally with very and from a range of culicine mosquitoes in Brazil,
high prevalence, up to 97% in some populations, Surinam, French Guiana, Guatamala, Honduras,
and including both children and adults. Antibody Trinidad, Panama or Mexico. Serological evidence
was also found in donkeys and a bird in South suggests that some of these viruses may also occur
Africa. Bwamba virus appears to be an important in Venezuela, Colombia and Peru. Pairs of the vi-
pathogen and the eight isolations in the Nigerian ruses which are closely related antigenically may
series represented 5% of all arbovirus isolations circulate in the same geographic location yet oc-
from febrile patients over a 7 year period, while 18 cupy separate ecologic niches. For instance, one
diagnoses (virological and serological) made in virus circulates in arboreal monkeys and mos-
similar patients in the Central African Republic quitoes which feed in the canopy layer, while a
BUNYAVIRIDAE 525
closely related virus circulates in rodents and mos- and the infection is seen most commonly in forest
quitoes which feed at the level of the forest floor. workers, and children who enter woodlands for
Other pairs of closely related viruses coexist in the recreational purposes, but also occurs focally in
same habitat simply by utilizing different vectors. rural and suburban residents. Males are more
Sporadic infections occur in persons who enter for- commonly infected than females, among both
ests. No large outbreaks of disease have been re- children and adults. Seroprevalence surveys and
ported, but disease is observed when susceptible prospective studies indicate that most infections are
outsiders such as military personnel enter endemic inapparent or benign. Probably less than 1% of
regions. Laboratory infections are relatively infected adults develop encephalitis, but the inci-
common. Disease, which lasts for up to a week and dence may be up to four times greater in young
runs a benign course, is characterized by fever, children.
rigors, headache, photophobia, conjunctivitis, After an incubation period of 3—7 days there is
tachycardia, myalgia, arthralgia, prostration, leuco- sudden onset of fever, headache, lethargy, nausea
penia and occasionally pain in the right upper and vomiting, pharyngitis and sometimes respir-
quadrant of the abdomen and jaundice. atory illness. There is seldom a cutaneous rash. In
mild cases of overt disease there may be transient
meningismus and disorientation, and recovery
Serogroup California within 1 week. In severe disease there may be
greater disturbance of consciousness, aphasia,
tremors, chorea, positive Babinski signs and other
California encephalitis, La Crosse,
abnormal reflexes, and hemiparesis in about 20% of
Snowshoe hare, Jamestown Canyon and
patients. Seizures may occur from the second day of
Keystone viruses
illness onwards: in about half of severely ill patients
California encephalitis virus, which is distributed there may be generalized, life-threatening convul-
across the western USA and into Canada, was iso- sions, and in a further 25% there are focal convul-
lated from mosquitoes in the early 1940s and short- sions associated with frontal or parietal brain
ly thereafter serological evidence was produced to lesions. Approximately one third of severely ill pa-
indicate that it causes encephalitis. However, from tients become comatose. There may be marked leu-
the mid 1960s onwards it became clear that most cocytosis, and examination of cerebrospinal fluid
cases of what are loosely termed ‘California en- reveals elevated mononuclear and polymorphonuc-
cephalitis’ are in fact due to infection with the La lear cell counts, but protein and glucose levels tend
Crosse subtype of virus, and that this agent is re- to remain normal. Electroencephalograms show
sponsible for the majority of the approximately 100 generalized slow-wave activity or localized changes
cases of arbovirus encephalitis diagnosed in the and sometimes epileptiform discharges. Treatment
USA annually, except in years when there are epi- is symptomatic and includes monitoring and con-
demics of St Louis encephalitis (a flavivirus). trol of intracranial pressure, and vigorous anti-con-
Most cases of disease due to La Crosse virus are vulsant therapy as indicated. Less than 1% of pa-
recorded in the mid-west states of Wisconsin, Iowa, tients with severe disease succumb and most are
Indiana, Minnesota and Ohio, but the virus is wide- discharged from hospital after about two weeks of
ly distributed and the infection is probably under- illness, but may remain irritable and emotionally
diagnosed elsewhere in the USA. The principal vec- labile for a few weeks. There are seldom residua, but
tor is Aedes triseriatus, a tree-hole breeding patients who suffer seizures in the acute illness may
mosquito, and accordingly the virus tends to be have recurrent convulsions over a period of 1 or
focally distributed in woodlands, but also occurs in more years, and lasting hemiparesis occurs in about
suburban situations where water that collects in 1% of patients. Virus has not been isolated from
discarded containers such as motor vehicle tyres, throat swabs, blood, stools or cerebrospinal fluid,
affords mosquito breeding sites. The virus is passed and only with difficulty from brain specimens. His-
transovarially in the vector and overwinters in mos- topathological lesions are not pathognomonic and
quito eggs; infection is amplified in the succeeding include cerebral oedema, perivascular cuffing and
spring and summer in small mammals such as chip- focal gliosis in grey matter. A variety of methods are
munks and squirrels. The vector is a diurnal feeder used for making a serological diagnosis, but demon-
526 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
stration of IgM antibody activity by means of en- or whether, as seems more likely, a cluster of closely
zyme-linked immunoassay holds greatest promise related viruses, subtypes or varieties is involved.
as a rapid diagnostic technique. There is no vaccine. The epidemiology of the disease has been studied
Snowshoe hare is a mosquito-borne subtype of most intensively in Moravia where seasonal flood-
California encephalitis virus with a distribution ex- ing of level woodlands provides extensive breeding
tending from north-western USA, across most of sites for mosquitoes. The virus is transmitted trans-
Canada to Alaska. The natural host appears to be ovarially in Aedes vexans which overwinters as eggs,
the snowshoe hare, Lepus americanus, and serologi- and in Culiseta annulata which overwinters as
cal evidence suggests that the virus is occasionally larvae. Amplification of infection in spring occurs in
associated with encephalitis in children and adults. small mammals such as hedgehogs, hares and rab-
Jamestown Canyon is a mosquito-borne virus which bits, as well as in domestic animals such as horses.
occurs widely in the USA, and high prevalence rates Hedgehogs may themselves serve as reservoir hosts
of antibody are found in white-tailed deer, and in for overwintering of virus in instances where they
humans where there are high concentrations of the undergo chronic infection during hibernation, with
deer. Serological evidence of an association with viraemia which persists for a few days after awaken-
encephalitis in humans has been found from the ing. Once infection of vertebrates occurs in summer
early 1980s onwards (Grimstad et al., 1986). Since other species of mosquito also become infected and
antigens and tests commonly used for the serodiag- serve as vectors for transmission of the virus.
nosis of ‘California encephalitis’ fail to allow re- Seroprevalence surveys indicate that infection is
sponse to Jamestown Canyon virus to be distin- much more common than overt disease in rural
guished from response to other members of the residents of Moravia. Nevertheless, the infection
serogroup, it is felt that the infection has probably accounts for up to 20% of patients hospitalized
been missed or underdiagnosed as a cause of en- with febrile illness in the region, including both
cephalitis in the past, both in the mid-west states adults and children. Patients may present with un-
and elsewhere where the deer occurs. In contrast to differentiated febrile illness with leucocytosis, but
La Crosse virus, Jamestown Canyon appears to pharyngitis, cough, and chest pain and infiltration
cause encephalitis more frequently in adults than in (demonstrable by X-ray imaging) may predomi-
children, and nervous disease is often preceded by nate, or gastrointestinal symptoms such as nausea,
respiratory illness. Keystone virus is associated with vomiting and abdominal pain may be dominant.
swamp-breeding aedines and cotton rats and rab- Aseptic meningitis occurs in a minority of patients
bits in south-eastern USA, and has been known to but fatal disease is unknown. Virus can be isolated
cause inapparent accidental infection in the labora- from blood early in the illness, but the infection is
tory. usually diagnosed by demonstration of an antibody
response. There is no vaccine.
Inkoo virus was isolated from aedine mosquitoes
Tahyna and Inkoo viruses
in Finland. Distribution of the virus extends to the
Tahyna virus is widely distributed in countries of Lapland region in the north of the country, and
central Europe, including Yugoslavia, Germany antibody occurs in the sera of humans, cattle, deer
and Italy, with antibody prevalence rates being par- and hares. Antibody prevalence rates of up to 25%
ticularly high in the Rhone Valley of France, the have been recorded in humans, and a few cases of
Danube basin in Austria, and in the southern febrile illness have been confirmed serologically.
Moravia region of the former Czechoslovakia,
where up to 95% of adults may be immune in some
Guaroa virus
communities. Viruses described as being Tahyna-
like have been isolated in the former USSR, while Guaroa virus has been isolated from twelve febrile
Lumbo virus, which was isolated from saltwater- patients in Colombia and the Amazon region of
breeding mosquitoes on the coast of Mozambique, Brazil, and from anopheline mosquitoes in Panama
is considered to be indistinguishable from Tahyna and Colombia. Antibody has been found in human
virus. Antibody to Tahyna virus has also been found sera in Colombia, Brazil, Argentina and Peru. Dis-
in southern China and Sri Lanka. It is not yet clear ease ascribed to infection with the virus was charac-
whether a single virus occurs throughout this range terized by fever, headache, myalgia, arthralgia,
BUNYAVIRIDAE 527
prostration and leucopenia. Virus was isolated from unidentified vector, and is introduced into urban
the blood of the patients, and from a liver biopsy in settings by infected travellers or by extension from
one instance. the sylvatic cycle. Humans serve as amplifier hosts
in the urban cycle, and the vector is the Culicoides
midge which breeds in decaying waste from tropical
Serogroup Guama agricultural products. Epidemics occur when there
are large concentrations of vectors and susceptible
Guama and Catu viruses humans. Aerosol infection is suspected to have oc-
curred in laboratory workers. The incubation per-
Catu virus has been isolated from sentinel monkeys iod is 4—8 days, and there is sudden onset of fever,
in Brazil, febrile humans and forest rodents in Brazil chills, headache, myalgia, arthralgia and prostra-
and Trinidad, and from culicine and anopheline tion. There may be a rash, and occasionally signs of
mosquitoes in the same two countries plus French meningitis or encephalitis, but there are no deaths
Guiana. Guama virus has been isolated from febrile or sequelae. Viraemia lasts from 2 to 5 days, as does
humans and sentinel monkeys in Brazil, and from illness, but myalgia persists for a further 3—5 days,
rodents and/or culicine mosquitoes in Brazil, and strenuous exertion in early convalescence can
Trinidad, Surinam, French Guiana and Panama. precipitate a relapse of symptoms.
The two viruses cause isolated cases of benign dis-
ease characterized by fever, headache, myalgia and
leucopenia, in persons who enter tropical forests. Akabane, Aino, Tinaroo and Shuni viruses
Catu has also been associated with laboratory infec- Most members of the Simbu serogroup do not oc-
tion. cur in the Americas, but are widely distributed in
Africa, Asia and Australasia. Of these latter viruses,
only Shuni has been marginally implicated in caus-
Serogroup Nyando ing human disease, but a few have been in-
criminated as causative agents in large outbreaks of
Nyando virus abortion, stillbirth and congenital defects (hy-
Nyando virus has been isolated from anopheline dranencephaly—arthrogryposis syndrome) in do-
and aedine mosquitoes in Kenya, Central African mestic ruminants, particularly sheep and cattle, and
Republic and Senegal. Antibody has been found in the suspicion exists that most members of the serog-
human sera in Kenya and Uganda, and the virus roup have the potential for producing this type of
was isolated from the blood of a single human disease. Akabane has been incriminated in out-
patient with biphasic fever, myalgia and vomiting in breaks of the disease in Japan, Australia and Israel,
the Central African Republic. Aino in Japan and Australia, Tinaroo in Australia,
and Peaton virus has been shown to be teratogenic
in experimental infections in Australia. The viruses
are transmitted by species of Culicoides midges
Serogroup Simbu
which mostly breed in dung, independently of avail-
able surface water, but which are favoured by the
Oropouche virus
humid conditions created by heavy rainfall. The
Oropouche virus was originally isolated from the natural hosts of the viruses are unknown, but anti-
blood of a febrile patient in Trinidad, but large bodies have been found in wild herbivores. Infec-
epidemics involving thousands of people have oc- tion is usually inapparent in domestic ruminants.
curred exclusively in northern Brazil over the past However, if infection occurs in pregnant animals at
20 years. In addition to humans, the virus has been critical stages of gestation, there may be embryonal
isolated from a sloth, a few species of culicine mos- or foetal death, or arrestation of brain development
quitoes and the midge Culicoides paraensis. Anti- (hydranencephaly), and the consequent lack of
body has been found in the sera of humans and/or trophic effect of nervous stimulation on skeletal
monkeys and birds in Brazil, Trinidad and Colom- muscles of the foetus results in postural defects of
bia. Virus is thought to be maintained in nature by the limbs, with joints locked in flexion (arthrog-
circulation in forest primates, sloths or birds and an ryposis). Once the stage of organogenesis in gesta-
528 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
tion is passed, foetuses are less susceptible to the feeders, but will feed in dark rooms where they rest
harmful effects of infection (the timing varies with during daylight hours. Human infection rates re-
length of gestation in different species). corded in outbreaks range from 3—75%, but the
Shuni virus has been isolated from cattle, sheep, attack rate varies focally and is influenced by back-
midges and once from the blood of a febrile human ground immunity in the human population. Large
in Nigeria, and from cattle and mosquitoes in South epidemics have often occurred in association with
Africa. It was also isolated from the brain of a horse socioeconomic upheavals, wars or natural disasters
with histopathological lesions of meningoen- such as earthquakes which create ideal breeding
cephalitis, which was submitted for laboratory conditions for sandflies and/or lead to widespread
examination for suspected rabies in Zimbabwe exposure of susceptible humans to sandflies.
(Foggin and Swanepoel, 1977). Experimental evidence suggests that most sand-
fly fever infections are symptomatic. Typically,
there is sudden onset of fever of 2—4 days duration,
GENUS PHLEBOVIRUS severe headache, sore eyes and photophobia, myal-
gia, arthralgia, anorexia and malaise. Occasionally
Serogroup Sandfly fever there may be sore throat, nausea and vomiting,
abdominal pain and diarrhoea, epistaxis and dizzi-
ness. Patients may have injected conjunctivae and a
Sandfly fever Naples, Sandfly fever Sicilian
flushed appearance, but there is seldom a rash, and
and Toscana viruses
meningeal signs are rare. The disease may be milder
Sandfly fever (also known as phlebotomus fever or in children. Treatment is symptomatic. Recovery is
pappataci fever from the vector, Phlebotomus complete and no deaths have been recorded. There
papatasi) has been known for at least two centuries appears to be lifelong immunity to the homologous
as a febrile illness encountered by armies invading virus. The diagnosis can be confirmed by isolation
the Mediterranean basin. It was demonstrated of virus from blood taken in acute illness, or demon-
shortly after the turn of the century that the disease stration of IgM antibody activity, or rising anti-
was caused by a virus transmitted by sandflies, but body titres in convalescence. Prevention of infection
it was only during the second world war that it was includes the use of insect repellants, but the treat-
shown that there are in fact two distinct viruses and ment of walls (sandfly resting sites) with residual
that there is no cross-immunity. Between them, the insecticides is highly effective.
two viruses are known to occur in Morocco, Tu- Toscana virus was first isolated in 1971 in Tus-
nisia, Egypt, Sudan, Somalia, Italy, Greece, Yugos- cany, Italy, from Phlebotomus perniciosus, a sandfly
lavia, Turkey, the former USSR, Israel, Saudi Ara- which breeds in forest litter. Antibody was found to
bia, Iraq, Iran, Pakistan, India and Bangladesh, and be common in the sera of rural and suburban resi-
are probably present in many intervening countries. dents of the region, and an association was estab-
Throughout this range Phlebotomus papatasi is the lished between the occurrence of aseptic meningitis
vector, and it breeds in moist soil in dark niches and serological evidence of infection with Toscana
such as in rubble, drains, cracks in soil and in ani- virus. Since then several cases of meningitis due to
mal burrows, equally successfully in large cities as in the infection have been encountered regularly each
remote rural locations. The viruses are transmitted year in summer months in the endemic region, and
transovarially in the vector, which overwinters in these can sometimes be diagnosed by isolation of
the larval stage, and adult flies emerge to assume virus from cerebrospinal fluid in acute illness, but
biting activity in summer. High infection rates have usually by demonstrating IgM antibody activity or
been recorded in newly emerged sandflies and it is rising antibody titres in sera taken during convales-
not known whether amplification in vertebrates is cence. The virus is transmitted transovarially in the
essential to ensure perpetuation of the viruses, but vector, P. perniciosus, and no vertebrate mainte-
humans develop sufficiently intense viraemia to nance host has been identified, but antibody has
serve as source for the infection of the vector. No been found in rodent sera, and the virus has been
wild vertebrate hosts of the viruses are known, but isolated from an insectivorous bat. The vector is
antibody has been found in gerbils (whose burrows widely distributed in Europe and the virus has been
are utilized by the vector). Sandflies are nocturnal isolated from cerebrospinal fluid from a meningitis
BUNYAVIRIDAE 529
Table 18.3 Abridged classification of the genus Phlebovirus showing members known to cause infection of humans and domestic
animals. Information derived from sources cited in the text
Sandfly fever
SANDFLY FEVER NAPLES (4)?
Sandfly fever Naples Phlebotomids ; Europe, Africa,
Asia
Toscana Phlebotomids ; Europe
CANDIRU (6)
Alenquer Phlebotomids? ; S. America
Candiru Phlebotomids? ; S. America
PUNTA TORO (2)
Punta Toro Phlebotomids ; C. America
RIFT VALLEY FEVER (3)
Rift Valley fever (Zinga) Mosquitoes ; ; ; Africa
Four other complexes (8)
Unassigned to complex (22)
Sandfly fever Sicilian Phlebotomids ; Europe, Africa,
Asia
Chagres Phlebotomids ; C. America
Uukuniemi
UUKUNIEMI (13)
Uukuniemi Ixodids ; Europe
Zaliv Terpiniya Ixodids ; Asia
Figures in parentheses indicate the total numbers of members of the relevant taxon.
patient in Portugal, so the disease may occur more Rift Valley fever virus
widely than at present recognized.
The literature on Rift Valley fever has been re-
viewed extensively (Henning, 1956; Weiss, 1957;
Easterday, 1965; Peters and Meegan, 1981; Shim-
shony and Barzilai, 1983; Meegan and Bailey, 1989;
Alenquer, Candiru, Punta Toro and Chagres Swanepoel and Coetzer, 1994). It is an acute disease
viruses of domestic ruminants in mainland Africa and
Madagascar, caused by a mosquito-borne virus and
Alenquer and Candiru viruses were isolated from the
characterized by necrotic hepatitis and a haemor-
blood of febrile patients in the Amazon region of
rhagic state, but infections are frequently inappar-
Brazil, but otherwise little is known of their biology,
ent or mild. Large outbreaks of the disease in sheep,
and it is surmised that they are transmitted by
cattle and goats are distinguished by heavy mortal-
phlebotomid flies. Punta Toro and Chagres viruses
ity among newborn animals and abortion in preg-
were isolated from febrile patients and from
nant animals. Humans become infected from con-
phlebotomids (Lutzomyia spp.) in Panama. These
tact with tissues of infected animals or from
two viruses are known to be transmitted trans-
mosquito bite, and usually develop mild to moder-
ovarially in phlebotomids, and antibodies to them
ately severe febrile illness, but severe complications
have been found in primates, sloths, porcupines and
occur in a small proportion of patients.
other rodents. Disease thus far associated with all
The disease was first recognized in the Rift Valley
four viruses fits the description of classical sandfly
in Kenya at the turn of the century, but the causa-
fever, with the difference that epidemics are un-
tive agent was not isolated until 1930. Since then
known and only isolated cases have been seen in
large outbreaks of the disease have been recorded in
persons who entered tropical forests for occupa-
Kenya, South Africa, Namibia, Mozambique, Zim-
tional or recreational purposes.
530 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
babwe, Zambia, Sudan, Egypt, Mauritania and which constitute drainage channels for surrounding
Senegal, while lesser outbreaks, periodic isolations high ground, and which are flooded by seepage after
of virus or serological evidence of infection have heavy rains. Vleis correspond to what are termed
been recorded in Angola, Botswana, Burkina Faso, dambos in the livestock rearing areas of central and
Cameroon, Central African Republic, Chad, eastern Africa. Sustained monitoring in Zimbabwe
Gabon, Guinea, Madagascar, Malawi, Mali, revealed that a low level of virus transmission to
Nigeria, Somalia, Tanzania, Uganda and Zaire. livestock occurred each year in the same areas
Epidemics may be extremely severe and, for where epidemics occurred. The generation of epi-
example, it is estimated that 500 000 ewes aborted demics, therefore, was associated with the simulta-
and a further 100 000 sheep died in the first out- neous intensification of virus activity over vast live-
break of the disease to be recognized in South Afri- stock rearing areas where it was already present,
ca in 1950—51. rather than lateral spread from cryptic endemic foci.
Prior to the 1970s, epidemics were seen only in Comparison of the distributions of canopy forests
eastern and southern Africa, where they tend to and vleis in Zimbabwe, plotted from satellite images
occur at irregular intervals of 5—15 years or longer and aerial photographs, with the distribution deter-
when above average rainfall favours the breeding of mined for endemic Rift Valley fever, revealed re-
the mosquito vectors. Meteorological conditions markable overlap between the endemic areas and
conducive to the occurrence of epidemics usually areas where vleis were common.
prevail over large tracts of Africa, so there has been A major advance in the understanding of the
some tendency for outbreaks in adjacent territories epidemiology of the disease was made when the
such as Zimbabwe and Mozambique, Kenya and virus was isolated from unfed Aedes mcintoshi mos-
Tanzania, or South Africa and Namibia to coincide. quitoes (:Aedes lineatopennis sensu lato) hatched
The fate of the virus during interepidemic periods in dambos on a ranch in Kenya during in-
was unknown for decades, but on the basis of obser- terepidemic periods in 1982 and 1984, confirming
vations made in Uganda, Kenya and South Africa, that the virus is endemic in livestock rearing areas
it was widely accepted that the virus was endemic in and indicating that it appears to be maintained by
indigenous forests which extend in broken fashion transovarial transmission in aedines. The available
from East Africa to the coastal regions of South evidence suggests that in Zimbabwe, as in Kenya,
Africa. The virus was thought to circulate in Eret- Aedes mcintoshi is the most important maintenance
mapodites spp. mosquitoes and unknown verte- vector of the virus while Aedes dentatus is probably
brates in the forests, and to spread in seasons of also a maintenance vector; the same two species and
exceptionally heavy rainfall to livestock-rearing possibly Aedes unidentatus and Aedes juppi are
areas where the vectors were believed to be flood- maintenance vectors on the inland plateau of South
water-breeding aedine mosquitoes of the subgenera Africa. Heavy rainfall and the humid conditions
Aedimorphus and Neomelaniconion, which attach which prevail during epidemics favour the breeding
their eggs to vegetation at the edge of stagnant of other biting insects besides aedine mosquitoes.
surface water. In contrast to other culicine mos- Following extensive flooding of aedine breeding
quitoes, it is obligatory that the eggs of aedines be sites, significant numbers of livestock become infec-
subjected to a period of drying as the water recedes ted and circulate high levels of virus in their blood
before they will hatch on being wetted again when during the acute stage of infection. Other culicines
next the area floods. Thus, the aedine mosquitoes and anopheline mosquitoes then become infected
overwinter as eggs which can survive for long per- and serve as epidemic vectors, particularly Culex
iods in dried mud, possibly for several seasons if the theileri in southern Africa, and biting flies such as
area remains dry. midges, phlebotomids, stomoxids and simulids
On the inland plateau of South Africa, where serve as mechanical transmitters of infection. Al-
sheep rearing predominates, surface water gathers though contagion has been demonstrated on occa-
after heavy rains in undrained shallow depressions sion under artificial conditions, non-vectorial trans-
(pans) and farm dams which afford ideal breeding mission is not considered to be important in
environments for aedines. On the watershed pla- livestock, as opposed to humans. Epidemics gen-
teau of Zimbabwe, where cattle farming predomi- erally become evident in late summer after there has
nates, aedines breed in vleis—low-lying grassy areas been an initial increase in vector populations and in
BUNYAVIRIDAE 531
circulation of virus, and terminate in late autumn African countries had long been known from anti-
when the onset of cold weather depresses vector body studies, and there had been periodic isolations
activity, or when most animals are immune follow- of the virus in West Africa, where it was sometimes
ing natural infection, or after there has been success- reported as Zinga virus, which is now known to be
ful intervention with vaccine. identical to Rift Valley fever virus. Various theories
Antibody surveys and laboratory studies have were advanced to account for the first known ap-
failed to prove that the virus is maintained in trans- pearance of the virus in Egypt in 1977, including the
mission cycles in rodents, birds, monkeys, baboons carriage of infected mosquitoes from the Sudan at
or other wild vertebrates, although it is felt that wild high altitude by prevailing winds associated with
ruminants could play a role similar to their domes- the inter-tropical convergence zone. The introduc-
tic counterparts in areas where they predominate. It tion of the virus through the transportation of infec-
is also believed that the possibility of endemicity of ted sheep and cattle on the Nile or overland from
the virus in forests cannot be dismissed entirely, and northern Sudan to markets in southern Egypt was
merits further investigation. considered to have been the strongest possibility,
It was recognized from the time of the original and the movement of slaughter animals by sea
investigations in Kenya that febrile illness in hu- could account for the evidence of infection detected
mans accompanied outbreaks of disease in live- in the northern and eastern coastal areas of Egypt.
stock, and that some patients experienced transient Although transportation on some routes would
loss of visual acuity, but the occurrence of serious take a long time in relation to the course of the
ocular sequelae was first reported in the 1950—1951 infection, Rift Valley fever virus has been shown to
epidemic in South Africa. Human deaths following persist for prolonged periods in various organs of
natural infection were first recorded in South Africa sheep, particularly the spleen, for up to 21 days after
during the epidemic of 1974—1976 when seven pa- infection. The same could be true for goats and
tients are known to have died of encephalitis and cattle, or even the camels brought in by overland
haemorrhagic fever associated with necrotic hepati- caravan routes. It is believed that humans slaugh-
tis. Subsequently deaths were also observed in Zim- tering or handling the tissues of such animals could
babwe. have become infected and served as the amplifying
Outbreaks of Rift Valley fever were reported in hosts for the infection of mosquitoes since the main
the Sudan in 1973 and 1976. In 1977 and 1978 a vector in the Egyptian epidemic, Culex pipiens, is
major epidemic occurred along the Nile delta and known to be peridomestic and anthropophilic. In at
valley in Egypt, causing an unprecedented number least one instance there were indications that hu-
of human infections and deaths, as well as numer- man infections centred on a location where intro-
ous deaths and abortions in sheep and cattle and duced camels were slaughtered.
some losses in goats, water buffaloes and camels. The occurrence of the epidemic in Egypt raised
Estimates of the number of human cases of disease the spectre that Rift Valley fever could be introduc-
range from 18 000 to more than 200 000 with at least ed to the mainland of Eurasia, and the possibility
598 deaths occurring from encephalitis and/or was underscored by the fact that the virus is appar-
haemorrhagic fever. A severe epidemic occurred in ently capable of utilizing a wide range of mos-
1987 in the Senegal river basin of southern quitoes as vectors. Extensive preventive vaccination
Mauritania and northern Senegal. In Mauritania of livestock was undertaken at the time in the Sinai
alone an estimated 224 human patients died of the peninsula and Israel. However, only isolated out-
disease, and there was a high rate of abortion in breaks of Rift Valley fever were recorded in Egypt in
sheep and goats. 1979 and 1980, and thereafter the country remained
The outbreaks of Rift Valley fever which occur- free of the disease for twelve years until ocular
red in North and West Africa differed in many complications of the infection in humans, and abor-
respects from the pattern of disease which had tions in cattle and water buffalo, were noted in the
hitherto been observed in sub-saharan Africa; in Aswan Governate in May 1993. On this occasion
particular they occurred independently of rainfall in there was not the same tendency for an explosive
arid countries, apparently in association with vec- outbreak of the disease to occur as in 1977—1978,
tors which breed in large rivers and dams. The but by October 1993 infections of humans and live-
presence of the virus in the Sudan and certain west stock, including sheep, had been recognized across
532 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
the length of the country in Sharqiya, Giza and El becoming infected while investigating the disease in
Faiyum Governates, and further infections were the field or laboratory, and the first known human
observed in 1994 (Anonymous, 1993; 1994; Arthur fatality was recorded in 1934 in a laboratory
et al., 1993; Botros et al., 1997). worker, but since the infection was complicated by
Fears have also been expressed in the past that thrombophlebitis and the patient died from pul-
the virus could be transported to Saudi Arabia with monary embolism, the potential lethality of the vi-
animals exported from Africa for ritual slaughter on rus for man was overlooked until fatal infections
the annual Islamic pilgrimage to Mecca. In West were recognized during the 1974—1976 epidemic in
Africa, Rift Valley fever virus remains active in the South Africa. The results of surveys following epi-
vicinity of the Manantali and Diama dams on the demics in southern Africa indicated that 9—15% of
Senegal river where the disease occurred in 1987, farm residents became infected, with a slight pre-
and with the human and livestock populations be- ponderance of adult males, although it appeared
ing migratory, there is poor herd immunity. that housewives also gained infection from hand-
From late October 1997 to February 1998, a ling fresh meat.
large outbreak of Rift Valley fever occurred in No outbreaks of the disease have been recognized
northeastern Kenya and adjoining southern in urban consumer populations and it is surmised
Somalia, following the occurrence of heavy rains that the fall in pH associated with the maturation of
and extensive flooding in what is essentially an arid meat in abattoirs is deleterious to the virus. More-
area where people had been receiving food relief over, highest infection rates were found in workers
owing to the extreme drought conditions that had in the by-products sections of abattoirs in Zim-
prevailed in the preceding 2 years (Anonymous, babwe and the implication is that the carcases of
1998). There were heavy losses of livestock and an infected animals which reach abattoirs are generally
estimated 500 human deaths, but many of the recognized as being diseased and are condemned as
deaths could have been to due the appearance of unfit for human consumption, and are then steriliz-
malaria in an area not normally affected by this ed in the process of preparing carcase meal which is
disease. Nevertheless, communications were severe- used as fertilizer.
ly hampered and starving people resorted to the Human infection presumably results from con-
slaughter and consumption of sick animals on a tact of virus with abraded skin, wounds or mucous
large scale. It was subsequently established that membranes, but aerosol and intranasal infection
extensive outbreaks of Rift Valley fever had also have been demonstrated experimentally and cir-
occurred elsewhere in Kenya and northern Tan- cumstantial evidence suggests that aerosols have
zania following heavy rains in the region, and a few been involved in some human infections in the lab-
human deaths in southern Kenya were also as- oratory, and in the field during the Egyptian epi-
cribed to the disease. demic. Many infections in Egypt are thought to
In contrast to the main vector in the Egyptian have resulted from the slaughter of infected animals
epidemic of 1977—1978, the principal mosquito vec- outside of abattoirs, and the fact that the mosquito
tors of Rift Valley fever virus in sub-Saharan Africa vector was anthropophilic is thought to explain the
tend to be zoophilic and sylvatic, with the result high incidence of infection which occurred in people
that humans become infected mainly from contact of all ages and diverse occupations. Low concentra-
with animal tissues, although there are instances tions of virus have been found in milk and body
where no such history can be obtained and it must fluids such as saliva and nasal discharges of sheep
be assumed that infection has resulted from mos- and cattle, and it appears that there may have been
quito bite. Occasional infections diagnosed in tour- a connection between human infection and con-
ists from abroad who visited countries in Africa are sumption of raw milk in Mauritania. In view of the
also thought to have resulted from mosquito bite. intense viraemia which occurs in humans and the
Generally, persons who become infected are in- fact that virus has been isolated from throat wash-
volved in the livestock industry, such as farmers ings, it is curious that there are no records of person
who assist in dystocia of livestock, farm labourers to person transmission of infection.
who salvage carcases for human consumption, vet- Despite the sudden and dramatic change per-
erinarians and their assistants, and abattoir ceived in the nature of the human disease in the
workers. There are numerous reports of humans mid-1970s, it was deduced from the 598 reported
BUNYAVIRIDAE 533
deaths and 200 000 estimated cases of disease that ual scarring of the retina, but in instances of severe
Rift Valley fever had a case fatality rate of less than haemorrhage and detachment of the retina there
1% in Egypt where a high prevalence of schis- may be permanent uni- or bilateral blindness.
tosomiasis may have predisposed the population to Probably less than 1% of human patients devel-
severe liver disease. The fatality rate may even have op the haemorrhagic and/or encephalitic forms of
been lower in relation to total infections, since an the disease. Underlying liver disease may predis-
antibody prevalence rate of 30% was detected and pose to the haemorrhagic form of the illness. The
the human population estimated at one to three haemorrhagic syndrome starts with sudden onset of
million in the areas affected by the epidemic. On the febrile illness similar to the benign disease, but with-
other hand, remarkably high estimates of 5 and in 2—4 days there may be development of a petechial
14% were made for case fatality rates in two separ- rash, purpura, ecchymoses and extensive subcu-
ate populations in the 1987 epidemic in Mauritania, taneous haemorrhages, bleeding from needle punc-
on the basis of the proportion of IgM antibody- ture sites, epistaxis, haematemesis, diarrhoea and
positive persons who actually reported illness con- melaena, sore and inflamed throat, gingival bleed-
sidered to be compatible with Rift Valley fever, but ing, epigastric pain, hepatomegaly or hepatos-
it can be deduced that the fatality rates in terms of plenomegaly, tenderness of the right upper quad-
total IgM antibody-positive persons are much rant of the abdomen and deep jaundice. This is
closer to the corresponding fatality rate in Egypt. followed by pneumonitis, anaemia, shock with rac-
The majority of Rift Valley fever infections in ing pulse and low blood pressure, hepatorenal fail-
humans are inapparent or associated with moder- ure, coma and cardiorespiratory arrest. Factors
ate to severe, non-fatal, febrile illness. After an incu- contributing to fatal outcome in the hepatic form of
bation period of 2—6 days, the onset of the benign the disease include anaemia, shock and hepatorenal
illness is usually very sudden and the disease is failure, with the kidney lesions possibly being as
characterized by rigor, fever that persists for several important as shock in producing anuria. A propor-
days and is often biphasic, headache with retro- tion of the less severely affected patients may make
orbital pain and photophobia, weakness, and a protracted recovery without sequelae.
muscle and joint pains. Sometimes there is nausea Encephalitis may occur in combination with the
and vomiting, abdominal pain, vertigo, epistaxis haemorrhagic syndrome. Otherwise, signs of en-
and a petechial rash. Defervescence and sympto- cephalitis in humans may supervene during the
matic improvement occur in four to seven days in acute illness, or up to 4 weeks later and include
benign disease and recovery is often complete in severe headache, vertigo, confusion, disorientation,
two weeks, but in a minority of patients the disease amnesia, meningismus, hallucinations, hypersaliva-
is complicated by the development of ocular lesions tion, grinding of teeth, choreiform movements, con-
at the time of the initial illness or up to four weeks vulsions, hemiparesis, lethargy, decerebrate postur-
later. Estimates for the incidence of ocular compli- ing, locked-in syndrome, coma and death. A
cations range from less than 1% to 20% of human proportion of patients may recover completely, but
infections, and possibly the differences stem from others may be left with sequelae, such as hemipar-
failure to record mild cases in populations where esis.
illiterate persons are less likely to report minor Abortion is the usual, if not invariable, outcome
disturbances of vision. The ocular disease usually to infection of pregnant ruminants, but an attempt
presents as a loss of acuity of central vision, some- to relate the occurrence of abortion in humans to
times with development of scotomas. The essential evidence of Rift Valley fever infection in Egypt pro-
lesion appears to be focal retinal ischaemia, gen- duced inconclusive results.
erally in the macular or paramacular area, asso- By analogy with the course of events believed to
ciated with thrombotic occlusion of arterioles and follow natural infection with other arthropod-
capillaries, and is characterized by retinal oedema borne viruses, it can be surmised that the pathogen-
and loss of transparency caused by dense white esis of the disease may involve some replication of
exudate and haemorrhages. Sometimes there is se- virus at the site of inoculation, conveyance of infec-
vere haemorrhage and detachment of the retina. tion by lymphatic drainage to regional lymph nodes
The lesions and the loss of visual acuity generally where there is further replication with spillover of
resolve over a period of months with variable resid- virus into the circulation to produce primary vir-
534 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
aemia, which in turn leads to systemic infection, and limits or abolishes production of coagulation pro-
that intense viraemia then results from release of teins and reduces clearance of activated coagulation
virus following replication in major target organs. factors, thereby further promoting the occurrence
Wild Rift Valley fever virus, which has not been of disseminated intravascular coagulopathy, which
subjected to serial passaging in laboratory host sys- in turn augments tissue injury by impairing blood
tems, is described as being hepato-, viscero- or pan- flow. Vasculitis and haemostatic failure result in
tropic, and immunofluorescence studies in labora- purpura and widespread haemorrhages.
tory animals indicate that replication occurs in The scant information available on clinical pa-
littoral macrophages of lymph nodes, most areas of thology findings in humans are compatible with
the spleen except T-dependent peri-arteriolar observations made in haematological and coagu-
sheaths, foci of adrenocortical cells, virtually all lation studies on monkeys, except that leucocytosis
cells of the liver, most renal glomeruli and some and anaemia may be more marked in severe human
tubules, lung tissue and scattered small vessel walls, disease. In most species there is an initial leucopenia
as well as in necrotic foci in the brains of individuals followed by leucocytosis, and the same may be true
which develop the encephalitic form of the disease. for humans. Monkeys may have prolonged ac-
These sites correspond to the lymphoid necrosis in tivated partial thromboplastin times and pro-
lymph nodes and spleen, hepatic necrosis and ad- thrombin times even in benign infection, and in
renal, lung and glomerular lesions seen in humans severe liver disease there may be depletion of coagu-
and livestock, and the brain lesions in humans (en- lation factors II, V, VII, IX, X and XII, throm-
cephalitis has not been described in natural disease bocytopenia and platelet dysfunction, increased
of ruminants). Titration of infectivity in organ schistocyte counts and depletion of fibrinogen to-
homogenates indicates that the liver and spleen are gether with raised fibrin degradation product levels.
the major sites of virus replication. Cell damage is Raised serum aspartate aminotransferase and
ascribed directly to the lytic effects of the virus, but alanine aminotransferase levels have been recorded
the inflammatory response seen in human brain even in benign disease in humans.
tissue suggests that there may also be an im- Treatment is essentially symptomatic, and sup-
munopathological element to the pathogenesis of portive therapy in the haemorrhagic disease in-
encephalitis. The same may be true for ocular cludes replacement of blood and coagulation fac-
lesions. Recovery is mediated by non-specific and tors. Results obtained in animal models suggest that
specific host responses, and the clearance of vir- the administration of immune plasma from re-
aemia correlates with the appearance of neutraliz- covered patients may be beneficial. The antiviral
ing antibody. No significant antigenic differences drug ribavirin inhibits virus replication in cell cul-
have been detected between isolates of the virus, tures and laboratory animals, and it has been sug-
although differences in pathogenicity for laboratory gested that it could be used even in benign disease in
rodents have been demonstrated, and immunity ap- order to obviate the potentially serious complica-
pears to be lifelong. tions which may occur in humans.
The haemostatic derangement which occurs in Specimens to be submitted for laboratory confir-
Rift Valley fever has been investigated in detail only mation of the diagnosis include blood from live
in rhesus monkeys, and the mechanisms involved patients, and tissue samples, particularly liver, but
remain speculative. Impairment of coagulation oc- also spleen, kidney, lymph nodes and heart blood of
curs even in benign infection of monkeys, and mod- deceased patients. Tissue samples should be sub-
erate thrombocytopenia has been observed in mitted in duplicate in a viral transport medium, and
benign infection in sheep, but haemostatic derange- in 10% buffered-formalin for histopathological
ment is most severe in the fatal hepatic syndrome. It examination.
is postulated that the critical lesions are vasculitis Viral antigen can frequently be detected rapidly
and hepatic necrosis. Destruction of the antithrom- in tissues and/or blood by a variety of immunologi-
botic properties of endothelial cells is thought to cal methods, including immunodiffusion, comple-
trigger intravascular coagulation, and the wide- ment-fixation, immunofluorescence and enzyme-
spread necrosis of hepatocytes and other affected linked immunoassay. Viraemia lasts for up to a
cells to result in the release of procoagulants into week. The virus is cytopathic and can be isolated
the circulation. Severe liver damage presumably readily in almost all cell cultures commonly used in
BUNYAVIRIDAE 535
diagnostic laboratories, and identified rapidly by attenuated vaccine confers lifelong immunity in
immunofluorescence. Virus can also be isolated in sheep, but is abortigenic and teratogenic in a small
suckling or weaned mice, or hamsters, inoculated proportion of pregnant ewes. The attenuated vac-
intracerebrally or intraperitoneally, and antigen cine is poorly immunogenic in cattle and they are
can be identified in harvested brain or liver by the immunized annually with the inactivated vaccine. A
immunological methods mentioned above. Defini- formalin-inactivated cell culture vaccine produced
tive identification of isolates is achieved by perform- in the USA is used on an experimental basis to
ing neutralization tests with reference antiserum. immunize persons such as laboratory and field
Histopathological lesions, particularly those in workers who are regularly exposed to Rift Valley
the liver, are considered to be pathognomonic, and fever infection.
are essentially similar in humans and domestic ru-
minants. The severity of the lesions varies from
primary foci of coagulative necrosis, consisting of
clusters of hepatocytes with acidophilic cytoplasms Serogroup Uukuniemi
and pyknotic nuclei, multifocally scattered
throughout the parenchyma, to massive liver de- Uukuniemi virus
struction in which the primary foci comprising Uukuniemi virus was originally isolated in Finland
dense aggregates of cytoplasmic and nuclear debris, in 1960 from Ixodes ricinus ticks, which parasitize
some fibrin and a few neutrophils and macro- livestock but also bite humans. The virus has subse-
phages, can be discerned against a background of quently been isolated from ticks, birds and field
parenchyma reduced by nuclear pyknosis, karyor- mice in Finland, Norway, Poland, Lithuania, and
rhexis and cytolysis to scattered fragments of cytop- the former USSR and Czechoslovakia. Antibody to
lasm and chromatin, with only narrow rims of de- the virus has been found in human sera in Finland,
generated hepatocytes remaining reasonably intact Hungary and former Czechoslovakia, but no evi-
close to portal triads. Intensely acidophilic cytop- dence has been presented to indicate that infection
lasmic bodies which resemble the Councilman bo- is associated with disease. The remaining members
dies of yellow fever are common, and rod-shaped or of the serogroup have been isolated from ticks asso-
oval eosinophilic intranuclear inclusions may be ciated with passerine or sea birds, and have no
seen in intact nuclei. Icterus may be evident. known medical or veterinary significance.
Antibody to Rift Valley fever virus can be demon-
strated in complement-fixation, enzyme-linked im-
munoassay, indirect immunofluorescence, haema-
gglutination-inhibition, or neutralization tests. GENUS NAIROVIRUS
Diagnosis of recent infection is confirmed by de-
monstrating seroconversion or a fourfold or greater Serogoup Crimean-Congo
rise in titre of antibody in paired serum samples, or haemorrhagic fever
by demonstrating IgM antibody activity in an en-
zyme-linked immunoassay.
Crimean-Congo haemorrhagic fever virus
Benign Rift Valley fever in humans must be dis-
tinguished from other zoonotic diseases such as The literature on CCHF is the subject of several
brucellosis and Q fever, while the fulminant hepatic comprehensive reviews (Chumakov, 1974; Hoog-
disease must be distinguished from the so-called straal, 1979; 1981; Watts et al., 1989). A disease
formidable viral haemorrhagic fevers of Africa: given the name Crimean haemorrhagic fever was
Lassa fever, Crimean-Congo haemorrhagic fever, first observed on the Crimean peninsula in 1944,
Marburg disease and Ebola fever. The occurrence and it was demonstrated through the inoculation of
of HFRS associated with hantavirus infections, is human subjects that the disease was caused by a
also a theoretical possibility in Africa. tick-transmitted virus, but the virus itself was only
An inactivated and a live attenuated vaccine are isolated in laboratory hosts, namely mice, in 1967.
available for immunization of livestock, but it is In 1969, it was shown that the agent of Crimean
usually difficult to persuade farmers to vaccinate haemorrhagic fever was identical to a virus named
livestock during long interepidemic periods. The Congo which had been isolated in 1956 from the
536 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 18.4 Abridged classification of the genus Nairovirus showing members known to cause infection of humans and domestic
animals. Information derived from sources cited in the text
?Figures in parentheses indicate the total numbers of members of the relevant taxon.
blood of a febrile child in Stanleyville (now Kisan- veloped haemorrhagic manifestations and died.
gani) in what was then the Belgian Congo (now Since then sporadic cases of haemorrhagic disease
Zaire), and since that time the two names have been and deaths have been diagnosed regularly each year
used in combination. in southern Africa, probably as a result of increased
The distribution of CCHF virus extends over awareness among clinicians, and severe disease has
eastern Europe, Asia and Africa: the presence of the also been recorded elsewhere in Africa.
virus or antibody to it has been demonstrated in the The virus has been isolated from at least 29 spe-
former USSR, Bulgaria, Greece, Turkey, Hungary, cies of ticks, but for most species there is no defini-
Yugoslavia, France, Portugal, Kuwait, Dubai, tive evidence that they are capable of serving as
Sharjah, Iraq, Iran, Afghanistan, Pakistan, India, vectors, and in some instances the virus recovered
China, Egypt, Ethiopia, Mauritania, Senegal, Bur- from engorged ticks may merely have been present
kina Faso, Benin, Nigeria, Central African Repub- in the bloodmeal imbibed from a viraemic host.
lic, Zaire, Kenya, Uganda, Tanzania, Zimbabwe, Members of three genera of ixodid ticks, Hyalomma,
Namibia, South Africa and Madagascar. However, Dermacentor and Rhipicephalus, have been shown
the evidence for France and Portugal is based on to be capable of transmitting infection trans-
limited observations and needs to be confirmed. stadially and transovarially, but Hyalommas are
In many instances virus or antibody was dis- considered to be the principal vectors, and the
covered in deliberate surveys, but in some countries known distribution of CCHF virus coincides with
of eastern Europe and Asia the presence of CCHF the world distribution of members of this genus of
first became evident in nosocomial outbreaks of ticks. Moreover, the prevalence of antibody to
disease, or in epidemics which arose in circumstan- CCHF virus detected in the sera of wild vertebrates
ces where humans were exposed to ticks and live- in southern Africa was highest in large herbivores
stock on a large scale, such as in major land recla- known to be the preferred hosts of adult Hyalomma
mation or resettlement schemes in Bulgaria and ticks, and in small mammals such as hares which
parts of the former USSR. In recent years the occur- are the preferred hosts of immature Hyalommas.
rence of the disease has been sporadic in Eurasia, Mammals of intermediate size, and passerine and
with most cases being recorded in Bulgaria and water birds, generally lacked evidence of infection,
Yugoslavia. Prior to 1981, a total of 15 cases of the but antibody was found in ostriches which are
disease had been reported in Africa, eight of them known to be parasitized by adult Hyalommas. Virus
laboratory infections, and only one patient had de- or antibody has also been demonstrated elsewhere
BUNYAVIRIDAE 537
in the sera of small mammals of Eurasia and Africa, contact with blood or other tissues of livestock, or
such as little susliks, hedgehogs, hares and certain having been bitten by ticks, but live in or have
myomorph rodents, and in some instances it has visited a rural environment where such exposure to
been shown that these hosts develop viraemia of infection is possible. Town dwellers sometimes ac-
sufficient intensity to infect ticks. quire infection from contact with animal tissues or
High prevalences of antibody occur in domestic tick bite while on hunting or hiking trips.
ruminants in areas infested by Hyalommas and the The majority of patients tend to be adult males
virus causes inapparent infection or mild fever in engaged in the livestock industry, such as farmers,
cattle, sheep and goats, with viraemia of sufficient herdsmen, slaughtermen and veterinarians. Serop-
intensity to infect ticks. It is doubtful whether trans- revalence studies indicate that infection of humans
ovarial transmission occurs with sufficient fre- is uncommon despite the widespread evidence of
quency in ticks to ensure indefinite perpetuation of infection in livestock, and this may be explained by
the virus in the absence of amplification of infection the facts that viraemia in livestock is short-lived,
in vertebrate hosts, and in particular, it is believed and of low intensity compared to that in other
that the infection of small vertebrates constitutes an zoonotic diseases such as Rift Valley fever, and that
important amplifying mechanism which facilitates humans are not the preferred hosts of Hyalomma
transstadial transmission of virus by adult ticks to ticks. The low prevalences of antibody generally
large vertebrates. found in populations at risk, and the paucity of
Young ruminants generally acquire natural infec- evidence of inapparent infection encountered
tion early in life and are viraemic for about a week. among the cohorts of cases of the disease, suggests
Humans become infected when they come into con- that infection is frequently symptomatic.
tact with the viraemic blood of young animals in the Incubation periods are generally short, ranging
course of performing procedures such as castra- from one to three days (maximum nine) following
tions, vaccinations, inserting ear tags or slaughter- infection by tick bite, and are usually 5 or 6 days
ing the animals. Animals which are raised under (maximum 13) in persons exposed to infected blood
tick-free conditions and moved to infested locations or other tissues of livestock or human patients.
later in life may acquire tick-borne diseases of live- Onset of the disease is usually very sudden. Patients
stock at the same time that they become infected develop fever, rigors, chills, severe headache, dizzi-
with CCHF virus, and consequently humans be- ness, neck pain and stiffness, sore eyes, photo-
come infected from contact with viraemic blood in phobia, myalgia and malaise, with intense backache
the course of treating sick animals or butchering or leg pains. Nausea, sore throat and vomiting
those that die. The available evidence suggests that commonly occur early in the illness and patients
the infection in humans is acquired through contact may experience non-localized abdominal pain and
of viraemic blood with broken skin, and this ac- diarrhoea at this stage. Fever is often intermittent
cords with the fact that nosocomial infection in and patients may undergo sharp changes of mood
medical personnel usually results from accidental over the next 2 days, with feelings of confusion and
pricks with needles contaminated with the blood of aggression. By the second to fourth day of illness
patients, or similar mishaps. Common source out- patients may exhibit lassitude, depression and som-
breaks involving more than one case of the disease nolence, and have a flushed appearance with injec-
can occur when several people are exposed to infec- ted conjunctivae or chemosis. By this time, tender-
ted tissues. Infection appears to be limited to those ness may be localized in the right upper quadrant of
who have contact with fresh blood or other tissues, the abdomen, and hepatomegaly may be discern-
probably because infectivity is destroyed by the fall ible. Tachycardia is common and patients may be
in pH which occurs in tissues after death, and there slightly hypotensive. There may be lym-
has been no indication that CCHF virus constitutes phadenopathy, and enanthem and petechiae of the
a public health hazard in meat processed and ma- throat, tonsils and buccal mucosa.
tured according to normal health regulations. A petechial rash appears on the trunk and limbs
Many human infections result directly from tick on day 3—6 of illness, and this may be followed
bite, and it has been observed that people can also rapidly by the appearance of large bruises and ec-
become infected from merely squashing ticks be- chymoses, especially in the anticubital fossae, upper
tween the fingers. Some patients are unable to recall arms, axillae and groin. Epistaxis, haematemesis,
538 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
haematuria, melaena, gingival bleeding and bleed- would contribute to haemostatic failure through
ing from the vagina or other orifices may commence stimulating platelet aggregation and degranulation,
on day 4—5 of illness, or even earlier. Sometimes a with consequent activation of the intrinsic coagu-
haemorrhagic tendency is evident only from the lation cascade. It is clear from the results of thera-
oozing of blood from injection or venepuncture peutic administration of platelets to patients that
sites. There may be internal bleeding, including ret- they are consumed, and evidence of depression of
roperitoneal and intracranial haemorrhage. Severe- thrombopoiesis in bone marrow has been reported.
ly ill patients may enter a state of hepatorenal and Widespread tissue damage in organs such as the
pulmonary failure from about day 5 onwards and liver would result in further release of
progressively become drowsy, stuporous and coma- procoagulants such as tumour necrosis factor into
tose. Jaundice may become apparent during the the bloodstream, and impairment of the circulation
second week of illness. The mortality rate is ap- through the occurrence of a disseminated intravas-
proximately 30% and deaths generally occur on cular coagulopathy would contribute to further tis-
day 5—14 of illness. Patients who recover usually sue damage. Damage to the liver would also impair
begin to improve on day 9 or 10 of illness, but synthesis of coagulation factors to replace those
asthenia, conjunctivitis, slight confusion and am- which are consumed.
nesia may continue for a month or longer. Lesions in the liver vary from disseminated foci of
Changes in clinical pathology values recorded coagulative necrosis, mainly mid-zonal in distribu-
during the first few days of illness include leucocyto- tion, to massive necrosis involving over 75% of
sis or leucopenia, and elevated aspartate trans- hepatocytes, and a variable degree of haemorrhage,
aminase, alanine transaminase, gamma-glutamyl with little or no inflammatory cell response. Lesions
transferase, lactic dehydrogenase, alkaline phos- in other organs include congestion, haemorrhage
phatase and creatine kinase levels, while bilirubin, and focal necrosis in the central nervous system,
creatinine and urea levels increase and serum pro- kidneys and adrenals, and general depletion of lym-
tein levels decline during the second week. Throm- phoid tissues. Fibrin deposits may be seen in small
bocytopenia, elevation of prothrombin ratio, ac- blood vessels in parenchymatous organs including
tivated partial thromboplastin time, thrombin time the liver, and thrombus formation and infarction
and fibrin degradation products, and depression of may contribute to the pathogenesis of the necrotic
fibrinogen and haemoglobin values are also evident lesions in these organs.
during the first few days of illness, indicating that Where possible, patients are treated by specially
the occurrence of disseminated intravascular co- trained staff in institutions equipped for handling
agulopathy is probably an early and central event in formidable viral haemorrhagic fevers, and barrier-
the pathogenesis of the disease. Changes are more nursing techniques are used for the protection of
severe in fatal than in non-fatal infections, and the medical personnel. Therapy appropriate for dis-
occurrence of certain markedly abnormal clinical seminated intravascular coagulopathy, such as the
pathology values during the first 5 days of illness are use of heparin, may be contemplated early in the
predictive of fatal outcome (Swanepoel et al., 1987; course of the disease by clinicians well versed in the
1989). treatment of haemostatic failure, but the procedure
It is surmised that peripherally introduced is considered to be risky, and generally only pa-
CCHF virus undergoes some replication at the site tients who acquire nosocomial infection come to
of inoculation, and that haematogenous and medical attention at a sufficiently early stage. Stan-
lymph-borne spread of infection occurs to organs dard treatment consists of replacement of red blood
such as the liver which are major sites of replication. cells, platelets, other coagulation factors, protein
Although it has not been shown conclusively that (albumin) and intravenous feeding as indicated by
there is infection of endothelium, capillary fragility clinical pathology findings. Immune plasma from
is a feature of the disease and there is evidence of recovered patients has been used in therapy, but
formation of circulating immune complexes with there is no firm evidence from controlled trials of
complement activation, and this would contribute the value of the treatment, and there has been a lack
to damage of the capillary bed and the genesis of of a uniform product with proven virus-neutralizing
renal and pulmonary failure. Endothelial damage activity. Ribavirin has been found to inhibit virus
would account for the occurrence of a rash and replication in cell cultures, and in suckling mice,
BUNYAVIRIDAE 539
and preliminary results of a trial in human patients which can be acquired from contact with animal
are promising. tissues, as well as tick-borne typhus (Rickettsia
On account of the propensity of the virus to cause conorii infection commonly known as tickbite fe-
laboratory infections, and the severity of the human ver), but many other conditions including bacterial
disease, investigation of CCHF is generally under- septicaemias may resemble CCHF.
taken in maximum security laboratories in coun- The control of CCHF through the application of
tries which have biosafety regulations. Specimens to acaricides to livestock is impractical, particularly
be submitted for laboratory confirmation of the under the extensive farming conditions which pre-
diagnosis include blood from live patients and, in vail in the arid areas where Hyalomma ticks are
order to avoid performing full autopsies, heart most prevalent. Pyrethroid preparations are avail-
blood and liver samples taken with a biopsy needle able which can be used to kill ticks which come into
from deceased patients. Virus can be isolated from contact with human clothing. Veterinarians,
blood and organ suspensions in a wide variety of slaughtermen and others involved with livestock
primary and line cell cultures, including Vero, CER should be aware of the disease and take practical
and BHK-21 cells, and identified by immunof- steps, such as the wearing of gloves, to limit or avoid
luorescence. Isolation and identification can be exposure of naked skin to fresh blood and other
achieved in 1—5 days, but cell cultures lack sensitiv- tissues of animals. Inactivated mouse brain vaccine
ity and usually only detect high concentrations of for the prevention of human infection has been used
virus present in the blood of severely ill patients on a limited scale in eastern Europe and the former
during the first 5 days or so of illness. Intracerebral USSR. Development of a safe and effective modern
inoculation of suckling mice is more sensitive and vaccine is inhibited by the limited potential demand
can be used to demonstrate low concentrations of for such a vaccine.
virus present in blood up to 13 days after the onset
of illness. Virus antigen can sometimes be demon-
strated in the blood of severely ill patients with
Serogroup Nairobi sheep disease
intense viraemia, or in liver suspensions, by en-
zyme-linked immunoassay.
Nairobi sheep disease virus
Antibodies, both IgG and IgM, become demon-
strable by indirect immunofluorescence from about Nairobi sheep disease virus was first isolated from
day 7 of illness (slightly earlier by enzyme-linked sheep blood in Kenya in 1910 and is known to be
immunoassay), and are present in the sera of all associated with disease of small ruminants, specifi-
survivors of the disease by day nine at the latest. cally sheep and goats, in a narrow band straddling
The IgM antibody activity declines to undetectable the equator from Kenya in the east to Congo in the
levels by the fourth month after infection, and IgG west. Antibody, but not disease, has also been found
titres may begin to decline gradually at this stage, to the north of Kenya in Ethiopia and Somalia, and
but remain demonstrable for at least 5 years. Recent southwards along the east of the continent to
or current infection is confirmed by demonstrating Mozambique and Botswana. The virus can be
seroconversion, or a fourfold or greater increase in transmitted transstadially by a range of ixodid
antibody titre in paired serum samples, or IgM ticks, including Amblyomma variegatum, but the en-
antibody activity in a single sample. Patients who demic vector appears to be Rhipicephalus appendi-
succumb rarely develop a demonstrable antibody culatus, in which transovarial transmission occurs.
response and the diagnosis is confirmed by isolation The disease in sheep and goats is characterized by
of virus from serum, or from liver specimens. Obser- fever, haemorrhagic gastroenteritis, and abortion in
vation of necrotic lesions compatible with CCHF pregnant animals. High mortality occurs when sus-
infection in sections of liver, provides presumptive ceptible sheep or goats are introduced into an en-
evidence in support of the diagnosis. demic area, but within such areas young animals
The disease must be distinguished from the other appear to undergo benign infection as maternal
so-called formidable viral haemorrhagic fevers: immunity wanes, and there is a high prevalence of
Lassa fever, Marburg disease, Ebola fever and immunity in adult animals. Attenuated live and
HFRS (hantavirus infections), other febrile illnesses killed vaccines for sheep and goats are available in
such as Rift Valley fever, Q fever and brucellosis East Africa. Small antelope are susceptible to the
540 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
disease, and rodents develop viraemic infection, but generic term haemorrhagic fever with renal syn-
seroprevalence studies have failed to identify wild drome (HFRS) is advocated, while the term han-
maintenance hosts of the virus. Larger ruminants tavirus pulmonary syndrome (HPS) is preferred for
such as cattle and buffalo, are not susceptible to the respiratory disease associated with hantaviruses in
disease. The virus has been isolated from human the Americas.
blood in association with febrile illness with arthral- The existence of a febrile disease with haemor-
gia and malaise in Uganda, and laboratory infec- rhagic and renal manifestations has been recog-
tion has been recorded. Antibody prevalence rates nized in Eurasia at least since the early years of the
of up 20% have been found in humans in endemic twentieth century and, in fact, descriptions of simi-
areas. lar disease can be traced back to antiquity. A dis-
Ganjam virus, first isolated from ixodid ticks in ease known by various names, including haemor-
India in 1954, is considered to be either closely rhagic nephrosonephritis, and which caused
related or identical to Nairobi sheep disease virus. It outbreaks among civilians and soldiers, was inves-
has been isolated from the blood of sheep and hu- tigated independently in the far eastern region of
mans with febrile illness in India, where it is asso- the former USSR and in Manchuria prior to the
ciated with ticks of the genus Haemaphysalis. There second world war, and by the early 1940s it was
is speculation that the virus may have been trans- established that the condition could be transmitted
located from India to Africa with ectoparasites on to human volunteers by inoculation of filtrates of
sheep and goats, which have been traded along sea patients’ blood or urine, or tissue extracts from
routes for centuries. Apodemus field mice (it had been observed that the
incidence of disease was greatest at the end of sum-
mer when the mice were most numerous). At the
Dugbe virus same time a febrile syndrome with abdominal pain,
There have been approximately 600 isolations of backache and renal manifestations was recognized
Dugbe virus from ixodid ticks, mainly Amblyomma in Scandinavia and numerous cases of this disease,
variegatum, in Nigeria, Central African Republic later named nephropathia epidemica (NE), were
and Ethiopia. The virus has also been isolated fre- observed in soldiers during the second world war.
quently from cattle blood in surveys, and from a Thousands of cases of a disease named Korean
giant rat (Cricetomys gambianus), aedine mos- haemorrhagic fever were observed in soldiers and
quitoes and Culicoides midges in Nigeria, and there civilians during the Korean war of the early 1950s,
is serological evidence of the occurrence of the virus and the disease continued to be seen after the war.
in Senegal and Uganda. There have been seven In 1976 it was found that convalescent sera from
isolations of the virus from the blood of persons patients in Korea could be used to demonstrate the
with benign febrile illness in Nigeria and Central presence of an antigen by immunofluorescence in
African Republic (including a laboratory infection). the tissues of Apodemus agrarius field-mice caught
One patient had mild meningitis and virus was near the Hantaan river. The antigen was shown to
isolated from cerebrospinal fluid. Surprisingly, be associated with a virus which could be subcul-
serosurveys have not revealed widespread human tured in field mice. The virus, named Hantaan, was
infection. successfully grown in cell cultures in 1981, and
shortly thereafter characterized as a member of the
family Bunyaviridae and placed in a new genus Han-
GENUS HANTAVIRUS tavirus. The virus is widely distributed as the causa-
tive agent of HFRS in Asia, particularly in the
eastern portion of the former USSR, China and
Hantaan, Dobrava, Seoul, Puumala, Sin
Korea. Virus associated with a severe form of
Nombre and related viruses
HFRS in the Balkans (Albania, Greece, former
Hantaviruses are associated with a range of neph- Yugoslavia and Bulgaria) has been found to be
rotic diseases in Asia and Europe known variously distinct from Hantaan, and has recently been desig-
as haemorrhagic nephrosonephritis, Korean haem- nated as Dobrava virus. It is associated with the
orrhagic fever, Songo fever, epidemic haemorrhagic yellow-necked field mouse, Apodemus flavicollis, as
fever and nephropathia epidemica, but use of the well as with Apodemus agrarius, and evidence is
BUNYAVIRIDAE 541
emerging to suggest that it may be more widely associated with a population explosion of the deer
distributed in Europe than previously realized. mouse, Peromyscus maniculatus, incriminated as the
In 1980 the presence of the causative agent of natural host of the virus. Sporadic cases of similar
nephropathia epidemica was demonstrated by im- disease were recognized elsewhere in the USA, some
munofluorescence in the tissues of Clethrionomys retrospectively, and by the end of 1993 a total of 53
glareolus voles in Finland, and subsequently the cases had been confirmed (Nichol et al., 1993; Brem-
agent, named Puumala virus, was grown in cell cul- ner, 1994; Duchin et al., 1994). After objections were
tures and shown to be related to Hantaan virus. raised to various names proposed for the new virus,
Although evidence obtained in former Yugoslavia, Sin Nombre (Spanish for ‘without name’) was adop-
Germany, Belgium, France and Britain suggests ted, and the disease was referred to as HPS.
that Puumala virus occurs widely in Europe, it is Isolated cases and outbreaks of HPS were subse-
most prevalent at northerly latitudes, extending quently recognized beyond the distribution range of
into the Arctic circle in Scandinavia and the adjoin- Peromyscus maniculatus in the USA, in Canada, and
ing western portion of the former USSR, where in several countries of South America. A succession
highest concentrations of the bank vole occur. of new hantaviruses was discovered in association
Seoul virus was isolated in 1980 in Korea from the with HPS, or in rodents tested speculatively in sur-
tissues of peridomestic rats, Rattus norvegicus and veys. Several new viruses were also discovered in
Rattus rattus, in association with human disease rodents in Europe and Asia, and unidentified vi-
which occurred in urban as opposed to rural envi- ruses which had previously been isolated from ban-
ronments. It has been incriminated as the cause of dicoots in Thailand and from suncid shrews in In-
human disease in Japan, China and Korea, but has dia were found to be hantaviruses. In general, the
been isolated from rats in Egypt, the USA and new viruses were discovered through the detection
elsewhere, and probably has a worldwide distribu- of cross-reactive antibody activity to hantavirus
tion. Isolation of the virus from rats led to specula- antigens, followed by the application of the poly-
tion that hantaviruses in general may have been merase chain reaction to detect viral nucleic acid,
disseminated worldwide with ship-borne rodents. and genetic characterization. Adaptation to cell cul-
However, the distribution patterns of most han- tures followed the initial identification of the vi-
taviruses within the interiors of continents, and the ruses, but in vitro culture has not yet been achieved
evolution of particular host relationships, consti- in all instances. It is suspected that in addition to the
tute evidence against recent spread of the viruses. hantaviruses currently known to be human patho-
The findings in Asia and Europe prompted inter- gens, some of the remaining viruses may also prove
est elsewhere, and as a result Prospect Hill virus was to be associated with disease (Table 17.5) (Schmal-
isolated from Microtus pennsylvanicus voles in the john and Hjelle, 1997; Kanerva et al., 1998; Monroe
USA, but no disease associations have been de- et al., 1999).
scribed for this virus. Serological classification of hantaviruses has
In May 1993, an outbreak of an acute respiratory lagged behind genetic characterization, primarily
disease in adults, with a high fatality rate, was rec- because the lack of in vitro culture systems for some
ognized in the Four Corners region of southwestern of the viruses has prevented the performance of
USA, where the borders of the states of Utah, Col- definitive cross-neutralization tests, but the extant
orado, Arizona and New Mexico meet, but the information on antigenic affinities is in agreement
initial occurrence of cases could be traced back to with the genetic clustering of the viruses, which in
late 1992. Antibody cross-reactive with the antigens itself conforms with the phylogeny of the rodent
of known hantaviruses was found in the sera of hosts (Table 18.5) (Arthur et al., 1992; Putha-
patients, and by means of reverse transcription and vathana et al., 1992; Chu et al., 1994; 1995; Schmal-
the polymerase chain reaction with consensus se- john and Hjelle, 1997; Kanerva et al., 1998; Monroe
quence hantavirus primers, it was possible to dem- et al., 1999). In brief, all hantaviruses are antigeni-
onstrate the presence of nucleic acid of a novel cally related, with greatest affinities existing within
hantavirus in the tissues of patients. The nucleotide clusters designated Hantaan-like, Puumala-Pros-
sequence of the entire genome of the virus was pect Hill-like, and Sin Nombre-like, while Thot-
determined even before it could be isolated and tapalayam virus from shrews in India is more dis-
grown in cell cultures. The outbreak was apparently tantly related to the others. Viruses of the Hantaan-
542 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 18.5 Classification of members of the genus Hantavirus. Information derived from sources cited in the text
Rodentia: Murinae
(Hantaan-like viruses)
Hantaan Apodemus agrarius HFRS Asia
Dobrava Apodemus flavicollis HFRS Europe
Apodemus agrarius
Seoul Rattus norvegicus HFRS Worldwide
Rattus rattus
Thailand Bandicota indica Asia
Rodentia: Arvicolinae
(Puumala—Prospect Hill-like viruses)
Puumala Clethrionomys glareolus HFRS (NE) Europe
Tobetsu Clethrionomys rufocanus Japan
Topografov Lemmus sibiricus Siberia
Khabarovsk Microtus fortis Siberia
Tula Microtus arvalis Europe
Prospect Hill Microtus pennsylvanicus N. America
Bloodland Lake Microtus ochrogaster N. America
Prospect Hill-like Microtus pennsylvanicus N. America
Microtus montanus
Microtus ochrogaster
Isla Vista Microtus californicus W. USA, Mexico
Rodentia: Sigmodontinae
(Sin Nombre-like viruses)
Sin Nombre Peromyscus maniculatus HPS W. and C. USA, Canada
(Grassland form)
Monongahela Peromyscus maniculatus HPS E. USA, Canada
(forest form)
New York Peromyscus leucopus
(eastern haplotype) HPS E. USA
Blue River Peromyscus leucopus C. USA
(S.W./N.W. haplotypes)
Bayou Oryzomys palustris HPS S.W. USA
Black Creek Canal Sigmodon hispidus HPS S.E. USA
(Eastern form)
Muleshoe Sigmodon hispidus S. USA
(Western form)
Can o Delgadito Sigmodon alstoni Venezuela
Andes Oligoryzomys longicaudatus HPS Argentina, Chile
Oran Oligoryzomys longicaudatus HPS N.W. Argentina
Lechiguanas Oligoryzomys flavescens HPS C. Argentina
Bermejo Oligoryzomys chacoensis N.W. Argentina
Hu 39694? Unknown HPS C. Argentina
Pergamino Akadon azarae C. Argentina
Maciel Bolomys obscurus C. Argentina
Laguna Negra Calomys laucha HPS Paraguay, Bolivia
Juquitiba Unknown HPS Brazil
Rio Mamore Oligoryzomys microtis Bolivia, Peru
El Moro Canyon Reithrodontomys megalotis W. USA, Mexico
Rio Segundo Reithrodontomys mexicanus Costa Rica
Insectivora: Crocidurinae
Thottapalayam Suncus murinus India
?Location where infection occurred is uncertain; virus will be named when the distribution and/or rodent is host known;
HFRS : haemorrhagic fever with renal syndrome; HPS : hantavirus pulmonary syndrome.
BUNYAVIRIDAE 543
like group are associated with rodent hosts of the like viruses occurs in persons who have occupa-
subfamily Murinae and with HFRS; Puumala- tional, residential or recreational exposure to ro-
Prospect Hill-like viruses are associated with the dent-infested buildings or to the outdoors, urban
subfamily Arvicolinae (voles and lemmings) and infection with Seoul virus occurs indoors in associ-
with NE, and Sin Nombre-like viruses with the ation with rat infestations, while all of the viruses
subfamily Sigmodontinae and with HPS (Table may cause infections associated with laboratory ro-
18.5). Despite the fact that Seoul virus appears to be dents. Rodents are subject to periodic population
very widely distributed, incontrovertible evidence explosions and crashes, and the incidence of human
of disease associated with hantaviruses (detection of infection with Hantaan-like, Puumala-like and Sin
virus) has been obtained only for Asia, Europe and Nombre-like viruses increases in years when the
the Americas, while elsewhere there has only been rodents are most numerous. Person-to-person
inconclusive serological evidence of infection. How- spread of hantavirus infection has been observed
ever, Seoul, Hantaan and Puumala viruses have been only in an outbreak of 20 cases of HPS caused by
encountered as contaminants of laboratory rodent Andes virus in southern Argentina, but it could not
colonies and are known to have caused infections in be established whether transmission was associated
laboratory workers in the former USSR, Korea, with direct contact, droplets, aerosols or con-
Japan, Belgium, France and England. In one in- taminated fomites (Wells et al., 1997). In excess of
stance virus was inadvertently preserved for years in 250 cases of HPS have been reported in the Amer-
rat tissues kept in frozen storage. Hence, the poten- icas, and up to 200 000 hospitalized cases of HFRS
tial exists for inadvertent dissemination of the vi- are recorded each year, with more than half occur-
ruses. ring in China (Schmaljohn and Hjelle, 1997; Mon-
The distributions of the hantaviruses, insofar as roe et al., 1999).
they are known, tend to overlap, but conform to the Four clinical forms of HFRS are recognized and
distributions of the rodent hosts. Individual viruses these vary in order of increasing severity from neph-
have been isolated from more than one type of ropathia epidemica associated with Puumala virus
rodent, but each tends to have a particular associ- infection, through mild or rat-borne HFRS asso-
ation with a single species of rodent. The viruses ciated with Seoul virus infection, to far eastern
appear to be apathogenic for their reservoir hosts. HFRS associated with Hantaan virus strains car-
After the rodents become infected there is an initial ried by Apodemus agrarius field mice, and so-called
viraemia followed by the persistence of infection, Balkan HFRS associated with Dobrava virus
probably for life, in lungs, kidneys and possibly strains carried by Apodemus flavicollis and
other organs, with chronic excretion of virus in Apodemus agrarius mice.
urine, faeces and saliva, despite the occurrence of a Far eastern HFRS occurs in China, the eastern
demonstrable immune response. There does not ap- part of the former USSR and Korea, mainly in adult
pear to be intrauterine transfer of infection, and males with occupational exposure to the outdoors,
transmission between rodents is thought to occur such as farmers, forest workers and soldiers
by bite, aerosol or contamination of dust, food and stationed in the field, and seldom occurs in persons
other fomites with excreted virus. Gamasid mite under 10 years of age. Most cases are seen in au-
parasites of rodents are suspected to be capable of tumn and early winter when crops are harvested
transmitting infection, but transmission occurs in and the rodents are most numerous, and subse-
the laboratory in the absence of mites. Foci with quently when the agricultural products are stored in
very high infection rates are observed among ro- proximity to homesteads. The incidence of asymp-
dents in nature. tomatic infection is unknown, but it was noted that
Humans become infected by the same means as American soldiers participating in an exercise in
rodents, but airborne infection from dust con- Korea who seroconverted, had all been ill, while in
taminated with rodent urine and faeces appears to parts of Korea high antibody prevalence rates with-
be the principal mechanism, and has been observed out corresponding levels of disease have been ob-
to occur even in persons who briefly visited infected served.
colonies of laboratory rodents. Infection occurs in The classical form of far eastern HFRS described
three main situations: rural or sylvatic infection in Korea has well-marked phases, but these may
with Hantaan-like, Puumala-like or Sin Nombre- overlap and be obscured in mild cases (Lee, 1982).
544 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
An incubation period of 2—3 weeks is followed by during the warmer months of the year. The disease
the abrupt onset of a febrile phase which lasts 3—7 is essentially similar to far eastern HFRS, but is
days and is marked by high fever, chills, malaise, more severe, with a higher proportion of patients
myalgia, anorexia, headache, dizziness, ocular pain, requiring renal dialysis, and with a greater tendency
and abdominal and back pain, which is felt particu- for the development of disseminated intravascular
larly in the renal area as a result of peritoneal and coagulopathy and haemorrhages. Reported death
retroperitoneal oedema. Proteinuria is marked dur- rates range from 5 to 35%.
ing this phase. Towards the end of the phase there is Natural outbreaks of mild or rat-borne HFRS, as
characteristic flushing of the face, neck and anterior opposed to outbreaks associated with laboratory
chest, with the conjunctivae, palate and pharynx rodent colonies, have been recorded in cities in
assuming an injected appearance, followed by the Japan, China and Korea. The disease occurs in
emergence of fine petechiae on the face, neck, soft urban residents who have no contact with field
palate and chest, together with conjunctival haem- rodents, and most cases are seen in spring and early
orrhages. Patients next enter a hypotensive phase summer. The disease is less severe and runs a shor-
which lasts hours to 2 days, and is marked by ter course than disease associated with Hantaan
classical shock: tachycardia, narrowed blood press- virus infections, and has less distinct clinical phases.
ure, cold and clammy skin, dulled senses and con- There is also less tendency for haemorrhages and
fusion. One-third of fatal cases enter irreversible renal failure to occur, and frequently signs of liver
shock at this stage. There is marked proteinuria, involvement are dominant: abdominal pain, hepa-
microscopic haematuria, raised haematocrit levels tomegaly and hepatic dysfunction. There are few
(haemoconcentration), leukaemoid reaction and deaths and mortality has been estimated at 1% or
thrombocytopenia. Capillary haemorrhages are less.
prominent. The patients then enter an oliguric Infection with Puumala-type virus occurs widely
phase which lasts 3—7 days. Blood urea and cre- in Europe, but the disease, nephropathia epidemica,
atinine levels increase, blood pressure begins to nor- is recognized most frequently in Scandinavia and
malize, but hypertension may result from a hyper- the neighbouring western region of the former
volaemic state. Bleeding tendencies increase USSR. The disease affects mainly adult males and
markedly, and there may be epistaxis, conjunctival, infection appears to be associated principally with
cerebral and gastrointestinal haemorrhages and ex- outdoor activities. Disease is seen most commonly
tensive purpura. There may be severe nausea and in late autumn and early winter, but many cases
vomiting, lung oedema and symptoms referable to occur in late summer following the traditional vaca-
the central nervous system. Most deaths occur at tion season. Cases seen during the colder months
this stage. A diuretic phase which follows may last are ascribed to the invasion of homes and barns by
days or weeks, and marks the start of clinical recov- voles at the onset of winter. The incubation period
ery. Diuresis of 3—6 litres per day is common, but is is thought to be about 1 month, but a range of 3
influenced by dehydration, electrolyte imbalance or days to 6 weeks has been reported. There is abrupt
secondary infections. Severely ill patients are at risk onset of fever, headache and malaise. By the third or
in this phase and may lapse into shock. A convales- fourth day of illness there is nausea, vomiting and
cent phase with progressive recovery of glomerular abdominal and lumbar pain. At this stage there may
filtration rate, renal blood flow and urine-concen- be azotemia, oliguria and proteinuria, which peaks
trating ability, may last 2—3 months. Mortality has 1 week after the onset of illness and declines over the
been reduced from the 10—15% observed during the next 3—6 days. Patients are extremely ill during the
Korean war to 5%, with intensive supportive ther- oliguric phase, and may manifest somnolence, rest-
apy and renal dialysis. lessness, confusion and meningismus. Transient
Balkan HFRS, associated with Dobrava virus, is myopia or blurred vision is regarded as pathogno-
also seen mainly in adult males, including wood- monic. Facial flushing and maculopapular rash of
cutters, shepherds and military personnel, but cases the neck and trunk are seen occasionally, as are
generally occur in spring and summer, possibly be- hepatomegaly, cervical lymphadenopathy, and
cause there is not the same type of cereal crop haemorrhages such as epistaxis and gastrointestinal
farming as in the Far East, and the reservoir host is bleeding. Patients seldom require renal dialysis. The
encountered in outdoor activities and at campsites oliguria is followed by polyuria of 3—4 litres daily
BUNYAVIRIDAE 545
for 7—10 days. At one stage it was thought that trophilia, plus increased myeloid precursors and
HFRS/NE and HPS were entirely distinct syn- atypical lymphocytes, haemoconcentration and
dromes, but it is now recognized that there is some thrombocytopenia, plus increased prothrombin
overlap, and in particular a proportion of NE pa- and partial thromboplastin times, although there is
tients may develop pulmonary infiltration similar no rash and seldom a tendency towards overt or
to HPS, and some may even exhibit respiratory internal bleeding. Within 2 days of being admitted
distress. Clinical improvement begins with the onset to hospital most patients develop diffuse bilateral
of polyuria, and 2 weeks after the onset of fever interstitial and alveolar pulmonary infiltration and
patients are subjectively well, but backache and pleural effusions demonstrable on radiographs,
lassitude may recur over weeks, and hyposthenuria with hypoxaemia which has necessitated intuba-
may persist for months. Recovery is usually com- tion, mechanical ventilation and oxygen supple-
plete, and mortality is consistently less than 1%. mentation in up to 88% of patients in some out-
The relatively high prevalence of antibody found in breaks. Renal insufficiency can occasionally follow
surveys suggests that inapparent infections may prolonged hypoperfusion, but early renal insuffi-
outnumber cases of overt disease by up to 20-fold. ciency and increased serum creatine kinase levels
It should be stressed that the hantaviruses over- (evidence of skeletal muscle inflammation) are not
lap in distribution and in the severity of HFRS uncommon in infection with Andes, Bayou and
which they induce, and for instance, neither rural or Black Creek Canal viruses. Death generally occurs
urban domicile of patients, nor season of occur- 6—8 days after the onset of illness, often within 48
rence of disease, allow Hantaan and Seoul virus hours of admission to hospital, but can range from 2
infections to be distinguished with certainty in Asia. days after the observed onset of illness to more than
Infections with Dobrava and Puumala type viruses 2 weeks. Fatality rates often exceed 40%, and incur-
in the Balkans may be equally difficult to distin- able shock and myocardial dysfunction may con-
guish. tribute to the high mortality. Autopsies reveal non-
Persons who develop HPS are often healthy cardiogenic pulmonary oedema and serous pleural
young adults, but may be of any age and either sex, effusions, with scant lymphoid infiltration of the
although the disease occurs infrequently in children. lung tissue. Some survivors manifested transient
Infection is acquired in similar manner to HFRS diuresis, but otherwise they make an uneventful
from occupational, residential or recreational expo- recovery without sequelae (Nichol et al., 1993;
sure to the outdoors or rodent infested buildings, Bremner, 1994; Duchin et al., 1994; Schmaljohn and
and in many instances infected rodents have been Hjelle, 1997; Kanerva et al., 1998).
found in the homes of victims. Incubation periods The underlying lesion in the pathogenesis of han-
are similar to HFRS, generally falling into the range tavirus syndromes appears to be vascular damage,
2—3 weeks, but the disease is characterized by severe and this is thought to be mediated by both viral
cardiopulmonary dysfunction rather than renal invasion of endothelial cells, and immuno-
failure and haemorrhage, despite the facts that there pathological mechanisms. Capillaries and small
is similar underlying capillary permeability and blood vessels dilate and there is extravasation of
marked thrombocytopenia. Onset of the prodromal plasma and cellular elements into tissues, and the
phase of the disease is marked by sudden develop- pathological changes observed in multiple systems
ment of fever, headache, severe myalgia and a cough all appear to be referable to the vascular damage
which may be productive in some instances. Gas- (Kanerva et al., 1998).
trointestinal manifestations in some patients in- Treatment of HFRS involves complex, phase-
clude abdominal pain, nausea, vomiting and diar- specific monitoring and support of homeostasis,
rhoea. After 3—6 days of illness there is progressive including fluid and electrolyte balances. Trials of
tachypnoea, tachycardia and hypotension, preced- ribavirin for the treatment of the disease in China
ing the onset of acute respiratory distress with pul- have been complicated by difficulties such as lack of
monary oedema. Patients are generally hospitalized uniformity of clinical status of patients at the time of
at this stage, but some may die before they can be protocol entry, but there are indications that use of
admitted. In addition to tachypnoea, tachycardia the drug reduces mortality and leads to improve-
and hypotension, on admission patients may be ment of objective markers of patient well-being, and
found to have proteinuria, leucocytosis with neu- clinical pathology values. Epidemiological evidence
546 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
suggests that there is lifelong immunity to han- dia, the former USSR, Yugoslavia, Bulgaria,
taviruses, at least to the homologous serotype. Slovakia, Somalia, Central African Republic,
There has been research on recombinant vaccines, Nigeria and Senegal. It has been suggested that the
but these are likely to find application only in Asia wide distribution of the virus could have resulted
where suitable populations at risk can be identified. from the carriage of immature ticks on migrating
Investigation of hantavirus infections is usually birds, although birds do not themselves appear to
undertaken in high security laboratories to mini- be susceptible to the virus. Isolations of the virus
mize the exposure of staff to infection. Isolation and have also been made from a hedgehog, ground
identification of hantaviruses is a notoriously diffi- squirrel and blood samples from cattle and sheep in
cult and time-consuming procedure, and is rarely Nigeria. Antibody has been found in cattle, sheep
successful on serum and urine specimens from pa- and goats parasitized by the ticks, and in human
tients. Demonstration of viral antigens in sera and sera in Slovakia, Yugoslavia, Italy and the Central
urine is equally unsuccessful. Viral nucleic acids of African Republic. Mild febrile illness was observed
hantaviruses can be detected in the tissues of human in two human patients who acquired laboratory
patients and experimentally and naturally infected infection, and serological evidence of infection was
rodents by means of reverse transcription and the obtained in a patient who suffered meningoen-
polymerase chain reaction with appropriate cephalitis in Yugoslavia.
primers (Arthur et al., 1992; Grankvist et al., 1992; Kasokero virus was isolated from fruit bats in
Xiao et al., 1992; Nichol et al., 1993; Monroe et al., Uganda, and caused four laboratory infections
1999). Detection of IgM antibody by enzyme-linked marked by febrile illness, headache, myalgia, ar-
immunoassay holds greatest promise as a rapid thralgia, abdominal pain, diarrhoea, chest pain,
diagnostic technique. Antibody activity appears to cough, as well as hyperactive reflexes in one patient.
be present in the sera of HFRS patients from the Bangui virus was isolated from the blood of a pa-
time of hospitalization, and titres increase rapidly tient with febrile illness, headache and rash in the
over the next 2 weeks. Owing to the antigenic cross- Central African Republic, and antibody was found
reactivity between hantaviruses, it may be difficult in the sera of local residents.
to determine the serotype of the virus responsible Issyk-Kul virus was isolated from several species
for the infection from antibody tests, but this can of insectivorous bat and from argasid tick parasites
sometimes be inferred by using a range of antigens of bats, birds and anopheline and aedine mos-
in enzyme-linked immunoassays (Feldmann et al., quitoes in the Central Asian Republics of the former
1993), or by performing neutralization tests with the USSR. It was demonstrated that aedine mosquitoes
full range of serotypes: antibody titres tend to be and argasid ticks are able to transmit the virus.
highest against the homologous infecting serotype. Antibody was found in human sera and virus was
In attempts to diagnose infection by an unknown isolated on at least 19 occasions from the blood of
hantavirus, particularly in locations where local vi- persons suffering from febrile illness with headache,
ruses are unknown, it is advisable that antigens dizziness, cough, nausea and vomiting. The cases
representative of all four antigenic types be included included laboratory infections. Keterah virus,
in the tests: Hantaan-like, Puumala-Prospect Hill- isolated from argasid tick parasites of bats and
like and Sin Nombre-like viruses and Thottapalyam from bat blood in Malaysia, has been shown to be
virus. closely related or identical to Issyk-Kul virus. It is
difficult to be certain whether the natural vectors
of Issyk-Kul/Keterah virus are argasid ticks or
POSSIBLE MEMBERS OF THE FAMILY mosquitoes.
BUNYAVIRIDAE Tataguine virus has been isolated from anophe-
line mosquitoes in Senegal, Nigeria, Cameroon and
Central African Republic. It appears to be a poten-
Bhanja, Kasokero, Bangui, Issyk-Kul,
tially important pathogen: antibody has been found
Tataguine and Wanowrie viruses
in human sera in Senegal and Nigeria, and there
Bhanja virus has been isolated from ixodid ticks of have been at least 31 isolations of the virus from the
five genera—Haemaphysalis, Amblyomma, Derma- blood of febrile humans in Senegal, Nigeria, Central
centor, Boophilus and Hyalomma—variously in In- African Republic and Cameroon. The infections
BUNYAVIRIDAE 547
Table 18.6 Abridged classification of possible members of the family Bunyaviridae. Information
derived from sources cited in the text
Bhanja
BHANJA (3)?
Bhanja Ixodids ; ; Europe, Africa, Asia
Yogue
YOGUE (2)
Kasokero Unknown ; Africa
Six other serogroups (17)
Ungrouped (22)
Bangui Unknown ; Africa
Issyk-Kul (Keterah) Mosquitoes? ; ; Asia
Tataguine Mosquitoes ; Africa
Wanowrie Ixodids ; Africa, Asia
?Figures in parentheses indicate the total numbers of recognized members of the relevant taxon.
were characterized by febrile illness with headache, Bishop, D.H.L., Calisher, C.H., Casals, J. et al. (1980)
rash and arthralgia. Bunyaviridae. Intervirology, 14, 125—143.
Botros, B.A., Swanepoel, R., Graham, R.R. et al. (1997) Genetic
Wanowrie virus has been isolated from Hy- characterization of Rift Valley fever isolates from the 1997 and
alomma ticks in India, Iran and Egypt. Little else is 1993—1994 outbreaks in Egypt. Abstracts of the 46th Annual
known about the virus except that it was isolated in Meeting of the American Society of Tropical Medicine and
Sri Lanka from the brain of a human patient who Hygiene, Colorado Springs Resort, Lake Buena Vista,
Florida, December 7—11, 1997.
succumbed to febrile illness with abdominal pain,
Bremner, J.A.G. (1994) Hantavirus infections and outbreaks in
vomiting, haematemesis and passing of blood per 1993. Communicable Disease Report, 4, R5—9.
rectum. Calisher, C.H. (1991) Bunyaviridae. Archives of Virology, 121
(suppl 2), 273—283.
Calisher, C.H. and Karabatsos, N. (1989) Arbovirus serogroups:
definition and geographic distribution. In The Arboviruses:
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
19
Arenaviruses
Colin R. Howard
Royal Veterinary College, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
552 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 19.1 The arenaviruses: host and geographical distribution
Worldwide
Lymphocytic Mus musculus, Aseptic meningitis Europe, N. and S.
choriomeningitis M. domesticus America
Old World
Lassa Mastomys natalensis Lassa fever West Africa
Mopeia Mastomys natalensis Infection possible Mozambique,
Zimbabwe
Mobala Praomys jacksoni Infection possible Central African
Republic
Ippy Arvicanthus spp. ? Central African
Republic
New World
Machupo Calomys callosus Bolivian haemorrhagic Bolivia
fever
Junin Calomys musculimus, C. Argentinian Argentina
laucha haemorrhagic fever
Akadon azarae
Sabia ? Brazilian haemorrhagic Brazil
fever
Guanarito Sigmodon alstoni, Venezuelan Venezuela
Zygodontomys brevicuda haemorrhagic fever
Tacaribe Artibeus literatus (bat) Infection possible Trinidad
Amapari Oryzomys gaedi,
Neocomys guianae
Pichinde Oryzomys albigularis Not recorded Columbia
Tamiami Sigmodon hispidus Not recorded Florida, USA
Latino Calomys callosus Not recorded Bolivia
Flexal Neocomys spp. Not recorded
Parana Oryzomys buccinatus Not recorded
Oliveros Bolomys obscurus Not recorded Argentina
virus and other arenaviruses from Africa. For this exception of Pichinde virus where a large number of
reason, they are loosely referred to as the ‘Old field isolates from Colombia have been character-
World’ arenaviruses, in contrast to those from the ized.
Americas, although now LCM can be found world- Nearly all 17 members of the Arenaviridae so far
wide except in Australia (Howard and Simpson, described have rodents as their natural reservoir
1980). The ‘New World’ arenaviruses show varying hosts. Although rodents are divided into over 30
degrees of serological relationships with Tacaribe families distributed worldwide, arenaviruses are
virus, first isolated in Trinidad. For this reason, predominantly found within two major families:
viruses from the Americas are frequently regarded Muridae (e.g. mice and rats) and Cricetidae (e.g.
as members of the Tacaribe complex. voles, lemmings, gerbils). The nature of the original
The Arenaviridae take their name from the sand- reservoir for LCM virus remains obscure, but it
sprinkled appearance when viewed in the electron appears to be mainly in species of the Muridae
microscope (Latin arena : sand). With the excep- which evolved in the Old World and subsequently
tion of LCM, all are referred to by names that spread to most parts of the globe. Interestingly,
reflect the geographical area in which they were there is a wide range of tropism and virulence
isolated (Figure 19.1). Various strain designations among laboratory strains of LCM virus originally
are also commonly used, in particular for LCM and isolated from laboratory mouse colonies.
arenaviruses isolated from humans. Multiple isola- The natural reservoir of Lassa virus and the re-
tions of non-pathogenic viruses that infect New maining Old World arenaviruses is the peridomes-
World rodents are made less frequently, with the tic rodent Mastomys natalensis. This is also a mem-
ARENAVIRUSES 553
Figure 19.1 Geographical distribution of (a) Old and (b) New World arenaviruses
ber of the Muridae and, in common with the host of filamentous strands up to 15 nm in diameter can be
LCM, frequents human dwellings and food stores. seen in preparations of spontaneously disrupted
In contrast, nearly all arenaviruses isolated from the Pichinde virus. These appear to represent globular
South American continent are associated with condensations which arise from an association be-
cricetid rodents whose members frequent open tween neighbouring turns of the underlying helix.
grasslands and forest. The exception is Tacaribe The basic configuration of the filaments shows a
virus, which was originally isolated from the fruit- linear array of globular units up to 5 nm in diam-
bat, Artibeus literatus. eter, probably representing single molecules of the
viral polypeptide. These filaments progressively
fold through a number of intermediate helical struc-
tures to produce the stable 15 nm diameter forms
Ultrastructure of Arenaviruses and (Young, 1987).
Infected Cells Arenaviruses replicate in experimental animals in
the absence of any gross pathological effect. How-
Negative-staining electron microscopy of extracel- ever, cellular necrosis may accompany virus pro-
lular virus shows pleomorphic particles ranging in duction, not unlike that seen in virus-infected cell
diameter from 80 to 150 nm (Figure 19.2). The virus cultures. The variable pathological changes asso-
envelope is formed from the plasma membrane of ciated with arenavirus infections are further compli-
infected cells. A significant thickening of both bi- cated by the occasional appearance of particles in
layers of the membrane together with an increase in tissue sections that react strongly with fluorescein-
the width of the electron-translucent intermediate conjugated antisera. Granular fluorescence with
layer is characteristic of arenavirus development. convalescent serum in the perinuclear region of
Little is known about the internal structure of the acutely infected Vero cells is often seen. In addition,
arenavirus particle, although thin sections of ma- intracytoplasmic inclusion bodies are a prominent
ture and budding viruses clearly show the ordered, feature in virus-infected cells both in vitro and in
and often circular, arrangements of host ribosomes vivo. These usually appear early in the replication
that are typical of this virus group and confer the cycle and consist largely of single ribosomes which
‘sandy’ appearance from which its name is derived. later become condensed in an electron-dense
Distinct well-dispersed filaments 5—10 nm in diam- matrix, sometimes together with fine filaments
eter are released from detergent-treated virus. Two (Murphy and Whitfield, 1975).
predominant size classes are present, with average
lengths of 649 nm and 1300 nm, respectively; these
lengths do not show a close relationship with the
two virus-specific L and S RNA species. Each is
circular and beaded in appearance. Convoluted
554 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 19.2 Electron microscopy of arenaviruses. (a) Negatively stained Lassa fever particle showing the whole surface covered in
projections. Few particles are less than 100 nm, and many are twice this size. (;300 000) (b) Mopeia virus from southern Africa. Here the
negative stain has disrupted the particle, the contents of which (x) have been extruded. (;150 000) (c) Lassa fever particles budding from
an infected Vero cell. The thick arrow shows a mature particle, the thin arrow a maturing particle at the plasmalemma. Nucleocapsids
and ribosomes line up immediately below the thickened membrane (white arrow). (;39 000)
Cell-mediated Immunity
Persistent Infection
The role of cell-mediated immunity during acute
LCM infection is manifested by a cytotoxic T cell
Antibodies
response associated with the clearing of virus; for
example, T cells cultured and cloned in vitro and Mice persistently infected with LCM virus produce
injected intravenously reduce the amount of virus antibodies to all the major structural proteins. This
100-fold in the spleens of acutely infected mice. finding was contrary to the view previously held
Cytotoxic T cell responses are restricted by the need that viral persistence is established or maintained in
for activated T cells to recognize both viral antigen vivo as a result of an absence of specific B cell
and host cell proteins encoded by the H-2 region, a responses to some or all viral antigens. As viral
concept developed in LCM-infected mice which has proteins continue to be produced in the tissues of
radically altered our concept of the mechanisms by such animals, circulating antigen—antibody com-
which the infected host clears virus from infected plexes are formed which can be detected by binding
tissues. The generation of specific cellular toxicity is Clq. It is worth noting that, despite the existence of
related to the replication of the virus in target or- antibody to all LCM virus structural polypeptides,
gans; inoculation with live virus appears necessary sera from persistently infected mice are negative by
as a primary cytotoxic T cell response is not seen if CF tests; this was the original basis for the belief
the virus is inactivated. This has implications for the that carrier animals do not produce a humoral
development of inactivated arenavirus vaccines response to this virus. Antibody in the sera of such
should the stimulation of cellular immunity prove animals binds to the surface of virus-infected cells,
essential for protection. T cell clones from mice but is unable to mediate complement-dependent
560 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
cytolysis, suggesting that viral antigens at the evidence that either immunopathological or aller-
plasma membrane may be either masked, thereby genic processes play any part in causing disease; it
preventing further immune reactions, or removed appears to be more likely that disease is caused by
by antigenic modulation. This notion would imply direct damage of cells by the virus. Postmortem
that persistently infected mice are deficient in viral studies on patients who died from Junin virus infec-
antibody of the complement fixing subclass of IgG, tion have shown generalized lymphadenopathy, en-
but this has not been proven. dothelial swelling in the capillaries and arterioles of
almost every organ, and depletion of lymphocytes
in the spleen. The virus first replicates in lymphoid
Cell-mediated Immunity tissue, whence it invades the reticuloendothelial sys-
Mice persistently infected with LCM virus should tem and those cells concerned in the humoral and
mount a normal T cell response to unrelated im- cellular immune responses; the host’s defence mech-
munogens, indicating a state of tolerance only to anisms are thus impaired. Fatal illness is invariably
specific antigens. However, it has been difficult to associated with capillary damage leading to capil-
distinguish T cell suppression from an absence of lary fragility, haemorrhages (Figure 19.3) and irre-
virus-specific T cell clones. Here it is pertinent to versible shock (Johnson et al., 1973). Disseminated
mention that persistence of LCM virus in mice in- intravascular coagulation is not a typical feature.
fected at birth or in utero was one of the important Although Lassa fever is often regarded as being
observations made by Burnet and Fenner to sup- hepatotropic, the extent of hepatic damage is insuf-
port the concept of tolerance to ‘self’ antigens. The ficient to account for the severity of the clinical
time of infection is critical, as LCM infection in- disease, and serum transaminase values often re-
duced 24 hours after birth results in a cytotoxic T main within normal limits except in severe cases.
cell response typical of acute disease. The failure of Studies of Lassa virus-infected rhesus monkeys
mice infected before this time to mount an adequate have shown that changes in vascular function may
cytotoxic response is presumably related to matura- play a much greater role in pathogenesis, as a result
tion of T cell function; it appears to be virus specific either of viral replication in the vascular epithelium
because adult carrier mice challenged with other or secondary effects of virus activity in different
unrelated arenaviruses mount normal cytotoxic T organs. Platelet and epithelial cell functions fail
cell responses. Thus the block appears to be either immediately before death and are accompanied by
in recognition of infected cells, or in their expression a drop in the level of prostacyclin; these functions
of type-specific antigenic determinants. The rela- rapidly return to normal in animals surviving infec-
tionship between the virus and the host immune tion (Fisher-Hoch et al., 1987). Impairment of the
response may be more complex than hitherto be- functions of vascular epithelium in the absence of
lieved, however, as there is evidence for arenaviral histological changes appears to be a common fea-
RNA being transcribed into complementary DNA, ture of the final stages of viral haemorrhagic dis-
presumably mediated by endogenous retroviral re- eases in general and suggests that hypovolaemic
verse transcriptase (Klenerman et al., 1997). This shock may be amenable to treatment with pros-
would imply that long term persistence of viral gene tacyclin.
sequences as retroviral elements results in continual The pathogenesis of Argentinian haemorrhagic
low level expression of viral proteins. Thus immune fever has been studied in guinea-pigs infected with
responsiveness is maintained by the continued pres- Junin virus, this being a suitable model of human
entation of viral sequences as MHC—peptide com- disease. There is a pronounced thrombocytopenia
plexes. and leucopenia characteristic of human infections,
and animals die of severe haemorrhagic lesions.
Bone marrow cells are destroyed, with release of
proteases and acid and alkaline phosphatases into
PATHOLOGY OF ARENAVIRUS the blood; this leads to consumption of the C4
INFECTIONS: GENERAL FEATURES component of complement. These effects may lead
in turn to progressive alterations in vascular per-
The mechanisms by which arenaviruses cause dis- meability and platelet function (Rimoldi and de
ease in humans are not fully understood. There is no Bracco, 1980). The most extensive histopathologi-
ARENAVIRUSES 561
Lymphocytic Choriomeningitis
Figure 19.4 Approximate endemic zone of Argentinian haemorrhagic fever. (From Howard, 1986)
crease in the number of CD4 ; cells, has not been conclusively established. The virus
thromobocytopenia and urinary casts containing may be carried in the air from dust contaminated by
viral antigen. Patients recover when the fever falls, rodent excreta or may enter by ingestion of con-
followed by diuresis and rapid improvement. Death taminated foodstuffs.
may result from hypovolaemic shock. Subclinical
infections also occur. Human-to-human trans-
Therapy
mission has not been observed.
In contrast to Lassa fever, antibodies play a major
role in recovery from Junin infection. Controlled
Epidemiology
trials of immune plasma collected from patients at
Argentinian haemorrhagic fever has a marked sea- least 6 months into convalescence have shown a
sonal incidence, coinciding with the maize harvest dramatic reduction in mortality if plasma is given
between April and July, when rodent populations within the first 8 days of illness (Maiztegui et al.,
reach their peak. Agricultural workers, particularly 1979). The efficacy of this therapy is directly related
those harvesting maize, are, not surprisingly, the to the titre of neutralizing antibody in the plasma;
most commonly affected. The main reservoir hosts as a result a dose of no less that 3000 ‘therapeutic
of Junin virus are Calomys field voles that live and units’ per kg body weight has been recommended
breed in burrows under the maize fields and in the (Enria et al., 1984). The late development of a neur-
surrounding grass banks (Figure 19.5). Other ro- ological syndrome is seen in up to 10% of patients
dent species may also be infected. Calomys spp. treated with immune plasma; it is often benign and
have a persistent viraemia and viruria, and virus is self-limiting but points to the possible persistence of
also present in considerable quantities in the saliva. viral antigens on cells of the central nervous system
The mode of transmission of Junin virus to humans well into convalescence. Treatment with immune
564 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Clinical Features
Bolivian haemorrhagic fever was first recognized in
1959 in the Beni region in northeastern Bolivia. The
disease continued in that region more or less an-
nually for a number of years in the form of sharply
localized epidemics. Its incidence has decreased
considerably since the late 1970s and human infec-
tions are now rarely reported. The mortality in
Figure 19.5 The habitat for the rodent Calomys sp. in the wet individual outbreaks varied from 5 to 30%. The
pampas of Argentina. (From Howard, 1986) most notable outbreak affected 700 people in the
San Joaquin township between late 1962 and the
plasma also restores the response of peripheral middle of 1964. The mortality was 18%. It is worth
blood lymphocytes to antigenic stimuli, suggesting noting that the discovery of a common morphology
that administration of plasma also results in the and serological cross-reaction between Machupo
modulation of cellular immunity. and LCM virus led to the concept of the arenavirus
family.
The clinical disease is similar to Argentinian
haemorrhagic fever. The incubation period ranges
from 7 to 14 days and the onset is insidious. About
Prophylaxis one-third of patients show a tendency to bleed, with
There have been attempts to produce a vaccine petechiae on the trunk and palate, and bleeding
against Argentinian haemorrhagic fever. The from the gastrointestinal tract, nose, gums and
XJC1 strain of virus grown in the brains of suck- uterus. Almost half the patients develop a fine
tremor of the tongue and hands, and some may
ling mice is relatively non-pathogenic and was ad-
ministered to 636 volunteers between 1968 and have more pronounced neurological systems. The
1970. Over a period of 3 years, 70 cases of Junin acute disease may last 2—3 weeks and convalescence
virus infection occurred among the population but may be protracted, generalized weakness being the
there were no cases amongst those immunized. most common complaint. Clinically inapparent in-
However, the vaccine often induced a mild febrile fections are rare. Machupo virus, the responsible
reaction or a subclinical infection, and its use was agent, is readily isolated from lymph nodes and
discontinued despite the fact that over 90% of vac- spleen taken at necropsy. Isolation of the virus from
cinees maintained neutralizing antibody for up to 9 acutely ill patients has, however, proved difficult,
years. There have been renewed attempts during the best results being obtained from specimens
recent years to develop a new vaccine strain suffi- taken 7—12 days after the onset of illness.
ciently attenuated for human use and meeting mod-
ern day requirements as to derivation, manufacture
Epidemiology
and potency. Several clones have been prepared
from the original XJ isolate, one of which exhibits The rodent reservoir of Machupo virus is the field
less neurovirulence than the XJCl strain yet pro- vole Calomys callosus; over 60% of animals caught
tected rhesus monkeys against challenge with wild during the San Joaquin epidemic were found to be
type Junin virus (McKee et al., 1993). This ‘Candi- infected. The distribution of cases in the township
date 1’ vaccine has been tested in a double blind was associated with certain houses and Calomys
study in volunteers. callosus was trapped in all households where cases
occurred. Transmission to humans is probably by
contamination of food and water or by infection
through skin abrasions. During the years when
Bolivian haemorrhagic fever was relatively
ARENAVIRUSES 565
Figure 19.6 Map of Nigeria showing the localities of the 1969 and 1970 outbreaks of Lassa fever. (From Howard, 1986)
EMERGING ARENAVIRUS
INFECTIONS Oliveros Virus
Brazilian Haemorrhagic Fever (Sabia This new agent has been isolated from a small ro-
Virus) dent Bolomys obscurus, within the endemic region of
Argentinian haemorrhagic fever (Bowen et al.,
This arenavirus was isolated in 1990 from human 1996). With a rodent host distinct from that of Junin
cases at autopsy (Lisieux et al., 1994). The source of virus, approximately 25% of captured Bolomys ob-
the infection was uncertain but is likely to have been scurus have been found to contain antibodies to this
acquired by exposure to infected rodents in an agri- virus. At present, there are no indications that this
cultural setting in an area immediately outside Sa o virus causes significant numbers of human infec-
Paulo. As a continuing reminder of the potential tions (Mills et al., 1996).
severity of these infections, a laboratory worker was
critically ill after having been accidentally exposed
to an aerosol containing Sabia virus. The virus was SUMMARY
first isolated from a fatal case of haemorrhagic fever.
A laboratory-acquired infection was characterized The increasing numbers of human infections due to
by a febrile illness accompanied by leucopenia and arenaviruses is beginning to require a greater vigi-
thrombocytopenia. There is little information re- lance on the part of public health workers. An
garding the epidemiology of this virus, although the arenavirus aetiology for febrile illnesses in individ-
extensive liver necrosis seen in the first case is a uals residing in endemic areas should be considered,
warning that this and other haemorrhagic fevers particularly those who are likely to have come into
may on first examination be mistaken for yellow regular contact with rodents by virtue of their life-
fever. style or occupation.
Although there is little or no evidence for human
arenavirus infection in North America, Europe and
Venezuela Haemorrhagic Fever Asia, this situation may change once there is greater
(Guanarito Virus) awareness of the potential for emerging infections,
particularly in geographical areas where the last
Between May 1990 and March 1991 an outbreak decades have seen clearance of woodland, forest
occurred among residents of Guanarito municipal- and scrub in advance of extensive changes in agri-
ity on the central plains of Venezuela. Originally cultural practices. The only certainty is that the
mistaken as dengue fever, a total of 104 cases were number of arenaviruses identified hitherto will in-
recorded with a mortality rate of around 25%. The crease as more becomes known regarding the natu-
Guanarito virus was subsequently isolated from the ral history of these agents.
spleens of such cases at autopsy. The principal ro-
dent hosts of this virus have been identified (Table
19.1) (Tesh et al., 1993, 1994). REFERENCES
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cent from the disease. Although initial reports sug- tivity with other arenaviruses. Virology, 113, 73—85.
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
20
Filoviruses
Colin R. Howard
Royal Veterinary College, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
572 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 20.1 Recorded outbreaks of filovirus infections 15 nm core surrounded by a helical, tubular capsid
of approximately 50 nm; the latter bears cross-stri-
Virus Year Location Cases Mortality
ations with a periodicity of about 5 nm. The latter
Marburg 1967 West Germany 29 7 (24) structures within infected cells are presumed to be
1967 Yugoslavia 2 0 virion nucleocapsids because their diameters are the
1975 Southern Africa 3 1 (33) same as those of the tubular structures found in
1980 Kenya 2 1 (50)
1982 South Africa 1 0 intracellular inclusions. These probably correspond
1987 Kenya 1 1 to the nucleocapsid structures with a density of
Ebola 1972 Caire 1? 0 1.32 g ml\ released from virions by detergent treat-
1976 Sudan 360 150 (42) ment.
1976 Zaire 237 211 (89) Virions are constructed from preformed nuc-
1977 Zaire 2 2
1979 Sudan 34 22 (65) leocapsids synthesized in the cytoplasm and envel-
1994 Gabon 44 29 (66) opes are added by budding through cellular mem-
1995 Côte d’Ivoire 1 0 branes. Thus virus-infected cells contain prominent
1995 Zaire 315 244 (77) cytoplasmic inclusion bodies consisting of viral
1995 Côte d’Ivoire 1 0
1996 Gabon 37 21 (57)
nucleoprotein material (Ellis et al., 1978). These
1996 Gabon 60 45 (75) bodies are complex and distinct, consisting of a
Ebola (Reston) 1989 USA 4@ 0 finely fibrillar or granular ground substance which
1990 Philippines 12@ 0 condenses into either tubular structures or nuc-
1992 Italy 0@ 0 leocapsids. As infection proceeds, these increase in
?Retrospective diagnosis.
size and become highly structured, even at sites
@Monkeys. remote from the cell membranes. The budding of
Values in parentheses are percentages. completed virions takes place at cell membranes,
into which virion spikes have been inserted. At the
which may initially be mistaken from other causes time of maturation from the cell surface a series of
of haemorrhagic fever. For general reviews of these individual nucleocapsids becomes enclosed within a
viruses, see Gear (1988), Lloyd (1998) and Murphy single continuous envelope; each individual nuc-
and Peters (1998). leocapsid is only about 700 nm long when produced
in the infected cell. It appears that nucleocapsids, at
the time of budding, may orientate relative to the
cell membrane, in any plane from perpendicular to
Morphology and Ultrastructure of parallel, and may undergo the branching character-
Infected Cells istic of these viruses. The final infective particle is
believed to be the torus form, made by the flexing of
The virions of Ebola and Marburg are very similar individual core length fragments into a circle or
in morphology. By electron microscopy, Marburg torus.
and Ebola particles are seen in a variety of forms;
these are generally long filaments, sometimes with
extensive branching, or as U-shaped, 6-shaped or
circular structures (Ellis et al., 1979). The particles Physicochemical Properties
vary greatly in length (up to 14 000 nm), but have a
uniform diameter of 70 nm and an outer membrane The extraordinary length of filoviruses makes puri-
covered with 15 nm projections spaced 10 nm apart fication difficult; with an estimated molecular
(Figure 20.1). Although the lengths of Marburg and weight in excess of 500 ; 10, particles migrate in
Ebola particles vary over a wide range, a recent density gradients very slowly at a sedimentation
study with rate-zonal, sucrose gradient-purified coefficient of around 1400 S. Filoviruses have a
particles indicates that the unit length associated buoyant density of 1.14 g ml\ in potassium tar-
with peak infectivity for Marburg is 790 nm. Ebola trate gradients, which reflect the lipid-containing
virus is some 1.2 times longer at 970 nm. nature of the outer envelope. Lipid solvents destroy
Beneath the virion envelope lies a complex, 20 nm viral infectivity and release the inner nucleocapsid
diameter nucleocapsid consisting of a hollow 10— with a density of 1.34 g ml\. This ribonucleo-
FILOVIRUSES 573
Table 20.2 Filovirus gene products
Nucleic Acid
Sudan
1976, 1979
Côte d’Ivoire
1995
Gabon Kenya
1994, 1996 1980, 1987
Zaire
1972, 1976, 1977, 1995
Ebola
Zimbabwe
Marburg 1975
The mechanism of transmission of infection in physician was also found to have been infected; he
the outbreaks was mainly by direct contact with had a history of a febrile illness in 1972 following
infected blood, close and prolonged contact with an accidental injury while performing an autopsy on a
infected patient, accidental inoculation or through patient who was also diagnosed as a case of yellow
the use of contaminated syringes and needles. There fever (Stansfield et al., 1982). A 7% seroprevalence
was no evidence to suggest respiratory spread in the of Ebola virus antibodies was found among the
community. All the major African outbreaks have local population.
been characterized by spread within hospitals. In early 1995, a charcoal-maker from near Kik-
A sporadic case was reported in 1977 in Zaire, wit in eastern Zaire became the first recognized case
325 km west of the centre of the first outbreak of an outbreak that, over the ensuing 6 months,
(Heymann et al., 1980). A 9-year-old girl was admit- resulted in 315 cases, 77% of which were fatal. The
ted to the Tandala Mission Hospital with a 3 day index case transmitted the virus to at least 12 family
history of fever, abdominal pain and haematemesis; members. One contact was hospitalized, which may
she died 1 day later. This case is interesting in that have seeded the outbreak. Shortly afterwards, a
the virus was isolated by inoculation of blood into resuscitation team became infected after treating a
guinea pigs even after shipment to the USA under patient misdiagnosed as having typhoid. The out-
less than ideal conditions. The case also stimulated break was inflamed by further infection of un-
a retrospective study of Ebola virus activity in this protected health personnel and infected patients
locality. Another case was a 45-year-old male who being returned into the community. The situation
had died some months previously after hospitaliz- was brought under control by the application of
ation with a clinical diagnosis of yellow fever. A barrier nursing techniques, disinfection and proper
578 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
disposal of the deceased. Once again, the failure of suggested that the virus is neither of the known
local health authorities to recognize the potential serotypes but crossreacts with the standard refer-
problem and to act swiftly led to a severe hospital- ence strains of Ebola virus.
focused outbreak.
Ebola virus has also been found in West Africa
within the last decade. A female ethnologist became
Is There an Animal Reservoir?
infected when conducting a postmortem examin-
ation on a chimpanzee found dead in the Tai Na- There is a strong suspicion that the disease is a
tional Park, Côte d’Ivoire. This case was only diag- zoonosis. Monkeys were originally implicated in
nosed retrospectively after repatriation to the three Marburg outbreaks, but there was a lack
Switzerland. Since 1994, three apparently indepen- of evidence suggesting that primates are among the
dent outbreaks have occurred in Gabon. In July natural reservoirs of the virus. After the South Afri-
1996, the largest of these accounted for over 40 can cases were identified in 1975, an extensive
deaths; one patient was treated in South Africa, search for a reservoir was carried out without suc-
where a fatal nosocomial infection occurred in a cess. In 1977, large numbers of small animals were
nurse. caught in the epidemic areas of the Sudan and
Ebola virus infection is clearly endemic in north- Zaire, and blood and tissues were removed for in-
ern Zaire. Between 4 and 10% of individuals in the vestigation in an attempt to throw some light on the
Tandala area were found to be antibody-positive by natural reservoir for these viruses. Some 200 mon-
immunofluorescence. Similar evidence of virus in- keys and more than 100 other animals were col-
fection has been recorded in other savannah regions lected in Zaire between 1976 and 1979. No speci-
of Central and West Africa. Antibodies to both mens yielded virus and attempts to find Ebola virus
filoviruses have been found in 1—2% of selected antibodies were unsuccessful. However, 26% of
populations in Nigeria, up to 10% among pygmies peridomestic guinea pigs in the Tandala region pro-
and farmers in the rain forest of the Cameroons and ved antibody-positive, although there was no corre-
13% in central Liberia. As with the arenaviruses lation between ownership of positive animals and
(Chapter 19), it is likely that severe haemorrhagic prevalence of antibody in the households con-
fever may represent an unusual human response to cerned. It appears that the guinea pig is an inciden-
these viruses. Cases appear to be most often mis- tal host that is unlikely to transmit the virus to
diagnosed as yellow fever. Virus isolation in a lab- humans.
oratory with suitable containment facilities should Unexpectedly, Ebola virus was found during an
be attempted whenever possible. outbreak of a respiratory disease among macaque
Johnson et al. (1983) undertook surveillance for monkeys (Macara fascicularis) housed in a commer-
Ebola virus infections following two cases of cial quarantine facility in Reston, Virginia. Within 3
Marburg infection in the west of Kenya in 1980. years, a similar outbreak occurred in Siena, Italy.
Evidence of human Ebola infection was obtained in The disease showed the clinical signs and pathologi-
two cases from the densely populated Mount Elgon cal lesions typical of Ebola, with the additional
region. Both were schoolgirls and serological evi- feature of large quantities of respiratory secretions.
dence of infection was obtained in a number of Despite the high titre of virus present (7 ; 10
contacts. It is surprising that no evidence of PFU ml\), there were, fortunately, no cases of hu-
Marburg virus infection was found among the 52 man illness among handlers and staff, despite evi-
suspected cases of acute viral haemorrhagic fever dence of seroconversion. As these animals orig-
identified in the 21 month study period. A further inated in the Philippines, the outbreak due to the
study between May 1984 and 1985 revealed that Reston subtype of Ebola may indicate a non-hu-
nearly 10% of 471 patients with suspected viral man primate reservoir in Asia. Extensive follow-up
haemorrhagic fever had demonstrable antibodies to studies in the Philippines have not confirmed this,
Ebola virus; the case fatality rate was 5%, no higher however, although serological evidence of infection
than that in those with no evidence of Ebola virus has been found in around 6% of people handling
infection. Antibody rates were higher toward the monkeys and working in collection areas. Thus, the
end of the two wet seasons, and were three times identity of any non-human reservoir of filoviruses
higher in males than in females. This study also remains as obscure as ever.
FILOVIRUSES 579
21
Rabies
Karl G. Nicholson
Leicester Royal Infirmary, Leicester, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
584 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 21.1 Members of the genus Lyssavirus of Rhabdoviridae
into four serotypes on the basis of serological and genotypes 5 and 6. Three other lassaviruses that
antigenic relationships. Genetic studies have con- belong to the rabies sero-group are Kotonkan virus
firmed and extended this classification and four and Obodhiang virus, which were isolated from Culi-
genotypes corresponding to the original four sero- coides mosquitoes in Nigeria and Sudan, and
types have been characterized. Serotype 1 con- Rochambeau virus, isolated from mosquitoes in
tained all street and vaccine strains of rabies virus French Guiana.
which infect a wide range of species in nature and ‘Rabid’ bats in Europe were reported as early as
include viruses responsible for historical disease 1954 in an unidentified species from Hamburg, Ger-
syndromes such as ‘oulo fato’ in African dogs, many, and in three Nyctalus noctula bats in Yugos-
Nigerian horse ‘staggers’, ‘derriengue’ in vampire lavia. Subsequently similarities were identified be-
bats, and ‘polar madness’ in Arctic foxes. tween Duvenhage and bat isolates from maritime
Lagos bat virus, the prototype strain of serotype Germany, which led to the assumption that
2, was first isolated from the pooled brains of bats in Duvenhage virus had been imported by bats carried
Nigeria and is probably enzootic in fruit bats by boat from South Africa. However, monoclonal
throughout Africa. It has been identified in a region antibodies have revealed a clear distinction between
encompassing Nigeria and Senegal in West Africa, Duvenhage and EBL1 and EBL2, which have been
the Central African Republic and Zimbabwe, South identified in European bats of at least eight different
Africa and Ethiopia in north-east Africa, principally species, ranging over a geographic area from the
in bats but also in the domestic cat. It is the only one Ukraine to Spain. Epticus serotinus appears to be
of the six genotypes not to have been associated the principal reservoir for EBL1 and Myotis dasyc-
with disease in humans. Mokola virus, the prototype neme and M. daubentonii the reservoirs for ELB2.
strain of serotype 3, was first isolated from the poole EBL2 caused the death of a zoologist in Finland in
d viscera of shrews in Nigeria and then from two 1985, and two deaths from infection with EBL1
Nigerian children with convulsions and a poliomy- have been recorded in the former Soviet Union.
elitis-like illness, one of whom died. Further isolates Over a period of 10 years, 1986—1995, 1882 bats
were made from shrews in the Cameroons and the belonging to 23 species of bat from all parts of
Central African Republic, from dogs in Zimbabwe, England were examined for the presence of rabies
and from domestic animals in Ethiopia. The proto- antigen; all were found to be negative, suggesting
type virus of serotype 4, Duvenhage virus, was first that EBL is not endemic in Britain. However, in
recovered from the brain of a human bitten on the June 1996, EBL2 was isolated from a Daubenton’s
lip by an insectivorous bat in South Africa. The man bat (M. daubentonii) in Newhaven, East Sussex. It is
died of a rabies-like illness, and the virus was subse- conceivable that this bat originated from mainland
quently isolated direct-ly from bats in South Africa. Europe and it is questionable whether EBL is en-
The recently identified European bat lassavirus 1 zootic in Britain.
and 2 (EBL1 and EBL2) have been classified into The European bat epizootic has been identified
RABIES 585
principally in north-central Europe and over recent fic for rabies virus variants that can not be distin-
years has shifted from Denmark to Northern Ger- guished by antigenic typing with currently available
many and the Netherlands. Sporadic cases have monoclonal antibodies, are being used for epi-
been identified in eastern Germany, Spain, the for- demiological monitoring of oral rabies vaccination
mer USSR and Czechoslovakia, France and Po- programmes of wildlife.
land. Evolutionary analysis of the nucleoprotein Analysis of several hundred isolates from animals
gene of EBL1 and EBL2 indicates that both viruses and humans in different parts of the world has
evolved into two genetically distinguishable lin- revealed both geographical and species patterns of
eages (a and b) following geographical drifting. It reactivity with monoclonal antibodies. Similarly,
has been suggested that subsequent lineages EBL1a live virus strains used for the immunization of ani-
and EBL1b were introduced into parts of northern mals can be distinguished from wild-type viruses.
Europe from different directions; EBL1b was intro- When the antigenic makeup of a large number of
duced most recently, probably from North Africa. field isolates were compared with that of the PM-
Although Australia is rabies-free, bat lyssavirus in- HDCS vaccine strain, the percentage of common
fection was identified in Queensland in two black antigenic determinants ranged from 44 to 100%.
flying foxes (Pteropus alecto, fruit bats) in 1996 and Despite these differences the current tissue culture
in one little red flying fox (P. scapulatis) in 1995. vaccines for use in humans have worldwide applica-
Apart from the three human cases of EBL infection, bility, as judged by the lack of reports of vaccine
there is no evidence of natural transmission of EBL failures in specific geographical locations or failures
to other species. The generally low prevalence of related to one or more animal species. Moreover,
EBL does not justify specific control measures. studies in mice indicate that immunization with
However, in both rabies-free and rabies endemic fixed Rabies virus provides protection against iso-
areas the public should be warned not to handle lates representing the spectrum of street rabies virus
bats that appear sick or behave strangely, e.g. day- variants found worldwide. Thus there is no evidence
time flying or entering houses, and bat handlers that antigenic differences account for vaccination
should be encouraged to wear gloves. Mouse pro- failures.
tection studies reveal little or no protection against
EBL viruses from inactivated Pitman Moore rabies
virus, which is used widely in rabies vaccine produc-
tion. Rabies vaccines also provide negligible or no Morphology
protection against Lagos bat virus, Mokola virus or
Duvenhage virus. Rabies virus is an enveloped, bullet-shaped, nega-
Members of the genus Lyssavirus contain a tive-stranded RNA virus with average dimensions
common cross-reacting N protein which can be of approximately 200 ; 75 nm. Variations in length
detected by standard fluorescent antibody, comple- are seen with different rabies virus strains. The par-
ment fixation and precipitation techniques. They ticles are composed of a compact helical nucleocap-
are distinguished by virus-neutralization and cross- sid of 30—35 coils, which are seen as cross-striations
protection tests, indicating that their surface by electron microscopy of negatively stained virus
glycoproteins are different. The application of particles. The nucleocapsid, or ribonucleoprotein
monoclonal antibody technology to the study of complex, is surrounded by a lipid bilayer, 7.5—10 nm
antigenic relationships among rhabdoviruses has thick, associated with two protein species, M2
confirmed the distinction between rabies and ra- which is internal, and G which is a glycosylated
bies-related viruses and has revealed extensive anti- transmembrane protein. The surface of the virion is
genic variation in both glycoprotein and nucleocap- covered with spike projections 8 nm long with
sid proteins of a number of laboratory and street knob-like structures at their distal end (Gaudin et
strains of rabies virus. There are marked differences al., 1992). By electron microscopy it appears that
between different strains of virus in their ability to the surface projection layer is arranged in triangles.
infect, spread within the body and produce disease, The protrusions are absent at the base, which is
and in vitro biological studies and nucleotide se- frequently invaginated, forming a hollow axial
quence studies also reveal distinct differences. Poly- channel. In addition to the typical virus particles,
merase chain reactions (PCRs), using primers speci- anomalous forms such as short, V- and Y-shaped
586 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
ecules of the phosphorylated N (nucleoprotein) that
it is resistant to RNase, and less tightly bound with
the M1 phosphoprotein (initially thought to be a
non-structural (NS) protein) and L protein. This
ribonucleocapsid complex is surrounded by a lipid
bilayer associated with the internal M2 protein and
G transmembrane protein.
N Protein
The nucleoprotein segment of the PV rabies virus
genome contains 1350 nucleotides and shares with
Vesicular stomatitis virus (VSV) four particularly
conserved regions (Tordo et al., 1986, 1988). N pro-
tein is a 450 amino acid long polypeptide. Studies of
Figure 21.1 Electron micrograph of the rabies virus the amino acid sequences of CVS-11, ERA and PV
strains reveal a high degree of similarity: the N
virions have been described. Rabies defective inter- proteins of ERA and PV differ by only four amino
fering (DI) particles measuring 70—100 nm in length acids (99% homology), and those of CVS-11 and
contain RNA and are similar antigenically to full- ERA differ by only nine amino acids, giving 98%
length particles. Although they interfere with the homology. N protein is produced abundantly dur-
replication of full-length virions they cannot repli- ing viral replication and lyssavirus group specificity
cate in their absence. The electron microscopic ap- is determined by its cross-reactivity. The antibody-
pearance of rabies virus particles is shown in Figure binding epitopes on N protein distinguish rabies
21.1. from the rabies-related viruses. Accordingly it has
an important role in diagnosis. Three antigenic sites
have been identified and several T helper epitopes
Chemical Structure
have also been identified, at least one of which is
The gross chemical structure of rabies virus has involved in protection. Studies with N and M1
been estimated. Each particle is composed of ap- proteins produced using baculovirus expression
proximately 24% lipids, 3% carbohydrates, 1% vectors show that N and M1 form complexes (Pré-
RNA and 72% protein. The single strand of RNA is haud et al., 1992). The N protein is less variable than
non-infectious. It has a molecular weight of the other proteins and is considered to be the best
3.8—4.6 ; 10, a sedimentation coefficient of 45S, a candidate for extending the protective spectrum of
buoyant density in Cs SO of 1.66 g m\, and when vaccines to include the rabies-related viruses.
uncoiled is about 4.2 m long. Neurotissue vaccines contain considerably more N
The non-segmented, single-stranded RNA protein than cell culture vaccines.
genome of the Pasteur virus (PV) strain contains
11 932 nucleotides (Tordo et al., 1986), whereas that
M1 Phosphoprotein
of the SAD-B19 strain contains 11 928 bases. These
encode in order from the 3 end, the leader RNA, The rabies virion contains approximately 1750 mol-
and five polyadenylated monocistronic mRNAs for ecules of M1 phosphoprotein, otherwise known as
nucleoprotein N, the phosphoprotein M1, the NS protein, which is highly hydrophilic and is 297
matrix or membrane protein M2, the glycoprotein and 303 amino acids long in rabies and Mokola
G and the large RNA-dependent RNA polymerase virus, respectively. Its nucleotide sequence is poorly
L protein. The relative molecular masses and conserved, especially in the central region. The role
number of copies of each protein per virion differ of the M1 protein is probably regulatory and is
between studies, possibly reflecting differences in believed to help the RNA-dependent RNA poly-
the methods used and the viral strains studied. The merase bind to the promoter for polymerization.
helical ribonucleoprotein core consists of the When these proteins were coexpressed via the vac-
genomic RNA so tightly bound to the : 1800 mol- cinia virus T7 RNA polymerase recombinant in
RABIES 587
mammalian cells, they formed a complex involving differences were not distributed equally throughout
the carboxy-terminal domain of L protein. the three domains of the glycoprotein. In the ec-
todomain, only a 6% difference was observed,
whereas there was 27% divergence in the cytoplas-
M2 Protein
mic domain and 41% in the transmembrane do-
The M2 protein, otherwise known as M protein, is main. Panels of monoclonal antibodies directed
202 amino acids long and each rabies virion con- against glycoprotein G have allowed the construc-
tains approximately 1650 molecules. It is located tion of functional antigenic maps. At least eight
between the viral nucleocapsid and viral mem- antigenic sites have been identified in the ectodo-
brane, probably interacting with both and playing main of different rabies strains and more than one
an important role in the morphogenesis of the virus. site is evidently involved in determining virulence.
A major antigenic determinant has been identified Site III, which is linear and extends from amino
between residues 1 and 78 and it may be involved in acid 330 to 338, is implicated in the recognition of
the host immune response to rabies. specific receptors present on nerve endings, and
mutations at amino acid 333 modify the host-range
spectrum of the virus (Kucera et al., 1985). Muta-
Glycoprotein G
tions at antigenic site II, especially those affected in
The glycoprotein G (molecular weight 70 500), amino acid 198, have reduced pathogenicity (Pré-
which is the only protein external to the virion, is haud et al., 1988). The glycoprotein has also been
required for virus infectivity. The rabies virion con- fragmented and the fragments tested for their ability
tains approximately 1800 molecules of the glyco- to evoke neutralizing antibody synthesis and reac-
protein G. It is also responsible for the recognition tivity with virus-primed T lymphocytes. Here again
of specific cell-surface receptors, bears the fusion it appears that specific immunological functions are
properties of the virus (Gaudin et al., 1993), and associated with different regions of the molecule.
induces and reacts with virus neutralizing antibody
and complement-fixing antibody which lyses infec-
ted cells. G protein also induces cellular immune L Protein
responses involving both T helper cells and The L gene of the PV strain encodes a single open
cytotoxic T cells. The amino acid sequence of the reading frame 2142 amino acids in length
glycoprotein from several laboratory strains of ra- (244 206 Da) that corresponds to the viral RNA-
bies virus and a number of mutants has been de- dependent RNA polymerase and shows a high de-
duced from the nucleotide sequence of cloned com- gree of homology with the VSV polymerase. The L
plementary DNA. The amino acid sequences of the protein of the SAD-B19 strain is 2127 amino acids
glycoproteins of human and canine street rabies long. One-third of the amino acids are in identical
virus strains have also been determined by se- positions to those of VSV polymerase and some
quencing PCR-amplified glycoprotein genes (Be- stretches are homologous with those of Sendai virus
nmansour et al., 1992). The deduced polypeptide for
and Newcastle disease virus, suggesting that rhab-
canine street rabies virus contains 524 amino acids
doviruses and paramyxoviruses are evolutionary
and is equivalent in size and organization to previ- related (Tordo et al., 1988). Each virion contains an
ously characterized glycoproteins. Similarly the G estimated 20—150 molecules of L protein.
protein of Mokola virus is 522 amino acids long.
The rabies G protein consists of a signal peptide
of 19 amino acids, an ectodomain of 439 amino
acids, a transmembrane domain of 22 amino acids, Replication
and a cytoplasmic domain of 44 amino acids (Ben-
mansour et al., 1992). The 19 residue terminal signal The binding of Rabies virus to its receptor is be-
initiates the translocation of the protein through the lieved to be mediated by the surface glycoprotein.
rough endoplasmic reticulum before being cleaved. The virus enters the cell by pinocytosis and the viral
The glycoprotein of a canine street rabies virus membrane fuses with the membrane of the lyso-
displayed 10% divergence in overall amino acid somal vacuole to release the ribonucleocapsid into
composition compared to CVS fixed virus, but the the cytoplasm. Virus replication takes place within
588 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
the cellular cytoplasm and the general scheme of In Vitro Growth
rabies RNA transcription is believed to be similar to
VSV and other non-segmented negative-stranded Rabies virus has been adapted to growth in a wide
RNA viruses. It proceeds via the formation of a variety of primary and continuous cell systems, not
full-length positive RNA copy that is an intermedi- only cells from warm-blooded animals but also
ate of genome replication, and five polyadenylated ones of poikilothermic vertebrate origin. The BHK-
complementary monocistronic messenger (m) 21 cell line is used most commonly in rabies re-
RNAs. Transcription proceeds under the control of search because of its high susceptibility to fixed
the L protein, the RNA-dependent RNA poly- strains of virus, high virus yields and lack of
merase, which is included in the nucleocapsid. The cytopathic effect. Street Rabies virus cannot be
mRNAs attach to membrane-bound polysomes or grown consistently in hamster kidney cells. Murine
cytoplasmic ribosomes for synthesis of their re- neuroblastoma (NA C1300) cells are the most sus-
spective protein molecules (translation). However, ceptible to street rabies virus infection and compari-
the G protein is produced in the rough endoplasmic son with the mouse inoculation test indicate that a
reticulum and is transported via the Golgi appar- rabies tissue culture infection test using mouse
atus to the cytoplasmic membrane of the cell before neuroblastoma cells is at least as efficient as the in
virion assembly. In common with other negative- vivo test, but the tissue culture based test is much
strand RNA viruses, the newly synthesized plus- quicker and less expensive.
stranded RNA eventually stops being generated Human diploid cells are used to produce rabies
into the form of multiple mRNA species; instead it vaccines with well-established records of safety, po-
serves as template to synthesize the progeny nega- tency and postexposure efficacy, but the poor
tive-stranded RNA molecules, the L protein prob- growth characteristics of virus in diploid cells make
ably acting as the replicase in such a reaction. Sub- vaccine production very expensive. Vero cells, a
sequently, the genomic RNA associates with N, M1 continuous line of African green monkey cells, yield
and L proteins to form the nucleocapsid which more virus than human diploid cells and the culture
migrates to cell membranes, these being converted of Vero cells on microcarriers in large scale bioreac-
into virion envelope by insertion of proteins G and tors has made possible the production of rabies
M2. New virions are formed by a process of bud- vaccines at an industrial scale at comparatively low
ding through the cell membrane. cost. Rabies virus has long been adapted to growth
in developing avian embryos for the purpose of
vaccine production, though only recently have
Effects of Physical and Chemical Agents modern purification techniques allowed the devel-
opment of a highly potent product. Virus infectivity
Rabies virus is inactivated below pH 4 and above in tissue culture is generally augmented by die-
pH 10. It has a half-life of about 4 hours at 40°C and thylaminoethyl-dextran or protamine sulphate.
is inactivated within 10 minutes at 60°C, but is Fluorescence appears about 12 hours after infec-
stable for several days at 0—4°C. It is also resistant tion, with higher yields of virus at 33°C than 37°C.
to repeated freezing and thawing. The virus is sensi- Cytopathic effects and plaque formation occur with
tive to quaternary ammonium disinfectants (e.g. some cell systems and virus strains, but in most cell
benzalkonium and cetrimonium at dilutions of 1/ types cytological changes are absent.
1000), 1% soap solutions, ionic and non-ionic de-
tergents, 5% iodine, most common organic solvents
(45% alcohol, ether and chloroform), formalin, - Epidemiology
propiolactone, acetylethyl-eneimine, tri(n-butyl)
phosphate, proteolytic enzymes, ultraviolet (UV)
Animal Rabies
light and ionizing irradiation. It is more resistant to
0.25—0.5% phenol used in Semple-type vaccines Rabies is enzootic in all continents except Austra-
where several days are required to completely inac- lasia and Antarctica. Most other disease-free areas
tivate the virus. An important component of are islands or, less commonly, peninsular land
postexposure treatment is the thorough cleansing of masses where stringent quarantine regulations can
the wound with soap or detergent. be regularly enforced. All warm-blooded animals
RABIES 589
are susceptible to the virus, including birds, al- infected population. Field trials to immunize foxes
though these are rarely if every infected in nature. against rabies began in Switzerland in 1978 and
The disease exists in two epidemiological forms: since then oral immunization of wildlife with baits
urban rabies, which is propagated chiefly in feral containing live attenuated rabies virus (or recom-
and domestic dogs and is prevalent in many devel- binant vaccinia virus expressing the glycoprotein of
oping countries of the world; and sylvatic rabies, rabies virus), dispersed by air, has been applied
which occurs in a wide range of species, principally widely throughout Europe. By this means the inci-
small carnivores and mustelids. dence of rabies in France declined by 98% during
Globally, rabies is most prevalent among wild the period 1989—1994 and large areas of France are
canids (foxes, wolves, jackals), followed by domestic now rabies free. Similarly in Belgium in 1995 the
dogs, skunks, farm animals, cats, bats, mongooses disease was eliminated from 80% of the areas infec-
and other species. However diagnostic and other ted initially, and in the Czech Republic, the number
facilities are limited in many developing countries of rabies cases fell from 1501 cases at the start of the
and the number of cases among domestic dogs is programe to 221 in 1994. The overall success of
probably grossly underestimated. these programmes is illustrated by figures on the
Extensive surveys show rodent rabies to be ex- number of rabies cases reported in Europe: in 1983
tremely uncommon. Rabies is unusual among la- the number totalled 23 002 cases; by 1995 it had
gomorphs (i.e. rabbits and hares) and insectivores fallen to 8134. However, experience has shown that
(i.e. shrews, moles and hedgehogs), though mus- the elimination of rabies depends upon repeated
telids (i.e. skunks, weasels, badgers and martens) vaccination programmes, continuous surveillance,
and the Viverridae (i.e. mongoose, suricate, ferret, with particular attention to cross-border contami-
genet, civet cat and polecat) are regularly found to nation, and subsequently restricted targeted vacci-
be infected. Non-human primates are infected occa- nation campaigns.
sionally and isolated cases have been reported The epidemiology of rabies in the United States
throughout Africa, South America and parts of has changed since the 1940s, from domesticated
Asia. Among the wild Felidae, sporadic cases have animals (mostly dogs) to wildlife vectors, principally
been identified in lions, hyaenas, leopards, cheetahs, skunks, bats and racoons. Racoon rabies, epizootic
lynxes, tigers and ocelots. Rabies among wild un- among racoons in the south-eastern and mid-Atlan-
gulates is identified regularly in central European tic states, has become an increasingly important
deer, and is recorded in camels in North and West problem in the eastern United States: the extension
Africa, India, and the Near and Middle East; kudu of the epizootic was largely responsible for a 43%
and eland antelopes in South-West Africa; and oc- increase in the total number of cases between 1990
casionally in buffalo and moose in North America. (4881) and 1991 (6975); in 1991, 3079 cases in rac-
The Chiroptera are of major importance as reser- oons were reported. The epizootic was probably
voirs and transmitters of infection in the Americas. introduced into the mid-Atlantic region in the mid-
In Latin America bites by haematophagous vam- 1970s when racoons were transported from the
pire bats cause occasional human deaths and losses south-eastern United States to the mid-Atlantic
of more than 100 000 cattle each year. In the United area to replenish hunting stocks. In 1994, 8224 cases
States bats represent the most widely distributed of rabies were reported in the United States, 93% of
vector, and the silver-haired bat variant has been which (7632 cases) were wild animals. The striped
identified as the aetiological agent of a number of skunk is a host of rabies in large areas of Canada
recent human rabies cases. In Europe and Australia and the United States. In Canada, foxes account for
infection with bat lyssaviruses has been reported 40% of all cases nationally. The success of cam-
but seems rare. paigns in Europe to eliminate fox rabies has encour-
In Europe the major reservoir of infection is the aged efforts in North America to control sylvatic
red fox: in 1992 there were over 11 000 reported rabies in coyotes, racoons and foxes. In the United
cases of animal rabies in Europe, 66% of which States, preliminary data indicate a significant re-
occurred in foxes. Martens, deer, badgers and other duction in the incidence of rabies in vaccinated
animals are involved much less frequently. As in the areas and expansion of a rabies epizootic involving
United States, domestic species in Europe account coyotes ceased.
for only a small percentage (10—15%) of the total In Africa, Mexico, Central and South America,
590 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Asia and Asia Minor the dominant reservoir is clinical presentation. Researchers in Columbia dis-
found in unvaccinated domestic dogs, which ac- covered that 27 of the 1596 people (1.7%) on whom
count for 85% of recognized cases. The degree of postmortems were carried out in Cali, in 1962, had
involvement of wildlife species in tropical regions is died of rabies, although only one or two cases had
largely unknown. To prevent or eliminate out- been diagnosed annually in years past. Similarly,
breaks of canine rabies, the World Health Organiz- 21% of all cases in the United States between 1960
ation (WHO) recommends that 70% of dogs in a to 1979 were not diagnosed until after death, and
population be immunized. This percentage has might have gone undiagnosed if postmortem patho-
been determined empirically from observations on logical or virological studies had not been under-
the relationship between vaccine coverage and the taken (Anderson et al., 1984).
incidence of rabies in dog populations around the Few tropical countries have rabies under control,
world. Since this level of coverage is not attainable and the risk of exposure in most developing coun-
readily and serological responses may be subopti- tries seems appreciable. A survey of missionaries
mal or of short duration, efforts are being directed and foreign aid members working in developing
at immunizing free-roaming domestic dogs using countries showed that the overall risk, based on a
baits containing rabies vaccine. The possible use of mean stay of 4—5 years, was 16% per household.
oral modified live rabies vaccine to control rabies in Similarly, a survey of 1882 foreign travellers to
jackals in Zimbabwe is also under consideration Thailand, who remained in the country for a mean
and a candidate virus has been tested for im- of 17 days, indicated that 1.3% were bitten by a dog,
munogenicity and safety in potential non-target and that almost 9% were licked by a dog. More
bait-consuming species. extensive analyses in 30 tropical countries show the
incidence of rabies to be 0.1—25.8 cases per million
population (mean, 3.7), and the number of treat-
Human Rabies
ments to be 2.7—4570 (mean, 867) per million popu-
There is little reliable information concerning the lation (Bögel and Motschwiller, 1986). However,
extent of human rabies or relative risk of exposure the incidence of rabies among travellers returning
in many regions of the developing world, so the true to Europe and North America is very low and it has
extent of the disease in humans is uncertain. In been estimated that the cost of routine pre-exposure
1992, a total of 36 001 cases of human rabies were immunization per case prevented per year of stay
reported worldwide to the WHO. Human rabies amounts to Canadian $275 000. Accordingly, pre-
occurs mostly throughout the Indian subcontinent, exposure immunization should be offered selective-
south-east Asia, much of Africa, Central and South ly to travellers deemed to be at the greatest risk.
America, and Mexico, where the virus is propagated
chiefly in feral and domestic dogs. Only a few en-
dogenous cases occur in Europe and North Amer-
ica despite the presence of sylvatic rabies in a numb- TRANSMISSION AND PATHOGENESIS
er of animal species. Nonetheless, the estimated
costs associated with the decrease in deaths in the Modes of Infection
United States is estimated at hundreds of million
dollars annually. Although the commonest mode of infection in hu-
The official reports of human rabies probably mans is by the bite of a rabid animal or the contami-
underestimate its true incidence, which may ac- nation of scratch wounds by virus-laden saliva,
count for as many as 100 000 deaths annually. The other means of infection are possible. Intact skin
official data should be interpreted with caution appears impenetrable to the virus, but infections
since many outlying villages in developing coun- have been reported following the handling of infec-
tries do not report rabies, yet as many as three- ted dog carcasses that were being prepared for
quarters of the cases are believed to occur in rural meals. Infection across undamaged mucous mem-
areas, where access to medical and diagnostic facili- branes of the mouth, conjunctiva, anus and geni-
ties is often poor. Of the cases that reach hospital, talia, either directly from rabid animals or indirectly
many undoubtedly are misdiagnosed, either through licking contaminated cord or material, are
through inadequate diagnostic facilities or atypical recorded. Infection by aerosol transmission has also
RABIES 591
been amply demonstrated in laboratory animals, to salivary glands and other tissues where viral
and has been implicated in human infection ac- replication and release occur. The survival of the
quired in bat-infested caverns in Texas and in sev- virus in nature is wholly dependent upon the behav-
eral laboratory accidents. ‘Rage de laboratoire’, the ioural changes caused by rabies encephalitis.
consequence of receiving incompletely inactivated
fixed rabies virus in human vaccines, is well
Entry Into Peripheral Nerves
documented in the older literature and an outbreak
involving 18 persons occurred in Brazil in 1960. Recent reports suggest that the rabies virus recep-
Human-to-human transmission by transplanta- tors on muscular cells coincide with the distribution
tion of infected corneas has been reported in five of acetylcholine receptors. -Bungarotoxin, a snake
instances, with the diagnosis in the donors only venom neurotoxin, also binds to acetylcholine re-
being made retrospectively. It has also reportedly ceptors , but the binding of both virus and -bunga-
occurred in an incident involving several young rotoxin can be inhibited by monoclonal antibodies
boys in the North-West Frontier Province of Pakis- raised against residues 190—203 of the rabies virus
tan who were infected by saliva applied to circum- glycoprotein. However, the virus can enter cells
cision wounds by a surgeon-barber with early ra- independently of the nicotinic acetylcholine recep-
bies. Occasional cases of human-to-human tor complex (Reagan and Wunner, 1985), but
transmission are described in the older literature, whether this is related to specific receptors is uncer-
and in a large Indian survey 1 of 11 134 contacts of tain. For neuronal and fibroblastic cells, it has been
human cases developed the disease. Judging by the found that oligosaccharides, sialic acids and gan-
absence of reports of cases in nursing and medical gliosides may be involved in virus binding. Using
staff, it seems to be a rare event. Nonetheless, medi- sequential tissue titrations, immunofluorescence
cal attendants are at genuine risk of being bitten or and electron microscopy, it has been shown that
contaminated by saliva, and especially to aerosols virus localizes initially at motor end-plates near the
generated by airway care, and should be considered site of inoculation, infects and replicates in my-
for postexposure therapy. Kissing and coitus as ocytes, is shed into extracellular spaces and involves
routes of human-to-human transmission have also neuromuscular and neurotendinal spindles to enter
been described in the older literature. peripheral nerves. Only during this early stage does
Vertical transmission after natural infection is the virus seem susceptible to postexposure therapy.
recorded in various species but not in humans, al-
though there are instances of women dying from
Translocation to the CNS
rabies shortly after parturition. Oral transmission
through cannibalism occurs in laboratory animals The first convincing demonstration that nerve
but has never been described in humans; its role in trunks provide the channel for the movement of
nature is unknown. Reference to infection in wean- virus to the brain was provided by Di Vestea and
ling animals, possibly through the consumption of Zagari in 1889. These investigators inoculated Ra-
infected milk, was made in a review by Afshar (1979). bies virus into sciatic and median nerves and
Although many patients can relate the circum- showed that virus was recovered first in the relevant
stances of their exposure, recent experience in the areas of the spinal cord and that its translocation
United States indicates that a large proportion are could be interrupted by section of the cord. In 1925,
unable to do so. Goodpasture noted that cytopathic changes in the
ganglia and CNS corresponded anatomically to the
sites of virus inoculation, again inferring neural
movement of the virus. Further studies gave sup-
Pathogenesis port to this hypothesis. Rabies virus moves from
peripheral sites to the CNS at a rate estimated
The pathogenesis of rabies involves the entry of crudely at 3 mm h\. Translocation of virus is unaf-
virus into peripheral nerves, centripetal movement fected by removal of the perineurium, epineurium
of virus to the cord and brain, viral replication or perineural epithelium, but can be blocked by
within the central nervous system (CNS), and cen- local anaesthetics, colchicine and vinblastine. No
trifugal movement of virus back along nerve routes replication occurs within the axoplasm, which is
592 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
devoid of the necessary organelles. However, ampli- secretions, are the olfactory end-organs in the nares
fication of virus may occur within neurons of the and the neuroepithelium of taste buds. Experimen-
dorsal route ganglia. tally, about 48% of dogs, 88% of cats, 42% of foxes
and 83% of skunks excrete virus in saliva, though
gland involvement of naturally infected animals
CNS Involvement
may reach 90%. The behaviour and appearance of
From the cord, virus ascent to the brain occurs animals are poor guides to their infectivity as re-
within 24—48 hours, with initial distribution in- ports of preclinical shedding of virus range from 3
fluenced by the site of inoculation. CSF is of doubt- days in cats to almost a month in foxes. Moreover,
ful importance in viral dissemination since in hu- there are well-documented instances of apparently
mans it is rarely infectious antemortem. There is healthy dogs excreting virus intermittently over
extensive viral replication in the brain, which prob- periods of many months, of apparently healthy ani-
ably supports several growth cycles before onset of mals outliving their victims, and of dogs recovering
clinical effects. The time taken for the virus to from rabies. These observations suggest that the
spread throughout the brain is roughly half that practice of discontinuing rabies postexposure treat-
taken to reach the CNS from the site of inoculation. ment if the biting animal is kept under observation
Terminally, there is widespread CNS involvement and remains healthy for P 5 days is potentially
in humans and animals, but surprisingly few neur- dangerous.
ons infected with street virus show structural abnor-
malities. The nature of the profound neural disorder
is thus obscure, although, according to one hypoth- Factors Influencing Infection
esis, the greater localization of virus to the limbic
system provides a clinicopathological correlate for Bites inflicted by rabid animals do not necessarily
the abnormal behaviour and aggression necessary cause disease. In humans, mortality rates of
for virus perpetuation. Interestingly, recent work 4—100% have been recorded when the infectivity of
suggests that opiate and acetylcholine receptors of the biting animal is established by the death of
infected cells are impaired, implying disordered another person or animal (Table 21.2). Apart from
neurotransmission as the cause for the progressive species differences in the susceptibility to the virus,
neural dysfunction. several factors modify the outcome. First, the infec-
tivity of the saliva may vary. Secondly, the virulence
of the virus may vary (differences in the amino acid
Movement from the CNS
composition of sites II and III of the glycoprotein
Following invasion of the CNS, ‘street’ and occa- affect both host range and virulence). Thirdly, the
sionally ‘fixed’ strains of Rabies virus move along site and severity (deep versus superficial, and
axoplasmic routes to salivary glands and other tis- multiple versus single) of the bites—severe head and
sues. Many sites may be involved, including lacri- neck bites carry the greatest risk, followed by bites
mal glands, myocardium, skeletal muscle, lung, to the upper limb (particularly the richly innervated
liver, adrenal glands, the retina, cornea, taste buds, hand), then the lower limb and trunk. However, the
sebaceous glands and hair follicles, particularly location and severity of wounds are influenced by
those of the head and neck. Sites with short neural the species of biting animal, which in turn affects the
connections to the CNS are usually the first to be amount of saliva deposited in the wound and, prob-
infected and immunofluorescence of corneal im- ably, its infectivity. Finally, the intervention of
pressions or skin biopsies may establish the diag- clothing reduces mortality by 60—80% compared
nosis antemortem. with wounds inflicted through bare skin.
The most important site for virus transmission is
the salivary gland mucosal epithelium which repre-
sents the major source of virus shed into secretions. CLINICAL FEATURES
Occasionally the titres of street virus in salivary
gland exceed those of brain, indicating efficient rep- Incubation
lication in mucous acinar cells. Other sites infected
commonly, which may shed virus into oronasal The incubation period is highly variable, ranging
RABIES 593
Table 21.2 Deaths following bites by rabid dogs and wolves 100
in the absence of rabies postexposure treatment (no account is
taken of the position, number and severity of bites)
80
Animals No. with rabies/
Reference species total number exposed
Percentage
60
Hunter (1793) Dog 1/24 (4)
Semple (1919) Dog 7/9 (78)
Cornwall (1923) Dog 148/423 (35)
Veeraraghavan (1969) Mostly dogs 109/195 (56) 40
Clinical Course Table 21.5 Patients in whom pain or paraesthesiae of the bitten
area was a presenting feature
The onset of human rabies is marked by 2—7 days of Dupont and Earle (1965) 23/49 (47)
prodromal symptoms that are almost entirely non- Suri and Chugh (1975) 19/30 (63)
specific, comprising fever, malaise, anorexia, Wilson et al. (1975) 4/23 (16)
Warrell (1976) 7/20 (35)
nausea, vomiting, diarrhoea, sore throat, cough,
Anderson et al. (1984) 19/38 (50)
myalgia and headache as the most common com-
plaints. Behaviour disturbances are often noted, in- Total 72/160 (45)
cluding anxiety, depression, stupor, hyperactivity, Values in parentheses are percentages.
aggression, delirium and intolerance to tactile, au-
ditory and visual stimuli. The patient may complain cipitated by other stimuli, e.g. eating, swallowing
of insomnia, nightmares, hallucinations, urinary re- accumulated saliva, or by a draught of air (aero-
tention, excessive libido, priapism and rarely recur- phobia). Many patients look intensely frightened,
rent ejaculation. An early symptom of diagnostic the appearance of fear being heightened by a pro-
significance is of abnormal sensation involving the ptotic stare, dilated pupils and an open mouth.
bitten area, most commonly pain or paraesthesiae, Often the respirations are shallow and rapid and
noted in about 45% of cases (Table 21.5). Few there may be chest pain and tightness.
objective signs appear early in the clinical course, so Approximately 50% of human cases exhibit hy-
unless the doctor’s attention is drawn to a recent drophobia, an overwhelming fear of water precipi-
exposure or healed bite wounds are noticed it is tated by attempts to drink, and, less frequently, by
unlikely that the diagnosis will be considered. the sight, sound or mention of water or other fluids.
Hydrophobia typically starts with uncontrollable
jerking movements of the hand, arm or body as
Patterns of disease
fluid is brought to the mouth; the head jolts back-
The prodrome is followed by one of two basic clini- ward, the arms upward, fluid is spilled and spasms
cal patterns in humans: the more common ‘furious’ are induced. Attempts to swallow are defeated by
form characterized by hyperexcitability, spasms coughing, retching, vomiting, aspiration, opis-
and hydrophobia; or ‘dumb’ rabies featuring an thotonous, asphyxiation and convulsions, which
ascending paralysis. For descriptive purposes they end in death in 28% of cases. Often the patient
are considered separately, although ‘furious’ rabies struggles violently, may attack attendants or bolt
is often accompanied by paralysis and ‘dumb’ rabies from the room.
may herald spasms and hydrophobia. Rabies The excitement phase sees an initial increase then
should therefore be regarded as having a broad decrease in the frequency and severity of spasms.
clinical spectrum rather than two distinct forms. The patient is usually febrile and may develop a
The onset of furious rabies is marked by increas- variety of abnormalities, including nuchal rigidity,
ing insomnia and periods of extreme agitation, de- photophobia, fasciculations and paresis, particular-
lirium, hyperactivity and purposeless movements, ly at the site of exposure, cerebellar signs, cranial
either occurring spontaneously or provoked by any nerve palsies, hypo- or hyperreflexia, extensor plan-
tactile, auditory, visual or olfactory stimuli. Such tar responses, focal or generalized convulsions and
patients may be mistakenly diagnosed as having a variety of autonomic disturbances. Deterioration
schizophrenia, acute mania or delirium tremens. is marked by the evolution of a flaccid paralysis and
Often these episodes of agitation are accompanied onset of coma, by irregular patterns of respiration
by frothing at the mouth, difficulty in swallowing and by potentially fatal complications. Untreated
and intense spasms affecting the muscles of degluti- cases survive for an average of 3—7 days once symp-
tion and accessory muscles of respiration and last- toms develop.
ing for 5—15 seconds. They are followed minutes Dumb rabies is far more likely to pose diagnostic
later by lucid intervals with the patient lying problems because the pharyngeal spasms and hy-
anxious and exhausted in bed. The spasms follow drophobia so characteristic of rabies hardly ever
characteristically attempts to drink but may be pre- appear. Paralytic rabies has an incidence of 14—60%
RABIES 595
in reported series of cases, there being approximate- Table 21.6 Clinical features and complications of human
ly four furious cases for every dumb one overall. The rabies
incidence is probably higher, since the onset of a Nervous system
flaccid paralysis some months after a minor or for- Cerebral oedema
gotten exposure is likely to be diagnosed as polio, Convulsions (focal or generalized)
transverse myelitis or Guillain—Barré syndrome. To Hypo- or hyperthermia
Diabetes insipidus
stress this point, it should be noted that at least five Hydrophobia
corneas have unwittingly been transplanted from Aerophobia
persons with paralytic rabies. Irregular respiration
‘Dumb’ or ‘paralytic’ rabies normally begins with Hyperactive episodes
typical prodromal symptoms, occasionally with hy- Paralysis
Anaesthesia/paraesthesia
peraesthesiae or pain localized to the bite site. An- Cranial nerve palsies
aesthesia may be present, but is seldom a dominant Priapism
feature. The paralysis often involves the bitten limb Photophobia
initially, then spreads rapidly and symmetrically. Nuchal rigidity
Sphincter control is typically lost and paralysis of EEG abnormalities
Cerebellar signs
the muscles of deglutition, articulation and respir- Inappropriate secretion of antidiuretic hormone
ation normally occur as a terminal event. The
course can be modified at any stage by the appear- Cardiovascular system
Arrhythmias
ance of spasms, hydrophobia and convulsions. Sur- Myocarditis
vival tends to be longer than for patients with furi- Hypotension
ous rabies, the average period from several series Heart failure
being 7—12 days. ECG abnormalities
Respiratory system
Pulmonary oedema
Irregular respiration
Inspiratory spasms
Complications Bulbar/respiratory paralysis
Bronchopneumonia
Complications involving the cardiovascular, respir- Respiratory arrest
atory and neurological systems become especially Atelectasis
prominent when the course is prolonged by inten- Gastrointestinal system
sive therapy, although disturbances affecting other Gastrointestinal tears
systems, e.g. renal failure, may also aggravate the Gastritis
clinical course (Table 21.6). Ulceration
Haematemisis
As a sequel to inflammatory or hypoxic cerebral Pancreatitis
oedema, raised intracranial pressure contributes to Ileus
the decreased level of consciousness and to focal
Other
and generalized convulsions. Other CNS complica- Hypovolaemia
tions include disturbances of thermoregulation, dia- Electrolyte imbalance
betes insipidus, autonomic dysfunction affecting pH abnormalities
fluid and electrolyte balance and blood pressure, Sialorrhoea and other excessive secretions
Renal failure
convulsions in two-thirds of cases, and irregular
breathing patterns consistent with lesions at the
midpontine level or pontomedullary junction. with pulmonary oedema, congestive cardiac failure
The clinical course is almost invariably compli- or acute renal failure is a common finding in the
cated by cardiac dysrhythmias of virtually any kind. latter stages of the disease and may be partly or
Several investigators have noted histological evi- wholly caused by myocarditis, hypoxia, autonomic
dence of a myocarditis; ECG changes supporting dysfunction or hypovolaemia.
the diagnosis of myocarditis have also been found Respiratory disturbances occur in all cases.
and, when searched for, virus has been isolated from Blood-gas and pH abnormalities complicate the
myocardium in 52% of cases. Severe hypotension early clinical course, partly because of hyperventila-
596 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
tion. Thereafter death is often precipitated by as- afebrile and prodromal symptoms are absent. They
phyxiation caused by hydrophobic spasms, convul- characteristically refuse to drink; if they do drink,
sions or bulber and respiratory paralysis. Certain they exhibit no jerkiness of hand, arm or head, and
complications, notably bronchopneumonia and no convincing inspiratory spasms. Fanning a pa-
collapse and consolidation, invariably occur after tient with genuine rabies often invokes inspiratory
tracheostomy and artificial ventilation and are po- spasms, a feature not seen with rabies phobia.
tentiated by increased secretions, heart failure and
aspiration. Spontaneous pneumomediastinum has
also been reported, presumably as a consequence of Tetanus
the inspiratory spasms. The respiratory disturban- The incubation period is shorter than for rabies,
ces together with the cerebral effects on ventilation although in many instances it cannot be deter-
significantly lower the arterial P , which is often mined. The presence of trismus and persistence of
found as a terminal event. increased muscular tone between spasms in tetanus
Hypovolaemia, electrolyte imbalance and pH normally differentiate it from rabies. The results of
disturbances, arising from fluid deprivation, inap- lumbar puncture are also helpful, since they are
propriate secretion of antidiuretic hormone, dia- abnormal in 60—90% of cases of rabies (Anderson et
betes insipidus, excessive secretions, ileus and venti- al., 1984) but rarely so in tetanus.
lation—perfusion defects, frequently complicate the
late clinical course. Some gastrointestinal complica-
tions are seen occasionally, including gastro- Poliomyelitis
oesophageal tears, frank ulceration, pancreatitis Unlike rabies, the fever of polio usually abates with
and ileus. the evolution of paralysis; sensory disturbances,
spasms and hydrophobia are also absent.
DIAGNOSIS
Vaccine Reactions
Differential Diagnosis
Neuroparalytic reactions to neural tissue vaccines
are usually seen 8—21 days after the onset of vacci-
With such non-specific prodromal features, rabies is
nation, well within the incubation period of rabies.
unlikely to be considered during the early stages
Spasms and hydrophobia are absent and recovery
unless a history of bite exposure is volunteered or
is the rule. There is also a notable absence of virus
obvious spasms or hydrophobia are present. Recent
and Negri bodies in the brains of those who die.
American experience showed rabies to be consider-
ed for only 3 of 21 patients on first visit to a phys-
ician and for only 7 or 23 on admission to hospital Guillain–Barré Syndrome
(Anderson, et al., 1984). The admission diagnoses
included viral encephalitis, polio, postinfectious en- This cannot be distinguished on clinical grounds or
cephalitis, vaccine reaction, Guillain—Barré syn- by examination of the cerebrospinal fluid (CSF)
drome, brain abscess, cerebrovascular accident, during the early stages. Recovery from Guillain—
brain tumour, tetanus, phenothiazine toxicity, psy- Barré syndrome is generally the rule, however, and
chosis, rabies phobia, pneumonia, myocardial in- consciousness is fully retained. Rabies should al-
farction, dissecting aortic aneurysm and arteritis. ways be considered if there is any deviation from the
Previous reviews mention ‘rage de laboratoire’, normal clinical course.
malingering, delirium tremens, botulism and poi-
soning by strychnine and other plant derivatives in Viral Encephalitis
the differential diagnosis. Important distinguishing
features for some of the above are outlined below. Hydrophobia and spasms are absent in other types
of encephalitis and are seen in only one-half of cases
with rabies. Since rabies is otherwise indistinguish-
Rabies Phobia
able from other encephalitides, it should always be
Most patients with rabies phobia or hysteria are considered for patients coming from endemic areas.
RABIES 597
Poisoning and Drugs How useful are these methods clinically? Recent
American experience showed the detection of serum
Strychnine poisoning may cause paroxysmal
antibody in unvaccinated subjects to be the most
spasms of respiratory muscles resembling those of
successful overall, with a cumulative detection rate
rabies, but there are no prodromal symptoms and
of 50% by days 5—8, 67% by days 9—12 and 100%
no fever. Similarly, the dystonia and oculogyric cri-
by days 13—16 (Anderson et al., 1984). Antibody
ses of phenothiazine toxicity are unlikely to be con-
appeared much later in CSF than in blood and was
fused with the spasms of rabies.
of no help in early diagnosis. As might be expected,
antibody detection and virus isolation were inverse-
ly related, with virus recovery from 60% on days
Diagnostic Testing 0—4, about 32% on days 5—8 and 9—12, and 18% on
days 13—16. Similarly, study of seven South Ameri-
Non-specific Examinations can cases showed virus shedding from 100% on
days 0—4, 50% on days 5—8, 33% on days 9—12, and
The blood white cell count is often elevated from no-one on days 13—16. It should be empha-
(10—20 000 per mm) with a polymorph predomi- sized that virus isolation is a time-consuming pro-
nance; occasionally it is normal or in excess of cedure and that it can be one or more weeks into the
30 000 per mm. CSF examination shows typically clinical illness before the diagnosis is established or
a normal opening pressure and a mixed pleocytosis refuted by either method.
rarely in excess of 300 per mm in 60—90% of cases In contrast, immunofluorescence testing, when
(Anderson et al., 1984). The CSF protein is modestly positive, establishes the diagnosis rapidly, although
elevated in one-quarter of cases during the first not all tests yield positive results. In the American
week of illness. Later it is raised to an average series, immunofluorescent staining for rabies anti-
concentration of about 100 mg dl\ in 80% of cases gen in corneal impressions or neck skin biopsy
(Anderson et al., 1984). CT brain scans are reported- specimens was diagnostic in only about 50% of
ly normal, and EEGs usually show diffuse slow- cases early in the clinical course. In addition, there
wave activity or an isoelectric recording. were instances when the corneal impression test
gave false-positive results. In the South American
study, daily examination of corneal impressions
Intravitam Diagnosis
gave negative results in all seven cases. A PCR with
Laboratory confirmation of the diagnosis is poss- nested primers has been used successfully to diag-
ible before death by finding specific fluorescence in nose rabies in brain tissue (see following section)
corneal impressions (obtained by gently abrading and it is highly probable that new molecular tech-
the cornea with a microscope slide), skin biopsy niques will play an important role in intravitam
(normally taken from the neck or face to show viral diagnosis.
antigen in sensory nerve endings) or brain biopsy
(rarely indicated); by recovery of Rabies virus from
Postmortem Diagnosis
saliva, throat swab, tracheal aspirates and excep-
tionally from tears, urine sediment and CSF; and The postmortem diagnosis of rabies in both animals
finally by detecting rabies antibody in serum or and humans for many years depended on the dem-
CSF. Virus can often be recovered from human onstration of Negri bodies in the brain, which ap-
saliva, throat swabs or tracheal aspirates during the pear as intracytoplasmic, eosinophilic inclusions
first 2 weeks of illness, but later attempts at virus generally measuring 0.2—27 m in diameter, but
isolation or immunofluorescence are often negative may be absent in as many as 30% of cases. They are
(Anderson et al., 1984), presumably because the vi- usually found in more or less undamaged nerve
rus has been ‘neutralised’ by antibody. CSF rarely cells, particularly in Ammon’s horn, cerebral cortex,
contains infectious virus ante mortem, although at medulla and cerebellum. After staining with Seller’s
autopsy it can occasionally be found. There are no methylene blue basic fuchsin, they show a hetero-
adequately documented cases of viraemia, and at- genous magenta-red matrix containing dark-blue-
tempts to isolate the virus from the blood of human to-black inclusions known as ‘Innerkörperchen’.
cases have been unsuccessful. Structures lacking this inner basophilic core but
598 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
otherwise similar to Negri bodies are known as typical bullet-shaped virus particles in postmortem
‘Lyssa’ bodies. Both structures contain rabies anti- brain. In addition to nervous tissue, rabies virus can
gen and are found in great numbers in rabid brain, be identified in a variable proportion of cases in the
but neither is pathognomonic for rabies. lacrimal and salivary glands, myocardium, skeletal
Intracerebral mouse inoculation with a homo- muscle, lung, liver, kidney, peripheral nerves and
genate of brain coupled with immunofluorescence is adrenal glands.
an extremely valuable confirmatory test, although if Once virus has been recovered, antinucleocapsid
the patient’s survival is prolonged until after the monoclonal antibodies provided by WHO can be
appearance of antibody, attempts at virus isolation used to distinguish between viruses belonging to
almost invariably yield negative results. NA C1300 Lyssa serotype 1 (positive with monoclonal anti-
murine neuroblastoma cells are the most suscep- body C-15, negative with antibody 422) and viruses
tible of cell lines for street Rabies virus and are at belonging to the other serotypes (negative with C-
least as efficient, and possibly more so than the 15 and positive with 422). Nucleocapsid mono-
mouse inoculation test. Moreover in vitro-based clonals also permit the identification of the geo-
culture techniques reduce the overall time required graphic origin or the species origin of the virus.
from several weeks to several days. Although no Glycoprotein monoclonals are of less value in this
cytopathic effects are observed, rabies antigen can respect due to the high variability of the GF epi-
be detected by immunofluorescence (IF). topes of field isolates.
Demonstrating rabies antigen in sections of hu-
man brain by IF is probably the most rapid specific
test, but again it is often negative in cases with
prolonged survival and requires the material for MANAGEMENT
examination to be in a good non-putrefied state.
However, IF can still be carried out if the specimen In contrast to most viral encephalitides there is a
has been fixed in formalin. An antigen detection relative lack of cytolysis and inflammation. The
ELISA, known as the rapid rabies enzyme im- changes noted are perivascular cuffing, neuro-
munodiagnosis (RREID), has been used success- nophagia and neural degeneration, and prolifer-
fully to detect rabies nucleocapsid in homogenized ation of the capsular cells surrounding ganglionic
brain suspensions that have not been fixed in for- neurons and Negri bodies. This inevitably fired
malin. The minimum amount of nucleocapsid de- speculation that intensive supportive care might
tectable by RREID is : 1 ng ml\ and a naked-eye eventually lead to recovery. The survival of two
reading is sufficient to confirm the diagnosis, there- patients with proven rabies for periods of 64 and
by obviating the need for sophisticated or expensive 133 days, together with the recovery of three pa-
equipment such as a photometer or IF microscope. tients with probable rabies, has raised further
Further increases in sensitivity are possible using hopes, although one survivor was left with severe
antirabies nucleocapsid globulin conjugated to bio- neurological impairment and all three were vac-
tin, and peroxidase-conjugated straptavidin. cinated prior to the onset of symptoms. Life can
Dot hybridization with P-radiolabelled probes undoubtedly be prolonged by meticulous support-
has also been used for rabies diagnosis but detects ive care, although it is equally clear from recent
rabies genome only in heavily infected samples. experience that it is the extent and severity of the
More recently, a nested PCR has been described encephalitis that are major barriers to survival.
(Kamolvarin et al., 1993) which is capable of detect- Many patients have been given the benefit of
ing as little as 8 pg of rabies virus RNA. The PCR treatment with antiviral agents or immunotherapy,
was able to confirm rabies infection in decomposing but the measures tried have all been singularly un-
specimens, left for 48 hours at room temperature, helpful. Steroids impair antibody production and
that were negative by IF. The whole procedure, depress immunity in laboratory animals and have
including reverse transcription, cDNA synthesis been used without success in humans and are gen-
and DNA amplification, was accomplished within erally contraindicated. No benefit has been gained
24 hours and will undoubtedly be of value in major from cytosine arabinoside, ribavirin, inosine
diagnostic centres. Finally, electron microscopy can prabonex, or administration of rabies antibody
establish the diagnosis by revealing the presence of either by the intrathecal or intramuscular routes, or
RABIES 599
from transfusion of a unit of immune whole blood. hard-water areas, as well as by soap. Wounds clean-
In fact, the discovery of ‘lytic’ antibody led to the sed with a soap solution should be thoroughly rin-
view that passively administered antibody after on- sed with water before application of one of the
set of disease might actually be harmful. Exchange chemical disinfectants. Gaping bite wounds should
transfusion was therefore tried; this too was unhelp- not be immediately sutured; if suturing is necessary
ful. Similar lack of success was noted with Freund’s then antiserum or immune globulin should be infil-
adjuvant and a polyanion interferon inducer. Inter- trated locally as stated below, and suturing should
feron itself has been tried in several patients by be delayed for as long as possible, up to 3 days.
intramuscular and intrathecal routes; none survived
and interestingly the virus could still be isolated
Passive Immunization
from one patient 5 days after onset of treatment.
Human treatment with antiserum began in 1891,
but it was not until 1954, when 29 Iranian villagers
were bitten by a rabid wolf and the mortality of
PREVENTION
vaccine-only and vaccine-plus-antiserum groups
were compared, that the value of passively adminis-
Postexposure Prophylaxis tered antibody was shown convincingly. In 1957,
the WHO Expert Committee on Rabies recognized
The essential components of postexposure prophy- the importance of prompt infiltration of wounds
laxis are the local treatment of wounds and active with antiserum and recommended its use. Selimov
and passive immunization. The potency of cell cul- et al. (1959), Fathi et al., (1970) and Fang-tao et al.
ture vaccines is determined by the NIH (National (1985) have provided further evidence for the benefi-
Institutes of Health) test and is expressed in interna- cial effect of antiserum following bites by rabid
tional units (iu) per millilitre. To satisfy the WHO animals, mostly wolves. Aggregated data from Iran,
requirements for human rabies vaccine, all cell cul- Russia and China indicate that antiserum saves the
ture vaccines should have a minimum potency of lives of approximately 85% of persons who would
2.5 iu per dose. Interferon and interferon inducers otherwise develop rabies and die, despite treatment
have yet to be evaluated in humans, though they are with neurotissue vaccine (NTV). The beneficial ef-
of proven value in laboratory animals. fect of combined active and passive immunization is
generally not in doubt, but there is now abundant
evidence that passively administered antibody
Wound Treatment
alone serves only to prolong the incubation period
Experimentally, the incidence of rabies can be re- and provides little, or no, protection.
duced markedly by local therapy alone. It is of WHO strongly recommends the use of hetero-
maximal value when applied promptly after expo- logous antirabies immune globulin, e.g. equine im-
sure but should not be neglected even if several mune globulin (ERIG) or human rabies immune
hours or days have elapsed. Recommended first-aid globulin (HRIG), for therapy of severe exposure, i.e.
procedures are the thorough cleansing of the wound licks of mucosa, multiple bites, or bites involving
for several minutes with copious amounts of soap the face, head, fingers or neck by a known rabid
and water, detergent, or water alone; the applica- animal, or following an unprovoked attack by an
tion of a virucidal substance, either 40—70% alco- animal in an endemic area unless proved negative
hol, tincture or aqueous solutions of iodine, or a by laboratory examination. It also considers that its
quaternary ammonium compound (QAC), e.g. use may be justified in some minor exposures, es-
1—2% benzalkonium chloride, 0.1% cetrimonium pecially when the skin has been deeply punctured. It
bromide (Cetavlon); passive immunization; and is customary in western Europe and North America
antitetanus procedures and antibiotics when in- to administer HRIG following bites by all rabid and
dicated. Puncture wounds should be probed gently suspected rabid animals.
with an appropriate disinfectant, taking care not to When employed, HRIG or ERIG is administered
cause further trauma. Wound toilet is possibly the only once at the beginning of antirabies prophylaxis
single most important procedure for preventing in- to provide antibodies during the early critical per-
fection. QAC may be neutralized by tap water in iod before development of active immunity. The
600 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
recommended dose of HRIG is 20 iu kg\ of body ciated with complete protection.
weight and 40 iu kg\ for ERIG. The immune The ‘unmyelinated’ neural tissues of newborn
globulin should be infiltrated into and around the mammals, frogs and fish are considered free from
wound and any remaining immune globulin should the encephalitogenic factor, and in 1955 Fuenzalida
be injected intramuscularly at a site distant from the and Palacios prepared a vaccine from the brains of
site of vaccine inoculation. ERIG is considerably suckling mice (SMBV). SMBV, inactivated by UV
less costly than HRIG. Most of the preparations of light, -propiolactone or phenol, is used extensively
ERIG available cunningly are highly purified, pep- in South America and is easily and cheaply pro-
sin-digested products and safe; however, serum duced. However, myelin is present in preparations
sickness may develop in up to 6% of patients 7—10 of brain from mice aged 9 days and, unfortunately,
days later. WHO advises that a skin test should be reports of neuroallergic reactions to SMBV have
carried out prior to its use. Although this may de- accumulated since 1967. Although the incidence of
tect rare cases of IgE-mediated hypersensitivity re- these reactions is approximately fivefold lower than
actions to equine antiserum most reactions result with conventional brain tissue vaccines, the case-
from complement activation and are not predicted fatality rate is higher, making SMBV only margin-
by skin testing. Although HRIG or ERIG should be ally safer overall. SMBV is now prepared using
given concurrently with the first dose of rabies vac- mice no older than 1 day at the time of inoculation.
cine, immune globulin preparations are not always Attempts were made to purify SMBV by centrifu-
available in the field. In this situation immune gation and chromatography, but events in the field
globulin can be administered up to 5 days after of tissue culture vaccine have overshadowed these
multisite postexposure immunization without sig- developments.
nificant depression of the active antibody response,
and may still provide antibodies before develop-
Duck Embryo Vaccine
ment of active immunity.
In countries like the United States and Britain,
vaccines prepared from fixed strains of Rabies virus
Neurotissue Vaccines
grown in duck embryos became available commer-
The infected brain tissue of adult rabbits, sheep and cially during the late 1950s and early 1960s. Al-
goats remains an important source of virus for vac- though far safer than NTVs, the high content of
cine production in some areas of the world. The avian protein in duck embryo vaccine (DEV) often
most widely used is the Semple type of vaccine, gave rise to troublesome local reactions and
which is completely inactivated with phenol. Due to anaphylaxis and to neurological accidents occur-
poor antigenicity, courses of treatment consist of up ring with an estimated incidence of 1 in 32 600 and
to 24 5-ml injections of a 5 or 10% brain suspen- fatality rate of 1 in 210 000. DEV was also poorly
sion, equivalent to 6—12 g of brain tissue. NTV antigenic but, despite shortcomings, it remained the
usage has long been associated with an unaccep- only vaccine available in Britain and the United
tably high incidence of postvaccinal reactions, in- States until licensure of human diploid cell vaccine
cluding inflammation, malaise and paralysis which (HDCV). Subsequently DEV was withdrawn from
develops 4—21 days after initiating therapy. Rates of the market by its American manufacturers but a
neurological reactions associated with the use of highly purified DEV (PDEV) is produced in Swit-
adult brain tissue vaccines vary greatly, with an zerland. PDEV contains only about 1% of the pro-
overall rate of around 1 per 1100. The case-fatality tein of previous DEV. Potency testing of consecu-
rate among these patients is about 10%. The en- tive batches has consistently shown antigenic values
cephalitogenic antigen has been identified in studies in excess of 5 iu ml\ and substantial antibody re-
of experimental allergic encephalomyelitis as myel- sponses are evoked in humans. Mild local reactions
in basic protein. Reports on the efficacy of NTV occur only slightly more frequently than with
after bites by animals causing rabies in untreated HDCV.
people indicate protection rates ranging from 0 to
87%. In two reports, treatment of bites by rabid
Human Diploid Cell Vaccine
dogs in Thailand (n : 77) and Dehli (n : 75) with
NTV, with and without antirabies serum, was asso- Strides made in the field of cell culture techniques
RABIES 601
and virus purification opened an entirely new ap- dian subcontinent and south-east Asia (Thailand).
proach for the preparation of rabies vaccines. From Purified Vero cell rabies vaccine (PVRV) is pre-
the early 1970s to the mid-1980s most attention pared using Vero cells grown on a microcarrier
focused on HDCV developed in WI-38 human di- system and infected with the same Pitman Moore
ploid cells which were isolated from human embry- strain of fixed Rabies virus used for HDCV produc-
onic lung. The virus used was the Pitman Moore tion (Montagnon et al., 1985). The use of such a
strain of fixed Rabies virus. HDCV is now prepared heteroploid cell substrate for producing human
using MRC-5 cells. Virus yields are concentrated by vaccines was excluded until recently because it was
ultrafiltration, or concentrated and purified by rate- thought that potentially oncogenic DNA might be
zonal centrifugation in sucrose gradient, and inac- released from the cells during viral replication.
tivated by -propiolactone. However, provided that appropriate tumorigenicity
Early trials in volunteers established rapidly that tests are carried out and the product is free from
HDCV was infinitely superior to neural and avian cellular DNA, continuous cell lines are now accep-
preparations. Not only were antibody levels far ted widely for human vaccine production. Inac-
higher than with other vaccines, but it appeared tivated polio vaccine was the first vaccine made
earlier and in practically 100% of subjects with using this cell substrate. PVRV satisfies the strin-
many fewer doses and with only mild local reac- gent safety requirements and has shown excellent
tions. In 1976, trials in Germany and Iran showed antigenicity in humans, again with antibody re-
HDCV together with serum or HRIG to afford sponses comparable to those of HDCV. Use of the
complete protection to persons bitten by known microcarrier technique and the greater yield of virus
rabid animals. The vaccination schedule consisted from Vero cells compared to human diploid cells
of six doses given on days 0, 3, 7, 14, 28 and 90; this has resulted in a considerable reduction in price.
regimen is now officially recommended by WHO, The vaccine is purified by zonal centrifugation and
though in some countries the day 90 dose has been chromatography and is well tolerated. Approxi-
dropped. More than 10 million doses had been used mately 11 million doses of PVRV had been used
to treat an estimated 2 million patients worldwide worldwide by December 1994.
by December 1994. In the former USSR, a UV light-inactivated vac-
The principal disadvantage of HDCV is its ex- cine has been prepared from primary hamster kid-
pense compared with other cell culture vaccines, ney cells (PHKCs) infected with the Vnukovo-32
which is partly related to the difficulty in handling strain of Rabies virus. A PHKC vaccine has also
diploid cells, and the need to concentrate virus har- been prepared using the Beijing strain of fixed Ra-
vests 10—20-fold to achieve vaccines of adequate bies virus inactivated with formaldehyde. Approxi-
potency. None the less, more than 10 million doses mately 50 million doses have been produced, princi-
had been used to treat several million people world- pally for postexposure use. Fetal rhesus monkey
wide by December 1994. Accordingly, vaccine diploid cell vaccine was developed in the United
manufacturers have explored new cell substrates, States and has been licensed there for both pre- and
culture systems, vaccine strains and technologies in postexposure treatment. Vaccines have also been
order to reduce costs and equal the high standards prepared using cultures of fetal bovine kidney cells
of potency and efficacy of HDCV. and dog kidney cells.
Pre-exposure Prophylaxis
Assessing Immunity to Rabies
The safety and potency of rabies vaccines of cell
culture origin allow persons who are regularly at The mouse neutralization test (MNT) provides a
high risk of exposure, such as veterinarians, labora- useful diagnostic tool and an important method of
tory workers, animal handlers and wildlife officers, assessing vaccines but it requires large numbers of
to be protected by pre-exposure immunization. It young adult mice and the facilities necessary to
should also be considered for persons, especially contain them, and is expensive and dangerous for
children, living in or visiting countries where rabies laboratory staff, who may accidentally inoculate
is a constant threat and where postexposure treat- themselves with challenge virus. The test also suffers
ment with potent vaccines is not readily available. with an inherent variability making it difficult to
Potent cell or avian culture vaccines (HDCV, correlate results between laboratories, even when
PCECV, PVRV or PDEV) are the vaccines of standard antiserum is included in each test. More-
choice. Immunization should preferably consist of over, the MNT has the disadvantage of requiring at
three intramuscular doses of vaccine of potency of least 14 days for completion.
at least 2.5 iu given on days 0, 7 and 28 (or on days 0, To overcome these problems a number of at-
28 and 56; a few days’ variation is unimportant). tempts have been made to develop an in vitro assay
The intradermal application of 0.1 ml volumes may for virus neutralizing antibody. A cytopathic effect
be used in place of the 1.0 ml volumes normally with Rabies virus, although reported by some
given intramuscularly. Antibody can be demon- authors, has not been sufficiently reliable to permit
strated in the sera of virtually 100% of subjects after routine tissue culture neutralization tests such as
three doses of potent cell culture vaccines and it is inhibition of cytopathic effect or plaque inhibition.
unnecessary to monitor the immune response ex- However, immunofluorescent staining techniques
cept in high-risk situations. In such people serum led to the rapid fluorescent focus inhibition test
samples should be collected 1—3 weeks after the last (RFFIT), in which foci of virus-infected cells are
dose and, if the titres are not equivalent to observed by fluorescent antibody staining. The test
0.5 iu ml\ or greater, boosters should be adminis- requires only 24 hours for completion and is more
tered until neutralizing antibody becomes demon- reliable and reproducible than a neutralization test
strable. in mice. However, it still requires the use of cell
Booster doses should be offered to persons at culture techniques and equipment and live virus.
continuing risk every 1—3 years or whenever necess- The complement fixation test, the haemag-
ary, as determined by antibody titration. In view of glutination inhibition test and the mixed haemad-
the allergic reactions recently described after sorption test have gained only limited acceptance
HDCV boosters (see above), efforts should be made for determination of rabies antibody, and radioim-
to avoid unnecessary boosters. munoassay is expensive, carries some risks and,
Local treatment of wound should always be car- because it requires expensive equipment, is retricted
ried out in exposed persons who have been vac- to central facilities.
cinated previously. Persons vaccinated previously Enzyme-linked immunosorbent assay (ELISA)
who have had full pre- or postexposure treatment provides a rapid, simple test for the measurement of
with cell culture or purified DEV, or who have had rabies antibodies and there is good agreement be-
a rabies neutralizing antibody titre of 0.5 iu ml\ tween the results of neutralization tests and ELISA,
in the past, should be given only two booster doses, especially if a rabies glycoprotein preparation is
one immediately and one 3 days later. Persons who used as antigen instead of purified inactivated ra-
RABIES 605
bies vaccines of cell culture origin. If a reference culture origin, animals that succumbed to infection
serum is included in the test the results can be showed high-titred antibody responses, comparable
expressed in equivalent units (eu ml\). The ELISA in height and time of appearance to those of surviv-
test also has the advantage of allowing separate ors. Several studies have shown that the antibody
titration of IgG and IgM antibodies. ELISA is profiles of patients dying after NTV treatment were
simple and quick to perform, with results being equivalent to those of survivors. Since postexposure
available in a few hours; however, it requires highly administration of passively administered antibody
purified antigen. A competitive ELISA for the de- generally prolongs survival but has little influence
tection of rabies virus neutralizing antibodies em- on mortality in laboratory animals, it is evident that
ploys a rabies neutralizing monoclonal antibody other limbs of the immune system have key roles in
conjugated to an enzyme and is not dependent on postexposure treatment.
highly purified antigen.
Cell-mediated Immunity
Antibody and Protection
The contribution of cell-mediated immunity to pro-
Virus neutralizing antibody has long been consider- tection afforded by postexposure immunization is
ed to be the key to successful rabies prophylaxis, as inconclusive as that of antibody. In approximate-
both before and after virus exposure, and inade- ly 80% of subjects immunized with potent tissue
quate antibody responses to neurovaccine and culture vaccines, lymphocyte transformation occurs
DEV have generally been held responsible for occa- and is a T cell response. Antibody-dependent cell-
sional treatment failures. Review of the literature in mediated cytotoxicity (K cell lysis) has also been
which pre-exposure immunization of various ani- demonstrated with sera and lymphocytes from hu-
mal species was followed by peripheral challenge mans receiving HDCV. Similarly, skin testing and
with street rabies virus reveals a 96% reduction in the inhibition of peripheral blood leucocyte migra-
mortality when antibody was present at the time of tion have demonstrated cell-mediated immune re-
challenge. In the absence of antibody, survival was sponses in volunteers immunized with SMBV. Nu-
57.5% if a humoral response had been demon- merous data in experimental animals are recorded,
strated previously, and 23.6% if it had not. Inspec- though few results suggest that cell-mediated im-
tion of the antibody titres of the 14 animals that munity rather than antibody is concerned with pro-
died revealed a range of 1:2 to 1:1750 (GMT 1:18), tection.
implying that even substantial titres of antibody do Experimentally, T lymphocytes, specifically cyto-
not always guarantee protection. Two recent stu- toxic for rabies-infected target cells, can be gener-
dies in preimmunized dogs and cats have shown a ated in vaccinated mice and rabbits, and in the
relation between the titre of neutralizing antibody former species it was, under certain circumstances,
and protection against rabies virus challenge: a neu- related to protection. Interestingly, cell-mediated
tralizing antibody titre of approximately 0.5 cytotoxicity was not generated in mice infected with
iu ml\ at the time of challenge is necessary for virulent street Rabies virus, suggesting that in natu-
uniform protection, and this titre is accepted by ral infection Rabies virus has an immunosuppres-
WHO as indicating a satisfactory response to vacci- sive effect. Support for this hypothesis has also been
nation. provided by various studies, including delayed-type
The association between antibody and protec- hypersensitivity reactions measured in mice by
tion is much less clear for treatment given after footpad swelling, and by in vitro studies of cytokine
exposure. Indeed, there is evidence to suggest that production by cells from rabies-infected mice.
the humoral response to rabies has a dual role— Moreover, study of seven proven cases of rabies
mostly favouring protection, but sometimes promo- (four furious and three paralytic) showed that natu-
ting the disease through immune cytolysis effected ral killer cells were diminished in almost all cases
through antibody and complement, or by antibody- and B lymphocytes were decreased in those with
dependent cytotoxicity or antibody enhancement of paralytic disease compared to those with encephali-
viral replication. In several studies in monkeys, in tis. Lymphocyte proliferation tests to rabies antigen
which challenge with street virus was followed by a on peripheral blood lymphocytes from nine pa-
single injection of potent rabies vaccine of tissue tients with the encephalitic form of rabies, and on
606 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
seven with the paralytic form, showed that six of the 294—329.
nine patients with encephalitis had proliferative re- Kamolvarin, N., Tirawatnpong, T., Rattanaswamoke, R. et al.
(1993) Diagnosis of rabies by polymerse chain reaction with
sponses, whereas all of those with the paralytic form nested primers. Journal of Infectious Diseases, 167, 207—210.
had no response. Kucera, P., Dolivo, M., Coulon, P. et al. (1985) Pathways of the
Until recently, most experimental attention con- early propagation of virulent and avirulent rabies strains from
cerning the protective immunity focused on the role eye to the brain. Journal of Virology, 55, 158—162.
Montagnon, B.J., Fournier, P. and Vincent-Falquet, J.C. (1985)
of the G protein, which induces neutralizing anti- Un nouveau vaccin antirabique a usage humain: rapport pre-
body. Purified rabies virus nucleoprotein has re- liminaire. In Rabies in the Tropics (eds E. Kuwert, C. Mereieux,
cently been found to provide resistance to lethal H. Koprowski et al.), pp 138—141. Springer-Verlag, Berlin.
challenge with both homologous and heterologous Nikolic, M. (1952) Die Ergebnisse der Tollwutschutzimpfung
virus strains and to evoke a strong T helper cell beim Menschen nach Wolfverletzung. Zeitschrift für Tropen-
medizin und Parasitologie, 3, 283—296.
response. This may provide an explanation for the Préhaud, C., Coulon, P., Lafay, C. et al. (1988) Antigenic site II of
protection afforded by some NTVs, which are poor the rabies virus glycoprotein; structure and role in viral viru-
in terms of neutralizing antibody induction, but lence. Journal of Virology, 62, 1—7.
none the less evoke high titres of antibody as meas- Préhaud, C., Nel, K. and Bishop, D.H.L. (1992) Baculovirus-
ured by ELISA. expressed rabies M1 protein is not phosphorylated: it forms
multiple complexes with rabies N protein. Virology, 189,
766—770.
Ragan, K.J. and Wunner, W.H. (1985) Rabies virus interaction
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Bögel, K. and Motschwiller, E. (1986) Incidence of rabies and on 30 cases. Journal of the Indian Medical Association, 65,
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World Health Organization, 64, 883—887. Tordo, N., Poch, O., Ermine, A. et al. (1986) Primary structure of
Cornwall, J.W. (1923) Statistics of antirabic inoculations in In- leader RNA and nucleoprotein genes of the rabies genome:
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del et al., pp 37—45. Institute of Public Health, Zagreb. and untreated. The Pasteur Institute of Southern India,
Fathi, M., Sabeti, A. and Bahmanyar, M. (1970) Séroprophylaxie Coonoor. Annual Report of the Director (1967) and Scientific
antirabique chez les sujets mordus par loups enragés en Iran. Report (1968), pp 35—42. Dioscesan Press, Madras.
Acta Medica Iranica, 13, 5—9. Wang, S.P. (1956) Statistical studies of human rabies in Taiwan.
Gaudin, Y., Ruigrok, R.W.H., Tuffereau, C. et al. (1992) Rabies Journal of the Formosan Medical Association, 55, 548—553.
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Gaudin, Y., Ruigrok, R.W.H., Knossow, M. et al. (1993) Low-pH actions of the Royal Society of Tropical Medicine and Hygiene,
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role in membrane fusion. Journal of Virology, 67, 1365—1372. Wilde, H., Sirikawin, S., Sabcharoen, A. et al. (1996) Failure of
Gremliza, L. (1953) Kasuistik zum Lyssa-problem. Zeitschrfit für post-exposure treatment of rabies in children. Clinical Infec-
Tropenmedizin und Parasitologie, 4, 382—389. tious Diseases, 22, 228—232.
Hunter, J. (1793) Observations, and heads of enquiry, on canine Wilson, J.M., Hettiarachchi, J. and Wijesuriya, L.M. (1975) Pre-
madness, drawn from the cases and materials collected by the senting features and diagnosis of rabies. Lancet, ii, 1139—1140.
society, respecting that disease. Transactions of the Society for
the Improvement of Medical and Chirurgical Knowledge, 1,
Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
22
Papillomaviruses
Dennis J. McCance
University of Rochester, Rochester, New York, USA
CLASSIFICATION
Genome
At present papillomaviruses are classified into five
supergroups, A (genital human papillomaviruses, The HPV virion contains a double-stranded DNA
HPV), B (associated with epidermodysplasia ver- molecule of 5 ; 10 da molecular weight with an
ruciformis), C (ungulate fibropapillomaviruses), D average of 7900 bp. The DNA, when in the virion,
(bovine papillomaviruses causing true papillomas), has a supercoiled circular configuration.
and E (animal and human cutaneous papil- The molecular organization of the papil-
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
608 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 22.1 Classification of the papillomavirus subfamily supergroups A—E
A B C D E
1 2 3 4 5 6 7 8 9 10 11 1 2 1 2
32 3 61 2a 26 30 18 7 16 6b 34 5 4 B1 E1 B3 1a
42 10 27 51 53 45 40 31 11 8 48 B2 D1 B4 41
28 57 69 56 39 43 33 13 9 50 B6 63
29 66 59 35 44 12 60 Co
68 52 55 14d 65 Ro
70 58 P1 15 Cr
67 17
R1 19
20
21
22
23
24
25
36
37
38
47
49
R : Rhesus monkey papillomavirus; B : Bone papillomavirus; Cr : Cottontail rabbit papillomavirus; Co : Canine oral papillomavirus; D : Deer
papillomavirus; E : Elk papillomavirus; Ro : Rabbit oral papillomavirus.
Adapted from the Los Alamos National Laboratory HPV database (Myers et al., 1997).
Figure 22.2 The genomic organization of HPV-16. The genome is circular, but represented as linear for clarity. The open reading
frames (ORFs) are bound by nucleotide numbers and the line within the ORF represents the starting ATG; its nucleotide number is
unlined. The pA and pA are the poly(A) signals for the early (E) and late (L) mRNAs, respectively. The URR is the upstream
# *
regulatory region which contains the major early promoter at p97 and the origin of replication (origin)
Table 22.2 Molecular weights of cause its rapid degradation through the ubiquitin
HPV-16 proteins
proteolysis pathway (Schneffer et al., 1990). E6 has
Protein Molecular weight also been shown to bind to a Ca>-binding protein
(kDa)? called E6BP, which has homology to a cellular
protein ERC-55 of unknown function (Chen et al.,
E1 70
1995). E7 binds another cellular protein, the re-
E2 45
E4 17 tinoblastoma gene product (pRB, Dyson et al.,
E5 8 1989) and de-represses the inhibitory activity of Rb
E6 16 for transcription factors that are important for ex-
E7 11 pression of genes, whose products are essential for
L1 57
L1 55
DNA synthesis. Repression of transcription may be
due to the binding by Rb of a histone deacetylase
?Molecular weight on SDS-PAGE gels may protein (Brehm et al., 1998), which functions to
be different, e.g. E7 runs at 15—16 kDa condense chromatin and restrict the access of tran-
because of charged amino acids in the
N-terminus of the protein, but codes for a scription factors to DNA. E7 appears to compete
protein of 11 kDa, and L1 runs at 75 kDa. for the binding site of the deacetylase on Rb. Both
these cellular proteins are negative regulators of the
properties which are consistent with the virus hav- cell cycle and so interference with their normal func-
ing to stimulate the infected cells into S phase so the tion may allow cells to divide in an uncontrolled
viral DNA has the cell’s replicative machinery manner. E7 has also been shown to bind to various
available for propagation of the genome. It has to members of the AP-1 family of transcription factors
be remembered that the virus is attempting to repli- and upregulate their activity (Antinore et al., 1996).
cate in cells that are programmed to differentiate E1 and E2 are involved in the replication of the
and so will have little or no replicative enzymes HPV genome (see Viral Replication). E2 has addi-
available to the virus. Both E6 and E7 are import- tional functions in that it can positively and nega-
ant for the efficient immortalization of human tively regulate transcription from the early promo-
keratinocytes and have functions which disrupt the ter. The full-length protein contains a trans-
normal control of G to S phase progression. E6 has activation domain at the 5 end and a DNA binding
been shown to bind the human p53 protein and domain in the 3 half, and the active complex is a
610 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
dimer. Negative regulation of the HPV early pro- viral-like particles (VLPs) produced using
moter is due to the fact that one of the E2-binding baculovirus technology has been used to carry out
sites lies next to the TATA box of this promoter and serological studies on the relatedness of HPV and
so E2 binding sterically inhibits binding of the on a small scale to determine serological responses
TATA-binding protein and therefore inhibits initi- in infected patients. VLPs are produced when the
ation of the early transcripts. major capsid protein, L1, is synthesized using
E5 is a very membrane-associated hydrophobic baculovirus vectors and insect cells, and form into
protein with transforming activity for rodent fi- an icosahedral structure similar to that of the virion
broblasts. The E5 protein inhibits the acidification (Kirnbauer et al., 1992). VLPs can also be produced
of endosomes (Straight et al., 1995) by binding to which contain both the major (L1) and minor cap-
the smallest subunit (16 kDa) of the vacuolar AT- sid protein (L2) and the latter appears to stabilize
Pase, a multicomponent proton pump. In human the icosahedral structure and also has been shown
keratinocytes this results in the delay of the epider- to possess neutralizing epitopes. To date, antiviral
mal growth factor receptor (EGFR) degradation antibodies have been produced to virions of HPV-
and a hyperstimulated cell. Since the EGFR is the 11 and -16 and these antibodies recognize VLPs of
major growth factor receptor on keratinocytes, this the L1 protein from HPV-11 and -16, respectively
activity may be important for stimulating cells into (Rose et al., 1994). Therefore, VLPs are recognized
S phase for viral DNA replication. E5 can trans- by antibodies to infectious virus particles, and via
form rodent fibroblasts and in the presence of the versa. The antibodies to VLPs or virions detect
epidermal growth factor there is an increase in the comformational epitopes, unlike earlier serological
efficiency of transformation, which may be due to studies, where disrupted particles, or fusion proteins
the delay in receptor degradation. of L1 or peptides of regions of L1 were used either
The late proteins L1 and L2 are the major and as targets or to raise antibodies to the L1 protein,
minor capsid proteins, respectively, of the virion. and produced extensive cross-reactivity. This cross-
The DNA and amino acid sequences are highly reactivity was so complete that antibodies to dis-
conserved between HPV types, especially in the L1 rupted BPV-1 particles recognized linear epitopes
protein. The amino acid homology can be as high as in most HPV types. Therefore, it is only with the
76% between HPV-16 and -33 (group A9). How- production of VLPs that the serological differences
ever, even within groups of papillomaviruses type- between HPVs, even those closely related, became
specific epitopes predominate, although some weak apparent. For example, within one group, say A9
cross-reactivity has been observed, so it is possible containing HPV-16, -31 and -33, polyclonal antibo-
to differentiate between types on a serological basis dies only react well with the type to which the
(see next section). antibodies were raised, although cross-reactivity
The mRNA for E4 is detectable late in infection, has been observed. The level of cross-reactivity is
although it has the prefix of an early gene. E4 inter- very low and is probably not relevant biologically
acts with the cell microfilaments, causing them to in cross-protection. Monoclonal antibodies to
collapse (Doorbar et al., 1991), but the motive for virions or VLPs have shown that there are pre-
this interaction is not clear, although it has been dominantly type-specific epitopes, although in
suggested that this might allow the virions to be closely related viruses such as HPV-6 and -11,
more easily released from the differentiated where L1 is over 80% homologous at the amino
keratinocyte after transfer to a new susceptible host. acid level, cross-reactive epitopes have been ob-
There are host coded histone proteins H2a, H2b, served.
H3 and H4 associated with the viral DNA within
the virion.
VIRAL REPLICATION
Figure 22.3 A section of biopsy containing CIN 1, showing nuclear staining of HPV-16 L1 antigen using a monoclonal antibody,
followed by alkaline phosphatase tagged antimouse secondary antibodies. Note the dark staining nuclei are found in the outer
differentiated part of the epithelium
ating epithelial cells, and by their nature these cells by cloning the origin region into a bacterial vector
do not grow in vitro. However, Frattini et al. (1996) and transfecting mammalian cells along with plas-
have shown that it is possible to transfect human mids expressing the E1 and E2 proteins of the re-
keratinocytes, the natural host cell, with HPV-31 spective virus. The E1 and E2 proteins are the only
DNA, select cell lines containing episomal copies of HPV-specific products necessary for the replication
HPV-31 DNA, and then differentiate the cells using of the origin-containing plasmid. The origin region
the raft culture system and show that virus particle of the DNA has been mapped to a region at the 3
production occurs in the few cells in the uppermost end of the URR (Figure 22.2) and both E1 and E2
part of the differentiated epithelium. The distribu- have specific DNA binding sites at the origin. In
tion of the virus particles is the same as that ob- addition to binding at the origin, E1 and E2 can
served in infected epithelium (Figure 22.3). The level bind together and E1 has been shown to have AT-
of virus production is very low and not enough to Pase and helicase activities, which are typical of
produce virus particles for serology or infectivity viral proteins, such as SV40 large T, that are in-
studies. However, propagation of HPV types has volved in the initiation of viral DNA replication.
been successful in a nude mouse kidney capsule
system and in human skin grafts on to the epi-
thelium of SCID mice. In the former system human
foreskin tissue fragments are mixed with a viral NATURAL HISTORY OF HPV
suspension of HPV and then the tissue is trans- INFECTIONS
planted under the kidney capsule of a nude mouse.
Over the next 60 days the tissue fragments grow and HPVs infect and replicate in squamous epithelium
produce infectious HPV virus. Both HPV-11 and on both keratinized and mucosal surfaces. Most
-16 have been propagated in this fashion; however, people are infected with the common HPVs (HPV-
this system is obviously complex, requiring skills 1, -2, -3, -4) in childhood and adolescence, usually
and animal facilities not available to all. on the hands and feet. A small group of individuals
The origin of replication of HPV-11 and -31 (Lu with the rare autosomal recessive disease epider-
et al., 1993; Frattini and Laimins, 1994) has been modysplasia verruciformis (EV) harbor a number of
mapped extensively and the region is conserved in virus types not often isolated from normal people
other HPV types. The mapping has been achieved (Table 22.1, group B1 and 2). Two common presen-
612 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
tations in EV patients are multiple warts, which involves hundreds of thousands of new cases each
may be so numerous as to produce coalescent areas year, usually among sexually active individuals,
and dry, scaly flat lesions, which may be red or with 18—30-year-olds having the highest incidence
heavily pigmented. These latter skin lesions, al- (cf. common hand warts). With certain of the genital
though not having a wart-like appearance, contain HPV types there is an increased risk of carcinomas,
many of the unusual types of HPV contained in especially of the cervix; what is known of the natural
Table 22.1, group B1 and 2. Squamous cell carcino- history and pathogenesis of the genital viruses is
mas develop in nearly one-third of patients with discussed below.
EV, usually in areas of skin exposed to the sun (face,
neck and hands being most commonly affected);
HPV-5 and -8 are found commonly in these lesions, PATHOGENESIS
while types -14, -17 and -20 account for a smaller
number. While the viruses found in EV patients are
HPVs cause benign and malignant changes in epi-
not isolated from normal individuals, some types,
thelial cells. It is the latter property that will be dealt
such as 5 and 8, have been detected in squamous cell with in this section, since certain types are the most
carcinomas in allograft recipients. Because the dis- important component of the aetiology of genital
ease EV is rare, and with the isolation of some of the cancers, and cervical cancer is the most common
EV-associated HPVs from transplant patients, it cause of cancer-related death in women worldwide.
suggests that these viruses must be circulating in the
community, infecting normal individuals perhaps
without any associated lesions.
As mentioned earlier there are mucotropic HPVs Oncogenic Potential of
which infect mostly the genitourinary tract. Papillomaviruses
Common isolates like HPV-6 and -11, which cause
benign condyloma, can also infect the oral cavity, Until recent epidemiological and laboratory-based
particularly the larynx. Infection of the larynx is studies, most of the evidence for an oncogenic po-
rare, but usually requires several episodes of surgery tential of HPVs came from research with animal
or laser treatment for removal of recurrent lesions. papillomaviruses. Work in the 1930s showed that
Frequent recurrences may be due to the fact that the Cottontail rabbit papillomavirus (CRPV) pro-
only the visible lesions are treated, although healthy duced benign tumors in this animal, its natural host,
looking areas of musocal tissue may harbour HPV and that these benign tumors in 25% of cases would
genomes. The oral cavity is also infected with other become malignant after 12 months. Benign tumours
HPV types associated with oral warts and hyper- produced in domestic rabbits became malignant
keratosis. more frequently and within a shorter time. Also,
Transmission is thought to occur from one epi- application of hydrocarbons or tar produced in
thelial surface to another in exfoliated cells contain- both animal species a higher and more rapid malig-
ing infectious virus, rather than free virus particles. nant conversion. The viral DNA was detected in
Therefore direct contact is the most efficient way to both the benign and the malignant lesions. These
transmit infection. Direct contact through sexual results suggested that the CRPV produced the be-
intercourse is the most important mode of trans- nign lesion, but other factors, genetic and environ-
mission of genital viruses. However, an infected mental, may be necessary for production of malig-
mother may transmit virus to her neonate during nant disease. More recently, oesophageal, intestinal
delivery through the birth canal. This route of infec- and bladder papillomas produced by Bovine papil-
tion is thought to be a major cause of laryngeal lomavirus 4 (BPV-4) were shown to become malig-
warts in babies and young children. It is possible nant when cattle were fed on a diet of bracken. In
that other modes of transmission occur for the geni- this case the BPV-4 DNA was detected only in the
tal viruses, since a number of studies have detected benign lesion and was not detectable after malig-
DNA in young children. However, the route of nant conversion. Recently, rhesus monkey papil-
transmission, apart from those mentioned above, is lomavirus 1 has been isolated from a lymph node
unclear. metastasis of a penile carcinoma. This virus is trans-
Infection of the genital mucosa is common and mitted sexually and is associated with both penile
PAPILLOMAVIRUSES 613
and cervical cancers. This animal virus may service cross contamination, has recorded levels of 80%
as a good, if expensive, model to investigate the positivity in women with a normal cervix. These
natural history of papillomavirus infections. results have been shown to be erroneous due to
In humans, one-third of patients with EV develop cross-contamination problems in the early days of
squamous cell carcinoma, usually in sun-exposed PCR. However, there is still a variation in isolation
areas. Over 30% of these lesions contain HPV depending on the age and life style of the individuals
DNA, most commonly types 5 or 8. This suggests studied. In studies of young women between 18 and
that, given the right environmental or genetic con- 25 years of age up to 46% will have detectable HPV
ditions, benign lesions may develop into carcinoma DNA by PCR (Bauer et al., 1991) in epithelial cells
with the help of HPVs. Other evidence of a helper from a normal cervix. HPV types 16 and 18, which
function associated with malignant conversion con- are commonly found in malignant disease, account
cerns laryngeal papillomas, which may convert for about one-third of the viruses detected. There-
from a benign to malignant state when treated by fore a large number of young women may be infec-
X-irradiation. With the increase in the number of ted with HPV types, which cause malignant disease.
allograft recipients there has been an increase in However, it is clear that only a small number will
reports of squamous cell carcinomas at many differ- ever develop disease, but recognizing those at risk is
ent sites. The cutaneous cancers are associated with not possible at present. HPV detection decreases in
those viruses isolated from similar lesions in EV older women (940 years of age) and is usually in
patients, and the genital cancers harbour the viruses the 5—10% range. While the level of infection is
found in similar cancers in immunocompetent indi- lower in older women, it has been shown that they
viduals. are more likely to have underlying disease (see next
It is the genital papillomaviruses, which are re- section).
sponsible for the most common HPV-associated
cancer, that will be discussed in more detail in the
following sections.
HPV Infection and the Abnormal Cervix
HPV-6a—f Condyolmata
acuminata Vulva
Vagina
Cervix
types. The premalignant lesions occur almost en- Penis (shaft, prepuce,
tirely on the transformation zone, the metaplastic urethral meatus)
Perianal
zone between native squamous epithelium of the Larynx
exocervix and the columnar epithelium of the en- LSIL/HSIL Cervix
docervical canal, and are white in appearance after VIN I—III Vulva
the addition of 5% acetic acid to the surface of the PIN I—III Penis
HPV—11a, b Condylomata
epithelium. Invasive cancer arises from these areas acuminata Vulva
of HSIL, and malignant cells migrate up into the Cervix
uterus and out to local lymph nodes. HPV-16 is the Perianal
Larynx
most common virus found in intraepithelial and LSIL/HSIL Cervix
malignant disease. It has been found in 70% of cases PIN I—III Penis
of HSILs in Germany and the UK and has been HPV-16 Condulomata
isolated in 80% of cases of invasive cancer of the acuminata Vulva, cervix and penis
LSIL/HSIL Cervix
cervix. VIN I—III Vulva
HPV-6 and -11 have not been found in malignant PIN I—III Penis
disease of the cervix, but have been isolated from Bowenoid papulosis Vulva and penis
Malignant carcinoma Cervix, vulva and penis
locally invading lesions of the vulva such as ver- HPV-18 LSIL/HSIL
rucous carcinoma. However, when the genome of Malignant carcinoma Cervix and penis
the viruses isolated were sequenced, it was found 31 LSIL/HSIL Cervix
that there were duplications in the long control Malignant carcinoma
33 LSIL/HSIL Cervix
region, which may be associated with the change in Malignant carcinoma
pathogenesis. The HPV types and associated 35 LSIL/HSIL Cervix
lesions are shown in Table 22.4. Malignant carcinoma
There is a difference in the state of the HPV-16 39 LSIL/HSIL Cervis and penis
PIN
DNA in premalignant and malignant lesions. In Malignant carcinoma
LSILs and HSILs the HPV-16 DNA is free and 40 LSIL/HSIL Cervix
unintegrated, while in the majority of malignant 42 LSIL/HSIL/VIN Cervix and vulva
43 LSIL/HSIL Cervix
cells the DNA is integrated. While integration is 44 LSIL/HSIL Cervix
random with the chromosomes, the HPV genome is 45 LSIL/HSIL Cervix
integrated in the E1 or E2 regions of the DNA, Malignant carcinoma
resulting in the retention of the expression of the E6 51 LSIL/HSIL Cervix
Malignant carcinoma
and E7 proteins, which appear essential for the 52 LSIL/HSIL Cervix
malignant phenotype. It is not clear how important Malignant carcinoma
integration is for invasive cancer to develop, since in 53 normal cervix
54 Conduloma Penis
a minority of cases the viral DNA is episomal in 55 Bowenoid papulosis Penis
malignant cells. 56 LSIL/HSIL Cervix
Malignant disease of the penis, while rare in de- Malignant carcinoma
veloped countries, is much more common in devel- 57 Intraepithelial Oral cavity, cervix
neoplasia
oping parts of the world. HPV-16 and -18 were 58 LSIL/HSIL Cervix
detected in over 50% of penile cancers in Brazil 59 VIN vulva
(McCance et al., 1986), where in one area of the 61 LSIL/HSIL cervix
northeast of the country the incidence of penile 66 LSIL/HSIL cervix
68 LSIL/HSIL cervix
cancer is 10 times the frequency seen in Europe.
Again in malignant disease of the penis the viral LSIL/HSIL : low-grade squamous intraepithelial lesion and high-grade
squamous intraepithelial lesion; VIN : vulvar intraepithelial neoplasia;
PIN : penile intraepithelial neoplasia.
?De Villiers (1989).
PAPILLOMAVIRUSES 615
DNA is integrated into the host cell chromosomes. site, although there is a considerable turnover of
While HPV types have a role in the aetiology of cells. However, not all cells in the basal epithelium
cervical cancer, there are certainly other factors have the same capacity to divide, so the viral DNA
involved that act with the virus to produce invasive may be sequestered in some quiescent basal cells,
disease, since not everyone infected and exhibiting which, when they divide subsequently, may activate
premalignant lesions will develop cancer. It is es- replication and produce lesions.
timated that up to 25% of women with LSILs will
progress to HSILs, while most will regress over a 3
year period. However, in older women (940 years DIAGNOSIS
of age) infection and persistence more commonly
leads to disease, and so they are potentially a group Apart from the familiar hand or verruca wart, the
to be closely monitored (McCance, 1998). The clinical appearance of papillomavirus infections va-
cofactors involved have not been delineated, al- ries considerably, from the scaly flat lesions on
though smoking maybe one such component. cutaneous epithelium of individuals with EV to the
aceto-white flat lesions on the cervix. The reader is
referred to specific papers for details of the clinical
presentation (Walker et al., 1983). This section will
Persistence of Infection deal with the laboratory diagnosis of HPV, in par-
ticular the genital isolates.
Epidemiological evidence suggests that HPVs may
persist in squamous epithelium without producing
recognizable lesions. Up to 50% of allograft recipi-
ents develop cutaneous warts within a year after Culture Methods
transplant, this proportion being high when com-
pared with the incidence in age-matched controls. Although several efforts have been made, no easily
This suggests that transplanted patients experience amenable cell type has been capable of supporting
either new infections or reactivation of persistent replication with production of infectious papil-
virus, the latter being supported by the finding of lomavirus particles. The nude mice kidney capsule
HPV DNA sequences in biopsies of normal areas of model system has been used to propagate some
larynx from individuals who have had episodes of HPV types, but cannot be considered an in vitro
laryngeal papillomas. As the recurrence rate of lar- system.
yngeal warts is high, this suggests that the virus is
capable of persisting somewhere in the respiratory
tract or oral cavity without producing recognizable Serological Methods
lesions—an inapparent infection.
Also the viruses associated with some of the Recently, with the advent of baculovirus-produced
lesions in EV patients are not found normally in VLPs, it is now possible to detect antibodies by
lesions from immunocompetent individuals, yet EV ELISA technique in the serum of patients infected
is such a rare disease that these patients cannot with HPV. At present there are limited numbers of
circulate the viruses among themselves. It would HPV types that have been used in these assays, but
seem that these viruses are circulating in the normal the initial results suggest that antibodies are detec-
population, causing inapparent infections. ted in approximately 50% of infected individuals,
In addition, during pregnancy genital warts can which is less sensitive than DNA-based detection
appear on the vulvar epithelium and then disappear methods. These assays have only been carried out
postpartum. It is not known if this is a hormonal for some of the genital isolates. The reason for the
effect or due to pertubations in the immune re- low positivity rates in infected people is unclear, but
sponse that often accompany pregnancy or the re- it has to be remembered that the virus is confined to
sult of the woman acquiring a recent infection from the stratified epithelium of the genital tract and few
her partner. In none of the above situations is there cells in a lesion support viral particle production. In
any direct evidence as to which cells harbour the addition, the mature viral particles are only pro-
virus. The basal epithelial cells are the most likely duced in the outer layers of the epithelium, where
616 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
degenerate primers are used—in fact it is possible to
detect even unknown HPV types (ones that have
not been cloned and sequenced); and (3) it is poss-
ible to test large populations. The major disadvan-
tage is that, because of the sensitivity of the method,
it is possible to amplify contaminating sequences
and so have false positives. This was a major prob-
lem in the beginning, but now that it is recognized
investigators have been more careful and included
strict controls.
One of the primer sets used widely amplifies a
region of L1, which is the most highly conserved
ORF of HPV. This set will amplify all the known
HPV types and then, to determine which one has
been amplified, the sequences are hybridized to a
DNA fragment made up of the intervening se-
quences of specific HPV types. These consensus
primers have been used by a number of investiga-
Figure 22.4 This represents the amplification of a specific re- tors for epidemiological studies.
gion of an HPV genome by the polymerase chain reaction (PCR)
using a set of primers, 1 and 2, which recognize homologous
areas of the HPV genome. The first cycle results in two new
double-stranded fragments; the second fragment makes four Hybrid Capture Method
double-stranded fragments and so on, so that after 20 cycles 10
double-stranded fragments are produced This method is available commercially in kit form
(Digene Diagnostics, MD, USA), and uses type-
the immune response is at its least effective. Taken specific RNA probes to detect viral DNA in
together, the results and the natural history of the samples. This method is not as sensitive as PCR but
virus suggest that it is not very immunogenic. How- does not have the problems of false positives asso-
ever, vaccine trials have taken place to immunize ciated with PCR, since it does not rely on the ampli-
dogs against oral papillomavirus infection (Suzich fication of the signal. It also detects only 16 mucosal
et al., 1995). The dogs were immunized intramus- types, whereas PCR can theoretically detect all
cularly with VLPs of canine oral papillomavirus HPV types.
and then challenged with live virus placed on scari-
fied areas of the oral cavity. Protection was 100%,
indicating that it is posible to immunize against
infection with intramuscular inoculations, although TREATMENT
it still does not argue that, in the natural infection,
confinement to the epithelium is a problem for rec- Although in most cases warts are a cosmetic nui-
ognition by the host defence mechanisms. sance which will disappear eventually spontaneous-
ly, they are notoriously difficult to treat. However,
as intraepithelial lesions, especially on the cervix,
may lead to malignant disease, treatment to elimin-
Polymerase Chain Reaction ate disease is important. This section will concen-
trate on the treatment of genital areas, as others
The most common and sensitive method for the (Bunney, 1982) have dealt extensively with common
detection of HPV infection is by PCR. This is a hand and plantar warts.
powerful technique which amplifies a specific piece
of DNA from a small amount of template (Figure
22.4). The advantages of this method are: (1) the Popophyllin
extreme sensitivity; (2) the versatility, in that it can
be used to detect more than one type of HPV when Popophyllin is a resin mixture obtained from the
PAPILLOMAVIRUSES 617
roots of podophyllum (American Mandrake) and is cervical lesions and makes use of a heated loop,
an irritant on cutaneous and mucous surfaces. It is which removes very precisely the complete lesion.
an antimitotic agent and should be used with care. One advantage over laser evaporation is that the
It is painted carefully on to the surface of warts and lesion is removed intact and the tissue can be used
should remain for no longer than 6 hours and then for pathological staging without having to biopsy
be washed off. It is adsorbed poorly by cutaneous the lesion first. Additionally, it allows the lesion to
surfaces and so has a limited effect. Several treat- be used for other studies, such as HPV typing.
ments are required and the continual inflammation
produced can lead to fibrosis of the areas treated,
without getting rid of the lesions. Podophyllin is Surgery
even less effective for treatment of plantar warts and
should never be used for treatment of hand warts. Curettage of common warts is not a common mode
of treatment. In any case, not all warts are suitably
sited for surgical removal. Furthermore, if all the
Cryotherapy abnormal tissue is not moved, small islands of warts
can recur around the site of the initial lesion.
Liquid nitrogen (9190°C) and dry ice (solid carbon
dioxide, 9 50°C) can be applied to warts to pro-
duce local destruction of the lesion. Care should be Interferon
taken to limit application to the lesion and not
surrounding areas, as this will lead to pain and Interferon has been used to treat recurrent laryn-
blistering of the healthy area. Cryoprobes are used geal warts and cervical intraepithelial neoplasia
to apply these cryogens to the cervix. (CIN). In the former cases, tumour load was first
reduced by surgery or by carbon dioxide laser—
interferon then being given parenterally. Although
Electrodiathermy recurrences were rare within 2 years of starting the
interferon course, it was necessary to maintain pa-
This is used for treating mucosal lesions, such as tients on interferon to prevent new lesions. The
those on the cervix, to destroy the diseased tissue by expense of this regimen and possible side-effects
heat. associated with interferon administration provide
drawbacks to this method of treatment. CIN lesions
have also been treated with interferon, but variable
Laser Evaporation results have been reported.
23
Human Polyomaviruses
Kristina Dörries
Julius-Maximilians University, Würzburg, Germany
CLASSIFICATION AND DETECTION tions contain at least two kinds of particles. Passage
of polyomaviruses at high multiplicity is followed
The human polyomaviruses are members of the by the generation of considerable amounts of empty
polyomavirinae subfamily in the family Pa- capsids. In addition, defective viral genomes con-
povaviridae. The small non-enveloped viruses of taining deletions, duplications and rearrangements
about 40 nm in diameter contain single genomes of of viral genetic information may be found. These
covalently closed superhelical double-stranded molecules may be encapsidated if they are within
DNA. Although mouse polyomavirus was detected the appropriate size limits (Hogan et al., 1984). In
almost 50 years ago, the existence of primate polyo- contrast, in vivo the amount of empty shells is con-
maviruses was not realized until the 1960s, with the siderably reduced, pointing to a highly effective vi-
detection of Simian virus 40 (SV40) in monkeys. Ten rus growth under natural conditions. Complete vi-
years later, the human polyomaviruses, JC (JCV) rus particles form a band at a density of 1.34 g ml\
and BK (BKV), were detected by isolation of JCV in CsCl equilibrium density gradients, whereas
from the brain of a patient with progressive multi- empty capsids have a density of about 1.29 g ml\
focal leucoencephalopathy (PML) in human fetal (Figure 23.1).
brain spongioblast cultures, and of BKV in tissue Although the structure of the human polyo-
culture inoculated with urine of a renal transplant mavirus capsids is not yet known completely, it was
recipient (Padgett and Walker, 1976; Zu Rhein, reported that recombinant JCV VP1 self-assembles
1969). into pentameric capsomers, and under appropriate
conditions these molecules will further assemble
into virus-like empty capsids (Chang et al., 1997;
VIRION STRUCTURE AND Frye et al., 1997). Calcium ions are required for
COMPOSITION capsid stability and disulphide bonds might exist
between capsid proteins, as reducing agents are re-
Polyomaviruses are non-enveloped viruses with quired to disassemble virus particles. The human
capsids containing three virus-encoded proteins— polyomaviruses are able to haemagglutinate eryth-
VP1, VP2, VP3. The icosahedral shell surrounds a rocytes at high virus concentrations. Each virus
DNA molecule that is stabilized by cellular histones also has distinct antigenic epitopes and can be dis-
in a chromatin structure. From studies on SV40 and tinguished by neutralization and haemagglutina-
Murine polyomavirus it is known that 360 molecules tion inhibition assays (Pass and Shah, 1982).
of the major capsid protein VP1 are associated with
approximately 30—60 molecules of each of the mi-
nor capsid proteins VP2 and VP3. Virus prepara-
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
620 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 23.1 JCV purification out of PML brain tissue. (a) Optical profile of the centrifugation with banding of virus particles in two
bands (*); intact virus particles at a density of 1.345 g ml\ CsCl . (b) Restriction endonuclease cleavage of DNA extracted from the
major peak of the gradient followed by Southern blotting and JCV specific radioactive hybridization. 1—4 cleavage with single-cut
enzymes EcoRI, BamHI, HpaI and PstI generating linearized form III DNA at about 5 kb in length; 5, 6 cleavage with single and more
cut enzymes in combination (BamHI/PvuII, BamHI/HindIII) generating DNA fragments (M 5,6) A—D and A—E. (c) Electron
micrograph of negatively stained JCV icosaedral virus particles of the small virus peak representing empty particles without
encapsidated viral DNA
Figure 23.2 Circular genomic map of the human polyomavirus JC subtype GS/B. Coding DNA segments are represented by shaded
arrows. Numbers designate the first nucleotide of the start and stop codons for the proteins (Loeber and Dörries, 1988). Position of
splice donor and acceptor sites are indicated. The position of the unique BamHI and EcoRI restriction sites are indicated on the inner
circle
lowed by continuing initiation of late RNAs for coding multiple overlapping genes. Each DNA
15—20 days. The virus has a stringent cell specificity strand carries about one half of the genetic informa-
and replicates efficiently in vitro only in primary tion. Early and late mRNAs are synthesized bidirec-
human fetal glial cell cultures, rich in spongioblasts, tionally from opposite strands of the genome. Pro-
a precursor cell of oligodendrocytes (Padgett, 1983: tein coding sequences consist of open reading
Frisque et al., 1984). In other cell lines from embry- frames for the early regulatory proteins and for the
onic kidney, amnion or urine-derived epithelium late proteins, the regulatory agnogene and the virus
virus growth is rather limited. In contrast, BKV has capsid proteins. Activity and specificity of virus
a broader cell specificity and can be grown in a wide multiplication is directed by the non-coding region.
range of human cell types. It is divided into two regulatory segments with a
single origin of DNA replication (ORI) and the
transcriptional control elements (TCR) within the
promoter region (Loeber and Dörries, 1988; Fris-
Molecular Structure of the Genome que and White, 1992).
The coding sequences exhibit high DNA se-
The genomic structure is highly related among the quence homology. Homology between the human
primate polyomaviruses. The circular DNA of the viruses is greater in all proteins than between JCV
human viruses is about 5100 bp in length (Figure and SV40 (69%). JCV DNA shares between 83%
23.2). The genome is divided into two regions en- and 59% amino acid homology with BKV for TAg
622 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 23.1 Human polyomavirus early and late proteins not yet clear, increased expression of these ‘17kDa’
species was correlated with enhanced transforming
BKV JCV
activity of JCV and might be involved in the control
Proteins Size Mol. wt Size Mol. wt of viral DNA replication. Additionally, it has been
(aa) (kDa) (aa) (kDa) proposed that the transactivating function of TAg
might be influenced by T’ as a transdominant re-
Early
tAg 172 20 172 20.2 pressor affecting the balance of viral persistence and
TAg 695 90 688 79.3 productive infection.
T’135 NR NR 135 17 The late region of the viral genome encodes two
T’136 NR NR 136 22 minor capsid proteins, VP2 and VP3, and a major
T’165 NR NR 165 23 capsid protein, VP1. Late mRNAs are generated
Late from a common precursor by alternative splicing.
Agno 66 NR 71 NR The coding sequences of the minor proteins VP2
VP1 362 46? 354 39.6
and VP3 are overlapping. VP2 contains the entire
VP2 351 40.5? 344 37.4
VP3 232 30.5? 225 25.7 VP3 sequence at its C-terminus and an additional
sequence of approximately 400 amino acids at its
?Molecular weight varies considerably between laboratories. N-terminus. In contrast, the major protein VP1 is
aa : amino acids; NR : not reported.
generated by an alternative reading frame. The pri-
mate polyomaviruses encode a fourth late protein,
and agnogene, respectively. The rates might be even the non-structural agnoprotein. The open reading
higher in functionally active regions of the virus frame is located in the late leader region and the
genes. The early region codes for the regulatory protein is probably involved in the morphogenesis
proteins, the tumour or T antigens (Table 23.1). of virus particles by inhibiting self-polymerization
Both human polyomaviruses encode two proteins of VP1 and the localization of the major capsid
small t and large T antigen, so-called on the basis of protein to the nucleus.
size. The multifunctional T antigen is essential for The non-coding part of the genome is framed by
control of the virus life cycle. Although the human the start codons for early and late genes. The ORI is
polyomavirus proteins are not studied to the same located between the TATA box and the initiation
extent as those of SV40, extensive sequence homol- codon for the early genes. This segment includes the
ogy points to similar functional activities, including conserved binding sites for large TAg. Correspond-
effects on nuclear localization, on viral DNA repli- ing to similar DNA replication strategies, the poly-
cation by direct DNA binding activities and bind- omavirus ORI DNA segment is highly conserved in
ing to DNA polymerase that initiates DNA repli- sequence and structure. In contrast, the promoter
cation, on cellular transformation by interaction region to the late side of the control region reveals
with tumour suppressor proteins, and as a tran- extensive differences. Heterogeneity of structure
scription factor known to be crucial for the regula- and sequence of individual transcription factor
tion of early and late promoter activities. binding sites is reflected in the divergent cell type
The tumour antigens are generated from a specificity and activity of transcription among the
common pre-mRNA molecule by one alternative species and human polyomavirus subtypes (Dör-
splicing event leading to identical N-terminal and ries, 1997).
different C-terminal DNA sequences. Recently,
three additional early virus proteins, T’135, T’136,
and T’165 were characterized in JCV-infected cells Control of Viral Gene Expression
(Trowbridge and Frisque, 1995). All T proteins use
the first alternate splice donor site leading to the
DNA Replication
same 132 N-terminal amino acids. For T’ proteins a
second splice donor site is combined with alternate Regulatory mechanisms leading to DNA replica-
acceptor sites. Whereas T’165 shares the C-termi- tion are closely related among the polyomaviruses.
nus with TAg, T’135 and T’136 have unique ends This is reflected in an origin of DNA replication
encoded by an alternate reading frame. Although being constructed by protein binding elements with
the role of these molecules in the virus life cycle is comparable sequence and spacing requirements
HUMAN POLYOMAVIRUSES 623
Figure 23.3 Alignment of the DNA sequence encompassing the origin of DNA replication in the non-coding genomic region of the
primate polyomaviruses SV40, BKV and JCV. All sequences are given in the sense of the early coding strand. TAg consensus
pentanucleotide recognition sites common to all virus strains are in shaded boxes
(Figure 23.3). Bidirectional replication takes place the virus species might be correlated with differen-
in the presence of the core ORI and TAg, proceed- tial affinity of binding proteins or post-translational
ing from the ORI elements with the T antigen bind- changes of the respective T antigen.
ing sites and terminating at a site about 180° from
the initiation site. The features shared by all ORI
Transcriptional Expression
regions are an inverted repeat on the early side, a
GC rich palindrome in the centre and an AT se- The transcriptional control region (TCR) of the
quence on the late side of ORI. All core ORIs human polyomaviruses is composed of a great
contain these elements, suggesting that common number of different regulatory protein binding mo-
mechanisms play a role in initiation of viral DNA tifs (Figure 23.4). Promoter activity is mediated bi-
replication. This assumption was confirmed by rep- directionally and even independently from the
lication studies on the human polyomaviruses, binding motifs by multiple interactions of transcrip-
showing that besides T antigen binding, site II and tion factors and associated cellular and viral pro-
the inverted repeat are essential for DNA replica- teins stimulating basal, cell type specific and, in
tion (Frisque and White, 1992). Whereas the pres- response of external stimuli, induced functions of
ence of flanking sequences stimulates DNA replica- the viral promoter (Raj and Khalili, 1995). The JCV
tion, the TCR does not affect DNA replication early (JCVE) and late (JCVL) promoter have been
activity directly (Daniel et al., 1996). In conse- most intensively examined in recent years, and it
quence, DNA replication is initiated if T antigen is can be assumed that the mechanisms leading to
provided by the host cell, irrespective of the viral human polyomavirus expression are directed by
promoter activity. However, the efficiency of DNA highly related mechanisms. Therefore, for better
replication directed from JCV ORI sequences is understanding of the complicated regulatory path-
substantially lower than that of SV40 and BKV ways controlling human polyomavirus growth, the
ORI. Subtle differences in the ORI sequence among function of the JCV promoter is examined more
624 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 23.4 JCV TCR with protein binding sites and interaction of cellular and viral transcription factors. TCR structure of
prototype JCV Mad-1 is shown between the start codons (arrows) for early and late genes. ORI : origin of DNA replication;
TA : TATA box; TR : tandem repeated promoter elements; similar shading represents identical DNA sequences, see Figure 23.5
legend; A—D : dissected promoter domains; MCP : minimal core promoter; LCE : lytic control element. Binding sites and the
respective proteins are indicated below the promoter domains; filled circles indicate interaction of transcription factors; TAT,
TAR : HIV related transactivation domains; arrows indicate interaction of inducing agents with the respective proteins; V, position of
SP1 binding site in JCV TCR type II genomes
closely. The JCV control region is artificially dissec- on the expression of factors involved in immu-
ted in the ORI domain and following four TCR nologically regulated signalling pathways to modu-
subdomains (A—D), in each of which a clustering of late JCV expression.
early and late interactive promoter sites is observed. It was shown that interference with B function
Outside of the TCR domain, on the early site of in late gene expression decreased, but did not abol-
ORI, a binding sequence is located for the potent ish, activity. This suggested that additional TCR
transcriptional enhancer nuclear factor B (NFB). elements may confer inducibility to the JCVL pro-
The site increases bidirectionally transcription from moter. At present the possibility that cross-com-
the late, and to a lesser extent from the early, pro- munication between motifs is mediated by a 40 kDa
moter in glial cells (Mayreddy et al., 1996). NFB is protein complex distinct from the classical NFB
constitutively expressed in B lymphocytes, one of subunits is discussed. In the presence of classifical
the sites of JCV persistence. NFB can be retained NFB binding proteins this complex may not effec-
in the cytoplasm by an inhibitor B protein. This tively interact with the B sequence. However, if the
binding can be released through stimulation by a NFB activators are not expressed, the 40 kDa pro-
number of agents as tumour promotors like phor- tein may bind and downregulate JCVL gene tran-
bol myristate acetate (PMA) or inflammatory scription. This may come into effect during the per-
cytokines. It results in the transport of NFB to the sistent state of infection.
nucleus, where the binding to the consensus DNA In addition, the GRS motif within ORI might
sites exerts activity. Upon PMA treatment both interact with the B motif (Raj and Khalili, 1995).
JCV promoters are responsive to NFB induction, GRS is similarly inducible by PMA and inflamma-
leading to increased JCV activity. In addition, tu- tory cytokines. The responsive region interacts with
mour necrosis factor (TNF) is able to enhance the protein GBP-i. GBP-i is induced in a wide range
binding of NFB to the JCV B site (Atwood et al., of cell types, thus it could play a role in mediating
1995). This stimulates the idea that activation of the JCV activation at all suspected sites of persistence.
transcriptional control in vivo might be dependent Comparable to the NFB class of proteins, the
HUMAN POLYOMAVIRUSES 625
GBP-i complexes probably represent a combina- cific late transcriptional silencer OP-1 contacting
torial assembly of various protein species that is directly downstream of the Tst-1 binding site points
changed upon induction. Duality of function could to the possibility that cross-talk between the two
involve the basal transcriptional machinery and sites could influence viral transcription. The OP-1
other transcription proteins associated with the late motif also interacts with the adjacent NF-1 site.
promoter activity. One potential factor is trans- OP-1 and NF-1 form a composite element that
forming growth factor acting through the GRS increases JCV early activity and reduced JCV late
and the NF-1 sites. In such a model the status of the activity by interaction of common proteins with
viral promoter could be modulated through the glial cell specific NF-1 protein binding (Raj and
GRS and variable interaction of cytokine-induced Khalili, 1995).
proteins. Although these cytokines might use differ- In addition, the poly(dA) stretch with the over-
ential signal transduction pathways for protein acti- lapping pentanucleotide motif differentially binds
vation, in consequence, the nuclear milieu will con- members of the LCP-1 protein family recognizing
tain the factors leading to expression by interaction alternative DNA structures. This influences early
with a promoter containing the respective contact and late promoter functions and is therefore desig-
sites. nated as JCV lytic control element (LCE). A YB-1/
The following domain C contains the JCV early pur binding site was found in close association
minimal core promoter (MCP) that is constituted with the LCE element. YB-1 is a member of a gene
by the TATA box region and the immediately ad- family isolated originally from a human B cell ex-
jacent binding site for the transcription factor Tst-1/ pression library. The essential contact site is located
Oct-6, a member of the POU-domain protein fam- in the C/T-rich sequence on the early strand of the
ily. In glial cells Tst-1 was found to be one of the LCE without affecting the adjacent NF-1 binding
permanently produced cell type selective transcrip- site. YB-1 binds to double-stranded and single-
tional regulators. Both JCV promoter orientations stranded DNA and activates transcription from
are stimulated by DNA binding of Tst-1 in glial both the viral early and late promoter. The function
cells to sequence motifs involving the TATA box is attributed to both its interaction with DNA and
and overlapping in part with adjacent OP-1 and its ability to interact with other proteins. The pen-
YB-1 binding sites. tanucleotide repeat on the late LCE strand is the
Like most pou proteins Tst-1/Oct-6 is an intrisi- target for the single-stranded DNA binding protein
cally weak transcriptional regulator. To compen- pur . It is associated with YB-1 early activation by
sate for this weakness, Tst-1 has to rely on viral or cross-communication of the proteins on the LCE,
glial cell specific coactivators. Large T antigen of mediating reduced or increased binding to YB-1.
JCV has been identified as a viral coactivator which Although TAg has no binding capacities to LCE
stimulates the function of Tst-1 synergistically by sequences, the close association of YB-1, pur and
direct interaction. The modular synergism of both TAg and the complexity of binding events suggest
proteins does not require DNA binding of JCV T that the interplay is important for a broad spectrum
antigen in the presence of a functional binding site of different functions during the JCV life cycle. The
of Tst-1. The high mobility group proteins HMG-I/ current model for the involvement of pur and
Y have been discussed as cellular coactivators. YB-1 in TAg-mediated transition of the early-to-
Though not a transcriptional regulator by itself, late promoter activity of JCV gene transcription is
HMG is believed to serve as a promoter-specific based on the following findings.
accessory factor modifying the transcriptional func- Pur increases early promoter activity with mi-
tion. The JCV AT-rich region with the Tst-1 site nor effects on the late sequences, TAg increases late
provides the binding site for HMG proteins that activity considerably, and YB-1 elevated basal ac-
substantially stimulate Tst-1 binding to the Tst-1 tivity on the early and late promoter with a higher
responsive element. rate in the late phase. Increasing concentrations of
Tst-1 and HMG are able to activate synergisti- TAg results in a gradual decrease in pur early
cally JC gene expression, clearly indicating cooper- enhancement. Therefore it is possible that pur
ation between the two proteins. However, the TST- might be involved in a negative feedback mechan-
1 site alone was not enough to mediate efficient ism for downregulation of JCVE during the late
cooperation. The identification of the glial cell spe- phase of infection.
626 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
In contrast to the early expression, cooperative actions at the binding sites involve NF-1 or NF-1-
action of pur and TAg leads to interference of pur like factors, ATF/CRE-related factors and Jun-re-
with the TAg late stimulatory effect, suggesting lated (AP-1-like) factors. Isolated binding sites do
that pur and TAg exert antagonistic effects on not mediate activity, suggesting an essential role for
each other’s transcriptional activity. The mechan- the surrounding binding sites and interactions of
ism by which the two regulators influence each related proteins for the activation process.
other’s activities is not yet clear; however, TAg may NF-1 sites A/B are involved in basal as well as in
destabilize indirectly the association of pur with glial cell specific modulation. This discrepancy was
the LCE. In this respect, TAg might cause dissocia- explained recently by the detection of a glial cell-
tion of pur from the LCE by increasing the rate of specific NF-1 form that considerably enhanced
YB-1 binding. transcription from the JCV promoter (Sumner et
From these findings it was proposed that at the al., 1996). It became clear that the level of transcrip-
initial phase of infection strong binding of pur to tional activity and specificity of the NF-1 sites is due
the LCE preferentially stimulates transcription of to a homodimeric/heterodimeric nature of different
the early genome. As the lytic cycle proceeds, the NF-1 molecules, and to the combinatorial interac-
composition of DNA—protein complexes in the tion of NF-1 with adjacent proteins. Apart from the
LCE is altered. The increasing level of TAg facili- prominent activity conveyed by NF-1 on to the
tates YB-1-mediated dissociation of pur from the JCVE, the sites are involved in JCVL cell-specific
pentanucleotide repeat by stabilizing the associ- activation by TAg transactivation (Kumar et al.,
ation of YB-1 with DNA. Removal of pur from the 1996a).
LCE results in a substantial decrease in early pro- Cell-specific activity in glial cells includes CRE.
moter activity. At the same time YB-1-associated JCVE expression is increased substantially by the
pur release of the late promoter allows T antigen second messsenger cAMP and forskolin. Activity is
to enhance the expression during the late phase of mediated by a CRE binding protein (CREB) and is
infection. not influenced by the NF-1 site. However, this does
The B domain in the early orientation contrib- not exclude the possibility that the binding of other
utes positively to expression in glial cells and is also proteins to CRE may modulate expression without
important for transcriptional activation of JCVL directly interacting with JCVE sites. Additionally, a
genes. The central motif is responding to glial factor glial cell-specific modification of CREB might be
1 (GF-1) (Chen et al., 1997). GF-1 protein expres- responsible for activation.
sion is most abundant in brain tissue. Interestingly, Thus a significant contribution to the enhanced
the amount of GF-1 is higher in kidney cells than in glial cell-specific expression of JCV appears to be
other cell types. Therefore it is considered that the made by inducible transcription factors interacting
level of GF-1 in kidney cells may be responsible for with unique motifs on the JCV promoter. Import-
the ability of JCV to replicate in urogenital tissue. antly, the mechanisms of induction for the factors
Binding to the GF-1 site stimulates transcription are through different signal transduction pathways.
from the JCVL promoter and to a lesser degree This combination allows a highly flexible JCV tran-
from the JCVE promoter. scriptional response to a large number of environ-
Pronounced high affinity NF-1 binding sites mental signals.
(NF-1 A/B) are located just upstream of the A/T At present, in domain A two potential promoter
rich region. Three more NF-1 sites (C, D, E) are elements are identified, a transactivator element as-
found in domain A and further into the late region. sociated to HIV TAR homologous sequences and
Three overlapping binding sites for nuclear factor Tst-1 binding sites. The D domain is localized close
Jun are associated closely. On the latter half of to the start codon of the late proteins in the leader of
NF-1 site A/B a consensus sequence similar to that the late RNAs. It spans binding sites for cellular
of activation transcription factor ATF or the cyclic proteins NF-1, c-jun, YB-1 and a NFB responsive
AMP-responsive element (CRE) and, overlapping region (Raj et al., 1996). YB-1 exhibits the ability to
NF-1 site E, a sequence with partial homology to modulate basal and activated levels of transcription
ATF/CRE sites is localized (Kumar et al., 1996b, of JCVL through its influence on p65 on the NFB
1996c). NF-1 site D coincides with a site resembling site. This points to a potential role for YB-1 as a
the sequence for the activator protein AP-1. Inter- transcriptional coactivator for regulation from the
HUMAN POLYOMAVIRUSES 627
NFB site. The YB-1 binding site on the B domain in a cell type-specific manner. Primary effect of Tat
does not account for such pronounced effects, thus on JCVL expression is to enhance the rate of tran-
the D domain respresents probably the more opti- scription, suggesting that interaction between HIV-
mal site for interaction of YB-1 and NFb/rel 1 and JCV in infected cells may facilitate JCV
subunits. growth by stimulation of viral late gene expression.
NFB/rel subunits modulate JCV late promoter Interaction of Human cytomegalovirus (CMV)
activity from the D domain, revealing an extensive with JCV expression has been studied less intensive-
duality of NFB/rel-mediated effects. The subunits ly (Heilbronn et al., 1993). In this case replicating
appear to have a mutually antagonistic function in CMV increased JCV DNA replication in non-per-
terms of their transcriptional activity from either missive cells. Although the CMV effector protein
the D domain or the NFB site. Basal uninduced was not characterized, it is likely that DNA replica-
transcription appears to be positively regulated by tion is affected indirectly. Since early expression of
p50/52 from the D domain, whereas induced tran- T antigen is indispensable for JCV DNA replica-
scription is positively regulated by p65 from the tion, initial steps might involve transactivation of
NFB site. At present, it appears likely that the the early promoter by an unknown CMV effector
interplay between NFB subunits and the D do- and subsequent expression of T antigen.
main is dependent on direct and specific interaction The mechanisms of human polyomavirus trans-
of YB-1 and the NFB/rel subunits influencing activation by heterologous viruses are not under-
each other’s binding capacity to their respective stood at present. However, the increasing number
target DNAs. Since each of the NFB dimers can of patients infected with the human immunodefi-
interact potentially with the NFB site, multiple ciency virus (HIV) who have active polyomavirus
levels of regulation can be mediated by the complex infections argues for a potential role of concomi-
interactions of NFB/rel subunits. This provides tantly infecting viruses for the activation of polyo-
the virus with an exquisite control mechanism over mavirus infections in men.
constitutive and induced NFB activity.
Figure 23.5 Structure of JCV TCR subtypes. JCV TCR subtype I: American JCV prototype Mad-1. TCR type II: European JCV
prototype GS/B. Archetype: European isolate JCV GS/K. The sequence is divided in seven boxed segments (A, B, C, D, E, F1, F2)
differing in number and/or length. A represents the TATA box. The initiation codon for the putative agnogene is indicated by an arrow.
Segment length is given in base pairs; AT : TATA box; ori : origin of DNA replication
patients groups points rather to the existence of Interestingly, in a number of PML patients JCV
stable TCR subtypes in the human population that V-T European/American genotype II genomes
might be less sensitive to rearrangements than to a linked to double repeat PML type TCRs were de-
high rate of rearrangements in all individuals (Els- scribed (Agostini et al., 1997a, 1997b). In conse-
ner and Dörries, 1998). quence, it was asked whether a specific genotype
On the basis of these findings, it is essential to might constitute a pathogenic strain. V-T genotypes
answer the question of the transcriptional activity 1 and 2 are both linked to archetypal and PML-
of subtype control regions (Ault, 1997). Initial ex- type TCR elements, and were both isolated from
periments point to a more complicated situation PML and persistently infected individuals. Al-
than assumed by the DNA sequence analyses of though at present no archetype genome has been
JCV promoter elements alone. In contrast to all described as the only variant in PML cases, from
assumptions, it was found recently that the rate of the relationship of archetype and PML-type regula-
early transcriptional activity mediated by du- tory regions it is conceivable that PML-type JCV
plicated TCRs can be similar to that of single pro- TCRs have been generated from single promoter
moter structures. It was also confirmed that the elements. Since geographical clustering of JCV type
overall cell type specificity of individual subtypes is II genomes cannot be excluded, it remains to be
not affected by structural changes (author’s obser- evaluated further whether one of the major JCV
vations). Therefore TCR heterogeneity of the hu- V-T types is associated dominantly with PML.
man polyomaviruses appears to be a determining
factor of reproductive growth rate, but is most
probably not related to the induction of disease.
630 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 23.2 Human polyomavirus-associated diseases
States and Target Organs of Human Japanese families in which both parents and
Polyomavirus Infection children excreted JCV in their urine. Typing of JCV
genomes in individual family members revealed the
Primary Infection presence of different viral subtypes (Kitamura et al.,
1994a; Sugimoto et al., 1997). In about 50% of
Primary contact with the human polyomaviruses persons the JCV strains originated from infection
occurs usually during childhood or early in adult- outside families. Children have many opportunities
hood, leading to lifelong infection in the im- to come into contact with urinary JCV infection,
munocompetent host. An association of primary suggesting urinary excretion as a prominent source
BKV infection with mild respiratory tract disease, of JCV in the human population.
mild pyrexia and transient cystitis is discussed occa-
sionally, but generally the course is asymptomatic
(Table 23.2). Viruses undergo normally an initial Persistent Infection
replication cycle in cells proximal to the site of entry The discovery of BKV came from the observation
and prior to viraemia. The site of JCV entry is not of cytological abnormalities in the urinary sediment
yet localized. In the case of BKV, the presence of of a kidney transplant patient. Later it became clear
viral DNA in nasopharyngeal aspirates and tonsils that BKV is a urotheliotropic virus affecting ep-
pointed to the oropharynx as the initial site of infec- thelia of the renal calyces, renal pelvis, ureter and
tion. Nevertheless, cell types supporting initial rep- urinary bladder (Shinohara et al., 1993). Similarly,
lication and routes of virus distribution remained JCV was detected in the urinary tract of im-
undefined. Viraemia is thought to occur, based on munosuppressed individuals and pregnant women.
the finding that virus reaches sites of persistence Polymerase chain reaction (PCR) analyses suggest
very early in infection (Figure 23.6). that the number of affected individuals parallels
Despite the knowledge that JCV infection is closely the percentage of persons with serological
widespread in the population, almost nothing is evidence for contact with JCV. Thus the kidney is
known about the way it is transmitted in humans. obviously an essential site of human polyomavirus
Studies in and around Tokyo suggest that popula- infection (Arthur and Shah, 1989).
tion density and environmental conditions may af- JCV DNA was detected in the kidney of two
fect the transmission of JCV. One route of trans- children with combined immunodeficiency disease
mission was elucidated by tracing JCV subtypes in who developed PML during primary JCV infection.
HUMAN POLYOMAVIRUSES 631
The presence of JCV DNA in spleen, lymph node Nevertheless, virus protein expression was restric-
and lung cells support the thesis that JCV persist- ted to less than 1% of the cells, thus pointing to an
ence in individual organs is established most prob- attenuated replication in lymphocytes. Although
ably during primary infection. Although repeated the virus is able to attach and penetrate into mono-
detection of JCV DNA in ureters of non-im- cytes, there was no expression. Therefore mono-
munosuppressed patients characterized the renal cytes may be involved in the degradation rather
tract as a site of persistence, the higher detection than in the replication of engulfed virus particles in
frequency in the renal medulla indicates that the natural infection. The presence of BKV DNA in
epithelial cells lining collective tubules are the ma- leucocytes of persistently infected individuals con-
jor target cells. The presence of JCV in urothelial firmed involvement of BKV in peripheral blood
sediments suggests that they are more often subject infection (Dörries, 1997).
to activation processes than other cells in the renal Haematogeneous spread of JCV to the CNS in
tract. PML had been suspected in early reports because of
In the tonsils, BKV DNA was found to be asso- the exceptional multifocal distribution of JCV foci
ciated specifically with the lymphoid tissue of Wal- in PML. Occasional involvement of spleen and
dayer’s ring, thus indicating involvement of lym- lymph nodes and the finding of virus particles in
phocytes in human polyomavirus infection. Lym- lymphocytes in a child with measles virus infection
phoid interaction of BKV was supported further by led to the assumption that lymphoid cells might be
a stimulatory effect of virus infection on human involved regularly in virus spread and establish-
lymphocytes in cell culture, and the demonstration ment of persistent infection. This was confirmed by
of specific BKV receptors on the surface of periph- demonstrating JCV DNA and capsid protein in a
eral blood cells by the rosetting technique. Interest- small number of mononuclear cells of bone marrow
ingly, only a small number of cells carried receptors and spleen in PML/AIDS patients. These cells were
for BKV. They were considered as B lymphocytes. characterized as B lymphocytes by cellular markers.
632 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
The presence of JCV-infected mononuclear cells in Although JCV is demonstrated easily in dis-
perivascular parenchyma and in Virchow—Robin seminated areas of PML autopsy material, the
spaces may support further the haematogeneous question of whether PML results from cytolytic
route of entry of JCV into the brain. In other re- invasion of the CNS under severe immunosuppres-
ports, only occasionally were monocytic cells re- sion or as a consequence of a preceding persistent
ported in PML brain, thus the question whether infection is under discussion (Dörries, 1997). In
glial cell infection originates from passage of virus early studies employing Southern blot analysis, in
through lymphocytes to parenchymal glial cells or situ hybridization or immunochemistry virus DNA
by another mechanism remains open. or protein had never been detected in the CNS.
Although infection rates in peripheral lym- However, application of the PCR revealed frequent
phocytes of PML patients were the highest, the JCV infection in the brain of patients without evi-
virus is found consistently during periods of im- dence of PML. In contrast to fetal brain tissue, JCV
munocompromise at rates of about 38% in HIV DNA sequences were present in about 30—70% of
patients without evidence of PML. Detection of full adults, depending on the experimental groups. JCV
length JCV DNA in peripheral blood from healthy obviously has no topographical preference, because
individuals added further support to the idea that dissemination of viral DNA is similar to that in
the human polyomaviruses are lymphotropic in na- PML tissue. Analysis of the physical state and gen-
ture. In situ hybridization confirmed common infec- etic complexity of virus sequences resulted exclus-
tion of human peripheral blood lymphocytes ively in unique, full length, episomal JCV genomes.
(PBLs) and experimental evidence demonstrating Compared to thousands of genome equivalents
interaction of JCV with human B lymphocyte cell present in affected PML samples, the amount of
lines argues for the involvement of these cell types in virus specific DNA in asymptomatic individuals is
natural infection (Dörries, 1997). much lower, with an estimated range of 1—100
Summarising the data, human peripheral leuco- genome equivalents per 20 cells. Thus persistent
cytes are obviously target cells for asymptomatic polyomavirus infection in the CNS is probably re-
polyomavirus infection in almost all adults by the stricted to isolated cells and most likely represents
age of 20 years. Therefore it is likely that polyo- chronic infection and not early stages of disease
maviruses are able to establish a persistent infection (Dörries, 1998).
in lymphocytes during primary infection or early in The findings prove that human peripheral polyo-
persistence. The amount of virus-specific DNA in mavirus infection is associated with subclinical vi-
the cells is regularly lower in healthy persons than rus entry into the CNS, probably long before the
in immunocompromised patients. This is consistent development of clinically overt PML. Virus activa-
with the finding that persistent polyomavirus infec- tion may lead to an increased number of infected
tion is restricted to a few virus genomes and to a low cells and a higher detection frequency in cases of
expression rate, if there is any. Nevertheless, the severe immunosuppression, in patients with malig-
number of viral DNA molecules increases occa- nant disease and in the elderly. This is in agreement
sionally, probably under impairment of the immune with the assumption that impairment of immune
system due to a timely restricted activation in competence favours involvement of the CNS in
healthy persons. Similarly, the higher incidence of polyomavirus infection.
virus DNA in multiple specimens of patients with
lymphoproliferative disorders, and the finding of
Concomitant JCV and BKV Infection
elevated virus specific antibody titres in the elderly,
in pregnancy or in patients with tumours, point to Double infection of JCV and BKV was established,
lymphocytes or their precursors as target cells for particularly after organ transplantation, in HIV-
virus persistence and continuous activation. The infected patients, in pregnancy and in a small
suggestion that infected B cells or their precursors number of immunocompetent individuals, by uri-
act as a reservoir for the virion in the diseased brain nary excretion or antibody rise against both viruses.
might be questionable; however, it is conceivable Molecular detection of JCV and BKV specific DNA
that JCV-infected lymphoid cells may act as a vec- confirmed that concomitant persistence frequently
tor for JCV CNS invasion and dissemination dur- occurs in kidney tissue. Extensive homologies of the
ing persistent infection (Gallia et al., 1997). genomic structure, similarities of virus spread and
HUMAN POLYOMAVIRUSES 633
state of infection started very early the discussion age group. Recently, it was reported that JCV DNA
on possible other sites of dual JCV and BKV infec- was present in the urine of about 30% of American
tion. After PBLs were found to be a target of polyo- and 60% of Japanese and European healthy indi-
mavirus infection, the presence of JCV and BKV viduals. The incidence of excretion was dependent
DNA in blood cells of the same individual was not on age, with lower rates in the young and then
astonishing. A high rate of concomitant infection gradually increasing in older age. Because of the
was evidenced molecularly in healthy and im- high frequency of JCV excretion throughout adult-
munosuppressed individuals. As JCV protein ex- hood, as detected by PCR, it is suggested that in
pression was detected in lymphoid cells of im- most healthy adults JCV infection is not in a latent
munosuppressed patients, and BK virus is able to but in a productive persistent state. Since all other
replicate in lymphocytes in vitro, it can be assumed data were accumulated by less sensitive methods, it
that both human polyomavirus infections are ac- is conceivable that a basic virus expression is main-
tivated periodically in persistently infected PBLs. tained permanently at an attenuated level.
Although BKV was not expected to invade the Pregnancy is among the most common condi-
CNS at a high rate, cell specificity of BKV is less tions that have been linked to viral activation. Inci-
stringent than that of JCV, therefore several labora- dence of viruria, at a level detectable by periodic
tories sought for double infection in the CNS. Clon- cytological examination, was about 3—7% for JCV
ing from CNS gene libraries and PCR revealed and 15% for BKV at some time during pregnancy.
frequent BKV dissemination to the brain. Indirect- The onset of viruria was late in the second trimester
ly, BKV infection of the human CNS was confirmed and during the third trimester. In excretors, virus
by the report of a subacute BKV-associated menin- shedding, once it was established, continued inter-
goencephalitis in a patient with the acquired im- mittently to term and then ceased in the postpartum
mune deficiency syndrome (AIDS). Physical state period. The detection rates are according to studies
and genetic complexity of BKV genomes were com- on high or increasing antibody titres in a compar-
parable to that of JCV DNA; however, low concen- able population, and therefore represent probably
tration of brain-derived BKV specific PCR prod- the true rate of activated persistent infections.
ucts suggests a considerably lower activity of BKV Renal transplant recipients experienced viruria at
in the CNS than that of JCV. The detection of a higher rate ranging from 9 to 19%, compared to a
amplification products belonging to both poly- rate of 2.4% in pregnant women with similar detec-
omavirus species gives strong evidence for concomi- tion sensitivities. Duration may be over a month,
tant polyomavirus infection in the CNS. Addition- even to years. In bone marrow transplant recipients
ally, it demonstrates that not only JCV but also almost all viruria was due to BKV, with an inci-
BKV is neurotropic in the human host, and estab- dence of up to 50% in the post-transplant period.
lishes frequently persistent CNS infection. Although these findings point to a significant role
The rate of adult healthy individuals found posi- of the immunosuppressive state in the activation of
tive for concomitant infection was astounding, but polyomavirus infection in the kidney, examination
the data correspond to recently discussed rates of of HIV patients revealed no significant changes in
infection with BKV of almost 100% by the age of virus activation compared to a comparable group
10, and JCV reaching more than 90% in adulthood. of non-immunocompromised controls; thus an en-
Therefore the number of positive cases probably hancement of urinary excretion by HIV-induced
reflects the true incidence of polyomavirus infection immunosuppression was not detectable. Similarly,
in the European population. PML patients do not necessarily have concomitant
JCV viruria. Aggressive chemotherapy does not in-
crease virus frequency, and in other immunosup-
Asymptomatic Activation of Infection
pressive diseases viruria can be intermittent, with
Transient polyomavirus viruria probably occurs at sparsely distributed infected cells in cytologically
the time of primary infection. However, in most positive urine, pointing similarly to a rather low
instances presence of virus in the urine is due to rate of virus production (Arthur and Shah, 1989).
activation processes. Activation of infection in the Activation at other sites of persistent infection is
healthy was assumed to be related predominantly less well recognized. This is due to the attenuated
to alterations of immunocompetence in the older growth of virus products in asymptomatic transient
634 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
activation states and to the low virus load in persist- rather associated with persistent BKV infection. In
ently infected healthy individuals. Although persist- immunological deficiency BKV is suspected of
ent infection in lymphocytes appears to be a causing graft rejection and renal dysfunction. Virus-
common event, activation can only be deduced so associated pathogenic effects are observed in uret-
far from the presence of higher amounts of virus eral stenosis as a late complication of renal trans-
DNA in PBLs of immunologically impaired indi- plantation, and in haemorrhagic cystitis in bone
viduals. In addition, in HIV-PML patients mono- marrow transplant patients. After BMT BK viruria
nuclear cells in PML tissue were characterized ex- is detected often during episodes of cystitis; how-
pressing JCV specific proteins. Thus it appears ever, it is not yet known to what extent virus growth
likely that JCV infection in lymphocytes is activated or therapeutic intervention is the cause for the tissue
under as yet unknown circumstances (Gallia et al., damage observed.
1997). The strongest evidence of BKV-induced systemic
The presence of JCV DNA in brain tissue of and CNS disease comes from an AIDS patient
immunocompetent patients in a distribution com- (Vallbracht et al., 1993). Histopathological evalu-
parable to that in PML, and increasing incidence of ation revealed association of BKV with affected
detection with age and in patients with malignan- lung, kidney and CNS tissue. The major pathologi-
cies, point to viral activation in the CNS. The detec- cal findings were a tubulointerstitial nephropathy,
tion of virus specific protein in a limited number of an interstitial desquamative pneumonitis and a sub-
glial cells in non-PML brain, and PCR amplifica- acute meningoencephalitis. In the kidney, alter-
tion of JCV specific products in CSF of the same ations consisted principally of focally accentuated
patient groups, further argue for asymptomatic tubular necroses. Virus products were detected in
JCV activation in persistently infected brain tissue epithelial cells along the entire nephron. Alterations
(Dörries, 1997). in the lung were characterized by intra-alveolar
If the current knowledge is summarized, it must aggregates of desquamated pneumocytes. Virus
be assumed that activation of BKV and JCV infec- products were detected in exfoliated pneumocytes
tion is species specific and occurs as a result of and in epithelial and smooth muscle cells of the
immune system alterations induced by pregnancy, bronchioli. Occasionally, isolated endothelial cells
older age, malignant tumour growth or AIDS. in the lung carried virus protein. A common feature
Some of these individuals may undergo sporadic in both organs was focal interstitial fibrosis with a
activation as a consequence of their genotypes or of mild inflammatory response. Whereas fibrocytes
incidental transactivation events by other viruses. were infected with virus, inflammatory cells were
In general, however, virus growth is dependent on free of virus products.
the impairment of immunological control resulting In the CNS no pathological changes apart from
in differentially regulated activation patterns in in- mild inner atrophy have been described. Thickened
fected organs. Although related mechanisms are fibrotic leptomeninges were infiltrated loosely by
unknown so far, the higher frequency of deficiencies mononuclear cells, indicating chronic inflamma-
correlated with T cell function in PML patients tion. In the cortex and adjoining white matter
points to deficient cellular immunity as a major oedematous tissue alterations were found. Reactive
virulence factor. astrocytes were localized in the outer layers of the
cerebral cortex. The ventricular system exhibited
focal degeneration of its ependyma and spongiform
Polyomavirus-Associated Diseases
destruction of subjacent brain tissue. The choroid
Activation of human polyomavirus infection may plexus revealed fibrosis of the stroma and atypical
either represent transient asymptomatic events or epithelial cells. In some areas, necrosis and exfoli-
pathological processes. The induction of fatal dis- ation of the plexus epithelium occurred. Small infil-
orders, however, is observed almost exclusively un- trates were seen in association with the lesions. The
der long-lasting severe impairment of the immune dominant target cells of BKV infection were fibro-
system. BKV is associated predominantly with blasts of the loose reticular connective tissue, en-
urogenital tract diseases (Arthur and Shah, 1989). dothelial and smooth muscle cells of blood vessels,
Mild clinical courses are reported in primary BKV astrocytes and infiltrating macrophages in pons and
infection, whereas cystitis and tubulonephritis are medulla oblongata. Epithelial cells in the choroid
HUMAN POLYOMAVIRUSES 635
plexus and astrocytes of the subependymal brain Table 23.3 Causes of JCV activation and induction of PML
tissue were additionally infected. Interestingly, the prior to the AIDS epidemic
only glial cell type involved in BKV-associated cen- Conditions associated with
tral nervous system disease was the astrocyte,
whereas the oligodendrocyte and nerve cells were Activation of persistent
not affected. The large number of cell types involved infection PML
(% of cases? except AIDS)
in BKV infection in vivo demonstrates the broad
BKV cell specificity and points to a greater relation- Age None
ship of BKV to SV40 than to JCV. The detection of Inflammation None
a BKV-associated disease affecting kidney, lung Pregnancy None
Diabetes Rare
and the CNS confirms the involvement of those Transplantation Rare
organs in BKV persistence and is in line with the Carcinomatous disease 2.2
persistence of BKV DNA in brain tissue of healthy Myeloproliferative disease 6.5
individuals. Granulomatous/inflammatory
In contrast to BKV, JCV-associated disease was disease 7.4
Immune deficiency states 16.1
never observed in the urogenital tract or in the lung. Lymphoproliferative disease 62.2
The only and most prominent disease associated
with JCV is the CNS disorder PML (Zu Rhein, ?Brooks and Walker (1984).
1969; Walker and Padgett, 1983; Berger and
Table 23.4 Development of PML lesions
Concha, 1995). Although SV40 was described as the
cause of PML in three American and Japanese I Early lesion
patients, it later became clear that JCV was most Altered oligodendrocytes in areas of demyelination
probably the only agent responsible for the disease. nuclei enlarged (2—3;)
PML is a degenerative demyelinating disorder regular chromatin pattern is lost
nuclei tint deeply with basophilic dyes
occurring as a late complication of pre-existing sys- irregular basophilic nucleic inclusions
temic diseases that impair immunological compet- Seldomly activated pleomorphic microglia
ence. Prior to the AIDS era, in about half of the Pinhead size (mm) beneath the cortical ribbon
cases malignant proliferative diseases were the II Aging lesion
dominant basic disorders in PML (Table 23.3). Reduced number of altered oligodendrocytes in the centre of
Nowadays, a steadily increasing number of cases is demyelination
associated with AIDS. Calculation of PML rates Shrinkage of nuclei at the rim of lesions
vary at between 5% and 20% of AIDS patients. A Widely distributed larger foci with coherent tissue
higher frequency of PML can also be observed III Late lesion
under immunosuppressive therapies. Several years Altered oligodendrocytes at the peripheral advancing zone
of treatment precede PML in such diseases as rheu- surrounding the area of myelin loss
In the centre
matoid arthritis, chronic asthma, sarcoidosis, lupus very sparse or absent oligodendrocytes
erythematosus, chronic polymyositis or renal trans- singly scattered reactive hypertrophic astrocytes
plantation. The duration of disease after onset of giant astrocytes in mitosis
neurological symptoms is described as having an astrocytes resembling cells in pleomorphic glioblastomas forming
irregular necrotic patches (cm)
average of 4—6 months; however, there are cases
with more than 12 months or so, with an intermit-
tently progressive or subclinical course over the but each new functional impairment becomes pro-
years. gressively more severe. Once clinical signs appear,
The onset of PML is often insidious. In the ear- the disease usually progresses steadily. Early neuro-
liest manifestation, multiple pinhead-sized de- logical symptoms regularly indicate multiple dis-
myelinating lesions are described beneath the corti- seminated lesions in the brain. The extent and top-
cal ribbon (Table 23.4). New small foci are being ography of lesions correlate well with the duration
added continuously in neighbouring tissue as and symptomatology of the illness.
growth centres of new lesions. This concept of the The pathognomonic feature of the disease is the
histological evolution of the disease is supported by striking alteration of oligodendrocytes in all lesions.
the clinical evolution: the onset might be gradual Oligodendrocytes are located in the peripheral rim
636 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
surrounding the zone of myelin loss. The central cally to the demyelinated lesions in PML (Frisque
area is composed of reactive astrocytes, including and White, 1992; Major et al., 1992; Dörries, 1997).
giant cells in mitosis, and astrocytes resembling Thus oncogenicity of JCV and BKV was examined
malignant cells of pleomorphic glioblastomas. In- almost from the beginning in experimental animal
fection of neurons, ependymal or endothelial cells systems. Developing tumour types reflected the cell
has never been convincingly demonstrated. type specificity of the viruses. As a consequence of
The basic cause of tissue destruction is a cytolytic the JCV infection, and corresponding to the speci-
JCV infection of the oligodendrocyte. Destruction ficity of JCV for glial cells, brain tumours are ob-
of these cells results in loss of myelin, tissue break- served predominantly. The highest yield is found
down and impairment of brain function. Infected after intracerebral inoculation of newborn Syrian
oligodendrocytes mediate highly effective virus hamsters, whereas peripheral nervous system tu-
growth in the range of more than 10 virus par- mours are prominent after intraocular virus admin-
ticles per gram of tissue. Viral expression products istration. Central nervous system tumours are
and virions are found in nuclei and cytoplasm of the located in the cerebrum, the cerebellum, the brain-
oligodendrocytes. Bizarre astrocytes may contain stem and the spinal cord. Mesenchymal tumours
JCV; however, the question of whether astrocytes within cerebral meninges are classified as malignant
may support actively infection is not yet answered. meningiomas. Ependymomas are the dominant
On the grounds of the morphological changes and type of intraventricular tumours, and occasionally
occasional reports on intracellular virus particles, it choroid plexus tumours are observed. The most
is considered that astrocytes may represent a common neoplasms are medulloblastomas, at a
semipermissive cell type mediating attenuated JCV rate of 95% in newborn hamsters. These are fol-
expression, or being sensitive to virus transform- lowed by malignant astrocytomas, glioblastoma
ation. multiforme, neuroectodermal tumours and
In contrast to classical PML, in the AIDS patient pineocytomas. Examination of the neuro-on-
inflammatory reaction with perivascular mononuc- cogenic potential in primates confirmed the occur-
lear cell infiltrates is observed often. Mononuclear rence of malignant glial brain tumours in about
cells in the Virchow—Robin spaces occasionally 50% of adult monkeys after intracranial JCV infec-
contain JCV specific DNA and capsid antigen, and tion, the predominant type being characterized as
in the subcortical white matter adjacent to blood astrocytoma grade 4. JCV T antigen expression in
vessels the density of infected cells appears to be tumour cells suggests that the early region of JCV
increased. In addition, beneath the ependymal layer DNA was present in most tumours. Rescue of JCV
JCV-infected cells were detected, indicating a close from tumour tissue was successful occasionally.
topographical association between infected cells Regularly, the virus genome was integrated in tan-
and the ventricular system. These findings may be dem copies at single or multiple sites, comparable to
explained by an essentially faster progress of disease the pattern seen in hamster tumours.
or by differences in the immunological control of The majority of JCV-induced CNS tumour types
JCV infection in HIV/PML. in hamsters have their human counterpart in tu-
mours of infants, older children or young adults. In
view of the giant glial cells being key features in
PML, the tendency to form malignant astro-
Oncogenicity of the Human cytomas and giant cells appears to be more a char-
Polyomaviruses acteristic of JCV than that of the animal species
inoculated. Although the course of PML does not
The discussion of a possible oncogenicity of the allow extensive tumour growth, it can therefore be
human polyomaviruses in humans had its origin hypothesized that a semipermissive persistent JCV
not only in the oncogenic potential of TAg but also infection might provide the cellular background for
in the bizarre pleomorphism of astrocytes within transformation and tumour induction in the human
PML lesions. This was described as a hallmark of host.
PML in the initial description of the disease, and The oncogenic potential of BKV appears to be
later oligodendroglioma and multifocal glioblas- less pronounced, inducing tumours in rodents but
toma were reported, corresponding topographi- not in non-human primates. The most prominent
HUMAN POLYOMAVIRUSES 637
cell types affected by BKV infection in humans— specificity of viral expression might be one of the
epithelial cells, fibrocytes, ependymal cells, as- major selection criteria for polyomavirus-asso-
trocytes and endothelial cells—are reflected by dif- ciated transforming activity in animal systems. In
ferent tumour types induced in experimental animal view of this assumption, it is considered that human
models. The type depends essentially on the route of polyomaviruses might also be responsible for the
infection, on the amount of virus inoculated and on respective tumour types in their natural host.
the BKV isolate used. In addition, as cofactors the Consequently, the role of JCV and BKV in the
age and immunocompetence of the host influence aetiology of human tumours was evaluated by
the rate of malignancy. many laboratories using most virological and mol-
After intraperitoneal inoculation, tumours are ecular methods available. However, despite the on-
observed rarely; subcutaneous infection yielded sar- cogenicity of these viruses in animal systems, an
comas with an incidence between 2% and 12%. association with human tumours remains contro-
Intravenous inoculation results in ependymomas versial (Major et al., 1992; Dörries, 1997; Gallia et
that can be associated with peripheral tumours, al., 1998).
insulinomas, osteosarcomas and tumours of the in- T antigen expression was analysed immunohis-
testine. In contrast, intracerebral inoculation result- tologically in medulloblastomas, ependymomas,
ed in choroid plexus papillomas and papillary epen- choroid plexus tumours and urinary tract tumours.
dymomas, without a sign of peripheral tumours in The results were inconsistent. Antibody titres to
88% of the animals. The disseminated localization TAg in sera from tumour-bearing patients exhibited
of tumours demonstrates a marked tissue tropism no significant difference to sera of healthy people.
and multioncogenicity of BKV in hamsters. Genetic From these findings the conclusion can be drawn
analyses of virus stock preparation with high trans- that TAg is not expressed regularly in human tu-
forming capacities and predilection for specific tu- mours. Since TAg must not necessarily be expressed
mour types revealed genetic differences. The tu- at all stages of tumour growth, the presence of
mour-inducing activity could be mapped to the polyomavirus-related DNA sequences was ana-
BKV transcriptional promoter elements, suggesting lysed repeatedly in a large number of tumours. So
that the transforming capacity might depend on the far, JCV DNA sequences were detected only in an
TCR specificity. This also explains the discrepan- oligoastrocytoma, in which neoplastic oligoden-
cies of the transforming capacity among various droglial cells expressed TAg and in a pleomorphic
BKV stocks used in tumour induction experiments. xanthoastrocytoma in which JCV DNA was iso-
In the tumours, BKV DNA is found either inte- lated by PCR.
grated tandemly in the cellular genome or in a free Similarly, in a series of human tumours repre-
episomal state. In most tumours, complex integra- senting tissue from cancers observed with high fre-
tion patterns of multiclonal origin were observed. quency in animal models and major categories of
Most cells express nuclear T antigen, virus can often human cancer, even sensitive DNA hybridization
be rescued by cell fusion with permissive cells, and methods did not demonstrate the presence of BKV
tumour-derived cell lines grow permanently, main- DNA. Although BKV DNA was occasionally de-
taining their tumour-inducing potential. Selection scribed in CNS tumours, osteosarcomas, tumours
of a defined integration pattern during prolonged of the pancreatic islet, urinary tract carcinomas and
tumour cell culture and decrease in the amount of Kaposi’s sarcoma (De Mattei et al., 1995; Monini et
BKV DNA may point to rearrangements of inte- al., 1995, 1996), the detection rate in persistently
grated BKV DNA in the course of tumour growth. infected control tissue increased similarly after PCR
This could give rise to cells with simpler integration analysis. This clearly shows that BKV cannot be
patterns, and even to the loss of viral DNA. The one of the major causes of human cancer.
observation of tumours without intact early gene Analyses of the state of BKV DNA in malignant
DNA sequences suggests that an intact T antigen tissue revealed tumours carrying mostly episomal
gene is not necessarily needed for maintenance of virus DNA. Virus mRNA or T antigen expression
cell growth, once the transformed state is estab- could be detected; even virus was rescued. This was
lished. indicative rather of a persistent state of infection
The spectrum of BKV-induced tumour types is than an association with transforming activities. In
comparable in all susceptible animal species. Thus contrast, in brain tumours and urogenital tract tu-
638 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
mours, integrated BKV-hybridizing DNA se- samples at the outer rim of active virus growth
quences are described, thus pointing to a different provides the best material for virus detection. Diag-
virus—host interaction being involved. Since virus nosis is based on the identification of virus products
DNA is present regularly in the human individual, and typical cellular changes in glial cells. The extra-
it remains difficult to differentiate between a persist- ordinary multiplication rate of the virus in diseased
ent virus DNA and DNA associated with neoplasia. tissue allows detection of viral nucleic acids and
Whereas in virus persistence intact virus genomes proteins by classical methods. Additionally, mol-
are exclusively in the episomal state, in transformed ecular detection of JCV by PCR is usually con-
cells intact as well as defective DNA can either be firmed by histopathology.
integrated or in the episomal state. At present, per- It is accepted widely that polyomaviruses persist
sistent and transforming episomal DNA molecules asymptomatically in the CNS of adults with former
cannot be differentiated, therefore it is not known virus contact (Major et al., 1992; Dörries, 1997;
whether transformation is associated with both or Weber and Major, 1997). In the persistent state,
either state of the DNA. virus DNA is not detectable regularly by dignostic
However, integrated human polyomavirus DNA, PCR examination of single brain samples. In the
as yet only observed in immortalized cells or tu- activated asymptomatic state of infection the virus
mour tissue, might evidence the accidental associ- load might increase and can then be detected in
ation of the virus with neoplastic cell changes. In immunoimpaired individuals; however, compared
addition, it cannot be ruled out that poly- to the thousands of genome equivalents present in
omaviruses interact synergistically with other fac- PML tissue, the amount of virus is considerably
tors to induce malignant growth in human cells, in lower. Consequently, in cases with doubtful results
which case the presence of BKV DNA in tumour quantitative PCR analysis can differentiate a per-
tissue could be a consequence of an event occurring sistently activated and a PML-associated JCV in-
much earlier in the natural history of the tumour fection. At present, a combination of stereotactic
when cell growth might be enhanced by T antigen. biopsy and PCR techniques ensures a rapid diag-
The virus genome might be lost in an advanced nosis of PML with the highest sensitivity and speci-
phase, when genetic alterations may activate cellu- ficity available.
lar pathways responsible for cell proliferation. This
may not only explain low amounts of viral DNA
sequences but also a defective state of viral DNA
and argues for an involvement of polyomaviruses Demonstration of Polyomavirus DNA in
during early stages of human tumour development. CSF
TREATMENT OF HUMAN
POLYOMAVIRUS INFECTIONS
Polyomavirus Specific Antibodies in
CSF Treatment of human polyomavirus infections con-
centrates on PML. A number of different treatment
The CSF is usually unremarkable in PML, and JCV regimens has been proposed on the basis of molecu-
HAI titres were detected only rarely. Thus the hu- lar findings and small series of patients. However,
moral immune response has been regarded essen- randomized therapeutic trials are mostly lacking
tially as unhelpful for diagnosis in the past and and the observation that PML may remain stable
studies on JCV antibodies and their dynamic for long periods of time, or even remit, highlights
changes in the CSF of PML patients are limited to the inadequacies of anecdotal reports suggesting
single cases. However, recently synthesized in- the value of a specific therapy (Major et al., 1992;
trathecal JCV specific HAI antibodies and oligo- Berger and Concha, 1995; Weber and Major, 1997).
clonal bands were detected in CSF of 67% of con- Nucleoside analogues have proven to interfere
firmed PML cases. Additionally, changes of HAI with viral DNA synthesis in virus infections, and
titre in the CSF were observed under PML treat- several compounds have been used for the treat-
ment, suggesting that JCV specific antibodies might ment of PML. Cytosine arabinoside (ARA-C,
additionally be responsive to therapeutic interven- cytarabine) has been used from the early begin-
tion. Thus JCV HAI antibodies produced in- nings: various degrees of improvement have been
trathecally appear to suggest active JCV multiplica- reported. Recently acquired molecular data showed
tion within the CNS. The range of titres in indi- that cytosine-b--arabinofuranoside suppressed
vidual PML patients appears to be highly depend- JCV replication in tissue culture, thus supporting
ent on the state of the disease and may also reflect the potential interference of ARA-C with PML.
HUMAN POLYOMAVIRUSES 641
However, first reports on the efficacy of ARA-C unlikely. Other agents have been tried, either alone
therapy by the AIDS Clinical Trials Group in the or in combination. No effect has been found with
treatment of PML showed no benefit of ARA-C. corticosteroid therapy or tilorone, an immune en-
Other analogues, such as adenosine arabinoside hancer. The use of antisense oligonucleotides to
(ARA-A, vidarabine), iododeoxyuridine or prevent virus specific protein expression has been
zidovudine, similarly do not appear to have an proposed but awaits development of appropriate
effect on PML. drugs. Thus unequivocal effective therapy of PML
Recently, it was found that the newly approved as yet remains elusive.
cidofovir diphosphate, a structural analogue of With 5—20% of AIDS patients succumbing even-
deoxycytidine triphosphate, is not only a potent tually to AIDS-associated PML, the disease is the
inhibitor of human herpes and papillomaviruses, third most common process producing focal CNS
but is also a selective antipolyomavirus agent. A lesions in these patients. In contrast to other oppor-
first report on clinical improvement of a PML pa- tunistic CNS disorders, the outcome remains fatal
tient in association with cidofovir/ARA-C therapy in almost all cases. Thus further investigation and
points to a potential role of this drug in AIDS- collaborative efforts are needed to decide which of
related PML treatment. Camptothecin, a DNA the different approaches might be effective for the
topoisomerase inhibitor shown to block JCV repli- treatment of PML.
cation in vitro, led to clinical improvement in a
patient with systemic lupus erythematosus, but im-
munosuppressive therapy was suspended concomi- ACKNOWLEDGEMENTS
tantly. Antiretroviral therapy has been reported an-
ecdotally to result in improvement of PML; Work in the author’s laboratory was supported by
however, improvement of cellular immunity might SFB 165/B3 of the Deutsche Forschungsgesell-
be the major cause for prolonged survival times in schaft and by Grant 91.061 of the Wilhelm Sander-
these cases. Similarly, introduction of protease in- Stiftung.
hibitors and triple drug therapies for AIDS did not
influence the outcome of PML.
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
24
Human Parvoviruses
John R. Pattison
Royal Free and University College Medical School, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
646 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 24.1 The Parvoviridae resistant to heat as other parvoviruses but still sur-
vives 48°C for 30 minutes (Young et al., 1984).
Subfamily Genera Hosts
Densovirinae Densovirus
Iteravirus Insects lar weight of approximately 1.8 ; 10. In general,
Brevidensovirus autonomous parvoviruses preferentially encapsi-
date single-stranded DNA of negative polarity but
B19 virus encapsidates positive and negative
strands with equal frequency. The genome of B19
virus is one of the largest among the parvoviruses,
with 5500 nucleotides. All parvovirus genomes have
palindromic sequences at both the 5 and 3 termini.
These segments of the DNA fold back on them-
selves to form hairpin loops which are stabilized by
hydrogen bonding. The hairpin termini of B19 virus
are substantially longer than those of most par-
voviruses and the palindromic sequences appear to
be more than 365 nucleotides in length. There is
some mismatching in the middle of the hairpin lead-
ing to the occurrence of two different sequence con-
figurations referred to as flip and flop, which is
Figure 24.1 The appearance of B19 virus in the serum of a case typical of parvovirus genomes (Deiss et al., 1990).
of aplastic crisis in a child with sickle cell anaemia. (Reproduced There are several possible promoter sites in the
with permission from Parvoviruses: medical and biological as-
pects, Pattison, J.R. in Fields Virology, 2nd edition 1990, p 1767) B19 genome but only that at map point 6 appears to
be functional, with all transcripts being initiated at
this point. As is usual with parvoviruses the 5 end
of the genome codes for the non-structural protein
B19 VIRUS and the 3 end of the genome for the capsid proteins.
There are nine RNA transcripts in infected eryth-
Structure roid cells but only three associated with the known
viral proteins described below (Ozawa et al., 1987a;
In electron micrographs of negatively stained prep- Luo and Astell, 1993).
arations parvoviruses appear as naked, spherical
particles with a diameter varying from 20 to 25 nm,
with a mean of 23 nm. The individual virus particles Viral Proteins
are icosahedral in form and disrupted fragments
and empty shells are a characteristic feature. The There is one non-structural protein, NS1, produced
appearance of B19 virus conforms to this descrip- by transcription of the B19 virus genome. The non-
tion (Figure 24.1). structural proteins of parvoviruses have been
Parvovirus particles band at a density of 1.31— shown to have many functions (Young, 1996) and
1.43 g ml\ in CsCl gradients. The buoyant density the NS1 of B19 virus is notable for its toxicity to
of B19 virus is at the top end of the range at 1.43, cells (Ozawa et al., 1988). There are two structural
whereas the empty particles have a density of proteins in B19 virus—VP1 (84 kDa) and VP2
1.31 g ml\. As with all parvovirus particles the in- (58 kDa)—which differ only in that VP1 has an
fectious particle is stable over a wide range of pH additional 227 amino acids at the amino terminus
and resistant to lipid solvents. It is not quite so (Shade et al., 1986). The capsid of B19 is composed
HUMAN PARVOVIRUSES 647
of approximately 5—10% VP1 and the remainder is
VP2. The structure of canine parvovirus (CAV) has
been determined in detail (Tsao et al., 1991) and
structural studies have been undertaken for B19.
They indicate that the central structural core of
eight antiparallel -sheets is present in B19 but the
prominent spikes on the threefold axis of CPV are
absent from B19 (Agbandje et al., 1994).
Virus Variation
PATHOGENESIS
Volunteer Studies
For a number of years following its discovery B19
virus appeared to be associated with, at most, a B19 virus has on occasion been detected in throat
mild, non-specific, febrile illness accompanied by swabs, and respiratory tract spread was confirmed
self-limiting leucopenia. In the early 1980s the cen- when a group of volunteers was successfully infec-
tral role of B19 virus in the aetiology of aplastic ted with B19 virus following intranasal inoculation
crisis in chronic haemolytic anaemias (Sergeant et (Anderson et al., 1985a). The sequence of events
al., 1981) was identified and then, in 1983, erythema following intranasal inoculation of susceptible indi-
infectiosum (fifth disease) began to emerge as the viduals is shown diagrammatically in Figure 24.2.
common manifestation of B19 virus infection (An- The virus sets up a systemic infection with copious
derson and Pattison, 1985). Subsequently the virus viraemia detectable for about 6 days, 1 week after
has been shown to cause chronic infection in the inoculation. At the same time virus is shed from the
immunocompromised (Kurtzman et al., 1988, 1989) respiratory tract. As the titre of virus in blood falls,
and to be responsible for a significant percentage of IgM antibody begins to appear and B19—IgM im-
cases of hydrops fetalis (Anand et al., 1987). It is mune complexes may be detected. Parvovirus-spe-
648 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
penia, neutropenia and thrombocytopenia do occur
(not shown in Figure 24.2), though slightly later,
with the lowest numbers being recorded 6—10 days
after inoculation and not so consistently as the
changes in reticulocyte numbers and haemoglobin
concentration.
The symptoms of erythema infectiosum occur
late in the course of events in volunteers, with the
rash appearing 17—18 days after inoculation and
joint symptoms of arthralgia a day or so later. Virus
is not detectable at this time except by the most
sensitive PCR assays. Although the exact mechan-
Figure 24.3 The detection of B19 virus in the serum of a volun- ism of production of these symptoms remains to be
teer infected on day 0 elucidated, an immune-mediated pathogenesis
seems likely. Studies of infected fetal tissue (Morey
cific IgM is first detectable towards the end of the et al., 1992) have failed to reveal infection of vascu-
second week after inoculation, with levels usually lar endothelial cells, whereas circulating immune
rising rapidly to reach a maximum within a few complexes have been detected in volunteers and at
days; thereafter this antibody declines but may be present these seem the most likely cause of the rash
detected for a total of about 2—3 months. There is a and arthralgia.
delay of some days in the production of IgG anti-
B19, which cannot be detected until the third week
after inoculation, and amounts increase more slow-
ly than is seen with IgM. Reinfection has been Cell Tropism
documented in one volunteer: 7 days after in-
tranasal inoculation, a brief low-titre viraemia was Autonomous parvoviruses are so called because
detected. The IgG response was accelerated so that they do not require the presence of a helper virus for
IgG and IgM anti-B19 appeared simultaneously. replication. However, they do depend on certain
Recent studies using in vitro culture for the detec- helper functions expressed transiently in cells dur-
tion of infectious B19 virus indicate that infectious ing the late S or early G phase of mitosis. Therefore
virus may be detectable as early as 3 days after virus replication will be relatively extensive in
inoculation. The presence of infectious virus in the rapidly dividing tissues and it is not surprising that
stored serum of the volunteers was relatively short diseases of the intestine, the haematopoietic system
lived and the disappearance correlates with the rise and the fetus feature frequently as consequences of
in IgM anti-B19. By contrast (Figure 24.3) B19 parvovirus infection in animals. However, it should
DNA can be detected for many weeks using a nes- be rememberted that parvoviruses are not pan-
ted PCR (unpublished observations). The likely ex- tropic, since there is evidence that susceptibility to
planation of this is that immune-complexed virus parvovirus infection is related to a particular stage
persists in phagocytic cells, since the addition of of differentiation of a cell.
mixtures of infectious virus and specific antibody to Brown and colleagues (1993) identified the cell
cell cultures from fetal liver tissue leads to strong receptor for B19 as globoside or erythrocyte P anti-
DNA hybridization signals within the cytoplasm of gen. This antigen is found on mature erythrocytes
phagocytic macrophages (Dr A.L. Morey, personal and erythroid progenitor cells. Purified P antigen
communication). This intracellular complexed virus (globoside) blocks the binding of virus to erythroid
will not be significant in the transmission of the cells and these cells can be protected from infection
infection, nor is it likely to be of clinical significance by preincubation with monoclonal antibody to
in a normal individual. globoside. Interestingly P antigen can also be found
During viraemia reticulocyte numbers fall to un- on megakaryocytes, endothelial cells, placenta and
detectable levels, recovering 7—10 days later. There on fetal cardiac myocytes and these findings may
is a consequent temporary fall in haemoglobin of have some implications for the pathogenesis of B19-
about 1 g dl\ in a normal individual. Lympho- related diseases. Propagation in vitro has been
HUMAN PARVOVIRUSES 649
achieved in erythroid cells of human bone marrow are often centred on primary schools, where up to
(Ozawa et al., 1987b), fetal liver (Yaegashi et al., 40% of the school may be clinically affected by the
1989), in cultured cells from a patient with eryth- rash illness of erythema infectiosum. If one includes
roleukaemia (Takahashi et al., 1989) and in a hu- subclinical infection then the attack rates may be as
man megakaryocytoblastoid cell line (UT-7) de- high as 60% of the susceptible population in a
rived from a patient with leukaemia (Shimomura et school (Plummer et al., 1985). During these out-
al., 1992). An early erythrocyte precursor is suscep- breaks, susceptible adults (parents and teachers of
tible to B19 virus infection. When bone marrow or cases) frequently become infected.
peripheral blood cells are cultured to develop BFUe Clinical observations of the frequency of cases of
colonies (haemoglobin-containing erythrocyte pre- erythema infectiosum among different age groups
cursor cells) in the presence of B19 virus the forma- reflect the serological profile of the population; anti-
tion of colonies is inhibited. The same is true of the bodies are most commonly acquired between the
later erythroid progenitor cell which goes on to ages of 4 and 10 years, after which the frequency
form CFU-E in culture. In all culture systems virus continues to rise, but more slowly, so that among
propagation is dependent on the presence of eryth- the blood donor population (aged 18—65 years)
ropoietin: even the UT-7 cell line will only support some 60% are seropositive (Cohen et al., 1983).
virus replication after growth in the presence of Most of these studies have been done in developed
erythropoietin for months. countries. In developing countries the acquisition of
Two cells are characteristic of B19 virus infection antibody occurs at an earlier age and infection ap-
of the erythroid lineage. Giant pronormoblasts are pears to be common in the third, fourth and fifth
characteristically seen in the bone marrow of pa- years of life.
tients at the height of transient aplastic crisis. These In addition to seasonality, the virus exhibits lon-
cells, which have been recognized for almost 50 ger-term cycles. For example, in Jamaica, peaks of
years (Owen, 1948), probably represent the virus incidence (monitored as cases of aplastic crisis) oc-
cytopathic effect of the non-structural protein and cur every 3—4 years. In the UK, the cycle seems
they do not contain complete virus particles. Later somewhat longer, with peaks occurring every 4—5
erythroid precursors, the normoblasts, do exhibit a years.
characteristic intranuclear eosinophilic inclusion Case-to-case intervals, determined by the time
body which displaces the nuclear chromatin to the elapsing between acquisition and excretion of the
margin. These inclusions are packed with complete virus, are independent of the type of disease. Volun-
virions (Young, 1996) and stain positively for B19 teer studies predict case-to-case intervals of 6—11
DNA. Productive infection in these cells is the only days, which accords well with case-to-case intervals
known source of the intense viraemia that is charac- observed both in outbreaks of erythema infec-
teristic of B19 infections. Megakaryocytopoiesis is tiosum and in aplastic crisis. Transmission between
also inhibited by B19 parvovirus in vitro but this is siblings is common, and in families where more
in the absence of virus replication and almost cer- than one child is affected by chronic haemolytic
tainly due to the cytotoxic effect of NS1 (Srivastava anaemia all susceptibles should be monitored for 2
et al., 1990). weeks if one develops an aplastic crisis.
Although spread from respiratory tract to respir-
atory tract is the common route of transmission of
B19 virus, the high-titre viraemia which occurs dur-
EPIDEMIOLOGY ing infection can lead to another mode of trans-
mission. Virus is found in donated units of blood,
Serological studies show that infection with B19 although the incidence is low (1 in 20 000—40 000) in
virus is worldwide in distribution, occurring in all epidemic periods), as would be expected of an acute
populations studied with the exception of some iso- infection circulating mainly among children. In
lated groups in Brazil and Africa (Schwarz et al., spite of this low incidence, blood-borne trans-
1989; De Freitas et al., 1990). In temperate climates mission has been shown to occur in recipients of
infection occurs throughout the year but outbreaks whole blood and of factor VIII concentrates (Mor-
are more common in late winter, spring and the timer et al., 1983). The frequency of seropositivity
early summer months. These outbreaks of infection among haemophiliacs is significantly higher than in
650 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 24.2 Relationship between nature of clinical disease to a rash illness in B19 virus infections. However,
and host factors in B19 virus infection individuals are symptom-free for some 7 days be-
Disease Host tween the minor febrile episode and the appearance
of the rash, and it is likely that this relatively long
Asymptomaic period between the biphasic symptoms has pre-
Respiratory tract illness Normal vented the recognition of a link between non-speci-
Rash illness
Arthralgia fic prodromal symptoms and a rash illness. Certain-
ly most individual patients will not give a history of
Chronic haemolytic anaemia
Severe anaemia Immunosuppressed any prodrome before the onset of the rash, and the
Intrauterine infection accumulated literature over the past 100 years con-
tains a conspicuous absence of any description of
Adapted, with permission, from Pattison, J.R. in Medical Microbiology,
14th edition, edited by Greenwood, Slack and Peutherer (1992). prodromal symptoms of erythema infectiosum.
The link between B19 virus infection and ery-
normals, except for those haemophiliacs who have thema infectiosum was made in 1983 (Anderson et
received factor VIII concentrates that have been al., 1984), even though in retrospect there was evi-
heated to 80°C for 72 hours (Williams et al., 1990). dence of a link between B19 infection and rash
Even so, B19 virus can be transmitted by treated illness from the time of the earliest descriptions of
blood products (Lyon et al., 1989). the virus (Cossart et al., 1975). One of the original
viraemic individuals had a rubelliform rash 4 days
after the viraemia, and the children of another of the
CLINICAL FEATURES original individuals had itchy rashes 3—5 weeks after
their mother was viraemic. During the last 10 years
The consequences of B19 virus infection range from the use of specific laboratory tests for the diagnosis
the wholly asymptomatic to serious and potentially of B19 virus infection has revealed a spectrum of
fatal conditions in a minority of the population that rash illness, due to B19 infection, in which the clas-
is particularly predisposed. This spectrum of clini- sic features of erythema infectiosum occupy a cen-
cal consequences of infection is illustrated in Table tral position.
24.2 and depends in part on the natural variation in Erythema infectiosum is most common in
symptomatology that occurs with common child- children aged 4—11 years and is sometimes called
hood infection and in part on recognizable host fifth disease because it was the fifth of six
factors. erythematous rashes of childhood in an old classifi-
cation. The exanthem occurs in three stages. The
first begins some 18 days after the acquisition of
Minor Illness infection and is characterized by a fiery red rash on
the cheeks—slapped-cheek appearance (Figure
Combined clinical and laboratory studies of infec- 24.4, see Plate IV). The edges of the involved areas
tion in children, in whom B19 infection is most may be slightly raised and there is relative cir-
common, have indicated that about half of all infec- cumoral pallor. At this stage the appearance may be
tions are asymptomatic. Non-specific respiratory suggestive of scarlet fever, drug sensitivity or other
tract illness is the next most common consequence allergic reactions, or collagen vascular diseases. The
of infection, at least in boys (Grilli et al., 1989). This second stage of the exanthem occurs 1—4 days after
can be mild or severe enough to mimic influenza the facial involvement with the appearance of an
and these respiratory tract illnesses coincide with erythematous, maculopapular rash on the trunk
the viraemic phase of the illness. and limbs (Figure 24.5, see Plate IV). This rash is
initially discrete but spreads to involve large areas.
Towards the end of this stage there is central clear-
ing of the rash from these areas to give the charac-
Rash Illness teristic lacy or reticular pattern (Figure 24.6, see
Plate IV). The third stage of the exanthem is highly
The non-specific, short-lived febrile episodes de- variable in duration, lasting from 1—3 or more
scribed above also occur as prodromal symptoms weeks, and is characterized by marked changes in
HUMAN PARVOVIRUSES 651
the intensity of the rash, with periodic complete although there are no estimates of how frequent this
evanescence and recrudescence. These fluctuations is.
are related to environmental factors such as the In adults the most common presentation is with a
exposure to sunlight and temperature (a hot bath sudden onset of symmetrical arthritis affecting the
may result in recrudescence in an apparently re- small joints of the hand. Proximal interphalangeal
covered child). The rash is often pruritic, especially and metacarpophalangeal joints are most often af-
in adults, and is generally more prominent on the fected, followed by wrists, ankles, knees and elbows.
extensor surfaces; the palms and soles are rarely Shoulders, cervical spine and lumbar spine as well
affected. as the hips may also be involved. There may be pain
While classic cases of erythema infectiosum are and stiffness in the joints, which may accompanied
easy to recognize clinically, especially during out- by minor swelling or synovitis. In cases in which a
breaks, there is a wide variation in the form of the rash occurs the joint involvement will have com-
exanthem, from a very faint, fleeting rash to a florid menced 1—6 days after the onset of the rash. In
exanthem, confluent over large areas. In many cases children the joint involvement may be asymmetrical
the illness is clinically indistinguishable from ru- and symptoms seem more severe than in adults and
bella. may be of longer duration.
Erythema infectiosum is essentially a benign dis- Important aspects of the B19 virus-associated
ease in which the major complication is joint in- arthropathy have been defined by two large studies
volvement (see below). Other reported complica- (Reid et al., 1985; White et al., 1985). In a large
tions include cases of transient haemolytic anaemia, outbreak of erythema infectiosum 42 patients pres-
encephalitis with recovery without residua, and en- ented with joint pain; 88% of the patients were
cephalopathy in a 9-month-old boy resulting in female and 90% were adults. The arthritis was most
permanent sequelae. Each of these cases occurred frequently symmetrical and resolved within 4 weeks
prior to the appreciation of B19 virus as the causa- except in the occasional patient in whom it persisted
tive agent for erythema infectiosum; in view of the for 6—7 months. In a second study B19 virus diag-
difficulty in diagnosing sporadic cases of erythema nostic tests were applied to 153 patients presenting
infectiosum on clinical grounds, it cannot be certain to an early synovitis clinic over a period of approxi-
that these cases were due to B19 virus infection. mately 1 year. Nineteen of these patients had evi-
There have been occasional cases of B19 infection dence of recent B19 infection; all were adult females
associated with a purpuric rash. In most the platelet and there was a prodromal rash in only four. It is
count is normal (Lefrere et al., 1985b) but throm- likely that this study selected the more severe cases
bocytopenia has been recorded (Mortimer et al., of B19 arthropathy. Symptoms persisted for more
1985). Sometimes the clinical diagnosis of than 2 months in 17, and for more than 4 years in
Henoch—Schönlein purpura is made but it is un- three. More characteristically, about two-thirds of
clear what percentage of cases of this condition are patients become symptom-free within 2 weeks of
caused by B19. The purpura in cases of B19 infec- the onset and the majority were fully recovered in 4
tion is transient in these patients and there is no weeks.
evidence that B19 causes idiopathic thrombo- Occasional cases of B19 arthropathy could be
cytopenic purpura. classified as early benign rheumatoid arthritis.
However, these cases are rheumatoid factor-nega-
tive and other evidence indicates that B19 is not the
cause of rheumatoid arthritis. The frequency of B19
Joint Involvement antibody is no greater in patients with seropositive
rheumatoid arthritis and B19 seroconversion oc-
The most common complication of B19 virus infec- curs in patients who have had rheumatoid arthritis
tion is joint involvement. It is relatively rare among for years (Lefrere et al., 1985a).
children but in adult females it is commonplace,
occurring in about 80% of infections with a rash. It
is now also clear, with the widespread application of Aplastic Crisis
B19 diagnostic tests, that the joint involvement may
occur in the absence of any evidence of a rash, An aplastic crisis is a transient, acute event that
652 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
complicates chronic haemolytic anaemia. It is char- B19 Infection in Pregnancy
acterized by a fall from steady-state values of hae-
moglobin concentration, disappearance of re- Properties of B19 virus—production of a high-titre
ticulocytes from peripheral blood and the absence viraemia and a requirement for dividing host cells—
of red blood cell precursors in the bone marrow. suggest that infection of the placenta and fetus may
The cessation of erythropoiesis lasts 5—7 days and occur if infection occurs during pregnancy. More-
patients present with symptoms of worsening anae- over, animal parvoviruses cause fetal loss in ham-
mia, namely fatigue, shortness of breath, pallor, sters (Kilham, 1961) and pigs (Mengeling, 1975) and
lassitude, confusion and sometimes congestive car- there are specific diseases associated with par-
diac failure. The event is serious in most patients vovirus infection in late pregnancy in cats (Kilham
and occasionally it is fatal. Blood transfusion is and Margolis, 1966) and dogs (Jeffries and Blake-
required in the acute phase but after about 1 week more, 1979). Thus the occurrence and effects of B19
the bone marrow recovers rapidly. There is a re- virus infection in pregnancy have always been of
ticulocytosis and the haemoglobin concentration interest.
returns to steady-state values. The first aspect of the possible effects to be inves-
The aetiological link between B19 virus infection tigated was the relationship between B19 virus in-
and aplastic crisis was first noted in patients with fection and birth defects. No excess of defects could
sickle cell anaemia in 1981 (Serjeant et al., 1981). be detected following clinically diagnosed out-
Studies since then have shown that more than 90% breaks of erythema infectiosum (Ager et al., 1966)
of all cases of aplastic crises in patients with chronic and no B19 antigen or specific IgM could be detec-
haemolytic anaemia are due to B19 virus infection ted in sera taken during the first month of life from
(Anderson et al., 1982; Rao et al., 1983; Kelleher et infants with birth defects (Mortimer et al., 1985).
al., 1984). Most cases occur in children under the Finally, in a large prospective study of B19 infection
age of 15 but adults who remain susceptible to the in pregnancy (Public Health Laboratory Working
virus infection may have B19-associated aplastic Party on Fifth Disease, 1990) all pregnancies which
crises in later life. B19-associated aplastic crises are reached term delivered a normal, healthy baby and
not confined to patients with sickle cell anaemia but all those that were followed to 1 year showed no
have occurred in those with hereditary spherocyto- serious problems. Thus there is no evidence to date
sis (Kelleher et al., 1983), pyruvate kinase deficiency that B19 causes birth defects, although it should be
(Duncan et al., 1983), -thalassaemia intermedia remembered that the sample sizes in the studies
(Rao et al., 1983) and the dyshaemopoietic anaemia, have been too small to detect a rare defect, i.e. one
HEMPAS (West et al., 1986). with a rate of 1% or less. Nevertheless there is no
B19 virus infection is worldwide in its distribu- reason to recommend termination of pregnancies
tion, and aplastic crises associated with the infec- complicated by laboratory-proven B19 virus infec-
tion have been described in Europe, North America tion provided the pregnancy remains normal and
and the West Indies. The crises are an expression of there is no evidence of hydrops fetalis (see below).
the tropism of B19 virus for early erythroid precur- Small studies have shown that laboratory-con-
sors occurring in individuals whose haemoglobin is firmed B19 infection does not occur as often as
already low and whose red blood cells have a short expected in pregnant women. This suggests that
lifespan of 15—20 days. In such circumstances an B19 infection may lead to early fetal loss, even
arrest of erythropoiesis in the marrow leads to a before pregnancy is confirmed. There has been one
sharp fall in haemoglobin and symptoms of anae- large study (Public Health Laboratory Working
mia. However, virus infection does not invariably Party on Fifth Disease, 1990) of the effect of con-
result in an aplastic crisis in patients with chronic firmed B19 infection in women who are pregnancy
haemolytic anaemia and even when it does occur test-positive but this UK study could not incorpor-
the severity of the episode varies between individ- ate a control group. Analysis of the 190 cases en-
uals. This is likely to reflect variation in erythrocyte tered into this study shows that fetal loss in the first
lifespan between patients but studies so far have trimester of pregnancies complicated by B19 infec-
failed to reveal an erythrocyte marker which is a tion was no greater than would be expected from
good predictor of this. historical controls (6% versus 5—12%). However,
HUMAN PARVOVIRUSES 653
there was a pronounced excess (12.4% versus 0.6%) that seen in aplastic crises complicating haemolytic
of fetal loss in the second trimester of pregnancies anaemia. Persistent parvovirus infection with asso-
complicated by B19 infection compared with those ciated anaemia has been seen in patients with con-
in a separate but comparable study in the UK. It genital immunodeficiency (Nezelof’s syndrome)
thus appears that B19 infection is a significant cause (Kurtzman et al., 1989), children with lymphoblas-
of second trimester fetal loss. The pregnancy is lost tic leukaemia and other malignancies (Graeve et al.,
most frequently 4—6 weeks after the onset of the rash 1989; Kurtzman et al., 1989), patients with the ac-
illness in the mother. quired immune deficiency syndrome (Frickhofen et
Clearly the majority of pregnancies complicated al., 1990) and following renal transplantation (Nield
by B19 continue to full-term delivery of normal et al., 1986). Some of the patients have been given
infants. However, occasionally serious effects do human normal immunoglobulin which contains
occur as a consequence of second or third trimester B19 antibodies and this has led to a fall in virus titre,
infection, and fetal hydrops appears to be a consist- reticulocytosis and in some cases rash illness pre-
ent feature in these cases (Anand et al., 1987; Ander- sumed to be due to immune complexes.
son et al., 1988). Maternal infection occurs 2—12
weeks prior to the diagnosis of hydrops fetalis. B19
is a likely cause if there is a low haemoglobin in the
LABORATORY DIAGNOSIS
fetus in the absence of haemolytic disease. In a
retrospective review of 50 cases of hydrops fetalis in
Aplastic crisis is the only one of the clinical syn-
a single department of pathology in the UK be-
dromes which can be assumed, with some accuracy,
tween 1974 and 1983 there was evidence of B19
to be due to B19 infection. Even so, in patients with
infection in four (Porter et al., 1988). Thus it is worth
chronic haemolytic anaemia moderate to severe de-
investigating B19 as a cause of non-immunological
grees of hypoplasia may be associated with systemic
hydrops fetalis by specific tests for virus infection on
bacterial infection or marrow-suppressive drugs,
fetal material, although raised maternal -feto-
and anaemia in the immunocompromised may
protein levels are a marker of intrauterine B19 infec-
have many other causes. Illnesses associated with
tion (Carrington et al., 1987).
maculopapular rashes with or without joint in-
Intrauterine transfusion has been used to manage
volvement also have a multiplicity of causes and
a number of cases of hydrops fetalis during preg-
only a minority of cases of hydrops fetalis will prove
nancy. Brown and colleagues (1994) describe three
to be due to B19 infection. Thus accurate diagnosis
such cases who at birth were found to have hy-
of B19 infection depends upon specific laboratory
pogammaglobulinaemia, and viral DNA was pres-
tests.
ent in the bone marrow, although absent from the
serum. One child died but the other two remained
persistently anaemic in spite of immunoglobulin
therapy. The authors conclude that persistent B19 Specimens
infection should be suspected in infants with con-
genital red-cell aplasia. Serum is the principal specimen used for the labora-
tory diagnosis of B19 infection (Pattison, 1995).
This approach is suitable for virus detection in cases
of aplastic crisis, persistent infection in immunosup-
B19 Infection in the Immunosuppressed pressed patients and persistent fetal infection. The
detection of specific IgM antibody in serum is the
A chronic B19 virus infection with associated anae- cornerstone of the diagnosis of rash illness and ar-
mia occurs in immunocompromised individuals thropathy, and is also as valuable as virus detection
who have failed to produce neutralizing antibody to in cases of aplastic crisis. Standard blood specimens
the virus. The anaemia is often severe and may be are all that are required and no special arrange-
persistent or intermittent, with the viraemia disap- ments are needed for transport to or storage in the
pearing during remission and reappearing when a laboratory. It should be remembered that viraemic
patient relapses. The patients often require blood samples contain much infectious virus. Care should
transfusions. The bone marrow picture is typical of be taken when handling samples, especially those
654 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
taken early in the course of an aplastic crisis, to B19 infection from cases of rash, rash and arthralgia
ensure that seronegative individuals in the labora- or arthralgia alone. The less sensitive techniques for
tory are not infected (probable cases of laboratory- virus detection would be negative in these cases
acquired infection have been described; Cohen et because this occurs later in the course of infection
al., 1988). The most hazardous manipulations ap- when virus is present in the blood in only very low
pear to be some of the washing steps necessary in concentrations. However, in such cases the nested
the serological tests. PCR was positive in 70% of samples. Nevertheless
Detection of B19 virus by in situ hybridization it did not diagnose any additional cases compared
can be performed on formalin-fixed, paraffin-em- with a combination of dot-blot hybridization and
bedded tissue, so that standard pathology protocols anti-B19 IgM tests.
can be used for dealing with fetal tissue. The diagnosis of B19 virus infection in a fetus also
depends upon the detection of virus. Maternal in-
fection will have occurred some weeks previously
and maternal serum may therefore by B19-specific
Virus Detection IgM negative. In most instances the fetus has also
been found to be specific IgM negative but there is
With B19 virus the propagation in vitro in erythroid frequently a persistent viraemia. Therefore the diag-
cells of human bone marrow and fetal liver remains nosis is best made by detection of virus in fetal
a research technique. Otherwise many of the stan- blood samples by the techniques described above.
dard techniques for the detection of viruses in clini- Equally virus can be detected in fetal tissues taken
cal practice have been applied to the diagnosis of at autopsy from which DNA has been extracted or
B19 virus infection. CIE, electron microscopy and detected by in situ hybridization on formalin-fixed,
immune electron microscopy are valuable because paraffin-embedded tissue sections. Biotin (Figure
they give a relatively rapid result. In addition vis- 24.7) and digoxigenin (Morey et al., 1992) have been
ualization of the virus by electron microscopy can evaluated for this purpose. The latter seems more
be a useful confirmatory test. The common clinical reliable when studying archival fetal tissues since
situation in which these tests are applied is to the false positives may be obtained with biotinylated
acute-phase specimen of cases of aplastic crisis. probes. If tissue samples such as placenta are ex-
CIE, which is capable of detecting approximately tracted and tested for B19 DNA, confirmation by
10—10 particles ml\, will reveal virus in approxi- Southern blotting with and without endonuclease
mately 30% of cases in which a specimen is taken resrtriction is mandatory, since weak, false-positive
within 24 hours of the onset of symptoms. More dot-blot signals do occur with such samples.
sensitive methods for the detection of virus are
radioimmunoassay or DNA hybridization using
cloned viral genome labelled with P (Cohen et al.,
1983; Anderson et al., 1985b). Application of these Specific Antibody Detection
techniques, which detect about 10 particles ml\,
to specimens taken within 24 hours of the onset of Standard solid-phase radiolabelled or enzyme-
an aplastic crisis will yield positive results in 60% of labelled immunoassays are the preferred methods
cases. Immune electron microscopy is only slightly for the serological diagnosis of B19 infection. If
less sensitive than this. these techniques are not available then CIE and
The most sensitive technique for the detection of immune electron microscopy may be used as rela-
virus requires amplification of the DNA using the tively insensitive tests of seroconversion.
PCR. These techniques, such as the nested PCR Current tests for class-specific B19 antibody (Co-
described by Patou and Ayliffe (1991), are capable hen et al., 1983) employ the antibody capture prin-
of detecting 1—10 virus particles. This technique has ciple in which class-specific antibody in the patient’s
not been applied to a series of cases of aplastic crisis serum is bound by a solid phase coated with anti-
but it is likely that almost all the sera would be body to human or chains. The viral specificity of
positive if taken within a week of the onset of the the bound antibody is determined by the addition
infection. The technique has, however, been applied of B19 virus antigen to the solid phase. In turn
to a series of specimens submitted for diagnosis of bound antigen is detected by the addition of mono-
HUMAN PARVOVIRUSES 655
Figure 24.7 Spleen from a fetus with hydrops fetalis stained with haematoxylin and eosin (a) and probed with a biotinylated B19
probe (b). Stain (a) reveals intranuclear inclusion bodies with margination of the nuclear chromatin and (b) shows that these cells
contain B19 virus DNA. (Adapted with permission from Parvoviruses: medical and biological aspects, Pattison, J.R. in Fields Virology,
2nd edition 1990, p 1771)
clonal anti-B19 antibody, which may itself be was taken on the day of onset of the aplastic crisis.
tagged with enzyme or radiolabel. Alternatively an An even higher percentage of cases of rash illness
extra step may be incorporated whereby the bound due to B19 will have such concentrations of specific
monoclonal anti-B19 is detected by the addition of IgM in sera taken when the patient first presents.
labelled antimouse immunoglobulin. Diagnosis us- However, in occasional cases (particularly of aplas-
ing these tests is dependent upon either the detec- tic crisis) specific IgM may not appear until 7—10
tion of B19-specific IgM or a significant change in days after the onset. Therefore if a negative result
the amount of B19-specific IgG present in paired for anti-B19 IgM is obtained with a serum taken
sera. As yet there are no national or international within 10 days of the onset of the illness, this serum
standard sera for B19 antibody tests so that labora- should be tested for viral DNA and/or a second
tories offering a diagnostic service usually report specimen to be taken about 14 days after the onset
their results in arbitrary units (au) by comparison of symptoms should be requested. Specific IgM is
with a local control serum. The quantification is detectable for 2—3 months after acute infection. In-
based on designating a highly reactive serum as terpretation of equivocal low concentrations of
containing 100 au and including a set of standards anti-B19 IgM in sera taken after this time is difficult
in each test. and in this circumstance testing for IgG may be
Sera containing 10 au or more of anti-B19 IgM helpful.
are unequivocally associated with recent infection. In the 12 months following infection patients
This relatively high threshold is set because sera have relatively high ( 50 au) concentrations of
containing high concentrations of antirubella virus anti-B19 IgG and the finding of such concentra-
IgM may give low false-positive results when tested tions supports the diagnosis of infection during that
for anti-B19 IgM, and vice versa (Kurtz and Ander- time. However, there is marked individual variation
son, 1985). This must be borne in mind when testing in the maximum amounts of specific IgG that can
sera taken within 2 weeks of a rubelliform illness. be demonstrated in a patient, so that the finding of
However, high concentrations of anti-B19 IgM only low values ( 20 au) does not exclude recent
usually appear within 3—4 days of the onset of infection. The assay used for the detection of anti-
symptoms. In a recent series of aplastic crises from B19 IgG is a capture assay and a positive signal
Jamaica (unpublished data), 100 au IgM were therefore depends on a minimum proportion of the
present in over 50% of patients in whom the serum total IgG being B19 specific. Sera with low concen-
656 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
trations may not bind sufficient antigen to be detec- haemolytic anaemia (or immunocompromised
ted by the indicator antibody and therefore the children) could be temporarily protected by the
anti-B19 IgG capture assay is not a sensitive test of administration of human immunoglobulin but to
past exposure to the virus. Previous infection (and date this has not been put into practice.
therefore immunity in most individuals) is indicated The prevention of parvovirus infections in ani-
by the detection of 1 au of anti-B19 IgG, pro- mals by vaccination is standard practice in veterin-
vided that the test serum:negative serum ratio is ary medicine. In the case of B19 virus it is unlikely
2. However, a negative result cannot be taken to that sufficiently large quantities of native virus will
be synonymous with susceptibility. ever be produced in cell culture systems. However,
There is a poor humoral immune response in self-assembling B19 virus capsids have been pro-
immunocompromised patients with chronic par- duced by recombinant DNA technology using a
vovirus infection and only low concentrations of baculovirus system (Kajagiya et al., 1991; Bansal et
anti-B19 IgG (and IgM) can be detected in these al., 1992). These genetically engineered capsids are
patients. None of the detectable antibody neutral- antigenically and immunogenically similar to na-
izes virus infectivity and this is taken to be an essen- tive virions and induce neutralizing antibodies. The
tial component of the pathogenesis of the chronic presence of VP1 in the capsid immunogen appears
infection. critical for the production of neutralizing antibo-
dies, and capsids with more than the normal VP1
content are more efficient in producing such anti-
body (Dr N.S. Young, personal communication).
TREATMENT AND PREVENTION Thus it seems possible that a B19 virus vaccine will
be available in the near future and decisions on
There is no specific antiviral chemotherapy for B19 whether or how to use it will need to be made.
infection. Symptomatic relief of troublesome joint
symptoms may be required for B19 virus-associated
arthralgia, and blood transfusion may be necessary
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
25
Angus G. Dalgleish
St George’s Hospital Medical School, London, UK
and
Clive Loveday
Royal Free and University College Medical School, London, UK
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
660 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
RNA
DNA
RNA
AAA
Figure 25.1 Simplified replication cycle of a retrovirus. The virion containing two RNA genome copies enters the virus via a specific
cell surface receptor. The single-stranded RNA genome is converted into a double-stranded NA provirus by the virion enzyme reverse
transcriptase. The provirus inserts into host chromosomal DNA in the same orientation as the original virion RNA. Transcription of
RNA from the integrated DNA provirus is mediated by cellular RNA polymerases, and this RNA serves both as messenger RNA for the
synthesis of viral antigens and as genomic RNA which becomes packaged into progeny virion budding from the cell surface
and the ability to acquire host genes which encode terminal repeats at both ends and the RNA form by
functions responsible for neoplastic transformation a polyadenylate tract at the 3 end. The long ter-
(oncogenes). There also exist endogenous minal repeats are responsible for integration of the
proviruses in the normal cellular DNA of many DNA provirus with cellular DNA, and contain im-
vertebrates, which represent ‘fossil’ infections of the portant promoter and enhancer sequences which
germline and which are passed from generation to bind cellular and viral proteins regulating viral gene
generation in a mendelian manner. expression.
All retroviruses carry at least three genes in the
order 5-gag-pol-env-3. Gag encodes a precursor
protein which is cleaved to yield three or four struc-
tural core and matrix proteins; pol encodes the re- CLASSIFICATION OF RETROVIRUSES
verse transcriptase, protease and integrase, syn-
thesized from a gag-pol precursor; and env encodes Retroviruses were taxonomically divided into three
a precursor cleaved to form the two disulphide- subfamilies: the Oncovirinae, which include those
linked envelope proteins. The core proteins are of- with oncogenic potential; the Lentivirinae, includ-
ten named by molecular weight (e.g. p24 and p17 of ing HIV, and the prototype Visna virus of sheep
HIV). The outer surface protein is glycosylated and which causes slow, progressive degeneration of the
is known as gp120 (e.g. for HIV with an approxi- central nervous system; and the Spumavirinae, or
mate molecular weight of 120 000) or gp70 (e.g. foamy viruses, which have not been shown to be
mammalian C-type oncoviruses). The anchored pathogenic. More recently, retroviruses of higher
transmembrane protein, to which the surface pro- vertebrates have been classified into seven distinct
tein is bound, is glycosylated in some retroviruses groups by dividing oncoviruses into five, as they are
(e.g. gp41 of HIV) but not others (e.g. p15E of only distantly related by genome sequence and
C-type oncoviruses). The genome is capped by long morphology (Figure 25.2 and Table 25.2). More
HUMAN IMMUNODEFICIENCY VIRUSES 661
Lentiviruses
inally detected in cultured nasopharyngeal car-
Spumaviruses SIV HIV-1
VMV
cinoma of a Kenyan patient (Achong et al., 1971).
HIV-2 Because the HFV genome is indistinguishable
SFV-3 EIAV Avian C-type from the of the Simian foamy virus type 6 (SFV-6)
HFV oncoviruses
RSV of chimpanzees, it may represent a single case of
zoonosis (Rosenblum and McClure, 1999).
MSRV HRV-5 Serological studies indicate that human popula-
HERV-K
GALV
SRV-2 tions are not infected by spumaviruses (Ali et al.,
PERV MPMV
MLV MuIAP
MMTV
1996), although they are endemic in many pri-
FeLV
BLV HTLV-II B and D-type mate species. Zoonotic SFV infection without
MLV-related HTLV-I
oncoviruses symptoms has been recorded in primate han-
C-type
oncoviruses HTLV-related dlers who have suffered bites or deep puncture
oncoviruses wounds (Heineine et al., 1998).
4. Human retrovirus 5 (HRV-5) is a retroviral
Figure 25.2 Phylogenetic tree of retroviruses genome occurring at very low viral load in nor-
mal subjects but particularly in patients with
generally, retroviruses are divided into those with arthritis and systemic lupus erythematosus
‘simple’ genomes, having gag, pol and env genes and (Griffiths et al., 1999). The virus has not yet been
perhaps one other, and those with ‘complex’ propagated in vitro; its genome is related to the
genomes, also possessing regulatory genes such as B-type and D-type retroviruses (Figure 25.2).
tat and rev of HIV and tax of HTLV and accessory 5. Human endogenous retroviruses (HERV) are
genes, such as nef, vif and vpr of HIV. mendelian loci in human chromosomes repre-
Five groups of retrovirus have been reported as senting ‘fossil’ infections of the germline. These
human infections but only the first will be described endogenous genomes derive from mammalian
in detail in this chapter: C-type and D-type (HERV-K) retroviruses. No
lentiviruses or spumaviruses are known to have
1. Human immunodeficiency virus types 1 and 2
become endogenous. HERV genomes are defec-
(HIV-1 and HIV-2) are the lentiviruses that
tive, that is, human endogenous retroviral
cause acquired immune deficiency syndrome
genomes have not been rescued in infectious
(AIDS). Before the term HIV was coined in 1986,
form, in contrast to BaEV of baboons and PERV
HIV was called lymphadenopathy virus (LAV)
of pigs, which threaten the safety of human
or HTLV-III. HIV and AIDS are the main topic
xenotransplatation from these sources (Weiss,
of this chapter.
1998). Some HERV genomes, however, express
2. Human T-lymphotropic virus types 1 and 2
envelope and other proteins, for example ERV-3
(HTLV-1 and HTLV-2), cause adult T-cell leu-
in the human placenta (Venables et al., 1995;
kaemia and neurological disease. They are re-
Löwer et al., 1996), HERV-K in type 1 diabetes
viewed in Chapter 25A.
(Conrad et al., 1997) and a HERV-W genome,
3. Human foamy virus (HFV) is a spumavirus orig-
MSRV, in multiple sclerosis (Perron et al., 1997).
Table 25.2 Classification of retroviruses of higher vertebrates
? B-type, D-type and spumaviruses have condensed cores visible in the cytoplasm of infected cells, whereas in the other retroviruses the cores condense as
crescent-shaped bodies during maturation and budding at the cell membrane.
662 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
EPIDEMIOLOGY
CLASSIFICATION OF HIV
As mentioned, in 1981 a handful of cases of Pneu-
HIV-1 was first isolated in 1983 (Barré-Sinoussi et mocystis carinii pneumonia and of Kaposi’s sar-
al., 1983), just 2 years after the identification of coma in young homosexual men in the USA heral-
AIDS. Further, independent HIV-1 isolates were ded the beginning of the AIDs era. Before HIV-1
reported in 1984 (Gallo et al., 1984; Levy et al., was first isolated (Barré-Sinoussi et al., 1983) it was
HUMAN IMMUNODEFICIENCY VIRUSES 663
Table 25.3 Global estimates of HIV infection in adults and
children (end of 1998)
Millions
Figure 25.4 UNAIDS world map showing numbers of adults and children (total 33.4 million) estimated to be living with HIV/AIDS
at the end of 1998
action (PCR)-amplified and cloned from a Zairian glutinin and interleukin 2 (IL-2). The SI strains
blood sample stored since 1959 (Zhu et al., 1998) generally adapt to growth in immortal CD4 ; T
indicates that the various subtypes have evolved cell lines, which become chronic virus producers.
since that date. Although it was thought that differ- NSI strains, however, need to be propagated in
ent subtypes might be more favourably transmitted PBMCs, where they are cytopathic, and in non-
by homosexual, heterosexual and parental routes, cytopathic macrophage culture. Isolation by cul-
by virtue of depending on Langerhans cell infection ture is used to assay infectivity and phenotypic
in the vaginal mucosa versus peripheral blood characters, including drug resistance. Figure 25.5
mononuclear cell (PBMC) infection in the rectum shows HIV particles produced by the CEM cell line
or intravenously, the data have not confirmed this in culture.
notion (Dittmar et al., 1997). Isolation of HIV in cell culture can be detected by
HIV-2 has had a lower transmission rate than several means: (1) a cytopathic effect, including syn-
HIV-1. In West Africa, where it remains endemic, cytia for SI strains; (2) detection of viral antigens in
HIV-1 infection is now surpassing HIV-2 in inci- infected cells by antibodies, e.g. immunofluores-
dent infections. HIV-2 has spread elsewhere, es- cence, enzyme-linked immunocytology of infected
pecially through Portusuese contacts, and it ap- cells, ELISA for p24 antigen in cells or in supernatnt
pears to be epidemic in India, although again less medium from infected cultures; (3) reverse tran-
prevalent than HIV-1. Mother-to-child trans- scriptase assay, either by enzyme activity or by
mission of HIV-2 is rare, perhaps owing to a gen- ELISA; and (4) genome detection, using PCR or
erally lower viral load. Southern blots. Virus isolation is useful for deter-
mining the phenotype of HIV strains, whether they
are SI or NSI, and whether they have developed
drug resistance.
VIROLOGY
Figure 25.5 HIV-1 particles produced by CEM cells. Note the crescent-shaped core in budding particles and the condensed cone-like
core in mature particles of approximately 100 nm diameter. (The black spots are indirect immunogold labelling of antibody from an
AIDS patient absorbed to the virus-producing cells)
and HIV-2. The gene maps are similar except tht enzymes protease, reverse transciptase and integ-
HIV-2 lacks vpu but carries vpx. rase. The env gene encodes the gp41 and gp120
The core and matrix proteins are encoded by gag. envelope glycoproteins, cleaved by cellular enzymes
The gag proteins of the mature virus are p17, p24, from the gp160 precursor.
p7 and p6, and are processed by cleavage of the p55 In addition to gag, pol and env, HIV-1 and HIV-2
precursor protein by the viral protease. The matrix carry seven regulatory and accessory genes (Emer-
antigen p17 is localized to the inner layer of the viral man and Malim, 1998). The tat gene encodes a
envelope and requires myristilation, which allows it protein that binds to TAR RNA sequences in the 5
to be tightly bonded to the inner envelope; matrix long terminal repeat to upregulate viral RNA tran-
antigen is indispensable for budding. The core shell scription through complexes of cellular transcrip-
is made of p24 capsid antigen. This is the viral tion factors and cyclin T. Rev serves to aid export of
protein most usually detected clinically as a long HIV transcripts from nucleus to cytoplasm
measure of antigenaemia. The pol gene encodes the through recognition of Rev-response elements
HIV-1 tat
vif
rev
5¢LTR gag vpu 3¢LTR
vpr
pol nef
HIV-2 tat
vif
vpr rev nef
5¢LTR gag
vpx 3¢LTR
pol env
Figure 25.6 HIV-1 and HIV-2 genome maps
666 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
(RREs) in unspliced or singly-spliced viral mRNA. Group N has only recently been discovered (Gao et
Nef has multiple functions in signal transduction al., 1999) and antibodies to it have not been rigor-
and downmodulation of CD4 cell surface expres- ously tested in commercial kits because so few hu-
sion. Vpr allows transport of newly infected prein- mans, confined so far to West Central Africa, are
tegration complexes of the HIV proviral DNA into group N carriers.
the nucleus for integration (by integrase) into host Neutralizing antibodies can be measured by re-
chromosomal DNA. Vpr also arrests cells in the G duction of infectious titre of HIV in cell culture
phase of the mitotic cycle, enhancing virus produc- assays. Such assays are not useful for screening or
tion. Vpu and vif have poorly understood functions. diagnosis but may be important in testing HIV
Vif may be incorporated into virus particles and candidate vaccines. Immunofluorescence assays are
helps infection in new target cells. Although SI no longer used for diagnostic purposes.
strains of HIV-1 can be propagated in T cell lines Differential assays for antibodies in sera to speci-
with deletions or non-functional mutations in nef, fic HIV antigens are generally performed by West-
vpu or vif, such mutations adversely affect HIV ern blot on antigens prepared from whole virions,
replication in PBMCs and macrophages so that infected cultures or recombinant viral proteins. In
attenuated infection or no replication occurs. Thus the early days of HIV diagnosis, Western blots were
all the viral genes are required for efficient infection considered important as confirmatory to ELISA
and pathogenesis in vivo. tests. They still have a place for sera yielding in-
determinant results, especially at the beginning of
seroconversion during primary infection. However,
genome detection is now preferred for such cases.
HIV Serology
b2-microglobin
CD4 lymphocytes
Total anti-HIV-1
P24 antigen
Anti-P24
0 12 24 36 48 60 72 84 96 108 120
Time (months)
Figure 25.7 The clinical, virological and immunological phases of HIV/AIDS. The clinical phases of the disease have been staged by
the Centers for Disease Control (1985, 1993) to define primary HIV infection (PHI, CDC stage 1), asymptomatic (CDC stages 2/3) and
symptomatic (CDC stage 4). Viruses are easy to isolate during PHI and are generally a relatively homogenous population of
macrophagotropic viruses. During the asymptomatic phase of disease viruses evolve into a heterogenous population but are difficult to
isolate; this becomes progressively easier as a population of lymphotropic viruses predominate in the host in symptomatic disease. CD4
lymphocyte counts (*) show distinctive changes during disease, with a rapid, short-term decline associated with the viraemia in PHI, a
recovery to near normal levels, a gradual loss of cells during the asymptomatic phase, and finally an accelerated decline associated with
late symptoamtic disease. The reciprocal of these changes is shown by -microglobulin (), a plasma measure of immune activation.
Plasma viral load () reaches a peak in PHI (mirrored by P24 antigen, ), declines to a ‘set point’ during early asymptomatic disease,
and then gradually increases after about 24 months of infection at 0.1 to 0.2 log copies ml\ per year. In symptomatic disease viral
load and P24 antigen increase progressively. Both viral load and CD4 cell counts in early asymptomatic disease are highly predictive of
disease progression. Anti-HIV-1 antibodies (s) increase to a maximum within 3—6 months of infection and remain detectable
throughout the natural course of disease. However, subpopulations of antibodies (i.e. anti-P24 antibodies, ) may decline in
association with, and predict, disease progression. With the introduction of potent antiretroviral therapy and persisently long-term
undetectable plasma virus ( 50 copies ml\), the antigenic stimulus for the immune system is deminished and quantitative declines in
plasma anti-HIV-1 antibodies are observed. This model of the natural history of the disease has been shown in practice to exhibit, in a
minority of patients, an accelerated course where viral load remains relatively high or rises, P24 antigen remains detectable. CD4 cell
counts decline rapidly and patients progress clinically in 5 years (rapid progressors), or a protracted course where the CD4 cell
counts remain in the normal range and viral load low or undetectable with asymptomatic disease for 15 years (long-term
non-progressors). These differences probably represent the limits of a normal distribution
HUMAN IMMUNODEFICIENCY VIRUSES 669
the host with one of the first observations that the immune system activated even in the presence of
patients with HLA-1 B8 DR3 were exceptionally highly active antiretroviral therapy.
fast progressors to disease. It is also noted that the
same haplotype has a higher activation resting level
than many other HLA types. In HIV-1 infected
Caucasians, patients with HLA B27 are slow or Cell Tropism and HIV Receptors
non-progressors to disease.
Chronic activation and progression to disease HIV infects mainly CD4 ; cells by binding to CD4
dependent on the HLA background is seen with as a receptor (Dalgleish et al., 1984). However,
viruses that encode superantigens. Initial reports many of these cells are not lymphocytes. For
that HIV did so have not been confirmed and, example, monocytes, macrophages, dendritic cells
although superantigen activity with regards to B (Langerhans cells) and some brain cells, such as the
cell epitopes have been reported, it is not thought to microglia, express the CD4 receptor, and many are
be sufficient to drive the panactivation seen infectible with HIV. It is likely that infection of these
throoughout the immune system in HIV-infected cell types plays a major role in the pathogenesis of
patients progressing to AIDS (Dalgleish et al., disease. Importantly, HIV can infect some macro-
1999). This panactivation is also similar to that seen phages and dendritic cells without killing them, and
in chronic HLA mismatched transplantation which hence they may form an important reservoir for
leads to chronic graft-versus-host disease. This re- persistent infection and be able to infect ‘passing’
mains a possibility, as HIV incorporates many CD4 lymphocytes. Some isolates preferentially in-
HLA molecules into its envelope in co-association fect monocytes compared to T cell lines, and vice
gp120. This property has been shown to enhance versa. The molecular changes that determine this
infectivity in vitro and could be one mechanism of remarkable difference in tropism are small and may
activating the immune system for the purpose of affect the envelope, including the V3 loop, as well as
establishing infection (Westby et al., 1996). nuclear factors if there are changes in the long ter-
It is thought that this co-association of HLA and minal repeat.
gp120 molecules may account for the early reports The CD4 cell surface antigen is necessary but not
of complete protection of macaques from simian sufficient for HIV-1 infection (Dalgleish et al., 1984;
immunodeficiency virus (SIV) infection after they Maddon et al., 1986). While CD4 is required for
were immunized with paraformaldehyde-treated high affinity binding of gp120, coreceptors are re-
human cells infected with SIV. The monkeys were quired for subsequent steps leading to fusion be-
only protected against challenge by SIV propagated tween the viral envelope and cell membrane. The
in human cells that clearly incorporated HLA mol- coreceptors were identified in 1996 to be chemokine
ecules, as the correlate of protection was anti-HLA- receptors and help to explain the differential
1 antibodies. Unfortunately, this seems to have been tropism for lymphocytes and macrophages. Pri-
a specific xenogeneic protection and there is no mary, NSI strains of HIV mainly utilize CCR5,
clear evidence as to whether patients who have which is the receptor for the chemokines MIP-1,
natural anti-HLA antibodies are protected from MIP-1 and RANTES, whereas SI strains utilize
HIV, although heavily exposed, seronegative pa- CXCR4, the receptor for stromal derived factor 1
tients tend to have more of these antibodies than a (SDF-1). The discovery that chemokine receptors
similar non-exposed population. act as coreceptors to CD4 (Feng et al., 1996; Weiss
Incorporation of HLA molecules into the mem- and Clapham, 1996) helped to explain why certain
brane should only provide an allogeneic stimulus, -chemokines can inhibit HIV-1 infection in vitro
which may be advantageous in order for a low virus (Cocchi et al., 1995). Modified chemokines and
load to infect as many cells at the site of entry as small molecular receptor blockers can potently in-
possible. This would not explain the ongoing acti- hibit HIV-1 entry and may have promise in therapy
vation, which appears to determine disease pro- (Schols et al., 1997; Simmons et al., 1997; Donzella
gression. As HLA sequences of a length sufficient to et al., 1998).
mimic an allo peptide are present in the envelope The importance of the CCR5 coreceptor for NSI
and nef proteins of HIV, it is possible that the viruses in HIV transmission and disease pro-
presentation of these epitopes is sufficient to keep gression is borne out in people with mutations in
670 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
the gene for this receptor. In Caucasian populations additional respiratory diseases and recently dis-
a deletion in CCR5, rendering it non-functional, covered malignancies, and Table 25.6 outlines the
occurs frequently; people homozygous for this classification most widely used to define entry into
‘delta 32’ mutation are resistant to NSI HIV-1 infec- contemporary clinical trials. The classification of
tion, even when exposed, for example, by regular paediatric HIV infection evolved independently to
sexual contact with an HIV carrier (O’Brien, 1997). reflect the unique features of the disease in this
A mutation in the promoter region of CCR5, which patient group (Table 25.7).
lowers the level of CCR5 expression, delays pro-
gression to AIDS in infected individuals (Kostrikis
et al., 1998).
Sequence analysis indicates that HIV in different Early Infection
organs and cell types may represent different sub-
populations in the various compartments of the Primary HIV infection (PHI) results in a well-rec-
body. Thus the dominant HIV substrains in the ognized constellation of clinical, virological and im-
blood and cerebrospinal fluid may differ, and within munological responses associated with rapid and
the blood, between CD4 ; cells and in CD8 ; widespread dissemination of virus following infec-
cells in late stage AIDS (Livingstone et al., 1996). tion. An initial illness occurs in 50—90% of cases, is
Whereas the depletion of CD4 ; cells accounts for classically described as mononucleosis-like, al-
the profound immune deficiency, and falling CD4 though in practice 20% have these features. A
cell counts are a useful marker of disease pro- typical presentation, in order of frequency, may
gression, the wasting disease and central nervous include: acute onset of fever, lethargy, maculopapu-
system (CNS) disease (AIDS dementia) are prob- lar rash, myalgia, headache, sore throat, cervical
ably more closely related to macrophage infection. lymphadenopathy, arthralgia, oral ulcers, photo-
This includes the microglial cells of the brain, which phobia, oral candida, and rarely meningoencephali-
are derived from monocytes rather than neurec- tis. The time from exposure to onset of signs and
toderm. Astrocytes may also become latently infec- symptoms is approximately 10 to 30 days.
ted, and changes to local cytokine secretion in the Lymphadenopathy is a frequent event during
brain lead to neuronal loss. PHI, usually in the second week of illness and is
often associated with a lymphocytosis. The lymph
nodes tend to decrease in size with time. The archi-
tecture remains relatively normal but HIV can be
HIV/AIDS: CLINICAL DISEASE AND visualized in association with both lymphocytes
MANAGEMENT and dendritic cells.
A distinctive feature of PHI is mucocutaneous
HIV/AIDS infection is characterized by an initial disease involving on the buccal mucosa, gingiva,
acute viral illness followed by a chronic, asympto- palate, oesophagus, anus and penis.
matic phase of disease associated with active viral The commonest neurological feature is an aseptic
replication and dissemination, and lasting as long meningoencephalitis reflecting the neurotropism
as 5—10 years. Ultimately, immune destruction re- associated with this group of viruses. Less frequent-
sults in end-stage disease (AIDS) associated with ly myelopathy, peripheral neuropathy, bronchial
opportunistic infections, malignancies and neur- neuritis and facial palsy and Guillain—Barré syn-
ological disorders. These clinical events correlate drome are seen. They are usually self-limiting. HIV
with virological and immunological changes and has been detected in the cerebrospinal fluid (CSF)
are shown in Figure 25.7. soon after infection, indicating the speed at which
Early classification of the disease by the Centers the virus penetrates the blood—brain barrier.
for Disease Control, Atlanta, USA, defined an Marked changes in the lymphocyte count are seen.
asymptomatic phase followed by a list of sympto- The CD4: CD8 ratio is reversed due to an increase
matic conditions that defined AIDS (Table 25.4). in the number of CD8 cells. An early CD4 lym-
This was later revised to reflect the effects of HIV/ phopenia is characteristic of PHI; it is associated
AIDS infection on clinical status (Table 25.5). Final- with very high plasma viral load levels that occur in
ly, in 1993 it was expanded to take account of early disease prior to immunomodulation. The
HUMAN IMMUNODEFICIENCY VIRUSES 671
Table 25.4 AIDS defining illnesses in HIV infection (defined in 1985, revised
1993)
Infections Neoplasms
Table 25.5 Centers for Disease Control (CDC): classification of the effects of
HIV-1 infection (CDC, 1985, 1993)
mechanism of this CD4 lymphopenia is explained The symptomatic phase of PHI lasts about 14
by a ‘predator—prey model; with HIV-1 undergoing days on average and is self-limiting; however, it is
multiple rounds of replication and ‘consuming’ now clearly evident that infection from a host with
available target cells. It is generally short lived, but advanced disease, protracted and wide ranging
on occasions precipitous early lymphopenia does symptoms, oral/oesophageal candida, neurological
occur and is thought to be one of the aetiological involvement, and persistently high plasma viral
factors associated with acute Pneumocyctis carinii loads and p24 antigenaemia are associated with
pneumonia, oral and oesophageal candida and mu- rapid disease progression.
cocutaneous ulceration. As a rule these early The differential diagnosis of PHI should include:
changes normalize with the development of an im- Epstein—Barr, cytomegalovirus, herpes simplex, ru-
mune response, CD4 cell counts return to normal bella and other acute viral infections; in addition,
range, plasma viral load drops to low levels of de- syphilis, disseminated gonorrhoea, toxoplasmosis
tection (‘set-point’) and p24 antigen becomes unde- and drug reactions should be considered.
tectable.
672 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 25.6 Centers for Disease Control (CDC) AIDS surveillance case
definition (CDC, 1993)
500 A1 B1 C1
200—500 A2 B2 C2
200 A3 B3 C3
Table 25.7 Centers for Disease Control (CDC) clinical categories for children with
HIV-1 infection (CDC, 1994)
The Asymptomatic Phase with Epstein—Barr virus infection and may respond
to acyclovir therapy. All these features are far less
The period after PHI and prior to symptomatic frequent in the era of combined antiretroviral ther-
disease has been described as the latent or asympto- apy.
matic phase. Although it is a clinically latent period, The progressive immune deficiency associated
in most cases virological latency is misleading, as with long-term HIV/AIDS infection results in op-
virus persistence and replication clearly occurs portunistic infections and malignancies which are
throughout the lymphoreticular and other tissues. often multiple and contribute to the rapid clinical
A review of factors which affect viral latency and deterioration in patients.
persistence is beyond the scope of this chapter
(Levy, 1998; Weiss, 1993).
The duration of the asymptomatic phase may be
variable and is related to the severity of PHI, the Opportunistic Infections
phenotypic characteristics of the infecting viruses,
the status of the host immunity, the life style of the Opportunistic infections seen in symptomatic HIV-
host and the use of antiretroviral therapies (see 1 disease (Tables 25.4 and 25.7) reflect adult and
below). childhood exposure; hence, fungal infections such as
Clinically, patients are relatively free of symp- histoplasmosis may be seen in persons who come
toms, although lymphadenopathy may be a com- from areas where the organisms are endemic. Infec-
plaint in some cases. Patients are offered a clinical tious organisms may be categorized into: (1) those
review every 3 months and regular plasma viral that do not cause disease in the immunocompetent
loads and CD4 lymphocyte counts are used to host (e.g. Pneumocystis carinii); (2) Those that cause
monitor disease status. Plasma viral loads meas- mild disease in the normal host (e.g. Herpes simplex
ured 6—12 months after PHI have been shown to be virus, HSV; Toxoplasma gondii); (3) Those that are
a powerful predictor of subsequent disease pro- conventional pathogens (e.g. Mycobacterium tuber-
gression, superseding CD4 lymphocyte counts culosis) but, as a consequence of immunosuppres-
taken at the same time, and in combination the two sion associated with HIV-1 infection, produce wide-
values are an even more accurate method for deter- spread debilitating disease in the host.
mination of patient prognosis (Loveday and Hill, Overall, the presenting features of symptomatic
1995; Mellors et al., 1996). HIV disease may be very different, according to the
This phase of clinical care is often supportive, geographical areas in which the hosts are found; for
involves surveillance of patients for features of early example, Pneumocystis carinii pneumonia and Ka-
progression and the optimum time to commence posi’s sarcoma are the commonest presenting dis-
antiretroviral therapy. eases in the UK, whereas Mycobacterium tuberculo-
sis and gastrointestinal infections are more frequent
in Central Africa.
Pneumocystis carinii pneumonia differs from the
Symptomatic Disease (AIDS) disease seen in other groups with non-HIV asso-
ciated immunosuppression, in that it is character-
In untreated patients early features of symptomatic ized by subacute onset involving a mild, persistent
disease were often non-specific and associated with cough and progressive chest discomfort and fatigue
dermatological manifestations, such as eczema and of 2—10 weeks duration. It is rare when CD4 cell
human papillomavirus eruptions, as well as oral counts are above 200 ; 10 l\. Subtle bilateral in-
and vaginal candidiasis, recurrent chest infections, filtrations with a batwing appearance may be seen
night sweats, weight loss and the appearance of on a chest radiograph but 50% are normal at pres-
lymphadenopathy. Oral lesions become more entation. Minimal hypoxia is present and diagnosis
common with gingivitis, candida, herpes simplex is made by detection of the pneumocystis organism
eruptions and aphthous ulcers. ‘Oral hairy leukop- in induced sputum or bronchoalveolar lavage. It is
lakia’ presents as white, ribbed lesions on the lateral treated with co-trimoxazole or pentamidine, and
margins of the tongue; it may be asymptomatic or the introduction of prophylaxis in patients with
produce soreness within the mouth. It is associated CD4 cell counts 200 ; 10 l\ has markedly re-
674 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
duced the incidence of this infection in HIV/AIDS, is available.
as have antiretroviral drugs. In addition viral (including herpes simplex) and
Cytomegalovirus (CMV) may also cause a diffuse bacterial infections are often implicated. Overall the
pneumonitis. It is also the commonest cause of pro- use of combined antiretroviral therapy has had a
gressive chorioretinitis in AIDS. The lesions are dramatic impact on the frequency and severity of
initially asymptomatic but, as the perivascular ex- such conditions.
udates and haemorrhages involve the macula, vi- Mycobacterium avium and M. intracellulare are
sion becomes impaired. Interestingly CMV pneu- two closely related species of ubiquitous environ-
monitis is rare in AIDS patients because it requires mental organisms which in the past had rarely been
active recruitment of the immune system to cause shown to be a cause of disseminated disease. How-
disease. In contrast, CMV retinitis is common in ever, it is common in patients with symptomatic
untreated AIDS patients as it is an uncontained HIV infection, suggesting they have an immu-
cytopathic disease not requiring immunocom- nological proclivity to these organisms. The organ-
petence. In addition, gastrointestinal ulceration, ad- isms are ingested or inhaled and produce dis-
renalitis and encephalitis may occur. Progression of seminated disease with non-specific symptoms,
CMV infection can be restricted by parenteral gan- including fatigue, fever, night sweats, weight loss,
ciclovir or foscarnet, and the institution of antiret- abdominal pain and diarrhoea, in patients with
roviral therapies has reduced the incidence and/or 50 ; 10 l\ CD4 lymphocytes. It is diagnosed
severity of CMV. by positive blood cultures using Lowenstein—Jensen
Cryptococcus neoformans also causes a diffuse solid medium (3—4 weeks) or a non-speciating broth
pneumonitis, although more commonly it causes medium (1—2 weeks), and confirmation by PCR, in
meningitis and is widely disseminated. It may also 90% of cases. Treatment and prophylaxis is with
present with fever, granulocytopenia, throm- specific antituberculous combination therapy. With
bocytopenic purpura, maculopapular rashes and the advent of highly active retroviral therapy
ulcerating gastrointestinal lesions. It may be iso- (HAART) these therapies may not be lifelong.
lated from many sites, including throat washings, Both this condition and the more classical my-
urine or blood. It is treated with fluconazole or cobacteria have become increasingly common in
amphotericin B. HIV-infected patients in the developed world, and
Toxoplasma gondii causes space-occupying antimicrobial resistant organisms are more evident
lesions in the CNS. These patients present with in this new tuberculosis epidemic.
fever and focal neurological signs and may have Oral candidiasis is commonly seen, and oesoph-
associated chorioretinitis. Serological tests for ageal involvement may be present with dysphagia,
toxoplasma are unreliable, and demonstration of odynophagia and retrosternal burning. Diagnosis is
the organism in the affected tissues by PCR may based on clinical findings and simple histology.
offer a more definitive diagnosis. The differential Treatment is by topical antifungal agents or intra-
diagnosis of cerebral lymphoma may be excluded venous therapy in the event of refractory and severe
using a highly sensitive and specific PCR for Ep- disease.
stein-Barr virus (EBV or Human herpesvirus 4, Other fungal infections like Aspergillus can pro-
HHV-4). It is treated with combinations of antimic- duce life-threatening respiratory disease, with white
robials. plaque-like colonies in the bronchi, and cavitation.
Other CNS-related conditions include progress- Infection is diagnosed by culture and treated with
ive HIV-associated encephalopathy, which involves itraconazole or amphotericin B; the outcome is
inflammatory changes in white and grey matter and poor, and time from diagnosis to death is short
is characterized by foci of inflammatory cells in- (months).
cluding macrophages and multinucleate giant cells Herpes simplex virus (HSV 2 HSV1) infection
with actively replicating HIV-1. commonly presents as a vesicular lesion on an
Progressive multifocal leucoencephalopathy erythematous base in oral, genital or perianal areas.
(PML) is a condition associated with JC virus Attacks are usually more widespread and of longer
producing oligodendritic lysis. It is diagnosed by duration, with more frequent recurrences in HIV-1
characteristic histological changes and confirmed if infection; lesions are associated with secondary bac-
possible by PCR for JC virus. No specific treatment terial infection and have been associated with wide-
HUMAN IMMUNODEFICIENCY VIRUSES 675
spread epidermal erosion, in some cases requiring Kaposi’s Sarcoma
skin grafts. Oesophageal and tracheobronchial in-
volvement is described. Diagnosis is by virus cul- Malignancy is a common feature of symptomatic
ture and/or PCR; treatment is with aggressive use of HIV/AIDS as a consequence of the profound cell-
oral or intravenous acyclovir therapy, depending mediated immunosuppression, and demonstrates
on the severity of the condition. the role of intact immunity in human cancer surveil-
Reactivation of Varicella zoster virus (VZV, lance. KS is the commonest AIDS-defining malig-
HHV-3) is common in HIV-infected patients but is nancy, but non-hodgkin’s lymphomas and
less often seen in advanced symptomatic disease, anogenital squamous carcinomas are of importance
probably reflecting the need for the immune re- (Weiss et al., 1999).
sponse in its pathogenesis. Ophthalmic zoster may It is striking that tumours present at late asym-
be complicated with a threat to vision, and any ptomatic or during the symptomatic phase of the
dermatomal presentation may become dis- HIV disease, at a time of diminishing immunocom-
seminated. Treatment is with high dose acyclovir, petence, and probably represent tumours of a viral
antibiotics for secondary infection and analgesia. aetiology.
Human papillomavirus (HPV-6 and -11)-induced KS is a rare vascular or lymphatic tumour
warts and molluscum contagiosum (Poxvirus) are (Dupin et al., 1999) that has been simply classified as
both common skin conditions in up to 25% of follows:
patients with HIV infection and may be of abnor-
mal presentation, and persistent. These manifesta- ∑ The endemic indolent form seen in elderly Jewish,
tions were often used in the past as clinical markers Greek and Italian men, African children and
to suggest that further investigation of HIV infec- adults in equatorial Africa, and transplant recipi-
tion might be indicated. ents;
Persistent or recurrent diarrhoea, which may be ∑ the aggressive form seen in symptomatic HIV/
copious in volume and watery in content, is a fre- AIDS patients.
quent problem in symptomatic patients. Giardia Early studies of the epidemiology of the aggres-
lamblia, Entamoeba histolytica, Shigella, Salmonella sive form defined clusters of cases in homosexual
and Campylobacter all cause symptomatic disease; and bisexual men in the developed world, with in-
however, appropriate treatment against these creasing cases at sites associated with the original
pathogens does not always eliminate the watery epidemic. Female cases were associated with bisex-
diarrhoea. Cryptosporidium parvum, an enteric coc- ual male partners. The sexual practice of oroanal
cidium, attaches to the epithelial surface of both contact was significantly higher in those with KS
small and large intestines and produces protracted and was proposed as a route of transmission of a
and severe diarrhoea in symptomatic HIV/AIDS potential infectious agent associated with this
patients. The diagnosis is by direct modified group. The disease has a lower prevalence in trans-
Ziehl—Neelsen staining of stool preparations; treat- fusion acquired HIV/AIDS (4%) and haemophil-
ment is difficult, with a wide range of antimicrobial iacs ( 1%), suggesting the blood-borne route as a
agents demonstrating marginal success. CMV, My- secondary pathway for transmission.
cobacterium avium and M. intracellulare, Isospora Using a modified PCR technique applied to sub-
belli, Microsporidium encephalozitozoon and Ka- stractive hybridization, Chang et al. (1994) analysed
posi’s sarcoma involving the bowel wall may also normal and KS-associated skin and identified novel
produce these clinical features. There is growing DNA sequences in the latter that had high amino
evidence that HIV-1 itself may cause enteropathic acid homology with proteins of EBV or HHV-4 and
signs and symptoms through infection and infiltra- Herpes virus saimiri. A new virus was identified
tion of local target cells. In such treatment of speci- related to the gamma herpesviridiae initially called
fic pathogens is unsuccessful and patients have both KS-associated herpesvirus or Human herpesvirus 8
malabsorption and loss of weight. The clinical con- (KSHV or HHV-8). DNA from HHV-8 was found
sequences of this condition may be profound; in all KS lesions, independent of HIV status (Chap-
HAART has been found to be of value in some ter 2F). PCR and serological assays are available for
cases. testing for HHV-8. The presence of HHV-8 predicts
676 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 25.8 TIS staging of Kaposi’s sarcoma
OI : opportunistic infection.
Karnovsky score : clinical scale of HIV/AIDS disability in units of 10, from 0 : dead to 100 : normal,
no signs or symptoms.
? B symptoms—see Table 25.6.
Non-Hodgkin’s Lymphoma
Anogenital Squamous Carcinoma
Most of the lymphoid malignancies are high-grade
Burkitt’s lymphomas and immunoblastic lym-
Rapidly progressive squamous intraepithelial car-
phomas. The diagnosis is 50 times more common in
cinomas develop in the anorectal junction and cer-
HIV/AIDS than the normal population. Its age
vical canals of men and women respectively, with
distribution is bimodal, with the former at a peak in
HIV/AIDS. These sites have a squamous-columnar
10—20-year-olds and the latter in 50—60-year-olds.
border in common and there may be a viral causa-
Non-Hodgkin’s lymphoma is becoming more
tion. These regions are commonly infected with
common in HIV/AIDS as patients live longer as a
Human papillomavirus (HPV) in these patient
consequence of prophylaxis for opportunistic infec-
groups, and, especially as immunosuppression in-
tions and HAART. They are usually widespread at
creases in symptomatic disease, it is proposed that
presentation and often occur in extranodal sites,
HPV-encoded oncoproteins (E6 and E7) known to
particularly the brain. Only about 50% of the lym-
promote genetic instability in cells may lead to tu-
phomas are EBV related, although higher figures
mour formation.
have been claimed in some populations. This sug-
Studies of those with HIV/AIDS confirm an in-
gests that the pathogenesis is more complicated
creased risk of in situ cervical cancer but not invas-
than the expression of an oncogenic virus in the face
ive disease (Weiss et al., 1999). Furthermore, once
of immunosuppression, although it is likely that
the latter is defined it follows a more aggressive
activation of virion stimulation has a role, just as in
course in this patient group. More frequent (annual)
KS (Weiss et al., 1999).
cervical screening is offered to patients with HIV/
Diagnosis is essentially by biopsy of systemic
AIDS but anal cytology in men has not yet been
non-Hodgkin’s lymphoma, but this is not routine
evaluated. These malignancies are treated conven-
for suspected primary cerebral disease. In the latter
tionally with chemotherapy and radiotherapy.
case a presumptive diagnosis of a CT lesion is made
Other tumours that appear to have a high fre-
in the absence of response to antitoxoplasmal ther-
quency in patients with HIV/AIDS include testicu-
apy, a negative thalium uptake scan and a positive
lar tumours, squamous cell carcinoma of the oro-
EBV PCR result in CSF.
pharynx tumours of the skin, and squamous
Treatment of systemic disease is stratified accord-
carcinoma of the conjunctiva in Africa. These may
ing to prognostic factors; generally those with ma-
be associated with immunosuppression and/or viral
jor adverse factors, like prior AIDS-defining illness,
aetiology, e.g. papillomaviruses, but further evi-
CD4 counts 100 ; 10 l\, primary cerebral dis-
dence is awaited.
ease and low Karnovsky score ( 70), receive palli-
ative care and low toxicity chemotherapy, focusing
on quality of life. The minority with a better prog-
nosis are treated with conventional ‘curative’ Clinical HIV/AIDS in Children
chemotherapy for non-Hodgkin’s lymphoma. Pri-
mary cerebral lymphoma is associated with Since 1982 cases of vertically transmitted paediatric
profound immunosuppression and very low CD4 HIV infection were recognized associated with
cell counts; a brief clinical improvement is derived mothers in traditional risk groups (intravenous
from brain radiotherapy and dexamethasone but drug users, sex workers, partners of haemophiliacs,
678 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
transfusion recipients and partners of bisexuals). and implement preventative measures and at pres-
Further large populations of infected children were ent this is not universally available. In the develop-
identified in sub-Saharan Africa, the seat of the ed world the uptake of HIV antibody testing in
epidemic. Rates of vertical transmission vary be- pregnancy is only about 50%, and in the developing
tween African (30—50%), North America (20—30%) world only an incomplete infrastructure exists to
and European (14%) studies. These differences are screen patients.
probably due to populations at different stages of
disease being investigated.
Theoretically the virus may be transmitted
throughout pregnancy, during delivery and as a Disease Presentation in Children
consequence of breast-feeding. HIV has been iso-
lated from first/second trimester aborted fetuses; Paediatric HIV-1 infection exhibits some distinct
twin studies show the first born has a higher rate of qualities in relation to adult disease; the natural
infection, presumed to be associated with longer history has not been fully defined but infection of
exposure to secretions; and breast-milk has been the host at a stage of immunological and biofunc-
demonstrated to carry HIV and be infectious as a tional immaturity will clearly influence the expres-
consequence of ingestion. Contemporary research sion of disease. Factors like time of infection, expo-
evidence suggests the majority of transmission sure to other pathogens, nutritional status, time of
events occur in late pregnancy and during breast- diagnosis and quality of care are all important.
feeding. Evidence suggests two groups of vertically infected
Strategies for interruption of vertical trans- infants: 30% with early clinical problems and life-
mission have had a dramatic impact on the numb- threatening illnesses in the first year of life; and 70%
ers of children infected with HIV-1 in the developed with few problems in early life and developing dis-
world. Evidence shows the treatment of the mother ease after several years. This has been explained by
with zidovudine during the last trimester of preg- early transplacental infection in the former and late
nancy and the infant for the first 6 weeks of life infection in the latter—a plausible view, but one
produced a 60—70% reduction in vertical trans- must also consider issues like pathogenicity of in-
mission (Study ACTG 076: 23% versus 8% trans- fecting viruses and immunological maturation,
mission; Connor et al., 1994). Studies of combina- which may vary widely from case to case.
tion therapies are now underway to optimize CDC disease classification for children is sum-
regimens in relation to drug toxicity and duration marized in Table 25.7.
of treatment. However, these drugs are not avail- Microbial infections are common in HIV-infec-
able worldwide and probably less than 10% of ted children. Herpes simplex, Varicella zoster and
HIV-infected mothers are in a position to benefit at Measles viruses all produce severe forms of the rec-
present. Caesarean section gave early inconclusive ognized presentations that require prompt and pro-
results, but a recent meta-analysis of European and longed therapy to avoid high morbidity and mor-
American studies showed a 55% reduction in risk of tality. Common paediatric bacterial infections, like
vertical transmission. Avoiding breast-feeding will Streptococcus pneumoniae, Haemophilus influenzae,
reduce the rate of transmission and this is advised in Escherichia coli and Salmonella species, exhibit un-
the developed world; however, in the developing usual presentations and severity; prompt treatment
world the risks to the baby of morbidity and mor- is required until culture results are available, and
tality associated with gastrointestinal infections due multiple infections should always be suspected in
to lack of conferred immunity from breast milk, and unresolving cases. Antimicrobial prophylaxis and
poor hygiene in preparation of bottle feeds, are immunoglobulin are instituted by some centres.
greater than those associated with HIV infection. As in adults, opportunistic infections with Pneu-
Thus at present the World Health Organization mocystic carinii, Mycobacterium tuberculosis, M.
advise breast-feeding in the latter and avoidance in avium and M. intracellulare, CMV, cryptosporidia
the former. Theoretically we have the technologies and non-oral candidiasis as a consequence of im-
to almost eradicate vertical transmission of HIV munodeficiency are common.
but it requires knowledge of maternal HIV anti- Failure to thrive is a common feature of paediat-
body status in early pregnancy accurately to advise ric HIV disease. The reasons are multiple: decrease
HUMAN IMMUNODEFICIENCY VIRUSES 679
Primary Confirmation Alternatives
screen
Perform three Consider:
further antibody Non-concordance
assays with different • HIV-2 infection
configurations • Seroconversion
(e.g. antiglobulin, • New subtype
competitive and Concordance • HIV-1 vaccination
PAA) • Prolonged use
Reactive of HAART
First-line • Non-specific
ELISA assay reaction
(HIV-1 and
HIV-2)
Negative
Check patient
details and confirm
all reactives in the Report
run from remaining
serum on the clot
food intake due to oral infections; decreased ab- adult infection, but a positive diagnosis involves
sorption due to intestinal disease and recurrent di- more complex algorithms that are dependent upon
arrhoea; and decreased calorie utilization for the mode of transmission (horizontal or vertical)
growth due to demands associated with inflamma- and the duration of the infection. The availability of
tory responses and tissue repair. antiretroviral therapies makes rapid diagnosis im-
HIV encephalopathy usually occurs in sympto- perative and strategies have therefore been devised
matic disease, presenting with developmental delay, to include PCR in the clinical algorithms.
motor deficits, and cognitive and behavioural dis- The algorithm for diagnosis of established adult
orders. It may be slow or rapid in progression, and disease is summarized in Figure 25.8. After the pri-
cerebral atrophy and ventricular enlargement is mary screen using a sensitive HIV-1 ; 2 ELISA
seen on CT. Other infectious causes like toxo- assay format, all negative results are reported once
plasma, JC virus, mycobacteria and cryptococcus the ‘reactive’ samples in the run (those with a signal
are unusual but should be excluded. greater than the mean of the negative) are con-
The use of antiretroviral therapies has improved firmed and the patient identification assured. Reac-
the prognosis for all these clinical conditions, but tive samples are entered into a confirmatory algo-
we do not know the long-term prognosis of acquir- rithm using three further ELISA assays with diverse
ing HIV at such an early phase of host development HIV-1 antigens on the solid phase and different
(Pizzo and Wilfert, 1994). principles of action (e.g. antiglobulin, particle agglu-
tination, competitive, etc.). A positive report is re-
leased if consensus results are obtained from all
three assays ( 90% of cases), and a second blood
Diagnosis and Monitoring of HIV is requested to confirm patient identity.
Infection If there is non-consensus in the confirmatory tests
the clinical virologist should consider HIV-2 infec-
Serology remains the cornerstone for the diagnosis tion, seroconversion, non-specific reactivity or rare-
of HIV infection in adults. The ELISA assays de- ly a new non-reactive HIV subtype. The proposed
scribed earlier in this chapter are based on different algorithm for follow-up of these samples generally
biological principles (e.g. antiglobulin, competitive, resolves 99.9% of cases, but a smaller number re-
etc.), have extemely low false-negative rates and are main that often produce alarming reactivity which
inexpensive. They may be used alone to exclude may persist for life or disappear over time; these
680 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 25.10 Typical serological and PCR changes associated with early primary HIV-1 infection
cases are often resolved by repeat negative results testing at 6-monthly intervals, and in the majority
with diagnostic PCR of proviral HIV-1 DNA from the reactivity will eventually wane.
PBMCs. The diagnosis of HIV-1 infection in the newborn
The Western blot assay is relatively insensitive in infant is confounded by the presence of maternal
relation to modern ELISA assays and is reserved antibodies in the baby’s circulation from approxi-
for difficult cases, HIV-2 confirmation, seroconver- mately 32 weeks of gestation onwards. Early diag-
ters and research. nosis is essential for early clinical interventions and
The adult seroconverter is confounded by initial considerable effort has been applied to establishing
low or non-detectable levels of anti-HIV-1 antibo- algorithms. One approach uses Western blot and
dies, and ELISA assays may be negative in the face titrated particle agglutination assays to measure
of clinical signs and symptoms of PHI. If a clinical qualitatively and quantitatively antibody concen-
diagnosis of PHI is proposed, the sample is tested trations from 3 days of life onwards, serum P24
with all four serological assays, a P24 antigen assay antigen and diagnostic PCR of infant-specific HIV-
and the diagnostic PCR test. In most cases a PCR 1 proviral DNA (Table 25.11). If available, PBMC
positive result is seen prior to, or associated with, culture may also be added to the algorithm. Rapid
low level seroreactivity, and in approximately 50% diagnosis is confirmed by two positive PCR and
of cases will show detectable plasma P24 antigen. one positive p24 antigen result in samples taken at 3
This is repeated at weekly intervals and evolving days, 3 weeks and 3 months. Titrated serology in an
signals in the serological assays confirm serocon- uninfected baby should demonstrate a halving of
version. The Western blot will demonstrate the passive antibody concentration every 28 days; if
evolution of a serological response to specific HIV antigen (virus) is present and evolving in an infected
antigens but that post-dates the sensitive serologi- baby this will reflect in a levelling off of the ex-
cal assays (Table 25.10). ponential decline in antibody concentrations or an
Patients with non-specific reactivity (presumed increase over time. It takes up to 18 months for all
to have cross-reacting antibodies that signal in cer- passive antibody to resolve in the majority of in-
tain or all assays) show a distinctive virological fants and the analysis and reporting of these events
pattern but present a difficult clinical problem. involves considerable virology experience.
They often present through the blood transfusion Diagnostic strategies are now well-defined but
service as a reactive sample on whom the donation the clinical decision to carry out the test(s) is less
has been withheld. The clinical follow-up usually straightforward. Indeed the diagnosis of any chro-
involves establishing they are PCR negative and nic, incurable and ultimately fatal disease requires
that they have non-evolving serology. They are fol- evaluation of the benefits for the patient and the
lowed over time for reassurance, support if they implications for others closely associated with that
proceed to any other medical investigations, repeat individual.
HUMAN IMMUNODEFICIENCY VIRUSES 681
Table 25.11a Typical serological and PCR changes from birth associated with an uninfected baby
Table 25.11b Typical serological and PCR changes from birth associated with an infected baby
In the late 1980s molecular virologists modified revealed the predictive value of such measures for
PCR-based technologies to quantify HIV-1 RNA clinical outcome (Mellors et al., 1996) viral load was
load (viral load) and genotypic resistance in plasma established as a marker, substituting for clinical
viruses (Kaye et al., 1992; Semple et al., 1993), and end-points, to define the efficacy of new antiret-
from 1990 this group and others used these ap- roviral drug combinations. In clinical practice pa-
proaches to monitor the efficacy of antiretroviral tients have regular viral load measures to assist in
therapy in trials (Schuurman et al., 1995; Katlama the determination of disease stage, progression, re-
et al., 1996) to demonstrate their predictive value for sponses to antiretroviral therapies and as evidence
disease progression (Loveday and Hill, 1995), to of early treatment failure.
demonstrate the association between failing vi- Early in vitro studies and investigations of pa-
rological response and evolving resistance in vivo tients receiving monotherapies revealed that, as vir-
(Loveday et al., 1995) and to demonstrate the rapid al load response failed, drug-resistant viruses evol-
initial decline in plasma HIV-1 (Loveday, 1994) that ved in the plasma. These were detected using both
defined the dynamics of virus replication and allow- drug sensitivity culture assays (phenotypic) and
ed the development of new theories of pathogenesis HIV genome sequencing strategies that identify
(Coffin, 1995). By 1994 first generation commercial base changes known to be associated with changes
assays were available based upon plasma HIV-1 in drug sensitivity (genotypic). The relative advan-
nucleic acid capture, reverse transcription and am- tages and disadvantages of these approaches are
plification and resulting complementaryu DNA sig- summarized in Table 25.12. The measurement of
nalling to quantify plasma viral load. These assays resistance in clinical practice is not yet universally
initially detected down to a level between hundreds accepted but the majority of virologists, clinicians
and thousands of copies ml\ plasma, but later and patients feel it will have value in optimizing the
second-generation assays defined cut-off values first and subsequent combination therapies (Hirsch
around 20—50 coipies ml\. The evolution of these et al., 1998).
assays is summarized in Loveday (1999). These viral
load assays were initially used experimentally to
define virological efficacy in clinical trials (Brun-
Vezinet et al., 1997), and when a large cohort study
682 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Potential therapeutics
7. Integration 6
8. Latency 7
Figure 25.9 The life cycle of HIV indicating the stages at which potential therapeutics might act
TREATMENT OF HIV-1 INFECTION have provided a foundation for the future of clinical
management of viral diseases.
The treatment of HIV/AIDS is subdivided into the
therapies and prophylaxis for conditions associated
with the profound immunosuppression (e.g. Pneu-
mocystis carinii pneumonia, KS, etc.) and the anti- History
retroviral therapy for HIV-1 itself. The former is
outlined in the appropriate sections. Since the The potential sites of drug action in the HIV-1 life
identification of AIDS in 1981, and HIV-1 in 1983, cycle are summarized in (Figure 25.9). In reality,
the rate of development of new antiretroviral drugs only the reverse transcriptase and protease activ-
to combat this disease has surpassed any other ities have so far yielded antiretroviral drugs in rou-
pharmacological developments in modern medical tine clinical use.
history. In addition, these discoveries have assisted Historically, the first antiretroviral drug was
in the understanding of retroviral pathogenesis and zidovudine (ZDV or AZT), a nucleoside reverse
transcriptase inhibitor (NRTI), identified in 1985
HUMAN IMMUNODEFICIENCY VIRUSES 683
Table 25.12 Comparison of the advantages and disadvantages of genotyping and phenotyping for
the determination of antiretroviral drug resistance
Advantages Disadvantages
? P3 : high containment laboratory for the culture of certain infectious pathogens like HIV-1.
and shown to exhibit a significant clinical benefit in The introduction of protease inhibitors in 1995
a placebo-controlled study in patients with sympto- provided a class of drugs with a new site of action
matic disease. CD4 lymphocyte counts showed a that had marked potency alone (not sufficient to be
modest and short-lived increase (30 ; 10 l\ over used as monotherapies) and, in combination with
3—6 months) and introduced CD4 counts as a two other RT inhibitors (HAART), had exceptional
marker of drug efficacy. High initial doses (1500 mg clinical effects and decreased viral load below levels
day\) were associated with side-effects and toxic- of detectability ( 50 copies ml\ for more than 12
ity, especially bone marrow toxicity. Nevertheless, months (Carpenter et al., 1997). The introduction of
use of ZDV was commenced for asymptomatic and these drugs also coincided with virologists examin-
symptomatic disease until a larger placebo-control- ing the early dynamics of viral load changes that
led study (Concorde Coordinating Committee, allowed the development of models of HIV
1994) showed early treatment with this drug was no pathogenesis (Ho, 1995; Wei et al., 1995; Phillips et
better than deferring therapy until patients were al., 1997b), and viral load responses to therapy were
symptomatic. Further nucleoside (ddI, ddC, 3TC, accepted by drug registration authorities as a pre-
etc.) and non-NRTIs (nevirapine, etc.) were devel- liminary alternative to clinical outcome as a marker
oped but had limited clinical efficacy as mono- of drug efficacy for registration. These events set the
therapies. Open virological studies in the early targets for modern antiretroviral therapy using
1990s, using quantitative PCR and culture tech- multiple drugs that produce a rapid decline in
nologies, demonstrated that this was associated plasma viral load to undetectable, with correspond-
with poor virological efficacy and the rapid evol- ing increases in CD4 cell counts, with minimal side-
ution of drug-resistant viruses in those receiving the effects.
monotherapies. Three large monotherapy control-
led studies (Delta, ACTG 0175 both with ZDV
versus ZDV ; ddI or versus ZDV ; ddC, and
NUCA 3001 with AZT versus AZT ; 3TC) be- Antiretroviral Drugs
tween 1992 and 1995 revealed the increased clinical,
immunological and/or virological efficacy of two There are currently 14 registered antiretroviral
NRTIs over ZDV alone (Delta Coordinating Com- drugs available for combination therapy (Table
mittee, 1996; Hammer et al., 1996; Katlama et al., 25.13) and new NRTIs (dideoxyfluorothiacytidine,
1996). FTC; fluorodideoxyadenosine, FddA), nucleotide
684 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 25.13 Antiretroviral drugs available to treat HIV-1 infection
NRTIs
Zidovudine (ZDV) 250—300 mg 9 0.5 to 0.7 log Nausea, headache Prodrug
b.d. codons 41, 67, 70, myopathy, bone Enters CNS
215 & 219, 151 marrow suppression Combivir-AZT/3TC
Didanosine (ddI) 200 mg b.d. 9 0.5 to 0.7 log Nausea, diarrhoea Prodrug
400 mg o.d. codons 65, 74, 75 rarely pancreatitis, Efficacy with
(no food) 184, 151. peripheral neuropathy hydroxyurea
Zalcitabine (ddC) 0.75 mg t.d.s. 9 0.5 log Oral ulcers and Prodrug
codons 65, 69 peripheral
74, 75, 184, 151 neuropathy
Lamivudine (3TC) 150 mg b.d. 9 0.5 to 0.7 log Nausea, bone marrow Prodrug
codon 184 suppression Well tolerated
Stavudine (d4T) 40 mg b.d. 0.6 log Peripheral Prodrug
codon 75 neuropathy
Abacavir (1592) 300 mg b.d. 9 1.5 log Hypersensitivity: Prodrug
codons 65, 74 fever, rash, and Rechallenge
115, 184 fatal rechallenge contraindicated
NNRTIs
Nevirapine 200 mg b.d. NR Rash on induction Long half-life,
initial dose codons 103, 106 15%, 5% grade 3, Induction with
escalation 108, 181, 188 Induces cytochrome P450 prednisolone
Delaviridine 400 mg t.d.s. NR Rash 20%, 5% grade USA only
600 mg b.d. codons 103, 181 3, headache, nausea Inhibits cytochrome P450
236 raised LFTs
Efavirenz 600 mg nocte NR Headache, dizzy, vivid Induces and
(minimize side- codons: 100, 103 insomnia, rarely inhibits cytochrome P450
effects) 108, 188, 190 psychoses, raised LFTs
Protease inhibitors
Indinivir 800 mg t.d.s. 9 1 to 2 log Nausea, nephrolithiasis ‘Early efficacy
fasting/low fat codons: 82, 46 haematuria, lipodystrophy late toxicity’
10 ; 2nd MTs hyperlipidaemia x-resistance PIs
Ritonavir 600 mg b.d. 9 1 to 2 log Nausea, vomiting, ‘Early toxicity’
(400 in combo) codons: 82 & taste changes, lipid and x-resistance PIs
10 ; 2nd MTs transaminase elevation
Saquinivir 1200 mg t.d.s. 9 1 log Nausea, diarrhoea Early formulation
(soft gel) 1800 mg b.d. codons: 48, 90 mild abdominal pain had poor absorpn
(with food) 7 ; 2nd MTs (self-limiting) x-resistance PIs
Nelfinavir 750 mg t.d.s. 9 1 to 2 log Diarrhoea: controlled x-resistance PIs
1250 mg b.d. codons: 30 by drug treatment
(with food) 8 ; 2nd MTs (self-limiting)
Amprenavir 1200 mg b.d. 9 1 log Nausea, vomiting, taste x-resistance PIs
codons: 50 changes, paraesthesia
4 ; 2nd MTs
LFT : liver function test; MT : mutations; NRTI : nucleoside reverse transcriptase inhibitor; NNRTI : non-NRTI; NR : not recorded;
PI : protease inhibitor.
Reduction of viral RIVA in plasma.
Position of mutations conferring resistance in reverse transciptase (NRTIs, NNRTIs) and protease.
25 A
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
696 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
(Hinuma et al., 1981). Using viral antigens from the (PHA) and IL-2. De novo infection of T cells and cell
MT-2 cells they developed a serological test and lines requires cocultivation using irradiated or
showed that nearly all ATLL patients and a high mitomycin-C treated HTLV producer cell lines.
proportion of the relatives had antibodies to this The HTLVs are not readily transmissible in cell-free
virus (Yoshida et al., 1982). Known initially in Ja- form and this is reflected in the observation that
pan as Adult T cell leukaemia virus (ATLV), se- only whole fresh blood transfusion and not plasma
quence analysis showed that ATLV was almost or other cell-free fractions leads to transmission
identical with HTLV-I. Serological studies revealed (Okochi et al., 1984). HTLV infection is not found
that ATLV/HTLV-I was endemic in southwest Ja- usually in haemophilia patients (who are generally
pan, with 15% of the population seropositive, rising treated only with cell-free plasma derivatives).
to 30% in some villages. In central Japan only 1% Susceptible cell lines can be identified by the for-
of the population were seropositive, with higher mation of syncytia (giant multinucleated cells) upon
rates again in the north. The inhabitants of central contact with virus-producing cells. This interaction
Japan are believed to have come from mainland requires the presence of the HTLV-I envelope and
Asia c. 300 , displacing the ‘older’ population to specific cell membrane ligands (receptors). The syn-
the north and southwest. The distribution of cytial assay has proved useful for studying HTLV-I,
HTLV-I in the population suggests that HTLV-I particularly in the detection of neutralizing anti-
has been present in Japan since before this migra- body and cellular receptors (Clapham et al., 1984).
tion. In vivo HTLV-I primarily infects CD4 ; cells,
A related virus, HTLV-II was isolated from the while HTLV-II infects CD8 ; cells. In vitro many
cells of a patient with an atypical hairy cell leukae- cell types from a broad range of species, including
mia (HCL) in 1982 (Kalyanaraman et al., 1982). astrocytes, can be infected. Although the cellular
Hairy cell leukaemias are usually of B cell origin, receptor has not been identified, the coding gene
while the cell line derived in culture had T cell has been localized to chromosome 17 and may be
markers which suggests that the HCL was unre- a member of the VCAM family. The envelope
lated to the HTLV-II infection. Although a second proteins of HTLV-I and HTLV-II are the target
isolation of HTLV-II from a patient with HCL was for neutralizing antibodies. Sera from British or
made, subsequently extensive investigation has fail- American patients and asymptomatic carriers of
ed to confirm a disease association. HTLV-II shares HTLV-I neutralize equally viruses from other
60—70% sequence homology with HTLV-I and is countries including Japan (Clapham et al., 1984);
detected by HTLV-I lysate or whole virus based this suggests that there is a single worldwide sero-
assays. type for HTLV-I.
Simian T-lymphotropic viruses have been found The 9 kb genome of HTLV-I contains the three
in macaques and other Old World monkeys, and major open reading frames (ORFs) gag, pol and env
STLV-I appears to be related more closely to of all retroviruses flanked by two long terminal
HTLV-I than to other STLV/PTLVs. HTLV-I/ repeats (LTRs) with an additional regulatory re-
STLV-I strains cluster geographically rather than gion, pX (Figure 25A.2). The names, product size
by host species. A newly identified PTLV has been and functions of the genes of HTLV-I are sum-
shown to be a distant relative of HTLV-II, while marized in Table 25A.1. The HTLV-II genome is
another recent isolate is not sufficiently similar to very similar except that five ORFs have been identi-
either HTLV-I/STLV-I or HTLV-II to be grouped fied in pX.
with these families. It has therefore been assigned to The first major reading frame codes for a gag
a discrete third family, PTLV-L (the L stands for precursor of 429 amino acids that is cleaved into the
Leuven where the virus was isolated; HTLV-III has three gag proteins: 19 kDa (matrix protein), 24 kDa
already been used as a former name for Human (capsid protein) and 15 kDa (nucleocapsid protein).
immunodeficiency virus type I, HIV-I) (Van Brussel A second major reading frame within the gag-pol
et al., 1998). complex codes for proteinase. This frame slightly
Morphologically HTLV-I and -II resemble C- overlaps with gag and is generated by a frameshift
type retroviruses (Figure 25A.1). They can be grown suppression of the gag terminator codon. The re-
in vitro in immortalized lines which are obtained by verse transcriptase (which uses Mg> as a divalent
culturing patients’ cells with phytohaemagglutinin cation) and integrase are encoded by a different
HUMAN T-CELL LYMPHOTROPIC VIRUSES 697
Figure 25A.1 Electron micrograph of HTLV-I particles produced by a T cell line transformed in vitro. Note the variable size of the
particles and the diffuse spherical core
II II
p30 p13
R U5 II U3 R
GAG POL
ENV IV
I III
tax
p40
U3 R
R U5
POL V IV
ENV I III
GAG II
Figure 25A.2 (a) HTLV-I and (b) HTLV-II genomes. (Reproduced with permission from Franchini G (1995) Blood, 86, 3619—3639)
698 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 25A.1 Products of HTLV-I genes
ORF and are cleaved from a 99 kDa precursor pro- duction of new virions and aids a shift from doubly-
tein. spliced pX RNA to gag-pol and env RNA.) Thus the
Env codes for a protein of 481 amino acids which, overall effect of Rex may be virion production soon
after glycosylation, has a molecular weight of after infection, followed by downregulation of viral
62 kDa and is processed into an outer surface pro- expression which would protect the infected cell
tein of 46 kDa and a transmembrane protein of from death due to the cytolytic effect of virion pro-
21 kDa. The envelope precursor is translated from a duction and protect both the infected cell and the
4.2 kb mRNA in which the gag and pol have been virus from the host immune response (Hidaka et al.,
spliced out. 1988). The Tax (p37) and Rex (p26) proteins of
The fifth genomic region referred to as pX is HTLV-II are slightly smaller than those of HTLV-
located between env and the 3 LTR. The region I. Px encodes three additional proteins, p10, p28
codes for tax and rex, which are translated from a and p11 from ORFs I, II and V respectively (re-
double-spliced 2.1 kb mRNA. In addition there are viewed in Ciminale et al., 1996).
at least four additional proteins encoded by ORFs
I, II and III, although their function is not clear.
Tax activates transcription from the LTR and is
therefore an important upregulator of viral replica- DIAGNOSIS
tion. In addition Tax transactivates a range of host
cellular genes through which it is thought to affect Detection of HTLV-I and -II infection is primarily
cell replication and play an important role in the by serology. A particle agglutination assay based on
pathogenesis of ATLL. The immunodominant whole viral lysate is sensitive for both viruses but
peptides for the host cytotoxic T lymphocyte re- limited by poor specificity. First generation com-
sponse are also found in the Tax protein, which may mercial enzyme immunoassays (EIAs) were more
be important both for the control of infection and in specific than the agglutination assays but less sensi-
the pathogenesis of inflammatory disease asso- tive, particularly for HTLV-II. The new generation
ciated with HTLV-I, of which the archetype is of EIAs incorporating recombinant peptides are
HTLV-I-associated myelopathy. The Rex protein highly sensitive and specific for HTLV-I and
determines the export of unspliced gag-pol mRNA HTLV-II. Immunofluorescence assays using fixed
and singly spliced env mRNA out of the nucleus, infected cells with uninfected cells as controls can be
thereby controlling the production of viral proteins used to confirm and type infections but is ‘operator’
and infectious virus. (Rex production results in in- dependent. Peptide-based assays which discrimi-
creased export of env mRNA and unspliced viral nate between HTLV-I and HTLV-II are available.
RNA for the gag/pol proteins essential for the pro- HTLV-I infection is usually confirmed by the detec-
HUMAN T-CELL LYMPHOTROPIC VIRUSES 699
tion of gag (p19 and p24) and env (gp21 and gp46) EPIDEMIOLOGY
antibodies by Western blot, although radioimmune
precipitation assays (RIPAs), radioimmune binding It has been estimated that 20 million persons world-
assays (RIBAs), line immunoprecipitation assays wide are infected with HTLV-I, including 1.2 mil-
(LIAs) and competitive ELISAs can also be used. lion in Japan, where up to 15% of the population in
p19 antibody is often absent in, and not required to the southwest are seropositive. Other endemic areas
confirm, HTLV-II infection. Recombinant env are the Caribbean, parts of the southeastern states
peptides have improved the sensitivity and specific- of USA, Melanesia, parts of sub-Saharan Africa,
ity of Western blots and type-specific antigens especially the West and Central Africa (Figure
rgp46-I and rgp-46-II enable the infections to be 25A.4a, see Plate V), Recently HTLV-I infection has
discriminated without resort to molecular methods. been found to be relatively common in southern
Western blots which reveal some virus-specific Africa, particularly Natal. Up to 0.5% of blood
bands but which are not sufficient to make a posi- donors in Brazil are HTLV-I seropositive, with con-
tive diagnosis are termed ‘indeterminate’. Further siderable interstate variation. HTLV-I infection is
investigation is then warranted, including DNA recognized among many native South American
amplification using generic or type-specific primers. indigenous peoples as well as among black and
Viral culture may be required to confirm an infec- Japanese immigrants. However, population mixing
tion but is costly, time consuming and must be has been extensive and in Brazil the proportion of
conducted in a category 3 laboratory (HTLV neurology patients with HTLV-I-associated
European Research Network, 1996). myelopathy/tropical spastic paraparesis (HAM/
TSP) is equal among the main racial groups. North-
ern Iran is another recently described area of
HTLV-I infection, with cases described in neigh-
VIRAL VARIATION bouring Middle East and central Asian countries.
The prevalence of HTLV-I among European Union
HTLV-I has a highly conserved genome with only (EU) blood donors is low (2—7 per 100 000) but
about 4% sequence variation between isolates from remarkably similar across the length and breadth of
around the world. The only exceptions are isolates the EU. Seroprevalence rates 50—100 times higher
from Australian aborigines and Melanesians, which have been found among women attending antenatal
form a distinct genotype or clade, HTLV-I , and clinics and among men and women attending sex-
+#*
which have up to 8% diversity in LTR and/or ually transmitted disease clinics. However the
env. Most other isolates from Africa, Japan or the numbers tested are much smaller and in many
Caribbean belong to the Cosmopolitan clade, countries such studies have not been conducted
HTLV-I . Two other clades have been recognized (Figure 25A.5). In Central and Eastern Europe very
!-1
from Central Africa and from Zaire. In phylogenetic few studies have been conducted, but cases of
analyses STLV-I, which is almost identical to HTLV-I related pathology have been diagnosed in
HTLV-I, clusters with HTLV-I by geography EU member states among patients from Bulgaria,
rather than by species (Figure 25A.3). This suggests eastern Germany and Romania. ATLL has been
that there have been a number of simian—human described in Georgia and a relatively high rate of
transmissions (Koralnik et al., 1994). HTLV-II HTLV EIA seroreactivity has been reported in Lat-
shares 60—70% sequence homology with HTLV-I. vian blood donors (reviewed by Taylor, 1996).
HTLV-II isolates belong to either genotype a or b HTLV-II infection is found among native Ameri-
but can be further discriminated into subtypes 1—6 can Indians in North, Central and South America
by sequencing or restriction enzyme analysis. A and was until recently considered a New World
number of new primate T-lymphotropic virus iso- virus (Figure 25A.4b, see Plate V). At some time
lates fall outside the recognized HTLV/STLV prior to the acquired immune deficiency syndrome
clades (Giri et al., 1994; Goulbau et al., 1994; Liu et (AIDS) epidemic, HTLV-II infection moved into,
al., 1994). and has been spread around the developed world
by, injecting drug users (IDUs) (Hall et al., 1996). In
Europe HTLV-II is common among IDUs in Eire,
Figure 25A.3 (a) The relationship of HTLV-I and STLV-I isolates as determined by an unrooted phylogenetic analysis of the STLV-I/HTLV-I 664 nucleotide consensus LTR
fragment using a neighbour-joining approach. The bootstrap statistical analysis was applied to the neighbour-joining and the Fitch and Wagner parsimony methods (wpars)
using 1000 bootstrap samples. The values on the branches represent the percentage of trees for which the sequences at one end of the branch are a monophylectic group. The
boostrap values for the wpars method are given in parentheses. The geographical origin of the species is indicated where known (? if unknown). The STLV-I host species are
indicated: Cercopithecus aethiops; -o- Macaca nemestrina; o Macaca tonkeana;
Papio hamadryas;
Pan troglodytes. (Reproduced by permission from Salemi, M. et al.
(1998) Virology, 246, 277—287.)
HUMAN T-CELL LYMPHOTROPIC VIRUSES 701
(b) The relationship between members of the HTLV-BLV family. (Modified from Van Brussel et al. (1998) Virology, 243, 366—379)
702 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Spain, Italy and Scandinavia, but rare in Germany The above family studies also indicated that in-
and France. The recent isolation of HTLV-II from a fection was likely to pass from husband to wife. In a
number of African pygmy tribes (Goubau et al., Japanese cohort study of 100 discordant couples
1992; Gessain et al., 1995) and of an HTLV-II-like practising unprotected sexual intercourse there
primate virus in Central Africa suggests that were seven seroconversions during 5 years of obser-
HTLV-II, like HTLV-I, originated in Africa. How- vation. Uninfected females were 3.9 times more like-
ever, a number of questions remain as HTLV-II is ly to become infected than uninfected males (Stuver
widely spread throughout the Americas but has et al., 1993). Higher rates of transmission have been
been found in few populations in Africa. To date reported: 60% of wives of seropositive husbands
PTLVs have not been identified in New World over 10 years in one study, but only 0.4% of hus-
primates and, with the exception of a report of bands of seropositive wives; 50% of wives during
HTLV-II in Mongolia, which may have been a 1—4 years of marriage in another. In a study of
recently imported infection, there is no population- HTLV-I and HTLV-II infected blood donors in the
based evidence of HTLV-II migrating from the old United States, those with higher HTLV-I proviral
to the new world. loads were more likely to have an infected partner.
Studies in Japan show an increasing serop- A similar association of transmission with high
revalence of HTLV-I with age. It has been sugges- proviral load was seen in HTLV-II, although the
ted that this as a cohort effect; however, similar viral burden is generally much less than in HTLV-I
observations have been made in the Caribbean, (Kaplan et al., 1996). Both HTLV-I and HTLV-II
Seychelles and Melanesia which cast doubt on this are more common in females, supporting the im-
explanation. portance of sexual transmission. It is reasonable to
For both viruses transmission may be by three assume that condoms will efficiently protect against
routes: from mother-to-child; through sexual inter- transmission.
course; and through blood—blood contact. Family HTLV-I is transmitted by cell-containing blood
studies in Japan suggested that HTLV-I was mainly products but not by plasma or plasma-derived
transmitted from mother-to-child (Kajiyama et al., products. This has been demonstrated in the rabbit
1986). HTLV-I was identified in lymphocytes in model and through clinical observation. Fresh
breask milk and transmission through breast-feed- blood is more infectious than older blood due to the
ing was demonstrated in marmosets and rabbits. short life of stored lymphocytes. Infection has oc-
The mother-to-child transmission rate in Japan was curred following transfusion of less than 50 ml
25% if babies were breast-fed, but only 5% if babies blood. The incubation period of HTLV-I-asso-
were bottle-fed. However, maternal anti-HTLV ciated myelopathy/tropical spastic paraparesis
antibodies seem to protect breast-feeding infants (HAM/TSP) following infection by transfusion is
until their titre starts to decline, so that in one study shorter than by other routes, and averages 3 years.
in Japan short-term breast-fed babies were no more Approximately 20% of HAM/TSP patients in Ja-
likely to be infected than bottle-fed babies pan have been transfused, suggesting that trans-
(Takahashi et al., 1991). Breast-feeding for 6 months fusion-acquired infection is an important cause of
is associated with higher rates of transmission HAM/TSP in endemic areas. The incidence of
which continue to increase is weaning is further HAM/TSP in Japan has fallen since the introduc-
delayed. Between 3 and 5% of babies are infected tion of blood donor screening. HTLV-I is also
despite avoidance of breast milk. Although HTLV-I transmitted through intravenous drug use but this
has been detected in cord blood, none of these route is more commonly associated with HTLV-II.
babies became infected. HTLV-I has been detected Indeed, outside of the endemic areas of America,
but not quantified in cervicovaginal secretions, and HTLV-II is primarily transmitted through reuse of
the contribution of in utero and during delivery injection paraphernalia. It seems likely that HTLV-
infections to the total infection rate is uncertain. II was introduced into the IDU population of the
There are conflicting data on the mother-to-child USA during the 1970s and into Europe slightly
transmission of HTLV-II but among the Kayapo later.
Indians in Brazil the risk of transmission from an
HTLV-II-infected mother to her child was found to
be 30—50% (Ishak et al., 1995).
HUMAN T-CELL LYMPHOTROPIC VIRUSES 703
Prevalence per 100 000 population
Austria
Belgium
Denmark
France
Germany
Greece
Holland
Italy
Norway
Portugal
Spain
Sweden
UK
Figure 25A.5 Comparison between HTLV-I/II seroprevalence rates in different risk groups in Europe. Blood donors; sexually
transmitted disease clinic attenders; s pregnant women; intravenous drug users. Rates of HTLV-I-I/II among European blood
donors are low but relatively uniform. Rates among the three other groups tend to have much wider 95% confidence intervals due to
the smaller sample size. The seroprevalence rate of (predominantly) HTLV-II among intravenous drug users are approximately
1000-fold higher than among blood donors. The seroprevalence rates of (predominantly) HTLV-I among pregnant women and among
men and women attending clinics for sexually transmitted infections are approximately 100-fold higher than among blood donors.
(Data from G.P. Taylor, 1996)
Figure 25A.6 A blood smear showing characteristic convoluted T cells in ATLL. (Courtesy of Dr E. Matutes and Professor D.
Catovsky)
is also defective frequently in ATLL. Tax sequences disturbing cellular genes important in oncogenesis,
are present in all these conditions. The absence of the transactivating characteristics of Tax assume
both humoral and cellular immune responses in importance. Although adult T-cell leukaemic cells
these exceptional cases makes an understanding of contain randomly clonally integrated HTLV-I
the pathogenesis of these inflammatory conditions which may be defective, tax is preserved (reviewed
even more elusive. by Taylor and McClure, 1998).
Examples of the cellular genes known to be trans-
activated by Tax are listed in Table 25A.2. These
include a number of interleukins, the chain of the
PATHOGENESIS IL-2 receptor, house-keeping genes, such as vimen-
tin, and several ongogenes. Tax has an inhibitory
Knowledge of the pathogenesis of ATLL is incom- effect on -polymerase, a DNA repair gene. This
plete, based to a large extent on in vitro studies of may increase the likelihood of mutagenesis. Tax
HTLV-I and Tax constructs and has to take into does not act directly on the cellular genes, nor in-
account a number of apparent discrepancies. deed on the promoter sequences in the (viral) LTR,
HTLV-I can immortalize T lymphocytes in vitro but binds to a number of specific transcription fac-
but there is low or absent expression of HTLV-I by tors with resulting enhancement of their interaction
adult T-cell leukaemia cells in vivo. Only a small with the target genes. Although others may exist,
proportion (2—4%) of those infected with HTLV-I the three recognized target sequences are the cyclic
develop ATLL, and then only after many decades of AMP-response element (CRE), the NFB binding
infection. Indeed the observation that only a pro- site and the serum responsive element (SRE). Tax
portion of the mothers of patients with HAM but all can bind to one or several members of a family of
the mothers of ATLL patients are carriers of cellular transcription factors, which in turn bind to
HTLV-I suggests that infection during infancy is the CRE promoters. Similarly Tax can bind to vari-
important for the development of ATLL. In ous members of the NFB family of transcription
common with other malignancies it is likely that activators.
transformation is a multistep phenomenon, with The interaction between the IL-2R gene and
the infection of a lymphocyte by HTLV-I being Tax has been of particular interest because of the
only one of several events leading to ATLL. Since high expression of IL-2R (CD25) by T lymphocytes
HTLV-I does not contain an oncogene and malig- in ATLL. IL-2R expression normally only occurs
nant transformation is not related to the integration after antigenic stimulation by the T-cell receptor.
706 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 25A.2 Cellular genes transactivated by HTLV-I Tax
NFB : nuclear factor; CRE : c-AMP response elements; SRE : serum responsive element.
However it appears that Tax is able to induce con- nantly CD8 ; , is followed by demyelination and
stitutive expression of the IL-2R gene. There is a atrophy. HTLV-I is rarely found in the lesions, and
12 bp sequence in the 5 IL-2R gene promoter when detected is probably in circulating CD4 ;
required for tax trans-activation which shows lymphocytes. The peripheral lymphocyte proviral
homology with the NFB binding site. The NFB load is approximately tenfold higher in HAM/TSP
precursor, a p105 heterodimer, is usually found in than in asymptomatic carriers, although there is
the cytoplasm because of masking of its nuclear considerable overlap between the ranges. HTLV-I
localization signal. Tax is able to cause NFB to Tax-specific cytotoxic T lymphocytes (CTLs) are
dissociate from its inhibitor, IB, which results in found in the majority of HAM/TSP patients
increased transport of NFB to the nucleus where (Jacobsen et al., 1990) and asymptomatic carriers
the NFB p50 is able to activate transcription of (Parker et al., 1992), and by limiting dilution assays
IL-2R. A secondary effect of Tax is that the disso- have been found in high frequency (Daenke et al.,
ciation of p50 from the heterodimer releases the p65 1996). Polyclonal expansion of HTLV-I-infected
protein, which is then free to dimerize with the lymphocytes has been reported in patients (without
product of the c-Rel oncogene. This p65-Rel hetero- malignancy) with high provial load, particularly
dimer is also able to activate the NFB motif. Thus patients with HAM/TSP. As in the oligo/mon-
Tax may further enhance the activation of the many oclonal proliferation of T lymphocytes in ATLL,
genes under the influence of NFB. viral replication in these cells is occurring through
One theory of T cell transformation by HTLV-I cell division, using cellular polymerases without the
has been that Tax, by promoting activity of both need for reverse transcription and integration.
IL-2R and IL-2 genes, gives rise to continuous pro- Thus, HTLV-I appears to be able to replicate
liferation of the cells by positive feedback—the through two quite distinct mechanisms, cell division
autocrine loop theory. However, stimulation of and virion production. The relative contribution of
Tax-transduced T cells can occur in an IL-2-inde- ‘cellular’ and ‘viral’ replication to HTLV-I proviral
pendent manner, tax-transfected cells continue to load in patients with HAM/TSP and in asympto-
grow after anti-CD3 stimulation in the absence of matic carriers is uncertain. The continuous presence
IL-2 and not all tax-transformed T cells express of a strong anti-Tax CTL response suggests conti-
IL-2, although they all express IL-2R. nuing expression of Tax by some cells. It is possible
The pathogenesis of HAM/TSP is also incom- that this may drive the proliferation of the clonally
pletely understood. It is rare to obtain histopathol- expanding cell populations.
ogy at the time of the initial symptoms but a There are three main theories concerning HAM/
perivascular lymphocytic infiltration of the spinal TSP pathogenesis: (1) the inflammatory response is
cord, which is at first CD4 ; and later predomi- directed against virus in the CNS; (2) the inflamma-
HUMAN T-CELL LYMPHOTROPIC VIRUSES 707
tory response against HTLV-I targets similar host Enhanced transcription of the multidrug resistance
CNS peptides: and (3) CNS tissue is an innocent by gene (MDR1) with significant P glycoprotein-me-
stander, damaged when CTLs recognize migrating, diated drug efflux in the T cells of HTLV-I-infected
HTLV-I-expressing, CD4 ; cells (Höllsberg, individuals, with and without malignancy, has been
1997). demonstrated, as has the ability of HTLV tax pro-
Although HAM/TSP is associated with high tein to activate the MDR1 promoter. Other treat-
proviral load, differences in the rates of HAM/TSP ments such as deoxycoformycin, interferons and ,
per HTLV-I infected subjects in different popula- topoisomerase II and, since CD25 is expressed by
tions suggests that other, possibly, host factors are all ATLL cells, anti-Tac (CD25) antibodies have
important. Of the candidate factors, HLA types been tried, with limited success. Y-labelled anti-
have attracted considerable interest. HLA types are Tac antibodies are currently under investigation.
known to be associated with other inflammatory Successful eradication, not only of ATLL cells but
diseases but could also influence outcome by con- also of HTLV-I infection, has been reported follow-
trolling viral replication. In Japanese ATLL pa- ing bone marrow transplantation (BMT) but pa-
tients HLA-A26, -B61 and -DR9 were found at an tients often fail to survive to or through BMT.
increased frequency, while HLA-A24 and HLA- Improved remission rates and survival have been
Cw1 were less frequently found than in controls. reported in uncontrolled studies by two groups us-
Conversely, in patients with HAM/TSP, HLA- ing the combination of interferon and zidovudine
Cw7, -B7 and -DR1 were found more commonly (Gill et al., 1995, Hermine et al., 1995). The mechan-
than in controls and patients with ATLL. It has ism of activity of these compounds in ATLL is not
been suggested that in Japan the clear; they are not effective when given indepen-
A26Cw3B61DR9DQ3 haplotype is representative dently and do not appear to have a cytotoxic effect,
of ATLL and is associated with a low immune although zidovudine was originally identified as a
response, while A24Cw7B7DR1DQ1 is the repre- potential anticancer therapy. Interest in this treat-
sentative haplotype of HAM/TSP and is associated ment followed anecdotal improvement in a patient
with a high immune response. Among different eth- with ATLL and HIV. The immune suppression seen
nic groups HLA class I haplotypes were variable in patients with ATLL is more severe than in other
but examination of class II suggested that some malignancies, and prophylaxis against Pneumocys-
haplotypes associated with disease are panethnic, tic carinii pneumonia is recommended.
while others are ethnospecific (Sonoda et al., 1996). HAM/TSP is also difficult to treat, and there
HLA-A02 seems to offer some protection against have to date been no controlled studies. Early re-
HAM/TSP by more efficient control of viral repli- ports of the use of pulsed high-dose steroids, ster-
cation (Jeffery et al., 1999). oid-sparing cytotoxics and plasmapheresis, all tar-
geting the immune response, were encouraging
(Osame et al., 1990) but no long-term improvements
have been reported (Matsuo et al., 1990). High-dose
TREATMENT vitamin C (Kataoka et al., 1993) and oxpentifylline
have also been favoured (Shirabe et al., 1997). Ex-
There are very few instances of successful treatment tensive research into the treatment of HIV-1 has
for ATLL reported, and no reports of controlled resulted in the licensing of three classes of antiret-
clinical trials. Until recently the overall manage- roviral therapy. Unfortunately the protease inhibi-
ment has been to treat the disease not as if it were tors have no activity against HTLV-I. The nucleo-
associated with a virus but as a clinical oncological side analogue reverse transcription inhibitors
problem, and the regimens for non-Hodgkin’s lym- (NRTIs) zidovudine and zalcitabine have been
phoma such as CHOP (cyclophosphamide, shown to be active against HTLV-I in vitro and in
adriamycin, vincristine and prednisolone) are fa- animal models. Unfortunately zalcitabine and two
voured. Although successive improvements in other NRTIs, didanosine and stavudine, are neur-
chemotherapy have increased remission rates to otoxic. Zidovudine was reported to improve mobil-
42%, ATLL is essentially a highly drug-resistant ity in ambulant patients in one study (Sheremata et
malignancy: survival time remains less than 12 al., 1993) but not in another (Gout et al., 1991).
months for acute and lymphomatous presentations. Neither reported proviral load measurements.
708 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
When considering the management of HAM/TSP it can be prevented by avoidance of breast-feeding
is important to realize that in longstanding disease (Hino et al., 1987). In Japan, carrier mothers are
demyelination and atrophy may rule out improve- identified through antenatal screening pro-
ment even if successful reduction of virus or of grammes, resulting in reduced transmission in so far
damaging cytokines has been effected. The bladder as these mothers refrain from breast-feeding. In the
spasticity, frequently, nocturia and incontinence are UK the prevalence of HTLV-I/II infection in
often distressing. The anabolic steroid Danazol has women attending metropolitan antenatal clinics is
been reported to improve these symptoms. Recent- approximately 1:200 (cf. HIV-1) but HTLV-I anti-
ly, intravesical instillation of capsaicin has been body testing has never been offered as a routine part
shown to reduce the bladder spasticity in these of antenatal care. Although breast-feeding is less
patients, with symptomatic improvement lasting common in the UK, there is now increasing concern
months (Dasgupta et al., 1996). to prevent the transmission of other viral infections
by this route and recognition that, when offered,
there is a high uptake by the mothers of interven-
tions to reduce transmission. Reduction of HTLV-I
PREVENTION OF DISEASE transmission from mother to infant is likely to pre-
vent most cases of end-stage HTLV-associated dis-
Although the association of HAM/TSP with high ease in the UK (ATLL by preventing infection of
proviral load suggests that proviral load reduction the infant, HAM/TSP by preventing both early and
with antiretroviral therapy may in the future be part later sexual transmission), although the effect on the
of the treatment or prevention of this disease, cur- incidence of disease would not be seen for several
rent strategies must be directed at preventing infec- decades.
tion. Vaccines are currently being tested in animal
models. Two transmission routes can be relatively
easily targeted. Many blood transfusion services ACKNOWLEDGEMENTS
now include HTLV-I/II antibody screening of all or
of new donors. This was first introduced in Japan in This chapter updates the section on HTLV from the
1985 where blood transfusion accounted for up to third edition. The cooperation of Robin Weiss and
60% of seroconversions in the Kyushu region of Gus Dalgleish in writing this version has been in-
southwest Japan (Kamihira et al., 1987). Blood do- valuable, as has the constructive criticism of Dr
nor screening has been standard practice in the Philip Mortimer of the Central Public Health Lab-
United States and Australia for many years and has oratory, Colindale.
more recently been introduced in South American
countries. With the commercial production of suit-
ably sensitive and specific screening assays, HTLV-
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type I. Acta Neurologica Scandinavica. Supplementum, 169, outcomes in human T lymphotropic virus types I- and II-
86—93. infected blood donors. Journal of Infectious Diseases, 176,
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Principles and Practice of Clinical Virology. Edited by Arie J. Zuckerman, Jangu E. Banatvala and John R. Pattison
Copyright © 2000 John Wiley & Sons Ltd
ISBNs: 0-471-97340-8 (Hardback); 0-470-84247-4 (Electronic)
26
Principles and Practice of Clinical Virology, Fourth Edition. Edited by A. J. Zuckerman, J. E. Banatvala and J. R. Pattison.
© 2000 John Wiley & Sons, Ltd.
712 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Table 26.1 The prion diseases
PrP1 is formed from cellular PrP (PrP!) by a post- disorders in which mutant PrP! is converted into
translational process in which PrP1 acquires a high mutant PrP1 and the accumulation of PrP1 pro-
-sheet content (Pan et al., 1993). Several lines of duces central nervous system (CNS) dysfunction
evidence argue that PrP1 formation occurs in (Gabizon et al., 1996). Thus, there is no evidence
caveolae-like domains (CLDs) near the surface of that a deficiency of PrP! is responsible for malfunc-
the cell. It seems likely that a protein provisionally tion of the brain in any of the prion diseases.
designated protein X facilitates the conversion of
PrP! into PrP1.
Although genetic ablation of the PrP gene caused
disease in two lines of PrP-deficient (Prnp) mice PRION DISEASES ARE SPORADIC,
at about 18 months of age, two other lines remained GENETIC AND INFECTIOUS
healthy (Büeler et al., 1992; Manson et al., 1994;
Sakaguchi et al., 1996). The Prnp mice were resis- The four human prion diseases, often referred to as
tant to scrapie and did not replicate prions (Büeler kuru, CJD, GSS and FFI, are variants of the same
et al., 1993; Prusiner et al., 1993). Restoring PrP disorder and thus share many features. The human
expression by genetic crosses rendered the mice prion diseases manifest as sporadic, genetic and
susceptible to prions and made them permissive for infectious disorders of the CNS. All three forms of
prion replication. The susceptible mice developed the human prion diseases have been transmitted to
disease with incubation times of less than 3 months. experimental animals (Gajdusek, 1977; Telling et
These findings are in accord with the inherited hu- al., 1996b). Kuru is thought to have been spread
man prion diseases, which are autosomal dominant exclusively through the infectious mechanism by
HUMAN PRION DISEASES 713
ritualistic cannibalism (Gajdusek, 1977). Table 26.2 Glossary of prion terminology
Although a few CJD cases can be traced to inocu-
Prion Proteinaceous infectious particle that lacks
lation with prions, i.e. human growth hormone nucleic acid. Prions are composed largely, if not
(HGH), cornea transplantation and cerebral elec- entirely, of PrP1 molecules. They can cause
trode implantation, most are sporadic, despite con- scrapie in animals and related
siderable efforts to implicate scrapie-infected sheep neurodegenerative diseases of humans such as
Creutzfeldt—Jakob disease (CJD). ‘Scrapie
as an exogenous source of prions (Cousens et al., agent’ is a synonym
1990; Fradkin et al., 1991). Although sCJD could be PrP1 Scrapie isoform of the prion protein. This
explained by prions being ubiquitous in our food protein is the only identifiable macromolecule
chain, with their efficiency of infection being very in purified preparations of scrapie prions
low, there is no evidence to support this hypothesis. PrP! Cellular isoform of the prion protein
PrP 27—30 Digestion of PrP1 with proteinase K generates
Of note, infection by the oral route is 10 times less PrP 27—30 by truncation of the N-terminus
efficient than intracerebral inoculation in hamsters. PRNP PrP gene located on human chromosome 20
Whether or not sCJD can arise endogenously with- Prnp PrP gene located on mouse chromosome 2.
out any exogenous prion source remains to be es- Prnp is congruent with the Sinc and Prn-i genes
tablished, but somatic mutation of the PrP gene or that control scrapie incubation times in mice
Pid-1 A locus on mouse chromosome 17 that appears
the infrequent spontaneous conversion of PrP! into to influence experimental CJD and scrapie
PrP1 seem to be likely explanations for sCJD incubation times
(Prusiner, 1998). Prion rod An aggregate of prions composed largely of
About 10—15% of CJD and virtually all cases of PrP 27—30 molecules. Created by detergent
GSS are inherited. Familial CJD (fCJD) and GSS as extraction and limited proteolysis of PrP1.
Morphologically and histochemically
well as FFI are caused by germline mutations in the indistinguishable from many amyloids
PrP gene. Five mutations of the PrP gene have been PrP amyloid Amyloid containing PrP in the brain of animals
genetically linked to the development of the human or humans with prion disease; often
prion diseases. Although some investigators argue accumulates as plaques
that PrP! is a receptor for the putative scrapie
‘virus’, and mutations in PrP! render people more
susceptible to this ubiquitous ‘virus’, considerable ferent biochemical and biophysical properties.
evidence militates against such a hypothesis. Limited proteolysis of PrP1 produces a smaller
protease-resistant molecule of : 142 amino acids
designated PrP 27—30; under the same conditions,
PrP! is completely hydrolysed. In the presence of
TERMINOLOGY detergent, PrP 27—30 polymerizes into amyloid
rods. These prion amyloid rods formed by limited
The term ‘prion’ is used to denote the small pro- proteolysis and detergent extraction are indistin-
teinaceous infectious particles that lack nucleic acid guishable from the filaments that aggregate to form
(Table 26.2) (Prusiner, 1998). Prions cause scrapie PrP amyloid plaques in the CNS. Both the rods and
and other related transmissible neurodegenerative the PrP amyloid filaments found in brain tissue
diseases of animals and humans (Table 26.1). Prions exhibit similar ultrastructural morphology and
are composed largely, if not entirely, of a protein green-gold birefringence after staining with Congo
designated as the scrapie isoform of the prion pro- red dye. To differentiate the amyloid plaques found
tein designated PrP1. A post-translational confor- in the prion diseases from those found in aged
mational change generates PrP1 from the normal, brains, Alzheimer’s disease (AD) and Down’s syn-
cellular isoform of the prion protein denoted PrP!. drome, it has been suggested that the former be
A major feature that distinguishes prions from vi- labeled PrP plaques and the latter be called A
ruses is the finding that both PrP isoforms are plaques.
encoded by a chromosomal gene. In humans, the Four diseases of humans are caused by prions or
PrP gene is designated PRNP and is located on the mutations in the PrP gene (Table 26.1). Whereas
short arm of chromosome 20. In mice, the PrP gene kuru is confined to the mountainous Fore region of
is designated Prnp and is located on chromosome 2. Papua New Guinea, the other three diseases are
PrP1 is readily distinguished from PrP! by its dif- found worldwide. Distinguishing between these
714 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
three disorders has grown increasingly difficult with hamsters accelerated purification of the infectious
the recognition that fCJD, GSS and FFI are particles (Prusiner et al., 1980). Bioassays for trans-
autosomal dominant diseases that are caused by mission of the human prion diseases to experimen-
mutations in the PRNP gene. Initially, we thought tal animals were initially confined to apes and mon-
that a specific PrP mutation was very frequently keys (Brown et al., 1994). The incubation periods
associated with a particular clinical and neur- were quite prolonged, which made experimental
opathological presentation. Although that is often studies difficult. Subsequently, CJD and GSS were
the case, an increasing number of exceptions are transmitted to laboratory rodents; only a few ani-
beginning to accumulate. In a single family with a mals developed disease after prolonged incubation
particular PrP mutation, different clinical and neur- times of 500 days or more on primary passage.
opathologic manifestations of the same genetic dis- Fewer cases have been transmitted to goats, mar-
ease can be seen (Hsiao et al., 1989; Johnson and mosets, cats and laboratory rodents (Gajdusek,
Gibbs, 1998). These different constellations of CNS 1977). On second passage, the incubation time was
symptoms, signs and neuropathological lesions reduced when homologous prions were inoculated.
would seem to render the old system of classifica- Before the molecular mechanisms responsible for
tion obsolete because the precise chemical cause is prion replication were elucidated, most investiga-
now known. Instead, it has been suggested that tors assumed that prions that had originated in
these disorders be labelled prion diseases followed humans but were subsequently passaged in mice
by the mutation. For example, many patients with a were different from those that had originated in
PrP mutation at codon 117 present with a dement- sheep and were then passaged in mice. We now
ing disorder, in which numerous PrP amyloid understand that, regardless of the origin, prions
plaques characteristic of GSS are found (Doh-ura et passaged in mice become mouse prions on primary
al., 1989; Hsiao et al., 1991); however, other patients passage (Prusiner, 1998). Having stated that prions
with the same mutation present primarily with passaged in mice become mouse prions on the first
ataxia (Mastrianni et al., 1995). passage, it is noteworthy that the original strain of
Although the above proposal for a new terminol- prions may persist or be changed when prions are
ogy may eventually prove preferable because it is passaged from one species to another.
based on the molecular lesion, some clinicians cur- Transgenic (Tg) mice have revolutionized the
rently prefer the old terminology in which CJD study of prion diseases. Mice expressing human
usually connotes a dementing illness and GSS an (Hu) or chimeric Hu/mouse (Mo) PrP transgenes
ataxic disorder in which numerous PrP amyloid provide a much more rapid and economical means
plaques are found at autopsy. In the interest of of studying Hu prions and permit the preservation
clarity, much of the older terminology is retained in of the strain of prions found in the Hu specimen
this chapter. (Telling et al., 1996b; Prusiner, 1998). Similarly,
mice expressing bovine (Bo) PrP transgenes allow
the study of BSE prions (Scott et al., 1997b).
MEASUREMENT OF PRION
INFECTIVITY
PRION DISEASES OF ANIMALS
The experimental transmission of scrapie from
sheep to mice (Chandler, 1961) gave investigators a Scrapie of Sheep and Goats
more convenient laboratory model, which yielded
considerable information on the nature of the un- Although scrapie was recognized as a distinct dis-
usual infectious pathogen that causes scrapie (Alper order of sheep with respect to its clinical manifesta-
et al., 1967). Yet progress was slow because quantifi- tions as early as 1738, the disease remained enig-
cation of infectivity in a single sample required hol- matic even with respect to its pathology for more
ding 60 mice for 1 year before accurate scoring than two centuries. Some veterinarians thought
could be accomplished (Chandler, 1961). that scrapie was a disease of muscle caused by para-
The availability of a more rapid and economical sites, whereas others thought that it was a dystro-
bioassay for the scrapie agent in Syrian golden phic process. An investigation into the aetiology of
HUMAN PRION DISEASES 715
scrapie followed the vaccination of sheep for loop- negative. Studies on the mapping of the site of inter-
ing ill virus with formalin-treated extracts of ovine action of PrP! with protein X provide an explana-
lymphoid tissue unknowingly contaminated with tion for this phenomenon (Kaneko et al., 1997).
scrapie prions (Gordon, 1946). Two years later, Substitution of an R in PrP! at the site where
more than 1500 sheep developed scrapie from this protein X binds abolished PrP1 formation. Those
vaccine. studies suggested that the mutated PrP! binds to
Even after the transmissibility of scrapie became protein X but is not released, and thus acts as a
well established, the spread of scrapie within and dominant negative.
among flocks of sheep remained puzzling. Parry
argued that host genes were responsible for the
development of scrapie in sheep. He was convinced Bovine Spongiform Encephalopathy
that natural scrapie is a genetic disease that could
be eradicated by proper breeding protocols (Parry, Prion strains and the species barrier are of para-
1983). He considered its transmission by inocula- mount importance in understanding the BSE epi-
tion of importance primarily for laboratory studies demic in Great Britain, in which it is estimated that
and communicable infection of little consequence in almost one million cattle were infected with prions
nature. Other investigators viewed natural scrapie (Anderson et al., 1996; Nathanson et al., 1997). The
as an infectious disease and argued that host gen- mean incubation time for BSE is about 5 years.
etics only modulates susceptibility to an endemic Most cattle therefore did not manifest disease be-
infectious agent. The incubation time gene for ex- cause they were slaughtered between 2 and 3 years
perimental scrapie in Cheviot sheep called Sip is of age (Stekel et al., 1996). Nevertheless, more than
said to be linked to a PrP gene restriction fragment 175 000 cattle, primarily dairy cows, have died of
length polymorphism (Hunter et al., 1989), a situ- BSE over the past decade (Figure 26.2a) (Anderson
ation perhaps analogous to the locus initially called et al., 1996). BSE is a massive common-source epi-
Sinc in mice. demic caused by meat and bone meal (MBM) fed
primarily to dairy cows (Wilesmith et al., 1991;
PrP gene Polymorphisms and Scrapie Nathanson et al., 1997). The MBM was prepared
from the offal of sheep, cattle, pigs and chickens as a
In sheep, polymorphisms at codons 136, 154 and high protein nutritional supplement. In the late
171 of the PrP gene have been studied with respect 1970s, the hydrocarbon-solvent extraction method
to the occurrence of scrapie (Figure 26.1, see Plate used in the rendering of offal began to be aban-
VI) (Goldmann et al., 1990a). Studies of natural doned, which resulted in MBM with a much higher
scrapie in the USA have shown that : 85% of the fat content (Wilesmith et al., 1991). It is now
afflicted sheep are of the Suffolk breed. Only those thought that this change in the rendering process
Suffolk sheep homozygous for Gln (Q) at codon 171 allowed scrapie prions from sheep to survive ren-
were found with scrapie, although healthy controls dering and to be passed into cattle. Alternatively,
with QQ, QR and RR genotypes were also found bovine prions were present at low levels prior to
(Westaway et al., 1994). These results argue that modification of the rendering process and, with the
susceptibility in Suffolk sheep is governed by the processing change, survived in sufficient numbers to
PrP codon 171 polymorphism. Indeed, sheep en- initiate the BSE epidemic when inoculated back
coding the R/R polymorphism at position 171 seem into cattle orally through MBM. Against the latter
to be resistant to scrapie (Hunter et al., 1997); pre- hypothesis is the widespread geographical distribu-
sumably, this was the genetic basis of Parry’s tion throughout England of the initial 17 cases of
scrapie eradication programme in Great Britain 30 BSE, which occurred almost simultaneously (Wile-
years ago (Parry, 1983). In Cheviot sheep, the PrP smith et al., 1991; Nathanson et al., 1997). Further-
codon 171 polymorphism has a profound influence more, there is no evidence of a pre-existing prion
on susceptibility to scrapie, as in Suffolks, but disease of cattle, either in Great Britain or else-
codon 136 also seems to modulate susceptibility. where.
Since sheep heterozygous for R171 appear
equally resistant as homozygotes, the substitution
of this basic residue appears to act as a dominant
716 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Figure 26.2 Disappearance of the kuru and BSE epidemics. (a) Number of annual cases of BSE in cattle in Great Britain. (b) Number
of biannual cases of kuru in Papua New Guinea. (Data compiled for BSE by John Wilesmith and for kuru by Michael Alpers; from
Prusiner, S.B. (1997) Science, 278, 245—251)
Origin of BSE Prions? phisms found in sheep, only one PrP polymorphism
has been found in cattle. Although most bovine PrP
The origin of the bovine prions causing BSE cannot
alleles encode five octarepeats, some encode six.
be determined by examining the amino acid se-
PrP alleles encoding six octarepeats do not seem to
quence of PrP1 in cattle with BSE because the
be overrepresented in BSE (Figure 26.1, see Plate
PrP1 in these animals has the bovine sequence,
VI) (Hunter et al., 1994).
whether the initial prions in MBM came from cattle
Brain extracts from BSE cattle cause disease in
or sheep. The bovine PrP sequence differs from that
cattle, sheep, mice, pigs and mink after intracerebral
of sheep at seven or eight positions (Goldmann et
inoculation (Bruce et al., 1997), but prions in brain
al., 1990b). In contrast to the many PrP polymor-
extracts from sheep with scrapie fed to cattle pro-
HUMAN PRION DISEASES 717
duced illness substantially different from BSE SPORADIC CREUTZFELDT–JAKOB
(Robinson et al., 1995). However, no exhaustive DISEASE
effort has been made to test different strains of sheep
prions or to examine the disease following bovine to The recognition that the neuropathology of a cere-
bovine passage. The annual incidence of sheep with bellar disorder of New Guinea natives was similar
scrapie in Britain over the past two decades has to that of scrapie prompted Hadlow to suggest that
remained relatively low (J. Wilesmith, unpublished transmission studies in apes be performed with ex-
data). In July 1988, the practice of feeding MBM to tracts of brain tissue from patients dying of kuru
sheep and cattle was banned. Recent statistics argue (Hadlow, 1959). The success of those studies (Gaj-
that the epidemic is now disappearing as a result of dusek et al., 1966) was followed by the transmission
this ruminant feed ban (Figure 26.2a) (Anderson et of CJD to apes (Gibbs et al., 1968), based on the
al., 1996), reminiscent of the disappearance of kuru earlier recognition that the neuropathological
in the Fore people of New Guinea (Gajdusek, 1977) changes in kuru were similar to those found in CJD
(Figure 26.2b). (Figure 26.4a, see Plate VIII) (Klatzo et al., 1959). In
1920, Creutzfeldt reported the case of a 23-year-old
woman who died of a neurologic disease, and the
Monitoring Cattle for BSE Prions following year Jakob reported five cases. Ironically,
some investigators doubt that Creutzfeldt described
Although more than 30 million cattle of age 30 the disease that now bears his name (Richardson
months or older have been culled from herds to and Masters, 1995).
minimize BSE-infected tissues from entering the hu- Infectious, sporadic and inherited forms of CJD
man food chain, it seems more important to moni- are now recognized. The only documented cases of
tor the frequency of prion infection in asympto- the infectious form of CJD are iatrogenic. The great
matic cattle. It is estimated that more than one majority of CJD cases are sporadic, whereas
million cattle in Great Britain were infected with 10—15% of cases are familial and inherited as an
BSE prions over the past 15 years (Anderson et al., autosomal dominant trait with variable penetrance.
1996). How much the slaughter of older cattle has
reduced the apparent incidence of BSE (Figure
26.2a) is unclear.
No reliable, specific test for prion infection in live Epidemiology
animals is available, but immunoassays for PrP1 in
the brainstems of cattle might provide a reasonable CJD is found throughout the world. The incidence
approach to establishing the incidence of subclini- of sporadic CJD is approximately one case per mil-
cal BSE in cattle entering the human food chain lion population (Masters et al., 1978). Although
(Safar et al., 1998). Determining how early in the many geographical clusters of CJD have been re-
incubation period PrP1 can be detected by immu- ported, each has been shown to segregate with a
nological methods is now possible because a re- PRNP gene mutation, which results in a non-con-
liable bioassay has been created by expressing the servative substitution. Attempts to identify a
BoPrP gene in Tg mice (Scott et al., 1997b). Prior to common exposure to some aetiologic agent have
development of Tg(BoPrP)Prnp mice, non-Tg been unsuccessful, both for the sporadic and famil-
mice inoculated intracerebrally with BSE brain ex- ial cases, to date (Cousens et al., 1990). Some fami-
tracts required more than 300 days to develop dis- lies have multiple cases of both CJD and AD. The
ease. Depending on the titre of the inoculum, the relationship, if any, between CJD and AD remains
structures of PrP! and PrP1 and the structure of to be established.
protein X, the number of inoculated animals devel- Ingestion of scrapie-infected sheep or goat meat
oping disease can vary over a wide range. Some as a cause of CJD in humans has not been shown by
investigators have stated that transmission of BSE epidemiologic studies, although speculation about
to mice is quite variable, with incubation periods this potential route of inoculation continues (Joh-
exceeding 1 year, whereas others report low prion nson and Gibbs, 1998). On the other hand, it is
titres in BSE brain homogenates compared to ro- assumed that the transmission of kuru among New
dent brain scrapie. Guinea tribesmen occurred after the consumption
718 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
of kuru-infected brain during ritualistic cannibal- Myoclonus
ism (Gajdusek, 1977). Studies with Syrian hamsters
provided convincing evidence that the oral route of Most patients (:90%) with CJD exhibit myoc-
inoculation, although extremely inefficient, can, lonus appearing at various times throughout the
with regularity, be a source of prion infection illness (Kirschbaum, 1968; Roos et al., 1973;
(Prusiner et al., 1985). Cathala and Baron, 1987). Unlike other involun-
tary movements, myoclonus persists during sleep.
Startle myoclonus elicited by loud sounds or bright
lights is frequent. It is important to stress that my-
General Clinical Features oclonus is neither specific nor confined to CJD.
Dementia with myoclonus can also be due to AD as
Non-specific prodromal symptoms occur in about a well as to cryptococcal encephalitis or Unver-
third of CJD patients and may include fatigue, sleep richt—Lundborg disease, etc.
disturbance, weight loss, headache, general malaise,
and ill-defined pain (Kirschbaum, 1968; Roos et al.,
1973; Cathala and Baron, 1987; Johnson and Gibbs,
1998). The majority of CJD patients present with Electroencephalography
deficits in higher cortical function. These deficits
virtually always progress to a state of profound The electroencephalogram (EEG) is often useful in
dementia, characterized by memory loss, impaired the diagnosis of CJD. During the early phase of
judgement and a decline in virtually all aspects of CJD, the EEG is usually normal or shows only
intellectual function (Nevin et al., 1960). A minority scattered theta activity. In most advanced cases,
of patients present with either visual impairment or repetitive, high voltage, triphasic and polyphasic
cerebellar gait and coordination deficits. Frequent- sharp discharges are seen. The presence of these
ly, the cerebellar deficits are rapidly followed by stereotyped periodic bursts of 200 ms duration,
progressive dementia (Gomori et al., 1973). Visual occurring every 1—2 s, makes the diagnosis of CJD
problems often begin with blurred vision and di- very likely (Nevin et al., 1960; Kirschbaum, 1968;
minished acuity, rapidly followed by dementia. Cathala and Baron, 1987; Johnson and Gibbs,
Generally, patients with CJD are between 50 and 1998). These discharges are frequently but not al-
75 years of age; however, patients as young as 17 ways symmetrical: there may be a one-sided pre-
and as old as 83 have been recorded (Masters et al., dominance in amplitude. As CJD progresses, nor-
1978; Cathala and Baron, 1987; Holman et al., mal background rhythms become fragmentary and
1996). slower. The appearance of these periodic electrical
Other symptoms and signs include ex- complexes during the clinical course of CJD is vari-
trapyramidal dysfunction manifested as rigidity, able and in many cases their presence is transient.
mask-like facies or choreoathetoid movements;
pyramidal signs (usually mild); seizures (usually ma-
jor motor) and, less commonly, hypoaesthesia;
supranuclear gaze palsy; optic atrophy; and vegeta-
tive signs such as changes in weight, temperature, Clinical Course
sweating or menstruation (Kirschbaum, 1968;
Cathala and Baron, 1987). One study indicates that In documented cases of accidental transmission of
lower motor neuron disease in association with a CJD to human subjects, an incubation period of
progressive dementing syndrome is not transmiss- 1.5—2.0 years preceded the development of clinical
ible to apes and monkeys (Salazar et al., 1983). disease (Duffy et al., 1974; Bernouilli et al., 1977). In
Based on these findings, the authors argue that the other cases, incubation periods of up to 30 years
term ‘amyotrophic Creutzfeldt—Jakob disease’ is have been suggested. Most patients with CJD live
not a useful label. 6—12 months after the onset of clinical signs and
symptoms (Kirschbaum, 1968; Masters et al., 1978;
Cathala and Baron, 1987), whereas some live for up
to 5 years.
HUMAN PRION DISEASES 719
Brain Biopsy ter, less expensive and probably more sensitive than
those in non-human primates.
If the constellation of pathological changes fre-
quently found in CJD is seen in a brain biopsy, then
the diagnosis is reasonably secure (Figure 26.4a,
and b, see Plate VIII). However, the enthusiasm for Immunological Studies
brain biopsies in patients with suspected CJD is
quite low for two reasons: first, there is no specific The rapid and reliable diagnosis of CJD post mor-
effective treatment for CJD, and second, decon- tem can be accomplished by using antisera to PrP.
tamination of surgical instruments requires special Initially, partial purification of 1—2 g of infected
protocols, described below. brain tissue using detergent extractions, differential
centrifugation and enzyme digestions was required
to detect protease-resistant PrP1 in cases of CJD
analysed by Western immunoblots (Bockman et al.,
Transmission to Animals 1987). Brains from control patients with anoxic en-
cephalopathy or AD did not contain these pro-
CJD has been transmitted to a variety of laboratory tease-resistant, immunoreactive prion proteins.
animals. Over 300 cases of human prion disease Subsequently, two additional procedures were de-
have been transmitted to apes and monkeys (Brown veloped for diagnostic evaluations of CJD; one pro-
et al., 1994). Fewer cases have been transmitted to tocol used a dot blot whereby the sample was first
goats, marmosets, cats and laboratory rodents. digested with proteinase K and then denatured with
Typically, only a minority of these non-primates GdnHCl (Serban et al., 1990). The other protocol
develop prion disease after inoculation with human was designated histoblotting and utilized the same
brain extracts from patients who died of prion dis- limited proteolysis followed by GdnHCl denatura-
ease. Moreover, the incubation times are greatly tion to enhance PrP1 antigenicity (Taraboulos et
prolonged upon transmission from humans to a al., 1992). These immunoassays have, for the most
relatively distant species. Generally, the more simi- part, replaced transmission studies using apes and
lar the PrP sequences are for two species, the more monkeys.
readily prions are transmitted between them. After Numerous Western blotting studies have consist-
the inoculated animals develop signs of neurologi- ently demonstrated PrP immunoreactive proteins
cal dysfunction, a progressive impairment of the that are proteinase K-resistant in the brains of pa-
CNS ensues, with death following in a few weeks. tients with CJD. Although there is wide agreement
With a few exceptions, the neuropathological about the specificity of PrP1, some investigators
changes in animals are roughly similar to those have tried to use migration and intensity of pro-
found in humans. teinase K-resistant PrP1 on Western blots. The
Extracts of brains from patients who died with usefulness of such approaches clinically remains to
sporadic CJD, fCJD(E200K), fCJD(V210I), and be established (Collinge et al., 1996; Parchi et al.,
FFI readily transmit disease to mice expressing a 1996), but it has proved quite efficacious in some
chimeric Hu/Mo (MHu2M) PrP transgene (Telling experimental studies (Telling et al., 1996b). It is
et al., 1995, 1996b). In contrast to non-Tg mice noteworthy that PrP1 is not uniformly distributed
inoculated with brain extracts that required 500 throughout the CNS so that the apparent absence
days for : 10% of the animals to develop CNS of PrP1 in a limited sample such as a biopsy does
disease (Tateishi et al., 1996), all of the Tg(MHu2M, not rule out prion disease. Although PrP1 is found
P102L)Prnp mice showed signs of neurological in the thalamus of patients who die of FFI, it is
dysfunction : 200 days after inoculation. Human frequently absent from other regions, such as the
CJD prions from British and American patients cerebral cortex and the cerebellum (Monari et al.,
seem to transmit disease infrequently to non-Tg 1994).
mice, whereas most Japanese cases readily transmit Since considerable data indicate that PrP1 is an
disease to : 10% of the inoculated animals essential, and very probably the only, component of
(Tateishi et al., 1996). Mice expressing appropriate the prion, we expect to find PrP1 in all cases of
PrP transgenes offer bioassays that are much shor- prion disease (Prusiner, 1998). Although many cases
HUMAN PRION DISEASES 721
of human and animal prion diseases are accom- inoculation, especially with neural tissues and in-
panied by the accumulation of a protease-resistant cluding formalin-fixed specimens, is potentially
form of PrP1, protease resistance is not an absolute very hazardous (Gorman et al., 1992). Electroen-
requirement (Telling et al., 1996a). cephalographic and electromyographic needles
Using a new immunoassay for PrP1 that does should not be reused after studies on CJD patients
not depend on the protease resistance of PrP1 to have been performed.
distinguish it from PrP!, it is likely that cases of There is no rational, scientific reason for surgeons
prion disease will be identified in which the deposi- or nurses to resist performing biopsies on demented
tion of protease-resistant PrP1 is minimal. This patients when this operation is warranted medi-
conformation-dependent immunoassay (CDI) de- cally. Likewise, there is no reason for pathologists
pends on the use of antibodies that react with epi- or mortuary attendants to resist performing au-
topes of PrP that are exposed in PrP! but become topsies on patients whose clinical diagnosis was
buried in PrP1 (Safar et al., 1998). The immunoas- CJD. Progress in the diagnosis, care and treatment
say measures the increase in immunoreactivity that of CJD patients requires the dedicated efforts of
occurs when the cryptic epitopes are exposed by physicians, nurses and other healthcare workers.
denaturation with GdnHCl. The assay is extremely The standard microbiological practices outlined
sensitive because the antibody is labelled with Eu, here, together with specific recommendations for
which can be measured by time-resolved fluor- decontamination, seem to be adequate precautions
escence (TRF); in fact, the assay is so sensitive that for the care of CJD patients and the handling of
about 10 PrP1 molecules in a sample can be meas- infected specimens.
ured, which is equivalent to one ID unit.
PrP immunostaining of amyloid plaques is diag-
nostic of prion disease, but a minority of CJD cases
exhibit PrP plaques. In 16 of 17 CJD cases with Decontamination of Prions
amyloid plaques (94%), we have found PrP im-
munoreactive plaques (Roberts et al., 1988). Be- Prions are extremely resistant to common inactiva-
cause not all cases of CJD have readily identifiable tion procedures (Gordon, 1946; Alper et al., 1967;
amyloid plaques, immunostaining of tissue sections Gajdusek, 1977; Prusiner, 1982).
fixed in formaldehyde and embedded in paraffin is Procedures for decontamination of CJD-infected
only useful when positive. The use of other fixatives materials have been defined (Gajdusek, 1977;
such as McLean’s appears to be superior to formal- Brown et al., 1984; Prusiner et al., 1984; Richardson
dehyde in preserving PrP antigenicity. and Barkley, 1988). Although there is general agree-
ment about the extreme resistance of prions to inac-
tivation, there is some disagreement about the opti-
mal conditions for sterilization. Little is known
Care of Patients about the resistance of the human CJD prion to
inactivation; most of our knowledge comes from
It is important to stress that CJD is neither a con- animal models of experimental CJD, scrapie and
tagious nor communicable disease, but it is trans- BSE (Manuelidis, 1997; Taylor et al., 1997). The
missible. Although the risk of accidental inocula- most widely studied murine CJD model uses an
tion by aerosols is very small, procedures producing isolate from a Japanese case of GSS (Tateishi et al.,
aerosols should be performed in certified biosafety 1996).
cabinets. Biosafety level 2 practices, containment Although some investigators argue that GSS or
equipment and facilities are recommended by the CJD prions passaged into rodents retain the prop-
Centers for Disease Control and National Institutes erties of the human CJD prion (Manuelidis, 1997),
of Health (Richardson and Barkley, 1988). this view is erroneous and seems to emanate from
The primary problem in caring for patients with the old idea that CJD is caused by a slow or uncon-
CJD is the inadvertent infection of healthcare ventional virus. Once human CJD prions are pas-
workers by needle and stab wounds, whereas the saged into mice, the resulting prion is a mouse
possible transmission of a contagion through the air prion. Whether or not this murine CJD prion accu-
has never been documented. Accidental parenteral rately reflects the properties of human CJD prions
722 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
remains to be established. used in the treatment of CJD but without any con-
Although some investigators recommend treat- vincing success (Terzano et al., 1983). When HPA-
ing CJD-contaminated materials once with 1 N 23, an inhibitor of viral glycoprotein synthesis, is
NaOH at room temperature (Brown et al., 1984), we given to scrapie-infected animals around the time of
believe this procedure may be inadequate for steril- inoculation but not later, it profoundly extends the
ization. Autoclaving at 132°C for 5 hours or treat- length of the incubation period. The effects of HPA-
ment with 2 N NaOH for several hours is recom- 23 in human CJD are uncertain (Cathala and Bar-
mended for sterilization of prions (Prusiner et al., on, 1987). DEAE, dextran and cortisone have also
1984). The term ‘sterilization’ implies complete de- extended the incubation period in experimental
struction of prions; any residual infectivity can be scrapie (Ehlers and Diringer, 1984). Interferon has
hazardous. been used in experimental scrapie of rodents but the
The greatest possible contamination of hospital incubation times were unaltered (Worthington,
equipment will most likely occur in the neurosurgi- 1972). Although amphotericin has been used to pro-
cal operating room. Sterilization of surgical instru- long scrapie incubation periods in rodents, it is not
ments by 2 N NaOH or autoclaving at 132°C seems effective in treating patients with CJD (Masullo et
mandatory, especially if the cranium has been al., 1992).
opened. By analogy to experimental scrapie, the Although antibodies have been raised against the
human CJD brain probably has higher titres of scrapie prion protein and these crossreact with
prions than any other organ. Furthermore, the in- prion proteins in CJD human brains, passive immu-
tracerebral route of inoculation of scrapie prions in nization or even vaccination would seem to be of
hamsters is approximately 10 times more efficient little value. CJD and scrapie both progress in the
than oral ingestion (Prusiner et al., 1985). These absence of any immune response to the offending
data emphasize the extreme danger of introducing prions; however, neutralization of scrapie prion in-
small numbers of prions into the CNS tissue during fectivity was accomplished when the infectious par-
neurosurgical procedures. ticles were dispersed into detergent—lipid—protein
It has been argued that subclinical cases of CJD complexes (Gabizon et al., 1988b).
will harbour high titres of the infectious prions, but As our understanding of prion propagation in-
these will not be manifest until some time after the creases, it should be possible to design effective
surgery is completed, when the patient develops a therapeutics. Because people at risk for inherited
neurological disorder. Although this is particularly prion diseases can now be identified decades before
disconcerting, the rarity of CJD clearly indicates neurological dysfunction is evident, the develop-
that very few, if any, patients develop CJD as a ment of an effective therapy for these fully penetrant
result of neurosurgical procedures. disorders is imperative (Chapman et al., 1994). Al-
The development of Tg mice that detect human though we have no way of predicting the number of
prions (Telling et al., 1995) and extremely sensitive individuals who may develop neurological dysfunc-
immunoassays for human PrP1 using TRF (Safar tion from bovine prions in the future (Cousens et al.,
et al., 1998) offers the possibility of assessing the 1997), it would be prudent to seek an effective ther-
resistance of human prions to various sterilization apy now (Prusiner, 1998). Interfering with the con-
procedures and of developing effective schemes for version of PrP! into PrP1 seems to be the most
complete inactivation. attractive therapeutic target. Either stabilizing the
structure of PrP! by binding a drug or modifying
the action of protein X, which might function as a
molecular chaperone (Figure 26.3, see Plate VII),
Prevention and Therapeutics are reasonable strategies. Whether it is more effica-
cious to design a drug that binds to PrP! at the
There is no known effective therapy for treating or protein X binding site or one that mimics the struc-
preventing CJD. There are no well- documented ture of PrP! with basic polymorphic residues that
cases of patients with CJD showing recovery, either seem to prevent scrapie and CJD remains to be
spontaneously or after therapy, with one possible determined (Kaneko et al., 1997; Shibuya et al.,
exception (Manuelidis et al., 1976) for which there is 1998b). Because PrP1 formation seems limited to
no confirmatory example. Amantadine has been caveolae-like domains (Gorodinsky and Harris,
HUMAN PRION DISEASES 723
1995; Taraboulos et al., 1995), drugs designed to implantation (Bernouilli et al., 1977) and surgical
inhibit this process need not penetrate the cytosol of operations using contaminated instruments or ap-
cells but they do need to be able to enter the CNS. paratus (Will and Matthews, 1982). Corneas un-
Alternatively, drugs that destabilize the structure of knowingly removed from donors with CJD have
PrP1 might also prove useful. been transplanted to apparently healthy recipients
The production of domestic animals that do not who developed CJD after prolonged incubation
replicate prions may also be important with respect periods. Corneas of animals have significant levels
to preventing prion disease. Sheep encoding the of prions, which makes this scenario seem quite
R/R polymorphism at position 171 seem to be resis- probable. The same improperly decontaminated
tant to scrapie (Westaway et al., 1994; Hunter et al., EEG electrodes that caused CJD in two young
1997); presumably, this was the genetic basis of patients with intractable epilepsy were found to
Parry’s scrapie eradication programme in Great cause CJD in a chimpanzee 18 months after their
Britain 30 years ago (Parry, 1983). A more effective experimental implantation.
approach using dominant negatives for producing Surgical procedures may have resulted in acci-
prion-resistant domestic animals, including sheep dental inoculation of patients with prions during
and cattle, is probably the expression of PrP trans- their operations (Gajdusek, 1977; Will and Mat-
genes encoding basic residues at the putative pro- thews, 1982), presumably because some instrument
tein X binding site (Figure 26.3, see Plate VII) or apparatus in the operating theatre became con-
(Kaneko et al., 1997). Such an approach can be taminated when a CJD patient underwent surgery.
readily evaluated in Tg mice and, once shown to be Although the epidemiology of these studies is highly
effective, it could be instituted by artificial insemi- suggestive, no proof for such episodes exists.
nation of sperm from males homozygous for the
transgene. More difficult is the production of PrP-
deficient cattle and sheep. Although such animals
Dura Mater Grafts
would not be susceptible to prion disease (Büeler et
al., 1993; Prusiner et al., 1993), they might suffer
Since 1988, more than 80 cases of CJD after implan-
some deleterious effects from ablation of the PrP
tation of dura mater grafts have been recorded
gene (Sakaguchi et al., 1996).
(Centers for Disease Control, 1997). All of the grafts
Whether or not gene therapy for the human prion
were thought to have been acquired from a single
diseases, using the dominant negative approach de-
manufacturer whose preparative procedures were
scribed above for prion-resistant animals, will
inadequate to inactivate human prions. One case
prove feasible depends on the availability of effi-
of CJD occurred after repair of an eardrum
cient vectors for delivery of the transgene to the
perforation with a pericardium graft (Tange et al.,
CNS.
1989).
INFECTIOUS CREUTZFELDT–JAKOB
Human Growth Hormone Therapy
DISEASE
The possibility of transmission of CJD from con-
Infection is a rare cause of prion disease in humans. taminated HGH preparations derived from human
Iatrogenic CJD, kuru and possibly variant CJD in pituitaries has been raised by the occurrence of fatal
Europe are human prion diseases caused by trans- cerebellar disorders with dementia in 112 patients
mission of prions from one host to another. ranging in age from 10 to 41 years (Fradkin et al.,
1991). While one case of spontaneous CJD in a
20-year-old woman has been reported, CJD in pa-
Iatrogenic CJD tients under 40 years of age is very rare. These
patients received injections of HGH every 2—4 days
Accidental transmission of CJD to humans appears for 4—12 years (PHS, 1997). Interestingly, most of
to have occurred with corneal transplantation the patients presented with cerebellar syndromes
(Duffy et al., 1974), contaminated EEG electrode that progressed over periods varying from 6 to 18
724 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
months. Some patients became demented during ceiving human pituitary gonadotrophin (Cochius et
the terminal phase of their illnesses. The clinical al., 1990).
courses of some patients with dementia occurring
late resemble kuru more than ataxic CJD in some
respects. Assuming these patients developed CJD
from injections of prion-contaminated HGH prep-
Variant CJD
arations, the possible incubation periods range
from 4 to 30 years. Incubation periods of 2—3 dec- In 1994, the first cases of CJD in teenagers and
ades have been suggested to explain cases of kuru in young adults that were eventually labelled new
recent years (Gajdusek et al., 1977). Many patients variant (v)CJD occurred in Great Britain (Will et
received several common lots of HGH at various al., 1996). Despite the young age of these patients
times during their prolonged therapies, but no (Bateman et al., 1995; Britton et al., 1995), their
single lot was administered to all the American brains showed numerous PrP amyloid plaques sur-
patients. How many lots of the HGH might have rounded by a halo of intense spongiform degener-
ation (Figure 26.4e, f, see Plate VIII) (Ironside,
been contaminated with prions is unknown. HGH
1997). Later, one French case meeting these criteria
from one suspect lot has been reported to transmit
disease to one monkey, but no confirmatory report followed (Chazot et al., 1996). These unusual neur-
has been published. opathological changes have not been seen in CJD
Although CJD is a rare disease with an incidence cases in the United States, Australia or Japan (Cen-
of approximately one per million population (Mas- ters for Disease Control, 1996). Both macaque mon-
ters et al., 1978), it is reasonable to estimate that it is keys and marmosets developed neurological disease
at least 100 times more common among dead several years after inoculation with bovine prions
people from whom pituitaries were taken for hor- (Baker et al., 1993), but only the macaques exhibited
mone extractions. Since 10 000 human pituitaries numerous PrP plaques similar to those found in
were typically processed in a single HGH prepara- vCJD.
tion, the possibility of hormone preparations con-
taminated with CJD prions is not remote. The con-
Have Bovine Prions Been Transmitted to
centration of CJD prions within infected human
Humans?
pituitaries is unknown; it is interesting that wide-
spread degenerative changes have been observed in The restricted geographical occurrence and chrono-
both the hypothalamus and pituitary of sheep with logy of vCJD have raised the possibility that BSE
scrapie. The forebrains from scrapie-infected mice prions have been transmitted to humans. Only
have been added to human pituitary suspensions to : 45 vCJD cases have been recorded and the inci-
determine if prions and HGH copurify. Bioassays in dence has remained relatively constant, which
mice suggest that prions and HGH do not copurify makes establishing the origin of vCJD difficult. No
with currently used protocols (Taylor et al., 1985). set of dietary habits distinguishes vCJD patients
Although these results seem reassuring, especially from apparently healthy people. Moreover, there is
for patients treated with HGH over much of the last no explanation for the predilection of vCJD for
decade, the relatively low titres of the murine teenagers and young adults. Why have older indi-
scrapie prions used in these studies may not have viduals not developed vCJD-based neur-
provided an adequate test. The extremely small size opathological criteria? It is noteworthy that epi-
and charge heterogeneity exhibited by prions may demiological studies over the past three decades
complicate procedures designed to separate pitu- have failed to find evidence for transmission of
itary hormones from these slow infectious patho- sheep prions to humans (Cousens et al., 1990). At-
gens. Even though additional investigations argue tempts to predict the future number of cases of
for the efficacy of inactivating prions in HGH frac- vCJD, assuming exposure to bovine prions prior to
tions prepared from human pituitaries using 6 mol the offal ban, have been uninformative because so
l\ urea, it seems doubtful that such protocols will few cases of vCJD have occurred (Cousens et al.,
be used for purifying HGH because recombinant 1997). Are we at the beginning of a human prion
HGH is available. disease epidemic in Great Britain, like those seen for
Four cases of CJD have occurred in women re- BSE and kuru (Figure 26.2), or will the number of
HUMAN PRION DISEASES 725
vCJD cases remain small, as seen with iCJD caused the remains of a near relative who had died of kuru,
by cadaveric HGH (PHS, 1997)? which presumably provided the source of infection.
That the kuru prions could remain apparently qui-
escent in these patients for periods of two decades
Strain of BSE Prions and then manifest themselves in the form of a fatal
Was a particular conformation of bovine PrP1 se- neurological disease is supported by incubation
lected for heat resistance during the rendering pro- periods of over 7.5 years in some monkeys in-
cess, and then reselected multiple times as cattle oculated with kuru agent.
infected by ingesting prion-contaminated MBM The incidence of kuru has progressively declined
were slaughtered and their offal rendered into more with the cessation of ritualistic cannibalism in the
MBM? Recent studies of PrP1 from brains of pa- highlands of New Guinea in the late 1950s (Figure
tients who died of vCJD show a pattern of PrP 26.2b). Each year the youngest kuru cases have been
older, in accord with the hypothesis that kuru was
glycoforms different from those found for sCJD or
transmitted by cannibalism (Gajdusek, 1977), even
iCJD (Collinge et al., 1996; Hill et al., 1997). But the
utility of measuring PrP glycoforms is questionable though this proposition has been challenged. The
in trying to relate BSE to vCJD (Somerville et al., lack of eyewitness accounts of cannibalism in the
1997) because PrP1 is formed after the protein is Fore region has caused some scientists to ask if
glycosylated (Borchelt et al., 1990; Caughey and cannibalism was in fact the route by which kuru was
Raymond, 1991) and enzymatic deglycosylation of transmitted.
PrP1 requires denaturation. Alternatively, it may be
possible to establish a relationship between the con-
formations of PrP1 from cattle with BSE and those
from humans with vCJD by using Tg mice, as was
Clinical Features
done for strains generated in the brains of patients
In one report describing 15 patients with kuru seen
with FFI or fCJD (Telling et al., 1996b; Scott et al.,
in 1978 and 1980, 13 of these received detailed neur-
1997b). A relationship between vCJD and BSE has
been suggested by finding similar incubation times ological examinations (Prusiner et al., 1982). Ten of
in non-Tg RIII mice of : 310 days after inoculation the 15 patients were women, and all were adults.
with Hu or Bo prions (Bruce et al., 1997). The mean age of all the patients was 40.2 years and
their ages ranged from 29 to 60. The mean age of the
10 women was 43.2 years and of the five men, 34.2.
Five men and four women were under 40; all those
KURU over 40 were female. The youngest patient, a male,
was 29 years old. All but three of the patients lived
Kuru devastated the lives of the Fore Highlanders in the South Fore, the others in the North Fore. The
of Papua New Guinea (Gajdusek, 1977). The high duration of clinical disease ranged from 5 to 22
incidence of the disease among women left a society months, the onset being taken as difficulty walking.
of motherless children raised by their fathers. It was All 15 patients related a history of joint pain
unusual in the Fore region to see an older woman. preceding the onset of difficulty walking by several
With the cessation of traditional warfare, older men months. Eleven of the 15 also reported diffuse head-
are now found. Many of these older men have had a ache as a prodromal symptom. The diagnosis of
succession of wives who each died of kuru, leaving kuru was first made by the patients themselves
several children. Because contamination during rit- upon recognizing that they were having difficulty
ualistic cannibalism appears to have been the mode walking. The rugged, mountainous terrain, which is
of spread of kuru among the Fore people and be- frequently muddy from tropical rains, provided an
cause cannibalism had ceased by 1960 in the Fore ample test for assessing their balance on a daily
region, the patients now developing kuru presum- basis.
ably were exposed to the kuru agent more than two Eleven of the 15 patients showed no signs of
decades ago (Gajdusek, 1977). In many cases, his- dementia at the time of examination, whereas four
tories have been obtained from patients and their were disoriented and confused and had loss of mem-
families of the episode in which they cannibalized ory. The latter individuals exhibited diminished
726 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
speech and frontal lobe release signs consisting of with all tests for coordination were observed. Rapid
suck, snout, bite, and both hand and foot grasps. alternating movements were of uneven amplitude
Three of the demented patients required a stick to and rhythm.
maintain balance while standing, and one was un-
able to stand. With advanced kuru, truncal ataxia
and tremor were so severe that the stick had to be Uniformity of Clinical Signs
implanted into earth, and patients with advanced
disease were unable to walk without the assistance The uniform clinical presentation of kuru is re-
of another person. In all patients still able to walk markable (Hornabrook, 1968). The prodromal
with the assistance of a stick or an observer, marked symptoms and onset of the disease were similar in
truncal ataxia was evident. all the patients of one study (Prusiner et al., 1982).
All the patients exhibited an apprehensive facial Even the time interval between the prodromal
expression, which remained unchanged for periods symptoms of headache and joint pain and the onset
as long as 30 minutes. It could be interrupted, how- of difficulty walking was always 6—12 weeks. In
ever, by laughter with other members of the village. most cases the disease progressed to death within 12
All patients were able to smile when requested to do months, and all patients were dead within 2 years of
so. Examination of the cranial nerves revealed no onset. The average duration of illness for the 15
abnormalities except for ataxic movements of the patients in this study was 16 months (Prusiner et al.,
eyes during tests of conjugate gaze. Optokinetic 1982). Invariably, signs of cerebellar dysfunction
nystagmus was diminished or absent bilaterally in dominate the clinical picture. All patients remain
most kuru patients, even at an early stage of the ambulatory with the aid of a stick for more than
disease. half of the clinical phase of their illness. These clini-
There was no evidence of muscle wasting or di- cal characteristics are similar to those reported for
minished strength, though one patient had coarse adult patients at the peak of the kuru epidemic
fasciculations intermittently in the triceps, quad- (Hornabrook, 1968).
riceps and gastrocnemius muscles. No fascicula- There has been debate about dementia in kuru
tions of the tongue were observed. (Hornabrook, 1968). Findings of memory loss and
Of the 13 patients who had neurological examin- disorientation accompanied by primitive reflexes,
ations, four showed increased resistance to passive such as snout, bite, suck, rooting and hand grasps,
movements, while an additional seven exhibited leave no doubt that patients become demented at
mild rigidity with demonstrable cogwheeling. The an advanced stage of the disease (Prusiner et al.,
initial clinical descriptions of kuru emphasized this 1982). The same patients also exhibited consider-
aspect of the disease as well as the cerebellar dys- able muscle paratonia or gegenhalten.
function. No patient was hypotonic. Nine of the 15 The uniformity of presentation and clinical
patients exhibited choreiform movements. All 15 course in kuru contrasts with that of CJD, in which
had normal strength. In 8 of 13 patients, hyperac- a wide spectrum of clinical manifestations is found
tive deep tendon reflexes were demonstrable but (Kirschbaum, 1968; Roos et al., 1973). Whereas
confined to the lower extremities. Seven patients most CJD patients exhibit dementia, myoclonus
had ankle clonus. Two showed unilateral extensor and pyramidal tract dysfunction at an early stage of
plantar responses, while two others exhibited bilat- the clinical illness, 10—20% present with an ataxic
eral responses. In one case a unilateral extensor illness (Gomori et al., 1973). However, the dementia
plantar response was accompanied by normal deep appears at an earlier clinical stage in ataxic CJD
tendon reflexes. In five patients in whom clonus at than in kuru. During the ambulatory phase of dis-
the ankles was readily elicited, no extensor plantar ease, no patients with kuru were found to be as
response was seen. severely demented as are most patients with CJD.
On sensory examination, responses to pinprick,
temperature, touch and vibration were normal.
Cortical sensory testing failed to reveal deficits. Incubation Periods That Exceed Three
Marked ataxia of the upper and lower extremities Decades
was pronounced in all patients, and all exhibited a
prominent intention tremor. Marked difficulties No individual born in the South Fore after 1959 has
HUMAN PRION DISEASES 727
developed kuru. Kuru has progressively disap- Origin of Kuru
peared, first among children and thereafter among
adolescents. The number of deaths in adult females It has been suggested that kuru began at the turn of
has decreased steadily and adult male deaths have the century as a spontaneous case of CJD that was
remained almost invariant. No one born in a village propagated by ritualistic cannibalism (Gajdusek,
since cannibalism ceased has ever developed kuru. 1977). Whether or not the Fore people and their
Each year the youngest new patients are older than immediate neighbours provide an especially per-
those of the previous year. These observations pre- missive genetic background that allows multiplica-
dict no kuru victims under the age of 40 in the tion of kuru prions remains to be established. Se-
future. quencing of the open reading frame of the PrP gene
Of several hundred kuru orphans born since 1957 from three kuru patients failed to reveal any muta-
to mothers dying of kuru, none has yet developed tions. A case of CJD outside the kuru region in
the disease. Thus, the many children with kuru seen Papua New Guinea is noteworthy.
in the 1950s were not infected prenatally,
perinatally or neonatally by their mothers. There is
no evidence for transmission in utero or by human
Transmission to Animals
milk. The regular disappearance of kuru is incon-
sistent with the existence of any natural reservoirs
Kuru has been regularly transmitted after in-
for kuru besides humans. Indeed, there is no evi-
tracerebral inoculation to apes and monkeys (Gaj-
dence for animal or insect reservoirs.
dusek, 1977). Occasional cases have been transmit-
While patients currently afflicted with kuru ex-
ted to cats but not to rodents. The prolonged
hibit greatly prolonged incubation periods, children
incubation periods in experimental animals are
with kuru who were observed 30 years ago provide
similar to those observed with CJD and GSS. Oral
some information on the minimum incubation per-
transmission of kuru to apes and monkeys has been
iod. The youngest patient with kuru was 4 years old
difficult (Gajdusek, 1977), but recent studies have
at the onset of the disease and died at age 5. It is not
demonstrated transmission to monkeys. Presum-
known at what age young children were infected.
ably, the difficulties in transmitting human kuru
Accidental transmission of CJD to humans has re-
prions to apes and monkeys orally are due to the
quired only 18 months after intracerebral or in-
inefficiency of this route (Prusiner et al., 1985) and
traoptic inoculation (Duffy et al., 1974; Bernouilli et
the crossing of a species barrier.
al., 1977). An incubation period of 18 months has
also been found in chimpanzees inoculated in-
tracerebrally with the kuru agent.
GERSTMANN–STRÄUSSLER–SCHEINKER
DISEASE
Genetics and Diagnostic Evaluation CNS disease (Tateishi et al., 1996), all of the
Tg(MHu2M, P102L)Prnp mice showed signs of
Most cases of GSS are familial (Masters et al., 1981), neurological dysfunction : 150 days after inocula-
exhibiting an autosomal dominant pattern with tion. Mice expressing appropriate PrP transgenes
nearly complete penetrance. The premortem diag- offer bioassays that are much shorter, less expen-
nosis of GSS is secure if a non-conservative muta- sive, and probably more sensitive than those in
tion of the PrP gene is found (Hsiao et al., 1989). non-human primates.
Before the discovery of PrP gene mutations causing
GSS, the diagnosis of GSS was rarely made prior to
autopsy. Biopsy of the cerebellum was occasionally
diagnostic when PrP amyloid plaques were found. Immunological Studies
Serological and CSF examinations are normal. CT
of the brain sometimes reveals cerebellar and brain- Using PrP antiserum, protease-resistant immuno-
stem atrophy. The EEG is normal or shows non- reactive proteins have been demonstrated in the
specific, diffuse changes. Periodic complexes have brain extracts of many patients with GSS (Bock-
not been reported in GSS except in those cases man et al., 1987; Tateishi et al., 1990). A dot blot
whose clinical manifestations resembled CJD. Elec- procedure for detection of PrP1 in brain
tromyography may reveal denervation potentials in homogenates provides a rapid and reliable method
lumbosacral myotomes. Sensory and motor nerve for the diagnosis of GSS, provided multiple regions
conduction velocities are normal. of the brain are sampled (Serban et al., 1990). Isola-
tion of amyloid plaques from the brains of GSS
patients and subsequent purification and se-
quencing of a major 11 kDa protein has shown that
Transmission to Animals this protein is a proteolytic fragment of PrP (Tag-
liavini et al., 1994).
In 1978, Tateishi and coworkers produced experi-
mental spongiform encephalopathy in mice and
rats by using brain tissue from a patient with a
chronic spongiform encephalopathy and kuru
plaques (Tateishi et al., 1996). Although this case NEUROPATHOLOGY OF THE HUMAN
was not thought to be familial at the time, its clini- PRION DISEASES
copathological features resembled those of GSS,
and subsequently Masters suggested that this case The triad of microscopic features that characterizes
be classified as GSS (Masters et al., 1981). In 1981, the prion diseases consists of: (1) spongiform degen-
transmission of GSS to monkeys using brain tissue eration of neurons, (2) severe astrocytic gliosis,
from a patient with familial disease was reported which often appears to be out of proportion to the
(Masters et al., 1981). Subsequently, brain tissue degree of nerve cell loss, and (3) amyloid plaque
from another afflicted member of the same family formation (Figure 26.4, see Plate VIII), (DeArmond
was used to transmit disease to marmosets. No and Prusiner, 1997).
diagnostic conclusions can as yet be made from Spongiform degeneration consists of intracellular
such transmission studies because transmissibility vacuoles that focally dilate neuronal processes,
of GSS appears to be variable, even within a single which gives the grey matter a microvacuolated ap-
pedigree (Masters et al., 1981). pearance by light microscopy. Ultrastructurally, the
Extracts of brains from patients who died with vacuoles display splitting of the unit cell membrane
GSS(P102L) did not transmit disease to mice ex- with the formation of blister-like membrane expan-
pressing a chimeric Hu/Mo (MHu2M) PrP trans- sions and multiple septa. In addition, the abnormal
gene. However, they did transmit disease if this membranes are focally thickened and necrotic. In-
chimeric MHu2M transgene carried the P102L mu- deed, these neuronal membrane alterations are con-
tation (Telling et al., 1995). In contrast to non-Tg sistent with the prion protein being a sialoglyco-
mice inoculated with brain extracts that required protein that, during the course of the disease, accu-
500 days for : 10% of the animals to develop mulates within neurons.
730 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
Tg196 mice expressing the mutant transgene pro- Several lines of evidence argue that the smallest
ved to be the best hosts. It is noteworthy that ex- infectious prion particle is an oligomer of PrP1,
tracts from the brains of patients who died with perhaps as small as a dimer. Upon purification,
GSS (P102L) have transmitted disease to PrP1 tends to aggregate into insoluble multimers
Tg(MHu2M-P102L) mice but not to Tg(MHu2M) that can be dispersed into liposomes (Gabizon et al.,
mice (Telling et al., 1995). In contrast, human prions 1988a).
from patients with sCJD, fCJD(E200K) and FFI In attempts to form PrP1 in vitro, PrP! was
have all transmitted disease to Tg(MHu2M) (Tell- exposed to 3 mol l\ GdnHCl and then diluted ten-
ing et al., 1995, 1996b). fold prior to binding to PrP1 (Kocisko et al., 1994).
Why mutations of the PrP gene that produce Based on these results, we presume that exposure of
seemingly unstable PrP! molecules require many PrP! to GdnHCl converts it into a PrP*-like mol-
decades in humans to be manifest as CNS dysfunc- ecule. Whether this PrP*-like protein is converted
tion is unknown. In Tg(MoPrP-P101L) mice, the into PrP1 is unclear. Although the PrP*-like pro-
level of expression of the mutant transgene is in- tein bound to PrP1 is protease resistant and insol-
versely related to the age of disease onset. In addi- uble, this protease-resistant PrP has not been reis-
tion, the presence of the wt MoPrP gene slows the olated in order to assess whether or not it was
onset of disease and diminishes the severity of the converted into PrP1. It is noteworthy that recom-
neuropathological changes. binant PrP can be refolded into either -helical or
-sheet forms, but none have been found to possess
prion infectivity as judged by bioassay.
PRION REPLICATION
In an uninfected cell, PrP! with the wt sequence Inherited and Sporadic Prion Diseases
exists in equilibrium in its monomeric -helical,
protease-sensitive state or bound to protein X (Fig- For inherited and sporadic prion diseases, the ma-
ure 26.6). We denote the conformation of PrP! that jor question is how the first PrP1 molecules are
is bound to protein X as PrP*; this conformation is formed. Once these are formed, replication presum-
likely to be different from that determined under ably follows the mechanism outlined for infectious
aqueous conditions for monomeric recombinant disease. Several lines of evidence suggest that PrP1
PrP. The PrP*/protein X complex will bind PrP1, is more stable than PrP!, and a kinetic barrier
thereby creating a replication competent assembly. precludes the formation of PrP1 under normal con-
Order of addition experiments demonstrate that for ditions. In the case of the initiation of inherited
PrP!, protein X binding precedes productive PrP1 prion diseases, the barrier to PrP1 formation must
interactions (Kaneko et al., 1997). A conformational be lower for the mutant (PrP!) than the wild type
change takes place wherein PrP, in a shape compet- (wt), and thus PrP* can spontaneously rearrange
ent for binding to protein X and PrP1, represents to form PrP1. Although the known mutations
the initial phase in the formation of infectious PrP1. would appear to be destabilizing to the structure of
736 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
PrP!, we lack useful information about the struc- polymerization (NP) formalism would be relevant.
ture of the transition state for either the mutant or In NP processes, nucleation is the rate-limiting step,
wt sequences. Studies of PrP in the brains of pa- and elongation or polymerization is facile. These
tients who were heterozygous for the E200K muta- conditions are frequently observed in peptide
tion revealed PrP1(E200K) molecules that were models of aggregation phenomena (Jarrett and
both detergent insoluble and resistant to limited Lansbury, 1993). However, studies with Tg mice
proteolysis, while most wt PrP was detergent insol- expressing foreign PrP genes suggest that a different
uble but protease sensitive (Gabizon et al., 1996). process is operative. From investigations with mice
These results suggest that in fCJD(E200K), insol- expressing both the SHaPrP transgene and the en-
uble wt PrP might represent a form of PrP* dogenous MoPrP gene, it is clear that PrP1 pro-
(Gabizon et al., 1996). In studies with CHO cells, vides a template for directing prion replication
expression of PrP(E200K) was found to be accom- (Prusiner et al., 1990). Inoculation of these mice
panied by the post-translational acquisition of re- with SHaPrP1 leads to the production of
sistance to limited proteolysis (Lehmann and Har- SHaPrP1 and not MoPrP1. Conversely, inocula-
ris, 1996), but such results could not be obtained tion of the Tg(SHaPrP) mice with MoPrP1 results
when other cell lines expressing PrP(E200K) were in MoPrP1 formation and not SHaPrP1. Even
tested, including cultured fibroblasts obtained from stronger evidence for templating has emerged from
a patient who was homozygous for the E200K mu- studies of prion strains passaged in
tation (Z. Meiner, R. Gabizon and S.B. Prusiner, Tg(MHu2M)Prnp mice expressing a chimeric
unpublished data). It is noteworthy that levels of Hu/MoPrP gene.
proteinase K used in the studies in which When a prion strain containing MHu2MPrP1
PrP(E200K) was expressed in CHO cells were derived from the brains of FFI patients is passaged
lower by a factor of 10—100 compared to digestions in Tg(MHu2M)Prnp mice, the molecular size of
of PrP1 derived from brain or ScN2a cells. the protease-resistant core is 19 kDa, as described in
Whether these alterations in the properties of more detail below. In contrast, when a prion strain
PrP(E200K) in CHO cells provide evidence for containing MHu2MPrP1 derived from the brains
PrP* or such changes lie outside the pathway of of fCJD(E200K) patients is passaged in
PrP1(E200K) formation remains to be deter- Tg(MHu2M)Prnp mice, the protease-resistant
mined. core is 21 kDa (Telling et al., 1996b; Prusiner, 1998).
Initiation of sporadic disease may follow from a Although the conformational templates were in-
somatic mutation and thus take a path similar to itially generated with PrP1 molecules having differ-
that for germline mutations in inherited disease. In ent sequences, these templates are sufficient to di-
this situation, the mutant PrP1 must be capable of rect replication of distinct PrP1 molecules when the
coopting wt PrP!, a process known to be possible amino acid sequences of the PrPs are identical. If
for some mutations (e.g. E200K, D178N) but less the formation of this template were rate limiting,
likely for others (e.g. P102L) (Telling et al., 1995, then an NP model could apply. However, studies of
1996b). Alternatively, the activation barrier separ- PrP1 formation in ScN2a cells point to a distinct
ating wt PrP! from PrP1 could be crossed on rare rate-limiting step.
occasions when viewed in the context of a popula- Cell biological and transgenetic investigations
tion. Most individuals would be spared, while pres- argue for the existence of a chaperone-like mol-
entations in the elderly, with an incidence of : 1 ecule, referred to as protein X, that is required for
per million, would be seen. PrP1 formation (Telling et al., 1995). As described
below, mutagenesis experiments have created domi-
nant negative forms of PrP! that block the forma-
tion of wt PrP1 by binding protein X (Kaneko et
Mechanism of Prion Propagation? al., 1997). This implies that the rate-limiting step in
vivo in prion replication must be the conversion of
From the foregoing formalism, we can ask ‘What is PrP! to PrP* because a dominant negative derived
the rate-limiting step in prion formation?’ If the from a single point mutation could only gate a
assembly of PrP1 into a specific dimeric or multi- kinetically critical step in a cellular process. In the
meric arrangement were difficult, then a nucleation- template-directed model, the conversion of PrP! to
HUMAN PRION DISEASES 737
PrP* is a first-order process. By contrast, NP pro- Table 26.3 Influence of prion species and strains on
cesses follow higher order kinetics ([monomer]
transmission across a species barrier
where m is the number of monomers in the nucleus). Incubation time [days < SEM]
The experimental implications of these rate rela- (n/n )
tionships are apparent in transgenic studies; if first-
order kinetics operate, halving the gene dose Inoculum Host Sc237 139H
(hemizygotes) should double the incubation time, SHa SHa 77 < 1(48/48) 167 < 1(94/94)
while doubling the dose of a transgene array should SHa non-Tg mice 700(0/9) 499 < 15(11/11)
halve the time to disease. This quantitative behav- SHa Tg(SHaPrP)81/ 75 < 2(22/22) 110 < 2(19/19)
iour has been observed in several studies in mice FVB mice
with altered levels of PrP expression (Prusiner et al., SHa Tg(SHaPrP)81/ 54 < 1(9/9) 65 < 1(15/15)
Prnp mice
1990; Carlson et al., 1994). The existence of prion
strains that are conformational isoforms of PrP1 Data from references in Prusiner (1998).
with distinct structures, incubation times and
neurohistopathology must also be considered in an recipient, to determine the tertiary structure of
analysis of the kinetics of PrP1 accumulation. Since nascent PrP1. These principles are demonstrated
the rate-limiting step in PrP1 formation cannot by studies on the transmission of Syrian hamster
involve the unique template provided by a strain, (SHa) prions to mice, showing that expression of a
differential rates of clearance seem most likely to SHaPrP transgene in mice abrogated the species
account for the variation in incubation times (Safar barrier (Table 26.3). Besides the PrP sequence, the
et al., 1998). strain of prion modified transmission of SHa prions
to mice (Table 26.3) (Scott et al., 1997a).
MHu2M share the same amino acid sequence at the and Hsp60 by two-hybrid analysis in yeast (Eden-
COOH-terminus. This also suggested that MoPrP! hofer et al., 1996; Kurschner and Morgan, 1996).
only weakly inhibited transmission of SHa prions Weiss and coworkers have used a similar approach
to Tg(SHaPrP) mice (Table 26.3) because SHaPrP to show that PrP binds the laminin receptor protein
is more closely related to MoPrP than is HuPrP. (Weiss, 1997). Although these studies are suggestive,
Using scrapie-infected mouse (Mo) neuroblas- no molecular chaperone involved in prion forma-
toma cells transfected with chimeric Hu/Mo PrP tion in mammalian cells has been identified.
genes, we extended our studies of protein X. Substi-
tution of a Hu residue at position 214 or 218 pre-
vented PrP1 formation (Figure 26.3, see Plate VII)
(Kaneko et al., 1997). The side chains of these resi- STRAINS OF PRIONS
dues protrude from the same surface of the COOH-
terminal helix, forming a discontinuous epitope The existence of prion strains raises the question of
with residues 167 and 171 in an adjacent loop. how heritable biological information can be en-
Substitution of a basic residue at positions 167, 171 ciphered in a molecule other than nucleic acid
or 218 prevented PrP1 formation; these mutant (Dickinson et al., 1968; Bruce and Dickinson, 1987;
PrPs appear to act as ‘dominant negatives’ by bind- Weissmann, 1991; Ridley and Baker, 1996). Strains
ing protein X and rendering it unavailable for prion or varieties of prions have been defined by incuba-
propagation. Our findings seem to explain the pro- tion times and the distribution of neuronal vacuola-
tective effects of basic polymorphic residues in PrP tion (Dickinson et al., 1968; Fraser and Dickinson,
of humans and sheep (Westaway et al., 1994; 1973). Subsequently, the patterns of PrP1 deposi-
Shibuya et al., 1998b). tion were found to correlate with vacuolation pro-
files and these patterns were also used to character-
ize strains of prions (DeArmond et al., 1987b; Bruce
et al., 1989).
Is Protein X a Molecular Chaperone? The typing of prion strains in C57Bl, VM and
F1(C57Bl x VM) inbred mice began with isolates
Because PrP undergoes a profound structural tran- from sheep with scrapie. The prototypic strains,
sition during prion propagation, it seems likely that called Me7 and 22A, gave incubation times of
other proteins such as chaperones participate in this : 150 and : 400 days in C57Bl mice, respectively
process. Whether protein X functions as a classical (Dickinson et al., 1968; Bruce and Dickinson, 1987).
molecular chaperone or participates in PrP binding The PrPs of C57Bl and I/Ln (and later VM) mice
as part of its normal function but can also facilitate differ at two residues and control incubation times
pathogenic aspects of PrP biology is unknown. In- (Carlson et al., 1994; Moore et al., 1998).
terestingly, scrapie-infected cells in culture display Until recently, support for the hypothesis that the
marked differences in the induction of heat-shock tertiary structure of PrP1 enciphers strain-specific
proteins (Tatzelt et al., 1995), and Hsp70 mRNA has information (Prusiner, 1991) was minimal except
been reported to increase in scrapie of mice (Ken- for the DY strain isolated from mink with trans-
ward et al., 1994). Although attempts to isolate missible encephalopathy (Bessen and Marsh, 1994).
specific proteins that bind to PrP have been disap- PrP1 in DY prions showed diminished resistance
pointing, PrP has been shown to interact with Bcl-2 to proteinase K digestion as well as an anomalous
HUMAN PRION DISEASES 739
Table 26.5 Distinct prion strains generated in humans with inherited prion diseases and
transmitted to transgenic mice
site of cleavage. The DY strain presented a puzzling tion time of : 130 days and a 19 kDa PrP1, where-
anomaly because other prion strains exhibiting as those inoculated with fCJD(E200K) prions ex-
similar incubation times did not show this altered hibited an incubation time of : 170 days and a
susceptibility to proteinase K digestion of PrP1 21 kDa PrP1 (Prusiner, 1998). The experimental
(Scott et al., 1997a). Also notable was the generation data demonstrate that MHu2MPrP1 can exist in
of new strains during passage of prions through two different conformations based on the sizes of
animals with different PrP genes (Scott et al., the protease-resistant fragments; yet the amino acid
1997a). sequence of MHu2MPrP1 is invariant.
The results of our studies argue that PrP1 acts as
a template for the conversion of PrP! into nascent
PrP1. Imparting the size of the protease-resistant
PrPSc Conformation Enciphers Variation fragment of PrP1 through conformational templat-
in Prions ing provides a mechanism for both the generation
and propagation of prion strains.
Persuasive evidence that strain-specific information Interestingly, the protease-resistant fragment of
is enciphered in the tertiary structure of PrP1 com- PrP1 after deglycosylation with an M of 19 kDa
es from transmission of two different inherited hu- has been found in a patient who died after develop-
man prion diseases to mice expressing a chimeric ing a clinical disease similar to FFI. As both PrP
MHu2MPrP transgene (Telling et al., 1996b). In alleles encoded the wt sequence and a Met at posi-
FFI, the protease-resistant fragment of PrP1 after tion 129, we labelled this case fatal sporadic insom-
deglycosylation has an M of 19 kDa, whereas in nia (FSI). At autopsy, the spongiform degeneration,
fCJD(E200K) and most sporadic prion diseases, it is reactive astrogliosis and PrP1 deposition were con-
21 kDa (Table 26.5) (Monari et al., 1994; Parchi et fined to the thalamus (Mastrianni et al., 1997).
al., 1996). This difference in molecular size was These findings argue that the clinicopathological
shown to be due to different sites of proteolytic phenotype is determined by the conformation of
cleavage at the NH -termini of the two human PrP1 in accord with the results of the transmission
PrP1 molecules reflecting different tertiary struc- of human prions from patients with FFI to Tg mice
tures (Monari et al., 1994). These distinct conforma- (Telling et al., 1996b).
tions were not unexpected because the amino acid
sequences of the PrPs differ.
Extracts from the brains of FFI patients trans-
mitted disease into mice expressing a chimeric Evidence for Different Conformations of
MHu2MPrP gene about 200 days after inoculation PrPSc in Eight Prion Strains
and induced formation of the 19 kDa PrP1, where-
as fCJD(E200K) and sCJD produced the 21 kDa A highly sensitive CDI that discriminated PrP1
PrP1 in mice expressing the same transgene (Tell- molecules among eight different prion strains
ing et al., 1996b). On second passage, Tg(MHu2M) propagated in Syrian hamsters was developed (Sa-
mice inoculated with FFI prions showed an incuba- far et al., 1998). This immunoassay for PrP1 does
740 PRINCIPLES AND PRACTICE OF CLINICAL VIROLOGY
not depend on the protease resistance of PrP1 to to protein X seems to be the rate-limiting step in
distinguish it from PrP! but instead utilizes antibo- prion replication (Kaneko et al., 1997), it follows
dies that react with epitopes of PrP that are exposed that the different incubation times of various prion
in PrP! but become buried in PrP1. The CDI strains should arise predominantly from distinct
measures the increase in immunoreactivity that oc- rates of PrP1 clearance rather than from the differ-
curs when the cryptic epitopes are exposed by de- ent rates of PrP1 formation. In accord with the
naturation with GdnHCl. The assay is extremely excellent correlation between proteinase K-sensi-
sensitive because the antibody is labelled with euro- tive PrP1 and incubation times, prion strains that
pium (Eu), which can be measured by TRF. seem to be readily cleared have prolonged incuba-
Brains from Syrian hamsters were collected when tion times, whereas those that are poorly cleared
the animals displayed signs of neurological dysfunc- display abbreviated incubation periods. However, it
tion; the incubation times for the prion strains var- is important to recognize that proteinase K sensitiv-
ied from 70 to 320 days. Most of the PrP in the ity is an imperfect model for in vivo clearance and
brains of Syrian hamsters with signs of neurological that only one strain with a long incubation time
disease was PrP1, as defined by the -sheet confor- exceeding 300 days has been studied.
mation. The level of PrP1 in the brains of these
clinically ill animals exceeded that of PrP! by three-
to ten-fold as determined by CDI (Figure 26.7a). PRIONS ARE UNPRECEDENTED
The highest levels of PrP1 were found in the brains
of Syrian hamsters infected with the Me7-H strain; Although the study of prions has taken several un-
in contrast, the lowest levels were found in the expected directions over the past three decades, a
brains of Syrian hamsters inoculated with the novel and fascinating story of prion biology is
SHa(Me7) strain (Figure 26.7a). Interestingly, the emerging. Investigations of prions have elucidated a
Me7-H and SHa(Me7) strains, which were both previously unknown mechanism of disease in hu-
derived from Me7 passaged in mice (Scott et al., mans and animals. Although learning the details of
1997a), possessed similar denatured/native PrP ra- the structures of PrPs and deciphering the mechan-
tios, but they accumulated PrP1 at quite different ism of PrP! transformation into PrP1 will be im-
levels (Figure 26.7a,b). The highest denatured/na- portant, the fundamental principles of prion biol-
tive PrP ratio of all tested strains was SHa(RML). ogy have become reasonably clear. Although some
In a plot of the ratio of antibody binding to investigators prefer to view the composition of the
denatured/native PrP graphed as a function of the infectious prion particle as unresolved, such a per-
concentration of PrP1, each strain occupied a spective denies an enlarging body of data, none of
unique position, indicative of a particular PrP1 which refutes the prion concept. Moreover, the dis-
conformation (Figure 26.7b,c) (Safar et al., 1998). covery of prion-like phenomena mediated by pro-
This conclusion was supported by a unique pattern teins unrelated to PrP in yeast and other fungi serve
of equilibrium unfolding of PrP1 found with each not only to strengthen the prion concept but also to
strain. When the incubation times of these eight widen it (Wickner, 1997).
strains were plotted as a function of the concentra- The discovery that prion diseases in humans are
tion of either PrP1 or PrP 27—30, no relationship uniquely both genetic and infectious greatly
could be discerned. Incubation times were also plot- strengthened and extended the prion concept. To
ted as a function of the ratio of denatured/native date, 20 different mutations in the human PrP gene,
PrP and again no correlation could be found. all resulting in non-conservative substitutions, have
In contrast to the lack of any correlation with been found either to be linked genetically to, or to
incubation times noted above, an excellent correla- segregate with, the inherited prion diseases (Figure
tion was found when the proteinase K-sensitive 26.1). Yet, the transmissible prion particle is com-
fraction of PrP1 ([PrP1] 9 [PrP 27—30]) was plot- posed largely, if not exclusively, of an abnormal
ted as a function of the incubation time for all eight isoform of the prion protein designated PrP1
prion strains (Figure 26.7d) (Safar et al., 1998). The (Prusiner, 1991).
proteinase K-sensitive fraction of PrP1 can be con-
sidered a surrogate for PrP1 clearance. Since the
binding of PrP! or a metastable intermediate PrP*
HUMAN PRION DISEASES 741
Figure 26.7 Eight prion strains distinguished by the conformation-dependent immunoassay. (a) Concentration of total PrP() and
PrP1(
). The columns and bars represent the average < SEM obtained from three different brains of LVG/LAK Syrian hamsters
infected with different prion strains and measured in three independent experiments. (b) Ratio of antibody binding to denatured/native
PrP and a function of concentration of PrP1 in the brains of Syrian hamsters infected with different prion strains. Concentration of
PrP1 (formula 1) and the ratio of antibody binding to denatured/native PrP were measured by the conformation-dependent
immunoassay. (c) Brain homogenates of Syrian hamsters inoculated with different scrapie strains and uninoculated controls, denoted
C, were digested with 50 g ml\ proteinase K for 2 h at 37°C prior to the conformation-dependent immunoassay. (d) Incubation time
plotted as a function of the concentration of the proteinase K-sensitive fraction of PrP1 ([PrP1] 9 [PrP 27—30]). (Data from Safar et
al., 1998)
Index