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Contributorsv
Prefacexi
Index427
Foot-and-mouth disease virus (FMDV) maintains impetus, and gradually diminishing the traditional
a continuing fascination not only because of its support for a slaughter-based control strategy. This
worldwide implications for economic develop- is reflected in renewed effort on vaccine designs
ment, but also because it makes us relive the events and the consideration of antiviral agents to control
of an outbreak of disease upon unsuspecting areas or prevent the infection, either by administering
of our planet. On top of this, many fundamental the agents alone or as a complement to vaccination
questions about its replication, transmission, or other immunization-based interventions. This
detection, spread and persistence do not yet have book reflects this trend by including a chapter on
an answer, since the virus displays unique features antiviral therapies that was not even considered in
even when compared with its closest picornavirus the 2004 version where small molecule inhibitors
relatives. As with other small viruses, FMDV is or RNA interference or silencing (to name just two
endowed with complex biological behaviour for an points) were not even mentioned.
apparently simple pathogen. In planning the new volume, we have done our
An invitation to produce a book similar in con- best as authors to invite those experts that in our
tent to the 2004 book is a clear sign that not only view have contributed either recently or histori-
the first edition was well received by the scientific cally to construct the body of current knowledge
community, but also that many problems and ques- on FMDV. Of course, they are not the only ones
tions remain, and that the unsolved issues have a in this endeavour, and we apologize for any omis-
very relevant scientific and economic impact in our sions of experts that could have been invited as
increasingly global world. Unanswered questions authors. Many names are listed in a remarkable
are, for example, the limited knowledge about host number of references that should serve as further
range determinants, or the lack of cost-effective reading to complement the core information
vaccines, as alternatives to the chemically inacti- gained by reading the 18 chapters. This book is not
vated conventional vaccines. The limited amount a reprint or even an updated version of the 2004
of funding devoted to FMDV research in the EU book. While many topics have been retained, each
is surprising considering the potential economic chapter has been written afresh, so as to include
impact of a disease outbreak within the EU or in recent progress as evidenced by the large number
neighbouring countries. of references to publications of the last decade.
A very important change regarding the social It is our hope that the present book will provide
perception of the disease has taken place since an updated overview of several interconnected
2004. Perhaps as a result of the terrible images of aspects of FMDV and its disease, including the
mass animal slaughtering during the 2001 European structure of the viral particle and encoded pro-
epidemic, witnessed on television by the public at teins, expression of the genetic material, natural
large, there is a growing trend to consider alterna- habitats of the virus, diagnostic procedures, epi-
tive means to deal with the disease. In particular, demiological spread, and control measures. As
the non-vaccination policy and the possibility of in the 2004 book, great attention has been paid
new types of antiviral interventions are gaining to what is known, and what is not, regarding the
innate and acquired immune responses elicited by as historical developments by many devoted scien-
the virus and their implications for classical vac- tists some of whom, unfortunately, are no longer
cines improvement and the development of new among us. Finally, our appreciation goes also to
immunization strategies. Caister Academic Press, and in particular to Hugh
We thank all authors for their timely contribu- Griffin, for his friendly and constructive work in the
tions, and for reflecting recent developments as well planning and production of this book.
molecular biology of the viral genome. Although complex in Kansas to replace the ageing facilities at
there has been significant progress in all/most Plum Island, New York, have been taken.
areas, FMD is still a continuing threat to the global In addition to these developments in the infra-
agricultural economy and there remains a pressing structure necessary to conduct work on FMD
need for better control measures, including diag- safely and effectively, there have been significant
nostics and improved vaccines. At the molecular movements to improve international coordina-
end of the spectrum there are still many aspects of tion of FMD on the global scale. For example, the
FMDV replication that are poorly understood. Global Foot-and-Mouth Disease Research Alliance
The trauma of the 2001 outbreak triggered a (GFRA) is a consortium of partners from FMD free
number of investigations into the state of FMDV and endemic countries which plays a valuable role in
research, its control and the response capability bringing together scientists who are trying to tackle
of the UK. These included a government enquiry FMD from very different perspectives. The role of
(Foot-and-mouth Disease 2001: Lessons to be international organizations in the co-ordination of
Learned), an enquiry sponsored and conducted approaches to deal with the global problems relat-
by the Royal Society (Royal Society Infectious ing to FMD are discussed in Chapter 17.
Disease in Livestock Inquiry Follow-Up Review) and
a policy commission report on the future of farm-
ing and food (Foot-and-mouth disease: Applying Progress and challenges in the
the Lessons). The general conclusions drawn from management of FMD
these enquiries were that there was considerable The disastrous epidemic in the UK in 2001 served
scope for improvement in virtually all aspects of to emphasize a number of specific issues of FMDV
FMD surveillance and control. At about the same outbreak management that required improvement
time (2002) the BBSRC, the UK research council or reappraisal. These include surveillance, diag-
responsible for maintenance and operation of the nostics, persistence, outbreak control, improved
UK high security laboratory at Pirbright, commis- vaccines, improved facilities and improved security
sioned a review of its status (Review of the Institute at national borders. Although the importance of all
for Animal Health – the Pirbright Laboratory). of these factors relating to FMD control was high-
One of the concerns expressed in this report was lighted by the UK outbreak they are of continued
that the Pirbright laboratory, one of the oldest and relevance to the global efforts to control the disease.
most respected research institutions working with In the last decade or so advances have been made
FMDV, had fallen into disrepair and required con- on all of these areas relating to FMD control, some
siderable investment to modernize its facilities. As a of which are briefly discussed below:
consequence of all these reviews into the shortcom-
ings of the support for veterinary viral diseases in Surveillance and diagnostics
general, and FMD in particular, extra funds were Effective surveillance is to a large extent dependent
allocated for a site redevelopment programme at on the availability of rapid, accurate and sensitive
Pirbright. However, it was not until thirteen years diagnostic tools. Cost too is an important factor
later these recommendations were realized with the in many parts of the world. Initial diagnosis is
opening of the BBSRC National Virology Centre: based on observation of the clinical presentations
the Plowright Building, which is now one of the typical of FMD and in classic infection in cattle the
most sophisticated high security research facilities symptoms are usually obvious. However, in other
in the world. species, sheep for example, the symptoms may
There have also been significant developments not be as simple to detect and it is vitally impor-
in the management and construction of high tant that livestock owners and veterinarians are
containment facilities for the handling of FMD appropriately trained to observe infected animals.
elsewhere in the world. For example, new and Expansion of international training and education is
improved laboratories were opened at the National of great importance in the local and global efforts to
Veterinary Institute at Lindholm in Denmark. Also, control the spread of FMD. International schemes
after much debate in the USA, the first steps in the to improve awareness of FMD and the importance
construction of a new high containment laboratory of rapid confirmation of the disease have expanded
in recent years with the involvement of several to shed virus (or at least virus/antibody complexes)
international organizations [e.g. European Com- long after clinical evidence of disease has gone (Sut-
mission for the Control of Foot-and-Mouth Disease moller et al., 1967). In addition, vaccinated animals
(EUFMD), the Food and Agriculture Organization can be infected to become carriers if their immune
(FAO) of the United States, World Organisation for status is sufficient to prevent clinical symptoms
Animal Health (OIE)] with the advent of GFRA but not to prevent all virus replication (Anderson
playing an important role et al., 1974). Interestingly, the acquisition of car-
Diagnosis of current infections has traditionally rier status is somewhat species dependent, being
required that virus in clinical samples is identified usually observed in bovines or ovines but not in
by serological techniques such as ELISA, often porcines but the reasons for these differences are
requiring the virus to be grown in tissue culture not known. In Africa the indigenous African buffalo
to provide sufficient antigen to perform the test. is particularly prone to persistent infection, often
This can require multiple blind passages and take with multiple serotypes simultaneously (Ayebazi-
several days. Diagnostic approaches that rely on bwe et al., 2010). The phenomenon of persistence
these techniques have many drawbacks includ- of FMDV in symptomless animals has long been
ing the time required to provide a conclusive a thorn in the side of regulatory authorities and
result, the problems attendant on maintaining has strongly influenced decisions on the use of
the sample in a sufficiently well preserved state to vaccination as an emergency procedure to control
recover viable virus and the occasional difficulty/ epidemics of FMD.
inability to grow field strains on conventional tissue There are three important questions relating to
culture cells. Among attempts to overcome these the problem of persistence of FMDV: how to detect
restrictions is the development of cells modified carrier animals, what is the relevance of persistence
to express the most effective FMDV (Yang et al., to disease prevention and control and finally what
1999) receptors (King et al., 2011). Along similar are the viral and host factors involved in the mainte-
lines there has been progress in the development of nance of a persistently infected state?
universal capture methods for ELISA by replacing
an immobilized antiviral capture antibody with How to detect potential carrier animals?
recombinantly expressed integrin receptor (Ferris In regions where the virus is endemic and control
et al., 2011). This has the advantage of potentially is by vaccination it has long been perceived that
capturing all FMDVs without the serotype restric- diagnostic procedures that could distinguish
tion inherent in the use of antibody capture. The between animals that had seroconverted follow-
development of sensitive and reliable pen-side tests ing vaccination from those that may have been
is another important research objective. infected, and are therefore potential carriers, would
There have been significant advances in the be of great value. The earliest attempt to provide
development of diagnostic procedures that rely on such a discriminatory test relied on detecting the
detection of the viral RNA irrespective of the abil- presence of antibodies to the so-called VIA (virus
ity to grow the virus. Nucleic acid based methods infection associated) antigen (Cowan and Graves,
have a number of advantages over the older more 1966). This was subsequently shown to be 3D, the
traditional diagnostic procedures including speed viral RNA dependent RNA polymerase which is
and sensitivity. In addition, full genome, deep surprisingly immunogenic in FMDV infections. It
sequencing methods (Wright et al., 2013) are pro- is also antigenically cross-reactive over the serotype
viding highly detailed information that is invaluable spectrum. A problem associated with the detection
for the study of viral transmission during the course of anti-VIA antibodies as an aid to discrimination
of epidemics and can inform predictive mathematic between vaccinated and infected animals is that
modelling of the patterns of spread of infection 3D, the VIA antigen, is frequently present in FMD
(Cottam et al., 2006). vaccines, unless these have been scrupulously puri-
fied, and so vaccinated animals would also develop
Persistence anti-VIA antibodies. There has been a lot of work to
It has long been known that a significant propor- try to identify other non-structural protein marker
tion of infected animals, especially cattle, continue assays that can successfully distinguish infected
from vaccinated animals and several of these such as a macrophage, an infection might ensue.
appear to work well and are being used in screen- The incredible infectiousness of FMDV makes this
ing procedures (Brocchi et al., 2006; Sorensen et scenario unacceptable, no matter how unlikely it
al., 1998). might be.
An alternative approach has been to develop
strains of vaccine virus (‘differentiating infected What are the viral and host factors
from vaccinated animals’ or DIVA vaccines) that involved in the maintenance of a
have been manipulated to assist distinction between persistently infected state?
the immune response to the vaccine or to infection Although the phenomenon of long-term persistent
with wild type virus. Removal of an epitope from shedding of FMDV from convalescent bovines
the vaccine virus without compromising its protec- has been known for many years, there has been
tive ability has been used for this purpose (Fowler considerable uncertainty regarding the mechanism
et al., 2014; Uddowla et al., 2012). Presence of anti- of persistence and the precise location of the tissue
body to the deleted epitope would therefore imply in which it is supported. Persistent virus can be
that the animals had been infected with wild-type isolated from probang sampling from the oro-
virus. pharynx and there is evidence to suggest that it is
mostly derived from the mucosal epithelia of the
What is the relevance of persistence to oropharynx (Alexandersen et al., 2002). It has been
disease prevention and control? proposed that basal cells in these areas can support
This is a major question for regulatory and control non-lytic viral replication possibly in conjunction
authorities. It is well established that infectious with down regulation of genes controlling innate
virus can be isolated from persistently infected immunity. Alternatively, it has been reported that
animals in the laboratory but how efficiently such FMDV particles can be retained in intact form
virus, normally present in immune complexes, at the surfaces of dendritic cells within germinal
can transmit infection to naive animals under field centres of regional lymph nodes ( Juleff et al., 2008,
conditions is not certain. Although there is consid- 2012). Presumably there must be a continuous
erable epidemiological evidence accrued over many reseeding of infectious virus particles held at den-
years to suggest that such transmission can occur dritic cell surfaces from a source of replication since
in the field numerous attempts to replicate this infectivity of FMDV is lost quite rapidly at body
under controlled laboratory conditions have failed. temperature.
However, it is impossible to discount the possibil- Although cell cultures persistently infected by
ity of transmission from a carrier and there is great FMDV can be selected in vitro (de la Torre et al.,
reluctance to risk the presence of clinically normal 1985), the relevance of these observations to the
persistently infected animals as a possible source mechanisms underlying persistent infection in vivo
of disease through transmission to uninfected is unclear. In the cell culture persistence studies a
animals. The virus shed from persistently infected very few residual surviving cells present after high
animals is mostly in the form of immune complexes level acute infection of BHK cell cultures were
and so effectively neutralized. However, infectious found to be infected with the virus. As has been
virus can be isolated directly from samples col- observed with persistently infected cell cultures
lected from the oropharynx of carrier animals by with other viruses (e.g. VSV) there is a continuous
probang sampling, although the infectivity titre of genetic battle between the host cell and the virus
such samples can be increased 10- to 100-fold by to maintain a quasi-stable equilibrium (Martin
extraction with organic solvents to remove inhibi- Hernandez et al., 1994) Interestingly, in long-term
tory materials such as antibodies (Alexandersen passage studies the ratios of the different forms
et al., 2002). In addition, it is know that FMDV/ of viral RNA in the cells changed dramatically
antibody complexes can be taken into and infect (Herrera et al., 2008). In a normal lytic infection
cells bearing fc receptors (Baxt and Mason, 1995; positive-strand RNA is in large excess over nega-
Mason et al., 1993). Consequently it is theoreti- tive strand RNA but as passage of the persistently
cally possible that, should an immune complexed infected cells progressed so this changed to reach
virus particle encounter an fc receptor bearing cell, a condition in which both strand are present at
similar levels, resulting in a high proportion of great that they became almost unsustainable and
double-stranded RNA. Intriguingly, a similar low engendered much public distress. Not only was
level viral persistence has been implied in examples the number of outbreaks occurring at the peak of
of chronic heart failure related to Coxsackievirus the epidemic exceedingly high but the ‘tail end’ of
infection (Archard et al., 1991). As interesting the outbreak continued for many months after the
as these studies are there is little evidence to sug- initial introduction of the disease (Chis Ster et al.,
gest that the observations made in the artificial in 2009). Interestingly at about the same time as the
vitro situations are comparable to the mechanisms UK outbreak FMD occurred in regions of South
underlying persistent virus infection and shedding America, such as Argentina, that had remained
in vivo. However, given the implication of some free of disease for several years. In contrast to the
studies that the site of persistent infection resides European situation the control measures adopted
in basal cells of the mucosal epithelia it is possible in South America were to revert to the practice of
that continual mutational response of the cells to mass vaccination that had been used to eliminate
infection, as seen in tissue culture, may play a role in FMD prior to its reintroduction. This was highly
the maintenance of infection. successful, resulting in a somewhat more rapid
Persistent infection is still an important and resolution of the epidemic than was achieved in
unresolved problem. The biological mechanisms the UK with ‘stamping out’ measures (Perez et al.,
that enable the virus to survive and continue 2004).
replicating are far from clear as is the means of trans- These and other global experiences of the effects
mission from carrier animals to naive stock. Given of FMD and its control have stimulated a great deal
that this can occur, albeit rarely and by undefined of research and debate concerning the best ways to
mechanisms, can we contemplate methods of clear- handle the disease in future. Improving our abilities
ing persistently maintained viral infection – maybe to understand and predict the scale and course of
with antiviral drugs? epidemic spread is of particular importance for
improved management following introduction of
Control in epidemic situations FMD into non-endemic regions. Retrospective
The basic principles of control of FMD epidemics analyses of outbreaks such as that in 2001 have
have remained unchanged for many decades. In been of great value for determining the mechanisms
non-endemic countries, such as the UK, control of spread between premises. For example, the role
of the spread of the disease following introduction of meteorological conditions; it has been known
from an external source is achieved by strict animal for many years that transmission via wind-borne
movement control, slaughter of stock that have droplets can occur under favourable conditions
been infected or are ‘at risk’ of infection together (Donaldson et al., 1970). In addition, the varying
with general decontamination and quarantine roles played by different host species (cattle, sheep
measures. In endemic regions vaccination is an and pigs) in maintaining and spreading infection
additional tool for disease control. However there could be analysed (Donaldson et al., 2001). The
are numerous and complex issues underlying these massive increases in recent years in the power of
simplistic descriptions of the availability and appli- genomic sequencing have enabled the fine defini-
cation of disease control measures. tion of transmission routes to be determined with
In 2001 the UK adopted the standard procedures unprecedented precision (Cottam et al., 2006).
for stamping out occasional outbreaks that have Furthermore, data from all of these sources have
been successful in the past and allowed the country enabled the development of increasingly sophisti-
to maintain a disease-free status, which is of great cated mathematical modelling approaches for the
importance for international trading purposes. prediction of epidemic progression (see Chapter
However a number of factors relating to that out- 16). Important improvements in the speed, accu-
break provided the ‘perfect storm’ situation in which racy, sensitivity and cost of diagnostic methods
the seeds of the epidemic had become widespread and devices have been made in recent years. Rapid
throughout the country before its full distribution and easily applied diagnostic tests are of special
had become apparent. As a consequence the scale importance to the control of FMD in the face of its
of the control measures that were applied was so extraordinary rate of spread. Recent advances in the
methods of detection and control are discussed in However, this was technically a serious breach of
Chapter 11. containment and other ‘within facility’ unwitting
transmissions have been reported. In the UK, in
Improved vaccines for global 2007, virus escaped from the high-level security site
protection at Pirbright (https://www.gov.uk/government/
Of course, vaccination has been and remains of uploads/system/uploads/attachment_data/
major importance for the prevention and control of file/250363/0312.pdf). The site incorporates
FMD. The importance of vaccination and vaccine both research facilities and a commercial vaccine
development is reflected in the fact that more than production plant and these share some common
one chapter in this book is devoted to the subject. containment infrastructure such as waste disposal
Although current vaccines are very effective if used drains. Although the exact means by which the
appropriately (in fact they are the biggest single virus escaped from the site to infect cattle on nearby
component responsible for the eradication of FMD farmland will never be known for sure, deficiencies
from many regions of the globe), there are many in maintenance of the infrastructure were almost
ways in which they might be improved. These certainly involved. Incidents such as these serve to
include potency, thermostability during distribu- highlight the importance of ensuring the continual
tion to point of use, duration of protection induced, safe maintenance and operation of high contain-
breadth of cover against variant strains of virus and ment facilities devoted to work with FMDV and
safety of production procedures and facilities. other high risk pathogens. Emergency steps were
In addition to improvements in conventional taken to improve biosecurity as a result of these
vaccine technologies there is a lot of interest and unfortunate occurrences and, as mentioned above,
progress into novel approaches for vaccine develop- these aged facilities have been (in the case of Pir-
ment. These fall into several categories including; bright) or are being (in the case of the USA facility)
the delivery of FMDV antigens by expression from replaced with new ‘state of the art’ laboratories.
a DNA virus vector, improvement of the stability of
seed viruses used to make conventional vaccines;
recombinant expression of stabilized virus-like par- Progress and challenges in
ticles (VLPs); in vitro assembly of E. coli expressed understanding the molecular
capsid proteins; the construction of live attenuated biology of FMDV
viruses and the reassessment of peptide vaccines. In addition to the issues discussed above, which are
These approaches are discussed in detail in other highly pertinent to the control of FMD, there are
chapters in this book. many intriguing aspects of the genome structure
and function that are still not well understood,
Security of facilities handling FMDV although significant progress has been made in
The serious consequences of escape of FMDV into the past 11 years. Some of these features are pecu-
the environment necessitate that work with the liar to or unique to FMDV and must contain the
virus, be it diagnostic surveillance, vaccine produc- molecular explanations of the specific pathogenic
tion or research, is carried out under conditions characteristics of the virus.
of maximum biosecurity. Consequently there is The following is an (incomplete) list of some
a limited number of high security facilities suit- of the aspects of FMDV structure, function and
able for FMDV work and the maintenance of their replication that are still poorly understood despite
biosecurity is of paramount importance. Breaches progress in recent years:
of biosecurity at a few institutions in recent years
have been the justifiable cause for public alarm. For The FMDV particle
example, in 1978 virus was found to have escaped The atomic structure of the FMDV particle was
from the high containment area at Plum Island. solved in 1989 and it provided molecular insight
Fortunately, the virus did not escape the confines into some of its biological properties (Acharya et
of Plum Island to reach the mainland (http://www. al., 1989). For example, the nature of the recep-
homelandsecuritynewswire.com/government- tor binding domain was revealed as loop-like
a d m i t s - a c c i d e n t s - p l u m - i s l a n d - b i o l a b) . projection from the virus surface; quite unlike
the groove or ‘canyon’ seen in the enteroviruses. virus for vaccine development purposes, there are
This provided an explanation of the extraordinary still important aspects of the biological function-
success of experimental simple peptide vaccines ing of the particle about which we know little.
in inducing protective immunity in laboratory In common with many other viruses we do not
animals compared with peptide vaccines against understand the assembly process. Genome RNA is
other viruses (Bittle et al., 1982). In addition, the preferentially/exclusively packaged within virions
location of specific amino acid residues, especially and the basis for this specificity is unclear. For other
histidines, at the interfaces of the pentameric subu- picornaviruses assembly involves the packaging of
nits of the particle provided a rational explanation nascent RNA and the same is likely true for FMDV.
of the extraordinary acid sensitivity of FMDV. The For poliovirus there is genetic evidence for the
structural insights into properties of the virion, involvement of the putative helicase, P2C (Liu et
such as acid lability, provided the background for al., 2010), but whether the same is true for FMDV
directed mutagenesis of the particle to modify these is not known. In addition, there is enormous con-
characteristics for the purposes of novel vaccine densation required to enclose the 8500 nucleotide
development. In particular, modifying the penta- genome into the 30-nm-diameter capsid and how
meric interfaces by introducing cysteine residues this is achieved is, as yet, a mystery.
to facilitate the formation of disulphide bonds to The function of a virus particle is to ensure
strengthen subunit interactions (Porta et al., 2013). safe delivery of the genome to a new host cell. For
Other mutations have been described in different the enteroviruses there is increasing evidence for
serotypes of the virus which also have stabilizing a mechanism by which the endocytosed virion
consequences (Kotecha et al., 2015). Recombinant undergoes receptor or acid mediated conforma-
expression of the structural proteins containing tional changes resulting in the externalization of the
stabilizing mutations at the pentamer interfaces, internal protein, VP4, and the N terminal domain
together with the viral protease, has produced of VP1. This results in a breaching of the endosomal
empty capsids (virus-like particles or VLPs) with membrane, most likely by formation of a protective
greatly increased resistance to heat or acid mediated channel through which the genomic RNA is trans-
disruption (Porta et al., 2013). This work has inter- ported into the cytoplasm to initiate infection while
esting potential for the development of safe, virus the capsid remains intact as an empty particle (Levy
free vaccine production, as discussed elsewhere in et al., 2010). It has been known for many years that
this book. acidification of the endosome is necessary and
Knowledge of the structure of the FMDV par- sufficient to trigger FMDV uncoating and penetra-
ticle has also proved useful in other vaccine related tion into the host cell. However, the details of the
studies. For example, modification of selected process are not understood. Exposure of FMDV
residues at subunit interfaces has resulted in viruses particles to pH levels found in endosomes, and
which maintain their infectivity but have increased necessary for uncoating, result in dissociation into
thermal stability, a useful potential advance for the subunits and RNA and it is difficult to envisage how
production of conventional vaccines that are better the RNA could be protected from hydrolysis in the
preserved in the field. Knowledge of the virus struc- lumen of the endosome and how it might be trans-
ture has also been exploited for the production of ported across the membrane. There is evidence for
marker vaccines in which specific epitopes have FMDV and the related equine rhinovirus A virus
been removed or replaced (Fowler et al., 2011). (ERAV) for the transient production of empty par-
It is intended that the immune responses to such ticles during acidification but whether these play a
modified viruses will help to distinguish vaccinated comparable role to the intermediate and empty par-
from infected animals. Finally, it has been shown ticles found during enterovirus cell entry is unclear
that epitope tags can be inserted at positions that (Tuthill et al., 2009).
do not interfere with the immunogenic potential of
the virus but can facilitate immuno-affinity-based Hidden features of the genome
procedure for facile purification and concentration sequence – GORS
of the particles (Seago et al., 2012). Detailed bioinformatic analysis of RNA virus
Despite the potential for modification of the genomes revealed the presence of genome-scale
ordered RNA structure (GORS), a previously analogy with enteroviruses, which have a highly
unrecognized feature inherent in viral genomes structured ‘clover leaf ’ at the 5′ end of the genome,
(Simmonds et al., 2004). Even more surpris- it is predicted that the S-fragment is involved in
ing was the comparative level of GORS in genome circularization and control of the switch
different virus groups, even within the same from translation to replication. There is some evi-
virus family. For example, the GORS score for dence to suggest that the S-fragment is involved
most picornaviruses is rather modest but this is in host species specificity but the structural and
completely different for FMDVs, which have an functional interactions of this distinctive fea-
extremely high GORS score, indicating a high ture with host and viral proteins and its role in
level of inherent and conserved RNA structure. viral replication are subjects of current research.
An interpretation of this phenomenon is that a Another striking feature is the poly(C) tract, a
viral genome with a high GORS score is likely contiguous stretch of pyrimidine residues, almost
to be degraded to produce small double stranded entirely cytidines, that varies in length between
RNA products within a restricted size range and different isolates and is in the range of 100–200
that these fragments have important interactions residues long. This feature was first recognized in
with, and evasion of, innate immune mechanisms the 1970s but its function is still obscure (Harris
with infected cells (Witteveldt et al., 2014). Other and Brown, 1976). Studies in the 1970s also sug-
viruses characterized by a high GORS score, such gested that it may have a role in attenuation but
as hepatitis C virus, are reported to be able to this is still unclear (Harris and Brown, 1977).
induce a persistent infected state and it has been However, in the cardioviruses, many of which also
proposed that this ‘hidden’ feature of the FMDV have a poly(C) tract, there is good evidence for
genome is related to the ability of the virus to a relationship between poly(C) tract length and
induce chronic infection. pathogenicity in mice. Mengovirus is highly path-
ogenic in mice but a form from which the poly(C)
Functions of the UTRs tract had been deleted was non-pathogenic, even
All picornaviruses have long 5′ untranslated though it grew well in tissues culture (Duke et al.,
regions (UTRs), largely due to the presence of a 1990). Clearly there is still a lot to learn about
large and complicated structure, the internal ribo- this ubiquitous FMDV feature. The final structural
some entry site (IRES) which enables translation domain found in the 5′ UTR is a string of pseudo-
of the viral open reading frame in the absence of a knots lying between the poly(C) tract and the cre
5′ CAP. However, the 5′UTR of FMDVs is extraor- element (Clarke et al., 1987). Variable numbers of
dinarily long, comprising approximately 1,300 these pseudoknots are found in different isolates
nucleotides and containing at least five apparently of FMDV, each being 43 nt in length. The pseudo-
independent domains (Belsham, 2005). The roles knot sequences are precisely deleted/inserted in
of two of these; the IRES (~450 nt) and a stem/ different viruses (Escarmis et al., 1995), support-
loop involved in templating the uridilylation of ing the suggestion that they are genuine structures
VPg and termed the cre (McKnight and Lemon, with functional roles. However, the role(s) of
1998) or bus (Tiley et al., 2003) are known but structures is unknown.
the functions of the remaining three domains are The 3′ UTR is much shorter than the 5′UTR
still not understood even though their presence and terminates in a polyA tail. There are two con-
has been recognized for many decades. The most served stem/loops within the heteropolymeric
5′ structure is the S-fragment (Sangar et al., 1980), region which clearly have roles in replication of
which is a generally well-conserved sequence of the genome. Interactions between these stem/
up to ~ 400 nt which is predicted to be largely loops and sequences within the 5′ UTR have been
double stranded and comprises a single long described, suggesting that they are involved in
stem/loop (Newton et al., 1985). Although usu- genome circularization during replication (Serrano
ally 350 or more nt in length, some isolates with a et al., 2006). However, the molecular ballet that
significantly shorter S-fragment have been identi- must accompany the assembly and function of the
fied (Valdazo-Gonzalez et al., 2013). The function replication complex and the role of circularization
of this extraordinary structure is unknown but by are still far from being understood.
understood. However, the precursor protein, 3CD, inform approaches to control and eliminate the
appears to improve uridilylation of VPg in in vitro disease.
assays, having some catalytic role in the process.
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Figure 2.1 Genome organization of FMDV. Important features of the FMDV RNA and its encoded proteins
are indicated and described in detail within the text. The FMDV RNA encodes a single polyprotein that is
processed mainly by the virus-encoded proteases (Lpro and 3Cpro; the cleavages generated by these proteases
are indicated by black arrows) to 15 mature products plus multiple precursors (only some of which are shown).
The break in the polyprotein that occurs at the 2A/2B junction is indicated by a green arrow. Abbreviations used
are: S, S-fragment; CCC(n), poly(C) tract; PK, pseudoknot; cre, cis-acting replication element; IRES, internal
ribosome entry site; L, Leader protein; VPg, virus protein genome-linked; VP1, VP2, VP3 and VP4 are the virus
capsid proteins which assemble around a single copy of the RNA to form a virus particle.
required by the viral RNA to initiate the infection other viral proteins, e.g. VPg (3B); the latter acts as
cycle. Hence, the virus particle essentially serves the primer for RNA synthesis, inside a membrane-
to protect the genome while it is outside of cells associated replication complex that contains several
and to bring about its delivery to the cytoplasm of other viral proteins. The infectivity of viral RNA
a cell where a new cycle of infection can be initi- demonstrates that the first phase of the infection
ated. The entire replication cycle of the virus occurs cycle has to be translation of the viral RNA so
in the cytoplasm of cells and, indeed, for certain that each of the viral proteins is made within the
picornaviruses it has been demonstrated that their infected cell. These viral proteins are required for
replication is maintained in enucleated cells (Follett the formation of RNA replication complexes, to
et al., 1975). The viral RNA contains all the infor- modify cellular activities and to generate new virus
mation required to allow the virus to take over the particles. At some point, it appears that there has to
cellular machinery. Most picornaviruses induce, be a switch in the function of the input viral RNA so
within infected cells, the shut-down of nearly all of that translation is blocked and RNA replication can
the host macromolecular synthesis and, hence, only commence. This seems necessary since the process
the production of viral RNA and viral proteins is of translation of picornavirus RNA (in which ribo-
observed. FMDV RNA is replicated with high effi- somes move along the RNA in a 5′–3′ direction) is
ciency within susceptible cells and after a few hours apparently not compatible with the initial step in
of infection the amount of viral RNA (all genome the replication of the viral RNA that is achieved by
length) within cells can reach a level similar to that the movement of the RNA polymerase from the 3′
of all the cellular cytoplasmic mRNAs (about 5% of end to the 5′ terminus (see Gamarnik and Andino,
total RNA). 1998).
The viral RNA is synthesized by the virus It is clear that the distinct processes of protein
encoded RNA-dependent RNA polymerase synthesis, RNA replication and RNA packaging
(3Dpol) (see Fig. 2.1). This process also requires (to form virus particles) need to be regulated and
possibly differentially localized within the cell. Sep- RNA replication elements
arating the various activities to different sites within
the cell could help to overcome potential incompat- The S-fragment
ibilities between these functions that each require At the 5′-terminus of the FMDV genome is a region
positive-sense RNA and hence may compete. The of about 360 nt, termed the S-fragment, which is
targets of any signals required for regulation of predicted to form a large hairpin structure (Clarke
function must be contained within the sequence of et al., 1987; Escarmis et al., 1992; Witwer et al.,
the genome. 2001). The S-fragment is the name given to the
This chapter will seek to examine the differ- smaller of the two RNA fragments that are gener-
ent features of the FMDV genome, which are ated when viral RNA is treated with oligo(dG)
responsible for directing the production of virus and RNaseH (Rowlands et al., 1978). This treat-
proteins and viral RNA within the infected cell. ment cleaves the RNA within the poly(C) tract
There is much that remains unknown about these and generates the 5′-terminal small (S)-fragment
processes and we will seek to identify these areas plus the major portion of the genome (the large or
and point out where relevant information is avail- L-fragment) that includes the rest of the 5′ UTR,
able from other related viruses. However, it should the polyprotein coding region and the 3′ UTR. It
always be borne in mind that the various members is assumed that the S-fragment is required for RNA
of the picornavirus family are distinct; hence, replication (since initiation of positive strand RNA
what is true for one virus may not always apply synthesis will commence at the complement of
to others. Several unique features of FMDV are this sequence on the negative strand). It has been
already known and undoubtedly more remain to reported that the cellular RNA helicase A (RHA)
be identified. interacts with the S-fragment and depletion of RHA
from cells inhibited FMDV replication (Lawrence
and Rieder, 2009). Interestingly the RHA also
The structure and function of co-immunoprecipates with FMDV 2C and 3A pro-
the FMDV RNA 5′ UTR teins, these also play a role in RNA replication (see
As indicated above, the 5′-UTR of FMDV RNA is below). Similarly, there is a considerable body of
about 1300nt long. This is significantly larger than data indicating that both cellular and viral proteins
most other picornavirus 5′-UTRs, e.g. the 5′-UTR interact with the ‘cloverleaf ’ structure that is located
of poliovirus (PV) RNA is about 740 nt in length at the 5′ terminus of the PV genome (Gamarnik and
while the encephalomyocarditis virus (EMCV) Andino, 1997; Parsley et al., 1997). This element
RNA 5′-UTR is about 830 nt long. The 5′-UTR (only about 80nt in length) binds to a cellular RNA
of each picornavirus RNA includes sequences binding protein termed the poly(rC) binding pro-
required for the initiation of protein synthesis on tein 2 and to the PV 3CD protein (by interaction
the viral RNA, for RNA replication and probably with sequences within 3C). It has been suggested
for other, currently unknown, functions. that they may play a role in the switch from transla-
The FMDV RNA 5′-UTR can be considered in tion to replication (Gamarnik and Andino, 1998).
several distinct regions, including the S-fragment, Additionally, these interactions may serve to circu-
a poly(C) tract, several pseudoknots and elements larize the RNA (since the poly(A) binding protein
termed the cis-acting replication element (cre) and can bind to both the 3′ terminal poly(A) tail and
the internal ribosome entry site (IRES) (see Fig. the poly(rC) binding protein) which may facili-
2.1). The IRES is about 450 nt in length and directs tate replication and/or translation (Herold and
the initiation of protein synthesis on the viral RNA Andino, 2001). It is also possible that the presence
(see Belsham and Sonenberg, 1996; Belsham and of proteins at (or near) the RNA termini serves to
Jackson, 2000; Martinez-Salas et al., 2001, 2015; protect the viral RNA from degradation (Murray
Belsham, 2009), its properties will be discussed et al., 2001; Kempf and Barton, 2008). Further
in detail below. The cre (about 55 nt in length) is information about the interaction between the 5′
required for FMDV RNA replication (Mason et and 3′-termini within FMDV RNA is included in
al., 2002; Nayak et al., 2005, 2006) and will also be ‘Implications of long-range 5′–3′ viral RNA interac-
discussed further below. tions on IRES activity’.
The poly(C) tract and pseudoknots animals (Duke et al., 1990). Surprisingly, the role
On the 3′ side of the S-fragment within FMDV of the poly(C) tract in EMCV, another cardiovirus-
RNA there is a long, almost homopolymeric, like mengovirus, appears quite different (Hahn
stretch of C residues termed the poly(C) tract. and Palmenberg, 1995). Mutant EMC viruses,
This is a common feature of the genomes of containing very short poly(C) tracts, were about as
FMDV and most cardioviruses (e.g. EMCV, but pathogenic in animals as wt EMCV and replicated
not Theiler’s murine encephalitis virus). There is at very similar rates in cells as judged by single-step
considerable variation in the length of this poly(C) growth curves.
tract amongst different strains of FMDV, from Between the poly(C) tract and the IRES ele-
about 80 nt to about 420 nt. In general, the shorter ment in FMDV RNA there is another stretch of
tracts are found in laboratory strains and typically sequence, about 250 nt in length, that is believed
field strains have a tract of about 200 nt (Brown et to contain multiple pseudoknots. Different FMDV
al., 1974; Harris and Brown, 1977). However, the strains have been predicted to contain 2–4 pseudo-
largest poly(C) tract identified was found in a strain knots (Clarke et al., 1987; Escarmis et al., 1995).
of FMDV (VR 100) recovered from persistently Interestingly the cardiovirus RNAs are predicted to
infected cells in culture (Escarmis et al., 1992) but contain multiple pseudoknots located on the 5′ side
the significance of the size of this homopolymeric of their poly(C) tract rather than on the 3′ side as
sequence is not clear. The VR 100 strain has been in FMDV RNA (Martin and Palmenberg, 1996). It
described as hypervirulent in BHK-21 cells (de la may be that the pseudoknots are involved, in some
Torre et al., 1988) but it is attenuated for growth in manner, in a joint function with the poly(C) tract.
mice and cattle (Diez et al., 1990). However, since
the VR100 genome is different from the parental A cis-acting replication element (cre)
virus at about 80 positions (1% of the genome), In addition to sequences at the termini of the
and these changes occur all across the genome viral RNA, it has been shown that internal RNA
including within the IRES region (Martinez-Salas sequences are required for RNA replication. One
et al., 1993), it is not clear which changes (including such feature has been termed a ‘cis-acting replica-
those within the long poly(C) tract) are responsible tion element’ (cre). The initial characterization of cre
for the different phenotypes. There is a strong selec- structures within picornavirus RNAs resulted from
tion pressure for the presence of the poly(C) tract the observation that a region within the P1-coding
within the viral RNA. When virus was recovered sequence was required to achieve efficient replica-
from infectious RNA transcripts, derived from plas- tion of replicons based on human rhinovirus-14
mids that initially contained as few as 6 C residues (HRV-14) (McKnight and Lemon, 1996). This
at this location, it was found that the length of the contrasts with studies on the closely related PV
poly(C) tract in the replicated RNA had increased RNA that have demonstrated that the entire capsid
to about 80 nt or more (Rieder et al., 1993). In coding sequence can be deleted without affecting
contrast, RNA transcripts containing just two C RNA replication (Barclay et al., 1998). Further
residues at the location of the poly(C) tract did not work (McKnight and Lemon, 1998) showed that
regenerate a poly(C) tract but these rescued viruses HRV-14 RNA replication required a specific stem–
grew fairly poorly in tissue culture cells. However, loop structure located within the coding region for
each of the rescued viruses retained virulence in the capsid protein 1D (VP1). This structure was
mice irrespective of the length of the poly(C) tract termed a ‘cre’ and subsequently, analogous ele-
(Rieder et al., 1993). The instability in the length of ments have been identified in other picornavirus
this tract in FMDV RNA is in marked contrast to genomes (e.g. Lobert et al., 1999, Goodfellow et al.,
observations made using mengovirus RNA (Duke 2000; Gerber et al., 2001). The cre elements from
et al., 1989, 1990). Modified mengoviruses, with HRV-14, HRV-2, cardioviruses and PV are within
differently sized poly(C) tracts, were rescued in the coding regions for 1D, 2A, 1B and 2C respec-
tissue culture and their virulence was determined tively. However, these cre structures can be moved
in mice. The viruses with short poly(C) tracts were without blocking function. Interestingly, Mason et
greatly attenuated but remarkably no selection for al. (2002) provided functional evidence for a cre,
viruses with enlarged tracts occurred within these located within the 5′-UTR of FMDV, just upstream
of the IRES (see Fig. 2.1). Each of the cre structures term ‘cre’ (cis-acting replication element) may not
defined within the entero-, rhino-, cardio- and be entirely appropriate, at least for the FMDV ele-
aphthovirus RNAs include a conserved sequence ment, and an alternative title of 3B-uridylyaltion
motif, AAACA, located within a loop at the top of site (bus) was proposed (Tiley et al., 2003) but the
a stable stem-structure (Rieder et al., 2000; Gerber original terminology has persisted.
et al., 2001; Mason et al., 2002). It has been demon-
strated that insertion of the wt FMDV cre into the
3′ UTR could restore replication ability to an RNA The IRES element – structure
transcript containing a mutated (and defective) cre and function
within its 5′UTR (Mason et al., 2002). Translation of FMDV RNA is initiated internally,
It was shown that the PV and HRV-2 cre under the control of a sequence known as the IRES
sequences act as a template for the uridylylation (Belsham and Brangwyn, 1990; Kuhn et al., 1990;
of VPg (3B) by the viral RNA polymerase in vitro Martinez-Salas et al., 1993). The FMDV IRES con-
(Paul et al., 2000; Gerber et al., 2001) and similar sists of a highly structured region, located several
observations have been reported for the analogous hundred nucleotides away from the uncapped 5′
FMDV structure (Nayak et al., 2005, 2006). This end of the genomic RNA (see Fig. 2.1). Initiation of
reaction generates the products VPgpU and/or translation mediated by IRES elements represents
VPgpUpU that act as primers for viral RNA syn- an alternative to the cap-dependent translation ini-
thesis. Thus, it is believed that this process is an tiation mechanism used for most cellular mRNAs.
essential first step in RNA replication. Each of the The 5′ end of all cytoplasmic eukaryotic mRNAs
three distinct FMDV VPg peptides can take part in has a cap structure (m7GpppN…) which plays a
this reaction in vitro (Nayak et al., 2005), consistent crucial role during translation initiation, it is recog-
with the use of each form of VPg within infected nized by the initiation factor eIF4F (Gingras et al.,
cells (King et al., 1980). 1999; Hershey and Merrick, 2000; Sonenberg and
The reaction, in vitro, requires a number of com- Hinnebush, 2009). This heterotrimer comprises
ponents; in addition to the VPg and the 3Dpol, it the translation initiation factors eIF4E (that binds
is necessary to include an RNA template including to the cap), eIF4A (an RNA helicase) and eIF4G.
the cre and the 3CD precursor protein (Nayak et The latter component has distinct binding sites
al., 2005). The role of the 3CD can be achieved by not only for eIF4E and eIF4A but also for other
3C alone, albeit less efficiently, and modification proteins including eIF3 (located on the small
of specific residues within 3C, that are involved in ribosomal subunit), the poly(A) binding protein
RNA binding, strongly inhibited VPg uridylylation (PABP) and Mnk - 1 (an eIF4E kinase). It is gen-
activity in vitro and also blocked virus replication erally believed that the small ribosomal subunit
within cells (Nayak et al., 2006). interacts with the eIF4F complex (bound at the 5′
When the cre structure in FMDV RNA was cap) and then migrates with it along the 5′ UTR in a
identified it provided an explanation for some 5′ to 3′ direction. The presence of eIF4A is thought
results that had demonstrated the presence of a to facilitate the unwinding of RNA secondary struc-
temperature-sensitive (ts) mutation within the ture (see Svitkin et al., 2001a). This migration is
5′-UTR of FMDV RNA. This mutation, located called scanning and continues until an AUG codon
within the stem–loop structure that constitutes the in an appropriate context (as defined by Kozak,
cre, destabilized the structure. A non-ts revertant 1989) is encountered. At this point, the ribosome
virus has a compensating mutation within the cre pauses, the large ribosomal subunit joins and poly-
that restores the stability of the stem (Tiley et al., peptide synthesis can commence. The ability of the
2003). An important feature of this ts mutant is that scanning process to recognize the correct initiation
the defect in replication can be complemented in codon can be inhibited by complex RNA structure
trans by other ts FMDVs (with defects elsewhere and by the presence of additional AUG codons. In
in the genome). This result seems compatible with contrast, internal initiation of translation mediated
the earlier observations that a pool of free VPg- by an IRES involves the direct recruitment of the
pUpU is generated within picornavirus-infected translational machinery to an internal position
cells (Crawford and Baltimore, 1983). Thus, the in the mRNA usually with the help of cellular
trans-acting factors. This process may, or may not, compare them to the structural features described
be followed by ribosome scanning (see below). for other IRES elements.
IRES elements were first identified within picor-
navirus RNAs (Pelletier and Sonenberg, 1988; Structure and function of the FMDV
Jang et al., 1988). Consistent with their role in IRES element
picornavirus translation, IRES-dependent transla- RNA virus genomes are reservoirs of structural
tion initiation can bypass stress conditions that are elements that perform specific steps in the viral rep-
inhibitory for cap-dependent translation initiation lication cycle, as illustrated by 3′ cap-independent
(Hellen and Sarnow, 2001; Martinez-Salas et al., translation enhancers (3′CITE), tRNA-like struc-
2001, 2008). Such conditions occur following tures (TLS), internal ribosome entry site elements
eIF4G cleavage (Gradi et al., 1998) that is induced (IRES), cis-acting replication elements (cre),
during picornavirus infection as a consequence etc. (Paul and Wimmer, 2015; Simon, 2015). In
of the action of viral proteases Lb (FMDV) or 2A particular, IRES elements adopt specialized three-
(poliovirus), or due to the dephosphorylation dimensional structures, which act in concert with
of the translational repressor 4E-BP1 in EMCV- host factors to recruit the translation machinery
infected cells (Gingras et al., 1996). internally on the RNA using a mechanism that is
The element in the viral RNA that constitutes independent of the 5′-terminus. However, IRESs
the IRES is believed to adopt a three-dimensional belonging to different groups of RNA viruses lack
structure that is essential for internal initiation of conservation of primary sequence and secondary
translation. However, IRES elements belonging RNA structure; they also differ in their host factor
to the picornavirus family do not show extensive requirements to recruit the translation machinery.
primary sequence conservation. Two major groups The sequences of picornavirus IRESs are generally
of picornavirus IRES structures were initially longer than other viral IRESs, and their secondary
described, from the entero-/rhinoviruses and structure is more heterogeneous (Martinez-Salas et
from the cardio-/aphthoviruses (Belsham and al., 2008). According to their secondary structures,
Jackson, 2000). Neither of these types of IRES picornavirus IRESs are classified into five different
has any apparent similarity to that of hepatitis C types (Sweeney et al., 2012). Furthermore, while
virus (HCV) (Honda et al., 1999). However, it has some motifs are found in various picornavirus
become apparent that at least five different types IRESs, others are exclusive for one type suggesting
of IRES element are present within the incessantly a specialized requirement for factors (Martinez-
growing family of picornaviruses (see reviews Salas et al., 2015).
by Belsham, 2009; Hellen and de Breyne, 2007; In spite of the diversity of primary sequence,
Martinez-Salas et al., 2015). One type, initially each type of picornavirus IRES harbours conserved
identified within the porcine teschoviruses (Kaku sequence motifs and a common RNA structure
et al., 2002; Pisarev et al., 2004; Chard et al., 2006a) core maintained by covariant substitutions. Evo-
is closely related to the HCV IRES and has now lutionary conserved motifs tend to preserve both
been identified in multiple genera of picornavi- RNA structures and RNA–protein interactions
ruses (Chard et al., 2006b, Bakhshesh et al., 2008; that determine the activity of the IRES element
Belsham, 2009; Hellen and de Breyne, 2007). (Lozano and Martinez-Salas, 2015). Compelling
The absence of apparent structural conservation evidence for the relevance of RNA structures for
between the different types of IRES element may IRES function was initially provided by functional
reflect the diversity of strategies used by IRES ele- studies. Indeed, mutations disrupting certain stems
ments to interact with the translational machinery. impaired IRES activity and, conversely, compensa-
However, it is noteworthy that the IRES elements tory mutations restored IRES function (Fernandez
from FMDV and PV, albeit of different types, each et al., 2011a; Haller and Semler, 1992; Haller et al.,
interact directly with eIF4G (López de Quinto and 1996; Hoffman and Palmenberg, 1996; Martinez-
Martinez-Salas, 2000; Stassinopoulos and Belsham, Salas et al., 1993, 1996; Serrano et al., 2007, 2009;
2001; de Breyne et al., 2009). We discuss here the van der Velden et al., 1995; Witherell et al., 1995).
features of the RNA structure of the FMDV IRES The RNA structure of the enterovirus IRES is
that are known to be functionally relevant and organized in modular domains (designated II to
VI). These IRES elements span about 450 nt in the enterovirus IRES harbours the Yn-Xm-AUG
which conserved pyrimidine tracts occur at the motif in which Yn (a pyrimidine tract of 8–10 nt) is
base of domain II, and in the central and basal part separated by a spacer (Xm, 10–20 nt) from an AUG
of domain V (Bailey and Tapprich, 2007). Domain triplet. This motif has been proposed to be the ribo-
IV harbours a C-rich loop and a GNRA motif some entry site, which in turn is separated from the
(N stands for any nucleotide, and R for purine) functional initiator codon by a long non-conserved
(Du et al., 2004). Domain V provides the binding spacer, which is scanned by the initiation complex
site for the polypyrimidine tract-binding protein (Hellen et al., 1994).
(PTB), which partially overlaps with the binding The RNA structure of the cardio- and aphtho-
sites for eIF4G and eIF4A during 48S complex virus IRES is arranged in modular domains (desig-
assembly (Sweeney et al., 2014). The 3′-border of nated H to L, or 1 to 5) (see Fig. 2.2). Uniquely
Figure 2.2 Schematic representation of the RNA structure of the FMDV IRES with indication of the binding sites
for host-factors. The secondary structure model was predicted by RNAstructure software imposing the values
of SHAPE reactivity. Nucleotides are coloured according to their SHAPE reactivity (grey < 0.2; yellow 0.2–0.5;
red > 0.5–1; dark red >1). The IRES domains (2 to 5, or H to L) are depicted at the bottom. Stem-loops (SL) and
junctions (J) referred to in the text are shown in blue, whilst conserved RNA motifs are depicted in black letters.
The sites on the IRES that interact with eIF4G, eIF4B, PTB, PCBP2 and Gemin5 are described in the text. The
first functional AUG (AUG1) is boxed.
for FMDV RNA, domain 1 of the IRES is located (6 mM). Differences in RNA conformation are
just downstream of the cre element (Mason et al., found in the hexaloop of SL3a, the loop of SL3b
2002). Domain 2 contains a conserved pyrimidine and the J1 junction that links SL1 with SL2 (see
tract that provides a binding site for PTB ( Jang and Fig. 2.2). Besides the higher flexibility of the apical
Wimmer, 1990; Luz and Beck, 1991). Domain 3 region of domain 3 at low Mg2+ concentration,
is a self-folding cruciform structure (Fernandez- it is also worth noting that the interaction of the
Miragall and Martinez-Salas, 2003) that harbours protein eIF4G with the FMDV IRES is impaired
three conserved motifs (GNRA, RAAA, and the at high Mg2+ concentration (Lozano et al., 2014),
C-rich loop) (Fernandez-Miragall et al., 2009). in agreement with earlier reports showing that
The GNRA motif is essential for IRES activity in concentrations of Mg2+ above 5 mM inhibit IRES-
both FMDV and EMCV (Lopez de Quinto and driven protein synthesis (Shenvi et al., 2005). Thus,
Martinez-Salas, 1997; Robertson et al., 1999). it appears that IRES activity is permissive at low
Domain 4 consists of a Y-shape RNA structure Mg2+ concentration, coincident with a more flex-
(containing subdomains J and K) and provides ible RNA conformation.
the binding-site for eIF4G (Bassili et al., 2004; Although the IRES of EMCV and FMDV differ
Kolupaeva et al., 1998; Lopez de Quinto and in primary sequence (ca. 50% identity), they have a
Martinez-Salas, 2000; Stassinopoulos and Belsham, similar secondary structure. In both cases, domain
2001; Clark et al., 2003). Finally, domain 5 consists 3 consists of two differentiated structural regions,
of a short hairpin with a conserved pyrimidine tract located in the basal and the apical parts of this
at its 3′ end that provides the binding site for eIF4B, domain. In the FMDV IRES, the apical region of
PTB, and other RNA-binding proteins (Lopez de domain 3 harbours stem–loops designated SL1
Quinto et al., 2001; Meyer et al., 1995; Pacheco et (absent in EMCV IRES), SL2 and SL3abc. In addi-
al., 2008). tion to the previously mentioned conserved motifs
Overall, the data derived from functional GNRA and RAAA, the hexaloop of SL3a and the
analyses are in agreement with the conservation of C-rich bulge (heptaloop) of SL2 are also conserved
structural motifs in highly variable viral genomes in both the EMCV and FMDV IRES elements
(Lozano and Martinez-Salas, 2015; Martinez-Salas, (Lozano et al., 2016a). Mutational analysis carried
2008). Likewise, the lack of accessibility towards out on SL1 and SL3b of the FMDV IRES showed
RNases was in accordance with the stems defined that disruption of the stems reduced IRES activity
by covariation data (Fernandez et al., 2011a). More whereas compensatory mutations restoring the sec-
recently, selective 2′-hydroxyl acylation analysed ondary structure recovered translation (Fernandez
by primer extension (SHAPE) reactivity data also et al., 2011a, 2013). Disruption of the SL3b stem
supported a modular organization of the IRES ele- abolishes IRES activity, and induces a reorganiza-
ment (Fig. 2.2), and reinforced the evidence for the tion of the entire apical region (Fernandez et al.,
localization of loops and internal bulges within the 2011a).
IRES structure in solution (Fernandez et al., 2011b; Based on the observations of inter- and intra-
Lozano et al., 2014, 2016b). Incorporation of molecular RNA–RNA interactions (Ramos and
SHAPE reactivities as constraints to RNA structure Martinez-Salas, 1999) it has been proposed that
software (Reuter and Mathews, 2010) generates domain 3 plays a fundamental role in dictating the
RNA structure models, which are in agreement formation of a constrained structure, likely provid-
with the data for the RNA accessibility to DMS ing the correct orientation to recruit the ribosome
and RNases T1 and T2 (Fernandez-Miragall et al., subunits to the initiation site (Martinez-Salas,
2009). 2008). A notable combination of structural motifs
Concerning the relevance of RNA structure for is found in this domain. The GNRA tetraloop
IRES function, the local RNA flexibility of specific motif is frequently found in folded RNAs (Correll
IRES regions is sensitive to the concentration of and Swinger, 2003); the loop–helix interactions
Mg2+ ions in the RNA folding buffer (Lozano et al., combine base pairing and stacking to determine
2014). Noteworthy, the apical loop of domain 3 is a tertiary conformation that stabilizes the global
more flexible at a near physiological concentration RNA folding. The GNRA motif adopts a tetraloop
(0.5 mM) of Mg2+ rather than at high concentration conformation in both FMDV and EMCV IRESs
(Dupont and Snoussi, 2009; Fernandez-Miragall and J2, within the apical region of domain 3 con-
and Martinez-Salas, 2003; Phelan et al., 2004). It nect the GNRA motif of SL3a and its potential
was proposed that the GNRA motif determines the receptor in SL2. Molecular dynamics simulation
folding of the apical region of domain 3, since its that explored the conformational variability in the
substitution by a stable UNCG tetraloop disrupts J1 junction revealed transitions between parallel
IRES function (Fernandez-Miragall and Martinez- and antiparallel conformations via a perpendicular
Salas, 2003). Additionally, structural analysis of intermediate that maintains the coaxial stacks,
GNRA substitution mutants (GUAA to GUAG, providing a modular structural platform that can
or to UCCG) showed a specific modification of be adjusted by the binding of cofactors and ligands.
the accessibility towards DMS and RNase T1 in Computational modelling of the entire domain
the distant residue G240, together with a change 3 suggests that the basal region is set apart from
in the local accessibility of the GNRA motif and a the apical region ( Jung and Schlick, 2014). How-
reorganization of SL3b. Conversely, substitution ever, biochemical RNA probing of a transcript
mutants affecting the G240 position induced recip- bearing solely this region showed a higher accessi-
rocal changes in the GNRA motif. These results bility to residues 120–133 within an internal bulge
led to the proposal of the existence of a potential (Fernandez-Miragall et al., 2006), suggesting that
GNRA receptor within the distant C-rich loop the stability and the global structure of the entire
(Fernandez-Miragall et al., 2006). domain depends on the apical region.
Computational modelling of domain 3 has been
achieved by a divide-and-conquer approach using Host factors critical for picornavirus
the secondary structure determined by mutational IRES activity
analysis and RNA probing to model RNA junction Picornavirus IRES-dependent translation ini-
topology ( Jung and Schlick, 2013). Candidates tiation bypasses stress situations that compromise
for three-dimensional models generated using cap-dependent translation initiation that occurs
MC-Sym (Parisien and Major, 2008) subjected to in FMDV-infected cells. Under normal situa-
molecular dynamics (MD) simulations identified tions, however, most eukaryotic mRNAs initiate
energetically favourable and stable conformational translation by a mechanism that depends on the
states compatible with the GNRA tetraloop–recep- recognition of the m7GpppN residue (or cap)
tor interactions (Fernandez-Miragall et al., 2006), located at the 5′ end of most mRNAs (Sonenberg
in which the adenosines A180 and A181 form hydro- and Hinnebusch, 2009). Many cellular translation
gen bonds with the receptors C230/G242 and G231/ initiation factors (eIFs) play an essential role in
C241 base pair, respectively. A similar tertiary inter- IRES-dependent initiation promoted by the FMDV
action was proposed to occur in the EMCV IRES and EMCV IRES elements (reviewed by Belsham
using a synthetic 16mer harbouring the GCGA and Jackson, 2000; Martinez-Salas et al., 2001). The
tetraloop and a 17mer corresponding to the C-rich canonical initiation factors eIF4A, eIF4G, eIF2 and
heptaloop (Mohammed et al., 2014). The GCGA eIF3, are each required for 48S complex formation
tetraloop of the 16mer folds into a standard GNRA in a reconstituted 40S ribosome-binding assay with
conformation (Correll and Swinger, 2003), with the EMCV or FMDV IRES elements (Pestova et
the A residue being in the form of a G: A sheared al., 1996; Kolupaeva et al., 1998; Pilipenko et al.,
base-pair. The C-rich loop of the 17-mer forms a 2000). In particular, reconstitution assays have
compact tertiary loop motif, in which Mg2+ ions shown that assembly of 48S initiation complexes
enhance RNA stability. Furthermore, stable RNA onto the FMDV IRES, extended through to the
structures were generated using synthetic oligo- second AUG, requires eIF4A, eIF1, eIF3, and
ribonucleotides that combine the GNRA tetraloop the C-terminal fragment of eIF4G resulting from
and the C-rich heptaloop of the EMCV IRES in the proteolytic cleavage induced by the L pro-
28-, 36- 44- and 48-mers as building blocks (Chan tease (Andreev et al., 2007). The region of eIF4G
and Ramesh, 2012). that interacts with the EMCV and FMDV IRES
In the dynamic simulations of the FMDV IRES, (residues 630–1560) contains the binding sites
SL3a forms an L-shape configuration ( Jung and for eIF3 and eIF4A (Gingras et al., 1999). Accord-
Schlick, 2014). Two four-way RNA junctions, J1 ingly, eIF4A stimulates binding of the central part
of eIF4GI (amino acids 746–949) to the EMCV negative mutants of eIF4A (which interfere with
and FMDV IRES (Pilipenko et al., 2000; Lomakin eIF4F function) block picornavirus IRES activity
et al., 2000). Consistent with the interaction of (Pause et al., 1994; Pestova et al., 1998; Svitkin et
the eIF4G-Ct with these picornavirus IRES ele- al., 2001a).
ments, internal initiation of translation promoted The 3′ region of the FMDV IRES has been
by these elements is highly efficient under condi- shown to interact with the eIF4B initiation factor
tions of eIF4G cleavage (Belsham and Brangwyn, (Meyer et al., 1995; López de Quinto and Martinez-
1990; Martinez-Salas et al., 1993; Ohlmann et al., Salas, 2000; Stassinopoulos and Belsham, 2001).
1996; Roberts et al., 1998; López de Quinto and Sequence comparison of field isolates of FMDV
Martinez-Salas, 2000). showed that domain 5 contains a conserved hairpin
A transcript corresponding to FMDV domain (see Fernandez-Charmorro et al., 2016) that is also
4 alone binds to eIF4G (see Fig. 2.2) as effectively shared with the EMCV IRES and a high-affinity
as the full length IRES, strongly suggesting that ligand for eIF4B that was selected in vitro (Methot
no additional sites for recognition of this protein et al., 1996; López de Quinto et al., 2001). How-
within the FMDV IRES are required. Furthermore, ever, substitution of conserved residues within
the carboxy-terminal end of the proteolytically pro- the hairpin structure did not impair FMDV IRES
cessed form of eIF4G (eIF4G-Ct), present in cells activity to the same extent as the mutations intro-
transfected with the FMDV Lb protease, binds as duced into the A-bulge of domain 4, or the GNRA
efficiently to the FMDV IRES as the unprocessed loop of domain 3. In spite of the strong binding
protein (López de Quinto and Martinez-Salas, capacity of the FMDV IRES for eIF4B, mutants
2000). Fully consistent with this result, the use impaired in eIF4B-binding only reduce IRES activ-
of RNA transcripts (ca. 150 nt) from the 3′ end ity two- to fourfold (López de Quinto et al., 2001).
of the FMDV IRES to deplete RRL extracts In agreement with this, the eIF4B initiation factor
lead to a strong reduction of factors required only stimulates 48S complex formation on the
for cap-dependent translation, including eIF4G EMCV IRES about two-fold (Pestova et al., 1996).
(Stassinopoulos and Belsham, 2001). Disruption In the early studies on IRES elements, a number
of the structural motif at the base of domain 4 is of proteins were found to associate with picornavi-
associated with the lack of binding of eIF4G to rus IRES elements, e.g. PTB and La (reviewed in
the FMDV IRES (López de Quinto and Martinez- Belsham and Sonenberg, 1996, 2000). Identifica-
Salas, 2000). Consistent with this observation, tion of these proteins as non-canonical initiation
eIF4G interacts with the related EMCV IRES in factors was particularly interesting since it seemed
a similar position (Kolupaeva et al., 1998), and possible they could contribute to IRES tropism
additional nucleotides within the J-domain have in different cell types, and hence determine viral
also been implicated in this interaction (Kolupaeva spread within an infected animal. Interestingly, a
et al., 2003; Clark et al., 2003). protein named unr (upstream of N-ras) is unique
The second species of eIF4G, termed eIF4GII, in its capacity to interact with the rhinovirus and
is also cleaved by the FMDV encoded Lb protease PV IRES elements (Hunt et al., 1999; Boussadia
in vitro (López de Quinto et al., 2001) and within et al., 2003) whereas PTB interacts with all IRES
FMDV-infected cells (Gradi et al., 2004). The elements tested (Luz and Beck, 1991; Hunt and
C-terminal fragment (Ct) also has a strong binding Jackson, 1999). PTB (also known as hnRNP I) was
affinity for the FMDV IRES, suggesting its involve- the first protein reported as an ITAF that stimu-
ment in IRES activity. Interestingly, a fragment of lated the activity of cardio- and aphthovirus IRES
the central region of eIF4GII interacts with the elements ( Jang and Wimmer, 1990; Luz and Beck,
EMCV IRES through a non-canonical RRM region 1991). This protein harbours four RNA recognition
(Marcotrigiano et al., 2001). The strong correlation motifs (RRM) that recognize U/C-rich sequences
found between the eIF4G–IRES interaction and (Conte et al., 2000). During early studies of the
IRES activity in transfected cells demonstrates that picornavirus IRES elements, it was noticed that a
eIF4G binding is an essential step in the recruitment polypyrimidine tract was the only motif conserved
of the translational machinery in vivo. Consistent between rhino-/enterovirus IRES elements and the
with these results, it has been shown that dominant cardio-/aphthovirus IRES elements (Meerovitch et
al., 1991; Pilipenko, et al., 1992; Meerovitch and de Quinto and Martinez-Salas, 2000; López de
Sonenberg, 1993). Both, FMDV and EMCV have Quinto et al., 2001, Stassinopoulos and Belsham,
two polypyrimidine tracts located at each end of 2001). The 5′ and central regions (domains 1–2
the IRES region. In agreement with the functional and 3, or H and I) are probably involved in the
role of each pyrimidine tract on IRES activity it has organization of the IRES architecture, directing
been found that PTB constrains the EMCV IRES intramolecular RNA–RNA interactions (Ramos
structure in a unique orientation by binding to the and Martinez-Salas, 1999). In agreement with this,
RNA with RRM1 – 2 contacting the 3′ end, and the PTB protein that seems to act as an RNA chap-
RRM3 contacting the 5′ end of the IRES (Kafasla erone has its main binding site near the 5′ end of
et al., 2009). the IRES but it also interacts with sequences near
In contrast to the Theiler’s murine encephalitis the 3′ end (Kolupaeva et al., 1996; Luz and Beck,
virus (TMEV) and EMCV IRES elements, the 1991).
FMDV IRES required binding to PTB and to the In addition to eIFs, several RNA-binding pro-
proliferation associated factor, ITAF45 (also known teins designated IRES-transacting factors (ITAFs),
as Ebp1 or PA2G4), for 48S complex formation stimulate the assembly of 48S complexes in vitro on
in vitro (Pilipenko et al., 2000). Therefore, even the cardio- and aphthovirus IRESs (Pilipenko et al.,
closely related IRES elements (i.e. from EMCV and 2000; Pilipenko et al., 2001; Yu et al., 2011). ITAFs
FMDV) that share secondary structure and primary are RNA binding proteins identified previously as
sequence in essential regions behave differently in factors involved in transcription regulation, splic-
terms of functional RNA–protein association. ing, RNA transport, RNA stability, or translational
Other members of the hnRNP family that control. Consistent with the fact that the entire
have been identified to associate with picornavi- picornavirus replication cycle occurs in the cell
rus IRESs are hnRNP K, PCBP1 (hnRNP E1) cytoplasm, many ITAFs are nuclear proteins that
and PCBP2 (hnRNP E2) (Gamarnik et al., 2000; shuttle to the cytoplasm in infected cells. In this
Lin et al., 2008; Sean et al., 2009). These proteins way, factors that normally participate in nuclear
recognize poly-r(C) regions and share the KH events associated with cellular RNA metabolism
RNA-binding domain. However, although PCBP2 become relocalized and participate in translation
binds to both EMCV and FMDV IRES, this modulation of viral RNAs lacking nuclear localiza-
factor only stimulates the first one (Walter et al., tion (Fitzgerald and Semler, 2011). Although early
1999), and is the only ITAF absolutely required to studies identified ITAFs as proteins stimulating
assemble 48S initiation complexes on enterovirus IRES activity, various examples of IRES down-
IRESs using purified components (Sweeney et al., regulators have also been found in later studies.
2014). Known IRES interacting proteins (unr, For instance, the cellular mRNA decay protein
PTB, La, ITAF45 and PCBP2) contain several AU-binding factor (AUF1) behaves as a negative
RNA binding motifs, and display multiple sites regulator of EV71 and HRV infections (Cathcart et
of interaction with the IRES molecule (Blyn et al., 2013). Another example of a repressor ITAF is
al., 1997; Hunt et al., 1999; Pilipenko et al., 2000; Gemin5 (see Fig. 2.2), a cytoplasmic protein that
Conte et al., 2000; Kim et al., 2000). Hence, it has binds directly to the FMDV IRES (Pineiro et al.,
been suggested that these proteins act as RNA 2013) and down-regulates translation (Pacheco
chaperones, directing or stabilizing the tertiary et al., 2009). An additional feature of many ITAFs
folding of the RNA. is their recognition as substrates of picornavirus-
The EMCV and FMDV IRES elements seem encoded proteases. In some cases, this leads to the
to possess a modular organization, in which the generation of truncated peptides with a different
different domains perform a precise function but capacity to modulate translation than the uncleaved
none of them is active on its own. The 3′ region polypeptide. Accordingly, proteolysis of PTB in
(domains J–K or 4–5) mediates the interaction PV-infected cells results in truncated polypeptides
with eIFs required for 48S complex formation in that repress IRES activity (Back et al., 2002), and
vitro (Kolupaeva et al., 1998; Pilipenko et al., 2000). proteolysis of Gemin5 in FMDV-infected cells
This region establishes RNA–eIF4G interactions, (Pineiro et al., 2012) leads to the appearance of
which are essential for IRES activity in vivo (López C-terminal fragments. These fragments, which
contain the IRES-repressor element on the most the RNA conformational changes induced by IRAB
distal domain, harbour a noncanonical bipartite could result in a misfolded IRES structure leading
RNA-binding motif (RBS1–RBS2) (Fernandez- to diminished internal initiation of translation.
Chamorro et al., 2014). In contrast, the FBP2
fragment lacking the C-terminal region behaves Implications of long-range 5′–3′ viral
as an IRES stimulator (Chen et al., 2013). In sum- RNA interactions on IRES activity
mary, most ITAFs are proteins that shuttle between The 3′UTR of the picornavirus genome plays a
the nucleus and the cytoplasm, and in many critical role in virus multiplication (Duque and
cases are targets for viral proteases or undergo Palmenberg, 2001; Melchers et al., 1997; Saiz et al.,
post-translational modifications leading to the 2001; Todd et al., 1997a). This region harbours a
reprogramming of gene expression within infected heterogeneous sequence, in addition to a polyA
cells. tail. An active role for interactions between the 5′
and 3′ ends of the viral RNA in controlling picor-
Small molecules perturbing the local navirus translation was supported by the fact that
flexibility of domain 3 inhibit IRES the activity of the FMDV IRES was stimulated by
activity its own 3′UTR (Garcia-Nunez et al., 2014; Lopez
Since RNA structure plays a critical role in de Quinto et al., 2002), irrespective of the presence
the function of regulatory elements control- of a polyA tail and the coexpression of the L pro-
ling expression of viral proteins, understanding tease. In addition to RBPs, 5′–3′ end bridges involve
the three-dimensional structure could help the direct long-distance RNA–RNA contacts (Serrano
development of antiviral drugs that inhibit virus et al., 2006), presumably leading to a pseudo-
multiplication. A variety of small molecules target- circularization of the viral RNA. It is worth noting
ing specific RNA motifs essential for picornavirus that the 3′end region, which is essential for FMDV
multiplication have been explored as antiviral agents infectivity (Saiz et al., 2001), can mediate two dif-
(de la Torre et al., 1987; Fajardo et al., 2012; Stone ferent long-range interactions (Serrano et al., 2006),
et al., 2008; Vagnozzi et al., 2007). Moreover, benzi- one with the IRES element and another with the S
midazole derivatives rearrange the SLIIa structural hairpin at the 5′end. These long-range interactions,
element of HCV-like IRESs, including those of in concert with the corresponding protein partners,
picornaviruses (Boerneke et al., 2014). Similarly, provide a mechanistic basis for the stimulation of
a novel benzimidazole compound (IRAB) resulted cap-independent translation of picornavirus RNAs
in a negative effect on both the HCV and FMDV resembling the synergistic stimulation of the cel-
IRES activities (Lozano et al., 2015), suggesting lular mRNA cap-dependent translation by the cap
some structural similarities between these distinct and polyA tail.
IRESs. Interestingly, following incubation of the
FMDV IRES transcript with IRAB, SHAPE prob-
ing revealed changes in the local flexibility of SL3A. The polyprotein coding region
This involved residues of the hexaloop and the The major portion (ca. 7000 nt) of the viral genome
GNRA motif, in addition to SL3C and SL1, while comprises the coding sequence for a polyprotein of
residues located at the junction J1 of the C-rich about 2330 amino acids. The length of the coding
loop were partially protected. Additionally, use of region can be increased to some extent; Seago et al.
aminopurine-labelled oligoribonucleotides showed (2013) showed that insertions of up to 300nt within
that the SL3A alone is enough to support binding the coding region could be accommodated by the
to this compound (Lozano et al., 2015). However, virus but inserts of 400 nt or more were unstable.
fluorescence data in conjunction with SHAPE The encoded polyprotein is never observed within
reactivity strongly support the hypothesis that the infected cells or within in vitro translation systems
entire apical region of domain 3 may be important (e.g. rabbit reticulocyte lysate, RRL) since this pro-
for IRAB binding, again supporting the idea that tein is rapidly processed by virus-encoded proteases
the entire apical region of domain 3 is a structural that are present within the polyprotein sequence
entity (Fernandez-Miragall and Martinez-Salas, (see Chapter 3). A large variety of precursors can
2003; Fernandez-Miragall et al., 2006). Therefore, be generated by alternative cleavage pathways (see
Fig. 2.1) and some of these precursors, as well as the codons, it was found that the 4 AUG codons were
mature products, may have distinct biological roles. each recognized, independently of whether transla-
The FMDV polyprotein yields 15 different mature tion initiation occurred by an IRES-dependent or
proteins including the two forms of the Leader (L) cap-dependent mechanism. Thus, the 84-nt region
protein and three different copies of VPg (3B1–3). between the two natural initiation codons has unu-
The function of some of these products is the sub- sual features. The most likely explanation given for
ject of other chapters within this volume and we the results was that the FMDV IRES directed ribo-
will consequently give only a brief outline of these somes to bind initially to the RNA just upstream
and thus will concentrate on other issues. of the Lab start site, as for EMCV. The recognition
of this first start site on FMDV RNA must be inef-
Selection of the initiation site for ficient, however, so that many ribosomes can then
protein synthesis scan downstream until the next initiation codon is
The sequencing of FMDV RNA (Forss et al., 1984; encountered (Belsham, 1992).
Carroll et al., 1984) indicated that two in-frame Subsequently, Cao et al. (1995) demonstrated
AUG codons, 84 nt apart, were present in the virus that the Lab initiation codon was not required for
genome that could potentially act as initiation virus viability but found that modification of the
codons. This feature is conserved across all seven Lb initiation codon was lethal. A requirement for
serotypes of the virus (Sangar et al., 1987). Each the Lb start site was also observed by Piccone et al.
initiation codon is preceded by a polypyrimidine (1995a), who constructed viable mutants of FMDV
tract; the arrangement of a polypyrimidine tract that lacked the Lb protein coding region but found
followed by an AUG codon is a general feature at that transcripts lacking the Lb start site were non-
the 3′ end of picornavirus IRES elements (Meero- infectious. To further analyze the initiation site,
vitch and Sonenberg, 1993). On FMDV RNA both López de Quinto and Martinez-Salas (1999) modi-
AUG codons are used as initiation sites for protein fied the context of the Lab start site and showed that
synthesis, generating two forms of the Leader (L) improving the context did increase the efficiency of
protein, termed Lab and Lb. Studies on EMCV translation initiation at this site. However, this had
RNA demonstrated that ribosomes initiated trans- no apparent effect on the efficiency of initiation at
lation at AUG-11 on the EMCV-R strain RNA the Lb start site. Hence, it was suggested that these
without encountering AUG-10 located just 8 nt two initiation codons could act independently.
upstream (Kaminski et al., 1990). The Lab start This view was supported by the observation that an
site on FMDV RNA is located at the same position antisense oligonucleotide, which annealed to the
relative to the IRES element as AUG-11. However, region of the Lab start site, blocked initiation at this
only a minor fraction of the ribosomes initiate site but had little effect on initiation at the Lb site.
protein synthesis at this point since Lb is made in Thus, it may be that some ribosomes are directed by
excess over Lab in infected cells (Lopez de Quinto the FMDV IRES to bind to the RNA downstream
and Martinez-Salas, 1999). of the Lab start site. However, this putative entry
To determine the mechanism of recognition of site must presumably be close to the Lab start site
the two start sites on FMDV RNA, Belsham (1992) since the additional AUG codons introduced just
analysed the usage of these start sites under a variety 18 nt downstream of the Lab site were efficiently
of conditions. RNA transcripts including the two recognized during IRES-directed translation initia-
FMDV initiation codons, either preceded by the tion (Belsham, 1992).
FMDV IRES element or not, were expressed within Reconstitution studies using purified compo-
cells and it was observed that both the Lab and Lb nents have shown a differential requirement of eIF1
start sites were used on each RNA. The Lb start and eIF1A, depending on whether the 48S initia-
site was recognized preferentially in both cases and tion complex is assembled at AUG1 or AUG2 using
the presence of the IRES resulted in greater usage the FMDV IRES extended to the second AUG
of the Lb site than occurred when the initiation (Andreev et al., 2007), suggesting the existence of
codons were recognized by scanning ribosomes. two distinct mechanisms operating at each of these
When two additional AUG codons were intro- codons. In summary, the FMDV IRES may direct
duced in-frame between the authentic initiation ribosome attachment to the viral RNA either just
upstream or just downstream of the Lab initiation Indeed both species of eIF4G, termed eIF4GI
site. Ribosomes landing upstream of the Lab site and eIF4GII (products of different genes), are
can initiate protein synthesis at this point but some cleaved in the presence of the L protease (see
may fail to do so and can then scan along the RNA below). The consequence of this cleavage is the
until the Lb site is reached. However, ribosomes inhibition of cap-dependent translation initiation
that land downstream of the Lab site can just and hence the loss of nearly all host cell protein
migrate along the RNA to initiate translation at the synthesis. The cleavage of eIF4G requires a very
Lb site. low level of the L protease and can be observed to
Different picornavirus IRES elements may occur within an hour of addition of virus to cells
direct ribosomes to the initiation codon with dif- (Belsham et al., 2000) and hence well before any
ferent degrees of precision (Ohlmann and Jackson, virus-encoded proteins can be detected directly.
1999). To analyse this process, chimeras (fused Indeed, replication-incompetent FMDV RNA
at the polypyrimidine tract) were made between transcripts that include the L coding sequence can
the FMDV IRES and a region of the EMCV RNA still induce efficient eIF4G cleavage when they
around the initiation codon (AUG-11). It was are introduced into cells by electroporation, even
shown that the FMDV IRES directed utilization of though protein can only be produced from the
AUG-11 (the correct initiation site) and AUG-12 input RNA (Belsham et al., 2000). A third activ-
(12 bases downstream) with similar efficiency in ity of the L protease is to stimulate the activity of
vitro. In contrast, the EMCV IRES linked to its own certain picornavirus IRES elements (Borman et
RNA directed almost exclusive use of AUG-11. al., 1997; Roberts et al., 1998; Hinton et al., 2002).
Furthermore, in the converse experiment, when It is not known whether this effect is a direct
the EMCV IRES was linked to the FMDV initia- consequence of the cleavage of eIF4G or whether
tion codons, the Lab site was predominantly used, the L protease also induces the cleavage of other
in contrast to the preferential use of the Lb site on proteins that modify IRES function [see Belsham
natural FMDV RNA. and Jackson (2000) for review and below].
Recombinant Lb protease can cleave isolated
The leader protease eIF4GI directly in vitro. A cleavage site has been
As indicated above, the use of two alternative identified on the C-terminal side of residue 674
initiation codons on the FMDV RNA results in (Kirchweger et al., 1994), which is close to that
the generation of two distinct forms of the Leader determined for the in vitro cleavage of eIF4GI by
protein. Unusually amongst the picornaviruses, the rhinovirus 2A protease (C-terminal side of
the Leader (L) protein of aphthoviruses, including residue 681; Lamphear et al. (1993). (The locations
FMDV and equine rhinitis A virus, is a protease of these cleavage sites are numbered according to
(Strebel and Beck, 1986; Hinton et al., 2002). The the system of Byrd et al. (2002) for the largest
L protease is a member of the papain-like cysteine form of eIF4GI, termed eIF4GIa; multiple initia-
proteases and is unrelated to other picornavirus tion codons are used resulting in multiple forms of
protease sequences. The active site residues have eIF4GI.) However, in both cases, high levels of the
been identified (Roberts and Belsham, 1995; proteases are required to achieve efficient eIF4G
Piccone et al., 1995b) and the 3D structure of cleavage in vitro compared to the very low levels of
FMDV Lb has been determined (Guarne et al., the proteases that are required within cells. Hence,
1998). Both the FMDV Lab and Lb proteins have there are legitimate concerns about whether the
been shown to cleave the L/P1 junction within cleavage of eIF4G induced by the FMDV L and
the polyprotein (Fig. 2.1), thus liberating the L rhinovirus 2A proteases within cells is a direct effect
protease from the rest of the molecule (Medina or is mediated through cellular protease(s) [see
et al., 1993). The protease can work in trans and Belsham and Jackson (2000) for review]. It is note-
probably also in cis (see Glaser et al., 2001). Both worthy that the addition of eIF4E to eIF4G greatly
forms of the L protease also induce the cleavage of enhances the efficiency of cleavage of eIF4G by the
the translation initiation factor eIF4G (Devaney 2A protease in vitro; this may suggest that the con-
et al., 1988; Medina et al., 1993), a component formation of the protein is important (Haghighat
of the cap-binding complex eIF4F (see above). et al., 1996). Evidence has recently been presented
that FMDV Lb only forms a stable complex with about 22,000 transcripts tested were up-regulated
a fragment of eIF4GII in the presence of eIF4E by at least two-fold in the cells infected with the
(Aumayr et al., 2015) and the HRV2 2A protease leaderless virus (Zhu et al., 2010) compared to
also forms a similar trimeric complex. the wt virus. Most of the apparently up-regulated
The identification of the cleavage site in eIF4G genes were known interferon-inducible genes.
that is generated by low levels of the Leader pro- However, a potential problem with such studies is
tease within FMDV-infected cells remains to be that the leaderless viruses grow less well than the
achieved. Such analyses should help to resolve the wt virus in some cells (Chinsangaram, et al., 1999;
issue of whether the FMDV L induced cleavage of de los Santos et al., 2007; Belsham, 2013) and
eIF4G is achieved directly or not. The L protease is hence it is difficult to ensure that the cells infected
not essential for virus viability since the complete with the different viruses are in the same stage
Lb coding sequence can be removed (Piccone of infection. This problem was well illustrated by
et al., 1995a). This leaderless FMDV (lacking the apparent difference in the degradation of the
Lb) was attenuated in cattle (Brown et al., 1996); p65/relA subunit of NFκB in wt and leaderless
however, proof that the attenuation was reversed virus infected cells (de los Santos et al., 2007);
by re-introduction of the missing sequence was although less degradation was apparent, there was
not obtained. In contrast to the deletion of Lb, it is also much less expression of viral protein (VP1).
not possible to delete the whole of the Lab coding Hence, the direct link between the L protease and
sequence and retain infectivity. However, the 84 nt loss of p65/relA in FMDV-infected cells was not
region between the two start sites (Belsham, 2013) clearly established. No cleavage products of p65/
can be deleted if the Lb coding region is still pre- relA have been reported within FMDV-infected
sent. Thus all of the Lab coding sequence can be cells.
deleted but not all at the same time. The basis for There is also a single report indicating that
this observation is not yet clear and is discussed the FMDV L protease has an additional means of
further below. blocking the host interferon response by acting
It may be that the primary advantage to the as a deubiquitinase (Wang et al., 2011). Several
virus of inducing the inhibition of host cell pro- components of the interferon signalling pathway
tein synthesis lies in reducing the ability of the are ubiquitinated within cells and expression of
cell to mount an antiviral response. It has been the L protease appears to block this modification.
shown that the mRNAs encoding the alpha/beta However, it can be difficult to separate out some of
interferons are induced within FMDV-infected the different effects of the L protease into direct and
cells, presumably this is a response to the presence indirect effects (e.g. due to loss of host cell protein
of dsRNA (Chinsangaram et al., 1999; Kaufman, synthesis) and there is a need to isolate mutants
2000). The shut-off of protein synthesis induced by of the protease which are clearly deficient in only
the L protease will inhibit synthesis of the alpha/ individual activities of this protein.
beta interferons within infected cells and hence As indicated above, two forms of the L protein
reduce the cellular antiviral response (Chinsan- are generated within infected cells, precise deletion
garam et al., 1999). Interferons trigger a variety of of Lb is tolerated by the virus (Piccone et al., 1995a)
responses within cells, these responses include an but deletion of the entire Lab coding sequence is
activation of the dsRNA-activated protein kinase not tolerated by the virus (see also Belsham, 2013).
(PKR, see review by Kaufman, 2000). This kinase The function of the 84 nt between the two initiation
phosphorylates the α-subunit of eIF2 and hence sites is not entirely clear. Recent studies have shown
blocks the initiation step of both host and viral that insertion of a 57 nt transposon or an epitope
protein synthesis. Thus, this process will result in tag within this ‘spacer’ region can be tolerated but
a reduction in virus production within infected resulted in attenuation of the virus, whilst a 51 nt
cells. The inability to block this host response deletion did not adversely affect the growth of the
could explain, at least in part, the apparent attenu- virus in cell culture or the virulence of the virus in
ation of the leaderless FMDV. A comparison of cattle (Piccone et al., 2010, 2011). In cell culture,
gene expression within cells infected with wild a virus which retains this 84 nt sequence with
type or leaderless FMDV found that 39 out of both functional initiation codons but lacks the Lb
region adapts rapidly during growth. The changes The capsid precursor P1–2A
that occurred meant that initiation at the Lab start The removal of the L protein from the N-terminus
site does not add additional amino acid sequences of the polyprotein reveals an N-terminal glycine
onto the P1–2A precursor (Belsham, 2013). Pre- residue on the P1–2A capsid precursor (see Fig.
cise deletion of the entire region between the two 2.1) that is present within the motif (GXXXS/T)
initiation sites can be tolerated when the Lb coding required for recognition of proteins by the cellular
region is retained (Belsham, 2013). It may be that myristoylation machinery. This modification is a
this region influences the efficiency of translation common (but not universal) feature of the picor-
initiation under different conditions. When the navirus capsid proteins. The addition of the C18
virus initially infects the cell, the RNA has to func- lipid to the N-terminus of 1A (VP4) is important
tion in competition with the host mRNAs using for the assembly and/or stability of the FMDV
the intact cap-binding complex (eIF4F, including and PV capsids (Chow et al., 1987; Abrams et al.,
eIF4G, eIF4A and eIF4E). However, once the L 1995). The processing of the FMDV P1–2A pre-
protein is produced eIF4G is cleaved very rapidly cursor to 1AB (VP0), 1C (VP3) and 1D (VP1)
(Belsham et al., 2000); then, the capped host cell is achieved by the 3C protease (see below) and
mRNAs are no longer used by the translation these products can self-assemble into empty capsid
machinery. Deletion of the Lb coding sequence particles (Abrams et al., 1995; Porta et al., 2013;
from the viral genome means that the viral RNA Gullberg et al., 2013). The cleavage of 1AB to 1A
has to continue to be translated under conditions and 1B normally occurs on encapsidation of the
similar to those encountered at the beginning of RNA genome but the precise mechanism of this
the infection cycle. Perhaps, under these condi- cleavage is not known. There is some evidence that
tions, there is a more efficient utilization of the VP0 cleavage can occur within assembled empty
RNA for translation if the sequence immediately capsid particles (Curry et al., 1995; Gullberg et
downstream of the Lab initiation codon is present. al., 2013, 2014). The structure and properties of
Hence, loss of this ‘spacer’ sequence, under condi- the FMDV capsid, including its ability to interact
tions of maximal competition with host mRNAs, specifically with cellular receptors, are discussed
may make the RNA sufficiently inefficient that it elsewhere in this volume (see Chapter 4). The
proves lethal (see Belsham, 2013). generation of the P1–2A precursor clearly requires
It has now been demonstrated that the L protease the loss of any linkage to the 2B protein and this
is able to induce the cleavage of the cellular protein process is mediated by the 2A sequence. However,
Gemin5 (Pineiro et al., 2012). Binding of Gemin5 the 2A sequence in FMDV is very short, just 18
to the FMDV and the HCV IRES elements inhibits amino acids. It seems unlikely that such a short
their activity (Pacheco et al., 2009). This protein, peptide could possess protease activity in its own
which was identified earlier as a component of the right. This peptide sequence is closely related to
SMN complex, has the capacity to interact directly the C-terminal region of the cardiovirus 2A protein
with the 3′end region of the FMDV IRES (Piñeiro (note that the cardiovirus 2A proteins are much
et al., 2013) via a non-canonical RNA-binding larger but do not contain any motifs characteristic
site located in its C-terminal domain (Fernandez- of known proteases either). It has been proposed
Chamorro et al., 2014). The proteolytic fragments that the FMDV 2A sequence may prevent the for-
of this protein, however, differ not only in their mation of the peptide bond at the 2A/2B junction
capacity to modulate IRES activity, but also in rather than acting as a peptidase to break a bond
their stability within FMDV-infected cells. While that has been formed (see Chapter 3). Recent
the larger C-terminal fragment (p85) binds to the evidence indicates that the cleavage of the 1D/2A
IRES and slightly stimulates translation, the short junction by the 3C protease is not essential for
distal C-terminal fragment harbouring the RBS2 capsid assembly or virus infectivity (Gullberg et
domain, is unstable in infected cells and harbours al., 2013, 2014). Thus infectious ‘2A-tagged’ virus
the repressor domain (Fernandez-Chamorro et al., particles containing VP1–2A can be produced.
2104; Piñeiro et al., 2015). Thus the cleavage of the Cleavage of the VP1/2A junction in the P1–2A
protein can be expected to enhance the IRES func- precursor is the last step in the capsid precur-
tion. sor processing (as indicated by the presence of
VP1–2A when no other precursors remain; Ryan et investigations of these activities in other picornavi-
al., 1989; Gullberg et al., 2013, 2014). Surprisingly, ruses. The 2C protein is the locus that determines
using in vitro assays with short peptide substrates, the sensitivity of viral RNA replication to the
the cleavage of this site is the most efficient (Birtley inhibitor, guanidine (Saunders and King, 1982).
et al., 2005). Different strains of FMDV vary in their sensitiv-
ity to this agent. Single amino acid substitutions
The P2 proteins within the protein are sufficient to confer resistance
The FMDV 2BC precursor is processed to 2B and to the inhibitor (Pariente et al., 2003; Belsham and
2C by the 3C protease (Fig. 2.1). Rather little is Normann, 2008). The precise role of 2C in RNA
known about these P2 proteins. Within PV-infected replication is not established.
cells, the P2 proteins are found within membrane- Biochemical studies on the FMDV 2C protein,
associated viral replication complexes (Bienz et expressed in E. coli, have shown that the protein
al., 1987, 1990). However, their functions are also forms hexameric structures in vitro in the presence
rather poorly understood. of ATP and RNA (Sweeney et al., 2010). These
Different members of the picornavirus family properties are characteristic of AAA + ATPases.
have very different 2B proteins, the FMDV and
EMCV 2B proteins show low levels of identity to The P3 proteins
enterovirus 2B proteins (Moffat et al., 2005; de The FMDV P3 precursor (Fig. 2.1) is processed by
Jong et al., 2008), hence it is not clear that they the 3C protease to 3A, the three distinct copies of
share functional activities. The enterovirus 2B pro- the 3B peptide (VPg1, VPg2 and VPg3), the 3C
tein can increase cell permeability, as judged by the protease and the 3D RNA polymerase plus vari-
susceptibility to the translation inhibitor hygromy- ous intermediates (e.g. 3CD). The 3A protein has
cin B (van Kuppeveld et al., 1997). However some hydrophobic sequences that serve to anchor it to
mutants within 2B affect cell growth without affect- membranes and this may be the means by which
ing either of these functions, hence other activities RNA replication is localized to membrane vesicles.
for the 2B protein may still need to be defined (van It is thought that the 3A protein also serves to deliver
Kuppeveld et al., 1997). The studies of de Jong et the 3B peptides to the sites of RNA replication.
al. (2008) indicated that enterovirus 2B proteins Various strains of FMDV have been isolated that
affect calcium homeostasis and intracellular protein contain in-frame deletions within the 3A coding
trafficking. sequence and these strains are attenuated in cattle
It has been demonstrated that the FMDV 2BC (O’Donnell et al., 2001) but remain pathogenic in
precursor is able to inhibit trafficking of proteins to pigs. This presumably reflects some differences in
the cell surface (Moffat et al., 2005). This activity the interaction of 3A with cellular factors between
has been associated with the 3A protein of other the two species. Expression of FMDV 3A alone
picornaviruses (Doedens and Kirkegaard, 1995) disrupted the Golgi apparatus of keratinocytes.
but the 3A of FMDV does not show this activity. It is interesting to note that some picornaviruses
The function of FMDV 2BC was not obtained with that disrupt Golgi function, e.g. PV, are extremely
the expression of either 2B or 2C alone but could be sensitive to the effect of brefeldin A (Maynell et al.,
reproduced by the dual expression of both proteins 1992). However, in contrast, it has been observed
(Moffat et al., 2007). It is believed that this effect that FMDV is rather insensitive to this agent
on protein trafficking may block the presentation of (O’Donnell et al., 2001), like EMCV (Iruzun et
viral proteins on the cell surface to the host immune al., 1992). Brefeldin A modifies vesicle transport
system. between the Golgi and the endoplasmic reticulum,
The various picornavirus 2C protein sequences and the different sensitivities of picornaviruses to
contain helicase and nucleotide binding motifs but this agent suggest that the different viruses recruit
until very recently no evidence for RNA helicase membranes to their replication complexes in differ-
activity has ever been reported. However, Xia et ent ways. The PV 3A protein has also been shown to
al (2015) have now reported that the 2C protein block protein secretion (Doedens and Kirkegaard,
of human EV71 has both RNA helicase and RNA 1995) whereas the 3A protein of FMDV does not
chaperone activity; no doubt this will encourage have this activity (Moffat et al., 2005).
Uniquely, FMDV RNA encodes three similar The precise location of the cleavage site generated
but distinguishable copies of the 3B (VPg) peptide. by the 3C protease in eIF4AI has been identified
The coding sequences for these different peptides (Li et al., 2001). The cleavage is specific for eIF4AI,
occur as a tandem array in the genome (see Fig. since the closely related eIF4AII (92% identical) is
2.1). Priming of RNA synthesis requires 3B (VPg) not modified. It has been suggested that the cleav-
and this explains the linkage of this peptide to the 5′ age of eIF4AI, which will inactivate the protein (Li
terminus of both positive- and negative-sense RNA et al., 2001), may contribute to the decrease in the
strands. As with the PV and HRV cre elements, the level of viral protein synthesis during the later phase
initial modification of VPg to VPgpU or VPgpUpU of virus infection (Belsham et al., 2000).
is achieved by the use of the ‘cre’ as a template (Paul As indicated above, the 3C protease also cleaves
et al., 2000; Gerber et al., 2001; Nayak et al., 2005). eIF4GI in some cells, e.g. BHK. This modification
Each of the FMDV VPgs has been found attached occurs on the C-terminal side of the site generated
to genomic RNA and thus each peptide is believed by the expression of the L protease (Belsham et al.,
to be functionally equivalent (King et al., 1980) and 2000). This result is consistent with the loss of intact
indeed each functions in the in vitro uridylylation eIF4GI within BHK cells infected by the leaderless
assay (Nayak et al., 2005). Even the closely related FMDV (Piccone et al., 1995a; Belsham et al., 2000).
equine rhinitis A virus, the only member of the Aph- Sequential cleavage of eIF4GI within FMDV-
thovirus genus other than FMDV, only has a single infected BHK cells by the L protease and then of
copy of this peptide. Using mutagenesis, deletion of the C-terminal cleavage product by the 3C protease
one or more FMDV 3B sequences can be achieved has been observed, both cleavages are complete by
but the mutant viruses replicate less efficiently than 3 hours post infection (Strong and Belsham, 2004).
the wt virus (Falk et al., 1992). Indeed, deletion of Thus at the time of peak viral protein synthesis the
3B3 alone destroyed virus viability but this appeared form of eIF4GI that supports IRES function is that
to result from a defect in polyprotein processing generated by FMDV 3C. Later on during infec-
rather than a direct effect on RNA replication. tion, further modification of eIF4GI, probably also
However, overall, it is not clear why the presence generated by FMDV 3C, occurs and these cleav-
of three different VPg sequences within the FMDV ages may contribute to the decline in viral protein
RNA enhances its replication efficiency (Falk et al., synthesis. It was found that human eIF4GI is not
1992). susceptible to cleavage by FMDV 3C (Strong and
Belsham, 2004) and this observation facilitated
The 3C protease the identification of the precise cleavage site for
The FMDV 3C protease is responsible for most 3C within eIF4GI. It has been shown that a single
of the cleavages within the polyprotein coding amino acid substitution (Pro in human and Thr in
sequence. It functions alone and, in contrast to the BHK eIF4GI) at residue 713 is sufficient to modify
PV 3C protease (see Ypma-Wong et al., 1988), it the sensitivity of the host protein to 3C (Strong and
does not require 3D sequences for any of its pro- Belsham, 2004).
cessing activities. The key catalytic residues of the In addition to its proteolytic activity, FMDV
FMDV 3C have been identified (Grubman et al., 3C also has RNA binding activity. This probably
1995) and the protease is a member of the chymo- accounts for the requirement of 3CD (or 3C itself)
trypsin-like family of serine proteases (except that within the in vitro uridylylation assay (Nayak et al.,
the active serine is replaced by a cysteine; see Chap- 2006). Three specific basic residues involved in this
ter 3). The 3D structure of the 3C protease has interaction have been identified and modification of
been determined (Birtley et al., 2005). In addition each of them individually either inhibits or blocks
to cleaving the viral polyprotein it has been found virus replication. These residues are located on the
that the FMDV 3C also modifies certain cellular opposite face of the molecule from the catalytic site
proteins. The histone H3 was shown to be cleaved (Nayak et al., 2006).
by this protease (Falk et al., 1990) and subsequently
it has been shown that the 3C protease also cleaves The 3D RNA polymerase
the translation initiation factors eIF4A and eIF4GI The 3D protein is the RNA-dependent RNA
within FMDV-infected cells (Belsham et al., 2000). polymerase. The replication of picornavirus RNA
has two distinct aspects to it. To replicate the an NH2-terminal segment that encircles the active
positive-sense genome, an antisense RNA has to be site (see Chapter 6). The enzyme has to perform
synthesized which then functions as the template multiple functions; it not only has to polymer-
for the production of new positive-sense infectious ize nucleotides to make the RNA strands (both
genomes (see Fig. 2.3). Within infected cells, a large positive and negative) but it also has to perform
excess of positive strands accumulates over nega- the uridylylation of VPg. Thus, it has a diverse set of
tive strands. Presumably this reflects a differential substrates to recognize.
recognition of the negative-sense template over the Interestingly the PV 3D molecule displays co-
positive strand template by the RNA polymerase operativity in its polymerase activity in vitro (Pata et
and/or differences in the stability of the transcripts. al., 1995) and may normally function as a multimer.
The 3′ terminus of the positive sense RNA (the The crystal structure of the PV 3D polymerase
poly(A) tail) and the negative-sense RNA tem- showed that there is considerable interaction
plate (antisense S-fragment) are very different in between adjacent molecules in the crystal (Hobson
sequence. Thus, the nature of the recognition pro- et al., 2001). Whether these properties are relevant
cess by the RNA polymerase is clearly complex but to the activity of the protein within an infected cell
is not yet defined. There are many different poly(A) remains to be determined.
containing mRNAs within the cell so presumably The precursor 3CD does not have RNA poly-
recognition of the positive strand FMDV RNA merase activity but it does have RNA binding and
must include sequences away from the immediate protease activity. The PV 3CD product has been
3′-terminus. shown to bind to the cloverleaf structure at the 5′
The structure of the FMDV RNA polymerase terminus of the PV genome (Gamarnik and Andino,
has been determined by X-ray crystallography 1998) and it is also required in the cre-dependent
(Ferrer-Orta et al., 2004, 2006). Like other RNA VPg uridylylation assay (Paul et al., 2000; Rieder
polymerases, it folds into characteristic fingers, et al., 2000). Similarly, the FMDV 3CD is also
palm and thumb subdomains, with the presence of required (in addition to 3D) for the uridylyation of
Figure 2.3 Distinct roles for positive-sense FMDV RNA. Following the entry of FMDV RNA into the cytoplasm
of the cell, the RNA has to be translated to generate the viral proteins required for viral RNA replication, protein
processing, capsid assembly and opposing host defence mechanisms. Newly synthesized positive sense (+)
RNA genomes can be used in translation, RNA replication or packaged into new virions as illustrated. The
negative sense (-) RNA is only used as a template for the synthesis of new positive strand RNAs.
VPg (Nayak et al., 2005) but this requirement can genome) are required to provide the specificity of
be met, albeit less efficiently, by 3C alone (Nayak PV RNA recognition by the replication machinery
et al., 2006). that would seem to be required, especially by the
PV 3CD is the protease required for the process- mutants lacking the usual 3′ UTR sequences.
ing of the PV P1 capsid precursor whereas the PV Studies have shown that the length of the
3C alone can process the P2 proteins (Ypma-Wong poly(A) tail has an effect on the infectivity of PV
et al., 1988). In contrast, there seems to be no RNA (Spector and Baltimore, 1974). This obser-
specific requirement for the FMDV 3CD protein vation may reflect modification of the stability of
in FMDV polyprotein processing. The FMDV 3C the RNA or possibly a requirement for interac-
protease is sufficient to process the P1–2A capsid tions between the 3′ and 5′ termini of the RNA
precursor (see, for example, Gullberg et al., 2013, potentially involving the poly(A) binding protein
2014). (PABP) that binds optimally to a sequence of at
least 25 A residues. It should be noted that PABP
interacts both with the translation initiation factor
Structure and function of the eIF4G (that binds directly to the FMDV IRES
FMDV 3′UTR element, see above) and with the poly(rC) bind-
The 3′ untranslated region of FMDV RNA con- ing protein 2 that itself binds to the FMDV IRES
sists of two components, a region of about 100 nt (Stassinopoulos and Belsham, 2001) and presum-
of heterogeneous sequence and the poly(A) tail. ably to the poly(C) tract. As indicated above, such
The poly(A) tail of picornavirus RNA is included interactions may serve to ‘circularize’ the RNA and
within the genome of the virus, this contrasts with it may be that such interactions affect the efficiency
cellular mRNAs in which this sequence is added as of RNA translation, at least in the early stages of
a post-transcriptional modification. There is rather infection prior to cleavage of eIF4G (see Svitkin et
little information about the role of the FMDV RNA al., 2001b). Cleavage of eIF4G by the FMDV pro-
3′ UTR except that deletion of the unique (het- teases can be expected to disrupt the circularization
erogeneous) sequence blocks infectivity (Saiz et al, of the RNA but also removes competition from the
2001). It has been shown that the FMDV 3′ UTR cellular capped mRNAs for the translation machin-
sequence can stimulate the activity of the FMDV ery. It has been suggested that circularization of the
IRES (Lopez-de Quinto et al., 2002); furthermore, PV RNA is required for RNA replication and is sup-
this effect is independent of the stimulation of IRES posed to be achieved through the interaction of the
activity by poly(A) observed previously (Svitkin et poly(A) binding protein (bound to the 3′ poly(A)
al., 2001b). These results suggest further possible tail) with the poly(rC) binding protein that binds
RNA–RNA interactions between the 5′ and 3′ to the cloverleaf structure at the 5′ end of the
UTRs or additional ‘bridging’ RNA–protein inter- genome (Herold and Andino, 2001). However, it is
actions. not entirely clear whether this would be achieved
There is evidence for complex RNA–RNA within the context of a cell containing many dif-
interactions within the heterogeneous sequence ferent polyadenylated mRNAs, each of which may
of the PV and HRV 3′ UTRs (Mirmomeni et al., have the poly(A) binding protein associated with
1997; Melchers et al., 1997) and also for protein it. As mentioned above, the cellular protein RHA
interactions with this element (Mellits et al., 1998). interacts with the FMDV S-fragment (Lawrence
However, surprisingly, it has also been shown that and Rieder, 2009) and this protein co-immunopre-
the unique 3′ UTR sequence of PV RNA can be cipitates with 2C and 3A which are components
deleted without loss of virus viability (Todd et al., of the replication complex. Thus, there is a link
1997) albeit that the virus replicated significantly between the S-fragment and the RNA replication
less well than wt virus. In contrast, studies on the 3′ mechanism. Furthermore, if the S-fragment does
UTR of cardiovirus RNA, have shown evidence for exist in a stable stem–loop structure, as predicted,
three stem–loop structures, one of which is essen- then the 5′ end of the RNA would be close to the
tial for virus viability (Duque and Palmenberg, poly(C) tract which is likely to be bound to the
2001). It is possible that other sequences within poly(rC) binding protein, potentially achieving the
the coding sequence (but towards the 3′ end of the same effect as proposed for the PV cloverleaf.
Unsolved issues there are several aspects of this process that remain
There are many questions about picornavirus biol- to be determined. To what extent do the capsid
ogy that remain to be answered and some of these proteins assemble prior to virion assembly? It is
issues are outlined below. well known that empty capsid formation can occur
in the absence of virion RNA but this may, or may
Interactions of viral proteins with not, be a dead-end product. Are there discrete RNA
cellular components replication complexes that are producing RNA
The interactions of the IRES elements with the genomes solely for packaging into virions? If so,
cellular protein synthesis machinery provides a how are these different from replication complexes
good insight into the complexity of virus–host that are producing RNA that will be translated and/
cell interactions but we are still a long way from or replicated? A recent report, based on work with a
understanding the mechanism of IRES action. It plant RNA virus, has indicated the presence of mul-
is apparent that cellular proteins that interact with tiple sequence elements within the viral genome
virus components can determine virus tropism. that facilitate virus assembly (Patel et al., 2015).
There are obvious examples, such as receptor mol-
ecules on the cell surface, but clearly any cellular
component that is required for the virus to replicate Concluding remarks
(either for RNA replication or protein production) Understanding of the structure and function of
may vary in its availability within different cell types. picornaviruses has grown considerably in recent
Hence, such factors may determine the competence years. However, the apparent simplicity of a picor-
of the cell to act as a host for the virus. These issues navirus genome belies the great complexity of
are important since they can determine the patho- picornavirus biology. Moreover, different picorna-
genicity of the virus. The differential sensitivity of viruses have their own individual properties that
cattle and pigs to FMDVs with deletions within the affect their interactions with the host cell. There-
3A protein (O’Donnell et al., 2001) is a good exam- fore, a deep understanding of the consequences of
ple of an unexplained host dependent determinant FMDV genome organization in co-ordinating the
of virus replication. synthesis of viral components that ultimately leads
to productive infection and virus spread demands
Compartmentalization a collaborative effort involving molecular biology,
Molla et al. (1991) described a system for the in cell biology and immunology.
vitro synthesis of infectious PV using HeLa cell
lysates. This system allowed the requirements for Acknowledgements
virus assembly and RNA replication to be ana- Studies in the EMS laboratory were supported by
lysed. No equivalent system has been reported for grants BFU2011-25437 and BFU2014-54564 from
FMDV but Svitkin and Sonenberg (2003) have MINECO, and by an Institutional grant from Fun-
described an in vitro replication system for EMCV dación Ramón Areces. Studies in GJB’s laboratory
and this holds out the possibility of achieving this were supported by the Danish Research Council
with FMDV RNA. The ability to produce infec- (DFF).
tious virus within a cell-free system does suggest
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FMDV polyprotein processing 1987; Fig. 3.1C). The second ‘primary’ processing
With regards to picornavirus polyprotein organiza- event occurs at the C-terminus of the 2A oligopep-
tion, the nature of the N-terminal and 2A regions tide region. In FMDV 2A is very short – just 18aa
represents the major differences between the and it has been proposed that this ‘cleavage’ is, in
various genera. The single, long ORF of FMDV fact, the result of a translational ‘recoding’ event,
encodes a polyprotein of some 2,330 aa, although rather than a proteolytic, mechanism (discussed
the full-length translation product is observed nei- below). In common with all other picornavirus
ther within infected cells nor translation reactions in polyproteins, the third ‘primary’ cleavage occurs
vitro – due to ‘primary’ proteolytic processing (Fig. between the 2C and 3A proteins, mediated by 3Cpro
3.1A). In the case of the FMDV polyprotein, three (Fig. 3.1A). The four products of FMDV primary
primary polyprotein cleavages are observed. The polyprotein processing therefore comprise; Lpro,
first occurs between the FMDV L and 1A (capsid) the capsid protein precursor [P1–2A] and the
proteins: FMDV L is a proteinase (Lpro), processing replicative protein precursors [2BC] and P3. Such
the polyprotein at this single site generating its own primary processing reactions occur in an intramo-
C-terminus. NOTE: In FMDV, the initiation of lecular manner (in cis). They are characteristically
translation occurs at two sites, separated by 84nts, very rapid (co-translational) and, since the cleav-
which gives rise to two forms of the L proteinase age site and the proteinase are parts of the same
– Labpro and Lbpro (Clarke et al., 1985; Sangar et al., molecule, the reaction is insensitive to dilution of
A
pro
Enterovirus 1A 1B 1C 1D 2A 2B 2C 3A 3C pro 3D pol
3B
[P1-2A] [2BC] P3
pro pol
FMDV L pro 1A 1B 1C 1D 2B 2C 3A 3C 3D
2A 3B1-3
B 1A 1B 1C 1D 3A 3C pro 3ABC
2A 3A
1A 1B 1C 1D 3A
2A 3AB ‘complex’
Secondary 3A
polyprotein 1A 1B 1C 1D 3A
2A
processing
1A 1B 1C 1D 3C pro 3D pol
3BCD 3C pro 3D pol
‘complex’ 3C pro 3D pol
3C pro 3D pol
C pro pol
Lab 1A 1B 1C 1D 2B 2C 3A 3C pro 3D
pro 2A 3B1-3
Lb
1A 1B
Figure 3.1 Primary and secondary polyprotein cleavages. The polyprotein domain organization of enterovirus
and FMDV are shown with capsid protein precursors shaded blue and replication proteins shaded green. The
sites of the ‘primary’, co-translational, cleavages and the virus proteins responsible are indicated by the curved
arrows. In the case of entero- and rhinoviruses, primary cleavages occur at the 1D/2A junction mediated by
2Apro, and at the 2C/3A junction mediated by 3Cpro. In contrast with enteroviruses, the FMDV polyprotein
undergoes a primary ‘cleavage’ at the L/1A junction mediated by Lpro, the translational ‘recoding’ event at the
2A/2B site (vertical arrow). Like all other picornaviruses, proteolysis at the 2C/3A junction is mediated by 3Cpro
(Panel A). Secondary processing of the primary cleavage products by 3Cpro gives rise to a series of alternative
processing products (Panel B). The mature, individual, 3Cpro processing products are shown together with the
two forms of Lpro, arising from alternative translation initiation sites, and the 1A/1B cleavage (vertical arrow)
which occurs concomitantly with vRNA encapsidation by a very poorly understood mechanism (Panel C).
translation reactions in vitro. Precursor forms span- mapped to protein 3C (Gorbalenya et al., 1979;
ning these primary cleavage sites are not observed. Palmenberg et al., 1979). Picornavirus 3C protein-
The [P1–2A], [2BC] and P3 precursor forms ases mediate a primary cleavage between 2C and
subsequently undergo ‘secondary’ processing 3A, although one report indicated an alternative
mediated by 3Cpro (Fig. 3.1B). Secondary process- primary cleavage between proteins 2A and 2B of
ing reactions occur in an intermolecular manner poliovirus (Lawson and Semler, 1992).
(in trans) and are characteristically slower. In con- In comparison with other picornavirus 3C
trast to primary cleavages, secondary polyprotein proteinases, FMDV 3Cpro cleaves a wide range of
processing reactions are sensitive to dilution, are amino acid pairs (E/G, Q/G, Q/T, Q/L, Q/I; Fig.
more sensitive to inhibitors and generally show a 3.2). The primary [2BC]/P3 cleavage occurs at
greater sensitivity to sequence variations flanking a conserved Q/I pair, whilst cleavages within P3
the scissile amino acid pair. Although sometimes all occur at E – G pairs. Similarly, the cleavage site
referred-to as processing ‘intermediates’, this may between 2B/2C is completely conserved, although
be misleading since some of these proteins have in this case at a Q/L pair. Cleavages with the capsid
specific activities in their ‘intermediate’ processing proteins precursor [P1–2A] show a wider range of
forms. The ‘mature’ processing products are shown pairs; E/G, Q/G, E/T, Q/T, Q/L and Q/M. Not all
in Fig. 3.1C. The cleavage of [1AB] to 1A + 1B such pairs present within the polyprotein are pro-
occurs concomitantly with encapsidation of virus cessed, however, – their position at the boundaries
RNA (vRNA), by a poorly understood mechanism. of polyprotein domains being the major determin-
In addition to cleaving the virus polyprotein, ing factor.
picornavirus proteinases have also been shown to FMDV 3Cpro is 213aa long (predicted
degrade specific cellular protein targets (discussed Mr = 23 kDa), whose N- and C-termini are defined
below). The following sections are organized by by its own proteolytic activity. The 3Cpro-mediated
the historical order in which their mechanisms of secondary processing of the [P1–2A], [2BC] and
action became understood. P3 precursors is shown in Fig. 3.1B. Whilst the
[P1–2A] precursor is processed to the end products
[1AB], 1C and 1D (the [1AB] ‘maturation’ cleavage
The 3C proteinase occurs concomitantly with vRNA encapsidation),
A virus-specific proteolytic activity – in contrast a multiplicity of different products are generated
with virus polyprotein processing by host cell during processing of P3, some of which are stable
proteinases – was first described for the cardiovirus products. For example, protein 3CD and those
encephalomyocarditis virus (EMCV; Lawrence comprising the 3BCD complex (3CD with 1, 2 or
and Thatch, 1975; Pelham, 1978) and subsequently 3 3B proteins still attached) are all stable products
L s 1A i1B ⇓ 1C ⇓ 1D ⇓ 2A
O1/K -QRKLK GAGQS-/-GALLA DKKTE-/-FPSKE GIFPV-/-DARAE TTSAG-/-APVKQ TLNFD-
R Q Q L
M
2A q 2B ⇓ 2C ⇓ 3A ⇓ 3B1 ⇓ 3B2
O1/K -ESNPG PFFFS-/-RAEKQ LKARD-/-PIFKQ ISIPS-/-QPQAE GPYAG-/-LPQQE GPYAG-
Figure 3.2 FMDV polyprotein cleavage sites. Cleavage at the L/1A site occurs at (K/R)/G pairs ( ). The capsid
protein 1A/1B cleavage (↓) occurs concomitantly with vRNA encapsidation by an unknown mechanism. The
primary 2A/2B translational recoding event ( ) occurs at a conserved G/P pair. 3Cpro-mediated processing ( ):
the primary 2C/3A cleavage occurs at a conserved Q/I pair whilst secondary processing of P3 occurs only at
E/G pairs and at the 2B/2C site at a conserved Q/L pair. A range of scissile pairs is cleaved during processing
of the capsid proteins precursor [P1–2A] at the 1B/1C, 1C/1D and 1D/2A sites.
(Ryan et al., 1989). In the case of poliovirus, the confirmed the predicted chymotrypsin-like fold
processing of the P1 capsid protein precursor is of the enzyme and catalytic mechanism. Detailed
mediated not by 3Cpro, but by 3CDpro ( Jore et al., discussion of the structural and mechanistic rela-
1988; Ypma-Wong et al., 1988). The 3Cpro from tionships between these enzymes and cellular
other genera are, however, able to process capsid proteinases can be found in Dougherty and Semler
protein precursors (Vakharia et al., 1987; Parks et (1993), Malcolm (1995), Babé and Craik (1997),
al., 1989; Jia et al., 1991) and although the FMDV Ryan and Flint (1997), Seipelt et al. (1999) and
3Cpro efficiently cleaves all 10 processing sites Skern et al. (2002).
within the FMDV polyprotein (Bablanian and Resolution of the atomic structure of FMDV
Grubman, 1993), processing of the capsid proteins pro
3C revealed a similar structural fold observed
precursor [P1–2A] was somewhat more efficient previously for other picornavirus 3C proteinases
with 3CDpro (Ryan et al., 1989). Like FMDV, the (Birtley et al., 2005 Sweeney et al., 2007; Fig.
EMCV 3C proteinase also retains proteolytic activ- 3.3A). The characteristic catalytic triad reported
ity in 3Cpro-containing precursor forms ( Jackson, in other picornavirus 3C proteinase structures was
1986; Parks et al., 1989). also observed in FMDV 3Cpro: His46, Asp84 and
Cys163, although the latter residue was mutated to
The catalytic mechanism of FMDV alanine to produce a proteolytically inactive form
3Cpro to facilitate expression, purification and crystalliza-
Early studies on 3Cpro using proteinase inhibitors tion – a strategy commonly adopted previously for
showed rather confusing inhibitor profiles. Inhibi- other picornavirus proteinases. In the initial report,
tion of proteolytic activity was observed with both the absence of a β-ribbon structure overlaying the
serine and thiol proteinase inhibitors (Summers substrate-binding cleft, as observed in other picor-
et al., 1972; Korant, 1972, 1973; Pelham, 1978; navirus 3C proteinase structures, was proposed to
Gorbalenya and Svitkin, 1983; Korant et al., 1985; contribute to the lower substrate binding specificity
Baum et al., 1991). A breakthrough came when sim- of FMDV 3Cpro, in that whilst other picornavirus 3C
ilarities between the large sub-class (trypsin-like) proteinases show a strong preference for glutamine
cellular serine proteinases and 3C proteinases were at the P1 position (-Q/X- scissile pairs), the FMDV
detected by sequence alignments (Gorbalenya et 3Cpro accommodates both glutamine and glutamate
al., 1986, 1989; Bazan and Fletterick, 1988, 1990). at this P1 position (-E/X- and -Q/X- scissile pair-
These analyses predicted both a chymotrypsin-like ings). In FMDV, however, this β-ribbon structure
(serine proteinase) fold and the identity of the appeared to be a disordered surface loop (Birtley et
residues which would form a catalytic triad analo- al., 2005). In the subsequent paper, however, a crys-
gous to that of serine proteinases (Fig. 3.3A). In tal form was reported in which residues comprising
the case of FMDV 3Cpro the putative catalytic triad this loop now adopted a β-ribbon structure, similar
would be composed of His46, Asp84, and, perhaps to other picornavirus 3C proteinases (Sweeney et
most interestingly, the active site nucleophile being al., 2007).
Cys163 (rather than serine, as in chymotrypsin). A recent publication showing the structure of
Subsequent site-directed mutagenesis experiments FMDV 3Cpro complexed with a synthetic peptide
confirmed the roles of these residues in rhinovirus (APAKQ/LLNFD – corresponds to the 1D/2A
(Cheah et al., 1990), polio (Hammerle et al., 1991; polyprotein cleavage site; Zunszain et al., 2010)
Kean et al., 1991; Lawson and Semler, 1991), hepa- showed that substrate binding produced an
titis A ( Jia et al., 1991) and FMDV 3Cpro catalysis enzyme structural re-arrangement which formed
(Grubman et al., 1995). an extended interaction with the substrate P4 to P2′
positions of the peptide APAKQ/LLNFD (under-
The structure of FMDV 3Cpro lined). Interestingly, the S1′ specificity pocket
The resolution of the atomic structures of the 3C which accommodates the P1′ leucine (APAKQ/
proteinases from human rhinovirus (HRV) 14 LLNFD; underlined) formed only once the pep-
(Matthews et al., 1994), hepatitis A (Allaire et al., tide substrate had bound (Zunszain et al., 2010).
1994; Bergmann et al., 1997), polio (Mosimann As observed previously for other picornavirus 3C
et al., 1997) and HRV 2 (Mathews et al., 1999) proteinases, the peptide substrate bound to the
A BB
N-terminal helix
Cys163
His46
catalytic Asp84
triad
C-terminal helix
FMDV enzyme in a linear, extended, conformation measures – emergency ring vaccination – involves
which oriented the P1 residue side-chain into the a considerable lag-time in the sero-conversion of
S1 binding pocket proximal to the catalytic triad. vaccinated animals and, therefore, the formation of
Whilst all the side-chains of the substrate P4-P4′ a barrier to further disease transmission. However,
residues form contacts with FMDV 3Cpro to one research aimed at the development of such small
extent or another, activity assays using a range of molecule anti-proteinase drugs for entero- and rhi-
synthetic substrates, together with structural data noviruses, for example, has not yet translated into
of enzyme–peptide complexes, revealed a marked the market place.
specificity for the P4, P2 and P1 positions – but a
lower specificity for the P3 and P1′ positions (Zun- FMDV 3Cpro: an RNA binding protein?
szain et al., 2010). An additional and quite unexpected property of
A case has been made for the development of this proteinase was discovered when mutations
FMDV antiviral drugs (3Cpro naturally being a suppressing the effect of a four base insertion within
target candidate) since one of the disease control the 5′ non-coding region of the RNA genome were
mapped within 3Cpro (Andino et al., 1990a). Subse- leader protein at the N-terminus of the polyprotein
quently it was demonstrated these mutations were is a feature possessed by the aptho- and erbovirus
affecting the binding of 3CDpro, rather than 3Cpro, genera alone. Lpro contains two in-frame translation
to positive- strand RNA (Andino et al., 1990b, initiation codons (84 nt apart) which results in two
1993). This vRNA-binding property of 3CDpro forms being produced; Labpro and Lbpro (Clarke et
was demonstrated for rhinoviruses and hepatitis A. al., 1985) The latter form undergoes self-cleavage,
Although mapping to multiple sites, when the data trimming the C-terminus by six to seven amino
were combined the mutations clustered to define acids, via a carboxypeptidase B-like activity pro-
an RNA binding site (Andino et al., 1990a,b, 1993; ducing a product Lb’pro (Sangar et al., 1988). This
Leong et al., 1993; Walker et al., 1995; Blair et al., C-terminal ‘trimming’ of Lbpro was reproduced
1996, 1998; Kusov and Gauss-Muller, 1997). by the truncation of sequences encoding Lbpro to
Mutations shown to affect RNA binding were remove the 6 C-terminal amino acids – termed
located within the atomic structure on the opposite sLbpro (Guarné et al., 1998, 2000) Note that this
side to the catalytic site. The majority of these muta- sLbpro form was used for structural studies (see
tions were observed in a domain interconnecting the below) and for comparative proteolysis analyses.
two lobes of the proteinase – located between the Additional substrate specificity or functionality for
two (anti-parallel) helices of the N- and C-termini sLbpro remains to be discovered, however, reports
– and also clustered on the (exterior) loop connect- suggest Lbpro and sLbpro differ in cleavage efficien-
ing βD2 and βE2 and centred about the conserved cies in trans (Cencic et al., 2007; Steinberger et al.,
entero-, rhinovirus –KFRDIR- motif of the domain 2014). The differential roles of these form of leader
linker. Interestingly, this interconnecting region lies proteins – if any – produced during infection is
in close proximity to the N- and C-terminal α-helices unknown. Critically, both AUGs are conserved in
of the proteinase. In the case of FMDV 3Cpro, both all natural FMDV isolates (Sangar et al., 1987; Cao
the structure (exterior loop) and a similar sequence et al., 1995; Carrillo et al., 2005) suggesting their
motif (–RVRDIT-, conserved amongst FMDVs) presence serves an as yet unknown function.
is similar to the RNA-binding site of entero- and Lpro generates its own C-terminus (Strebel and
rhinovirus 3C proteinases (Fig. 3.3B). Similarly, Beck, 1986), the cis cleavage occurring between
in FMDV 3Cpro this interconnecting region lies in Lys/Arg201 and Gly202. Both the Labpro and Lbpro
close proximity to the N- and C-terminal α-helices. forms have been shown to cleave at the L/P1 junc-
An RNA-binding activity has not been examined tion either in cis or in trans (Medina et al., 1993;
for FMDV 3CDpro, but the secondary structure of Cao et al., 1995). Besides cleaving itself from the
the FMDV 5′ terminal region of the non-coding nascent viral polyprotein, Lpro cleaves the host cell
region is quite different from enteroviruses, in that translation factor eIF4G (Devaney et al., 1988;
FMDV has a much longer stem–loop. Medina et al., 1993). Proteolytic cleavage of eIF4G
The significance of this RNA-binding activ- occurs between residues Gly479 and Arg480 – only
ity of picornavirus 3C/3CD proteinases has not seven amino acids upstream from the enterovirus
been fully determined, but the conservation of the 2A proteinase site at Arg486 and Gly487 (Kirchweger
structure and the electrostatic nature of this region et al., 1994). The closely separated Lpro and 2Apro
amongst 3C proteinases of viruses within differ- cleavage sites are both thought to lie in a hinge
ent genera of picornaviruses support the notion region between the two major domains of eIF4G.
that this is an important aspect of virus replication Cleavage activity within this region results in the
worthy of further investigation. separation of these two domains: the N-terminal
portion required for eIF4E (binds the 5′-terminal
7meG host cell mRNA cap structure) and poly(A)
genome to proceed via binding to the viral IRES, Lbpro by reducing the number of substrates cleaved
whilst progressively ‘shutting-off ’ cap-dependent via product inhibition.
translation of host cell mRNAs (Lamphear et al., In the crystal structure of Lbpro, the C-terminus
1993). of one molecule is bound into the substrate bind-
ing site of the adjacent molecule. This gave an
Atomic structure and catalytic additional insight into the function of the enzyme
mechanism since half of the substrate was bound into this site.
Amino acid sequence analysis of Lpro suggested a The branched hydrophobic P2 residue (L/V) forms
similarity to papain-like thiol-proteinases which an important interaction with a deep hydrophobic
comprise active site cysteine and histidine residues cavity, although (unlike papain) Lbpro has appreci-
(Gorbalenya et al., 1991). This was later confirmed able binding pockets for the P1 and P1’ residues,
via inhibitor and site-directed mutagenesis stud- which make a substantial contribution to its sub-
ies, with Cys51 and His148 identified as active site strate binding specificity. Subsequent work has
residues (Kleina and Grubman 1992; Piccone et al., highlighted the substrate specificity of Lpro in com-
1995a; Roberts and Belsham, 1995). parison to other papain-like enzymes: Lbpro exhibits
Resolution of the atomic structure of Lbpro an extended substrate binding site (up to P7), with
was solved via molecular replacement using the substitution of peptides at each position within the
shorter sLbpro as a model. The crystal structure of substrate affecting enzyme activity (Santos et al.,
Lbpro showed a compact globular form with a flex- 2009). High prime-side specificity has also been
ible C-terminal extension (CTE) comprised of 18 documented (Nogueira Santos et al., 2012).
amino acid residues from D184 to K201 (Guarné et
al., 1998, 2000). The active site is located between Replication characteristics of
an α-helical domain and a β-sheet domain. The ori- L-deleted viruses
entation of the histidine component of the active Despite the Lbpro form being the predominant form
site is optimized by its interaction with Asp163, produced (Sangar et al., 1987; Cao et al., 1995),
rather than an asparagine residue which performs deletion of this region is not important for virus
this role in thiol proteinases such as papain. Lpro viability. Piccone and colleagues found that viruses
cleaves at its own C-terminus: the substrate binding lacking the portion of L directly after the Lbpro ini-
site must, at some stage, therefore, accommodate its tiation codon replicated less efficiently in BHK-21
own C-terminus (the ‘P’ side residues of the scissile cells (Piccone et al., 1995b). This replication was
bond). This is consistent with the observed flexibil- only slightly slower than the wild-type (WT) virus,
ity of the CTE in that it is disordered in both the characterized by smaller plaque sizes, lower titres,
crystal structure and in solution (NMR analyses; slower host cell shut-off and slight attenuation in
Cencic et al., 2007). Interestingly, the NMR data mice; however, when tested in cattle the ‘partially
confirmed the formation of Lbpro homodimers in leaderless’ virus had greatly reduced pathogenicity
solution (in agreement with the crystal studies), and was less widely disseminated in the lung at 24
which did not dissociate by increasing the ionic hours post infection (p.i.), with no lesions or virus
strength, dilution, or through binding to a peptide detectable in secondary sites at 72 hours p.i. (Brown
substrate corresponding to the eIF4GI cleavage et al., 1996). In a later study (Mason et al., 1997)
site. Although complete dissociation of dimers two of three animals inoculated with the leaderless
(into monomers) was not observed following bind- virus did not develop lesions when challenged with
ing to the peptide substrate, shifts in NMR spectra WT virus, but showed mild signs of infection. The
suggested movement of the monomers which third inoculated animal developed some lesions,
corresponded to the dimer interface, proposing but these were less severe than in the un-inoculated
Lbpro might function as a dimer under physiologi- control animal, which showed classical FMD.
cal conditions; however, a later study found Lbpro When this virus was later tested in swine, similar
functioning as a monomer until enzyme concen- results to those observed earlier in cattle were found
trations reached > 2 µM (Santos et al., 2009). The (Chinsangaram et al., 1998). The differences in rep-
authors of the previous study (Cencic et al., 2007) lication of the leaderless virus within BHK cells and
proposed dimerization may regulate the activity of reduced virulence in animals reported in the above
studies appears to lie in the type I interferon (IFN) chloramphenicol acetyl-transferase (CAT) were
response. The inability of FMDV-infected cells to linked via FMDV 2A (plus the N-terminal proline
translate type I IFN mRNA, due to Lpro-mediated of 2B) to β-glucuronidase (GUS) in a single ORF
shut-off of host cell translation, makes a substantial ([CAT-2A-GUS]). Analysis of this polyprotein
contribution to the virulence of the virus. The BHK system using in vitro translation systems showed,
cells commonly used to propagate FMDV are, how- indeed, that the 20 amino acid FMDV sequence
ever, defective in their response to IFN. was able to mediate a co-translational cleavage
producing the cleavage products [CAT-2A] and
GUS (Ryan and Drew, 1994). In this heterolo-
The 2A oligopeptide gous context, therefore, the FMDV 2A sequence
The second primary cleavage of the FMDV mediated a highly efficient cleavage at its own
polyprotein occurs between the 2A and 2B pro- C-terminus – just as in FMDV polyprotein pro-
teins – markedly different from the 2A proteinase cessing (-DxExNPG⇓P-). Consistent with these
mediated polyprotein processing observed in the observations, other studies showed that deletion
entero- and rhinoviruses NOTE: rhinoviruses are of the N-terminal 66% of the cardiovirus 2A
now included within the genus enterovirus (Fig. did not abrogate cleavage at the 2A/2B site, and
3.1). Precursor forms spanning the FMDV 2A/2B that mutations within the conserved – NPGP –
cleavage site are not detected during native poly- sequence at the extreme C-terminus of EMCV
protein processing. The 2A region of the FMDV 2A abolished cleavage activity (Palmenberg et
polyprotein is very short (18aa), with a C-terminal al., 1992). Cleavage at the 2A/2B site of Theilers
sequence motif (-DxExNPG-) conserved amongst murine encephalitis virus (TMEV) was highly
all FMDVs: the N-terminal proline residue of efficient when only 2A and 2B sequences were
FMDV protein 2B is completely conserved. present (Batson and Rundell, 1991). Later it was
Indeed, this motif (together with the N-terminal shown that the C-terminal 19aa together with
proline of protein 2B) is now known to be con- the N-terminal proline of 2B from either FMDV,
served amongst 2A proteins of many picornavirus EMCV or TMEV, when inserted into an artificial
genera (e.g. Aphtho-, Aquama-, Avihepato-, Avisi-, polyprotein, are able to mediate a co-translational
Cardio-, Cosa-, Erbo-, Hunni-, Kunsagi-, Mischi-, ‘cleavage’ with high efficiency (~95%; Donnelly et
Mosa-, Pasi-, Seneca-, Sicini- and Teschoviruses, al., 1997).
plus the proposed new genera Limnipvirus and Based upon the observed kinetics of the cleav-
Potamipivirus) – indeed, some viruses have mul- age reaction, inability to inhibit the reaction with
tiple iterations of the -NPGP- motif within what proteinase inhibitors plus data from site-directed
must be multifunctional 2A proteins. Cleavage at mutagenesis of the 2A sequence, we have proposed
the 2A/2B site of FMDV or cardiovirus polypro- that the 2A/2B cleavage is not a proteolytic cleavage
teins was shown to require neither the L nor 3C (by either a virus or host cell proteinase), but the
proteinases (Clarke and Sangar, 1988; Roos et al., result of a translational ‘recoding’ activity – latterly
1989; Ryan et al., 1989). Early studies showed the termed as ‘ribosome skipping’ (Ryan et al., 1999;
FMDV 2A region was not simply a substrate for a Donnelly et al., 2001a). Briefly, we have proposed
virus proteinase (Lpro, 3Cpro), and the very rapid, that the nascent 2A sequence interacts with the exit
complete, ‘cleavage’ suggested it did not function pore of the ribosome to bring about a reorienta-
as a substrate for a host cell proteinase. A series tion of the tRNA-peptidyl ester linkage precluding
of in-frame deletions of the FMDV polyprotein it from nucleophilic attack by the prolyl-tRNA.
were made such that the upstream or downstream Hydrolysis of this bond would lead to release of the
context of 2A was altered (Ryan et al., 1991). peptide such that elongation may continue from
These analyses suggested that the 2A/2B ‘cleavage’ the prolyl-tRNA, producing two, discrete, ‘cleavage’
was, indeed, associated with this 2A oligopeptide products (Fig. 3.4). The N-terminal proline of 2B
alone (plus the N-terminal proline of protein is, therefore, a crucial part of the proposed mecha-
2B). To test this hypothesis, the 2A sequence nism. Site directed mutagenesis of this residue
was used in the creation of an ‘artificial’ reporter abrogates cleavage: it is completely conserved in all
gene polyprotein in which sequences encoding picornavirus 2B proteins which use this ‘cleavage’
egress of
prolyl-tRNA
Nascent pep1de
P
OH OH G G P OH G
E P A E P A E P A
eRF1/3
E P A E P A E P A
eEF2
eEF2
(7) (8) (9)
OH OH P OH P OH P
E P A E P A E P A
Figure 3.4 2A-mediated translational ‘recoding’: Current Model. When the nascent FMDV capsid proteins [P1]
upstream of 2A emerge from the ribosome, 2A is positioned in the ribosome exit tunnel (step 1). The nascent
[P1–2A]-tRNAGly is translocated from the A- to P-site and prolyl-tRNA enters the A-site (step 2). The nascent
2A peptide interacts with the exit tunnel of the ribosome such that the C-terminal portion of 2A (-DVESNPG-) is
sterically constrained within the peptidyl-transferase centre (PTC) of the ribosome. Nucleophilic attack of the
ester linkage between [P1–2A] and tRNAgly by prolyl-tRNA in the A-site is inhibited – effectively stalling, or
pausing, translation. The failure to form a new peptide bond leads to dissociation of prolyl-tRNA from the A-site
of the ribosome (step 3), required for entry of release factors into the A-site (step 4). We have shown that this
block is relieved by the action of translation release factors eRF1 and eRF3, hydrolyzing the ester linkage (step
5) and releasing the nascent protein (step 6). eRF1 leaves the complex, eRF3 being involved in this process
(step 6). Two, mutually exclusive, outcomes may then arise: (i) translation terminates at the C-terminus of 2A,
or (ii) prolyl-tRNA (re)enters the A-site (step 7), is translocated by eukaryotic elongation factor 2 (eEF2) from the
A- to the P-site (step 8), allowing the next amino-acyl tRNA to enter the A-site to permit the synthesis of the
sequences downstream of 2A (step 9).
mechanism and in ‘2A-like’ sequences from other encoding three proteins; (i) DNαF – yeast proalpha
viruses (see below). factor with, instead of its native signal sequence, the
Preliminary studies showed the FMDV 2A signal recognition particle (SRP)-dependent signal
sequence-mediated translational recoding in yeast sequence of dipeptidyl amino peptidase B (Dap2p)
(we now know this system is active in all eukary- (ii) FMDV 2A and (iii) green fluorescent protein
otic cells tested to date). An interesting test of this (GFP). Here, SRP binds to the Dap2p leader
hypothesis – using yeast expression systems – came sequence, arrests translation, and leads to the nas-
from the analysis of an artificial polyprotein system cent protein being translated in a ribosome: protein
in which the first protein bore a leader sequence. conducting channel complex (formed in the process
A single open reading frame was constructed of translocating proteins across the membrane into
the lumen of the endoplasmic reticulum – ER). In see ‘2A-like’ sequences, below) of viruses encod-
this situation, therefore, the nascent protein would ing an ‘NPGP’ type of 2A –suggests that this form
not be accessible to cytosolic protein(ases) and of 2A evolutionarily predates the appearance of a
the folding of the nascent protein would not occur proteinase-type of 2A.
until passing out of the protein conducting channel
into the lumen of the ER. Analysis of the process- The ‘translational’ model of
ing properties of this artificial polyprotein and the 2A-mediated cleavage: implications
sub-cellular localization of the ‘cleavage’ products for the biology of FMDV
showed that (i) 2A-mediated ‘cleavage’ was unaf- Our work on the cleavage of a [GFP-2A-GUS]
fected; (ii) the first protein, DNαF, was translocated polyprotein system using in vitro translation sys-
into the lumen of the ER; and (iii) GFP was located tems showed that a molar excess of the [GFP-2A]
in the cytoplasm (de Felipe et al., 2003). ‘cleavage’ product accumulated above the GUS
That the FMDV 2A sequence was active in product (Donnelly et al., 2001a). If the gene order
yeast enabled the power of yeast genetics, notably was reversed ([GUS-2A-GFP]), more [GUS-2A]
the extensive library of yeast mutants, to screen accumulated than GFP. We showed this was not
for host cell proteins that could be involved in due to different rates of protein degradation or
the 2A translational recoding mechanism. These non-specific dissociation of either the T7 RNA
studies identified eukaryotic release (termination) polymerase (during transcription of the template),
factors eRF1 and eRF3 affecting this mechanism or ribosomes (during translation). The molar excess
(Doronina et al., 2008a,b; Sharma et al., 2012). The was due to different levels of synthesis: the two dif-
translational model of 2A recoding activity was ferent parts of the single ORF were being translated
refined to propose these factors brought about the at different levels. This implies that in the FMDV
hydrolysis of the nascent peptide-tRNA ester link- polyprotein, more capsid proteins could be synthe-
age in a manner directly analogous to the ‘normal’ sized than replicative proteins. In a FMDV-infected
termination of translation, but that (i) these factors cell at the latter stages of replication one might
bound in a stop codon independent manner; and expect the depletion of cellular factors or, more
(ii) that once these factors exited the ribosome ter- probably, a reduction in activity due to phosphoryl-
mination could either terminate at the C-terminus ation/dephosphorylation, to become rate-limiting
of 2A, or prolyl-tRNA could re-enter and re-initiate for virus replication. Indeed, a decrease in the rate
the synthesis of the downstream (replication) pro- of ribosome elongation is typical during the course
teins (Fig. 3.4). of picornavirus infections (Summers et al., 1967;
In contrast with this model, a recent study using Hackett et al., 1978; Ramabhadran and Thatch,
a translation system reconstituted with human 1981). It could be that under these conditions,
translation factors showed, however, that transla- FMDV has a evolved a mechanism for ‘switching’
tional recoding at the EMCV 2A/2B site did not those remaining cellular resources present at the
require supplementation of the reaction with eRFs latter stages of replication into the synthesis of
(Machida et al., 2014). Obviously, further work on capsid proteins – and not replication proteins. If
the detailed mechanism of this translational recod- this were true, both particle yield and the kinetics
ing event is required! of vRNA encapsidation could be increased – which
In summary, the huge expansion of the picor- might, in part, account for the rapidity of the growth
navirus sequence database shows that this type of and highly contagious nature of this amazing virus.
translational recoding event at the 2A/2B protein
junction is quite common amongst the different ‘2A-like’ sequences
genera of the Picornaviridae, and that encoding a At the outset of our work on FMDV polyprotein
proteinase-type of 2A is (currently) found only processing, this type of 2A sequence was known
in a few genera: certainly enteroviruses and quite only for FMDV and cardioviruses. In addition to
possibly sapelo- and saliviruses. The number of viruses within the range of picornavirus genera
genera – but perhaps more importantly the wider listed above, database probing with a [D-(V/I)-E-
range of hosts (mammals, birds, fish, insects: also X-N-P-G-P; ‘X’ = any amino acid] motif revealed
expression – due to the inability of FMDV-infected by 3Cpro (Grigera and Tisminetzky 1984; Falk et
cells to translate type I IFN mRNA (Chinsangaram al., 1990; Tesar and Marquardt, 1990; Capozzo
et al., 1999) – as well as significant induction of et al., 2002). Again, the significance of these
interferon IFN-β and IFN stimulated gene (ISG) observations with regards to FMDV replication
mRNA in cells transfected with ‘leaderless’ FMDV is not clear. FMDV 3Cpro also induces proteoly-
(de los Santos et al., 2006). Additionally, Lpro has sis of nuclear RNA-binding protein Sam68 (the
been implicated in degradation of the p65/RelA Src-Associated substrate in Mitosis of 68 kDa,
subunit of nuclear factor kappaB (NF-κB) (de los also called KHDRBS1 – KH domain containing,
Santos et al., 2007, 2009) and as a virally encoded RNA binding, signal transduction associated 1).
deubiquitinase thought to be involved in degrada- Sam68 localizes predominantly to the nucleus
tion of host cell innate-immune proteins (Wang et and its major function in the nucleus is to regulate
al., 2011a,b). alternative splicing by recognizing RNA sequences
Interestingly, in addition to Lpro, FMDV 3Cpro flanking the included/excluded exons. FMDV
has also been shown to degrade host cell translation 3Cpro cleaves away the C-terminus containing the
factors. 3Cpro degrades eIF4G producing cleavage nuclear localization sequence (NLS) – causing the
products different from those generated by Lpro truncated form to redistribute to the cytoplasm.
(Belsham et al., 2000). 3Cpro also degrades the ini- Knock-down of Sam68 in LFBK cells was shown
tiation factor eIF4A1, a DEAD-box helicase which to greatly reduce titres of FMDV and decrease
undergoes ATP hydrolysis-coupled conformational IRES-driven in vitro translation. In the same study,
changes to unwind mRNA secondary structures binding of full-length Sam68 to the IRES of FMDV
during translation initiation (Belsham et al., 2000; was observed, suggesting a role for this protein in
Li et al., 2001). enhancing translation of the vRNA genome (Law-
Early studies showed histone H3, the most rence et al., 2012).
extensively modified of the five histone proteins, 3Cpro has been documented to antagonize
was degraded in FMDV-infected cells and subse- the IFN-α/β response through proteolysis of
quently it was demonstrated that this was mediated nuclear transcription factor kappaB (NF-κB)
essential modulator (NEMO). The cleavage site Batson, S., and Rundell, K. (1991). Proteolysis at the 2A/2B
junction in Theiler’s murine encephalomyelitis virus.
within NEMO was mapped to Gln383, resulting in Virology 181, 764–767.
removal of the C-terminal zinc-finger domain – a Baum, E.Z., Bebernitz, G.A., Palant, O., Mueller, T., and
region essential for full activation of NF-κB, and Plotch, S.J. (1991). Purification, properties, and
subsequently interferon-stimulated gene (ISG) mutagenesis of poliovirus 3C protease. Virology 185,
140–150.
expression (Wang et al., 2012). Reporter assays Bazan, J.F., and Fletterick, R.J. (1988). Viral cysteine
showed that fragments of NEMO produced by proteases are homologous to the trypsin-like family of
3Cpro proteolysis were inefficient at activating the serine proteases: structural and functional implications.
IFN-β promoter, completely abolishing, or impair- Proc. Natl. Acad. Sci. U.S.A. 85, 7872–7876.
Bazan, J.F., and Fletterick, R.J. (1990). Structural and
ing expression. catalytic models of trypsin-like viral proteases. Seminars
FMDV proteinases not only play a central role in Virol. 1, 311–322.
in the biogenesis of virus proteins, but are of great Belsham, G.J., McInerney, G.M., and Ross-Smith, N.
interest in disease control. Firstly, they may well form (2000). Foot-and-mouth disease virus 3C protease
induces cleavage of translation initiation factors eIF4A
drug targets – it could be argued that in certain situ- and eIF4G within infected cells. J. Virol. 74, 272–280.
ations (such as contingency ring vaccination) drug Bergmann, E.M., Mosimann, S.C., Chernaia, M.M.,
administration could protect susceptible animals Malcolm, B.A., and James, M.N. (1997). The refined
until a protective immune response is established. crystal structure of the 3C gene product from hepatitis A
virus: specific proteinase activity and RNA recognition.
Secondly, it is apparent that establishment of per- J. Virol. 71, 2436–2448.
sistent infections involves changes in the activity of Birtley, J.R., Knox, S.R., Jaulent, A.M., Brick, P.,
the virus proteinases. Thirdly, over the past decade Leatherbarrow, R.J., and Curry, S. (2005). Crystal
it has become clear that these virus-encoded structure of foot-and-mouth disease virus 3C protease.
New insights into catalytic mechanism and cleavage
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host cell proteins (e.g. eIF4G) involved in cellular Blair, W.S., Nguyen, J.H., Parsley, T.B., and Semler, B.L.
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suppress the host cell innate immune system. S1-specificity pocket affect substrate recognition and
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foot-and-mouth disease virus polyproteins expressed in
Structure of the FMDV virion with a cell receptor fragment versus the uncom-
In the last ~35 years many high-resolution plexed virion (Fry et al., 1999, 2005b); and (viii)
structures of spherical viruses, including differ- engineered empty capsids of increased thermosta-
ent picornaviruses, have been obtained by X-ray bility versus the parent capsids (Porta et al., 2013b;
crystallography or, in a few cases, by cryoelectron Kotecha et al., 2015).
microscopy (cryo-EM). These studies have pro- The crystal structures of complexes between
vided atomic-detail views of virus particles that FMDV-neutralizing antigen-binding antibody
reveal structural resemblances as well as many fragments (Fab) and an FMDV capsid peptide
differences underlying their diverse properties (the VP1 GH loop) that contains a major antigenic
and biological functions. The reader interested site and the natural cellular receptor binding site
in placing work with FMDV in a more general were determined by Ignacio Fita, Nuria Verdaguer
structural context may want to consult recent struc- and collaborators (Verdaguer et al., 1995, 1996,
tural virology books by Agbandje-McKenna and 1998; Ochoa et al., 2000). Structures of complexes
McKenna (2011), Rossmann and Rao (2012), or between the same Fabs and the FMDV virion were
Mateu (2013a), or the overviews by Johnson and obtained by Elizabeth Hewat and collaborators
Speir (2008), Prasad and Schmid (2012), Harrison using cryo-EM (Hewat et al., 1997, Verdaguer et
(2013), Castón (2013) or san Martín (2013). Gen- al., 1999). Studies on the solution structures of the
eral overviews on picornavirus structure include VP1 GH loop have been carried out by a number of
those by Rossmann (2002) or Fry and Stuart groups, and the interaction between the VP1 GH
(2010). Books and reviews on picornaviruses that loop and a natural integrin receptor of FMDV using
contemplate structure–function relationships nuclear magnetic resonance (NMR) spectroscopy
include those by Semmler and Wimmer (2002), was studied by DiCara et al. (2007).
Ehrenfeld et al. (2010), Tuthill et al. (2010) or
Racaniello (2013). General structure of the FMDV virion
The first crystal structures of picornaviruses, Comparison of the crystal structure of FMDV
those of the enteroviruses human rhinovirus (Acharya et al., 1989; Lea et al., 1994; Curry et al.,
(HRV) and poliovirus (PV), were respectively 1996) with those of other picornaviruses confirmed
solved by Michael Rossmann’s and Jim Hogle’s the expected broad structural similarities between
groups as early as 1985 (Rossmann et al., 1985; animal viruses of a same family (summarized in this
Hogle et al., 1985). The structure of Mengovirus subsection). However, it revealed also unexpected
followed in 1987 (Luo et al., 1987) and, shortly structural differences (as outlined in the next two
thereafter, David Stuart and collaborators deter- subsections) whose biological relevance has, in
mined the first crystal structure of a FMDV several cases, been investigated (reviewed in later
(Acharya et al., 1989). sections).
The crystal structures of other FMDV virions The small (30 nm in diameter), roughly
and capsids followed, all of them solved by D. spherical FMD virion (Fig. 4.1a) is formed by a
Stuart and coworkers as a part of collaborative stud- nonenveloped, hollow protein capsid of icosahedral
ies. This extensive crystallographic work allowed symmetry (Fig. 4.1b) that contains the genomic
atomic-detail comparisons of (i) virions of different RNA molecule. As in all picornaviruses, the mature
serotypes, O (Acharya et al., 1989), C (Lea et al., FMDV capsid is made of 60 copies of each of three
1994), A (Curry et al., 1996) and SAT1 (unpub- major structural proteins termed VP1 (1D), VP2
lished; Protein Data Bank ID 2wzr); (ii) virions of (1B), VP3 (1C) (Fig. 4.1b), and a smaller polypep-
a same serotype (Lea et al., 1995; Fry et al., 2005b); tide termed VP4 (1A).
(iii) neutralizing antibody-resistant virions versus As in PV, HRV and most picornaviruses, VP4
their parent virions (Parry et al., 1990; Lea et al., is myristoylated at its N-terminus (Chow et al.,
1995); (iv) reduced versus non-reduced type O 1987), and can be regarded as a long N-terminal
virion (Logan et al., 1993); (v) FMDV variants extension of VP2 that is released by proteolytic
adapted to different growth conditions (Curry et cleavage during virion maturation (see ‘Morpho-
al., 1996); (vi) empty (RNA-free) capsid versus full genesis of FMDV’, below). VP1, VP2 and VP3,
virion (Curry et al., 1997); (vii) a virion complexed each about 210–220 residues in length, share a
Figure 4.1 (a) Structure of the FMDV virion. VP1, VP2 Figure 4.2 (a) Schematic fold of VP1, VP2 and VP3
and VP3 are respectively coloured blue, green and of FMDV. Single letters and letter pairs respectively
red. (b) Icosahedral organization of the VP proteins identify β-strands and loops. Capsid surface is
in the FMDV capsid. VP1, VP2 and VP3 belonging up (arrow). (b) Approximate positions of most
to one biological protomer are coloured as in (a); surface-exposed loops and C-termini of VP1, VP2
cyan and violet thick lines outline two neighbouring and VP3 in one biological protomer in the FMDV
pentamers. (Reproduced from Rincón et al., 2014.) capsid. The black circle indicates the approximate
size of an antibody footprint drawn to the same scale
as the viral capsid. (Reproduced from Sobrino, F. and
Domingo, E. (eds). 2004. Foot-and-mouth Disease:
common fold consisting of a trapezoidal, eight- Current Perspectives (Wymondham, UK: Horizon
Bioscience); originally adapted from Harrison, S.C.
stranded β-barrel (Acharya et al., 1989; Fry et al., 1989. Nature 338: 205–206, with permission).
2005a) (Fig. 4.2a).
The eight β-strands (labelled alphabetically
as they appear in the polypeptide chain from the longer GH, EF and CD loops, together with the
N-terminus to the C-terminus) form two four- protein C-terminus, make up a large part of the two
stranded β-sheets (respectively including strands C, other, more irregular sides of the trapezoidal VP
H, E, F, and B, I, D, G) that together adopt a wedge- structure, and of the capsid outer surface, with the
like shape. These two β-sheets together make up a GH and EF loops being the most exposed (Fig. 4.2a
large part of two, relatively flat sides of the trapezoi- and b).
dal VP and of the capsid inner surface, where the The similar size and shape of the structurally
VPs N-termini are also located (Fig. 4.2a). homologous VP1, VP2 and VP3 allows these three
The β-strands are connected by loops of variable proteins to fit quite closely as trapezoidal ‘bricks’ to
length (each labelled according to the two strands build a capsid with pseudoT = 3 (P = 3) icosahedral
they connect, Fig. 4.2a). The relatively short BC, symmetry (Figs. 4.1b and 4.2b). Five VP1 subunits
HI, DE and FG loops are located at the narrow are arranged around each capsid five-fold axis, and
end of the wedge, closest to a capsid symmetry axis three copies of VP2 and three copies of VP3 alter-
(five-fold for VP1 and three-fold for VP2 and VP3), nate around each capsid three-fold axis. The VP4
with the BC loop closest to the capsid outer surface, polypeptide adopts an extended, partly disordered
and the FG loop closest to the inner surface. The conformation, with the N-terminus close to a
capsid five-fold axis and the C-terminus close to a or animal viruses (e.g. the T = 1 parvoviruses), in
capsid three-fold axis. most picornavirus structures no segments of the
The capsids of FMDV or any other picornavi- viral RNA molecule are seen to adopt similar, icosa-
rus can be subdivided into 60 equivalent, roughly hedrally ordered folds that bind equivalent sites at
trapezoidal substructures, each containing one the capsid inner wall. In some crystal structures of
copy of each VP (Figs. 4.1b and 4.2b). The VPs are PV (Filman et al., 1989) or HRV (Arnold and Ross-
associated through extensive noncovalent interac- mann, 1990), a few nucleotides were tentatively
tions, with the largely extended VP4 polypeptides identified in a similar location, and in a PV structure,
running along the inner surface. This substructure, nucleotide bases stacked with aromatic residues
termed the biological protomer, constitutes the of VP4 were detected (Lentz et al., 1997). Other,
building block from which the capsid is assembled disordered portions of VP4 might be interacting
(see ‘Morphogenesis of FMDV’, below). Each trian- with segments of the RNA molecule. Icosahedrally
gular facet of the icosahedral capsid (in which VP1, ordered density corresponding to parts of the viral
VP2 and VP3 are related by a pseudothree-fold RNA and some capsid-RNA contacts have been
axis) was defined as a crystallographic protomer. It identified in HRV virions and uncoating intermedi-
is important to remember that it is the (trapezoidal) ates and in enterovirus 71 (Verdaguer et al., 2000;
biological protomer, with its particular quaternary Wang et al., 2012; Pickl-Herk et al., 2013). Thus, the
organization, and not the (triangular) crystallo- picornaviral RNA appears to be loosely tethered
graphic protomer, the one that has a free existence to the capsid, at least in enteroviruses. However,
as a capsid building block. no ordered nucleotides could be observed in any
In FMDV and picornaviruses in general, five FMDV structure so far. Positively charged polyam-
biological protomers associate around each capsid ines that may help to neutralize the negative charge
five-fold axis to form a higher-order, pentagonal of the viral RNA have been detected in HRV and
capsid substructure termed the pentamer (Fig. PV virions (Fout et al., 1984), and they may be
4.1b). The protomers in each pentamer are held expected to be present also in FMDV virions.
together mainly by multiple non-covalent interac-
tions that are, however, less extensive than those Specific structural features of the
involved in intraprotomer interactions (Arnold and FMDV virion
Rossmann, 1990; VIPER database, Carrillo-Tripp The features of the FMDV virion described above
et al., 2009). The association of the N-termini of are shared with PV, HRV and other picornaviruses.
VP3 and VP4 around each five-fold axis contribute However, several structural features distinguish
to connect the protomers in the pentamer. The FMDV from other picornaviruses; some of them
picornaviral pentamer has a free existence as a are unique.
capsid assembly intermediate (see ‘Morphogen- [Note: in this chapter, residues in FMDV cap-
esis of FMDV’, below). In the picornaviral capsid, sids are identified by using four-digit numbers, in
including that of FMDV, the pentamers are non- which the first digit identifies the capsid protein
covalently associated with neighbouring pentamers (VP1, VP2, VP3 or VP4) and the three other digits
mainly through rather flat protein–protein inter- correspond to the residue number in the polypep-
faces, energetically weaker than the interprotomer tide chain (Lea et al., 1994)].
interfaces within each pentamer (Arnold and
Rossmann, 1990; VIPER database, Carrillo-Tripp The smooth outer surface of the capsid
et al., 2009). Formation of β-annuli made of VP2 The FMDV capsid is thinner than the capsids of
N-termini, and of extended β-strands crossing the other picornaviruses because the surface loops
interpentamer interfaces contribute to connect the are generally shorter. The average thickness of
pentamers in the capsid. the FMDV capsid is about 3.3 nm (75% of the
Also in common with other picornavirus struc- HRV capsid thickness) if VP4 is excluded. The
tures, no ordered RNA could be observed in the capsid surface is quite smooth (Fig. 4.1a), with
crystal structure of any FMDV virion. In contrast no conspicuous topographic features such as the
to what was found for some other icosahedral plant large depressions or protrusions found in other
picornaviruses (the long and mobile VP1 GH loop inactivated FMDV at 37°C but not at 25°C, which
being an outstanding exception). The biologically suggests that the pores in the FMDV capsid may be
relevant, deep canyons or pits found in the capsids conformationally dynamic and may open enough
of enteroviruses or Mengovirus are filled in FMDV to allow penetration of this relatively large com-
by the C-terminal segment of each VP1 subunit as pound (Broo et al., 2001).
it runs along the capsid surface.
The capsid three-fold axes
Absence of capsid pockets In the FMDV virion the N-termini of three VP2
The 60 equivalent, large hydrophobic pockets subunits form a short internal β-annulus at each of
behind the canyon floor in enterovirus capsids that the 20 capsid three-fold axes, that fills a depression
can accommodate the so-called pocket factors, and on the capsid inner wall. This β-annulus contrib-
which have critical roles for HRV and PV infec- utes to the association of three different pentamers
tions, do not exist in FMDV. together around each three-fold axis. The presence
of a calcium ion bound to Glu2006 residues within
The capsid five-fold axes the VP2 β-annulus was suggested (Acharya et al.,
Compared to other picornaviruses, the VP1 1989).
β-barrels in the FMDV capsid are more canted up
towards the five-fold axes, which leads to a higher Interpentamer β-sheets
exposure of the short BC, HI, DE and FG loops In the FMDV capsid extended, six-stranded β–
of VP1 on the capsid surface, and a quite different sheets cross the interfaces between neighbouring
local topography. pentamers. These sheets are formed by the C, H, E,
As in other picornaviruses, the N-termini of F strands from VP3 of one pentamer, and the two
five VP3 subunits form an internal β-annulus strands that form a β-hairpin in the N-terminal seg-
(cylinder) at each of the twelve five-fold axes in the ment of VP2 of the adjacent pentamer (Fry et al.,
FMDV capsid. In FMDV, this annulus delineates 2005a). In HRV and PV, the equivalent extended
a largely hydrophobic channel (pore) with an aver- β-sheets include a seventh strand contributed by
age inner diameter of 10 Å that communicates the the VP1 N-terminal segment.
virus interior with the environment. VP3 residues
including Phe 3003, Val3005 and Cys3007 form a Serotype-specific disulfide bonds
constriction, with the side chains (sc) of Cys3007 In serotype O FMDVs, but not in other serotypes,
acting as a diaphragm with an aperture about 6 Å a disulfide bond is formed under non-reducing
in diameter. (extracellular) conditions between Cys1134
In PV, an inner layer formed by the VP4-linked (located at the beginning of the VP1 GH loop) and
myristyl groups and N-terminal amino acid resi- Cys2130 in VP2, covalently linking VP1 and VP2
dues of five VP4 subunits is observed below each in each biological protomer. Reducing conditions
five-fold axis, but these elements are not visible in reversibly break this bond, leading to conforma-
any FMDV crystal structure; no ordered residues tional changes that involve the VP1 GH loop (see
obstruct the five-fold axis pore (Acharya et al., below), VP2 EF loop and VP3 GH loop.
1989), although the myristyl groups could still
form a cluster at the base of the five-fold axis which The highly mobile GH loop of VP1
may interact with the VP3 β-annulus. In FMDV, the GH loop of VP1 (also called the
Under non-reducing conditions, some of the ‘FMDV loop’) contains both a major antigenic and
five cysteines around each five-fold axis are linked immunogenic region (see ‘Structural insights into
by disulfide bridges, which could limit the dynam- the recognition and neutralization of FMDV by
ics of the pore region. The existence of these pores antibodies’, below, and Chapter 10) and the inte-
in the FMDV capsid and their basal free diameter is grin receptor binding site (see ‘Structural insights
consistent with the penetration of caesium ions and into the recognition of cell receptors by FMDV’,
small organic compounds into the virion. Interest- below, and Chapter 5). This long loop encom-
ingly, the RNA-alkylating agent N-dansylaziridine passes approximately residues 1130–1160 (type O
Figure 4.3 Structure and location of the VP1 GH loop. (a) in a reduced type O virion (loop ordered in the ‘down’
orientation; Logan et al., 2003). (b) in a complex between a type C virion and the Fab fragment of neutralizing
antibody SD6 (Hewat et al., 1997). In (a) and (b), left and right images respectively show ribbon diagrams of a
pentamer (front view) and a protomer (side view) in the virion. The capsid VP subunits are coloured as in Fig.
4.1. (c) close-up view of the VP1 GH loop structure (ribbon diagram) in reduced type O virion. (Reproduced
from Sobrino, F. and Domingo, E. (eds.). 2004. Foot-and-mouth Disease: Current Perspectives (Wymondham,
UK: Horizon Bioscience)).
its infectivity was markedly inferior to that of the Camarero et al., 1993; France et al., 1994; Pegna et
non-reduced virion (Fry et al., 2005a). al., 1996a,b; Haack et al., 1997; de Prat Gay, 1997;
In the VP1 GH loop of reduced type O virion Petit et al., 1999; Furrer et al., 2001; DiCara et al.,
(Fig. 4.3c), a stretch of residues (1144–1146) in 2007; Wagstaff et al. 2012). In general, these stud-
the N-terminal part of the VP1 GH loop adopts a ies revealed that the loop-mimicking peptides are
β-strand conformation adjacent to β-strand C of essentially disordered in aqueous solution, but
VP2, extending the CHEF β-sheet of this protein. in structure-inducing solvents they have strong
The central Arg-Gly-Asp (RGD) motif (residues conformational propensities. In some studies,
1145–1147), critical for integrin binding, adopts the conformational preferences found include a
an open turn conformation very similar to those β-turn conformation in the RGD region, followed
observed for the same motif in γ-crystallin, other by a helical segment (France et al., 1994; Haack
integrin-binding proteins, and a complex between a et al., 1997; DiCara et al., 2007; Wagstaff et al.,
RGD peptide and integrin αvβ3 (Xiong et al., 2002), 2012), similar to what was observed in the crystal
which is also a receptor for FMDV. The segment structures of reduced type O virus and of VP1 GH
that follows the RGD turn (residues 1148–1155) loop–antibody complexes (next).
in the loop folds as a short 310-helix (Fig. 4.3c). The
same overall conformation of the VP1 GH loop is Structure of the VP1 GH loop as a
adopted in type O viruses differing in up to four functional synthetic peptide bound to
amino acid residues within the central 15-residue virus-elicited neutralizing antibodies
stretch in this loop (Lea et al., 1995). The immo- A 15-mer synthetic peptide (A15) that represents
bilized VP1 GH loop fits a shallow depression on the central sequence of the VP1 GH loop (residues
the surface of VP2 of the same biological protomer. 1136–1150) of a serotype C virus (strain C-S8)
binds different virus-neutralizing monoclonal
Structure of the VP1 GH loop as an antibodies (MAbs) elicited against the whole
unliganded, functional synthetic peptide virion. Comparative quantitative immunochemi-
Studies by different groups showed that some syn- cal analysis indicated that the conformation of the
thetic peptides representing the sequence of the A15 peptide in a complex with a virus-induced
VP1 GH loop faithfully mimicked the antigenic- neutralizing antibody should accurately mimic the
ity and immunogenicity of this loop in the virion conformation of the VP1 GH loop in a complex
(see ‘Structural insights into the recognition and between the complete virion and the same anti-
neutralization of FMDV by antibodies’, below) body.
and could be used as experimental FMD vaccines This observation prompted the determination
(Chapter 13). Those peptides could also efficiently of the crystal structures of complexes between
inhibit binding of the virion to host cells and viral peptide A15 variants representing the sequences of
infection. Moreover, the effects of amino acid two type C virus isolates, and the Fab fragments of
substitutions on recognition of the VP1 GH loop neutralizing MAbs (SD6 and 4C4) that recognize
in the virion by antibodies could be also faithfully overlapping continuous epitopes in the VP1 GH
mimicked using synthetic peptides (see ‘Structural loop (Verdaguer et al., 1995, 1998; Ochoa et al.,
insights into the recognition and neutralization of 2000; Fig. 4.4a). In every complex, the VP1 GH
FMDV by antibodies’). These results showed that loop-mimicking peptide adopted a conformation
the functionality of the VP1 GH loop is largely self- that resembles closely that of the unliganded VP1
contained, and have justified the extensive use of GH loop in the reduced type O virion (compare
synthetic peptides to investigate in detail its struc- Figs. 4.3c and 4.4b). The peptide conformation
ture and function. is quasi-circular, being stabilized by several intra-
Studies of either linear or cyclized (conforma- peptide hydrogen bonds, van der Waals interactions
tionally restricted) synthetic peptides representing and some buried hydrophobic area. The stretch
(partial) sequences of the VP1 GH loop of different immediately before the RGD motif (shorter by
FMDV variants have been carried out in solution, four residues in FMDV type C than in type O)
using circular dichroism, NMR spectroscopy and/ is in an extended conformation. The RGD motif
or molecular modelling (Siligardi et al., 1991; (residues 1141–1143 in type C) adopts an open
Figure 4.4 (a) Structure (ribbon diagram) of the complex between the Fab fragment of FMDV-neutralizing MAb
SD6 and the VP1 GH loop-mimicking peptide A15 (top; coloured yellow). The side chains of the RGD motif
are depicted (Verdaguer et al., 1995). (b) Close-up ball-and-stick model of the VP1 GH loop peptide in the
Fab–A15 complex. The RGD adopts an open turn conformation very similar to that in the unliganded VP1 GH
loop in a reduced type O virus (compare Fig. 4.3c); Leucines at RGD+1 and RGD+4 are on the same face of a
short helix, as in the NMR structural models of the VP1 GH loop of different serotypes under structure-inducing
conditions (compare Fig. 4.9). (Reproduced from Sobrino, F. and Domingo, E. (eds). 2004. Foot-and-mouth
disease: current perspectives (Wymondham, UK: Horizon Bioscience)).
turn conformation, almost identical to those in the conformation of the VP1 GH loop, leading to a
unliganded VP1 GH loop of the reduced type O change in the predominant orientation of the loop
virion or in other integrin-binding proteins. A short towards the ‘down’ conformation. Differences in
helical stretch follows the open turn, in a direction the local conformations and orientations of the
that is essentially coincident with that of the longer visible stretches at the beginning and end of the dis-
helix in the VP1 GH loop of the reduced type O ordered VP1 GH loop between types O (Acharya
virion. et al., 1989; Parry et al., 1990), C (Lea et al., 1994)
and A (Curry et al., 1996) virions indicated that
Different conformations and orientations this highly mobile, antigenic loop adopts different
of the VP1 GH loop on the virion sets of conformations and orientations in different
surface serotypes (Curry et al., 1996).
In the crystal structure of unperturbed (non- The structures of complexes between type
reduced) type O virion, bifurcated electron C FMDV and the Fab fragments of neutralizing
density for the disulfide bond linking Cys1134 MAbs SD6 and 4C4 were later solved by cryo-
with Cys2130 suggested the presence of multiple EM (Hewat et al., 1997; Verdaguer et al., 1999).
conformations for the disordered VP1 GH loop Fitting into the low-resolution electron density
(Acharya et al., 1989). Comparison of the crystal maps obtained by cryo-EM of the high-resolution
structures of an antibody-resistant type O mutant crystal structures of unbound virion and A15-SD6
virion with its parent virion (Parry et al., 1990) or A15–4C4 complexes provided quasi-atomic
indicated that the VP1 GH loop could adopt two models of FMDV-antibody Fab complexes, and
extreme orientations on the capsid surface, termed revealed the approximate positions the VP1 GH
‘up’ (closer to a capsid five-fold axis) and ‘down’ loop adopts in those complexes. In the type C
(closer to a interpentamer interface; Fig. 4.3a). virion–SD6 complex (Hewat et al., 1997; Fig.
Mutations in the VP1 BC loop (Parry et al., 4.3b), the VP1 GH loop was bent towards the
1990) or disruption of the VP1-VP2 disulfide ‘up’ position proposed for the non-reduced type
bonds (Logan et al., 1993) destabilized the ‘up’ O virion. In the type C–virion–4C4 complex the
loop was oriented radially, fully protruding from, This latter observation suggests that the entropic
and very loosely connected with the rest of the penalty that must be paid for binding the free,
capsid, with the RGD motif at its apex, farthest conformationally disordered peptide to some anti-
from the capsid surface. FMDV antibodies must also be paid, with only a
The fluctuations in position and conformation small ‘rebate’, when binding the VP1 GH loop as
of the unliganded VP1 GH loop in serotype O a part of the virion to those same antibodies. The
FMDV have been simulated by all-atom molecular somewhat lower entropic penalty in the latter case
dynamics (MD) (Azuma and Yoneda, 2009). The would be achieved through limited conformational
coordinates of a crystallographic protomer were restrictions (covalent linkage with the rest of the
used, the simulation was started with the reduced capsid). As with some intrinsically (partially)
state, the disulfide bond was formed in silico, and disordered proteins, binding the ligand (antibody
the atomic trajectories were followed on a nano- or cellular receptor) or other capsid residues (in
second timescale. The results were compatible the reduced type O FMDV) would fully stabilize
with the experimental observations using type O the transiently structured VP1 GH loop into one
FMDV or type C FMDV–antibody complexes. In defined conformation.
the simulations, three states of the VP1 GH loop In addition to any internal conformational plas-
were identified: (i) the initial folded state in the ticity, it is clear that the VP1 GH loop undergoes
‘down’ position; (ii) an intermediate state in which extensive, global movements, and can populate
the unliganded loop is conformationally flexible very different orientations on the capsid surface
and moves not as a hinged rigid body, but like a depending on the specific virus variant and the
‘tentacle’, protruding from the capsid surface as in conditions. Some functional consequences of the
the virion–antibody complexes; (iii) a final, equi- peculiar structure and dynamics of the VP1 GH
librium state close to the ‘up’ position, in which the loop are considered in ‘Structural insights into the
loop still fluctuates somewhat between different recognition and neutralization of FMDV by anti-
conformations and orientations. bodies’ and ‘Structural insights into the recognition
of cell receptors by FMDV’ (below).
A model for the structure and dynamics
of the VP1 GH loop
To recapitulate, the available evidence supports Morphogenesis of FMDV
a model in which the long, dynamic, mobile VP1 As the pseudoT = 3 icosahedral capsid of FMDV
GH loop has a strong intrinsic propensity to tran- (like those of other picornaviruses) is actually
siently adopt some conformational variations of a assembled from 60 identical protomers, it can also
consensus strand-turn-helix motif. Several observa- be regarded as a very simple T = 1 capsid made of 60
tions argue against a permanently, fully folded and heterotetramers (each containing one copy of each
stable intrinsic conformation of the unliganded capsid protein). There is no quasiequivalence, and
VP1 GH loop on the unperturbed surface of viri- no conformational switches are required during
ons of different serotypes (C, A and non-reduced its assembly. However, in vivo morphogenesis of
type O): (i) the absence of a defined conforma- even the structurally simplest viruses is a complex
tion of the VP1 GH loop as a free, either linear or process that involve a number of viral and cellular
conformationally restricted peptide in aqueous proteins, nucleic acid and other biomolecules, and
solution (consistent with the lack of a fully folded that requires tight spatial and temporal coordination
conformation for nearly any other protein-derived of multiple recognition events at the molecular and
free peptide of similar length); (ii) the very loose cellular levels (Mateu, 2013b; Almendral, 2013).
connection of the VP1 GH loop in the virion with Morphogenesis is still a poorly understood stage in
the rest of the capsid, especially in some functional the infectious cycle of FMDV, and of most icosahe-
(antibody- or receptor-binding) orientations; (iii) dral viruses.
the results of all-atom MD simulations; (iv) a com-
parable MAb-binding affinity of free, disordered Steps in FMDV morphogenesis
VP1 GH loop-mimicking linear peptides, and the Most studies on picornavirus morphogenesis have
VP1 GH loop as a part of the virion. been carried out with enteroviruses, and with
Figure 4.5 A scheme of the model for enterovirus morphogenesis described by Jiang et al. (2014). See text
for a detailed explanation. Steps where glutathione (GSH) is required are inhibited by L-buthionine sulfoximine
(BSO). Pentamers are recruited by viral protein 2C to the replication complex made of several different viral
proteins and bind the VPg-linked viral RNA (red line).
those in the assembled virus particle (Hogle et al., each made of five protomers (Putnak and Phillips
1985; Rossmann et al., 1987). 1981b) (Fig. 4.5). These pentamers can be found
In HRV, PV and other picornaviruses including in cells infected with any picornavirus. For FMDV
FMDV, extensive intraprotomer interactions and there is clear evidence for the progression from
intertwining of N- and C-termini of the VP subunits stable 5S protomers, to highly stable 12S pentam-
clearly indicate that the quaternary organization of ers, to complete viral particles (e.g. Grubman,
the capsid building block corresponds closely to 1984; Grubman et al., 1985).
that of the trapezoidal biological protomer in the A high-resolution structure of the free penta-
virion (Fig. 4.1b). For FMDV, further evidence meric assembly intermediate is not available for
was provided by specific MAbs that efficiently bind any picornavirus. However, a low-resolution (30 Å)
both the assembled FMDV virion and the unpro- structural model has been generated for the free
cessed capsid protomer (Saiz et al., 1994; Goodwin pentamer of bovine enterovirus, based on EM
et al., 2009). These antibodies recognize discontin- imaging and three-dimensional reconstruction of
uous epitopes in antigenic sites that can be formed negatively stained pentamers (Li et al., 2012). The
in the biological protomer (Lea et al., 1994), but model suggests that very large structural rearrange-
not in the crystallographic protomer. In addition, ments may not occur during assembly of pentamers
both the virion and the unprocessed protomer into enteroviral particles.
are similarly recognized by integrin αvβ6, a natural In the crystal structures of all picornaviruses,
receptor of FMDV (Goodwin et al., 2009). Thus, highly complementary interprotomer interfaces
different regions of the free unprocessed protomer are formed within each pentamer. In addition, the
and the mature protomer in the virion must display β-annulus formed by the N-termini of five VP3
conformations similar enough to be recognized by subunits around each five-fold axis knits together
proteins whose binding activity is very sensitive to the five protomers in the pentamer, and VP4-linked
minor structural changes in the ligand. myristates and the N-termini of VP4 (as a part
As in HRV, PV and other picornaviruses, in the of VP0) enhance the assembly of PV pentamers
FMDV virion the VP termini that are joined in the (Ansardi et al., 1992). Likewise, in FMDV both
unprocessed protomer are not contiguous in the VP4 and the myristyl group were shown to be
virion structure. The C-termini of VP2 and VP3 important for the correct in vitro self-assembly of
are on the capsid outer surface, while the N-termini pentamers. Deletion of VP4 prevented the associa-
of VP3 and VP1 are in the capsid interior. In the tion of protomers; absence of the myristate led to
folded unprocessed picornaviral protomer, the two association of protomers into capsid assembly-
covalent linkages between VP0 and VP3, and VP3 incompetent 17S particles, different from the
and VP1 remain on the surface, fully accessible to assembly-competent 12S pentamers that were
proteases. In FMDV at least, the cleavages may be formed when VP4-containing, myristoylated
dependent on the protomer local or global struc- protomers were used (Goodwin et al., 2009).
ture, as a mutation on the protomer surface, but
not in the cleavage site, affected the processing of Step 4: Assembly of immature viral
the VP3-VP1 linkage (Escarmís et al., 2009). After particles
cleavage, some rearrangements of the N-terminal In PV and other enteroviruses (but not every picor-
segments of VP3 and VP1 and the C-terminal seg- navirus), 75S empty capsids are formed during the
ments of VP0 and VP3, and perhaps other minor infection process (Hummeler et al., 1962), and can
local rearrangements, would suffice to achieve the also self-assemble in vitro (Putnak and Phillips,
final structure of the mature, assembly competent 1981a,b). Inhibition of RNA synthesis in infected
5S protomer. cells by low guanidinium chloride concentrations
led to the accumulation of procapsids that were
Step 3: Assembly of the pentameric converted into virus particles when RNA synthesis
intermediate was restored (Fernandez-Tomas and Baltimore,
During picornavirus morphogenesis, mature 5S 1973). These and other observations are consistent
protomers form relatively stable 14S intermediates, with the view that, in infected cells, the pentamers
may self-assemble into RNA-free capsids, and the cell-free translation systems in vitro (Grubman
viral RNA would be later packaged in the preformed 1984; Grubman et al., 1985). Early studies on
empty capsid. FMDV morphogenesis by Eduardo Palma and
Other evidence, however, supports an alterna- colleagues using pulse-chase experiments revealed
tive model of enterovirus assembly that is currently a precursor–product relationship between capsid
favoured. 14S pentamers (but not empty capsids) precursors, procapsids (empty capsids) and viri-
are able to bind RNA in vitro, which leads to their ons, with an observed rate of virion formation from
structural rearrangement (Nugent and Kirkegaard, procapsids that was identical to the observed rate
1996; Verlinden et al., 2000). After a block caus- of empty capsid assembly from capsid precursors
ing accumulation of pentamers in infected cells (Gomez-Yafal and Palma, 1979). These results led
was removed, the pentamers could be chased into the authors to propose a model in which the FMDV
mature virions (Rombaut et al., 1990). The viral empty capsid is self-assembled first, and the viral
2CATPase, a component of the membrane-associated RNA is packaged later in the preformed capsid,
viral RNA replication complex, was found to bind using some unknown mechanism. The authors also
VP1 and/or VP3 and recruit the preassembled considered the alternative possibility that empty
pentamers to the site of virus particle assembly (Liu capsids could serve as a reservoir of capsid pro-
et al., 2010). Recent studies revealed that the pres- teins and could dissociate into pentamers which,
ence of an inhibitor of PV morphogenesis causing in turn, would coassemble with the RNA to form
reduced glutathione depletion did not inhibit the the provirion. However, they suggested that for
formation of empty capsids, but these were unable FMDV this latter possibility is unlikely, based on
to form virions (Ma et al., 2014). These and other the observed quantitative transformation of empty
observations support for PV the hypothesis that the capsids into virions, and on stability considerations.
capsid and viral RNA are simultaneously coassem- Further studies are required to decide whether
bled. In this model of enteroviral assembly, specific FMDV empty capsids are (on-pathway) productive
recognition between pentamers and the viral RNA intermediates of virion assembly, or (off-pathway)
molecule, and condensation of twelve pentamers dead-end products or capsid protein reservoirs.
around the RNA, perhaps with the help of 2CATPase,
directly yields an immature, noninfectious 150S Step 5: Maturation of the provirion
provirion ( Jiang et al., 2014) (Fig. 4.5). The noninfectious, relatively stable enterovirus pro-
It has been suggested that the empty capsids virion is converted into a mature, infectious 150S
formed in infected cells may constitute an off- virion by a RNA-dependent, probably autocatalytic
pathway assembly product that does not act as cleavage of VP0 into VP4 and VP2 (Fig. 4.5). This
a direct precursor of the assembled virion, but maturation process entails a substantial conforma-
that could act as a physiological reservoir of tional rearrangement of the PV particle, including
capsid proteins. Nonetheless, the possibility that repositioning and organization of the VP4 and
an on-pathway empty capsid is formed first and VP2 termini (Hogle et al., 1985), which provide
the enteroviral RNA is later packaged into the further interactions between pentamers and likely
preassembled capsid has not been entirely ruled contributes to stabilize the mature virion. His2195,
out. Many other important aspects of this step in conserved in all picornaviruses, was identified as
enterovirus morphogenesis remain unclear. For being essential in PV for efficient cleavage of the
example, picornaviral RNA packaging signals have VP4–VP2 linkage; mutations of this residue led
been searched with no success to date, except for to assembly of VP0-containing, RNA-containing,
the Aichi virus of the genus Kobuvirus (Sasaki and immature 150S particles that were highly unstable
Taniguchi, 2003); it is unclear whether the RNA- (Basavappa et al., 1994; Hindiyeh et al., 1999).
bound VPg could interact with capsid proteins Morphogenesis of FMDV also includes as a
during morphogenesis; etc. ( Jiang et al., 2014). final step the cleavage of VP0 into VP4 and VP2
As for other picornaviruses, FMDV empty to yield an infectious mature virion. As in other
capsids are naturally formed in infected cells picornaviruses, this process entails a consider-
(Rowlands et al., 1975), and can also be assembled able conformational reorganization of the virion,
inside cells (reviewed in Dong et al., 2014) and including the repositioning and reorganization of
VP2 and VP4 termini and the formation of second- for assembly upon its proteolytic processing
ary structure elements (β-annuli at the three-fold by 3Cpro that converts the three domains into
axes and extended β-sheets) that reinforce the individual proteins VP0, VP3 and VP1.
connection between pentamers (Acharya et al., 3 The three proteins remain non-covalently
1989; Lea et al., 1994). No stable, non-infectious associated in the mature protomer, whose
FMDV provirions have been detected so far, but conformation closely resembles that of the
a combination of mutations around the VP4–VP2 immature protomer, except for the reposition-
cleavage site led to production of VP0-containing, ing of the released, solvent-exposed termini of
non-infectious provirions (Knipe et al., 1997). VP0, VP3 and VP1.
Empty capsids that are naturally produced 4 Five mature protomers associate to form a
during infection by PV or other picornaviruses lack highly stable pentameric intermediate. The
viral RNA, and usually contain uncleaved VP0. In formation of pentamers that are competent
contrast, in a serotype A FMDV empty capsid used for self-association into complete particles
for structural studies, most VP0 subunits had been involves the establishment of many protomer–
cleaved into VP4 and VP2 (Curry et al., 1995). protomer interactions, including association
However, this cleavage likely occurred downstream of the VP3 N-termini in a β-cylinder around
of the cleavage site observed in mature virions. the central five-fold axis, and requires the par-
Comparison of the crystal structure of this FMDV ticipation of the VP4 N-termini and myristate
empty capsid (Curry et al., 1997) with that of PV groups. This morphogenetic step may be
suggested that the RNA-dependent, autocatalytic, helped by chaperone Hsp90.
His2195-mediated mechanism proposed for VP0 5 Twelve assembly-competent pentamers asso-
cleavage in PV operates also in FMDV. ciate to form complete empty capsids. These
A structural comparison of the empty capsid viral RNA-free capsids present an outer surface
and the corresponding virion (serotype A) (Curry that is quite similar to that of the virion, but
et al., 1997) allowed an appraisal of the structural differ from the latter in some structural features
effects the RNA exerts on the mature capsid of its inner surface.
structure (after VP0 processing). The differences 6 RNA-containing, non-infectious virus parti-
observed are confined to the interior of the capsid. cles are formed. FMDV empty capsids may
The N-terminus of VP1 and C-terminus of VP4 are constitute on-pathway assembly intermediates
less well ordered in the empty capsid. This higher into which the viral RNA is encapsidated by
disorder suggested a more loose association of an unknown mechanism; however, evidence
these segments that help connect the capsid subu- obtained with enteroviruses favours the pos-
nits, protomers, and pentamers, and a role of the sibility that picornavirus empty capsids are
viral RNA in the stabilization of some intersubunit dead-end products or reservoirs of capsid pro-
interfaces, including those between pentamers. teins. If this pathway occurs, assembled empty
capsids should eventually dissociate again into
An (incomplete) model for FMDV pentamers, and free pentamers (or partially
morphogenesis disassembled empty capsids) would associate
To recapitulate, the limited evidence available tends with the newly synthesized viral RNA to form
to support a model for FMDV morphogenesis that non-infectious provirions in a single coassem-
resembles the model proposed for enterovirus bly plus encapsidation step.
assembly by Jiang et al. (2014) and schematized 7 The non-infectious, unstable provirion
in Fig. 4.5, with some important differences and immediately matures into a relatively stable,
uncertainties: infectious virion; this process involves the
RNA-dependent, probably autocatalytic
1 The unprocessed P1–2A polypeptide folds as cleavage of VP0 into VP4 and VP2, and a
a three-domain (VP0, VP3, VP1) protein, the substantial conformational reorganization of
immature biological protomer, which consti- parts of the inner capsid surface in the viral
tutes the elementary capsid building block. particle, that may contribute to stabilize the
2 The immature protomer is made competent mature virion.
genome’, below). The trade-off of such energetically Production of FMDV empty capsids
weak interfaces would be an extreme sensitivity to using baculovirus-based expression
disruption by even single mutations that remove systems
a few interpentamer interactions. This sensitivity, In 1990, Just Vlak and collaborators described a
however, may be not detrimental to rapid FMDV procedure based on the introduction of the P1–2A
evolution: the virus manages to accept the intro- and 3C protease coding regions of FMDV in a
duction of many individual lethal mutations at the baculovirus expression vector to produce in insect
interpentamer interfaces through fixation of com- cells small amounts of 70S particles that resembled
pensatory mutations that frequently occur at a few FMDV empty capsids (Roosien et al., 1990). In
‘compensation hotspots’ in the viral capsid (Luna recent years, a number of articles have described
et al., 2009). other baculovirus-based approaches to produce
FMDV empty capsids. Li et al. (2008, 2011) used
Production of FMDV empty capsids recombinant baculovirus containing the FMDV
for the development of novel P1–2A and 3C coding regions to obtain empty
anti-FMD vaccines capsids in silk worms. The capsid preparations
The early discovery that empty capsids are formed obtained protected bovines against viral chal-
during FMDV morphogenesis, and that they share lenge. Cao et al. (2009) constructed recombinant
the same antigenic and immunogenic character- baculovirus to simultaneously express P1–2A
istics as the virion (Rowlands et al., 1975) led the and 3C of FMDV in insect cells. The 70S empty
development of different approaches to produce capsids obtained were characterized in immu-
FMDV empty capsids, either for fundamental noassays and imaged by EM, and were shown to
studies or as a basis for novel vaccines. The design induce anti-FMDV neutralizing antibodies in
of alternative anti-FMD vaccines is a quite intense guinea pigs. Cao et al. (2010) avoided the need to
area of applied research (Rodriguez and Grubman, use the 3C protease by expressing separate VP0
2009; Chapters 13 and 14). Empty capsid-based and VP1–2A-VP3 that were processed and assem-
vaccines would avoid any risk of virus escape or bled into capsid-like particles in insect cells. Porta
deficient inactivation during vaccine production, et al. (2013a, 2013b) described the expression of
while preserving the full immunogenicity and anti- FMDV empty capsids in insect cells using recom-
genic spectrum of current virion-based vaccines; binant baculoviruses expressing P1–2A–3C with
capsid-based vaccines would also allow differen- a number of modifications to reduce 3C activity,
tiation between vaccinated and infected animals thus decreasing its toxic effects and increasing
(DIVA assays). empty capsid production. Empty capsids were
The development of procedures to obtain sub- obtained in yields high enough to be purified and
stantial amounts of recombinant FMDV empty crystallized. They were characterized immuno-
capsids in different cell types (mammalian, insect chemically and by EM and genetically modified
or bacterial) or transgenic plants for vaccination to increase their stability (see next section), and
purposes started about 25 years ago, and is being their structure was solved by X-ray crystallogra-
actively pursued today (Roosien et al., 1990; Lewis phy (Porta et al., 2013b; Kotecha et al., 2015). The
et al., 1991, Abrams et al., 1995; Li et al., 2008, purified empty capsids were shown to induce high
2011; Pan et al., 2008; Cao et al., 2009, 2010; Lee et levels of neutralizing antibodies in guinea pigs
al, 2009; Polacek et al., 2013; Gullberg et al., 2013; and bovine, and to protect cattle against challenge
Guo et al., 2013; Porta et al., 2013a,b; reviewed by with virus (Porta et al., 2013b). Ruiz et al. (2014)
Dong et al., 2014). Such strategies are recapitulated described a modified baculovirus-based expres-
here. DNA vaccines based in the use of recombinant sion system that has also been used to produce
adenovirus carrying the FMDV capsid polyprotein FMDV empty capsids in insect larvae, leading to
P1–2A and 3C genes for expression of immuno- high yields (about 30 micrograms/g of insect bio-
genic empty particles directly in the vaccinated mass) that compared to those obtained by Li et al.
animals (reviewed by Grubman et al., 2010; Fowler (2008, 2011) and Porta et al. (2013a,b).
and Barnett, 2012) are described in Chapter 14.
Production of FMDV empty capsids fusion proteins formed a complex, and after specific
using vaccinia virus-based expression proteolytic cleavage, capsid-like particles were
systems observed by EM.
In 1995, Graham Belsham and associates described
the use of a recombinant vaccinia virus containing Production of FMDV empty capsids in
the P1–2A and 3C coding regions for production in transgenic plants
mammalian cells of FMDV 70S empty capsids that Pan et al. (2008) introduced the P1–2A and 3C
were characterized by EM and immunoassays with coding regions of FMDV into a plant binary vector
anti-FMDV MAbs directed against discontinu- and transformed tomato plants using Agrobacterium
ous epitopes (Abrams et al., 1995). Very recently, tumefaciens. Guinea pigs vaccinated with transgenic
different modifications of vaccinia virus-based plant extracts were protected against a challenge
expression systems have been described to sub- infection.
stantially increase empty capsid yields. Polacek To recapitulate, critical advances have been
et al. (2013) showed that cotransfection using a made on the production of FMDV empty capsids
plasmid encoding P1–2A and lower amounts of using different expression systems. Production
a plasmid encoding 3C was sufficient to obtain in insects or cultured insect cells, or in cultured
efficiently processed capsid proteins. Gullberg et mammalian cells, respectively, using recombinant
al. (2013) described two alternatives to reduce 3C baculovirus or vaccinia virus-based systems in
activity in the transfected cells: the use of a mutated which 3C activity has been adequately modulated
3C (Cys142Ser) with reduced specific activity; has been thoroughly investigated and shows great
or the use of internal ribosome entry site (IRES) promise. However, there are still several impor-
elements with reduced ability to direct internal tant issues that must be solved to allow the use
initiation of translation to produce lower levels of recombinant empty capsids as a basis for anti-
of 3C. They showed that P1–2A was efficiently FMD vaccines. They include the need to establish
expressed and processed, and that 70S empty a routine, large scale production and purification
capsids were produced. EM imaging was used to of FMDV empty capsids, and the fact that empty
obtain a three-dimensional reconstruction of the capsids are even more thermosensitive than virions
assembled empty capsids at low (36 Å) resolution, (Doel and Baccarini, 1981). To address this latter
which showed the expected size, icosahedral sym- issue, that also concerns current virion-based vac-
metry and general morphology. The capsids bound cines, both FMDV virions and empty capsids have
the receptor integrin with the same activity as the been engineered to increase their stability against
intact virion, and were recognized by anti-FMDV heat-induced dissociation (see next section).
antibodies. Porta et al. (2013a,b) also produced
FMDV empty capsids in mammalian cells using a
vaccinia virus-based expression system in which the Structural determinants of
3C activity was modulated. FMDV (in)stability
Compared to other picornaviruses, FMDV virions
Production of FMDV empty capsids in and empty capsids are remarkably labile. FMDV
Escherichia coli virions rapidly lose infectivity when subjected to
In 1991, Marvin Grubman and associates obtained mild acidification, moderate heating, subzero tem-
similar FMDV 70S particles by expressing the peratures or very high pressures or, at a slower rate,
P1–2A polyprotein and 3C protease in E. coli, and even when stored at 4°C. The weak acid stability of
showed that these particles were recognized by the FMDV virion plays a critical role in infection;
anti-FMDV MAbs against discontinuous epitopes, its weak thermal stability may also be a selective
were immunogenic in guinea pigs and swine, and trait; its heat-induced dissociation into pentamers
protected the later against viral challenge (Lewis et constitutes a serious problem in FMD control by
al., 1991; Grubman et al., 1993). Lee et al. (2009) vaccination. Identifying the structural determinants
simultaneously expressed in E. coli SUMO protein of the very low physical stability of FMDV is criti-
fused to VP0, VP1 and VP3 of FMDV. The three cally important not only for a better understanding
of morphogenesis and genome uncoating, but also productively infect another animal, the relatively
for the development of improved vaccines and anti- low survival rate in even mildly acidic environ-
viral approaches. ments may not impose an insurmountable negative
It is important to stress that acid stability and selection pressure. In contrast, such extreme acid
thermal stability of FMDV particles are not nec- sensitivity appears to have a critical adaptive value
essarily linked: (i) empty capsids can be more by facilitating uncoating of the viral genome in the
acid-resistant than full virions, but they are even mildly acidic environment of endosomes.
less thermostable (Doel and Baccarini, 1981; Inspection of the crystal structure of FMDV
Curry et al, 1995); (ii) FMDV virions from differ- revealed a relatively high density of histidine resi-
ent strains and different sensitivity to acid-induced dues lining the pentamer interfaces. The pKa value
inactivation showed similar sensitivity to thermal of the sc of a free histidine is close to the range of
inactivation (Maree et al., 2013); (iii) an engineered pH at which dissociation of the FMDV virion
FMD virion with a highly increased resistance into pentamers occurs. Thus, it was suggested that
against thermal dissociation into pentamers was, interpentamer electrostatic repulsions involv-
however, as sensitive as the parent virion against ing protonated histidines could underlie the acid
acid-induced dissociation (Rincón et al., 2014). sensitivity of this virion (Acharya et al., 1989).
Likewise, experimental evidence shows that for Subsequent structural and biochemical analysis of
the FMDV virion, stability against thermal inacti- virions and capsids of several serotype A isolates
vation of infectivity, and stability against thermal led to the suggestion that electrostatic repulsions
dissociation into subunits, do not necessarily occur close to the capsid two-fold axes between proto-
at the same time and may be uncoupled: (i) both nated His3142 in each pentamer and the intrinsic
at 4°C and at 42°C, purified FMDV virions are dipole of an α-helix (residues 2189–2198) in the
inactivated much faster than they are dissociated neighbouring pentamer could influence the acid
into pentamers (Mateo et al., 2008); (ii) some engi- lability of the FMDV virion (Curry et al., 1995,
neered virions with a much increased resistance 1997). Twomey et al. (1995) analysed the positions
against dissociation into pentamers are, at most, of charged residues around histidines at the inter-
only slightly more stable than the parent virion pentamer interfaces and suggested that His3142
against thermal inactivation at 42°C (Mateo et al., and His3145 could be major determinants of acid
2008). lability.
Simplified titration calculations, using a model
FMDV sensitivity to acidification composed of two crystallographic protomers
Investigation of the structural basis of the acid labil- related by a two-fold symmetry axis, reproduced
ity of FMDV particles is especially important for a the decrease in stability of the FMDV capsid at
better understanding of the genome uncoating step acidic pH, but not the difference in sensitivity
during the infectious cycle (Chapter 5). between two type A viruses (van Vlijmen et al.,
1998; Schaefer et al., 1998). According to these
Histidine residues close to interpentamer calculations, only residues within 15 Å of the inter-
interfaces as determinants of acid lability face could influence acid lability, with His3142 and
Of all picornaviruses, the FMDV virion is the most His3145 in serotype A (corresponding to histidines
sensitive to dissociation into pentamers under acidic 3141 and 3144 in serotype A and 3140 and 3143
conditions. The acid sensitivity varies somewhat in serotype C) being major contributors, as previ-
between different virions, but in nearly all reported ously suggested. The calculations predicted that the
cases the pH at which half of the viral particles specific interaction between His3142 and the helix
lose integrity (pH50) in vitro is close to neutrality dipole in the neighbouring pentamer contributes
(around pH 6–7 for natural virions, depending on about 20% of the total destabilization energy.
viral isolate and experimental conditions). This Mutational analysis was used to ascertain the
acid sensitivity must be highly disadvantageous effects of replacements of His3142 in the assembly
for the virion in the environment. However, as the and stability of FMDV recombinant empty capsids
number of virions shed by an infected animal can produced in mammalian cells. A His to Arg muta-
be quite large, and very few virions are enough to tion prevented capsid assembly, while a His to
Asp (charge reversal) mutation allowed assembly could be traced to an increased resistance against
of empty capsids which showed a significantly acid-induced dissociation of the virions into pen-
increased stability against acid-induced dissocia- tamers. Experiments carried out with the mutant
tion into pentamers (Ellard et al., 1999). This result virion Asn1017Asp and the control virion showed
provided experimental evidence for a electrostatic that increased resistance against acidification may
destabilization of pentamer–pentamer interactions be achieved without a significant reduction in
at acidic pH determined by His3142 in the FMDV infectivity and biological fitness at physiological
capsid. pH, at least in cultured cells (Martín-Acebes et al.,
2011). Also, neither mutation altered the antigenic
Structural determinants of decreased specificity of the virion. Both mutations involve an
or increased acid resistance of selected increase in negative charge and in bulkiness of the
FMDV mutants sc, and are located at a considerable distance from
Infectious FMDV mutants with either decreased or the interpentameric interfaces and from each other.
increased resistance against acid-induced inactiva- Residues mutated in an acid-resistant variant of a
tion of infectivity have been readily isolated from different serotype selected in an earlier study are
viral populations of different serotypes subjected to also located far from the interpentameric interfaces
the appropriate selection pressure (Twomey et al., (Twomey et al., 1995).
1995; Martín-Acebes et al., 2010, 2011; Vázquez- Inspection of the FMDV virion structure
Calvo et al., 2014; Caridi et al., 2015). A series of suggested some tentative explanations for the
studies by Francisco Sobrino’s group have identified virion-destabilizing or stabilizing effects at
a substantial number of capsid residues that modu- acidic pH of the selected mutations. For exam-
late acid stability of FMDV, and provided new tools ple, the destabilizing mutations located close to
to study FMDV uncoating (Vázquez-Calvo et al., the interfaces could exert their effect by locally
2012a,b; see ‘Structural insights into uncoating of distorting the interfaces and altering the charge
the FMDV genome’, below, and Chapter 5). distribution around the pH-sensing histidines,
FMDV mutants with increased sensitivity to leading to increased electrostatic repulsion between
acid inactivation were readily selected by impairing pentamers at acidic pH. The destabilizing or sta-
acidification in the endosomes of host cells treated bilizing mutations located at the VP1 N-termini
with NH4Cl (Martín-Acebes et al., 2010; Caridi could provoke some local rearrangement of these
et al., 2015). Individual mutations responsible of segments, which could propagate to the interfaces,
increased acid lability were mapped to VP2 or VP3 leading to either increased or decreased repulsion
residues located very close to the interpentamer between pentamers at acidic pH. These and other
interfaces, or within a stretch of residues (1011– tentative structural explanations proposed remain
1022) in the VP1 N-terminal segment. Of the five to be tested.
mutations close to the interfaces, all preserved the
electric charge, but most led to the introduction of A role of the viral RNA in determining
a bulkier sc (e.g. Ala3118Val or Ala3116Val). The acid lability of the FMDV virion
6 mutations at the VP1 N-terminus were chemi- FMDV empty capsids are between 0.3 and 0.7 pH
cally and sterically varied, but all of them affected units more resistant to acid-induced dissociation
residues clustered at some distance from the inter- than the corresponding RNA-filled virions (Curry
pentameric interfaces. et al., 1995). In contrast, they appear to be less resist-
FMDV mutants with increased resistance against ant to dissociation than the virions at nonacidic pH
acid inactivation were selected by incubation of and moderate temperatures (Doel and Baccarini,
viral populations under mildly acidic conditions 1981). Thus, the RNA itself must contribute, either
(Martín-Acebes et al., 2011; Vázquez-Calvo et al., directly or indirectly, to the increased acid lability
2014). The two mutations individually responsible of the virion relative to the empty capsid. The inter-
of the acid-resistance phenotype were Thr1017Asp pentamer interfaces in the virion and empty capsid
at the VP1 N-terminus (very close to mutations are nearly identical, but they differ in the organiza-
responsible for the opposite, acid-labile phenotype) tion or degree of disorder of internal features that
and His2145Tyr. In both cases, the stabilizing effect involve VP terminal segments (Curry et al., 1997).
A tentative explanation is that this reorganization and usually confer adequate protection of animals.
could modify the electrostatic potential close to the Unfortunately, a number of problems severely limit
interpentamer interfaces specifically at acidic pH, its safety and efficacy. One problem arises from the
thus increasing the electrostatic repulsion between high sensitivity of the FMDV virion to heat-induced
pentamers in mildly acidic conditions; a possible dissociation into poorly immunogenic pentam-
change in the dielectric constant has also been ers (Doel and Baccarini, 1991; Doel and Chong,
invoked (Curry et al., 1995). 1982), leading to unacceptable reductions in vac-
cine potency during storage and transportation.
FMDV sensitivity to thermal An expensive cold chain is routinely implemented,
inactivation of infectivity but this chain is frequently disrupted, eventually
The mechanism by which FMDV subjected to leading to vaccine failure. Different methods of
moderate heating irreversibly loses infectivity chemical thermostabilization of FMD vaccines
without particle dissociation is still unclear. Early have been tested along the years, but so far they
observations suggested that mild heating, even at appear to be inadequate, insufficient and/or incon-
physiological temperature (37°C), facilitates deg- sistent enough to tolerate a significant relaxation of
radation of the RNA genome inside the virion by the cold chain.
an endogenous viral nuclease (Brown and Wild, Empty capsid-based vaccines are being seriously
1966; Newman and Brown, 1997). In addition, considered and investigated as potential novel
more recent studies showed that many mutations vaccines against FMD (see above). Unfortunately,
that involve substitution of different residues in there are still several important issues with this
the FMDV capsid either decrease or increase the approach, and an important one is the fact that the
resistance of the virion to thermal inactivation of FMDV empty capsid is even more sensitive than
infectivity (e.g. Mateo et al., 2003, 2008; Martín- the virion to heat-induced dissociation (Doel and
Acebes et al., 2011). Likewise, shortening the viral Baccarini, 1981).
RNA led to increased resistance against thermal
inactivation of infectivity (Ojosnegros et al., 2011). Engineering FMDV virions with
High pressure and subzero temperatures also inac- increased thermostability for improving
tivate the FMDV virion without disrupting the viral current anti-FMD vaccines
particle (Ishimaru et al., 2004). As one possible uni- The demand to improve the thermostability of
fying explanation, it could be tentatively suggested current anti-FMD vaccines and, eventually, of
that any of these factors may influence the energy potential empty capsid-based vaccines led our
barrier of a same unidentified, inactivating con- group to attempt the rational engineering of FMDV
formational rearrangement of the virion without virions with increased stability against thermal
capsid disruption. There is of course the possibility dissociation into pentamers, in order to develop
that virion inactivation may occur by several differ- genetically stabilized anti-FMD vaccines that could
ent mechanisms triggered by different agents. be prepared using current production procedures,
but that are less dependent on a fail-safe cold chain.
FMDV sensitivity to thermal The thermostabilizing mutations found could also
dissociation into pentamers, be introduced in recombinant empty capsids in
and engineering of FMDV virions future developments of capsid-based vaccines of
and capsids with increased adequate thermostability.
thermostability for the development As a result, Mateo et al. (2008) first provided
of improved vaccines proof of concept that fully infectious FMDV viri-
ons with highly increased stability against thermal
Sensitivity of FMDV particles to dissociation into pentamers can be rationally
heat-induced dissociation is a problem engineered, providing a solid starting point for the
for FMD control by vaccination thermostabilization by genetic methods of current
Current commercial anti-FMD vaccines based on anti-FMD vaccines (see comments by Hedge et al.,
chemically inactivated virions (Chapter 12) are 2009). In a successful approach, different amino
economically produced using time-tested methods, acid substitutions to increase the net electrostatic
attraction between pentamers were introduced at site-directed mutations. Single removal of most
or close to the interpentamer interfaces in a sero- of the six targeted solvent-exposed carboxylates
type C FMDV model virion (Fig. 4.6a). Residues (Fig. 4.6a, top left image) preserved viral infectiv-
chosen for replacement included those adequately ity and increased as predicted the stability of the
positioned and whose sc established limited virion against thermal dissociation into pentamers
interpentamer interactions that could be removed (Rincón et al., 2014). Introduction of the Ala-
(by mutation to Ala) without causing substan- 2065His mutation into one of these thermostable
tial reductions in viral infectivity (Mateo et al., variants (Asp3195Asn) led to no further increase
2003). Mutations Ala2065His and Asp3069Glu/ in stability, consistent with the hypothesis that
Thr2188Ala separately led to dramatic increases in His2065 (with a raised pKa due to its interactions)
virion stability against heat-induced dissociation acts by neutralizing repulsions between Asp3195
into pentamers (Mateo et al., 2008). The half-life and other acidic residues nearby. Simplified elec-
of the two purified mutants was much (severalfold) trostatic potential calculations were qualitatively
higher than that of the parent virion, both under consistent with the experimental results and the
storage at 4°C (see graph in Fig. 4.6a) or at high repulsive effect of the identified carboxylates (Fig.
ambient temperatures, e.g. 42°C. Both mutants 4.6a, bottom left image), supporting the use of such
preserved the full infectivity and antigenic specific- calculations to guide the electrostatic stabilization
ity of the parental virion, and were genetically fairly of protein complexes (Rincón et al, 2014; see com-
stable during propagation in cell culture (enough ments by Sivertsson and Itzhaki, 2014).
for large-scale production during vaccine prepara-
tion) (Mateo et al., 2008). One of the thermostable Engineering empty capsids with
virions (Ala2065His) was tested for acid-induced increased stability for developing safer
dissociation and found to be as sensitive as the anti-FMD vaccines
parent virion, consistent with its normal infectivity Some of the virion-thermostabilizing mutations
(as required for vaccine production). Moreover, identified using the electrostatics-based strategy
these mutants could be normally inactivated by reviewed above (Mateo et al., 2008; Rincón et al.,
binary ethyleneimine without losing their higher 2014) were introduced in the FMDV empty capsid.
structural thermostability (Rincón et al., 2014). As expected, they increased also the resistance of
the empty capsid against thermal dissociation into
Identification of structural determinants pentamers (Rincón and Mateu, unpublished obser-
of the sensitivity of FMDV to thermal vations).
dissociation Very recently, a consortium of groups has
The engineered thermostable virions described described two additional strategies aimed at the
above were used to investigate the thus far unknown engineering of recombinant empty capsids with
structural determinants of the sensitivity of FMDV increased thermostability (Porta et al., 2013b;
virions and capsids to heat-induced dissociation Kotecha et al., 2015). As only empty capsid-based
into pentamers. Rincón et al. (2014) found that vaccines were (in principle) contemplated in
charge screening at very high ionic strength had their designs, the choice of adequate mutations
little effect on the stability against thermal dissocia- was based only on their possible effects on capsid
tion of the purified engineered virions, but actually assembly and stability; there was no need to con-
increased that of the parent virion. This observation sider possible unwanted effects on virus infectivity
suggested that the mutations introduced in the or genetic stability, unlike the original approach
thermostable mutants were increasing the net elec- followed by Mateo et al. (2008).
trostatic attraction between pentamers by reducing The strategy followed by Porta et al. (2013b)
electrostatic repulsions. was based in the introduction of disulfide bonds
Carboxylate groups close to the Ala2065His sta- between pentamers, an approach that had been
bilizing mutation and to the interpentamer interface successfully used before for thermostabilization
were identified in the parent FMDV structure (Lea of phage MS2 under nonreducing conditions
et al., 1994), and individually removed in the parent, (Ashcroft et al., 2005). The FMDV structure was
thermolabile virion by isosteric charge-to-neutral manually inspected to identify geometrically
UNCORRECTED PROOF
Figure 4.6 Rational strategies for thermostabilizing FMDV virions and/or empty
capsids. (A) An electrostatics-based approach for stabilizing FMDV particles
without impairing infectivity and genetic stability of the virion (Mateo et al., 2008;
File: FMDV 4P
Rincón et al., 2014). Top left image: two neighbouring pentamers are coloured
cyan or violet. Inbuilt electrostatic repulsions between the carboxylates of some
acidic residues (coloured yellow) close to the interpentamer interfaces contribute
to the low resistance of FMDV particles against thermally induced dissociation
into pentamers (right image). Individual removal of the negative charges of any
of these carboxylates through isosteric mutations (Asp to Asn or Glu to Gln), or
partial neutralization by introducing a positive charge through mutation of Ala2065
adequate positions. Substitution His2093Cys cell culture), but yielded titres comparable to those
(see Fig. 4.6b) was chosen and introduced in a of the parent viruses.
recombinant empty capsid (serotype A) to form The relative stability of the mutant virions
a disulfide bond between the Cys2093 residues of was determined in temperature gradients, both
neighbouring pentamers across each of the capsid at neutral and slightly acidic pH. Four out of five
two-fold axis. Parent and mutant empty capsids mutations tested in type O and two out of three
were produced both in mammalian cells (using a mutations tested in type SAT2 thermostabilized
vaccinia virus-based expression system), and to the virions to different extents. The stability of one
higher yields in insect cells (using a baculovirus- of the most stable type O mutants (Ser2093Tyr)
based expression system). The crystal structures of was additionally determined by incubation at fixed
both recombinant empty capsids were determined. times and temperatures. Albeit a controlled com-
The geometry of the disulfide bond was found to parison is not possible, similar analyses of particle
be correct. No other differences were observed, integrity on prolonged storage at 4°C suggests that
except that VP4 was fully disordered in the mutant. the thermostabilizaton achieved by Ser2093Tyr in
The capsids produced in mammalian cells were a type O virion in this study compares to that found
analysed in qualitative stability assays. Heating in the previous study by Mateo et al. (2008) by Ala-
at 56°C for 2 hours disrupted the purified parent 2065His or Asp3069Glu/Thr2188Ala in a type C
capsids, while most mutant capsids were not dis- virion using a different approach.
sociated. Likewise, acidification to pH 5.2 for 15 The stability of three type O and one type A
minutes dissociated the parent capsids, while the mutant empty capsids were also determined, again
mutant capsids remained intact. Thus, introduction by incubation at fixed times and temperatures.
of the disulfide bonds led to capsids with increased All were more thermostable than the parent virus
stability against dissociation by both heat and acid. and also showed increased acid resistance. The
Both capsids induced comparable antibody titres in structures of the thermostable mutants type O
cattle, and protected two (parent capsid) or three Ser2093Tyr, type A His2093Phe and type SAT2
(mutant capsid) out of four animals against infec- Ser2093Tyr were determined by X-ray crystallog-
tion with the homologous virion. raphy or cryo-EM. Overall, the structures revealed
The strategy followed by Kotecha et al. (2015) the expected stacking interactions between the
used simplified all-atom MD simulations to predict substituted aromatic residues, consistent with the
changes in binding energy across the interpentamer predicted role of these interactions in stabilizing
interface caused by pre-selected mutations. A panel the viral particles.
of potentially stabilizing amino acid substitutions Immunization of calves with freshly prepared
within VP2, close to the capsid two-fold axis, vaccines based on type O or type SAT2 inactivated
were first manually chosen based on inspection of virions (parent and Ser2093Tyr mutant) yielded
FMDV crystal structures. The somewhat different similar neutralizing antibody titres. Immunization
structural contexts and residues in the capsids of of guinea pigs with vaccines based on SAT2 inacti-
different serotypes (O, SAT2, A) were considered. vated virions (parent and Ser2093Tyr mutant) and
Final MD simulations were performed using sim- stored for 1 or 6 months at 4°C resulted in higher
plified models which included only those atoms neutralizing antibody titres for the thermostabi-
from two crystallographic protomers related by a lized mutant.
two-fold axis, and located within 13 Å of the inter- To recapitulate, different structure–based
pentamer interface (Fig. 4.6b). Positional restraints rational approaches based on removal of electro-
were increased with the distance of the atoms to the static repulsions between pentamers, introduction
interface. Based on the stabilizing effects predicted of disulfide bonds, or introduction of additional
by the simulations, some chosen mutations were stacking interactions at the interpentamer interfaces
actually introduced in recombinant empty capsids led to substantial stabilization of a considerable
produced in mammalian or insect cells. Using infec- number of FMDV virions and empty capsids of
tious clones, the mutations were also introduced in different serotypes (C, A, O and SAT2) against
virions, which presented small-plaque phenotypes heat-induced dissociation into pentamers (Mateo
(suggestive of clearly reduced biological fitness in et al., 2008; Porta et al., 2013b; Rincón et al., 2014;
Kotecha et al., 2015). Other desirable features the interaction between virus-neutralizing antibod-
were tested for some mutants only. When tested: ies and a particular, important antigenic region in a
(i) the thermostabilizing mutations introduced virion, and revealed some remarkable mechanisms
in virions had no or moderate (small plaque) of virus escape without compromising viral func-
detrimental effects on infectivity; (ii) the virions tion.
were genetically fairly stable (allowing their large-
scale production); (iii) the virions conserved their The VP1 GH loop is a major antigenic
increased thermostability after chemical inactiva- region of FMDV
tion; (iv) the viral particles were found to conserve As a part of the pioneering work on FMDV by Fred
the antigenic specificity and/or were as immuno- Brown and collaborators, it was found that trypsin
genic as the parent; (v) they conferred protection treatment of virions (serotypes O or A) greatly
against infection; and (vi) they conserved their diminished their infectivity, attachment to host
immunogenic potency as vaccines for a longer time cells, antigenicity and immunogenicity (Wild and
than their parents. Brown, 1967; Wild et al., 1969). Trypsin cleavage
led to excision of a capsid peptide fragment from
type O virus particles (Strohmaier et al., 1982),
Structural insights into the and it was concluded that this peptide contains
recognition and neutralization of both the cell receptor binding site and a major
FMDV by antibodies antigenic region in the FMDV virion, a conclu-
Antibodies are major effectors in the protection sion fully confirmed later. Determination of the
of host animals against FMD (Chapter 10). Thus, FMDV structure (Acharya et al., 1989) showed
actions for better control of this disease, including that this peptide corresponds to the VP1 GH loop
immunological surveillance, updating of current in the virion (see ‘Structure of the FMDV virion’,
vaccines and development of new vaccines, may above).
greatly benefit from a deep understanding of the Chemically synthesized peptides that represent
mechanisms of virus recognition by neutralizing (a part of) the sequences of the VP1 GH loop of
antibodies and virus escape, in the context of the different FMDV serotypes were efficiently rec-
high genetic variability and quasispecies structure ognized by serum antibodies elicited against the
of FMDV populations (Chapter 7). As a conse- virus, and could themselves elicit a potent antiviral
quence, in the last four decades these aspects have humoral response (Kaaden et al., 1977; Bachrach et
been intensively studied, mainly using polyclonal al., 1979; Strohmaier et al., 1982; Pfaff et al., 1982;
antibodies and MAbs, virus variants of different Bittle et al., 1982). Amino acid substitutions in the
serotypes, antibody-escape virus mutants and syn- VP1 GH loop of viruses of different serotypes,
thetic peptides representing viral sequences. subtypes or variants, when introduced in those
Structural aspects of the recognition of FMDV synthetic peptides, mimicked the specificity of the
by antibodies and the mechanisms of virus escape immune response elicited by the complete virion
have been thoroughly reviewed by Mateu (1995) in animal models (Bittle et al., 1982; Clarke et al.,
and Mateu and Verdaguer (2004). These reviews 1983; Rowlands et al., 1983).
have not been fundamentally outdated by later Geysen et al. (1984, 1985) used variant synthetic
studies. Here a more condensed, updated overview hexapeptides with all possible single replacements
on this subject is provided. to explore in detail the antigenicity of this region of
the FMDV capsid using polyclonal antibodies. An
Structural basis of the interaction of interesting observation was that conserved residues
virus-neutralizing antibodies with a in the RGD(L) sequence, later identified as the cell
major antigenic site in the VP1 GH attachment site, were antigenically important in the
loop three serotypes tested. Unfortunately, these pep-
A large number of functional and structural studies tides proved to be too short to reproduce complete
by different groups have focused in the VP1 GH epitopes, and thus provided an incomplete, biased
loop as a major, independent antigenic element. portrait of consensus residues involved in polyclonal
These studies provided a uniquely detailed view of antibody binding.
Discontinuous and continuous epitopes two VP1 stretches (Parry et al., 1990). Similar
that involve the VP1 GH loop conformation-dependent mechanisms of escape
Later antigenic analyses of the VP1 GH loop from neutralization was proposed for two different
generally used neutralizing MAbs elicited against epitopes in serotype A viruses (Thomas et al., 1988;
virus particles to characterize individual epitopes. Bolwell et al., 1989a,b; Curry et al., 1996).
Antigens tested included virions and/or VP1 of Unlike serotype O virus, multiple continuous
field isolates of known sequence, and VP1 GH epitopes (i.e. formed by residues within one short
loop-mimicking peptides of nested or overlapping peptide segment) within the VP1 GH loop of
sequences, eventually containing single or multiple serotypes C or A viruses defined an independent
amino acid substitutions. In addition, many mutant antigenic site (termed site A for type C viruses)
viruses that escaped recognition by neutralizing (Mateu et al., 1987, 1988, 1989, 1990; Bolwell et
MAbs were selected under antibody pressure, and al., 1989a). These epitopes are true native epitopes,
the escape mutations were identified by sequencing. as they are defined by neutralizing MAbs elic-
Comparisons of the crystal structures of different ited against the virion. In type C viruses the VP1
FMDVs provided detailed structural insights into C-terminus also contain continuous epitopes that
the epitopes identified. define another antigenic site (site C). Site C and
The epitopes thus detected in viruses of different site A are topologically independent, as binding to
serotypes (O, A, C) differ in fundamental ways. In the virion of a type A MAb did not sterically inhibit
serotype O FMDV (strain O1BFS), the VP1 GH binding of a type C MAb, and vice versa (Lea et al.,
loop is involved in discontinuous epitopes (i.e. 1994).
epitopes formed by residues that are located far
apart in the primary structure, but that come spa- Functional and structural dissection of
tially close in the folded protein). These epitopes continuous epitopes that define a major
include residues from both the VP1 GH loop and antigenic site in the VP1 GH loop
the VP1 C-terminal segment (Parry et al., 1985, Because of the remarkable structural compactness
1989). of folded proteins, most B-cell epitopes on their
Remarkably, some escape mutations selected by surfaces are discontinuous. Some authors even sug-
those MAbs mapped in the VP1 BC loop. The same gested that true continuous B-cell epitopes may not
mutations inhibited the interaction between the exist in folded proteins. Thus, a full characterization
virion and serum antibodies elicited against a VP1 of the seemingly continuous epitopes defined by
GH loop-mimicking peptide, whose epitopes could neutralizing antibodies elicited against the FMDV
not include residues outside that loop. Disruption virion was conceptually important. Definitive
of VP1 GH loop epitopes by residues located else- proof of the existence of continuous B-cell epitopes
where was noted also in another study with a type in FMDV (and other pathogens) has also a clear
O virus (Krebs et al., 1993). biotechnological relevance when evaluating the
Structural studies provided an explanation for potentiality of peptide-based vaccines against FMD
those puzzling results. In the crystal structure of or other diseases.
the parent O1BFS virion, the disordered, mobile A uniquely extensive and detailed series of
VP1 GH loop in the ‘up’ position is close to the structure–function studies on epitopes within the
VP1 C-terminus and BC loop. In contrast, in the VP1 GH loop was started by Esteban Domingo’s
structure of an escape virion with a mutated VP1 group using serotype C FMDV, and soon became a
BC loop, the VP1 GH loop appeared to be mainly multidisciplinar collaboration that involved a con-
towards the ‘down’ position (Fig. 4.3a), far away siderable number of additional groups. It was found
from the VP1 C-terminus and BC loop (Parry et that 15-mer to 21-mer linear peptides encompass-
al., 1990). The authors proposed that mutations in ing a core sequence of the type C VP1 GH loop
the VP1 BC loop stabilize the ‘down’ conformation approached, on a molar basis, the reactivity of the
of the VP1 GH loop away from its original posi- loop in the virion when bound to the same antibod-
tion close to the VP1 C-terminus, thus disrupting ies. The same single or multiple replacements in the
the discontinuous epitopes that involve these peptide and the virion caused similar variations
in the binding of several virus-neutralizing MAbs field isolate of serotype C was also very similar
(Mateu et al., 1989, 1992). Thus, free linear pep- to the conformation of the reference (C-S8) A15
tides proved to be excellent quantitative mimics of peptide. Comparison of the unliganded and
the antigenic activity and specificity of the VP1 GH A15-bound structures of Fabs SD6 and 4C4
loop for binding multiple antibodies. Individual suggested that peptide recognition involves sub-
replacements at any of nine positions within a stantial conformational rearrangements in the
10-residue stretch affected the recognition by each antibody paratope for a better accommodation of
MAb tested (Verdaguer et al., 1998). These and the peptide (Verdaguer et al., 1996, 1998). Some
other results allowed a precise functional definition substantial local differences in the mc torsion
of multiple continuous epitopes within a 15-residue angles were, however, observed between the Fab-
segment (1136–1150) of the VP1 GH loop of sero- bound peptides, particularly around the RGD
type C FMDV (Mateu et al., 1989, 1990; Verdaguer motif. Changes in the torsion angles in Gly1142
et al., 1995, 1998; Mateu, 1995). (in the RGD triplet) compensated the differences
The RGD motif and residue 1146 at position in other torsion angles, explaining the overall
+3 downstream of the RGD (RGD+3), within the structural similarity despite the local differences
contiguous short helical stretch were critical for (Verdaguer et al. 1995, 1998; Ochoa et al., 2000).
binding all MAbs tested, while some replacements Consistent with the structural similarities
of some other residues severely affected binding of between SD6- and 4C4-bound peptides, many
only a few MAbs (Mateu et al., 1990; Novella et al., specific peptide–antibody interactions, including
1993; Verdaguer et al., 1995, 1998). Thus, despite a critical contacts with the RGD motif, were shared
substantial overlap of the epitopes, and the critical by the three complexes. However, in agreement
roles of some residues within site A in binding most with the local structural differences observed
antibodies, the individual epitopes in the VP1 GH between the bound peptides, and between the
loop defined by different MAbs are not functionally antibody paratopes, some interactions did not
identical. occur in every complex. The crystallographically
Comparisons on the recognition of variant VP1 defined contact epitope (i.e. the residues involved
GH loop peptides by polyclonal sera obtained from in interactions with the antibody paratope) and the
convalescent or vaccinated swine indicated that the immunochemically defined functional epitope (i.e.
antibodies elicited against the VP1 GH loop in a the residues that affect the MAb-binding affinity)
natural host recognize epitopes that are similar to accurately matched each other, even in details at the
those recognized by neutralizing MAbs, supporting residue level (Verdaguer et al., 1995, 1998; Ochoa
the biological relevance in the field of continuous et al., 2000).
epitopes quite similar to those structurally and The pseudoatomic models of the SD6- or 4C4–
functionally characterized in detail using neutral- virion complexes provided a direct view on how
izing MAbs (Mateu et al., 1995b). these antibodies interact with the VP1 GH loop in
The crystal structures of complexes between the context of the complete viral particle (Hewat
the Fab fragments of MAbs SD6 or 4C4 and the et al., 1997; Verdaguer et al., 1999; Fig. 4.3b). The
A15 peptide (Verdaguer et al., 1995, 1998; Ochoa orientation of the loop is very different in both
et al., 2000; see also the section on the structure complexes (see section on the structure of the VP1
of the VP1 GH loop, above) (Fig. 4.4) showed GH loop, above), providing further evidence of
that SD6 contacts 10 out of the 15 residues in the extreme mobility of this functionally critical
peptide A15, and 4C4 contacts 8 out of the 11 loop in FMDV. In both complexes, the antibodies
A15 residues ordered in the complex. Thus, in appear to interact almost exclusively with the VP1
both cases the structural epitope consisted of an GH loop, irrespective of the different orientations
almost continuous stretch of amino acid residues. of the latter, and make no other contacts with the
The conformations of the VP1 GH loop in the capsid. This observation provided additional struc-
SD6-A15 or 4C4–A15 complexes are very similar tural evidence for the true continuous nature of
to each other. The conformation of a variant A15 the epitopes defined in the FMDV virion by these
peptide that represents the sequence of a different neutralizing antibodies.
A model of the interaction of the or (even better) cyclized peptides (Valero et al.,
VP1 GH loop with virus-neutralizing 2000) representing the VP1 GH loop sequence are
antibodies excellent qualitative and quantitative antigenic and
To recapitulate, the evidence supports the following immunogenic mimics of this antigenic site (and also
model for the interaction of the VP1 GH loop in the potent inhibitors of virion-cell and virion-integrin
native virion with virus-induced neutralizing anti- recognition). Both as a part of the virion or as a free
bodies. As already discussed, the unliganded loop is peptide, binding of the VP1 GH loop to antibodies
very loosely connected to the rest of the capsid, and or integrins may be achieved at the cost of compa-
can sample a wide range of similar conformations rable reductions in the conformational entropy of
and widely different orientations, behaving much the unliganded antigen. If the unliganded VP1 GH
like a free linear peptide whose ends had been loop on native virions were already fully and stably
immobilized and conformationally restricted. folded (instead of only transiently folded), the bind-
In viruses of some serotypes (type C and, to a ing affinity of anti-VP1 GH loop antibodies to free
limited extent, type A) the different predominant peptide mimics should be much lower (because of
orientations tend to exclude any interaction of the the much higher entropic penalty), which was not
VP1 GH loop residues with other capsid residues. observed.
As a consequence, host antibodies can recognize this The model for antibody recognition of the VP1
loop as an independent antigenic region. Because GH loop of serotype O virions differs in some
of the limited surface area of the VP1 GH loop and points from the model just described for serotype
its propensity to adopt a protruding, convex fold C (and possibly A) viruses. In native type O virions,
on the virion surface, binding of antibodies with the preferred ‘up’ orientations of the VP1 GH loop
concave paratopes (as observed for MAbs SD6 and on the capsid surface may place this loop particu-
4C4) is favoured because it maximizes contact area, larly close to, and may be transiently interacting
providing higher binding affinity. This situation with, the VP1 C-terminus. Thus, antibodies that
strongly resembles the recognition of free peptides recognize both VP1 segments as one discontinuous
by anti-peptide antibodies that also have concave epitope with a larger surface area are favoured in
paratopes, instead of the flatter paratopes typical of type O viruses over antibodies that would bind con-
antibodies that recognize the abundant, relatively tinuous epitopes within either the VP1 GH loop or
flat discontinuous epitopes in proteins. the VP1 C-terminus. This situation provides type
As also discussed above, the unliganded VP1 O viruses with the selective advantage of escaping
GH loop in the native virion may not be fully folded neutralization through an additional mechanism:
in a stable conformation. The limited intraloop mutations outside the epitopes that favour a dif-
enthalpic interactions and internal hydrophobic ferent orientation of the VP1 GH loop separate it
effect may barely compensate the reduction in from the VP1 C-terminus, leading to the physical
conformational entropy on folding of this ‘inde- disruption of the epitopes (Parry et al., 1990; Logan
pendent’ loop. This would lead to a very small free et al., 1993).
energy difference between its folded and unfolded
states that could be easily overcome, even by ther- Immunodominance of the VP1 GH loop
mal fluctuations. Recognition of the VP1 GH loop in FMDV
by an antibody (or by an integrin receptor; see Multiple observations have revealed that the VP1
section ‘Structural insights into the recognition of GH loop constitutes one, but not the only major
cell receptors by FMDV’, below), would provide immunogenic region in FMDV (summarized and
the additional enthalpic interactions and increased extensively referenced in Mateu and Verdaguer,
hydrophobic effect required to finally overcome 2004; see next subsection). Moreover, the degree
the entropic cost of fully stabilizing the unliganded of immunodominance of the VP1 GH loop is
loop into a relatively rigid strand–turn–helix con- dramatically variable. For example, immunoaffin-
formation, for which this loop has a strong intrinsic ity fractionation of anti-type C FMDV sera from
conformational propensity. convalescent or vaccinated pigs revealed that the
This scenario also explains why free, linear pep- percentage of the virus-neutralizing activity corre-
tides (Mateu et al., 1989; Verdaguer et al., 1995) sponding to antibodies that bind antigenic site A in
GH loop (Group 1), the VP1 C-terminus (Group Fab competition experiments to define topo-
2), and the VP2 BC, VP3 BC, HI, EF loops and logically independent sites in FMDV have been
BB ‘knob’ (Group 3); similar (but not identical) carried out with FMDVs of serotype A (A10Hol-
groupings could be made for the sites detected in land; Thomas et al., 1988) and serotype C (C-S8;
other type A isolates. Lea et al., 1994). The results of these experiments,
In a serotype Asia1 virus (Grazioli et al., 2013), the locations of all of the escape mutations found
four functionally independent sites respectively for FMDV of any serotype, and the relatively large
involved the VP1 GH loop, VP2 BC loop, VP3 BB size of an antibody footprint (Fig. 4.2b), support
‘knob’ and VP3 C-terminus. the existence of at most three physically separate
antigenic regions on each protomer in the generic
Topologically independent antigenic FMDV virion (compare Figs. 4.2b and 4.7). These
sites in FMDV consensus sites were termed structural antigenic
The definition of functionally independent anti- sites I, II and III in Mateu and Verdaguer (2004).
genic sites based on cross-neutralization assays is Site I involves the mobile VP1 GH loop and
widely used, convenient and useful, but has some the VP1 C-terminus; depending on virus strain or
limitations. These limitations arise especially when serotype, and the relative locations of these two
one wishes to understand FMDV-antibody recog- elements, they behave as structurally independent
nition in structural terms, or for some applications sites containing continuous epitopes, or as a single
(e.g. the design of novel anti-FMD vaccines). site made of discontinuous epitopes. Site II con-
Evidence obtained with FMDV and other tains discontinuous epitopes that are between the
viruses (Mateu, 1995; Mateu and Verdaguer, 2004) three-fold axis and the junction between VP1, VP2
has shown that: and VP3 on the protomer surface. Antigenic struc-
tural elements may predominantly include the VP2
1 As more MAbs and escape mutants were BC loop and/or the VP3 BB ‘knob’, and/or also
included in the assays, antigenic sites originally the VP2 HI and EF loops, VP3 BC and GH loops,
defined as functionally independent based on and a turn at the beginning of the VP1 C-terminal
lack of cross-neutralization had to be redefined segment. Not all of these elements are involved in
as a single functional site. This was the case, for all site II contact epitopes, which probably overlap
example, with sites 2 and 4 in FMDV type O only to a limited extent. Site III contains discon-
that were later functionally connected (Barnett tinuous epitopes located close to the five-fold axis
et al., 1998). that collectively involve at least the VP1 BC and HI
2 Mutual steric inhibition between pairs of Fabs loops.
in competition experiments for binding the Functional or topological antigenic sites are
virion showed that antibodies that recognize both operational definitions, because they depend
functionally independent sites actually bind on the use of limited panels of escape mutants and/
structurally overlapping epitopes in the viral or MAbs as probes. A quasi-continuum of epitopes
particle, and thus define only one topological may exist on the smooth FMDV capsid surface (as
antigenic region. For example, functionally observed with some thoroughly analysed protein
independent antigenic sites D1, D2 and D3 in antigens). Indeed, many short peptides represent-
FMDV type C are contained a single topological ing the sequence of many segments of VP1, VP2
site, site D, at the junction between VP1, VP2 or VP3 bound anti-FMDV antibodies, which sug-
and VP3 on the surface of each biological gested that their epitopes are scattered all over the
protomer (Lea et al., 1994). Functionally virion surface (Meloen et al., 1986). The three dif-
independent sites 1 and 5 in serotype O FMDVs ferent topological (structural) antigenic sites I, II,
are defined by escape mutations located very III proposed could be viewed as wide areas of each
close within the VP1 GH loop; it is likely that protomer that constitute three centres of higher
they could be functionally connected if more epitope density. However, no clear borders would
escape mutants and MAbs were used to test exist between them (Fig. 4.7), unlike antigenic areas
cross-neutralization, and that they constitute a in enteroviruses that are more clearly separated by
single topological antigenic site. high protrusions and deep canyons (Minor, 1990;
Mateu, 1995). Thus, thermostabilized FMDV had limited or no effect on attachment to cells and
capsids may prove to be a best option in case one infectivity (Mateu et al., 1996). Moreover, they
wishes to develop non-infectious anti-FMD vac- abolished recognition by nearly all tested site A
cines able to reproduce the full immunogenicity of MAbs and anti-site A antibodies present in sera
the FMDV virion. from host animals. Remarkably, analyses of many
type C field viruses isolated in two continents over
Biological functions of the FMDV six decades revealed that only very rarely the viruses
virion severely constrain viral had ‘chosen’ to evade the antibody response through
strategies for escaping antibody a single mutation at those two ‘ideal’ residues. The
recognition general route apparently followed to escape neu-
The extensive studies on the recognition of the VP1 tralization in the field involved accumulation of
GH loop of serotype C FMDV by virus-neutralizing substitutions at other site A positions (Martínez
antibodies (see above) provided a paradigm for the et al., 1991) despite the fact that, individually,
presence and consequences of functional restric- replacements at these latter positions had a limited
tions to mutation in a viral antigenic site (reviewed potential to evade a polyclonal response against
in Mateu, 1995; Domingo et al., 2003; Mateu and this antigenic site (Mateu et al., 1990, 1995b). Viral
Verdaguer, 2004). Several of the 10 contiguous resi- competition experiments revealed that antibody
dues in the SD6 contact epitope and overlapping escape mutations in the VP1 GH loop of FMDV, or
epitopes in antigenic site A within this loop were in other antigenic sites, are frequently acquired at
never found mutated in a very large collection of the expense of reduced biological fitness (Domingo
SD6-escape mutants (Mateu et al., 1989; Martínez et al., 2003). The rare occurrence of antigenically
et al., 1997), and were highly conserved in many drastic mutations in the VP1 GH loop during cir-
type C field variants (Martínez et al., 1991). It was culation of type C FMDV in the field may be due
found that most of these residues are required for to considerably reduced biological fitness, perhaps
integrin-mediated attachment of the virus to cells because of a somewhat reduced affinity for the
and viral infectivity (Mateu et al., 1996; see also integrin receptor (Mateu et al., 1996; see also next
next section). Thus, residues critically involved in section).
antibody recognition are kept invariant in the face Restrictions to sequence variation at the FMDV
of antibody pressure through the action of nega- capsid surface are not limited to the VP1 GH
tive selection (Verdaguer et al., 1995; Mateu, 1995, loop at all. In fact, indirect evidence suggested
Mateu and Verdaguer, 2004). restrictions to variation also in site D, the other
Accordingly, when the negative selection pres- major antigenic site identified in type C (Lea et al.,
sure on these residues was removed by creating a 1994; Mateu et al., 1994; Mateu 1995; Mateu and
binding site for a different receptor elsewhere in Verdaguer, 2004) and in other antigenic sites of
the capsid after virus propagation in cell culture, FMDV (Mateu, 1995). Further genetic and anti-
a completely different spectrum of SD6-escape genic analysis suggested that antigenic variation of
mutants was obtained (Martínez et al., 1997; FMDV type C in the field in two continents over six
Ruiz-Jarabo et al., 1999; Domingo et al. 2003). decades has occurred mainly through accumulation
Many of these variants contained mutations in of mutations confined to very few, highly exposed
residues critically involved in binding to integrins positions on the capsid surface. These positions are
(no longer used as receptors for these variants), remarkably coincident with those mutated in MAb-
that had not been found in SD6-escape mutants escape variants that define different functional
derived from the parental virus or in type C field antigenic sites (Martínez et al., 1992; Mateu et al.,
variants. 1994; Feigelstock et al., 1996; Mateu and Verda-
Restrictions to variation within neutralization guer, 2004).
epitopes were also found for residues not critically As the effects of more mutations introduced in
involved in receptor recognition. Single substitu- the FMDV capsid by site-directed mutagenesis of
tions of highly conserved residues 1145 (RGD + 2) infectious clones are tested, it is becoming more
and, especially, 1146 (RGD + 3) in antigenic site clear that the vast majority of non-natural capsid
A within the VP1 GH loop of serotype C FMDV mutations are detrimental for viral infectivity and/
or biological fitness; the same is being found for et al., 1997, 1999) show that antibodies that bind
other animal viruses. The capsid of FMDV and of the VP1 GH loop do neutralize FMDV infectivity
small, nonenveloped animal viruses in general may by binding some of the same residues that bind the
have been streamlined through evolution to occupy integrin receptor (including the RGD), and steri-
quite narrow peaks of higher fitness in the sequence cally inhibiting attachment to cells.
space. Only a few, relatively small ‘hotspots’, even
on the viral capsid surface, may tolerate a relatively
large spectrum of amino acid substitutions with- Structural insights into the
out causing unacceptable reductions in biological recognition of cell receptors by
fitness. FMDV
In this scenario, the structurally confined, func- FMDVs isolated in the field, and most strains
tional antigenic sites defined by escape mutations adapted to growth in tissue cultures, infect cells by
correspond to small capsid surface ‘hotspots’ where binding any of several integrins of the αv subgroup
amino acid substitutions are better tolerated by the (αvβ1, αvβ3, αvβ6, αvβ8), with αvβ6 probably being the
virus. For the same reason, these same ‘hotspots’ primary cognate receptor in host animals. In addi-
probably constitute the places most frequently tion, some FMDVs propagated in cultured cells have
‘used’ by FMDV (as well as other picornaviruses acquired the capacity to bind HS proteoglycans as
and nonenveloped viruses) to escape the antibody alternative receptors. Some evidence indicates that
response during circulation in the field. It has other receptors may also mediate FMDV infection
long been noted that these ‘hypervariable’ regions of cells, but they have not been identified yet.
generally correspond to quite exposed tips of long Molecular aspects of the recognition of cell
loops that abound in the nonenveloped capsids of receptors by FMDV and virus entry into cells are
many animal viruses. It is tempting to speculate extensively covered in Chapter 5. For other recent
that exposed loops on those capsids may have reviews on structural and/or functional aspects of
been enlarged by positive selection partly to act FMDV receptors see Jackson et al., 2003; Fry et
as ‘decoys’ to confront the immune system. These al., 2005a; Ruiz-Sáenz et al., 2009; Fry and Stuart,
relatively long, mutation-tolerant loops promote 2010; Han et al., 2015. Here only a few aspects
antibody binding, but provide at the same time an regarding the important, but still rather limited
easy escape route, because they can accept muta- structural knowledge gained so far on FMDV-
tions that impair antibody recognition without receptor recognition are summarized.
severely compromising virus viability and fitness.
FMDV-integrin receptor recognition
Neutralization of FMDV infectivity by After the VP1 GH loop was identified as a recep-
antibodies tor binding site (Wild and Brown, 1967; Wild et
Despite the wealth of structural and functional al., 1969; Strohmaier et al., 1982; Acharya et al.,
information on the recognition of FMDV by 1989; previous section), RGD-containing peptides
different antibodies, not much is known on the (Surovoi et al., 1988; Fox et al., 1989; Baxt and
mechanisms of antibody-mediated FMDV neu- Becker 1990) and FMDV mutants (Mason et al.,
tralization. Early work by Baxt et al. (1984) revealed 1994; McKenna et al., 1995; Rieder et al., 1996)
that some anti-FMDV MAbs neutralize infectivity were used to show that the conserved RGD motif
mainly through aggregation of viral particles, and within that loop is involved in receptor recogni-
others act by blocking virus attachment to cells; tion. Subsequently, Barry Baxt, Peter Mason and
one neutralizing MAb caused, however, little collaborators identified the RGD-binding integrin
aggregation and had no effect on attachment. A αvβ3 as a receptor for FMDV (Berinstein et al., 1995;
different MAb could neutralize FMDV by trigger- Neff et al., 1998). Later, a series of studies by Terry
ing some conformational change in the virion that Jackson, Andrew King and collaborators identi-
facilitated RNA release (McCullough et al., 1987b). fied other RGD-binding integrins as additional
The observed binding of Fabs SD6 and 4C4 to the receptors of FMDV: αvβ6 ( Jackson et al., 2000b),
RGD and other studies on FMDV attachment to αvβ1 ( Jackson et al., 2002) and αvβ8 ( Jackson et al.,
cells and neutralization by these Fabs (Verdaguer 2004) (Chapter 5).
Unfortunately, no structure of any complex of free or bound to integrin αvβ3 (DiCara et al., 2007;
FMDV virion or VP1 GH loop-mimicking peptide Wagstaff et al., 2012). The results of these studies
with any integrin has been determined to date. are summarized next.
Current structural knowledge on FMDV-integrin The crystal structure of the ectodomain of integ-
recognition derives mainly from: (i) The crystal rin αvβ3 bound to a RGD peptide provided the first
structure of the ectodomain of integrin αvβ3 bound view on an integrin-RGD ligand (Xiong et al., 2002;
to a RGD peptide (Xiong et al., 2002); (ii) studies compare Fig. 4.8b). The bound RGD adopts an
on the interaction of VP1 GH loop-derived pep- open turn conformation. The peptide is bound at
tides or FMDV mutants with host cells expressing the ectodomain apex, inserted in a crevice between
different integrins, and/or with isolated integrins; two domains, one from the α subunit and the other
(iii) NMR studies on the conformation and inter- from the β subunit. The Arg sc is involved in salt
actions of VP1 GH loop-mimicking peptides either bridges, and the Asp sc is at the centre of a network
(A)
(B)
Figure 4.8 Structural aspects of the recognition between the FMDV virion and cellular receptors. (A) Structure
of a complex between a type O virion and its heparan sulfate receptor (Fry et al., 1999). The left image
shows a blue-colour spacefilling model of one pentamer in the virion bound to HS; the right image shows
a blue-colour ribbon diagram of one protomer in the virion bound to HS. The HS receptor is represented as
a ball-and-stick model and coloured green. (B) A docking model of a complex between a type C virion and
integrin αvβ3 (Verdaguer et al., 2004). The model was obtained by docking the integrin structure bound to the
RGD tripeptide (Xiong et al., 2002) onto the structure of the virion with the VP1 GH loop in the position observed
in a virion-neutralizing Fab complex (Hewat et al., 1997). The left image shows a front view of one pentamer
(blue-colour spacefilling model) docked to five integrin molecules (green-colour ribbon models); the right image
shows a side view of one protomer (blue) docked to an integrin molecule (green). The RGD peptide is shown as
a ball-and-stick model. (Reproduced from Sobrino, F. and Domingo, E. (eds). 2004. Foot-and-mouth disease:
current perspectives (Wymondham, UK: Horizon Bioscience)).
of polar interactions, including coordination with of the RGD have been identified as being important
a Mn2+ ion. The extended conformation of the for recognition between FMDV of different sero-
central Gly forms a bridge at the interface between types and integrin receptors. Serotype A mutant
the two integrin subunits. The RGD conformation FMDVs with enhanced ability to bind BHK cells
in the peptide–integrin complex is very similar to showed substitutions at residues RGD+1 and +7
that observed in the unliganded VP1 GH loop in within the helical stretch (Rieder et al., 1994). In
reduced type O FMDV (Logan et al., 1993), and in a systematic analysis to dissect the individual role
the A15 peptide–antibody complexes (Verdaguer of VP1 GH loop residues in infection of cultured
et al., 1995, 1998; Ochoa et al., 2000). Thus, it can BHK cells by a type C FMDV, each residue in
be presumed that, in a complex between the FMDV the VP1 GH loop-mimicking A15 peptide (see
virion and the αvβ3 receptor, the RGD in the VP1 above) was individually replaced by any of 16 other
GH loop would establish interactions with this amino acids. The inhibitory activity of each of the
integrin similar to those observed in the RGD pep- variant peptides on virus infection was tested. The
tide–αvβ3 complex. inhibitory action of A15 on FMDV infectivity had
A complex between the VP1 GH loop of FMDV been previously shown to be due to the efficient
presented on the hepatitis B core surface and integ- and specific inhibition of virus attachment to cells
rin was visualized by EM at low resolution (Sharma (Hernández et al., 1996), which validated this
et al., 1997) but yielded no structural details on approach to study the determinants of the interac-
the loop–integrin interaction. Docking models tion between the virion and the receptor on the cell
of a complex between FMDV and αvβ3 were later membrane. The results revealed a nearly absolute
obtained by combining the structural models of requirement for R, G and D of the RGD motif,
the FMDV-VP1 GH loop peptide–Fab complex and of the highly conserved Leu residues located
and of the integrin–RGD complex, relying on the at positions RGD + 1 and RGD + 4 in the helical
very similar RGD conformations common to those stretch contiguous to the RGD turn (and to a minor
models for a best superposition (Verdaguer et al., extent, the less conserved residue at RGD + 2)
2004). The final models (Fig. 4.8b) suggest that (Mateu et al., 1996).
integrin αvβ3 can only bind the virion through the More recently, inhibition by peptides of FMDV
VP1 GH loop when the latter is in a highly exposed binding to cells expressing different integrins and
orientation, similar to the ones that allow binding infection revealed that Leu residues at RGD + 1
to antibodies. This docking model is not precise and RGD + 4 are specifically relevant for recogni-
enough to predict which other capsid residues tion of type O FMDV by integrins αvβ6 and αvβ8
(apart from the RGD), either in the VP1 G-H loop (Burman et al., 2006). Analysis of the recognition of
or elsewhere, could establish interactions with the variant peptides by integrin αvβ6 identified the same
integrin. RGD+1 and RGD+4 residues as important deter-
Functional studies on the interaction between minants for a stable serotype O peptide–integrin
variant peptides or virus mutants with cells and interaction (DiCara et al., 2007, 2008). Differences
isolated integrins have revealed specific determi- in the sequence at the RGD + 1 and RGD + 4, and
nants of the interaction. The three residues in the other positions in the segments flanking the RGD
RGD triplet are strictly required, with very few motif mediate the specificity of different FMDV
exceptions (Chapter 5). Studies on virus or peptide isolates/serotypes for different integrin receptors
binding to purified integrins confirmed the critical ( Jackson et al., 1997, 2000a, 2002; Burman et al.,
role of the RGD motif for recognition by individual 2006; DiCara et al., 2007).
integrins ( Jackson et al., 1997; Sharma et al., 1997). Apart from the RGD motif itself, the confor-
In quite exceptional occasions, FMDV propagation mation adopted by the VP1 GH loop when bound
in cell culture led to variants that could still use to an integrin receptor is not known. However, the
integrins but had modified RGD motifs, such as strong propensity of this loop to adopt a strand-
RSGD (Rieder et al., 2005) or KGE (Berryman et turn-helix conformation, either in unliganded
al., 2013). form or bound to antibody (see above; Figs. 4.3a
In addition to the RGD triplet, other amino acids and b and 4.4) suggests that it may adopt a similar
at the helix located immediately to the C-terminus conformation when bound to an integrin. This
global fold is also preserved in different FMDV rigid helix preceded by a RGD motif exhibiting
serotypes and isolates despite large variations in slow internal motion.
loop sequence (including insertions/deletions), To recapitulate, the strong intrinsic propensity
and some local differences in conformation that of the VP1 GH loop of FMDV to adopt a strand-
are adequately compensated through different turn-helix conformation appears to be the result
intraloop interactions. Consistent with this view, of a selective pressure to preserve binding to αvβ6
introduction of different conformational restric- and other receptor integrins on the surface of host
tions on VP1 GH loop-mimicking peptides by cells. The conformation of this loop is, then, similar
different cyclizations led to qualitatively similar when bound as an ‘independent’ capsid element
effects on antibody and cell recognition (Mateu to integrins or antivirus antibodies, or when it is
et al., 1996). It seems reasonable to hypothesize immobilized in reduced type O FMDVs. Both the
that the strong propensity of this loop to adopt a RGD motif and residues at positions RGD+1 and
conserved strand–turn–helix conformation may RGD+4 in the exterior face of the helical element
have been biologically selected precisely to allow adjacent to the RGD turn in the folded VP1 GH
productive binding of FMDV, irrespective of sero- loop may directly interact with αv integrins includ-
type, to integrin receptors. ing αvβ6, the principal receptor of FMDV in host
It had been noted that in the VP1 GH animals (Monaghan et al., 2005; Burman et al.,
loop-derived peptides complexed to antivirus anti- 2006; Chapter 5), and critically determine affinity
bodies, the Leu residues at RGD + 1 and RGD + 4 and specificity by influencing structure and confor-
are located on the same face of the helical stretch mational dynamics. Other residues in the VP1 GH
adjacent to the RGD turn (Mateu et al., 1996; Fig. loop, especially within the helical segment, appear
4.4b) and make hydrophobic contacts with the anti- to have some role in binding integrins or modulat-
body paratope (Verdaguer et al., 1995, 1998). In a ing conformational propensities of the loop.
remarkable structure–function study by DiCara et
al. (2007), NMR spectroscopy was used to probe FMDV-HS receptor recognition
the conformation and interactions of VP1 GH Jackson et al. (1996) found that a serotype O
loop-mimicking peptides with integrin αvβ6. The FMDV strain that had been originally adapted to
results revealed that (i) in the presence of structure- grow in tissue culture could use HS as a receptor to
inducing solvents, the VP1 GH loop peptides do efficiently infect cultured cells. HS is a negatively
adopt turn-helix conformations similar to those charged, glycosaminoglycan component of pro-
determined for the unliganded loop in reduced type teoglycans found on the membranes of many cell
O virus (Fig. 4.3c) and in loop peptide-antibody types. It was subsequently found that propagation
complexes (Fig. 4.4b). (ii) the RGD forms the turn of FMDV strains of different serotypes in cultured
at the tip of the loop; downstream residues form a cells sometimes lead to the selection of variants with
helical stretch with residues RGD+1 and RGD+4 a high affinity for HS that show increased virulence
are presented as adjacent residues on the exterior and expanded tropism in cell culture, but that are
face of the loop; (iii) In the peptide-αvβ6 integrin attenuated in cattle (Sá-Carvalho et al., 1997; Bara-
complex, these two residues bind very closely to the nowski et al., 1998, 2000; Escarmís et al., 1998).
integrin surface, possibly through a hydrophobic The ability of the adapted viruses to bind HS was
interaction with the integrin; (iv) Other residues acquired through only one or two mutations on the
in the loop may influence the propensity to adopt capsid surface that generally involved an increase in
a helical conformation, and thus indirectly contrib- positive charge (e.g. His3056Arg in type O strains).
ute to the strength of the interaction between the The crystal structures of HS bound to two
VP1 GH loop and the integrin. In a further study, FMDV strains (of serotypes O and A) propagated
NMR structures and dynamics of a VP1 GH loop in cultured cells (Fry et al., 1999, 2005b) provided
peptide (both in linear and cyclic forms) and other direct information on the interaction between
RGD-containing, αvβ6-binding peptide in the pres- FMDV and this alternative receptor (Fig. 4.8a). In
ence of trifluoroethanol were analysed (Wagstaff et both cases, the HS binding site is located in a con-
al., 2012) (Fig. 4.9). The results suggested that αvβ6 cavity close to the junction between VP1, VP2 and
specificity requires the formation of a structurally VP3 at the centre of the biological protomer. The
(A)
(B)
(C)
(D)
Figure 4.9 NMR structures (closest to the mean) for peptides mimicking the VP1 GH loop of FMDV O1 BFS and
other RGD-containing, integrin αvβ6-binding peptides. (A, B) two cyclized forms of the VP1 GH loop peptide.
(C) linear VP1 GH loop peptide. (D) linear LAP2 peptide (derived from the latency-associated peptide from
transforming growth factor β1). For each peptide the assigned NOE contacts, hydrogen-bond restraints are
indicated at right (Reproduced from Wagstaff et al., 2012, with permission).
HS site overlaps with relatively variable spots on the making contacts with some nine amino acid resi-
capsid surface that form a part of the topologically dues belonging to any of the three capsid proteins.
mapped antigenic site D in type C viruses and in In both complexes, Arg3056 (a residue responsi-
which escape mutations that define antigenic sites ble for adaptation of type O viruses to bind HS) is
D3 in type C and 4 in type O viruses were identified involved in ionic interactions with sulfate groups,
(see previous section). while most contact residues are not conserved
In the complex, a carbohydrate motif made of in the two HS-bound virus isolates analysed.
up to five sulfated residues is bound to the capsid, Residues at the beginning of the VP1 C-terminal
segment also contact HS, and it has been sug- uncoating occurs in endosomes (Vázquez-Calvo et
gested that interactions of other residues within al., 2012).
this exposed segment and other spatially close The biochemical and structural studies on the
capsid residues may indirectly contribute to shape acid lability of the FMDV virion and capsid in vitro
the HS binding site. Binding of HS occurs with (see above), and the limited evidence obtained so
very little local or global structural reorganization far on FMDV uncoating in the cell (Vázquez-Calvo
of the virus capsid. No evidence for a physical or et al., 2012), support a simple model in which the
spatial connection between the integrin binding moderately acidic environment in endosomes
site (the mobile VP1 GH loop) and the HS bind- directly leads to the complete dissociation of the
ing site was apparent, thus providing structural viral capsid into pentameric subunits, and both
support for the observed independent function of VP4 and the viral RNA molecule are just released
the two sites in binding the respective receptors in the endosomal cavity. However, this proposed
on host cells. mechanism leaves as an open question how the
released RNA is protected from acid degradation
in the endosome, and how it is transferred to the
Structural insights into cytosol.
uncoating of the FMDV genome Studies on acid-dependent viral entry have
There is abundant evidence that, after FMDV binds revealed that acid-mediated uncoating of some
and integrin receptor in a susceptible cell, the virion viral genomes in endosomes may lead to disrup-
is internalized via clathrin-mediated endocytosis tion of the latter, allowing the direct release of the
and delivered to endosomes, where the acidic pH viral nucleic acid (and the endosomal content) in
mediates uncoating of the viral genome (reviewed the cytosol. Alternatively, some viral RNAs may
by Vázquez-Calvo et al., 2012; Chapter 5). Unfortu- be translocated into the cytosol through the for-
nately, the mechanism of RNA uncoating is one of mation of pores in endosomal membranes. Both
the less understood steps of the FMDV infectious mechanisms have been documented for HRV
cycle. (Vázquez-Calvo et al., 2012). For enterovirus like
Binding the receptor does not compromise the HRV and PV, it is hypothesized that pores in the
integrity of the virions (Baxt and Bachrach, 1980). endosomal membrane are formed after an acid-
Natural integrin receptors could be artificially induced conformational change of the virion in
bypassed by redesign of virus-cell recognition in the endosome, which leads to the externalization
the laboratory, providing alternatives for targeting of five myristoylated, hydrophobic VP4s and the
the endosomes (Mason et al., 1993, 1994; Rieder N-terminal segment of one VP1 molecule through
et al., 1996). These and other observations indi- a capsid opening (a pore or fracture). The exter-
cate that natural FMDV receptors allow specific nalized hydrophobic segments would be inserted
virus-cell recognition, and may also play a role in in the endosomal membrane, forming a channel
internalizing the viral particle and facilitating entry through which the viral RNA could be directly
into the endosome, but they are not involved in translocated from the viral capsid cavity into the
genome uncoating. cytosol (Vázquez-Calvo et al., 2012, and references
Shortly after entry into cells, the 140S virion dis- therein).
sociates into 12S pentameric subunits and releases The FMDV uncoating model based on the pH-
the viral RNA (Cavanagh et al., 1978; Baxt and induced dissociation of the capsid and the direct
Bachrach 1980, 1982; Baxt, 1987). Weak bases and release of the viral RNA into the endosomal lumen
ionophores that block acidification in endosomes excludes the channel-based translocation mecha-
inhibit infection by FMDV (Carrillo et al., 1984, nism proposed for enteroviruses: this mechanism
1985; Baxt, 1987). Internalized FMDV particles requires preservation of the integrity of the viral
colocalize with markers of endosomes (Berryman particle until the RNA has been transferred to the
et al., 2005; O’Donnell et al., 2005), and expression cytosol through the pore generated in the endo-
of a dominant negative mutant of the Rab5 quinase somal membrane. However, a few observations
impairs FMDV infection ( Johns et al., 2009). suggest that uncoating of the aphthovirus genome
All these observations indicate that viral RNA may be more complex. FMDV provirions obtained
by mutation, in which the VP0 to VP4+VP2 cleav- suggestions the viral structures have induced await
age did not occur, could still adsorb to cultured cells verification by performing appropriate biophysical,
and were as acid-sensitive as the non-mutated infec- biochemical or biological experiments. Conversely,
tious mature virions, but they were non-infectious structural interpretations of the results of some bio-
(Knipe et al., 1997). Thus, acidification may not be chemical or biological analyses have not yet been
enough for allowing a productive infection. verified by solving the appropriate viral structures.
Recent studies by David Rowlands and col- The organization of the viral RNA inside the capsid,
laborators on equine rhinitis A virus (ERAV), an the conformational dynamics of FMDV particles,
aphthovirus closely related to FMDV (Tuthill the mechanisms of FMDV assembly, integrin
et al., 2009; Groppelli et al., 2010) are opening a recognition, and viral genome uncoating are still
new perspective on aphthovirus uncoating. As for little understood. Our limited current knowledge
FMDV, productive infection by ERAV is dependent on the structural biology of FMDV compromises
on clathrin-mediated endocytosis and endosome the rational design of improved vaccines and the
acidification (Groppelli et al., 2010). Remarkably, development of antiviral approaches. Continued
when the ERAV virion was subjected in vitro to a structure–function work may be instrumental to
weakly acidic treatment under conditions that sta- effectively control and even eradicate FMD, an
bilize uncoating intermediates in PV, it first released old but re-emerging disease (Chapter 18) that still
the viral RNA, becoming a relatively unstable 80S inflicts severe economic losses to many countries,
empty particle before being disassembled into and poses a permanent threat to the world trade
free pentamers. The crystal structure of the ERAV economy.
empty particle is very similar to that of the virion,
but significant rearrangements and a higher dis- Acknowledgements
order were observed in several internal loops and The author acknowledges M.A. Fuertes for help
VP termini, that were proposed to destabilize the with figures. I wish to express my gratitude also
empty particle relative to the virion (Tuthill et al., to former and present members of my group, and
2009). The authors tentatively suggested that acidi- to our many collaborators in the study of relation-
fication of ERAV in the endosomes, and perhaps ships between structure, properties and function
of FMDV, could lead to the transient formation of of viruses. Present work in the author′s laboratory
an unstable viral particle. Such particle could act as is funded by grants from MINECO/FEDER EU
an uncoating intermediate to release the viral RNA (BIO2012-37649 and BIO2015-69928-R), and
through a channel-mediated mechanism similar to by an institutional grant from Fundación Ramón
that proposed for enteroviruses, before dissociating Areces. M.G.M. is an associate member of the Insti-
into pentamers (Tuthill et al., 2009). Further exper- tute for Biocomputation and Physics of Complex
iments are needed to test this enticing possibility. Systems, Zaragoza, Spain.
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W., Shen, X., Porta, C., et al. (2012). A sensor-adaptor and McCullough, K.C. (1987). Neutralization of
mechanism for enterovirus uncoating from structures of foot-and-mouth disease virus can be mediated through
EV71. Nat. Struct. Mol. Biol. 19, 424–429. any of at least three separate antigenic sites. J. Gen. Virol.
Wild, T.F., and Brown, F. (1967). Nature of the inactivating 68, 1637–1647.
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Wild, T.F., Burroughs, J.N., and Brown, F. (1969). Surface structure of the extracellular segment of integrin
structure of foot-and-mouth disease virus. J. Gen. Virol. alphaVbeta3 in complex with an Arg-Gly-Asp ligand.
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varying global distributions. The virus possesses two auto-proteinases (Lpro, 2Apro), and a third
a single-stranded positive-sense RNA genome proteinase, 3Cpro, which performs the majority
of approximately 8.5 kilobases (kb) in length. of the polyprotein cleavages (Kleina and Grub-
The coding sequence spans approximately 7000 man, 1992; Klump et al., 1984; Strebel and Beck,
nucleotides comprising a single open reading frame 1986; Vakharia et al., 1987). The VP0 precursor
(ORF), from which a polyprotein precursor is molecules are cleaved by an unknown mechanism
translated and subsequently cleaved into a series of to produce VP2 and VP4, transforming the parti-
intermediate and mature viral proteins. Translation cles into mature (infectious) virions. In cells, the
is initiated from one of two potential AUG start presence of viral receptors is the first level of sus-
codons upstream of the leader proteinase (Lpro), ceptibility to productive infection, while NS viral
thus producing one of two possible isoforms: Lab proteins create conditions within the cell, which
or Lb. Classically, the coding sequence downstream favour replication of the viral RNA and impose a
of Lpro has been sub-divided into three regions second level of susceptibility.
P1, P2, and P3. P1 encodes the four structural FMD had been effectively controlled in
proteins: 1A (VP4), 1B (VP2), 1C (VP3), and developed countries for many years through the
1D (VP1). The virus NS proteins are encoded by implementation of rigorous import controls and
P2 (2Apro, 2B, and 2C) and P3 (3A, 3B1, 3B2, 3B3, vaccination. However, within the past several
3C protease or 3Cpro, and the 3D RNA-dependent years, outbreaks of FMD have occurred in coun-
RNA polymerase or 3Dpol). The single ORF is tries such the United Kingdom, Taiwan, Japan,
flanked on both sides by highly structured 5′ and 3′ and South Korea, with unusual host restrictions.
untranslated regions (UTRs). The representative These events have emphasized both the need for
stable secondary structures found in the UTRs control of the disease and the inadequacies of the
have been implicated in viral protein synthesis and current vaccine (see Grubman and Baxt, 2003,
RNA replication, though the functions of some and references therein). Traditional inactivated
structures remain unknown. The 3′ UTR possesses whole virus vaccines generally induce protection
a poly-adenylated (poly-A) tail, encoded within by seven days post-inoculation (Doel et al., 1994;
the genome. One of three copies of a small peptide Graves et al., 1968; Sellers and Herniman, 1974),
(VPg or 3B), of viral origin, is covalently attached which is adequate for countries in which the dis-
to the 5′-terminal nucleotide. Additionally, two ease is endemic. However, in emergency outbreak
closely adjacent stem loop structures have also been situations, it is important to either block or reduce
predicted in the 3′ UTR in between the termination virus shedding as rapidly as possible to contain the
of the P3 coding region and the poly-A tail. The 5′ outbreak. Future containment strategies will need
UTR is approximately 1200 nucleotides in length to employ antiviral therapy in conjunction with
and contains a crucial cis-acting replication element vaccination for effective control of an outbreak.
(cre) required for genome replication, and a region In order to develop effective antiviral agents, it is
to direct internal ribosomal binding (internal imperative to identify specific viral or host targets
ribosomal entry site or IRES). Additional RNA ele- and to understand how they may be exploited.
ments of unknown function in the 5′ UTR include This article addresses only those elements where
an approximately 360 nucleotide segment that folds available data indicates their involvement in either
into a long stem–loop (called the S-fragment), a virulence or host range within susceptible ani-
segment of greater than 100 nucleotides containing mals. Readers will find clear descriptions of recent
mostly cytosine residues (poly-C tract), and three advances made in the understanding of FMDV–
or four RNA pseudoknots (see Mason et al., 2003, host interactions, focusing on the cognate receptor
and references therein). molecules for FMDV and other non-receptor mol-
As stated above, the mono-cistronic FMDV ecules implicated in the attachment, uptake, and
genome encodes a single polyprotein, which propagation of the virus. Other sections in this
undergoes a series of proteolytic cleavages book examine elements involved in viral replica-
yielding a total of 14 structural and NS proteins tion and the pathogenesis of the disease, so we will
(Grubman et al., 1984; Robertson et al., 1985). discuss these only as they pertain to the issues we
Cleavage occurs as a result of the activity of are considering.
a
Confirmed expression.
b
Inferred expression (Kraft et al., 1999).
c
Molecule heavily expressed.
a
Ruoslahti (1996).
b
Kraft et al. (1999).
c
Isolated immobilized α5β1 can bind virus (Jackson et al., 2000a), but does not mediate infection when expressed on
cells in tissue culture (Jackson et al., 2000b; Neff et al., 1998).
the cell lines used for these studies, was unable to all integrin β subunits (Calvete et al., 1991; Green
function as an FMDV receptor in cell culture even et al., 1998; Horton, 1997; Hynes, 1992). The
though the virus was shown to bind to the isolated cysteine-rich region has been implicated in inte-
immobilized integrin ( Jackson et al., 2000a). grin activation (Faull et al., 1996; Kashiwagi et al.,
When a similar experiment was performed with 1999; Yan et al., 2000; Yan and Smith, 2000, 2001),
type O1BFS, however, virus was able to replicate and sequence differences between the human and
in cells which were not transfected with the αvβ3 bovine β3 subunit in this region, which include
integrin, and neither antibodies to α5β1 nor RGD one less cysteine in the bovine subunit (Neff et al.,
peptides were able to inhibit viral replication (Neff 2000), may explain the increased activity of the
et al., 1998, 2000), suggesting that this virus was not bovine receptor.
utilizing an integrin receptor. Interestingly, when While these studies identified a receptor for the
plasmids encoding either human or bovine homo- virus, they left many unanswered questions. In sus-
logues of αvβ3 were transfected into αvβ3-negative ceptible species, FMDV infects primarily epithelial
cells, the bovine homologue was more efficient in cells. Initial sites of viral replication are the lung and
mediating infection than its human counterpart. In pharyngeal areas followed by rapid dissemination to
addition, this phenomenon appeared to be a func- oral and pedal epithelial sites (Alexandersen et al.,
tion of the bovine β3 subunit (Neff et al., 2000). To 2001, 2003; Brown et al., 1992; Brown et al., 1995,
gain insight into increased activity of this subunit, 1996; Burrows et al., 1981; Sutmoller and McVicar,
chimeric bovine/human β3 subunits were gener- 1976; Zhang and Kitching, 2001). In humans, αvβ3
ated and analysed for receptor activity. Surprisingly, is generally expressed on vascular endothelium and
the increased efficiency of the bovine β3 subunit smooth muscle cells (Brooks et al., 1994; Felding-
mapped, not to the subunit ligand-binding domain Habermann, 1993; Liaw et al., 1995). Studies of
(LBD), but to the C-terminal region of the ecto- αvβ3 expression in susceptible animals (cattle
domain (Neff et al., 2000). This region has a high and pigs) revealed it is expressed in epithelium of
number of cysteine residues, which is common to a number of organs, mainly of the small intestine,
kidney, bile duct (Merono et al., 2002; Singh et al., acted as a selective pressure leading to the emer-
2001), and also in endometrium (Kimmins and gence of soluble integrin resistant (SIR) FMDV
MacLaren, 1999). Thus, it was presumed that other mutants that subverted the anti-adsorptive effects
integrin receptors might be involved in disease of the soluble αvβ6 treatment (Lawrence et al.,
pathogenesis (Neff et al., 1998). Of all of the other 2013). Cumulatively, these findings strongly imply
RGD-dependent integrins, αvβ5 has a tissue dis- that, as a receptor molecule, αvβ6 plays a greater
tribution in humans, which is the most consistent role in FMDV pathogenesis than αvβ3.
with tissues affected by FMDV in susceptible spe-
cies (Goldman and Wilson, 1995; Kim et al., 1994). αvβ1
Despite this, studies utilizing antibody inhibition Shortly after the identification of αvβ6 as another
or transient expression of recombinant αvβ5 have integrin heterodimer that would support FMDV
demonstrated that it is not a functional receptor for attachment to host cells, αvβ1 was also reported to
FMDV (Berinstein et al., 1995; Duque and Baxt, function as an FMDV receptor molecule ( Jackson
2003; Jackson et al., 2000b). et al., 2002). Subsequent infectivity assays utilizing
transient expression of the cloned bovine homo-
αvβ6 logues of αvβ6 and αvβ1 confirmed these results
The hypothesis, that there must be more than one (Duque and Baxt, 2003), but did not examine the
integrin receptor for FMDV, was proven correct efficiency of these receptors compared with the
when it was shown by Jackson and co-workers human homologues. As described above, different
that the epithelial integrin αvβ6 was utilized as an efficiencies of receptor usage between serotypes
FMDV receptor ( Jackson et al., 2000b), followed in A and O FMDV was observed. With respect to
rapid succession by studies from the same labora- αvβ1, it appears that serotype O viruses utilize
tory implicating both αvβ1 ( Jackson et al., 2002) αvβ1 with relatively higher efficiency than serotype
and αvβ8 ( Jackson et al., 2004) as receptors for A viruses (Duque and Baxt, 2003). Exchanges of
the virus (Table 5.2). Interestingly, a study using LBDs between the different β subunits suggested
cell lines transiently expressing different integrin that the subunit LBD appeared to play a role in the
heterodimers indicated that different FMDV sero- efficiency of receptor usage, but exchanging the
types may exhibit preferential binding to different G-H loop of the type A virus with one from type
integrin receptors (Duque and Baxt, 2003). In that O (Rieder et al., 1994a) did not change the recep-
study, serotype A FMDVs showed efficient bind- tor utilization of the type A virus, suggesting that
ing to αvβ3 and αvβ6, but only moderately so with sequences outside of the loop were responsible for
αvβ1. In contrast, serotype O FMDVs exhibited these results (Duque and Baxt, 2003).
greater efficiency binding αvβ1 and αvβ6 than αvβ3.
Subsequent studies examining the role of αvβ6 αvβ8
in the pathogenesis of FMDV have suggested that Later, Jackson and co-workers reported that integrin
αvβ6 may be the more relevant integrin heterodi- heterodimer αvβ8 was a fourth receptor molecule
mer for the propagation of virus in the animal host. for FMDV ( Jackson et al., 2004). Interestingly, the
Investigations of the level of integrin expression swine based IBRS2 cell line is enriched in αvβ8 and
in tissue at the sites of FMDV replication in both not in the other three FMDV integrin receptors
pigs and cattle have repeatedly shown higher levels (Burman et al., 2006; Johns et al., 2009). Thus, this
of αvβ6 relative to αvβ3 (Monaghan et al., 2005). adds another cell culture system with which to char-
Indeed, the production and examination of soluble acterize FMDV variants that have greater preference
secreted bovine αvβ3 and αvβ6 revealed that both for this integrin heterodimer over others (Table
serotype A and O FMDVs bind to αvβ6 with greater 5.1). The RGD+1 site has been described as an
affinity than αvβ3 (Duque et al., 2004). Moreover, important determinant of FMDV receptor affinity,
pre-incubation of virus with soluble αvβ6, but not and is frequently occupied by a leucine (L) residue,
αvβ3, significantly impeded the progression of though occasionally arginine (R) and methionine
infection by blocking virus adsorption in cell cul- (M) have been found to occupy this site in isolated
ture. In addition, serial passaging of FMDV in cell viruses. Viruses displaying an RGDR sequence in
culture in the continuous presence of soluble αvβ6 the VP1 G-H loop were observed to have reduced
affinity for αvβ8 as a receptor molecule in a study spleen express αvβ3 (Singh et al., 2001) leading to
employing IBRS2 cells for virus characterization speculation that such cells might be a mechanism of
(Burman et al., 2006). As was observed between disseminating virus from sites of initial replication
FMDV serotypes A and O with αvβ1, αvβ3, and to secondary replication sites through lymphoid
αvβ6, a serotype-specific differential integrin recep- organs, although it is doubtful that virus can pro-
tor preference for αvβ6 was also detected in the SIR ductively replicate in lymphoid cells (Alexandersen
mutant investigation (Lawrence et al., 2013). et al., 2003; Baxt and Mason, 1995; Rigden et al.,
It is not clear why FMDV should need four inte- 2002).
grin receptors to cause disease, or if it utilizes all, As with other viruses, FMDV is subject to a
or some, of the integrins in the susceptible hosts. variety of environmental stresses that function
Infection with different FMDV strains may cause as selective pressures forcing the virus to adapt.
different degrees of disease severity, but there are The most perilous adaptation for a virus is a shift
generally no differences in clinical symptoms within in receptor tropism. Any mutation rendering the
a species. There are, however, differences in the virus incapable of binding to its traditional recep-
clinical course of disease among different suscepti- tor without a corresponding mutation allowing for
ble species. Additionally, FMDV can utilize integrin attachment to another site on the host cell surface
receptors from humans, hamsters, and monkeys, will condemn the virus to a non-infectious state
species not known to be susceptible to the disease. left to drift into non-existence in the extracellular
While the virus can utilize the bovine αvβ3 integrin milieu. Many strategies have been developed in the
more efficiently than the human homologue (Neff laboratory setting to drive virus-receptor plasticity,
et al., 2000), no comparisons of homologues of the and viruses have evolved natural back up strategies
other known integrins from different species have when the primary cell receptor is limiting or una-
been conducted. Table 5.2 includes the known vailable. In the next section, artificial and natural
tissue distribution of the RGD-binding integrins in FMDV alternative receptors will be described with
humans. Each of the integrins identified as recep- particular focus on the FMDV secondary receptor
tors for FMDV are expressed in tissues that are not and the elusive third FMDV receptor.
involved in disease pathogenesis. Also, as described
above, different serotypes of FMDV exhibit altered Artificial FMDV receptors
efficiencies of integrin utilization, and all serotypes Using modern cloning technologies and online
tested utilized the αvβ6 integrin with relative high repositories of genomic sequence information,
efficiency (Duque and Baxt, 2003). The possibility affinities for specific receptor molecules have been
exists that the virus uses different receptors during rationally designed and engineered into labora-
various stages of the disease. That is why definitive tory viral constructs. For example, an alternative
information on integrin expression in the suscepti- FMDV receptor has been engineered by joining
ble species would be helpful in correlating integrin the antigen-binding domain of an FMDV-specific
utilization in tissue culture with sites of viral rep- antibody to a cell-surface receptor specific for
lication in vivo. We were able to generate cDNAs another virus. This was accomplished by cloning
encoding the bovine αv, β1, β3 and β5, subunits the antigen-binding portion of an anti-FMDV
from primary bovine tongue keratinocytes (Duque type A12 monoclonal antibody (mAb) in the form
and Baxt, 2003). In contrast, β6 sequences could of a single chain antibody (scAb) (Mason et al.,
not be amplified from a number of different FMDV 1996), and fusing it to ICAM-1, the receptor for
target organs, but was amplified from bovine thy- the major group of human rhinoviruses (Rieder et
roid cells (Duque and Baxt, 2003), which are highly al., 1996). When Chinese hamster ovary (CHO)
sensitive to FMDV infection (House and House, cells were transfected with this engineered recep-
1989; Snowdon, 1966). However, the bovine thy- tor molecule, they supported productive infection
roid gland does not appear to be a target organ for of type A12 virus, while non-transfected CHO
infection as evidenced by the absence of FMDV cells were resistant to infection. This receptor was
antigen in thyroids from infected animals (D. only functional for viruses carrying the epitope
Gregg and B. Baxt, unpublished). It is interesting to recognized by the scAb, and the RGD sequence
note that bovine mononuclear cells in the lung and was unnecessary for infection with these viruses
(Rieder et al., 1996). Using cells expressing this resembled viruses isolated from infected animals in
receptor, large amounts of type A12 virus, lacking the field (Sa-Carvalho et al., 1997).
the RGD sequence, were produced, which were The cDNAs encoding the capsids of these
shown to be non-infectious in cells normally two variants were inserted into a type A12 infec-
susceptible to infection with RGD-containing tious cDNA clone and used to produce viruses
viruses (Rieder et al., 1996), and unable to infect exhibiting the phenotypes of the original variants
cattle (McKenna et al., 1995). (Sa-Carvalho et al., 1997). Receptor utilization of
these viruses, and type O1BFS, was examined in
Alternative FMDV receptor: heparan wild-type (K1) and GAG-deficient (677) CHO
sulfate cells, and was compared with type A12. Cells were
In 1996, it was demonstrated that serotype O1 transfected with cDNAs encoding human αvβ3
FMDV could utilize heparan sulfate (HS), a ubiq- integrin subunits, resulting in cell lines expressing
uitous cell-surface glycosaminoglycan (GAG) specific integrin and GAG receptors (Neff et al.,
consisting of a sulfated polysaccharide covalently 1998). Viral replication in this panel of CHO cells
linked to a protein core, as an alternative receptor showed that tissue culture adapted type O1BFS
molecule ( Jackson et al., 1996). While the involve- and the 3056R viruses only replicated in cells
ment of co-receptors in FMDV infection had not expressing HS, independent of αvβ3 expression.
been ruled out, the data indicating that the integrin A 3056R virus with an RGD → KGE mutation
receptor or alternative receptors like HS were suf- also only replicated in the HS-expressing cells,
ficient for binding the virion to cells, argued against indicating that this virus was not utilizing another
the existence of co-receptors in cultured cells. The RGD-dependent integrin. In contrast, the A12 and
results of Jackson et al. (1996) correlated well with O1 3056H viruses only replicated in cells express-
earlier studies suggesting that some FMDVs utilized ing the transfected integrin cDNAs, and did not
a second receptor, present in high copy number on need HS for replication. Furthermore, a type O1
cultured cells (Baxt and Bachrach, 1980; Sekiguchi 3056H virus containing an RGD → KGE mutation
et al., 1982), a property descriptive of an HS recep- was unable to replicate in cells expressing either
tor. In addition, these results helped to explain HS or the integrin, identical to results obtained
earlier findings that type O1BFS replicated in cells with RGD-mutated type A12 (Mason et al., 1994a).
which did not express the αvβ3 integrin (Neff et al., Therefore, while the ‘field-type’ virus utilizes an
1998). integrin as a receptor, tissue culture adaptation of
type O1 virus results in the utilization of HS as an
Critical amino acid substitutions alternative secondary receptor. The tissue culture
associated with HS receptor usage adapted phenotype results from genetic changes at
To further examine the role of HS in viral infec- amino acid residue 56 in VP3, distant from the VP1
tion, genetically defined derivatives of a serotype RGD motif, and may also be dependent on residue
O1 Campos (Sa-Carvalho et al., 1997) which are 134 in VP2 (Sa-Carvalho et al., 1997). The impor-
similar to the viruses (O1Kaufbueren and O1BFS) tance of these residues in binding HS was directly
utilized by Jackson et al. (1996) were used. This supported by X-ray crystallographic structural data
study demonstrated that passage of FMDV in cell of types O1BFS and A10 virus crystals infused with
culture resulted in selection of viruses capable of HS (Fry et al., 1999; Jackson et al., 2003). This
growing in CHO cells. This adaptation appeared showed amino acid residue 3056R of both viruses
to be mediated by selection of an arginine (R) made contact with two sulfate groups within HS. In
residue at position 56 of VP3 (here referred to as addition, a conserved R residue at position 135 of
3056R) (Sa-Carvalho et al., 1997). The added VP2 (2135R) also made contact with one of the HS
positive charge on the virion surface would medi- disaccharides. This latter residue is next to K134 in
ate electrostatic binding to negatively charged HS. VP2, which we showed was necessary for the tissue
Additionally, heparin inhibited plaque formation culture adapted phenotype (Sa-Carvalho et al.,
by this virus (3056R), but had no effect on plaque 1997). Interestingly, two residues at the C-terminus
formation by a second O1Campos variant with a of VP1 (residues 1193K and 1195H) of both type
histidine (H) at this position (3056H) and more O1BFS and A10 also make contact with HS (Fry et
al., 1999; Jackson et al., 2003). It had been previ- strains of FMDV serotype O viruses (O/Fujian/
ously reported that removal of C-terminal residues CHA/9/99wt and O/Tibet/CHA/6/99wt)
201–211 of VP1 of type O1, or antibodies directed involved the acquisition of several amino acid sub-
against this region, inhibited virus attachment to stitutions in capsid proteins VP4, VP2, and VP1.
cells in culture (Fox et al., 1989). It is still unclear if However, the positively charged VP1 (E103K in O/
this region mediates binding to the integrin or HS Fujian/CHA/9/99tc) and neutral VP2 (L2080N
receptors, although the virus–HS complex showed in O/Tibet/CHA/6/99tc) amino acid substitu-
no interactions between HS and this region of VP1 tions were determined to be critical for adopting
(Fry et al., 1999). HS as receptor (Bai et al., 2014). The acquisition
Studies described above with type O1Cam- of positively charged residues has been also found
pos variants showed the importance of the associated with cell culture passaged type C viruses
virus–integrin interaction in vivo. Inoculation of (Baranowski et al., 2000, 2001a,b; Martinez et al.,
cattle with either the HS-binding 3056R variant 1997).
or the integrin-binding 3056H variant revealed FMDV serotypes SAT1, SAT2, and SAT3 are
that the 3056H virus was highly virulent in cattle, notoriously difficult to adapt to cell culture (Pres-
while the 3056R virus was at least 100,000-fold ton et al., 1982). Studies with cell culture adapted
less virulent in cattle (Sa-Carvalho et al., 1997). SATs identified positively charged amino acid
Moreover, two cattle inoculated with large amounts substitutions in the F-G (SAT1) and D-E (SAT2)
of HS-binding virus eventually showed signs of loops of VP1, positions proximal to the five-fold
FMD. Virus isolated from these animals could no axis of symmetry on the virus particle (Maree
longer replicate in CHO cells, interact with HS in et al., 2011; Maree et al., 2010). A recent report
vitro, and had amino acid substitutions at either describing the cell culture adaptation of a vaccine
residue 56 of VP3 (R → C), or at residue 134 of VP2 variant of FMDV serotype A (A/IRN/87) identi-
(K → E), which removed positive charges from the fied positively charged amino acid substitutions
virion surface (Sa-Carvalho et al., 1997). Finally, in a novel location in the VP2 that was different
in the context of the full-length infectious clone from the previously determined HS-binding sites
of O1 Campos (Bra/58 strain), the substitution of in the depression between VP1, VP2, and VP3 as
H56R in VP3 was reinforced as the critical amino well as the five-fold symmetry axis (Chamberlain
acid leading to cell culture adaptation and virus et al., 2015). Interestingly, two isolated variant
attenuation, in the absence of the K134E VP2 viruses, termed A+ and A-, have been described
substitution described above (Borca et al., 2012). where the latter virus possessed a large deletion
In this study, viruses identical genetically except for within the G-H loop of VP1 that encompassed the
H or R at position 56 in VP3 grew similarly in cell highly conserved RGD motif for integrin binding
culture, but showed significantly different degrees and yet remained infectious in cell culture (Fowler
of pathogenicity in cattle and pigs. The VP3-H56 et al., 2011) and in cattle (Fowler et al., 2014).
virus exhibited the characteristic clinical course Furthermore, this unique set of mutations in VP2
of FMD in both host species, while the VP3-R56 allowed for growth of the A- virus on both CHO K1
counterpart only showed disease symptoms if the (HS-positive) and CHO 677 cells (HS-negative)
R reverted to an H or C. Additionally, in a recently indicating that this virus can utilize a pathway to
described soluble integrin study, the H56R muta- infection that is both integrin and HS independent
tion in VP3 was consistently acquired in every (Chamberlain et al., 2015). Moreover, type C1 vari-
serotype O SIR mutant that adopted HS binding in ants have also been isolated which appear to bind
lieu of integrin receptors in the continued presence to host cells and replicate in both an integrin and
of soluble bovine αvβ6 (Lawrence et al., 2013). HS-independent manner, suggesting the possibil-
Interestingly, variants of serotype A SIR mutants ity of a third natural receptor system for FMDV
that developed an affinity for HS binding exhibited (Baranowski et al., 2000; Baranowski et al., 1998).
amino acid substitutions exclusively in VP1, with Such non-integrin non-HS targeted type C viruses
a single L150R in the RGD + 4 position for A-SIR were obtained after continuous serial passaging in
#45 (Lawrence et al., 2013). In another instance, cell culture for 100 passages (FMDV C-S8c1p100)
the cell culture adaptation of PanAsia-1 field or 213 passages (FMDV C-S8c1p213).
if they were introduced to the animal via aerosol. the exact role of the A-particle in enterovirus entry
Importantly, tissue cross-sections were examined by and uncoating (Dove and Racaniello, 1997), the
immunofluorescent microscopy for co-localization initial conformational change which generates this
of FMDV antigen and JMJD6. In tissue from particle is mediated by the interaction of the virion
animals infected with the wild-type virus, VP1 with the cellular receptor, which binds into a pit or
could occasionally be observed in JMJD6-positive canyon on the surface of the virion (Belnap et al.,
cells. However, in strong contrast, in tissue from 2000b; He et al., 2000; Hogle et al., 1985; Kolatkar
JMJD6-FMDV-infected animals, VP1 was detected et al., 1999; Luo et al., 1987; Muckelbauer et al.,
exclusively in JMJD6-positive cells. Based on the 1995; Rossmann et al., 1985; Xiao et al., 2001),
cumulative findings, it was proposed that JMJD6 as determined from studies using soluble-secreted
could function as an alternative FMDV receptor enterovirus receptors (Gomez Yafal et al., 1993;
both in vitro and in vivo (Lawrence et al., 2016a,b). Kaplan et al., 1990; Tsang et al., 2001).
Attachment is only the first stage of the In contrast to enteroviruses, A-particles have not
virus–host cell interaction. Without subsequent been detected in cells following FMDV infection.
internalization, the FMDV particle would be left After adsorption to the cell surface, the 140S virion
tethered to the cell surface in a precarious state, breaks down into 12S pentameric subunits releas-
unable to commandeer the host cell translation ing the RNA (Baxt, 1987; Baxt and Bachrach, 1980,
machinery within the cytoplasm to begin the pro- 1982; Cavanagh et al., 1978). This breakdown does
duction of viral proteins necessary for assembly not occur on the cell surface, since particles which
of more virus capsids and replication of the RNA are eluted from the cell after adsorption are fully
genome. In the next section, we discuss what has infectious and still sediment at 140S (Baxt and
been delineated regarding the receptor-mediated Bachrach, 1980). The intracellular uncoating of
endocytosis of FMDV. FMDV occurs within an acidic endocytic vesicle
(Baxt, 1987; Berryman et al., 2005; Carrillo et
al., 1984, 1985; O’Donnell et al., 2005). FMDV
Mechanisms of FMDV host cell provirions were generated by a site-directed muta-
entry tion in VP0, resulting in an inability to perform
Studies of the entry and uncoating of FMDV into the maturation cleavage of VP0 to VP2 and VP4.
infected cells have suggested that the processes The provirions were analysed, and found to be
have some similarities and differences with other non-infectious, could adsorb to cells in culture,
Picornaviruses. In context of enteroviruses, the and were acid-sensitive (Knipe et al., 1997). Thus,
first step in the uncoating occurs prior to entry of breakdown of 140S virus to pentameric subunits,
the virion through the membrane. The interaction by itself, does not lead to productive infection, sug-
with the receptor causes a conformational rear- gesting there are additional steps subsequent to the
rangement of the virion resulting in the release virion breakdown.
of VP4 and the externalization of the N-terminal Internalization of vitronectin by αvβ3 has
extension of VP1 to form an altered (A) particle been reported to require the ligation of a second
(Carson, 2014; Everaert et al., 1989; Fricks and integrin, α5β1 via the β3 cytoplasmic domain
Hogle, 1990; Lonberg-Holm and Whiteley, 1976; (Blystone et al., 1995; Pijuan-Thompson and
Organtini et al., 2014). A-particles express new Gladson, 1997). However, deletions to either the
antigenic determinants and have a lower sedimen- αv or the β3 cytoplasmic domains were found to
tation rate (135S) in sucrose gradients than the not affect the ability of αvβ3 to function as a viral
native virus (160S) (De Sena and Mandel, 1977). receptor (Neff and Baxt, 2001). This contrasted
These particles are hydrophobic resulting from the with the results of Miller and colleagues (Miller
presence of bound saturated fatty acid ‘pocket fac- et al., 2001), who showed that deletion of the
tors’ and have an affinity for liposomes (Fricks and β6 cytoplasmic domain abolished the ability of
Hogle, 1990), an interaction which results in the the αvβ6 integrin to mediate FMDV infection,
conversion of the A-particle to an 80S particle and while still retaining the ability to bind the virus.
the release of the RNA into the cell (Belnap et al., However, this study did not directly examine the
2000a). While there is still controversy regarding internalization of the virus by these cytoplasmic
domain-deleted receptors. It is possible that cyto- the redistribution of JMJD6 in the presence of
plasmic domain-deleted αvβ3 internalizes FMDV hypertonic sucrose and chlorpromazine, but
by either interacting with another cell surface not nystatin as observed by immunofluorescent
molecule, or by an integrin-independent mecha- microscopy (Lawrence et al., 2016a). That both the
nism, similar to that which has been described for integrin- and JMJD6-involved pathways were sensi-
the internalization of cyclic RGD peptides by this tive to CCP inhibitors is consistent with findings
integrin (Castel et al., 2001). suggesting that αvβ3 (and potentially αvβ6) and
Internalization of parechoviruses or adeno- JMJD6 occupy a similar membrane microdomain
viruses, mediated by αvβ3 or αvβ5, was shown to seen in studies with C. elegans implicating JMJD6 as
be clathrin-mediated and required dynamin ( Joki- a PSR involved in recognition of PS on the surface
Korpela et al., 2001; Meier et al., 2002; Wang et of apoptotic cells (Hisatomi et al., 2003; Hsu and
al., 1998). Similarly, subsequent studies have dem- Wu, 2010; Wang et al., 2003).
onstrated that integrin-mediated entry of FMDV Therefore, it can be inferred that the uptake
occurs by the same mechanism. Immunofluorescent pathway employed by FMDV is pre-determined by
microscopy experiments elegantly demonstrated the receptor molecule utilized by the virus for the
that FMDV particles bound to host cells via the initial attachment step. Collectively, the informa-
integrin primary receptor underwent receptor- tion reviewed to this point supports a model where
mediated endocytosis through clathrin-coated FMDV is internalized in complex with its cognate
pits (CCPs) (Berryman et al., 2005; O’Donnell et receptor, it is delivered to an acidic vesicular com-
al., 2005). Consistent with these observations, the partment, where capsid de-stabilization leads to the
internalization of FMDV bound integrin molecules delivery of the FMDV RNA genome to the host cell
was sensitive to the effects of chemical inhibitors of cytoplasm where the bulk of its replication cycle
the CCP uptake pathway including chlorpromazine transpires. These critical post-attachment and entry
and hypertonic sucrose. steps are driven by a series of non-receptor protein
In contrast, cell culture adapted FMDV particles interactions involving FMDV proteins and RNA
(O1 Campos with a 3056R substitution), which elements that are described further in the last sec-
utilize the HS secondary receptor, were visualized tion of this chapter.
by immunofluorescent microscopy to enter host
cells through the caveolae-mediated uptake path-
way (O’Donnell et al., 2008). Cell culture adapted FMDV interactions with
FMDVs were endocytosed via caveolae as evidenced non-receptor host factors: roles
from the co-localization of VP1 with the signature in infection, host range, and
marker of caveolae: caveolin-1. The caveolae con- virulence
taining FMDV eventually merged with endosomes Beyond the initial engagement between the
consistent with the observed co-localization with FMDV capsid with the host cell surface receptor
endosomal markers. Additionally, chemical uptake molecule(s), a variety of non-receptor host factors
inhibitors of caveolae-dependent internalization play important roles in the progression of infec-
(such as nystatin) effectively blocked the progres- tion, host range, and virulence. The limited coding
sion of FMDV infection through the HS receptor. capacity of small RNA viruses necessitates that
Similarly, knockdown in caveolin-1 expression the virus acquire and re-purpose host factors in its
through RNA interference also impeded infection sub-cellular replication environment to efficiently
by cell culture adapted FMDVs. complete a replication cycle. Significant progress
Finally, in the context of the JMJD6-involved has been made in the understanding of the molecu-
attachment and entry pathway, the JMJD6-FMDV lar and cellular biology of FMDV replication,
variant appeared to be internalized through the where multiple host proteins have been identified
CCP-dependent pathway. Productive infection as as important contributors to the progression of
measured by non-structural protein translation FMDV infection (summarized in Table 5.3). The
(3Dpol) was blocked when hypertonic sucrose molecular communication between the virus com-
or chlorpromazine were present, but not nys- ponents and the host cell machinery can be broadly
tatin. Similarly, JMJD6-FMDV failed to affect divided into two categories: viral protein-host
protein interactions and viral RNA-host protein and Grubman, 1992; Piccone et al., 1995a; Rob-
interactions. erts and Belsham, 1995; Skern et al., 1998), and
is auto-catalytically cleaved from the polyprotein
Viral proteins at its C-terminus (Guarne et al., 1998; Strebel
and Beck, 1986). It is the N-terminal component
Viral proteases: Lpro and 3Cpro of the polyprotein, and the region of the genome
Infection of cultured cells with FMDV induces a encoding this protein contains two in-frame AUG
rapid shut-off of host cell macromolecular synthe- initiation codons that result in the generation of
sis. Both the 3Cpro and Lpro proteins are involved in two L proteins, Lab and Lb (Robertson et al., 1985;
these events. Lpro is a papain-like protease (Kleina Sangar et al., 1987). Both forms of the protein are
Sam68 RBP IRES, 3Cpro, 3Dpol RNA replication Lawrence et al. (2012), Rai et al.
(2015)
JMJD6 RBP VP1, RHA Site of attachment, Lawrence et al. (2014, 2016a,b)
RHA demethylation
PABP1 RBP Poly-A tail – Rodriguez-Pulido et al. (2007)
PCBP2 RBP IRES Affects translation Pacheco et al. (2008)
Gemin5 RBP IRES Inhibits translation Fernandez-Chamorro et al. (2014)
PTB RBP IRES Promotes Niepmann et al. (1997)
translation
Beclin1 Autophagy 2C Promotes Gladue et al. (2012)
translation
Vimentin Autophagy 2C Membrane Gladue et al. (2013)
rearrangement
DCTN3 Dynactin 3A Intracellular Gladue et al. (2014)
component movement
N-Myristoyl Lipid modifier VP4 Capsid assembly Belsham et al. (1998)
transferase
LYPLA/APT1 Lipid modifier – – Guo et al. (2015)
eIF-4G Translation factor Lpro Block cellular Devaney et al., 1988
translation Kirchweger et al., 1994
NF-KB, IRF3, Transcription Lpro Modulation of De los Santos et al. (2007), Wang
IRF7, RANTES factors innate immunity et al. (2011a,b)
PKR, OAS IFN-induced Lpro Antiviral activity Chinsangaram et al. (1999)
genes
Histone H3 DNA packaging 3Cpro Block overall Grigera and Tisminetzky (1984),
cellular Tesar and Marquardt (1990), Falk
transcription et al. (1990)
eIF-4A and eIF-4G Translation 3Cpro Block cellular Belsham et al. (2000)
factors translation
Ebp1 RBP IRES Transcription Monie et al. (2007)
regulation
NEMO Transcription 3Cpro Modulation of Wang et al. (2012)
Complex Adaptor innate immunity
synthesized in infected cells (Clarke et al., 1985), with leaderless virus showed no clinical disease at
though data strongly suggest that Lb may be the 72 hours and no pulmonary changes at either 24
biologically relevant protein in vivo (Cao et al., or 72 h. These animals had only limited positive
1995; Piccone et al., 1995a). virus-specific ISH signals in respiratory bronchi-
This protease is responsible for inhibition of oles by 24 hours and no evidence of lesions or ISH
cellular protein synthesis in infected cells. Unlike signals in epithelial tissue by 72 hours (Brown et al.,
most host mRNAs, actively translated viral mRNA 1996). Thus, the leaderless virus did not appear to
does not contain a 7-methyl-G cap structure at its 5′ replicate well at the site of primary infection and
end (Grubman and Bachrach, 1979) and initiates was not able to spread to other sites within the
protein synthesis internally, at the IRES, by a cap- host. It was subsequently shown that infection with
independent mechanism (Belsham and Brangwyn, wild-type or leaderless virus induced the synthesis
1990; Jang et al., 1988; Kuhn et al., 1990; Pelletier of type I interferon (IFN) mRNA in both tissue
and Sonenberg, 1988). Lpro inhibits cap-dependent culture (Chinsangaram, 2001; Chinsangaram et al.,
mRNA translation by cleaving the protein synthe- 1999), and in lung mononuclear cells from aero-
sis initiation factor, eIF4G (Devaney et al., 1988; sol exposed cattle (Brown et al., 2000). In tissue
Kirchweger et al., 1994), which bridges the mRNA culture, however, IFN-associated antiviral activ-
cap to the 40S ribosomal subunit. In contrast, ini- ity was only detected in leaderless virus infected
tiation of FMDV RNA translation only requires the cells (Chinsangaram, 2001; Chinsangaram et al.,
Lpro generated C-terminal eIF4G cleavage product, 1999). This latter observation can be attributed
which binds to the FMDV IRES and interacts with to the inability of the leaderless virus to inhibit
the 40S ribosomal subunit (Lopez de Quinto and host translation, including IFN synthesis, and
Martinez-Salas, 2000; Saleh et al., 2001). the production of IFN within the infected animal
The proteolytic activity of Lpro was inhibited probably inhibited initial amplification and spread
in both a cell-free translation system and in cell of the virus. In contrast, wild-type virus infection
culture (Kleina and Grubman, 1992) by the blocks IFN mRNA translation allowing the virus to
compound E-64 and its membrane-permeable rapidly spread to neighbouring cells, and systemi-
analogue E-64d; both of which have been shown cally, prior to the induction of the adaptive immune
to specifically inhibit thiol proteases (Hanada et response. A similar leaderless virus containing the
al., 1978a; Hanada et al., 1978b). Inhibition of Lpro capsid-coding region from serotype O1Campos in
effectively blocked virus capsid assembly, presum- the genetic background of type A12 was also aviru-
ably because N-terminal myristoylation of VP4 lent in cattle, but caused a mild disease in swine and
and processing of P1–2A by 3Cpro were blocked. was transmitted to a naive animal (Almeida et al.,
Animal experiments with genetically engineered 1998).
Lpro-deleted (leaderless) type A12 virus showed Lpro The main function of viral 3Cpro is to perform
to be a virulence determinant, which was thought most of the cleavages of the viral polyprotein
to be due to the inability of the virus to shutoff (Clarke and Sangar, 1988; Vakharia et al., 1987).
cellular protein synthesis. While leaderless virus However, it has also been implicated in cleavage of
replicated at only a slightly lower rate than wild- some host cell components. 3Cpro has been shown
type virus in BHK-21 cells (Piccone et al., 1995a), to cleave another member of the cap-binding com-
it was markedly avirulent when injected into cattle plex, eIF4A (Belsham et al., 2000), and thus may
and pigs and was unable to spread to co-housed also be involved in the inhibition of cellular protein
animals (Chinsangaram et al., 1998; Mason et synthesis. It also cleaves eIF4G late in infection,
al., 1997). Post-aerosol exposure, wild-type virus although at sites different from those cleaved by the
infected cattle had histologically altered respiratory Lpro (Belsham et al., 2000). Histone H3 is cleaved
bronchioles and virus-specific in situ hybridization in FMDV-infected cells (Grigera and Tisminetzky,
(ISH) signals in bronchioles by 24 h. By 72 hours 1984), and 3Cpro was later shown to be responsible
these animals developed clinical disease includ- for this cleavage, suggesting that it is also responsi-
ing fever and vesicles on the feet and positive ISH ble for the inhibition of host cell transcription (Falk
signals in epidermal sites corresponding to visible et al., 1990; Tesar and Marquardt, 1990). Addition-
lesion development. In contrast, cattle infected ally, 3Cpro was demonstrated to cleave histones as
both co-immunoprecipitation and observed susceptible animals. This 153 amino acid protein is
co-localization through immunofluorescent cleaved from the C-terminus of 2C and N-terminus
microscopy. This finding is consistent with a previ- of 3B by 3Cpro. It is considerably longer than 3A
ous report that FMDV 2C co-localized with the proteins found in enteroviruses, and consists of an
LC3 autophagy marker along with non-structural N-terminal hydrophilic region followed by a hydro-
proteins 2B and 3A. This same study showed that phobic domain of about 28 residues that allows for
viral replication was impeded through inhibitors association with intracellular membranes where
of the autophagy pathway while an autophagy RNA replication occurs (O’Donnell et al., 2001).
inducer increased viral yields (O’Donnell et al., The precise role of the protein in FMDV replica-
2011). The authors suggest that this interac- tion is still being delineated. However, FMDV 3A
tion promotes FMDV replication by subverting forms a complex with the 3B protein (Falk et al.,
the fusion of lysosomes with autophagosomes. 1992; O’Donnell et al., 2001), and the enterovirus
Moreover, FMDV was demonstrated to induce the 3AB forms a complex with 3CDpro and binds to a
formation of autophagosomes, which appeared to cloverleaf structure at the 5′ end of the viral RNA
facilitate infection by the virus but not at the level (Xiang et al., 1995).
of RNA replication (Berryman et al., 2012). This The 3A protein was reduced in size by a 19- or
study showed a significant departure from what had 20-codon deletion in the C-terminal portion of the
been observed with other picornaviruses, which protein (Giraudo et al., 1990) when FMDV was
utilize autophagosomes as sites of RNA replication, serially passaged in embryonated eggs, during an
and whose formation is concomitant with non- attempt to produce an attenuated vaccine virus
structural protein production, particularly 3A and (Giraudo et al., 1987; Sagedahl et al., 1987). The
3D polymerase. In contrast, neither FMDV 3A nor role of 3A in both viral virulence and host range
3Dpol were detected in association with autophago- was demonstrated during studies of the FMDV
somes triggered by FMDV infection, but rather responsible for an outbreak of FMD in Taiwan
FMDV VP1 was found to co-localize with these in 1997 (designated O/Taw/97). This outbreak
vesicles. Additionally, formation of autophago- was unusual in that only pigs, and not cattle, were
somes could be induced by UV-inactivated FMDV affected, and the disease had an unusually high
and empty FMDV capsids. This latter point sug- mortality rate (Dunn and Donaldson, 1997; Huang
gested that the induction of autophagosomes by et al., 2000). Molecular characterization of the virus
FMDV occurred during the process of cell entry, revealed that it contained a 10-codon deletion in
and happened irrespective of whether the virus the C-terminal half of the 3A protein (Beard and
particle bound the primary integrin receptor or the Mason, 2000), similar to the deletion found in
alternative HS receptor (Berryman et al., 2012). FMDV passaged in chick embryos. The role of this
Additionally, the 2C protein was also identified deletion in the bovine-attenuated phenotype was
as a binding partner for the cellular protein vimentin confirmed using reverse genetic analysis (Beard
(Gladue et al., 2013). Although the significance of and Mason, 2000; Pacheco et al., 2003a), and
this interaction remains to be elucidated, vimentin subsequent analysis of viruses circulating in the
appears to form a structure surrounding FMDV 2C region suggested that in addition to the deletion,
that is eventually dissipated. While increased levels mutations in the 3A protein in the region surround-
of vimentin did not affect virus yields, FMDV repli- ing the deletion, may also be responsible for the
cation was impeded separately by treatment with a observed phenotype (Knowles et al., 2001). The
vimentin disrupting agent and the overexpression molecular basis for the porcinophilic phenotype
of a dominant-negative isoform of vimentin. These appears to be related to a reduction in viral RNA
results suggest that the 2C–vimentin interaction synthesis, which is manifested to a greater degree
may be essential for virus replication. in bovine cells than in swine cells (O’Donnell et
al., 2001). The 3A proteins from either the porci-
Non-structural protein 3A nophilic or a bovine-virulent isolate co-localized
Several lines of evidence have shown that non- to RNA replication complexes in either bovine
structural protein 3A is an important host-range or porcine cells, and also caused a disruption of
determinant for FMDV in both tissue culture and the Golgi apparatus (O’Donnell et al., 2001).
Viral genetic elements length of the poly-C tract was associated with viru-
lence (Harris and Brown, 1977), other studies have
S-fragment been unable to correlate poly-C length with this
Upstream of the poly-C tract at the extreme 5′ property of the virus (Costa Giomi et al., 1984). It
terminus of the FMDV genome exists a large stem has been shown that the poly-C length of natural
loop structure that has been postulated to be critical viral isolates are elongated by repeated passage in
for replication of the FMDV RNA genome. RNA cell culture (Escarmis et al., 1992), as is the length
pull-down experiments indicated a large ~ 120 kDa of this segment in genetically engineered viruses
unidentified RNA-binding protein pulled down (Rieder et al., 1993). It is possible to replicate virus
with the S-fragment portion of the 5′ UTR (Serrano with essentially no poly-C, and while this virus
et al., 2006). A similarly large RNA-binding protein, is virulent in suckling mice, it has a much higher
RNA Helicase A (RHA), was also described as a particle/PFU ratio than viruses containing longer
critical host factor involved in virus RNA replica- poly-C tracts (Rieder et al., 1993). The virulence
tion of the positive-sense single-stranded RNA of this virus in susceptible animals has not been
genome of hepatitis C virus (Isken et al., 2007). determined.
Through a combination of RNAi-induced knock-
down of RHA and biochemical and microscopy cre
experiments, RHA was confirmed to be a critical Just upstream from the IRES there is a short hairpin
host factor in the replication cycle of FMDV, and loop structure termed the cre (Mason et al., 2002).
the first known RNA-binding protein to interact The cre, which has been identified in the genomes
with the S-fragment (Lawrence and Rieder, 2009). of other picornaviruses, has a stem–loop with a
Intriguingly, FMDV infection triggered the subcel- conserved AAACA sequence in the loop region.
lular rearrangement of RHA from the nucleus to the In contrast to other picornaviruses, where the cre is
cytoplasm, and this redistribution was determined located within different regions of the ORF, the cre
to be contingent upon the de-methylation of argi- of FMDV is located within the 5′ UTR. However,
nine repeats in the C-terminus of the protein. The it has been shown to be positionally independent
de-methylation was later determined to be caused (Mason et al., 2002), and also has been demon-
by the arginine de-methylase, JMJD6, which is also strated to be functional in trans (Tiley et al., 2003).
an RBP (Lawrence et al., 2014). During the course The cre is essential for RNA genome replication (see
of FMDV infection, RHA co-precipitated with viral Grubman and Baxt, 2003, and references therein).
proteins 2C and 3A as well as cellular PABP, which Although the cre has not been shown to be involved
were also observed to localize in close proximity in virulence or host range, its role in replication
to each other via immunofluorescent microscopy makes it a good candidate for antiviral intervention.
(Lawrence and Rieder, 2009). Although the func-
tional significance of these interactions remains to IRES
be fully delineated, 2C, 3A and the S-fragment are The FMDV IRES contains a high degree of second-
all purportedly involved in RNA replication, and ary and tertiary structure (Belsham and Brangwyn,
the helicase activity of RHA would be of benefit to 1990; Kuhn et al., 1990). The IRES element for
melting the interaction between the positive and Aphthoviruses, similar in structure to the cardio-
negative RNA strands during viral RNA replication. virus IRES, is about 450 nucleotides in length and
has been modelled into a five-domain structure
Poly-C tract (Pilipenko et al., 1989). IRES elements contain a
The poly-C tract comprises over 90% C-residues pyrimidine-rich region at their 3′ ends immediately
with a small number of U and A residues. This preceding the AUG translation initiation codon
segment is over 100 bases in length; however, the and FMDV contains pyrimidine-rich regions
length of the poly-C tract can be extremely vari- directly upstream of each of the alternative AUG
able (Costa Giomi et al., 1984) and, in one case, a initiation codons (Belsham and Brangwyn, 1990;
tissue culture adapted virus has been shown to have Kuhn et al., 1990; Pilipenko et al., 1992). The
a poly-C length of over 400 bases (Escarmis et al., FMDV IRES interacts with a number of cellular
1992). Although an early study suggested that the proteins, including initiation factors important for
normal cellular mRNA translation. A host factor of based approach to identify an array of previously
57 kDa, the polypyrimidine tract binding protein unreported host proteins as potential co-factors
(PTBP), was shown to interact with at least two with FMDV upon infection of BHK-21 cell line.
regions of the IRES (Luz and Beck, 1990; Pilipenko The SILAC assay revealed several host proteins
et al., 2000). Deletion of these two sites inhibited that were up-regulated and several that were
both the binding of the protein and in vitro trans- down-regulated upon infection of host cells with
lation (Luz and Beck, 1991). Another host factor FMDV. These proteins are of diverse functions,
has been identified which is required for FMDV and in many cases, their relative significance to
IRES-driven translation, but not for translation of the progression of FMDV infection has yet to
cardiovirus mRNA (Pilipenko et al., 2000). This be determined. A few of the reported proteins
45 kDa protein, IRES-specific trans-acting factor were further examined for potential roles in
(ITAF45), along with PTBP, is required for the for- FMDV infection, virulence, and host range. One
mation of the 48S translation–initiation complex novel protein demonstrated to be up-regulated
(Martinez-Salas et al., 2001; Pilipenko et al., 2000). in response to FMDV infection by the SILAC
More recently, Sam68, previously shown to interact method was: LYPLA/APT1, which has been char-
with virus proteins during poliovirus infection acterized as a protein acyl thioesterase responsible
(McBride 1996), was found to interact with the for the removal of palmitoyl groups (Veit and
FMDV IRES (Lawrence et al., 2012), and showed a Schmidt, 2001). Since reversible protein palmi-
preference for a UAAA motif within domain 4 (Rai toylation is frequently associated with membrane
et al., 2015). Similar to RHA, Sam68 was observed interactions, this is of interest to FMDV research
to undergo a partial redistribution from the nucleus as FMDV infection is known to promote the re-
to the cytoplasm, and was found to interact with organization of membranes in the cytoplasm of
viral 3Cpro and 3Dpol. In a cell-free system, Sam68 infected cells with implications for viral RNA
was found to promote FMDV RNA replication replication. The many proteins detected in this
(Rai et al., 2015), and this role is likely functionally study provide attractive targets for future research
important as RNAi-induced Sam68 knockdown into the complex network of FMDV non-receptor
significantly diminished virus titres (Lawrence et protein interactions.
al., 2012). Additionally, Gem-associated protein 5
(Gemin5) was identified in large proteomic screens
to interact with the IRES of FMDV, an association Final remarks
that was demonstrated to inhibit viral translation While significant progress has been made in
(Pineiro et al., 2013). Therefore, the functionality elucidating the molecular biology of FMDV,
of this large RNA structure in the FMDV 5′ UTR many unanswered questions remain regarding
can be significantly modulated through a variety of FMDV pathogenesis, virulence, and host range in
host protein interactions. susceptible animals. The delineation of the roles
It has been suggested that IRES elements, and that multiple receptors play in disease as well as
possibly the host factors that bind to them, effect the identification of the as yet undescribed part-
pathogenicity and virulence of other Picornavi- ner molecules involved in the processes of viral
ruses (Evans et al., 1985; Kawamura et al., 1989; attachment and entry will continue to be of great
Malnou et al., 2002; Moss et al., 1989; Ochs et al., interest. Moreover, the developing knowledge of
2003; Skinner et al., 1989). In FMDV, a virus res- the plasticity of these interactions and the shifting
cued from persistently infected BHK-21 cells had of downstream pathways will contribute to the
two mutations within the IRES, which the authors rationale design of novel vaccine platforms and
suggest might have resulted in increased virulence antiviral therapeutics. In addition, further analysis
of the virus in tissue culture (Martinez-Salas et al., of the interactions between viral proteins, genome
1993). segments, and intracellular host components, in
addition to their role in the virus life cycle, will
Novel binding proteins increase both our understanding of the pathogene-
Recently, Guo et al. (2015) used a stable isotope sis of FMD, and help to determine ways for rational
labelling with amino acids in cell culture (SILAC) control of this disease.
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Figure 6.1 (A) Ribbon diagram of the structure of FMDV 3D polymerase in complex with a template-primer
RNA (PDB: 1WNE). The 3D molecule (grey) is shown in the conventional orientation with the seven conserved
structural motifs of palm and fingers domains depicted in different colours and explicitly labelled. The RNA
bound to the central cavity is shown in orange. (B) Structure of the ternary complex FMDV 3D-RNA-RTP (PDB:
2E9R). Side view of the polymerase surface (grey) that has been cut to expose the three channels by which the
different substrates and products of the polymerization reaction go in (template and rNTP channels) or leave
the active site (central channel). The conserved structural motifs are coloured as A, the template and primer
strands of the bound RNA molecule are shown in orange. (C) (Top panel) conformation and interactions in
the 3D active site on RTP binding (PDB: 2E9R). The 3D active site is in open conformation with the incoming
nucleoside analogue (stick representation in atom type colour with carbons in blue) stacked to the 3′ terminus
of the primer (orange) and base paired to the template acceptor base. The position of the RTP base is further
stabilized by interactions with residues of motifs A (red), and the motif B loop β9α11. The triphosphate moiety is
hydrogen bonded to different residues of motifs A and F (blue) and interacts with one metal ion (light blue ball).
The bottom panel show an example of a picornavirus 3D palm in closed conformation. This corresponds to the
PV 3D-RNA-CTP ternary complex after nucleotide incorporation and pyrophosphate release (PDB 3OL7). The
catalytic aspartic acids of motif A and C, as well as, the motif B residues interacting with the incoming nucleotide
are shown as sticks. The two metal ions are shown as balls in light blue. (D) Structure and interactions in the
template channel of FMDV 3D–RNA complexes, the molecular surface of the polymerase is shown in grey with
the acidic residues of the active site in red and the RNA depicted as sticks in orange (wt 3D–RNA complex;
PDB 1WNE). The RNA corresponding to the FMDV 3D N-terminal mutant K18E is superimposed in green (PDB
4WZM). The left and right insets show close-ups of the interactions involving the N-terminal residues (from M16
to K20) responsible of the largest conformational changes occurring at the template channel entrance.
this motif (β9-α11 in FMDV) is directly participat- uncertain (see Paul and Wimmer 2015 for an exten-
ing in template binding and translocation of the sive review). Biochemical and structural studies
nascent dsRNA (Ferrer-Orta et al., 2007; Gong performed for different members of the family: PV
and Peersen 2010; Garriga et al., 2013; Sholders (Lyle et al., 2002), HRV16 (Appleby et al., 2005),
and Peersen, 2014). Until recently, the only func- FMDV (Ferrer-Orta et al., 2006b), CVB3 (Gruez
tion associated with motif D was scaffolding for et al., 2008) and EV71 (Chen et al., 2013) revealed
the palm domain. Recent NMR data provided evi- three distinct VPg binding sites on 3Dpol (Fig.
dences that motif D is highly dynamic and that this 6.2A). Strikingly, whereas the most picornaviruses
dynamics appears to be essential for the closure of express only a single VPg protein, FMDV possesses
the catalytic complex when the correct nucleotide three similar but not identical copies of VPg: VPg1,
is placed in the active site (Yang et al., 2012). Motifs VPg2 and VPg3 (Forss et al., 1982), all of which
E and F are located at the boundary of the palm and are found linked to viral RNA (King et al., 1980).
thumb domains (motif E) and in the fingers domain Although not all the copies are needed to maintain
(motif F). These motifs participate in primer and infectivity (Falk et al., 1992, Pacheco et al., 2003),
rNTP binding, respectively. Finally motif G, is also there are no reports of naturally occurring FMDV
located in the fingers and forms the entry of the strains with fewer than three copies of 3B, suggest-
template channel (Fig. 6.1A and B). ing that there is a strong selective pressure towards
Furthermore, mutational analyses together maintaining this redundancy (Carrillo et al., 2007).
with the structural characterization of the mutant The structures of two complexes between
enzymes in FMDV and also in PV have demon- FMDV 3D and VPg1 were determined by X-ray
strated that some substitutions in residues located crystallography. They depicted both uridylylated
far from the active site have significant effects on and non-uridylylated forms of VPg (Ferrer-Orta
catalysis and fidelity. All of these observations sug- et al., 2006b). Both structures revealed that the
gest that nucleotide binding and incorporation are VPg occupies the central cleft of the polymerase,
modulated by a long-distance network of interac- with the hydroxyl group of Tyr-3 positioned so as
tions (Pfeiffer and Kirkegaard 2003; Thompson to mimic the free 3′-hydroxyl group of a nucleic
and Peersen 2004; Arnold et al., 2005; Agudo et acid primer at the active site of 3D (Fig. 6.2B and
al., 2010; Ferrer-Orta et al., 2010; Moustafa et al., C). Several amino acid contacts between 3D and
2011; Ferrer-Orta et al., 2015b). VPg, predicted to be important for initiation of
RNA synthesis, were confirmed by site-directed
mutagenesis of 3D polymerase and by using
Structural and functional studies chemically synthesized mutant versions of VPg.
of VPg binding and uridylylation Twelve out of 16 3D residues that were identified
Picornaviruses initiate RNA replication by the suc- as interacting with VPg, are strictly conserved
cessive addition of two uridine-monophosphate among different picornaviral polymerases, suggest-
molecules to the Tyr-3 residue of VPg. VPg uri- ing that they may play an important role during the
dylylation is catalysed by 3D using as template a critical VPg-uridylylation step (Ferrer-Orta et al.,
cis-replicating element (cre) that is located at differ- 2006b). In the 3D–VPg-pU complex, the hydroxyl
ent positions of the RNA genome, in the different group of the Tyr-3 side chain was found covalently
picornaviridae genera (e.g. the FMDV cre is located attached to the α-phosphate moiety of the uridine-
in the 5′-UTR of the genome). The picornaviral monophosphate (UMP) molecule. Two divalent
proteins: VPg, 3D and 3C, alone or in the 3CD cations, together with the catalytic aspartic acid
precursor form, together with the viral RNA cre residues of motifs A and C, participate in the uridy-
elements comprise the so-called ‘VPg uridylylation lylation reaction, following a similar mechanism to
complex’ responsible of VPg uridylylation in vivo. that described for the nucleotidyl transfer reaction
The extensive structural and biochemical studies in other polymerases (Steitz, 1998). A cluster of
provided several different models for the interac- positively charged residues of motif F also partici-
tions established between VPg and 3D or 3CD in pate in the uridylylation process, stabilizing Tyr3
the uridylylation complex, however, the precise and UMP in a proper conformation for the catalytic
mechanism of uridylylation in picornavirus remains reaction (Ferrer-Orta et al., 2006b) (Fig. 6.2C).
Figure 6.2 (A) Comparison of identified VPg binding sites in picornavirus 3Ds. As all reported structures of
picornavirus 3Ds share high structural similarities, we used the structure of the FMDV 3D–VPg-UMP complex
(PDB: 2F8E) as a representative model. The molecular surface of the FMDV 3D polymerase (grey) is shown
in two different views: the conventional orientation, as if looking into a right hand (left) and a side view (right).
The bound VPgs of FMDV (pale blue), CVB3 (yellow; PDB: 3CDW) and EV71 (red; PDB: 4IKA) are shown as
sticks. The 3D residues essential for VPg binding in FMDV, PV and EV71 are coloured as blue, wheat and
magenta, respectively, in the surface representation. (B) Structure and interactions of the FMDV 3D–VPg-UMP
complex. Top-down view of the FMDV polymerase represented with its molecular surface (grey) that been cut
to expose the central channel where the primer protein VPg (pale-blue) binds. The conserved structural motifs,
lining the central cleft of the polymerase are shown as cartoons in different colours (Motif A red, B green,
C yellow, E cyan, F blue and G magenta). The VPg residue Tyr3 is located at the active site, positioning its
hydroxyl group as a molecular mimic of the free 3′ hydroxyl group of a nucleic acid primer for nucleotidylylation.
In the structure, the hydroxyl group of Tyr3 side chain was found covalently attached to an UMP molecule
(orange) by a phosphodiester linkage. Two metal ions (green spheres) participate in the uridylylation reaction.
(C) Detail of the interactions in the 3D active site. Metal 1 bridges the catalytic aspartate of motif C and the O–
of tyrosine side chain, now covalently bonded to the α-phosphate of UMP. Metal 2 coordinates the O1 oxygen
of phosphate α and the hydroxyl group of the conserved serine of motif B. The positively charged residues of
motif F also participate in the uridylylation process.
This ‘front-loading’ mode of VPg binding to model described for FMDV was further supported
the FMDV 3D polymerase is compatible with a cis by the crystal structures of HRV16 3Dpol (Appleby
mechanism of VPg uridylylation. The VPg priming et al., 2005) and of the PV 3CD precursor (Marcotte
et al., 2007). In the latter structure, the extensive The important sequence homology between
crystal packing contacts found between symmetry- the picornaviral VPg sequences (Ferrer-Orta et al.,
related 3CD molecules and the proximity of the 2006b; Sun et al., 2014) and the high similarities
N-terminal domain of 3C to the VPg binding site, existing in the 3D polymerase structures prompts
in the way that VPg was positioned in the FMDV to hypothesize that the three VPg binding sites
3D–VPg complex, suggests a possible role of the observed in the different crystal structures would
contacting interfaces in forming and regulating the reflect distinct binding positions of VPg to both 3D
VPg uridylylation complex (Marcotte et al., 2007). or its precursor 3CD at different stages of the virus
After VPg nucleotidylylation, a number struc- replication initiation process. As mentioned above,
tural rearrangements of the 3D polymerase occur, the FMDV genome codes for three VPg molecules,
marking the transition from initiation to the elonga- all of them are present in naturally occurring viruses.
tion phase of RNA synthesis. There is experimental
evidence supporting possible structural differences
in 3D when involved in the priming reaction versus Structural elements regulating
elongation of RNA. i.e. The nucleoside analogue the replication elongation
5-fluorouridine triphosphate (FUTP) is a potent process
inhibitor of VPg uridylylation but not of RNA elon- The replication elongation process can be roughly
gation (Agudo et al., 2008). divided in three steps, including nucleotide
A second binding site for VPg was found in the selection, phosphodiester bond formation and
structure of the CVB3 polymerase (Gruez et al., translocation to the next nucleotide for the subse-
2008). The VPg fragment solved, corresponding quent round of nucleotide addition. A number of
to the C-terminal half of the peptide, was bound at structures during RNA elongation in FMDV were
the base of the thumb sub-domain in an orientation obtained in the presence of natural nucleotides and
that did not allow uridylylation by its own 3D car- the nucleotide analogues FUTP and RTP, using
rier (Fig. 6.2A). This binding mode, partially agreed different RNAs as template-primer molecules
with previous data reported for PV, showing that a (Ferrer-Orta et al., 2007). The structures revealed
number of amino acids located in motif E of polio- the critical polymerase residues involved in the
virus (PV) were required for VPg or their precursor, correct positioning of the template and primer
3AB, binding and affecting VPg uridylylation (Lyle nucleotides and those responsible of the recogni-
et al., 2002). In light of these data, authors proposed tion and binding of the incoming nucleotide for
that a VPg bound at this position might be either catalysis (Fig. 6.1). In all complexes analysed, the
uridylylated by another 3D molecule or playing a single stranded 5′-end of the template extends across
structural stabilizing role within the uridylylation the face of the fingers domain towards the active
complex (Gruez et al., 2008). site cleft, contacting with different residues in the
Finally, a third VPg binding site was discovered polymerase N-terminus and motifs G and F, which
in the structure of EV71 3D–VPg complex. In this drive the template chain towards the active site
complex, VPg is anchored at the bottom of the cavity. The acceptor base of the template (position
palm domain of the polymerase, showing a V-shape t + 1) is located adjacent to the nucleotide binding
conformation that crosses from the front side of the pocket, sitting above the active site and stacked
catalytic site to the back side of the enzyme (Fig. on the upstream duplex and ready for the binding
6.2A). As in the previous viruses, the mutational of the incoming rNTP (Fig. 6.1D). Furthermore,
analyses of the interacting residues evidenced the downstream t + 2 template nucleotide is also
a reduced binding of VPg to the EV71 3D, also pointing towards the active site cavity, stacked with
affecting uridylylation (Chen et al., 2013; Sun et al., the t + 1 nucleotide. The interactions established
2012). However, the structure of the EV71 3D–VPg between t + 2 and the 3D N-terminal residue R17
complex showed that the VPg Tyr3 is buried at the substantially contribute to maintain the observed
base of the polymerase palm (Fig. 6.2A) indicat- template orientation (Fig. 6.1D). In the active site,
ing that a conformational change should occur to the 3′-hydroxyl group of the primer strand interacts
expose the side chain of Tyr3 for the uridylylation directly with the catalytic aspartic acid D338 of
reaction. motif C (Ferrer-Orta et al., 2004; 2007).
The structures of the ternary complexes FMDV 3D after the incorporation of standard
3D-RNA-rNTP, where the incoming nucleotides nucleotides or the mutagenic nucleoside analogue
were trapped close or at the polymerase active site, FUTP (Ferrer-Orta et al., 2007). However, no
showed the central role of amino acid D245 of motif structures of translocation intermediates are cur-
A, N307 of the motif B helix, and S298, G299 and rently available for picornaviral polymerases and the
T303, of the motif B N-terminal loop (β9-α11), in precise mechanism is not yet known. Very recently,
nucleotide recognition and correct positioning of structural and functional data in PV indicate that
the sugar in the ribose-binding pocket (Fig. 6.1C). steric clashes between the motif-B loop and the
Structural comparisons of the different FMDV template RNA would also promote translocation
elongation complexes revealed that the loop β9-α11 (Sholders and Peersen 2014).
is able to adopt different conformations in response Structural comparisons between different
to different template and incoming nucleotides, 3D–RNA–rNTP complexes indicate that some
being the most flexible element of the active site of interactions are common to standard nucleotides
the FMDV polymerase (Ferrer-Orta et al., 2009). and to nucleotide analogues, while others are
In all these complexes the polymerase active site specific for a particular analogue (Ferrer-Orta et
was trapped in an open conformation, character- al., 2007; Fig. 6.1C). This structural information
ized by a partially formed three-stranded β-sheet of is essential to understand the molecular basis of
the palm domain motifs A and C and the catalytic template-copying fidelity and may help in the
residue of motif A, D240, not properly oriented for design of new mutagenic nucleotides targeted spe-
the catalytic reaction. In this open conformation, cifically to viral RdRPs.
the incoming rNTP reaches the active site and
establishes base-pairing interactions with the tem-
plate base (t+1) but catalysis has not taken place. Structural and functional studies
In addition, only one metal ion was found in con- with the 3D polymerase of
tact with the triphosphate moiety of the incoming ribavirin-resistant mutants
nucleotide (Fig. 6.1C) (Ferrer-Orta et al., 2007). Viral RdRPs are considered to be low fidelity
The structural analyses of other replication– enzymes, generating mutations that allow the rapid
elongation complexes in the enteroviruses PV and adaptation of these viruses to different tissue types
CVB3 trapped the polymerase active site in a closed and host cells. One consequence of the low fidelity
conformation. This conformation was achieved by of RdRPs is the enhanced sensitivity of the viral
the realignment of the palm β-strands that include population to accumulate additional mutations
motifs A and C after correct nucleotide binding, during replication. A new strategy against RNA
resulting in the repositioning of the motif A aspar- viruses consists in using mutagenic nucleotides.
tate to allow interactions with the two metal ions The objective is to provoke an excessive number
required for catalysis (Fig. 6.1C) (Gong et al., 2013; of mutations, to deteriorate the viral functions to
Gong and Peersen 2010). Furthermore, growing the point that the virus cannot survive (reviewed
amounts of biochemical and structural data in in Domingo, 2005). One of the mutagens used in
PV also indicated that additional conformational research on lethal mutagenesis is ribavirin (1-β-d
changes in motif D, including the repositioning of -ribofuranosyl-1-H-1,2,3-triazole-3-carboxamide),
a strictly conserved lysine (K369 in FMDV; Fig. extensively employed in clinical practice. Unfortu-
6.1C) determined both efficiency and fidelity of nately, viral mutants that are resistant to ribavirin
nucleotide addition (Castro et al., 2007, 2009; Yang have been selected, thus facilitating escape from
et al., 2012). This lysine is presumed to coordinate lethal mutagenesis.
the export of the PPi group from the active site Ribavirin-resistant mutants have been isolated
once catalysis has taken place, triggering the end of both in PV and FMDV and the resistance phe-
the reaction cycle and allowing enzyme transloca- notype mapped in 3D (Pfeiffer and Kirkergaard,
tion (Shen et al., 2012). 2003; Sierra et al., 2007). Interestingly, the amino
A number of crystal structures have been solved acid substitutions that confer ribavirin-resistance
in a post-translocation state (Gong et al., 2013; are different for the two viruses, whereas G64S is
Gong and Peersen 2010), including those of the located within the fingers domain of PV 3D M296I
resides in the motif B loop β9-α11 of FMDV. template, the dynamic nature of residues lining
Biochemically, the two mutant enzymes showed the template channel should permit the access of
significant differences, the purified mutant poly- the t + 1 nucleotide into the 3D catalytic site. The
merase 3D(G64S) displayed an increase in copying base of the template channel is built mainly by
fidelity relative to wild type enzyme, at the cost residues of the β9-α11 loop that are involved in
of producing mutant spectra of lower complexity interactions with the t + 1 nucleotide, as well as,
than wt virus (Pfeiffer and Kirkergaard et al., 2005; with the incoming rNTP (Ferrer-Orta et al., 2007).
Vignuzzi et al., 2006). In contrast, the 3D(M296I) Altogether these data prompts to hypothesize that
mutant FMDV restricted the incorporation of the rearrangements in the template channel and
ribavirin monophosphate (RMP) into the RNA the β9-α11 occur in a concerted manner and that
through subtle rearrangements in the loop β9-α11, these concerted changes would regulate both RNA
close to the polymerase active site that did not have replication processivity and fidelity.
a significant effect on the rate of misincorporation
of the standard nucleotides (Sierra et al., 2007;
Arias et al., 2008; Ferrer-Orta et al., 2010). Conclusions
A third mechanism of ribavirin-resistance The ever growing availability of structures of
in FMDV was elucidated by the functional and picornavirus 3D catalytic complexes provided
structural characterization of a mutant obtained an increasingly accurate picture of the functional
the in the presence of increasing concentrations steps and regulation events underlying viral RNA
of the mutagen (from 800 μM to 5000 μM), result- genome replication. Data currently available
ing in the sequential incorporation of three amino provides high-resolution pictures for a range of
acid substitutions (M296I, P44S and P169S) in interactions associated to VPg binding and uridy-
3D, 3D(SSI) (Agudo et al., 2010). The in vitro lylation and for the different conformational states
polymerization assays performed with the purified associated to template and primer binding, rNTP
3D(SSI) enzyme indicated that the main biological recognition and binding, catalysis and chain trans-
effect of these substitutions was to attenuate the location. Such structural information is providing
consequences of the mutagenic activity of ribavirin new insights into the fidelity of RNA replication,
through the alteration of the pairing behaviour of and for the design of antiviral compounds.
RTP (Agudo et al., 2010). Structural comparisons Protein primed mechanism of replication initia-
of the wild type and mutant polymerases suggested tion mediated by VPg appears to be a process that
that the amino acid substitutions altered the posi- involves more than one VPg binding site in 3Dpol
tion of the template RNA in the entry channel of possibly at different stages of the virus replication
the enzyme, thereby affecting nucleotide recogni- initiation process. Although the number of 3D-VPg
tion. The observed structural changes affect mainly structures available show individual snapshots
the 3D N-terminal residues M16-K20 which appear of the process, to obtain a global picture of the
completely reorganized, resulting in the formation assembly, regulation and dynamics of complete
of a hydrophobic pocked where the t + 2 nucleotide replication initiation complexes, requires further
binds (Fig. 6.1D) (Agudo et al., 2010). analyses of high order assemblies formed not only
Surprisingly, similar changes in the template by the polymerase and VPg but also involving dif-
binding channel together with rearrangements in ferent viral and host proteins, protein precursors
the β9-α11 loop were also observed in the charac- and RNA templates. Such structures should pro-
terization of other FMDV 3D mutants, harbouring vide a more detailed view of the molecular events
substitutions at the 3D N-terminal residues K18 underlying the initiation of picornavirus genome
and K20 which belong to a nuclear localization replication.
signal of the enzyme. All these mutants exhibit The structures of a large number of replication–
low RNA binding activity, low processivity and elongation complexes of FMDV 3D as well as other
alterations in nucleotide recognition, including picornaviruses captured subtle conformational
increased incorporation of RMP compared with changes associated with nucleotide selection, active
the wild-type enzyme (Ferrer-Orta et al., 2015b). site closure and chain translocation. Among these
Besides facilitating specific contacts with the RNA movements, the motif B-loop β9-α11appears to
play a crucial role assisting in the positioning of the Castro, C., Smidansky, E., Maksimchuk, K.R., Arnold, J.J.,
Korneeva, V.S., Götte, M., Konigsberg, W., and Cameron,
template and incoming nucleotides in the active C.E. (2007). Two proton transfers in the transition
site, in chain translocation and presumably in the state for nucleotidyl transfer catalyzed by RNA- and
discrimination between natural nucleotides and DNA-dependent RNA and DNA polymerases. Proc.
mutagenic analogues as ribavirin. Natl. Acad. Sci. U.S.A. 104, 4267–4272.
Chen, C., Wang, Y., Shan, C., Sun, Y., Xu, P., Zhou, H., Yang,
The structural and functional studies with C., Shi, P.Y., Rao, Z., Zhang, B., and Lou, Z. (2013).
a number of FMDV 3D mutants resistant to Crystal structure of enterovirus 71 RNA-dependent
ribavirin suggest that subtle rearrangement in the RNA polymerase complexed with its protein primer
template channel, mainly affecting the polymerase VPg: implication for a trans mechanism of VPg
uridylylation. J. Virol. 87, 5755–5768.
N-terminus, may occur in concert with the confor- Domingo, E., Ed. (2005). Virus entry into error catastrophe
mation changes in the β9-α11 loop and that these as a new antiviral strategy. Virus Res. 107.
concerted changes would regulate both RNA rep- Falk, M.M., Sobrino, F., and Beck, E. (1992). VPg gene
lication processivity and incorporation of correct amplification correlates with infective particle formation
in foot-and-mouth disease virus. J. Virol. 66, 2251–2260.
versus incorrect nucleotides. Ferrer-Orta, C., Ferrero, D., and Verdaguer, N. (2015a).
RNA-Dependent RNA Polymerases of Picornaviruses:
Acknowledgements From the Structure to Regulatory Mechanisms. Viruses
Núria Verdaguer acknowledges funding from the 7, 4438–4460.
Ferrer-Orta, C., de la Higuera, I., Caridi, F., Sánchez-Aparicio,
Spanish Ministry of Economy and Competitive- M.T., Moreno, E., Perales, C., Singh, K., Sarafianos, S.G.,
ness (BIO2011–24333 and Maria de Maeztu action Sobrino, F., Domingo, E., and Verdaguer, N. (2015b).
MDM-2014-0435). Multifunctionality of a picornavirus polymerase
domain: nuclear localization signal and nucleotide
recognition. J Virol. 89, 6848–6859.
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Figure 7.1 A simplified representation of an evolving viral quasispecies that maintains an invariant consensus
sequence. Genomes are depicted as horizontal lines and mutations as symbols on the lines. Genomes
represented by discontinuous lines are not perpetuated due to an excess of mutations; the invariant consensus
sequence is represented by a horizontal line at the bottom of each distribution. In reality, viral populations
consist of hundreds or thousands of genomes in each replicative unit, as evidenced experimentally with
many viruses. Figure reproduced from Domingo et al. (2012), with permission from the American Society for
Microbiology, Washington DC, USA.
of viral pathogenesis. As a classic example, simian lability of virus particles, differences in plaque
immunodeficiency virus (SIV) evolved in monkeys morphology, and atypical pathogenic manifesta-
from an initial phase without disease symptoms tions of viral isolates. Indeed some FMDV variants,
into a phase with AIDS-like pathology. Naive mon- often depending upon their passage history in cell
keys developed disease when they were inoculated culture, could be associated with fulminant or
with virus expressed from clones retrieved from degenerative forms of myocarditis, neuropathology
the disease phase but not from the asymptomatic or diabetes; atypical symptoms of viral infections
phase of the infection (Kimata et al., 1999). The have been described for many viral pathogens
result means that evolution of SIV played a direct (Bachrach, 1968; Domingo et al., 1990; Holland et
role in disease development. Recent observations al., 1992, and references therein). Therefore, con-
on the molecular epidemiology of pathogenic trary to an extended belief that goes back to early
viruses have documented genetic changes that times of influenza virus research, viral diseases are
have modified their virulence profile. Genetic not necessarily ‘invariable’ manifestations of ‘vari-
heterogeneity and diversification are also major able’ viruses. This point was addressed by J. Holland
challenges for disease prevention by vaccination and colleagues who wrote the following statement:
and antiviral treatment because of the selection of ‘Therefore, the acute effects and subtle chronic
mutants that are not significantly affected by the effects of infections will differ not only because
vaccine-induced immune response or by antiviral we all vary genetically, physiologically, and immu-
agents (reviewed in Domingo, 2016; Domingo and nologically, but also because we all experience a
Schuster, 2016). different array of quasispecies challenges. These
FMDV, the most important viral pathogen in facts are easily overlooked by clinicians and scien-
veterinary medicine, fully participates of quasispe- tists because disease syndromes are often grossly
cies dynamics and its biological consequences, similar for each type of virus, and because it would
affecting viral pathogenesis, virus persistence and appear to make no difference in a practical sense.
difficulties for disease prevention and control. However, for the person who develops Guillain–
Phenotypic plasticity of FMDV was suggested Barré syndrome following a common cold, or for
by early studies on variations of acid and thermal the individual who remains healthy despite many
years of HIV-1 infection, for example, it may make number of substitutions per nucleotide and year
all difference in the world’ (Holland et al., 1992). (s/nt/y) that are incorporated in the consensus
New findings in the last decade have unveiled a sequence of an evolving viral population. If a hori-
degree of population complexity of FMDV which is zontal time axis were added to the scheme of Fig.
even greater than suspected a few years ago. In this 7.1, a zero evolutionary rate (invariant consensus
chapter we first review briefly the molecular basis sequence) would be calculated despite a dynamic
of genetic diversity of viruses, its consequences for mutant spectrum. Rates of evolution can be calcu-
virus population dynamics, and, finally, observa- lated within a single host or among multiple hosts
tions with FMDV on intra-population interactions (during virus spread in an outbreak, epidemic or
and new antiviral strategies. pandemic).
Figure 7.2 Scheme of main types of genetic variation in viral genomes. (A) Mutation due to miscopying of
template residues by viral replication complexes. (B) Hypermutation (high frequency of biased mutation types)
often due to cellular editing activities. (C) Genome segment reassortment. (D) Homologous, replicative (left),
and non-replicative (right) recombination. (E) Genome segmentation as documented with FMDV (described in
‘Fitness gain and limits to fitness gain. A transition towards genome segmentation’). Figure reproduced from
Domingo et al. (2012), with permission from the American Society for Microbiology, Washington DC, USA.
mechanisms of antigenic variation of FMDV (Acha- for serotype classifications, history of long-term
rya et al., 1989; Curry et al., 1996; Lea et al., 1994, virus circulation in nature, or constraints to varia-
1995). Comparison of amino acid sequences of a tion of some antigenic sites [reviewed in (Domingo,
major antigenic site located within the G-H loop of 2016)]. The number of serotypes may reflect the
capsid protein VP1 [a protruding and mobile loop constraints that operate in viruses to tolerate amino
that includes also an integrin-recognition domain acid replacements that affect their antigenic profile.
(Acharya et al., 1989)] of serotype C viruses,
together with virus reactivity with MAbs, revealed Antigenic variation in the absence
two mechanisms of antigenic diversification of of immune selection: coevolution of
FMDV in nature: (i) a gradual increase in antigenic antigenicity and cell tropism
distance brought about by amino acid replacements While selection by antibodies and cytotoxic T-cells
at two hypervariable regions within the antigenic probably plays a major role in antigenic variation
site, and (ii) an abrupt antigenic change, associ- of viruses, evidence with several viruses, includ-
ated with a replacement at some critical positions ing FMDV, suggests that antigenic variation is not
within the same antigenic site that resulted in loss necessarily the result of immune selection (Bolwell
of several epitopes (Martínez et al., 1991b). These et al., 1989; Curry et al., 1996; Díez et al., 1990;
two mechanisms are similar to antigenic drift and Domingo, 2016; Domingo et al., 1993, 2001;
shift, extensively characterized for human influenza Haydon and Woolhouse, 1998; Holguín et al., 1997;
viruses, but without participation of genome seg- Martínez et al., 1991a; Sevilla and Domingo, 1996).
ment reassortment. The underlying mechanism is the acceptance of
FMDV has at least four different antigenic amino acid replacements generated at random at
sites some of which may interact with each other, the antigenic sites because surface residues of viral
depending on the viral serotype, and by mecha- capsids and envelopes are less subjected to struc-
nisms which are not well understood (Baranowski tural constraints than internal residues in the same
et al., 2001b; Feigelstock et al., 1992; Mateu, 1995; proteins. Following its generation by mutation, an
Mateu et al., 1994; Parry et al., 1990). Furthermore, antigenic variant may rise to dominance by the fol-
there are non-additive effects of amino acid substi- lowing mechanisms:
tution in antigen–antibody recognition (Mateu et
al., 1992), thus contributing to complex antigenic 1 The relevant antigenic site may perform
profiles of FMDV isolates. functions additional to the interaction with
A general point is in place here. High mutation components of the immune response. In
rates do not entail a correspondingly high antigenic particular amino acid residues of antigenic
diversity. Within the Picornaviridae family the sites may also be involved in recognition of
number of serotypes varies remarkably: there is cellular receptors (Baranowski et al., 2001a,
a single serotype of Mengo virus or of hepatitis A 2003; Verdaguer et al., 1995, 1998; reviewed
virus (HAV), three of poliovirus, seven of FMDV in Domingo, 2016). The use of an entirely new
and around one hundred of human rhinoviruses. receptor by a virus may render the obsolete
Yet in all these viruses the frequency of monoclo- receptor-binding site highly tolerant to varia-
nal antibody (MAb)-escape mutants is similar tion with obvious consequences when the old
and in the range of 10–3 to 10–5, and the specific receptor recognition residues overlap with an
frequency values are dependent on the nature of antigenic site. These mechanisms underlie a
each individual epitope rather than on the degree possible coevolution of antigenicity and host
of antigenic diversity attained by the virus in cell tropism, with consequences for the adap-
nature. Several mechanisms have been proposed tive potential of the virus (Baranowski et al.,
to account for differences in the number of viral 2003; Domingo, 2016).
serotypes among viruses that share similarly high 2 An additional mechanism for antigenic
mutation rates. They include the presence in some variation in the absence of immune selection –
viruses of dominant and invariant antigenic sites which does not necessitate that residues of an
that obscure the contribution of other variable antigenic site are involved in other functions –
antigenic sites, differences in the procedures used relates to the classical concept of ‘hitch-hiking’
virus–host interactions, rendering an understand- PanAsia-II and some of its sub-lineages have been
ing of viral pathogenesis a challenging endeavour. associated with recent outbreaks in the Middle
East and West Eurasia (see Mahy and Belsham, this
book). It is interesting that some lineages and sub-
Diversification in the field: lineages are endowed with the capacity of extensive
power and limitations of dominance, while others such as FMDV serotype C
phylogeny; epidemiological are nearly extinct. At least for the time being.
fitness
Phylogeny connects the evolutionary history of The molecular clock controversy:
related virus isolates, and it is a basic technique for intra-host versus inter-host
molecular epidemiology and virus classification. Its evolutionary rate
main weakness is that a phylogenetic position is a Early work on the rate of FMDV evolution during a
poor predictor of important viral features such as disease outbreak in a limited geographical area pro-
pathogenic potential. In other words, modifications vided evidence that the rate of evolution (defined
of virulence may occur without alteration of the as the accumulation of mutations in the consensus
position of a virus in a phylogenetic tree. Despite sequence of sequential isolates as a function of time;
being based on specific nucleotide sequences, trees see ‘Mutation and antigenic variation’) is not con-
for viruses should be regarded as consisting of a stant. The rate varied not only among viral genomic
cloud of points at the tip of the branches, to recog- regions of the same isolates but also depending
nize the simplification implied by the connection on the time interval between the viral isolations
one virus–one sequence. Many different genomic on which calculations were based (Sobrino et al.,
sequences from the same cloud display different 1986). Values ranged from < 4 × 10–4 to 4 × 10–2
phenotypic traits. s/nt/year. Rate differences had been previously
Phylogenies express the dominance of viral described for the haemagglutinin gene of influenza
lineages in the field but not its causes. The term virus type C, that were attributed to circulation of
epidemiological fitness was coined to describe different evolutionary lineages, so that the sampled
an ensemble of properties (often difficult to sequences could not reflect a linear accumulation
specify) that determine the prevalence of some of mutations in an individual lineage (Buonagurio
variants at the epidemiological level. The standard et al., 1985). Related observations have been made
fitness concept, that is, the relative replication with other viruses, notably human immunodefi-
(multiplication) capacity of virus variants in a given ciency virus type 1 (HIV-1) and hepatitis B virus
environment is just one of several parameters that (HBV).
contribute to dominance in the field. Epidemiologi- The two main observations are:
cal fitness encompasses several features in addition
to intracellular replication capacity: stability of viral 1 The rate of evolution decreases as the time
particles in infected animals and in the environ- interval between the isolation of the viruses
ment, transmissibility, host range, symptomatology used for the nucleotide sequence comparison
– including capacity of produce subclinical infec- increases.
tions that delay virus detection – and others that 2 The intra-host rate of evolution is higher than
apply to all pathogenic viruses (Domingo, 2016). In the inter-host rate.
Chapter 18, B.W.J. Mahy and G.J. Belsham describe
the spectacular expansion of FMDV O PanAsia These findings have been explained as follows:
since its first isolation in India in 1990. This virus
replaced other circulating strains in many countries 1 For viruses that remain infectious in the exter-
during the 1990s, and produced in the year 2000 nal environment (outside the infected host),
an outbreak in Japan, a country that had been free calculated rates of evolution are lower when
of the disease since 1908! FMDV O Pan Asia is periods in which the virus does not replicate
an example of high epidemiological fitness due to – and thus no significant number of mutations
mechanisms that are not understood. In support are introduced – are included in the calcula-
of the continuous capacity of FMDV to diversify tion (Fig. 7.4) (see Chapter 1 for studies on
while maintaining its dominance, a virus termed air-borne transmission of FMDV).
Date: 10:24 Monday 12 September 2016
UNCORRECTED PROOF File: FMDV 4P
Quasispecies in FMDV | 155
Figure 7.5 A model for slower inter-host than intra-host rate of evolution. A subpopulation from an infected
individual (left) is preferentially transmitted. Intra-host virus replication (in the individual with a brown outline)
generates a complex quasispecies. The subpopulations transmitted to the individual with the green outline
(right) resemble those of the first transmission. The result is that rates of evolution at the epidemiological level
appear lower than the rates within individual hosts. Boxes highlight the main points. Figure reproduced from
Domingo (2016), with permission from Academic Press, Elsevier, Amsterdam.
Date: 10:24 Monday 12 September 2016
UNCORRECTED PROOF File: FMDV 4P
156 | Domingo et al.
memory loss, which implied a deterministic behav- treatment-planning purposes, mainly for chronic
iour of an evolutionary outcome (Ruiz-Jarabo et infections that may require treatment changes. The
al., 2003b). A deterministic feature of quasispecies presence of inhibitor-resistant mutants at memory
(as opposed to a random or stochastic behaviour) levels should discourage administration of the same
had been previously documented by the repro- or a related inhibitor in subsequent treatments. A
ducible dominance of wild type over a neutral current trend towards considering antiviral agents
mutant of vesicular stomatitis virus during growth- in veterinary medicine (as an alternative or comple-
competition experiments in cell culture (Quer et ment to vaccination, to avoid massive slaughtering
al., 1996, 2001). It is suspected that deterministic of animals; see Chapter 15) may render the memory
or stochastic features are manifested depending on concept and the capacity to extend diagnosis to
parameters such as population size, constraints to minority components of mutant spectra relevant to
variation, and others, although the issue is far from animal viruses in the field.
settled (Domingo, 2016; Herrera et al., 2008; Rouz-
ine et al., 2001). Intra-mutant spectrum interactions
A second FMDV memory marker took advan- Viral quasispecies are not mere aggregates of mutant
tage of an extended internal oligoadenylate tract genomes, each acting independently of the others.
–that elongates four adenylate residues found Within a replicative unit a cross-talk between
immediately preceding the second functional genomes is established, mainly through trans-acting
AUG protein synthesis-initiation codon– present proteins expressed by individual genomes. A func-
in clones that had been subjected to serial plaque- tional protein may complement a non-functional
to-plaque bottleneck transfers. The elongation counterpart expressed by a mutant genome. Con-
was probably due to slippage mutagenesis of viral versely, a non-functional protein may interfere with
polymerase or replicating complex (Escarmís et replication of other genomes of the same ensemble.
al., 1996) (see ‘Fitness decrease due to Muller’s The first indication of the modulating capacity of a
ratchet’). Large population passages resulted in quasispecies was provided by J.C. de la Torre and
reversion of the extended oligoadenylate tract to the J. Holland, who showed that the mutant spectrum
standard sequence, with accompanying fitness gain. of a vesicular stomatitis virus population could sup-
Genomes with a larger number of adenylate resi- press replication of a mutant of superior fitness (de
dues remained at memory levels that, as in the case la Torre and Holland, 1990).
of the RGD marker, were present at frequencies at Work with FMDV has contributed to the under-
least 10-fold higher than found in populations with- standing of intra-mutant spectrum interactions
out a prior history of dominance of genomes with that render an ensemble of mutants the unit of
the extended oligoadenylate (Arias et al., 2001; selection. Specifically, mutagenized whole FMDV
Ruiz-Jarabo et al., 2000, 2002). Memory remained populations (González-López et al., 2004), as well
constant for at least 150 serial large population as individual, RNA replication-competent capsid
infections, albeit with a shift towards genomes with and polymerase mutants (Perales et al., 2007),
a shorter oligoadenylate tract; in the course of pas- suppressed replication of the standard virus, as
saging, memory genomes gained fitness at a rate evidence by co-electroporation experiments. Com-
comparable to the entire population, in agreement plementation and interference can be regarded as
with the Red Queen hypothesis (Arias et al., 2004; two sides of the same coin, in the sense that comple-
Ruiz-Jarabo et al., 2002). mentation generally may occur during replication
Memory is a collective property of the entire under standard mutation rates while interference
quasispecies, and the introduction of a population becomes increasingly prevalent at higher muta-
bottleneck resulted in memory loss. Memory has a tion rates such as during lethal mutagenesis; this
number of biological implications for virus behav- interference mechanism is the basis of lethal defec-
iour. It may allow a quasispecies to respond more tion, one of the mechanisms that participate in the
effectively to selective constraints that have been transition towards error catastrophe (reviewed in
previously experienced by the same viral lineage, Domingo et al., 2012).
provided no population bottlenecks intervened. A selective force can act either at the level of
Quantification of memory genomes is relevant for an individual genome (independent of other
Figure 7.7 Schematic representation of accumulation of mutations and fitness decrease associated with serial
bottleneck events. Bottlenecks are experimentally realized as plaque-to-plaque transfers on cell monolayers,
and are depicted from the central mutant spectrum towards the right (thin, discontinuous arrow). In contrast
to plaque-to-plaque transfers, large population passages result in fitness gain, as shown to the left of the
central mutant spectrum (large blue arrow). Consensus sequences are shown below the corresponding
mutant distributions. Fitness gain can occur with or without a modification of the consensus sequence.
Figure reproduced from (Domingo et al., 2012), with permission from the American Society for Microbiology,
Washington DC, USA.
formation is the standard criterion to quantify Muller’s ratchet in vivo reflects the potential impact
FMDV infectivity in cell culture, the specific infec- of transmission events in generating virus variants
tivity (ratio between infectivity and amount of viral with altered behaviour.
RNA) was at least 140-fold lower than that of the
parental clone. Decreases of specific infectivity are FMDV extinction through lethal
also observed during extinction by lethal mutagen- mutagenesis or the transition into error
esis (compare with ‘FMDV extinction through catastrophe
lethal mutagenesis or the transition into error catas- Another consequence of high mutation rates and
trophe’), and in viruses with deoptimized codon quasispecies dynamics is the proximity of RNA
usage (reviewed in Domingo, 2016). genome replication to an error threshold for main-
A study by C. Carrillo, D.L. Rock and colleagues tenance of the core genetic information that must
demonstrated that the profound effects of bottle- be transmitted for virus survival. This very relevant
necks noted in cell culture apply also in vivo. They concept was based on theoretical calculations
designed an experiment consisting on 20 serial dealing with survival of simple replicons (Alves
swine-to-swine contact transmissions of FMDV and Fontanari, 1998; Eigen, 2002; Eigen and Bie-
that resulted in diminished virus virulence and the bricher, 1988; Nilsson and Snoad, 2000; Nowak
establishment of a carrier state (inapparent infec- and Schuster, 1989; Swetina and Schuster, 1982).
tion) in pigs (Carrillo et al., 2007). The extension of It asserts that for any replication system that must
maintain inheritable information, there is a maxi- Loeb and Mullins, 2000; Severson et al., 2003;
mum error rate above which the information is lost. review by Eigen, 2002). These and other studies
The critical error rate is termed the error threshold, have rendered lethal mutagenesis an interesting and
and violation of the error threshold results in a tran- active field of antiviral research, based on the use of
sition into error catastrophe. Several parameters virus-specific mutagenic nucleotide analogues as
determine the position of the error threshold. One antiviral agents.
of them is the complexity of the genetic information One of the most remarkable findings in the
that must be maintained. In the case of RNA viruses studies on lethal mutagenesis was that the broad-
(with compact genomes and absence of or limited spectrum antiviral agent ribavirin, licensed for use
redundant information) complexity can be equated in humans, may in some cases exert its antiviral
with genome length. Crossing the error threshold activity through mutagenesis, following its intracel-
should result in a sharp transition from a productive lular conversion into the triphosphate form, and
into an abortive infection and virus extinction (Fig. incorporation by some viral polymerases (Airak-
7.8). The first experimental evidence of such a tran- sinen et al., 2003; Crotty et al., 2000, 2001; Maag
sition was obtained by J. Holland and his associates et al., 2001; Vo et al., 2003; reviewed in Graci and
who documented an adverse effect of chemical Cameron, 2002). A mutagenic activity of ribavirin
mutagenesis on the infectivity of vesicular stomati- and its triphosphate derivative is supported by
tis virus and poliovirus (Holland et al., 1990; Lee et studies with replicating viruses and with assays
al., 1997). Results were extended to several viruses with purified RNA-dependent RNA polymer-
that use different replication strategies (Airaksinen ases. Therefore, it is possible that, inadvertently,
et al., 2003; Crotty et al., 2000, 2001; Grande-Pérez ribavirin has been exerting clinical benefits through
et al., 2002; Lanford et al., 2001; Loeb et al., 1999; enhanced mutagenesis. Mutations that confer
Figure 7.8 Representation of the connection between the error threshold concept of quasispecies theory and
virus extinction by enhanced mutagenesis. The central horizontal axis marks the theoretical limits of copying
fidelity (1, perfect fidelity; 0, no fidelity; both extremes are impossible for biological systems). The upper part
shows the steps that have been identified in viral populations as the mutation rate increases: first the region
of quasispecies distributions; then lethal defection in which an increase in the frequency of some classes of
defective genomes jeopardizes replication of the ensemble; this is followed by overt lethality due an excess
of mutations that prompts the viral population to cross the error threshold leading to virus extinction (loss of
information in quasispecies nomenclature). The weights on the scales indicate factors that can prevent or
contribute to virus extinction. Figure copied from Domingo et al. (2012), with permission from the American
Society for Microbiology, Washington DC, USA.
resistance to ribavirin have been identified in the administration of these drugs at adequate con-
polymerases of poliovirus (Pfeiffer and Kirkegaard, centrations results in similar intracellular pool
2003), FMDV (Agudo et al., 2010, 2016; Ferrer- imbalances. In contrast to mycophenolic acid,
Orta et al., 2010, 2015; Sierra et al., 2007) (see ribavirin in its triphosphate form can be a substrate
‘FMDV mutants resistant to mutagenic nucleotide for viral polymerases. Comparison of mutation
analogues’) and hepatitis C virus (Young et al., frequencies in the mutant spectra of FMDV upon
2003). The last occurred in patients subjected to treatment with either ribavirin or mycophenolic
ribavirin monotherapy, suggesting that the hepatitis acid supports a direct mutagenic activity of the
C polymerase was a target of the antiviral agent. A analogue in the process of elimination of the virus
second, encouraging finding for the design of anti- from the persistently infected cells (Airaksinen et
viral strategies based on error catastrophe, has been al., 2003). The result is in agreement with conclu-
the demonstration that the mutagenic pyrimidine sions with other viruses regarding a mutagenic
analogue 5-fluorouracil (FU) prevented the estab- activity of ribavirin (Contreras et al., 2002; Crotty
lishment of a persistent LCMV infection in mice et al., 2000, 2001; Severson et al., 2003; Zhou et
(Ruiz-Jarabo et al., 2003a). This result constitutes al., 2003, among other studies).
a proof of principle that the new antiviral approach Pre-extinction FMDV populations are defined
is feasible in vivo. as those from the passage preceding the one from
The studies on lethal mutagenesis using FMDV which infectivity and FMDV-specific RT-PCR-
have been aimed at understanding the molecular amplifiable material can no longer be rescued after
basis of virus entry into error catastrophe, and at three blind passages in the same cells, in the absence
evaluating the effect of viral fitness and viral load of mutagens and inhibitors (Pariente et al., 2001;
(number of infectious particles involved in an Sierra et al., 2000). Such populations depicted
infection) in FMDV extinction by mutagenic base increases of 2- to 10-fold in the complexity of their
analogues. For a given viral load, a low relative mutant spectra. This increase was not accompanied
fitness favoured FMDV extinction. Likewise, for by a modification of the consensus sequence of the
a given relative fitness, a low viral load favoured population in its way towards extinction, denoting,
FMDV extinction (Pariente et al., 2001; Sierra et as expected, the absence of adaptive mutations in
al., 2000). These results suggested that combina- the transition into error catastrophe. The invari-
tions of mutagenic agents and antiviral inhibitors ance of a virus population consensus nucleotide
(to lower the viral load) could be more effective sequence in its way to extinction illustrates that
than mutagenic agents alone in driving the virus constancy of a consensus sequence provides no
to extinction. Indeed, systematic extinctions of argument against quasispecies, and that no conclu-
high fitness FMDV in cell culture were achieved sions regarding quasispecies can be derived from
by combinations of FU and the antiviral inhibitors an examination of consensus sequences. A mutant
guanidine hydrochloride and heparin (Pariente et spectrum can be highly dynamic and heteroge-
al., 2001). Whenever the combined mutagenic and neous without any signs of the dynamics being
inhibitory activities were insufficient to produce revealed by a comparison of successive consensus
extinction, inhibitor-resistant mutants of FMDV sequences (Fig. 7.1).
were selected (Pariente et al., 2003). What is the critical difference between the con-
Ribavirin can eliminate FMDV from BHK-21 siderable resistance of FMDV to extinction despite
cells that are persistently infected with the virus accumulation of mutations in serial bottleneck
(de la Torre et al., 1987). A comparison of the transfers (see ‘Fitness decrease due to Muller’s
effects of mycophenolic acid and ribavirin on ratchet’) and the very efficient extinction associ-
persistent FMDV indicated that ribavirin exerted ated with enhanced mutagenesis? Our current
its curing activity mainly through enhanced view is that with a continuous mutagenic action,
mutagenesis of the FMDV genome (Airaksinen the virus cannot find the low frequency advanta-
et al., 2003). Ribavirin and mycophenolic acid geous mutations that permitted eluding extinction
are both inhibitors of the cellular enzyme inosine (Lázaro et al., 2002; Manrubia et al., 2003; Martin
monophosphate dehydrogenase, a key enzyme et al., 2008; O’Dea et al., 2009). With a continuous
of the GTP biosynthesis pathway and, therefore, mutagenic pressure, negative selection is so intense
that no survivors can maintain infectivity. Transient protocols for other viruses (C. Perales, S. Manru-
survivors are soon hit by additional mutations. The bia et al., unpublished work). A theoretical model
ultimate imposition of negative selection is a way of developed by S. Manrubia and her colleagues
viewing lethal mutagenesis. showed that the advantage of a sequential versus
combination treatment is due to key parameters
FMDV mutants resistant to mutagenic related to the virus response to mutagens and
nucleotide analogues inhibitors. Some of the parameters are: concentra-
Several viral mutants that display resistance to tion of standard and defective viruses resistant or
base or nucleoside analogues have been isolated sensitive the inhibitor, mutation rate, number of
and characterized (Borderia et al., 2016), includ- infected cells, and rate of generation of inhibitor-
ing FMDV mutants resistant to ribavirin or resistant mutants (Iranzo et al., 2011; Perales et
5-fluorouracil (Agudo et al., 2010, 2016; Arias al., 2009, 2012). Another contributing factor is
et al., 2008; Ferrer-Orta et al., 2010, 2015; Sierra that the simultaneous presence of a mutagen and
et al., 2007; Zeng et al., 2013; de la Higuera et al., inhibitor may result in the latter impeding the
manuscript in preparation). There are at least four interfering activity of the defectors produced by
mechanisms of viral escape to mutagenic nucleotide the mutagen; indeed, defectors need to replicate
analogues: (i) replacements in the viral polymerase to contribute to negative intra-mutant spectrum
that increase the general template-copying fidelity interactions (see ‘Intra-mutant spectrum interac-
thereby restricting the incorporation of the muta- tions’) (Perales et al., 2007).
genic nucleotide; (ii) polymerase replacements that In a more direct fashion, a combination of an
limit specifically the incorporation of the muta- inhibitor and mutagen can be suboptimal due to
genic nucleotide without affecting significantly the interactions such as (i) a mutagen may increase the
fidelity of incorporation of the standard nucleo- frequency of inhibitor-escape mutants, and (ii) an
tides; (iii) polymerase replacements that modulate inhibitor can prevent the replication of defectors
nucleotide incorporation in a way that compensate that contribute to extinction.
the incorporation bias produced by the mutagen; Clearly, much remains to be done before lethal
(iv) replacements in viral proteins other than the mutagenesis can find an application in human or vet-
polymerase that affect either the fidelity or modula- erinary medicine. New, viral specific and non-toxic
tion properties of the polymerase. FMDV has been mutagenic agents must be found, and protocols for
key in the establishment of mechanisms (iii) and their optimal use with available inhibitors must be
(iv) (Agudo et al., 2010, 2016; de la Higuera et al., investigated. Effective concentrations of mutagens
manuscript in preparation) (structural aspects are and compatible inhibitors must be reached at
in Chapter 6). Thus, viruses can find adaptive path- the replication sites of the viruses in the infected
ways to resist the action of mutagenic nucleotides, organisms, to avoid selection of extinction-escape
as they do to resist standard inhibitors. viral mutants. Despite these limitations, research
on error catastrophe is increasing its impetus, as a
Alternative lethal mutagenesis-based reward to those of us who believe that basic science
antiviral interventions is the fuel for practical applications in ways that are
Combination therapy with two (or ideally more) generally unpredictable.
inhibitors has advantages over monotherapy to
control a virus and avoid (or delay) selection of
inhibitor-resistant mutants. Experiments with Fitness gain and limits to fitness
FMDV suggested that when a mutagenic agent gain: a transition towards
is a component of therapy, a sequential inhibitor- genome segmentation
mutagen administration may have an advantage In contrast to fitness decrease driven by bot-
over the corresponding combination (Perales et al., tleneck transfers (see ‘Fitness decrease due to
2009). A similar observation was made with lym- Muller’s ratchet’, above), large population passages
phocytic choriomeningitis virus (Moreno et al., of FMDV result in fitness increase through a variety
2012), and experiments are in progress to explore of mutational pathways (Escarmís et al., 1999).
the efficacy of sequential lethal mutagenesis Previous studies with vesicular stomatitis virus
documented exponential fitness increases during higher probability of beneficial mutations when
large population passages (Novella et al., 1995). fitness is very low.
Subsequent investigations have amply confirmed FMDV was used to further explore the con-
these early observations, and fitness increase is sequences of driving the virus to a limiting high
generally accepted as a direct consequence of fitness by hundreds of large population passages
selection of the fittest variants from a replicating at high MOI. The unexpected outcome was that
virus ensemble (Fig. 7.7). However, fitness does the standard genome was replaced by a segmented
not increase continuously and when it reaches high version with two distinct RNAs encapsidated into
values it displays fluctuations (Novella et al., 1999). separated particles; the RNA segments included
The fluctuations were interpreted by the limitation an internal deletion, one within the L-coding
of population size that would be required for fitness region and the other within the VP3, VP1-coding
to continue increasing. A similar pattern of fluctua- region (García-Arriaza et al., 2004, 2006; Moreno
tions was observed with FMDV at low fitness levels et al., 2014; Ojosnegros et al., 2011) (Fig. 7.9). The
(Escarmís et al., 2002; Lázaro et al., 2003). In this transition towards segmentation was mediated by
case the underlying mechanism is probably the several point mutations that accumulated in the
Figure 7.9 Scheme of the main events involved in segmentation of the FMDV genome when the virus was
subjected to 260 passages at high MOI (vertical arrows). The parental biological clone C-S8c1 (equivalent to
pMT28 expressed from a plasmid) is depicted by a filled square, and subsequent passaged populations as
empty circles; the parental genome is drawn in the upper box, with indication of genomic regions and encoded
proteins. The segmented version C-s8p260, composed of ∆417 (with an internal deletion at the L-coding region)
and ∆999 (deletion within the VP3, VP1-coding region), is displayed in the bottom box. The standard genome
can be rescued by low MOI passages of the segmented virus (above the bottom box). Figure reproduced from
Domingo (2016), with permission from Academic Press, Elsevier, Amsterdam.
viral genome during passages and it was favoured reasons, profusely described in the literature on
by increased stability of viral particles that encap- quasispecies, the combined use of three or more
sidate shorter RNAs. Segmentation represents an antiretroviral inhibitors prevents or delays the
example of biologically relevant recombination selection of multidrug resistant HIV-1. With the
as opposed to inconsequential recombination investigations on lethal mutagenesis, it is also
(Perales et al., 2015), and illustrates the innovative becoming apparent that it is possible to improve
capacity of replicating mutant swarms. protocols to obtain more effective results upon
administration of mutagenic and non-mutagenic
antiviral agents. This is an area in which FMDV has
Summary, conclusions and largely contributed to, and can still contribute, with
prospects useful information.
The research on the population dynamics of FMDV
must be viewed in the broader context of additional Acknowledgements
studies carried out with human, animal, and plant The contributions of colleagues from Centro de
RNA viruses. The main concept derived from these Biología Molecular ‘Severo Ochoa’ (CSIC-UAM),
investigations is that the consensus nucleotide Centro Nacional de Biotecnología (CNB, CSIC),
sequences that we routinely determine for surveil- Centro de Investigación en Sanidad Animal (CISA-
lance or research purposes represents only a rough INIA) and Centro de Astrobiología (CSIC-INTA)
approximation of what the genetic information whose names appear in the bibliography of this chap-
contained in a viral population really is. Minority ter are gratefully acknowledged. Work supported
components of mutant spectra cannot be ignored by grants BFU2011-23604 and SAF2014-52400-R
because they act as an underground commanding from Ministerio de Economía y Competitividad,
police that controls virus behaviour. The presence and grant S2013/ABI-2906 (PLATESA from
and role of mutant spectra is why the molecular Comunidad Autónoma de Madrid/FEDER). C.P.
evolution theory of quasispecies was welcomed by is supported by Miguel Servet program of the Insti-
virologists. tuto de Salud Carlos III (CP14/00121) cofinanced
Although the studies on FMDV population by the European Regional Development Fund
dynamics discussed in preceding sections may (ERDF).
seem distant from the real problems afflicting the
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A B C
Figure 8.1 Clinical signs and lesions in and around the mouth due to foot-and-mouth disease in cattle. (A)
Drooling from the mouth. Ruptured vesicles leaving erosions (reddish areas) on the dental pad and hard palate
(B) and in the nostril (C) of a cow infected with FMDV.
may also be observed on the muzzle, gums, dental dairy cattle, making it difficult to milk cows whose
pad and palate (Fig. 8.1B) and in or around the nos- lactation is often already declined due to the dis-
trils (Fig. 8.1C). Ruptured oral lesions are usually ease. Neonatal deaths of calves and abortions may
painful, causing animals to avoid eating. Lesions on be observed in cows (Kitching, 2002).
the feet are particularly prominent on the coronary In an FMD outbreak in the Republic of Korea
band and interdigital spaces (Fig. 8.2A and B). Foot in 2010/2011, dairy cows had more severe clinical
lesions also commence as blanched areas, which signs than beef cattle. The frequency of subclinical
progress to vesicles and eventually rupture resulting disease was higher for beef farms compared to dairy
in erosions. Feet lesions often cause cattle to shift farms (Yoon et al., 2012).
their weight from one foot to another. Vesicles and Clinical signs of FMD are often less severe in
erosions can also occur on the teats, especially in indigenous cattle breeds in regions endemic for
A B
Figure 8.2 Foot lesions in bovine foot-and-mouth disease: A ruptured vesicle on the coronary band (A) and
interdigital space (B) of the foot of FMDV-infected cattle.
A B C
Figure 8.3 Foot-and-mouth disease lesions in pigs. Vesicles on the snout (A) seen as a fluid-filled elevation and
the coronary band extending to the heel bulb (B) seen as blanched elevations. (C) Ruptured vesicle on the heel
bulb of a pig infected with FMDV.
FMD such as Africa and Asia (Kitching, 2002). in pigs (Yoon et al., 2012). Clinical signs such as
Also, (re-)exposure of vaccinated and/or cattle that lameness or unsteady gait were clearly more severe
have recovered from FMD to live virus may result in pigs than in other affected species. Due to exten-
in asymptomatic infection. Additionally, cattle may sive vesicle formation around the coronary band,
recover from acute infection, with or without obvi- followed by rupturing and a severe inflammatory
ous clinical signs, yet continue to harbour virus in reaction, pigs with severe FMD may lose their hoofs
the oro-pharyngeal mucosa. Such animals that con- (Fig. 8.5). Spontaneous abortion and stillbirths
tinue to have detectable virus in the oro-pharynx have also been observed in pregnant sows. Sudden
beyond 28 days after infection in the absence of death, often without clinical signs, was observed in
clinical signs, other than possibly at the acute stage, piglets. This is attributed to acute myocarditis as the
are known as carrier animals (Alexandersen et al., virus invades the heart muscle in young animals
2002). Furthermore, some cattle have been known (Kitching and Alexandersen, 2002). Additionally,
to never fully recover from the effects of FMD, vesicles/erosions on the teats (Fig. 8.5) and/or
which leaves them with impaired breathing and mastitis were suspected to account for some of the
rough hair coat (hairy panters), often attributed to
thyroid gland pathology (Kitching, 2002).
Pigs
In pig FMD, vesicles can be observed on the snout,
around coronary bands and heel bulb of the feet
(Fig. 8.3A, B and C), with foot lesions being the
most common observation (Alexandersen and
Donaldson, 2002). Lesions at other sites, especially
in the mouth, are less frequent. When they do occur,
they are found on the dorsal surface in the distal
portion of the tongue or at the tip of the tongue Figure 8.4 Foot-and-mouth disease lesions on
(Fig. 8.4) and usually very small in size (Alexan- swine tongue. The image shows numerous erosions
dersen and Mowat, 2005). Field reports from the in an advanced stage of healing. These would have
started as small vesicles that eventually ruptured and
2010/2011 FMD outbreak in the Republic of Korea caused erosions. The lesion at the distal part of the
confirmed vesicle formation on the feet as the most tongue is more extensive and may have resulted from
commonly observed clinical manifestation of FMD coalesced vesicles.
infected, but are not highly susceptible and appear unapparent infection in the African buffalo (Syn-
to be unable to transmit the disease. Llamas were cerus caffer; Vosloo et al., 2007) to acutely fatal
difficult to infect under experimental conditions, infection in mountain gazelles (Gazella gazella;
and when infected, developed only very mild dis- Shimshony et al., 1986). This wide variation is due
ease. These studies have shown that NWCs can be to a combination of factors such as the type and
infected with various serotypes and through differ- quantity of virus, route of infection, host species,
ent routes that result in mild-to-severe clinical signs as well as individual differences including the level
(Wernery and Kinne, 2012). of immunity (Alexandersen et al., 2003; Thomson
et al., 2003). Thus, diagnosis of FMD in wildlife is
Foot-and-mouth disease in wildlife more difficult than in domestic species because of
Natural and experimental FMDV infection has the greater variability in clinical signs and disease
been reported in numerous species of true wild- severity (Thomson et al., 2003).
life, captive wildlife, and feral species (reviewed
in (Weaver et al., 2013; Arzt et al., 2011; Alexan-
dersen and Mowat, 2005; Pinto, 2004; Thomson Pathology
et al., 2003; Schaftenaar, 2002). Susceptibility has The early events of FMD pathogenesis differ
been reported, primarily, among wild members of slightly between species and also depend on the
the order Artiodactyla (even-toed ungulates) in route of inoculation. Cattle are highly susceptible
the families Bovidae, Cervidae, Suidae, Tayasuidae, to infection by aerosolized virus via the respiratory
Camelidae, Giraffidae and Antilocapridae (Arzt route (Pacheco et al., 2010; Donaldson et al., 1987).
et al., 2011). However, natural infection has also In cattle inoculated with aerosolized virus, the
been described in species in other taxa includ- virus first infects cells of the pharyngeal mucosa-
ing elephants (order Proboscidea; Pyakural et al., associated lymphoid tissue of the nasopharynx
1976, Schaftenaar, 2002), bears (order Carnivora; (Alexandersen et al., 2003; Burrows et al., 1981).
Officer et al., 2014), hedgehogs (order Insectivora; In particular, the mucosa-associated lymphoid
McLauchlan and Henderson, 1947) and others tissue of the nasopharynx may be involved (Arzt
(reviewed in Weaver et al., 2013; Wernery and et al., 2010). In contrast, pigs have been shown to
Kinne, 2012; Alexandersen and Mowat, 2005). be more refractory to aerosol exposure and more
The pathogenesis of FMDV and clinical pres- susceptible to infection via cuts and abrasions in
entation of FMD in most susceptible wildlife skin or mucosae or possibly by the gastrointestinal
species has not been studied extensively (Arzt et route (Sellers and Gloster, 2008; Donaldson and
al., 2011). Many earlier reports of FMD in wildlife Alexandersen, 2001). In the case of oral infection
are based on clinical examination alone without in pigs the paraepiglottic tonsils have been shown
confirmation by virology or serology. Experimen- to be a primary site of FMDV infection with con-
tal studies have been conducted for many species, tinuing replication occurring in the oropharyngeal
usually on a small number of animals and with a tonsils (Stenfeldt et al., 2014). In all domestic spe-
limited number of virus types. In general, when cies, mechanical transfer of virus from infected to
present, typical clinical signs in wildlife are simi- susceptible animals with virus entry through cuts
lar to those in domestic animals (Thomson et al., or abrasions in the skin or mucosae may occur
2003). Development and rupture of vesicular (Alexandersen and Mowat, 2005) with primary
lesions on skin and mucosae can occur at multiple replication occurring at the site of infection. After
sites, but are most commonly found on the feet initial replication, there is widespread viraemic
(e.g. coronary band and interdigital positions) dissemination to surface epithelium, and visceral
and in the oral cavity (e.g. tongue and dental organs with subsequent development of vesicular
pad). Lameness, difficulty eating, as well as other lesions (Alexandersen et al., 2001; Burrows et al.,
behavioural changes due to damage to the epithe- 1981).
lium and secondary infections are often observed Vesicular lesions are most often observed in and
in severe cases. The reported pathogenicity and around the mouth and on the feet, however, they
clinical presentation of FMD among wildlife is may also be seen on the snout/muzzle, teats, mam-
highly variable and ranges widely from subclinical, mary gland, prepuce, vulva, and other sites of the
skin. Lesions may also develop in the squamous Mortality in young animals may be due to acute
epithelium of the rumen and can be observed as myocarditis which is observed macroscopically as
vesicles or erosions on the ruminal pillars during multifocal white to grey spots or stripes throughout
post-mortem examination. In pigs experimentally the myocardium, primarily in the left ventricle and
infected by the oropharyngeal route, micro-vesicles interventricular septum (Fig 8.6B). Heart lesions
have been observed within crypt epithelium of the are characterized by lymphohistiocytic myocarditis
tonsil of the soft palate (Stenfeldt et al., 2014). The with hyaline degeneration and necrosis/fragmen-
characteristic histopathologic change seen in early tation of cardiomyocytes (Fig 8.6C) (Gulbahar
vesicular lesions is ballooning degeneration of cells et al., 2007; Alexandersen et al., 2003). On occa-
in the stratum spinosum of the stratified squamous sion, skeletal muscles may also be affected. Using
epithelium. As the lesion progresses there is inter- immuno-microscopical methods, viral antigen
cellular oedema with necrosis of epithelial cells has primarily been detected within keratinocytes
and mild infiltration of mononuclear cells and neu- of vesicular lesions (Fig 8.6D) but also has been
trophils. Subsequently, there is a loss of cohesion observed in other tissues including cardiomyo-
between epithelial cells (acantholysis) with loss of cytes. In persistently infected cattle, FMDV RNA
cells in the stratum spinosum and replacement by and antigen could be detected by in situ hybridi-
fluid and neutrophils to form vesicles (Fig 8.6A). At zation and immunohistochemistry in clusters of
later stages vesicles rupture and secondary bacterial nasopharyngeal epithelial cells (Zhang et al., 2001;
infections may occur. Pacheco et al., 2015).
Figure 8.6 Pathology findings in animals experimentally infected with FMDV. (A) Bovine skin showing
intraepidermal vesicle (arrow). Note the presence of acantholytic keratinocytes (arrowheads) with neutrophil
infiltration. (B) Pig heart showing white streaks and pale areas in the myocardium (arrows). Inset: On
cross-section multifocal pale necrotic areas are visible in the left ventricle (arrowheads). (C) Histological
examination of affected pig heart shows swelling and fragmentation of cardiomyocytes (arrow) with moderate
inflammatory infiltrate. (D) Heel of white tailed deer showing immunohistochemical detection of FMDV antigen
within keratinocytes (arrow).
exceptionally valuable animals. Rev. Off. Int. Epizoot. Vosloo, W., de Klerk, L.M., Boshoff, C.I., Botha, B.,
21, 613–623. Dwarka, R.M., Keet, D., and Haydon, D.T. (2007).
Sellers, R., and Gloster, J. (2008). Foot-and-mouth disease: Characterisation of a SAT-1 outbreak of foot-and-mouth
a review of intranasal infection of cattle, sheep and pigs. disease in captive African buffalo (Syncerus caffer):
Vet. J. 177, 159–168. clinical symptoms, genetic characterisation and
Shimshony, A., Orgad, U., Baharav, D., Prudovsky, S., phylogenetic comparison of outbreak isolates. Vet.
Yakobson, B., Bar Moshe, B., and Dagan, D. (1986). Microbiol. 120, 226–240.
Malignant foot-and-mouth disease in mountain gazelles. Weaver, G.V., Domenech, J., Thiermann, A.R., and Karesh,
Vet. Rec. 119, 175–176. W.B. (2013). Foot and mouth disease: a look from the
Stenfeldt, C., Pacheco, J.M., Rodriguez, L.L., and wild side. J. Wildl. Dis. 49, 759–785.
Arzt, J. (2014). Early events in the pathogenesis of Wernery, U., and Kinne, J. (2012). Foot and mouth disease
foot-and-mouth disease in pigs; identification of and similar virus infections in camelids: a review. Rev.
oropharyngeal tonsils as sites of primary and sustained Off. Int. Epizoot. 31, 907–918.
viral replication. PLOS ONE 9, e106859. Yoon, H., Yoon, S.S., Wee, S.H., Kim, Y.J., and Kim, B.
Stenfeldt, C., Pacheco, J.M., Singanallur, N.B., Ferreira, H.C., (2012). Clinical manifestations of foot-and-mouth
Vosloo, W., Rodriguez, L.L., and Arzt, J. (2015). Clinical disease during the 2010/2011 epidemic in the Republic
and virological dynamics of a serotype O 2010 South of Korea. Transbound. Emerg. Dis. 59, 517–525.
East Asia lineage foot-and-mouth disease virus in sheep Zhang, Z.D., and Kitching, R.P. (2001). The localization of
using natural and simulated natural inoculation and persistent foot and mouth disease virus in the epithelial
exposure systems. Vet. Microbiol. 178, 50–60. cells of the soft palate and pharynx. J. Comp. Pathol. 124,
Thomson, G.R., Vosloo, W., and Bastos, A.D. (2003). Foot 89–94.
and mouth disease in wildlife. Virus Res. 91, 145–161.
by wildlife independent of domestic livestock. strategies need to be more compatible than cur-
Another is that SAT viruses, SAT-2 particularly, rently is the case, i.e. requiring a more integrated
exhibit considerably more antigenic variation than approach. That is perhaps the most fundamental
Eurasian serotypes which complicates control based challenge confronting FMD management in sub-
on vaccination. However, outbreaks of SAT-type Saharan Africa today.
FMD in cattle – certainly in southern Africa – have a
history of causing mild, slow-spreading disease that
has limited direct impact on livestock, especially in Introduction
extensive rangeland systems. The implication is that Foot-and-mouth disease (FMD) viruses occur
the direct impact of FMD on livestock producers in as seven serotypes within the Aphthovirus genus
southern Africa is considerably less than indirect of the family Picornaviridae (Vallée and Carré,
impacts. The latter are comprised of the cost of dis- 1922; Waldmann and Trautwein, 1926; Brooksby
ease control measures (usually borne mostly by the and Rogers, 1957; Brooksby, 1958 and references
public sector in southern Africa), trade restrictions therein; Brooksby, 1982 and references therein).
and adverse socio-economic and environmental Based on genome analysis, serotypes O, A, C and
impacts of control measures. Moreover, FMD con- Asia 1 constitute a clear evolutionary lineage dis-
trol measures have proven inadequate in southern tinguishable from the other lineage comprising
Africa over the last decade and a half, while the the South African Territories (SAT) serotypes (i.e.
performance of countries with FMD-free zones in serotypes SAT-1, 2 and 3) (Robson et al., 1977; Pal-
the international beef export market has declined in menberg, 1989; Tully and Fares, 2008; Yoon et al.,
recent years. 2011). The O, A, C and Asia 1 lineage has tradition-
Geographic approaches to management of FMD ally been associated with Europe and Asia where its
based on creation of FMD-free zones with fenced existence has apparently been recognized for 2000
boundaries, especially in extensive rangeland sys- years (cited by Casas-Olascoaga, 2003). A disease
tems where mobility is vital for both wildlife and syndrome that could be FMD was described in
pastoralists, inevitably result in a clash between the India in the 11th century (Nene 2007), and also
interests of livestock production and wildlife con- in the mid-sixteenth century in what is now Italy
servation. This is unfortunate because both are vital (Bulloch, 1927). Serotype A, O and C viruses
for future healthy rural development in southern subsequently spread to the Western Hemisphere
and eastern Africa. Therefore ways of reducing this in the late nineteenth century (Brown, 1986). The
conflict are necessary. occurrence of A, O and C viruses in Africa has been
While it is true that some countries in southern associated in recent times with imports of livestock
Africa have established large FMD-free zones that from Europe and South America but it is not known
enable access to international beef markets, so far whether or not one or more of these serotypes has
– over a period of 30 years or more – that approach a more ancient association with sub-Saharan Africa
has not proven implementable by other countries in (Vosloo et al., 2002a). Based on their historic, geo-
sub-Saharan Africa. The net result is that only about graphical and species associations, it is clear that the
15% of cattle raised in countries of the Southern SAT serotypes evolved in sub-Saharan Africa, most
African Development Community (SADC) main- likely in association with African buffalo Syncerus
land are currently located in FMD-free zones. This caffer caffer (Fig. 9.1). The history of FMD in South
means that the vast majority of livestock producers Africa indicates that indigenous people in the hin-
are excluded from high-value markets. terland of the country were aware of the disease by
Alternative, non-geographic approaches to sani- 1780, i.e. before it could have been introduced by
tary trade standardization (e.g. commodity-based Europeans (Henning, 1956).
trade [CBT]) have been advanced as a solution to Based on the above and Bayesian evolutionary
this problem and recently amended international analysis of the 1D coding region of FMD viruses
standards have begun to provide new opportunity of all serotypes, it is likely that a common progeni-
on this basis. This chapter summarizes the current tor (named SAT X), that infected African buffalo,
situation in that respect and also points out that gave rise to all extant serotypes at around AD
sanitary trade standards and FMD management 1138 (Knowles, 2013). Serotype O probably arose
400–900 years ago from a SAT-like buffalo virus, et al., 2010a; Tekleghiorghis et al., 2014b). The
followed by the evolution of serotypes A and C from remaining 5 serotypes have different geographic
one serotype O lineage (Knowles, 2010). However, distributions and according to this, as well as the
it is not known whether or not this took place ini- genetic relationships between these viruses, Africa
tially in Africa or in Eurasia because cattle have been has been divided into three virus pools (Paton et
domesticated in Africa for many thousands of years al., 2009). In East Africa (pool 4) and West Africa
and one lineage may have first been domesticated in (pool 5), serotypes O, A, SAT-1 and SAT-2 are
Africa (Stock and Gifford-Gonzalez, 2013; Decker found, while in southern Africa (pool 6), SAT-1,
et al., 2014). A recent bottle-neck in the evolution SAT-2 and SAT-3 predominate. Considerable over-
of FMD viruses in Africa was caused by the Great lap in the presence of viruses from East and West
Rinderpest Pandemic that afflicted most of sub- Africa indicate that the boundaries between these
Saharan Africa from 1887–1904; it decimated both pools are affected by animal movement and trade
cattle and African buffalo and could have resulted in (Tekleghiorghis et al., 2014a).
viral extinctions (Knowles, 2013). Since the Great This chapter will focus on work conducted over
Rinderpest Pandemic there have been a number the last 40 years, mainly in southern Africa, aimed
of introductions of type O and A viruses to Africa at elucidating the maintenance of SAT viruses by
but representatives of African lineages or earlier re- wildlife populations and the transmission of these
introductions could also be present. viruses between wildlife and domestic species. In
Whatever the case, there is no evidence for addition, control options and trade possibilities in
historic associations between aphthoviruses and the face of a free ranging maintenance host of FMD
wildlife in regions other than sub-Saharan Africa, virus will be explored.
although a wide range of wildlife species endemic
to various regions of the world may become
infected, and sometimes subsequently transmit the African buffalo Syncerus caffer:
infection, following contact with infected domes- the maintenance host of the SAT
tic animals (Hedger, 1981; Thomson et al., 2001, type FMD viruses
2003a; Weaver et al., 2013). The general observation has been made that wher-
Currently six of the seven serotypes are prevalent ever FMD has been eradicated or spontaneously
in Africa; only Asia-1 has never been recorded. The disappeared among domestic animals in a particu-
last occurrence of serotype C was in 2004 in Kenya lar area, infection also disappeared from wildlife
and genetic comparisons suggest poorly inactivated (Roeder, 2009; Thomson et al., 2001; Thomson et
vaccine as the source (Sangula et al., 2011). Recent al., 2003a; Weaver et al., 2013). The African buffalo
serological indications that serotype C may be is the only wildlife species unequivocally shown
present in East Africa still need to be confirmed to be capable of independently maintaining FMD
by virus isolation (Rufael et al., 2008; Ayebazibwe viruses for long periods of time (Hedger, 1972,
1976; Hedger et al., 1972; Condy et al., 1985; (Hedger, 1968, 1972, 1976; Condy et al., 1969;
Thomson, 1994; Thomson et al., 2001, 2003a; Hedger et al., 1969, 1973; Thomson, 1994; Thom-
Thomson and Bastos, 2004). Bearing in mind that son and Bastos, 2004). However, buffalo in the
all cloven-hoofed animals are potentially suscepti- southern-most contemporary distribution of the
ble to infection with FMD viruses, the observation species are an exception (Esterhuysen et al., 1985).
above does not preclude the possibility that other The populations in the Hluhluwe and Umfolozi
wildlife species may in certain circumstances be Game Reserves as well as nearby small groups of
able to maintain FMD viruses for relatively short buffalo were found to lack serological evidence of
periods of time. infection with SAT viruses, which confirmed the
Owing to the extreme variability in morphology long-established view that FMD does not occur
of the African buffalo, there is controversy about endemically in the KwaZulu/Natal Province of
the validity and taxonomic status of the various South Africa (with the recent exception of the
recognized subspecies. Previously three or four Ndumo Game Reserve – www.daff.gov.za/docs/
subspecies of the African buffalo were recognized, media/FMD_Report20May2011.pdf). A similar
namely the large, black savannah buffalo Syncerus situation occurs in the Addo National Elephant
caffer caffer (from southern Africa), S. c. brachyceros Park in the Eastern Cape in South Africa and in
(from West Africa), the central African savannah isolated populations in Namibia. The reason for this
buffalo S. c. aequinoctialis and the forest buffalo S. c. has been speculated upon but there is no certainty
nanus. A recent study, using a set of mitochondrial on the matter (Esterhuysen et al., 1985). Occasion-
D-loop sequences indicated that there are two ally, groups of animals have been encountered
major lineages, with one containing all previously where only one SAT type was present such as those
identified subspecies from West and Central Africa in the Madimbo Strip (a strip of land along the
(S. c. nanus, S. c. brachyceros and S. c. aequinoctialis) South African border with Zimbabwe) where infec-
and the other from East and southern Africa (S. c. tion with SAT-3 virus exclusively was found in the
caffer) (Smitz et al., 2013). Regardless of the clas- resident buffalo population (Records of the Onder-
sification, little is known about the susceptibility of stepoort Veterinary Institute, unpublished 1991).
all these subspecies to FMD virus infection, and this Evidence of FMD infection disappearing from
constitutes a major gap in our knowledge regarding small groups of buffalo (43 and 70 free-ranging ani-
FMD epidemiology (see below). Although African mals, respectively) has also been found and it was
buffalo are found throughout sub-Saharan Africa, postulated that this occurred due to the failure of
they are increasingly being confined to conserva- recruiting more animals over successive years (cited
tion islands with the largest populations in East and by Anderson, 2003). As a result of extensive breed-
southern Africa (East, 1999; Casey et al., 2014). For ing programmes in southern Africa, especially in
example, the numbers of buffalo estimated in Tanza- Zimbabwe and South Africa, substantial numbers
nia, Uganda and Kenya in 1998 was approximately of FMD-free buffalo have been raised. Elsewhere in
382,000, whilst in southern Africa (Zimbabwe, southern Africa, serological evidence indicates that
Zambia, South Africa and Botswana) the combined the three SAT serotypes are the prevalent serotypes
populations were estimated at 148,000, compared in buffalo in Zambia (Sikombe et al., 2015), which
with central Africa (Gabon, Central African Repub- is most probably the situation for all buffalo popu-
lic and the Democratic Republic of Congo), where lations in southern Africa (Thomson and Bastos,
the number was 78,180. In most areas the popula- 2004; Penrith and Thomson, 2012). Viruses from
tions were deemed as stable or decreasing; only in all three SAT serotypes were isolated from buffalo
southern Africa were numbers reported as increas- probangs from national parks in Mozambique,
ing (East, 1999). Zambia and Tanzania (Kasanga et al., 2014).
In other regions of sub-Saharan Africa, studies
Association between African buffalo into the relationship between wildlife and FMD
and FMD viruses viruses have been less extensive. In eastern Africa,
In southern Africa most populations of African buf- for example, where large populations of wildlife
falo have been shown to harbour SAT type viruses; including buffalo occur, most studies focused on
generally all three serotypes simultaneously serological assays to determine the prevalence of
has been observed that in calves raised in captivity It should be remembered that 10–14% of animals
with their mothers, detectable antibody levels may in breeding herds may comprise calves less than a
persist for up to 17 months in a few individuals year old and that such herds may contain more than
while in approximately 50% of the calves antibody a thousand animals. Although there are seasonal
levels are detectable between 8–15 months of age calving peaks, calves are born throughout the year,
(W. Vosloo and R.G. Bengis, unpublished results). providing a constant supply of susceptible animals
Protection of calves from infection may not persist (Hedger, 1972). Thus a significant subpopulation
beyond 3–4 months, presumably because high of naive animals may be present in breeding herds
antibody levels are required to provide protec- at any given time. Infection in one animal would
tion from infection (Condy and Hedger, 1978). likely spread rapidly to other susceptible animals
The latter observation has been used to raise buf- of the same approximate age (Fig. 9.2). It has been
falo calves free from FMD infection by removing shown that infection of naive buffalo with SAT
the calves from their mothers immediately after viruses results in excretion of infectivity by routes
birth or within a few weeks (Condy and Hedger, similar to those of cattle, although of a lower order,
1978; Laubscher and Hoffman, 2012). However, and viral excretion appears to persist for longer
transmission from dams to calves is also an erratic than in acutely infected cattle (Gainaru et al., 1986;
occurrence. This was demonstrated in an experi- Thomson, 1994; Thomson and Bastos, 2004).
ment where calves free of FMD were bred, and 2 Following the acute stage of infection that lasts
of 17 calves failed to obtain detectable colostral less than two weeks, as is the case with a number
antibodies from their dams and did not become of ruminant species, detectable virus disappears
infected for the 5 months during which they were from all secretions and excretions of individual
kept with persistently infected cows (W. Vosloo and animals with the exception of those of the pharynx,
R.G. Bengis, unpublished results). Similarly, calves where low-level viral replication persists in ≤ 60% of
born to a group of captured buffalo cows that were individuals (Hedger, 1972, 1976; Anderson et al.,
held in pens in the KNP for more than a year failed 1979; Thomson, 1996). The levels of virus recov-
to become infected by these cows (Thomson et al., erable from oesophageo-pharyngeal secretions of
1985). persistently infected buffalo (often referred to as
Most buffalo calves in the KNP become infected ‘carriers’) are usually undetectably low, although
during their first year of life and probably soon after levels >104 TCID50 have been reported (Anderson
their passively acquired antibody levels fall below et al., 1979; Dawe et al., 1994b). Individual animals
the protective threshold (Hedger, 1972; Thomson may remain persistently infected for at least 5 years
et al., 1992; Thomson, 1994; Thomson and Bastos, (Condy et al., 1985) but it is probable that a signifi-
2004). Condy and Hedger (1978), working in cant number of animals fail to maintain infection
the WNP, found that all 17 captured calves aged for a prolonged period of time because the pro-
between 6 and 8 months were infected. In East portion of persistently infected animals falls after
Africa, buffalo also seem to sero-convert within reaching a peak in the 1–3 year age group (Hedger
the first 1 to 2 years with sero-prevalence higher 1976; Anderson, E.C. and Knowles, N.J., personal
to SAT-2, followed by SAT-1 and lastly by SAT-3, communication, 1994). The frequency and titre
although it was not clear whether or not the SAT-3 of the virus recovered decrease over time (Vosloo
results were due to serological cross-reactions et al., 2007) and it has also been shown that per-
(Bronsvoort et al., 2008). The observation that sistently infected buffalo can clear infection under
calves do not necessarily become infected by their experimental conditions over a 15-month period
mothers and that, in the KNP at least, infection ( Juleff et al., 2012a).
with SAT-1 usually precedes that with SAT-2 and A further site of viral persistence in buffalo has
3 (Condy and Hedger, 1974; Thomson et al., 1992, been shown to be germinal centres of lymphoid tis-
2003a; Thomson, 1994; Thomson and Bastos, sues ( Juleff et al., 2012a,b), similar to cattle where it
2004), contributed to the conclusion that infection was shown that virus locates to and is maintained in
of most calves in breeding herds probably occurs a non-replicating state in the light zone of germinal
as a result of ‘childhood’ epidemics, i.e. horizontal centres within the dorsal soft palate, pharyngeal
transmission between calves less than one year old. tonsil, palatine tonsil, lateral retropharyngeal lymph
On first infection young buffalo excrete large Following recovery from acute infection >50%
quantities of SAT viruses for 2-3 weeks although of infected calves become persistently infected
clinical signs are mild or absent (carriers) for months to years, i.e. low levels of
virus present in the oro-pharynx.
Figure 9.2 Diagrammatic representation of the theory for transmission of SAT serotype FMD viruses within
breeding herds of African buffalo in southern Africa (where buffalo breed seasonally) and transmission of these
viruses from acutely infected- (efficient pathway) and carrier buffalo (inefficient pathway) to other cloven-hoofed
species. Source: Foot-and-mouth disease: Southern Africa, Bulletin #4, March 2014 (http://www.afrivip.org/
node/3857/zip_download).
node and mandibular lymph node following pri- addition, because buffalo populations have been
mary infection ( Juleff et al., 2008). The authors fragmented for many years now, different buffalo
postulated that it is possible that maintenance of populations can be differentiated on the basis of
non-replicating FMD virus in these sites represents SAT-type viruses recovered from persistently
a source of persisting antigens and contributes to infected animals representative of those popula-
the generation of longer-lasting antibody responses tions (Vosloo et al., 2001). Even within the buffalo
against neutralizing epitopes of the virus than can population of the KNP (approximately 30,000
be generated by inactivated vaccines. individuals), clear intratypic differences in the
More than one type of SAT virus may be genomes of SAT-1, -2 and -3 viruses obtained from
maintained by individual buffalo (Hedger, 1972; different regions of the KNP have been shown
Anderson et al., 1979). This is despite the fact that (Vosloo et al., 1995, 2001; Bastos et al., 2000,
high levels of antibody to the viruses concerned 2001, 2003).
occur in pharyngeal secretions (Hedger, 1976; The fragmentation of southern Africa’s buf-
Francis and Black, 1983). It was demonstrated falo population occurred as a result of the drastic
that persistently infected buffalo are refractory to reduction (approximately 10,000-fold) in buffalo
re-infection with the same strain of virus (Hedger numbers following the Great Rinderpest Pandemic
et al., 1972). of 1887–1904 (Rossiter, 1994) as well as human
expansion into formerly deep rural areas. That is
Genetic diversity of FMD viruses likely the reason why some small populations within
derived from buffalo in sub-Saharan the southern-most buffalo distributional range are
Africa free of FMD (Esterhuysen et al., 1985). The present
Genomic analysis of viruses isolated from domestic geographic isolation of discrete buffalo populations
and wildlife species for the three SAT serotypes in ‘conservation islands’ probably explains the
divides each serotype into a number of topotypes, locality-specific distribution of viral topotypes
i.e. genotypes that occur within a specific geo- apparent today. High mutation rates (Vosloo et al.,
graphic region (Bastos et al., 2001; Samuel and 1996, 2007) and continuous, independent virus
Knowles, 2001). To date, nine topotypes have cycling within discrete buffalo populations (Condy
been assigned for SAT-1 (Sangula et al., 2010; et al., 1985) probably account for the extensive
Sallu et al., 2014), 14 for SAT-2 (Ayelet et al., 2009; intratypic variation evident today.
Habiela et al., 2010) and six for SAT-3 (Bastos et al, For all three SAT serotypes in southern Africa it
2003) (Table 9.1). Since a number of these studies has been demonstrated that the genetic differences
included historical viruses, some topotypes may between viruses from different topotypes is such
be extinct as no recent, related isolates have been that outbreaks should be traceable to specific coun-
found. The number of viruses recovered from wild- tries, game parks and even to specific regions within
life in most regions of sub-Saharan Africa is limited, game parks (Bastos, 2001; Bastos et al., 2001, 2003;
the exception being southern Africa, making accu- Vosloo et al., 2001, 2002a,b). However, some
rate inferences on genetic relationships difficult. countries such as Botswana and Zimbabwe have
Outside southern Africa, viruses from wildlife gen- more than one topotype within their borders and if
erally fall within topotypes previously described for uncontrolled movement of buffalo occurs, distribu-
domestic animals, but there is evidence, for SAT-1 tion of topotypes will become commingled (FMD
at least, that unassigned topotypes are present Bulletin, 12 December 2011). The high levels of
(Ayebazibwe et al., 2010b; Kasanga et al., 2014; genetic diversity will most likely be reflected in
Wekesa et al., 2015). antigenic differences and it has been shown that for
In southern Africa, where most detailed vaccination to be effective, the viruses incorporated
genetic analyses have been performed, SAT-type into vaccines need to be antigenically related to
viruses have been shown to be constantly evolv- viruses circulating in the field (Hunter et al., 1996;
ing in buffalo populations (Vosloo et al., 1996; Hunter, 1998). Therefore, the uncontrolled move-
Bastos et al., 2001, 2003), as would be expected ment of buffalo within the southern African region
from what is known of the quasispecies dynam- could have severe implication for the control of
ics of FMD viruses (Domingo et al., 2003). In FMD (Maree et al., 2014).
Table 9.1 Summary of the historical topotype distribution across countries for SAT-1, SAT-2 and SAT-3. The
current topotype distribution is not certain due to lack of sample submission from both livestock and wildlife
for further characterization (Information kindly provided by NJ Knowles from records at the World Reference
Laboratory, The Pirbright Institute)
Serotype Topotype Countries
SAT-1 NWZ/I Kenya, Malawi, Mozambique, Tanzania, Zambia, Zimbabwe (north-west)
SEZ/II South Africa, Swaziland, Zimbabwe (south-east)
WZ/III Botswana, Namibia, South Africa, Zambia, Zimbabwe (west)
IV Uganda
V Niger, Nigeria
VI Israel, Nigeria, Sudan, Yemen
EA-2/VII Uganda
EA-3/VIII Uganda
IX Ethiopia
SAT-2 I Botswana, Malawi, Mozambique, South Africa, Zimbabwe (south-east)
II Botswana, Namibia, Zimbabwe, Zambia
III Botswana, Namibia, Zambia, Zimbabwe
IV Bahrain, Burundi, Ethiopia, Kenya, Malawi, Tanzania, Uganda, Yemen, Zambia
V Ghana, Nigeria, Senegal
VI Gambia, Senegal
VII Cameroon, Egypt, Eritrea, Ethiopia, Libya, Mali, Mauritania, Niger, Nigeria, Oman,
Palestinian Autonomous Territories, Saudi Arabia, Sudan, Uganda
VIII Burundi, Democratic Republic of the Congo, Rwanda
IX Kenya
X Democratic Republic of the Congo, Uganda
XI Angola
XII Uganda
XIII Ethiopia, Sudan
Sudan
XIV Ethiopia
SAT-3 SEZ/I South Africa, Zimbabwe (south-east)
WZ/II Namibia, Botswana, Zimbabwe
NWZ/III Malawi, Zimbabwe (north-west)
ZAM/IV Zambia
EA/V Uganda
VI Uganda
Knowledge of the occurrence and distribution behaviour of buffalo harbouring SAT-type viruses
of SAT virus lineages and topotypes in buffalo (Condy et al., 1969; Hedger et al., 1969, 1972;
populations has enabled the transmission of SAT Condy, 1971, 1979; Hedger, 1972, 1976; Condy
viruses from buffalo to other species to be unequiv- and Hedger, 1974; Hedger and Condy, 1985).
ocally proven (Bastos et al., 2000; Brückner et al.,
2002; Vosloo et al., 2002b; Thomson et al., 2003a). Ability of acutely and persistently
Further details are provided below. This confirms infected buffalo to transmit SAT
the early observations made by J.B. Condy and R.S. viruses to cohorts
Hedger into the association between the occur- Transmission of SAT-type viruses between indi-
rence of FMD in cattle and the distribution and vidual buffaloes appears to occur in two ways: (1)
contact transmission between acutely infected and following loss of maternally derived immunity in
susceptible individuals that is likely to account the calves and then fall as the number of susceptible
for the majority of infections and (2) occasional individuals decreased. Furthermore, periods of
transmission between persistently infected buffalo time where no new infections occurred within the
and susceptible individuals (Fig. 9.2). Although herd were predicted resulting in the infection ‘dying
precisely how persistently infected buffalo transmit out’. The only way in which viral circulation could
the infection to susceptible cohorts is unknown be re-established was through the introduction of
(Thomson, 1996), a possibility for which the new acutely infected individuals at a time when a
evidence remains tenuous is sexual transmission new cohort of susceptible young animals had been
(Bastos et al., 1999; Thomson et al., 2003a). In a created by the next batch of calves losing their
separate study in buffalo 3–5 years of age, virus was maternal immunity or by re-activation of the virus
isolated from only one of 108 sheath washes and by transmission of the virus from a ‘carrier’ to the
one of 23 testes samples. No virus was isolated from new group of susceptible calves (Thomson et al.,
the reproductive tracts of 25 adult female buffalo 1992). It should be remembered, however, that, in
(W. Vosloo, B. Botha and C.I. Boshoff, unpublished reality, transmission of SAT viruses within buffalo
results). It is also not known whether these animals herds is more complex than this because such herds
were actively or persistently infected. However, usually maintain all three SAT serotypes simultane-
these results indicate that the presence of virus in ously.
the reproductive tract of buffalo is a rare occurrence. It is likely therefore that there is a period of time
Therefore, unless further and more convincing evi- each year, probably covering 2–3 months, when
dence for sexual transmission can be produced, it significant numbers of young buffalo in breeding
can only be considered an interesting theory. herds will be excreting SAT viruses that pose a
The close contact that occurs between buffalo threat of infection for other species that may come
in breeding herds, especially among juveniles, is in close contact with buffalo breeding herds.
likely to ensure efficient transmission of SAT-type
viruses, bearing in mind the levels of excretion that Transmission of SAT viruses from
have been demonstrated in naive animals infected buffalo to other species
for the first time (Gainaru et al., 1986). Transmis- In the KNP outbreaks of FMD among impala
sion between persistently infected buffalo and Aepyceros melampus within the Park are a regular
their cohorts has been shown (Condy and Hedger, occurrence, although strangely, other species are
1974) but is less efficient than that involving acutely rarely affected. This could be due to the low mini-
infected animals that excrete large quantities of virus mum infectious dose (approximately 1 TCID50 was
(Thomson et al., 1985). Generally, these infections shown to be capable of infecting adult impala by the
are ‘silent’ because buffalo generally suffer few ill respiratory route – R.G. Bengis and G.R. Thomson,
effects from infection (see below). Therefore, infec- unpublished data) required to infect impala, in con-
tion can only be inferred from serology or other trast to cattle and sheep that require 10–25 (Gibson
laboratory-based investigations. and Donaldson, 1986; Donaldson et al, 1987).
To better understand the complex interac- Since 1938 there have been ~ 60 recorded outbreaks
tions between breeding herds of buffalo and SAT of FMD in impala the KNP and of the 25 outbreaks
viruses, the above factors were incorporated into a recorded between 1976 and 2008, 62% were caused
stochastic model for the behaviour of a single SAT by SAT-2, followed by SAT-1 (28%) and a single
virus in a breeding herd of buffalo numbering up to outbreak due to SAT-3. These outbreaks lasted 4.4
500 individuals. Included were variable transmis- months on average (range 1–13 months) (Vosloo
sion coefficients for spread of the infection within et al., 2009). Unlike the situation in buffalo, these
subgroups of the herd (such as family sub-groups) outbreaks have been identified by the fact that, usu-
as well as within the herd at large (Thomson ally, at least a proportion of impala developed clear,
et al., 1992). Using values for the transmission although transient, clinical signs of FMD. Sequence
coefficients that are plausible based on current analysis of the SAT-2 viruses involved has shown
understanding, the model predicted that each year that these outbreaks were causally distinct (Vosloo
new infections in the herd would rise to a peak et al., 1992; Bastos et al., 2000). Infection and attack
rates have varied in outbreaks of FMD in impala that the fact that impala are more frequently infected
have been studied, with the latter sometimes much than other species supports the probability that
lower than the former, indicating that subclinical impala are more susceptible than wildlife species
infection is common (Keet et al., 1996), as has been other than buffalo. It was furthermore speculated
seen in experimentally infected impala (R.G. Bengis that the buffalo: impala ratio may explain differing
and G.R. Thomson, unpublished data). Serological patterns of infection in impala in this study (Vosloo
evidence has indicated that impala in other regions et al., 2009). At the two study sites where impala
of sub-Saharan Africa, where impala are abundant, were relatively abundant, SAT infection occurred,
have also been infected but clinical disease has not whereas where relatively fewer impala were present
been reported (Anderson et al., 1993) except for relative to buffalo, infection did not occur. It would
anecdotal, unconfirmed reports (S. Cleaveland, therefore appear that the higher the proportion of
personal communication, 2015). That outbreaks impala relative to buffalo, the more likely SAT virus
of FMD in wildlife have only been recorded in the transmission to impala becomes.
KNP could be due to the fact that surveillance in Persistent infection in impala has not been
this Park is more thorough than elsewhere. demonstrated (Hedger et al., 1972; Anderson et
Between 1997 and 2007 a serological survey was al., 1975; C. de W. van Vuuren, personal commu-
performed on impala in the KNP in three defined nication, 1997). However, FMD epidemics caused
locations located in the north, centre and south of by identical viruses have recurred in impala 6–18
the Park. Animals were sampled on a three-monthly months after the original outbreak ceased (Vosloo
basis in each of the locations, involving collection of et al., 1992; Keet et al., 1996), indicating that the
blood for serum preparation as well as examination virus may have been maintained within the impala
for clinical disease. About 40 randomly selected population. The more likely explanation is that the
animals were darted with a tranquillizer at each same virus was transmitted on more than one occa-
sampling. In the most northern sampling site, no sion from buffalo to impala in the same vicinity.
serological or clinical evidence of infection was Evidence based on genome sequencing of
found, while in the central site, serological data appropriate viruses indicates that impala in the
indicated that both SAT-1 and SAT-2 infections KNP usually first become infected with SAT
had occurred on at least three occasions, although viruses derived from buffalo in the vicinity (Keet
no virus was recovered because lesions were not et al., 1996; Bastos et al., 2000; Bastos et al., 2003).
detected. However, mixed infection in impala herds It is presumed that this results from direct or indi-
has been confirmed by virus isolation previously rect contact between resident impala and buffalo
(Vosloo et al., 2009). The most southern sampling breeding herds. However, there is a paradox in
site had serological evidence of at least six SAT-2 understanding the transmission of FMD viruses
outbreaks. A regression model indicated that from buffaloes to impala because while SAT-1
summer and autumn were the highest risk periods viruses appear to circulate more rapidly within
associated with sero-prevalence, which was in buffalo herds in the KNP (see above) and would
contrast with clinical data that indicated most out- therefore be expected to be associated more
breaks occur at the end of the dry season (Bengis frequently with impala outbreaks, most FMD
et al., 1994; Bastos et al., 2000; Vosloo et al., 2006). outbreaks in impala within the KNP have been
Vosloo et al. (2009) postulated that this discrepancy caused by SAT-2 viruses (Thomson, 1994; Bastos
could be explained by a slow progression of infec- et al., 2000; Thomson and Bastos, 2004; Vosloo et
tion from the dry season into summer, i.e. the wet al., 2009). A possibility is that impala are more sus-
season. This slow progression of infection in impala ceptible to infection with SAT-2 viruses than with
herds had previously been shown by Keet et al. SAT-1 or 3 but there is no direct evidence for that.
(1996). Females and older animals also had a higher Sequence data derived from historic South Afri-
risk of sero-positivity that could be explained by can FMD outbreaks in cattle indicate that there were
impala herd structure and behaviour (Vosloo et al., occasions when related SAT isolates were obtained
2009). It has been observed that contact between from cattle before they were recovered from impala.
buffalo and impala constitutes < 10 % of impala This suggests that the impala may have acquired
interactions with other species (Whyte, 1976), so the infections from contact with cattle rather than
buffalo (Vosloo et al., 2006). This is more likely intact FMD virus particles are retained on the fol-
to have occurred in the past than in recent times, licular dendritic cell network may explain increased
because only since the early 1970s has the whole shedding of virus from ‘carriers’ experiencing
KNP been game-fenced and, since the early 1980s, immune activation involving enhanced lympho-
cattle immediately outside the Park are vaccinated cyte migration through lymph nodes. In contrast,
on a biannual basis. Conversely, as explained above, corticosteroids are known to inhibit lymphocyte
there is evidence that on occasion SAT viruses have migration and therefore, immunosuppressed hosts
been transmitted first from buffalo to impala and would possibly experience decreased shedding in
then from impala to cattle (Sutmoller et al., 2000; secretions ( Juleff et al., 2012b).
Hargreaves et al., 2004). In this way impala, and pre- The observation that male buffalo and female
sumably other antelope species less frequently, can cattle were consistently involved where carrier
provide a conduit of infection between buffalo and transmission was suspected, raised the spectre of
livestock. It seems therefore that although impala sexual transmission (Bastos et al., 1999). This was
and other antelope species are unable to maintain rendered plausible by the reported observation by
SAT viruses independently, they sometimes serve farmers in Zimbabwe and South Africa that buffalo
as transmission intermediaries between buffalo and bulls sometimes mount domestic cows in the field.
livestock. As explained above, however, the issue of sexual
Much experimentation has attempted to transmission remains a paradox.
explain how persistently infected buffalo transmit There is no doubt that contact between buf-
the infection to cattle with which they come in falo and cattle occasionally results in outbreaks
contact (Hedger, 1970; Condy and Hedger, 1974; of FMD caused by SAT viruses among cattle in
Anderson et al., 1979; Condy et al., 1985; Hedger southern Africa (Condy, 1979; Thomson, 1994,
and Condy, 1985; Bengis et al., 1986; Dawe et al., 1996; Thomson and Bastos, 2004; Miguel et al.,
1994a,b; Vosloo et al., 1996; Juleff et al., 2012a). 2013; Kock, 2014). The involved circumstances
Unlike the confusion surrounding the possibility whereby outbreaks of FMD in cattle originate from
that ‘carrier’ cattle may rarely transmit the infection buffalo/cattle interaction was demonstrated by
to cohorts in close contact (Salt, 1993; Thomson, FMD occurrence in Swaziland in 2001. The origin
1996), it has been shown unequivocally that per- was traced back to South Africa through cattle that
sistently infected buffalo are able to transmit the became infected by contact with buffalo. The cattle
infection not only to other buffalo (Condy and were subsequently exported to Swaziland and some
Hedger, 1974), but to cattle as well (Dawe et al., found to be infected at slaughter (Brückner et al.,
1994a,b; Vosloo et al., 1996). This was shown by 2002; Vosloo et al., 2002b; Thomson et al., 2003a).
demonstrating that the viruses causing FMD in the A recent discrete-traits analysis using 1D gene
cattle were identical to those ‘carried’ by buffalo sequences of SAT-2 isolates where the species of
with which they were in contact based on nucleo- origin is known, supported viral transitions from
tide sequencing. African buffalo to cattle at a high level of confidence
The mechanism whereby this occurs is still and from buffalo to impala and vice versa as well as
uncertain although it is known that direct contact from cattle to buffalo, although the posterior sup-
between persistently infected buffalo and cattle is port for these did not reach 95% (Hall et al., 2013).
required over a period of several weeks or months However, the authors warned that these results
(Dawe et al., 1994a,b; Vosloo et al., 1996). Initially, should be interpreted with care due to sampling
it was speculated that persistent infection (either bias. Nevertheless, the findings support epidemio-
cattle or buffalo) could be reactivated by stress, logical observations. Studies in Zimbabwe at the
and an instance where this was thought to have wildlife/domestic interface have provided strong
occurred was reported (Hedger and Condy, 1985). evidence that contact rates between cattle and buf-
Subsequent experimental work in cattle failed to falo significantly influenced FMD dynamics, but
substantiate this possibility; treatment of ‘carrier’ that factors influencing the contact-rate differed
cattle with corticosteroids resulted in the disap- between study sites (Miguel et al., 2013). It is there-
pearance of the virus from oesophago-pharyngeal fore impossible to predict which factors will apply
samples (Ilott et al., 1997). Recent findings that in any particular locality.
In East Africa, in contrast to the above, it would mortality could therefore not be proven. It seems
appear that the transmission of FMD from buffalo that the disease was transmitted from cattle to the
to cattle is less frequent. However, studies into this wildebeest because FMD in domestic animals was
connection require extension before firm conclu- reported in February when the wildebeest were
sions can be reached (Wekesa et al., 2015). in the southern plains where there is little contact
with cattle. As soon they migrated into western
Serengeti in June, where they came in contact with
The role of wildlife species other livestock, the problem in wildebeest became evi-
than buffalo in the epidemiology dent. Surprisingly, however, other antelope species
of FMD in southern Africa in the vicinity remained apparently unaffected (T.
Weaver et al. (2013) listed over 100 species that Mlengeya, personal communication, 2003). How-
have been shown to be susceptible to FMD virus; ever, outbreaks are seemingly not as common in
however, many of these were artificially infected wildlife as in cattle in the Serengeti, where frequent
and it is not clear whether or not they would be sus- outbreaks are documented in communities sur-
ceptible to infection by natural routes and able to rounding the national park (S. Cleaveland, personal
transmit the infection. Many wildlife species have communication, 2015).
been reported as having been infected (Macaulay, Essentially all cloven-hoofed animals and
1963; Hedger, 1981) and a wide range of species in Camelidae (i.e. members of the order Artiodactyla)
southern Africa have been shown to have antibodies are susceptible to infection with FMD viruses.
to FMD virus (Lees May and Condy, 1965; Condy FMD infection is dependent on the species and
et al., 1969). Recently, a serological study in central even breed of animals, the strain and dose of virus
and West Africa added Buffon’s Kob Kobus cob and causing infection and the level of immunity of the
Oribi Ourebia ourebi, to the list of naturally infected animals (Thomson, 1994; Thomson and Bastos,
animals (Di Nardo et al., 2015). However, the bulk 2004). The relative susceptibility of most wildlife
of evidence suggests that wildlife species other than species is unknown, as are the levels of virus excre-
buffalo play only a minor role in the maintenance tion that occur during infection. With the exception
and spread of FMD viruses (Roeder, 2009; Weaver of impala, no studies have been conducted on the
et al., 2013). Elsewhere in sub-Saharan Africa, the minimum infectious dose of FMD viruses required
information available on this issue is too limited to to initiate infection (see above).
allow an opinion to be formed. Impala seem to be The only species in which virus persistence has
the most susceptible of these species (see above) been unequivocally demonstrated to be impor-
in South Africa and are considered an indicator tant in maintenance and transmission of the SAT
host for the presence of SAT viruses, because serotypes FMD is the African buffalo (see above).
infection in impala in the past often presaged the However, persistence of FMD viruses in the oro-
occurrence of FMD in livestock (Meeser, 1962). pharynx for more than 4 weeks following infection
In other countries in the region kudu Tregalaphus – the criterion that is usually used to define carriers
strepsiceros have been shown to be involved in – probably occurs in many ruminant species (Salt,
transmission of the disease (Hargreaves et al., 2004; 1993). Persistent infection developed experimen-
Letshwenyo et al., 2006). In the Serengeti, where tally in kudu and virus was isolated for up to 140
wildebeest are by far the most numerous large days after infection (Hedger et al., 1972). Excretion
mammal species, FMD is infrequently reported of FMD virus for a little over 4 weeks has also been
although a severe outbreak caused by SAT-2 was shown in wildebeest (Anderson et al., 1975) and
recorded in wildebeest in 1999. At least 20% of the sable antelope Hippotragus niger (Ferris et al., 1989).
migratory herd of wildebeest was affected. These In impala attempts to identify persistently infected
animals showed limping as a result of foot lesions animals have failed (see above). FMD infection
located on the coronary band and interdigitally and occurred in wildebeest, hartebeest Alcelaphus buse-
in some cases the hooves sloughed off. However, laphus, eland, gemsbok Oryx gazella and springbok
typical mouth lesions were not observed. Deaths Antidorcas marsupialis in the Kalahari region of
in calves occurred although no fresh carcasses Botswana after the widespread aphthisation of
for sampling were obtained and the cause of calf cattle during 1957 (Falconer, 1972). However, the
infection in the Kalahari did not persist, presum- 2003a). FMD in wildlife varies from ‘silent’ infec-
ably because African buffalo do not occur in that tion, as seems usually to be the case in African
region. For other antelope species there is simply buffalo, to acutely lethal, as was seen among moun-
no information on this issue. Outside Africa, fallow tain gazelles Gazella gazella in Israel (Shimshony
deer Dama dama, sika deer Cervus nippon and et al., 1986; Shimshony, 1988). Mortality due to
white-tailed deer Odocoileus virginianus have been FMD infection has also been described in impala
shown to become persistently infected with the (Hedger et al., 1972), blackbuck Antilope cervicapra
European serotypes (Forman et al., 1974; McVicar (Kar et al., 1983), saiga Saiga tatarica (Kindyakov et
et al., 1974; Gibbs et al., 1975). The epidemiologi- al., 1972; Khukorov et al., 1974), white tailed-deer
cal relevance of persistent infection of ruminants Odocoileus virginianus (McVicar et al., 1974), and
of all species with the exception of African buffalo warthogs (R. G. Bengis, personal communication).
remains doubtful. FMD infection appears to be subclinical in most
It was shown in experimental studies that in African buffalo based on observations made during
kudu and buffalo, among which ‘carriers’ occur culling operations in the KNP (cited by Thomson
(see above), antibody responses were higher and et al., 2003a). Experimentally, however, typical
of longer duration than in impala, warthogs Phaco- small lesions appeared, particularly on the feet of
choerus aethiopicus and bush pigs Potamochoerus some animals (Anderson et al., 1979; Gainaru et
spp. in which, like domestic pigs, the ‘carrier’ state al., 1986). Two instances of clinical disease due to
has not been demonstrated (Hedger et al., 1972). natural infection were reported in captive buffalo
This suggests a relationship between persistent kept for unrelated experiments (Young et al., 1972;
infection and the height and duration of antibody Thomson et al., 2003a). In a further outbreak of
levels in susceptible species. FMD among buffalo held in captivity for experi-
Giraffe Giraffa camelopardalis in the KNP have mentation on tuberculosis, mature animals, with
been observed with clinical disease, supported by high antibody levels to all three SAT serotypes,
serological results. However, despite experimental developed severe lesions on the dorsal and lateral
evidence that infected giraffe can transmit the dis- surfaces of the tongue, the insides of the cheeks
ease to in-contact animals, no virus was detected and hard palate with foam appearing at the corners
in probangs up to 60 days post infection and they of the mouth. However, no drooling of saliva was
are not deemed to play an important role in the observed. The animals avoided feeding and ‘soaked’
dissemination of FMD in the wild (Vosloo et al., their mouths in the water troughs. No foot lesions
2011). African elephants Loxodonta Africana, in were evident (Vosloo et al., 2007). In contrast to
contrast to Indian elephants Elephas maximus, do that, Young et al. (1972) described painful gait and
not appear to be susceptible to natural infection, salivation occurring during a separate outbreak in
although they were shown to be susceptible to captive buffalo. In the event described by Vosloo
needle inoculation (Howell et al., 1973; Hedger et al. (2007), the lesions healed within 2 weeks
and Brooksby, 1976; Bengis et al., 1984). Hippo- but remained visible as pale, weakly circumscribed
potamuses Hippopotamus amphibius do not seem to areas with poorly developed papillae. The infected
play a role in the epidemiology of the disease on the animals lost an average of 6% of their pre-infection
African continent. This has been discussed in more weight. Approximately 2 months after the clinical
detail elsewhere (Thomson et al., 2003a). onset, the buffalo began to recover their weight loss.
Even in sub-Saharan Africa, where wildlife are Less mature animals did not lose weight during the
clearly involved in the maintenance of FMD, live- outbreak; their weight gain was merely interrupted
stock sometimes transmit the infection to wildlife for a period of about a month. There was a signifi-
rather than the converse (Hedger, 1976; Thomson cant decline in red cell and lymphocyte counts, as
et al., 1984; Anderson et al., 1993). well as haematocrit and lymphocyte percentages
during the outbreak that lasted approximately 100
days. Conversely, the mean red cell volume and
Clinical signs of FMD in wildlife mean haemoglobin values were significantly raised
The clinical manifestation of FMD in wildlife has during the FMD outbreak (Vosloo et al., 2007).
been described elsewhere (Thomson et al., 2001, During an experiment involving buffalo
persistently infected with SAT-1, where SAT-2 followed by testing only the positive sera for anti-
virus was inoculated intra-dermolingually, small bodies directed against structural viral proteins (i.e.
interdigital lesions on one or more feet and single serotype-specific tests) that are able to differenti-
small ruptured lesion on the dental pad of two ate serotypes (Bronsvoort et al, 2008; Ayebazibwe
out of six animals were seen (Hedger et al., 1972). et al., 2010a, 2012; Di Nardo et al., 2015). This
SAT-1 was isolated from a mouth lesion. Dawe et may be acceptable on the basis of cost-saving, but
al. (1994b) also noted a single small interdigital given that so little is known about the duration of
lesion and a small tongue lesion in one buffalo that the antibody response to SAT NSPs, especially in
became naturally infected. It is therefore possible wildlife, it is also possible that the interpretation of
that lesions do occur naturally in buffalo, but are of results will be misleading. In addition, since only
such a limited size and duration that they have little a subset of sera is tested to determine serotype-
or no obvious effect on the animals concerned and specific reactivity, the apparent prevalence of
are generally inapparent. serotype-specific antibody in the case of wildlife
may also be unreliable. Moreover, increased sero-
logical cross-reactivity that occurs when animals
Diagnosis of FMD in wildlife have been infected with more than one serotype,
Clinical diagnosis of FMD in wildlife is more provides a further potential source of inaccuracy.
difficult than in domestic animals because most For example, some studies have found antibodies
animals need to be immobilized for close examina- to serotype C in wildlife and domestic species
tion. The tendency towards development of mild or (Rufael et al., 2008; Ayebazibwe et al., 2010a;
subclinical infections in wildlife also complicates Tekleghiorghis et al., 2014b), a serotype last
the diagnosis. However, it needs to be remembered confirmed by virus typing in 2004. These issues
that domestic livestock, especially sheep and goats, need to be considered carefully when interpreting
also suffer mild or inapparent disease. For these serological data emanating from wildlife.
reasons it is usually more difficult to recover lesion
material for viral identification in suitably equipped
laboratories. Control of FMD in sub-Saharan
The issue of laboratory diagnosis has been Africa where wildlife are
addressed by Thomson and co-workers (2003a). involved
More recently, the use of tests based on the detec- As indicated above, independent maintenance
tion of antibodies to the NSP of FMD virus has of SAT serotypes by wildlife presents a serious
been used extensively for surveillance and preva- complication for the control of FMD in parts of
lence determination, both in domestic animals Africa where these virus serotypes and abundant
and in wildlife (see above). For the latter, the wildlife occur together (Thomson et al., 2003a,
interpretation of results is simpler because wild- 2013a; Weaver et al., 2013; Maree et al., 2014). The
life are not normally vaccinated and therefore evidence is that SAT viruses are in fact African buf-
presence of antibodies directed against NSPs is a falo viruses which periodically spill-over into other
clear indication of infection. However, the dura- cloven-hoofed species – both wild and domestic
tion of these antibodies in wildlife has not been – in their close vicinity. Moreover, SAT serotypes
studied. In cattle the indications are that there are have a wider array of antigenic variants than other
differences in the duration and strength of NSP FMD virus serotypes, rendering vaccination against
responses where SAT-type viruses are involved, FMD caused by SAT serotypes less effective than is
i.e. in comparison with Eurasian serotypes (W. the case for FMD elsewhere in the world (Maree
Vosloo and J.J. Esterhuysen, unpublished data). et al., 2015). An additional complication in areas of
This is further complicated by the availability Africa where pastoralism is common (the Horn of
of a number of commercial as well as in-house Africa for example) is that spread of FMD viruses by
serological tests, few of which have been validated the uncontrolled movement of sympatric livestock
for use in wildlife (Bronsvoort et al., 2008). Most and wildlife is impossible to control effectively.
recent serological studies in wildlife first screened The net result is that management of FMD has not
sera for the presence of antibodies to the NSPs, improved in Africa in recent years and, in southern
Africa at least, has deteriorated demonstrably over limited spread, reflected in low apparent morbidity
the last decade and a half (Thomson et al., 2013a; and mortality rates reported to the World Organi-
Maree et al., 2014). sation for Animal Health’s (OIE’s) World Animal
Perhaps for understandable reasons, the meth- Health Information System (WAHIS – www.oie.
odologies by which FMD in situations outlined in int; Table 9.2). Furthermore, the mildness and
the paragraph above can or should be managed, are slow spread of FMD outbreaks in southern Africa
not yet well established, i.e. existing international is not a new phenomenon (Du Toit, 1932). For
recommendations and standards for FMD manage- these reasons it is likely that in southern Africa at
ment do not address these issues effectively. least, the indirect costs of FMD far outweigh those
In southern Africa at least, this confusion is occasioned directly.
further compounded by the fact that the actual The low incidence of disease associated with
economic impact of FMD is largely unknown. many, if not most cattle outbreaks caused by
Knight-Jones and Rushton (2013) have, on the SAT serotypes in southern Africa, is reflected in
basis of a literature review and expert opinion, Table 9.2. This shows ‘apparent morbidity rates’
estimated the overall cost of FMD in Africa to be (AMRs) of FMD within ‘disease events’ reported
between US$1–5 billion per annum; production by countries around the world to the OIE for a
losses were estimated at US$2.3 billion and vac- 4-year period. Most striking amongst these was the
cination costs at $20 million. The inference of this apparently completely subclinical infection event
publication is that direct impact outweighs indirect in South Africa’s FMD-free zone – consisting of
costs, an aspect that requires more focused investi- 46 related outbreaks – caused by a SAT-1 virus in
gation. In southern Africa at least, the probability is February 2011 that was only detectable serologi-
that costs occasioned by FMD are largely indirect; cally (WAHID – www.oie.int). Consequently, the
made up of (ignoring ‘externalities’ – Knight-Jones AMR in those 46 outbreaks was zero. Many other
and Rushton, 2013) the following: (1) the high outbreaks of SAT-type FMD in southern Africa
cost and logistical difficulty of controlling FMD, have been reported where the AMR was below
especially creation and maintenance of FMD-free 1% and a few below 0.01% (Table 9.2). This situ-
zones; more particularly erection and maintenance ation, surprisingly, appears to have occurred with
of the fencing systems for their biosecurity (accu- roughly equal frequency in previously vaccinated
rate costing of this aspect is impossible because and unvaccinated cattle populations. Table 9.2 also
disaggregated animal disease control costs borne shows that in events caused by O, A and Asia-1
by the public sector are not in the public domain), serotypes, the AMRs were consistently higher
(2) constraints to trade (e.g. South Africa’s meat than for SAT serotypes, i.e. for SATs the AMR was
industry claimed a figure of R 4 billion [equivalent below 1% in 25/42 (60%) events while in 100%
to $400 million at the time] annually between of events involving Eurasian serotypes, the AMR
2011–2014 when exports from the country’s FMD- was greater than 1% and in 70%, greater than 10%.
free zone were suspended due to a FMD outbreak), However, these conclusions need to be interpreted
(3) socio-economic and environmental impacts with caution because there was lack of clarity as to
that ensue from control actions (Barnes, 2013; how the denominator (i.e. susceptible population)
Cassidy et al., 2013; Thomson et al., 2013a) and (4) was arrived at in order to derive the AMRs in these
exclusion of livestock owners who are not located reports. Nevertheless, it does appear that reported
within the FMD-free zone from market access. In AMRs of SAT outbreaks that occur in endemic
Namibia, for example, the so-called red-line fence situations in Africa, are unusually low and inde-
has precluded more than half the country’s cattle- pendent of vaccination history.
owning population from access to export markets It is intuitively obvious that where mild disease
for decades with resultant political tensions (Iita, occurs with such low prevalence, detection of the
2014). presence of infection through physical inspection
Direct impacts certainly occur and occasion- is unlikely to be reliable. It must be remembered
ally have been reported as being appreciable but, moreover that physical inspection forms the back-
in general, FMD outbreaks in southern Africa are bone of FMD surveillance in southern Africa. The
characterized by mild disease and generally slow, corollary to this observation is that the incidence
Table 9.2 Immediate notifications by countries to the World Organisation for Animal Health’s information
system (OIE – WAHIS) of FMD events involving cattle showing apparent morbidity rates for each event between
2011–2014
Presence of Apparent
Reported preventative morbidity
events vaccination rate (AMR%)
involving programme associated with
Year Country cattle Serotype(s) (Yes/No) each event Comment
2011 Botswana 4 SAT 2 Yes 5.5; 0.6; 0.2; 1.3
China (People’s 1 O ? 8.0
Republic)
Israel 3 O ? 3.4; 4.7; 37.5
Kazakhstan 2 O ? 6.7; 34.4
Korea (Democratic 1 O ? 36.8
People’s Republic)
Korea (Republic) 1 O ? 2.2
Libya 2 O ? 9.6; 23.0
Mozambique 1 SAT 2 ? 0.3
Myanmar 1 A ? 47.2
Namibia 1 SAT 1 Yes 5.8
Paraguay 2 O No 1.6; 9.2
Russia 1 O Yes 12.1
South Africa 3 SAT 1/SAT 2 Yes/No 23.8; zero; 0.9 The outbreak with AMR=0
(subclinical) occurred in a
non-vaccinated area; the 2
others were vaccinated areas
Tajikistan 1 Asia 1 ? 14.8
Vietnam 1 Asia 1 ? 78.2
Zimbabwe 1 SAT 2 ? 0.3
2012 Botswana 4 SAT 2 2 Yes; 2 No 0.6; 0.2; 1.3; 0.1 Vaccination had no apparent
influence on AMR
China ((PDR) 2 O No 8.0; 22.4
Egypt 1 SAT 2 No 40.9 SAT 2 outbreak resulting from
an incursion from the Horn
of Africa
Israel 1 O No 9.4
Libya 2 O/SAT 2 ? 25.5; 23.9 SAT 2 outbreak as a result of
spread from Egypt
Namibia 1 SAT 1 Yes 5.1
Palestinian 1 O/SAT 2 ? 23.5; 2.5 SAT 2 outbreak as a result of
Authority spread from Egypt
Paraguay 1 O ? 9.2
Russia 1 O Yes 14.9
South Africa 2 SAT 2 Yes 0.9; 0.7
Zambia 1 SAT 2 ? 0.4
2013 Botswana 2 SAT 2 ?/Yes 0.6; 0.3
China (DR) 6 Ax4; Ox2 ? 23.9; 0.6; 14.7;
10.5; 26.0; 13.1
Israel 1 O ? 25.0
Kazakhstan 1 A ? 3.0
Libya 1 O ? 67.6
of FMD outbreaks in endemic areas of southern cattle populations together with serotypes O and A
Africa is likely to be higher than diagnosed and (OIE/FAO Annual Report of the Foot-and-mouth
reported hitherto. disease Reference Laboratories Network, 2014).
A positive aspect to these observations is that A few southern African countries have estab-
there is evidence that clinical disease is necessary for lished FMD freedom for the country as a whole
efficient transmission of FMD by cattle (Charleston (Lesotho and Swaziland – small countries where
et al., 2011); consequently, the ‘weight of infectiv- infected wildlife do not occur) or in large zones
ity’ associated with outbreaks where AMRs are low (Botswana, Namibia and South Africa). These
could be concomitantly low. That may contribute zones were established through elimination of
to the slow and limited spread of many cattle out- infected buffalo and, to maintain separation of
breaks in southern Africa first observed in 1932 ‘infected’ from ‘uninfected’ animal populations,
(Du Toit, 1932). extensive veterinary cordon fencing (VCF) systems
were erected, together with the imposition of sup-
The current FMD management porting zoo-sanitary measures.
situation The zoning approach to FMD management
Some countries in southern Africa have managed has, for decades, enabled Botswana and Namibia
to prevent FMD from becoming endemic to their to access high-value international beef markets,
cattle populations despite SAT viruses being present principally in the European Union (EU) and
in wildlife. Conversely, in East, West and Central Norway. South Africa has so far had more limited
Africa SAT1, and SAT2 particularly, are endemic in access to high value markets outside Africa but that
is mainly due to factors unrelated to FMD (www. 0.6% of global exports while it has about 6.7% of
farmersweekly.co.za/article.aspx?id=44228); the world’s cattle (data extracted from FAOSTAT).
furthermore negotiations for access to EU A contributory factor to the above situation is
meat markets continued 11 months after South that the Progressive Control Pathway for FMD
Africa regained OIE recognition of its FMD- (PCP-FMD – www.fao.org/ag/againfo/commis-
free zone in February 2014 (www.moneyweb. sions/docs/PCP/PCP-26012011.pdf), which
co.za/news-fast-news/south-africa-negotiating- forms part of the joint FAO/OIE Global Frame-
eu-market-access-for-meat/). Despite these work for the Control of Transboundary Animal
achievements, the competitiveness of livestock Diseases (TADs), does not adequately deal with
production in the southern African region has the issues related to FMD management in Africa
declined in recent years (Meyer and Davids, 2012). outlined above, because its prime objective is
For example, both Botswana and Namibia are only elimination of FMD from defined areas. Regional
competitive in European beef markets as a result elimination of SAT serotypes has been shown to be
of bilateral tariff protection (Rich and Perry, 2011; difficult, if not impossible (Thomson et al., 2015).
Thomson et al., 2013a). The reasons for declining Modification of the PCP-FMD is needed so that it
competitiveness are, however, complex and multi- is both appropriate to circumstances that prevail in
factorial and only partly related to animal diseases, sub-Saharan Africa, while retaining compatibility
including FMD (Thomson et al., 2013a,b). with the overall global objective.
In spite of the success of the above three coun-
tries in establishing FMD-free zones, no new Considerations in development of
FMD-free zones have been created in sub-Saharan an appropriate FMD management
Africa in the last 30 years. Zimbabwe’s former strategy for southern Africa
FMD-free zone that enabled beef exports to EU The fundamental question related to FMD facing
markets, ceased to exist more than a decade ago. sub-Saharan Africa is whether or not the estab-
The net result is that only approximately 15% of lishment of additional or expansion of existing
the cattle population of mainland Southern African FMD-free zones1 (where vaccination is not prac-
Development Community (SADC) countries tised, i.e. the only successful approach adopted so
are raised in locations that are within internation- far) continues to be appropriate? In this context
ally recognized FMD-free areas (i.e. countries or it needs to be appreciated that countries can also
zones). Furthermore, the widely assumed impera- be recognized by the OIE as free from FMD ‘with
tive for creation and expansion of FMD-free zones vaccination’ but no country or zone in Africa has
has been called into question by the fact that India attempted that so far; the reason probably being
– which is not free from FMD and has no inter- that vaccination against SAT viruses presents
nationally recognized FMD-free zones – has the greater difficulties than for the Eurasian serotypes.
largest share by volume of the international beef In the past the wisdom of creating FMD zones in
export market. To further illustrate the point, India’s which vaccination was not practised was accepted
beef exports increased by 31% in volume terms in as axiomatic because there were essentially no
2013–14 while the value of those exports rose by alternatives and it was not widely appreciated in
52% (http://articles.economictimes.indiatimes. the animal health world that negative, albeit unin-
com/2014-06-25/news/50856076_1_buffalo- tended, socio-economic and/or environmental
meat-beef-exports-meat-export). Brazil, the second consequences can result from the establishment
largest beef exporter by volume, is also not com- of FMD-free zones, i.e. the overall cost of zonation
pletely free of FMD and there are zones within the can outweigh the benefits.
country where vaccination against the disease is The nature and extent of these considerations
routine (www.oie.int – see animal disease situation have been an area of investigation in the recent past
in the world). It is therefore unclear whether or not with interesting outcomes:
FMD is the real reason for poor market access and
lack of competitiveness on the part of southern Afri-
1 It is assumed in this discussion that eradication of
can countries, or merely an excuse. It is telling that buffalo as a FMD control option is untenable for moral
southern Africa’s beef exports make up only around and ecological reasons.
Alternative mechanisms for FMD risk vaccination programmes have proven increasingly
management ineffective, i.e. most cattle outbreaks have occurred
A number of alternatives have become available over in locations where the cattle are vaccinated regu-
the last few years that potentially enable safe trade larly against FMD (data extracted from the OIE’s
from locations that are not recognized as FMD-free. WAHIS – www.oie.int; Table 9.2) in addition to
These can broadly be divided into four categories the difficulty of incorporating vaccine strains into
(1) FMD-free countries or zones where vaccination vaccines that cover against a broad spectrum of
of cattle is systematically conducted [i.e. FMD- field viruses in circulation, particularly for SAT 2,
freedom with vaccination – Article 8.8.3 of the that renders prophylactic vaccination currently
OIE’s Terrestrial Animal Health Code (TAHC)], unreliable (Maree et al., 2014); (2) compartmen-
(2) processing of raw commodities in ways that talization for FMD in southern Africa is currently
inactivate the infectious agent under consideration of limited value because the relevant international
or otherwise abrogate its presence, e.g. heating the standard (Article 8.8.4 of the TAHC) precludes the
product (Article 8.8.23 of the OIE’s TAHC), (3) use of vaccine in compartments for FMD or even
systems based on compartmentalization where the introduction of cattle that have recently been
integrated biosecurity systems are applied for an vaccinated (in endemic areas of southern Africa
establishment or set of connected establishments cattle are usually vaccinated 2–4 times a year and
whereby FMD viruses can be excluded (Article therefore are disqualified from introduction into
8.8.4 of the TAHC) and (4) risk mitigation along compartments) and (3) at the moment the TAHC
value chains for animal products where hazard does not provide standards that can unambiguously
analysis critical control point (HACCP) and/ be applied directly to sanitary risk mitigation along
or commodity-based trade (CBT) principles are value chains, although Article 8.8.22 has elements
applied (Article 8.8.22 of the TAHC2; FAO, 2011; appropriate to value chain management of sanitary
Thomson et al., 2013b). Although Article 8.8.22 of risk.
the TAHC is an example of a value chain standard, It should be appreciated that in production
it contains elements which makes the principle(s) systems of all kinds, including for products derived
upon which it is based difficult to define. from animals, modern-day focus is on optimiza-
These various options have advantages and dis- tion of the value chain in all respects. Therefore,
advantages but an overriding problem is that they the sooner sanitary risk management is rendered
are complicated and therefore not well understood, compatible with other aspects of value chain man-
much less recognized by competent authorities of agement, the better.
countries involved in trade in animal commodities
and products. For that reason the alternatives have Potentially adverse socio-economic
recently been presented in the form of a guideline and environmental consequences of
focusing on beef trade (Thomson and Penrith, zonation
2015). That guideline is in the process of revision Various studies have shown that zonation, which
to take account of a recent positive change to a rel- conventionally includes the use of VCF erected to
evant international standard (Article 8.8.22 of the protect the FMD-free zone’s border, can and does
TAHC – see also below) that now contains a clause have serious negative repercussions for both people
that enables quarantine – traditionally widely living within or near such areas as well as the envi-
employed in southern Africa – to be incorporated ronment (Fergusson and Hanks, 2010; McGahey,
into value chain management of FMD risk. 2011; Barnes, 2013; Cassidy et al., 2013; Thomson
To briefly illustrate the advantages and disad- et al., 2013a; Woodroffe et al., 2014). The envi-
vantages of the different management options for ronmental effects are mostly related to effects on
FMD as far as trade is concerned, the following are wildlife and biodiversity conservation. However,
mentioned: (1) FMD-free zones where vaccination it also needs to be acknowledged that some VCFs
is practised is unlikely to be practical under pre- have protected the environment and also enabled
sent circumstances because routine prophylactic access to markets that otherwise would be difficult.
Livestock agriculture in southern Africa has few
2 This Article is not described as a value chain standard comparative advantages and, as indicated above,
in the TAHC but, in essence, that is what it is.
Date: 10:24 Monday 12 September 2016
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Natural Habitats where FMDVs are Maintained | 199
competitiveness in this area has been declining assurance that FMD viruses do not contaminate the
progressively (Thomson et al., 2013a). Conversely, final traded commodity or product and ensuring
tourism based on wildlife is a far bigger income that FMD’s effects on livestock are managed so that
generator for most southern African countries than the productivity and profitability of the relevant
animal agriculture (Thomson et al., 2013a) render- value chain are not compromised. The bottom line
ing wildlife conservation vital, quite apart from is that for value chains aimed at producing tradable
aesthetic, ecological and other considerations. animal commodities/products, animal diseases in
It can therefore no longer be assumed that general – not just FMD – need to be managed such
development of new or expanded FMD-free zones that the productivity of the livestock concerned is
will have a generally beneficial outcome for most maintained. This does not necessarily imply that
stakeholders. Furthermore, as pointed out above the infections concerned need to be absent from
with reference to India, there is clear evidence that the locality of production, only that their effects are
achievement of geographic freedom from FMD is managed adequately.
not a prerequisite for trading success when it comes In the context of value chains located in areas
to commodities and products derived from cloven- where FMD (SAT serotypes particularly) is
hoofed animals. endemic to wildlife in the general vicinity, the aim
is therefore to minimize occurrence of infection by
The relationship between animal a variety of measures while at the same time ensur-
disease control measures and sanitary ing that if infection does occur, the virus concerned
trade standards for FMD would not contaminate the final product.
For geographic and partially geographic approaches This issue was recently addressed by a pilot
to sanitary risk management (disease-free coun- project3 conducted in the Zambezi Region (ZR) of
tries/zones and compartmentalization) OIE Namibia. The ZR is a relatively small geographical
standards exist for establishment and maintenance area in the centre of the Kavango-Zambezi Trans-
of the FMD control system as well as trade in frontier Conservation Area where people and their
commodities produced within such systems. For livestock have long lived in harmony with wildlife
non-geographic systems – with the exception of (KAZA TFCA – www.peaceparks.co.za/tfca.
processed products (e.g. those that are cooked) – php?pid=19&mid=1008). However, despite the
the OIE defines neither the system nor appropriate fact that the ZR cattle population has been vacci-
sanitary trade standards (with the exception of Arti- nated against SAT 1–3 two or three times annually
cle 8.8.22 for trade in beef produced in locations that for many years, it has suffered repeated FMD out-
are not free from FMD but where an official control breaks in cattle since 2007. This resulted in loss of
programme exists). So while commodity-based the traditional market (South Africa) for the beef
approaches are accepted and promoted by the OIE produced in the ZR with devastating effects on the
(see www.oie.int/international-standard-setting/ local population.
overview/commodity-based-approach/) the prin- In an attempt to solve this problem, a value chain
ciples are not defined and few standards based on approach was instituted on a trial basis whereby
CBT, other than where cooking is involved, are FMD risk mitigation was applied at all stages of the
provided. This makes institution of non-geographic ZR beef value chain, i.e. from (1) cattle belonging
approaches difficult and complicated. to contracted owners using the communal grazing
In instances where non-geographic sanitary system, (2) transportation of slaughter cattle to a
trade approaches are needed as a mechanism for quarantine station (3) actions conducted during
achieving market access for commodities and prod- the 21-day quarantine period, (4) transport of the
ucts derived from animals (e.g. application of CBT cattle to holding pens of the abattoir (5) slaughter
and/or HACCP as risk mitigation measures along
value chains where FMD is endemic in wildlife),
3 Development of Export Opportunities for Beef Products
the effect of FMD on productive efficiency of the from the Zambezi Region (MCAN/LMEF/2010/02 and
animals providing the source commodities (gener- MCAN/LMEF/2012/04), co-ordinated by the Meat
Board of Namibia, funded by the Livestock Marketing
ally milk or meat) also needs to be considered. Thus Efficiency Fund. and Commissioned by the Millennium
there are two separate but complementary issues: Challenge Account Namibia (www.mcanamibia.org/
files/files/Final%20Report%20LS.pdf).
Date: 10:24 Monday 12 September 2016
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200 | Vosloo and Thomson
and carcass handling, including beef maturation and (Article 5 of the SPS Agreement). This means that
deboning and (6) packaging, storage and dispatch currently, application of value chain approaches that
of boxed beef cuts. The FMD risk mitigation meas- incorporate HACCP and/or CBT methodologies
ures associated with each step of the value chain need to be based on a risk assessment. This makes
have been outlined (Thomson et al., 2013b). These this approach not only complicated but expensive
mitigation steps were furthermore integrated with and time-consuming.
food safety measures by application of HACCP and However, based on results from the above pro-
CBT in parallel to achieve ‘an appropriate level of ject’s findings, an amendment to Article 8.8.22 of
protection’ for all sanitary risks (Fig. 9.3). In addi- the OIE’s TAHC dealing with beef exports from
tion, a quantitative risk assessment with respect to locations that are not free from FMD virus infec-
FMD was conducted on the whole value chain to tion, but where an official control programme exits,
prove that an appropriate level of protection was was proposed to and adopted by delegates to the
achieved for FMD (Fosgate, Penrith and Thomson, OIE General Session held in May 2015. This change
in preparation). In that way the effectiveness of the in the standard, providing acceptance of quarantine
approach was proven. systems as part of the value chain risk management
Where an appropriate international trade process, has potentially rendered export of beef
standard is lacking, the World Trade Organization’s from areas of the region not recognized as free from
(WTO) Agreement on the Application of Sanitary FMD considerably more attainable.
and Phyto-sanitary Measures (SPS Agreement –
through which the OIE receives its mandate for FMD control in extensive rangeland
setting trade standards) recommends that as long systems
as a risk assessment shows that the risk mitigation Extensive rangeland systems, in Africa at least, are
measures attain an appropriate level of protection, mostly located in arid and semi-arid areas where
such trade is ‘safe’ and therefore should be enabled mobility of animals, whether wild or domestic,
Figure 9.3 Diagrammatic representation of integrated sanitary risk (food safety and animal disease risk)
management along a beef value chain. Source: Foot-and-mouth disease: Southern Africa, Bulletin #4, March
2014 (http://www.afrivip.org/node/3857/zip_download).
Date: 10:24 Monday 12 September 2016
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Natural Habitats where FMDVs are Maintained | 201
is crucial to effective adaptation to their environ- providing ways in which livestock producers are
ment (Notenbaert et al., 2012; Cumming et al., enabled to farm in the presence of disease (until
2015; Durant et al., 2015). Managing contagious such time as new technologies evolve). Integral to
diseases like FMD where animal movement cannot that approach is enabling ‘safe’ trade despite the
be reliably controlled, on the other hand, presents endemicity of FMD. The simple fact is that sani-
a fundamental problem. The need for mobility in tary risks posed by FMD (or any other disease risk
some ecological circumstances is not recognized for that matter) can be managed effectively in a
by current recommendations for FMD manage- variety of ways and such approaches need to be
ment; in fact little guidance is available to those facilitated.
parts of the world confronted by endemic FMD It has been argued that acceptance of non-
other than where the creation of FMD-freedom geographic trade standards will go a long way
is possible. The underlying presumption is that towards providing alternatives to the current
co-existence of animal agriculture and FMD are approach (Thomson et al., 2004, 2013a; Rich
incompatible. However, systems like attainment of and Perry, 2010; Paton et al., 2010; FAO, 2011).
country or regional freedom from FMD (with or However, that will only occur if appropriate trade
without vaccination) or compartmentalization – a standards as well as compatible FMD manage-
methodology ideally suited to intensive, vertically ment practices are developed, recommended by
integrated production systems for poultry and international agencies and accepted by trading
pigs – are not apt for managing diseases like FMD nations. Clinging to geographic approaches for
on extensive rangelands where susceptible wildlife FMD management and trade is irrational in some
are present in large numbers as explained above. circumstances, particularly in sub-Saharan Africa.
The absence in the past of alternative approaches It is vital that livestock production, including
to managing FMD has resulted in animal disease pastoralist systems, and wildlife conservation are
authorities in such locations making illogical deci- rendered more compatible.
sions, with inevitable results.
For some TADs where wildlife are important in
the maintenance of the infection (e.g. highly patho- Fences and fencing
genic avian influenza and classical swine fever) the Fences and fencing systems have been accepted
approach of the OIE has been, in essence, to dis- as fundamental to the effective management of
regard the presence of these infections in wildlife FMD and other TADs for more than a century in
when it comes to determining the safety of animal southern Africa; the first fences for this purpose
commodities or products for trade purposes. For (although not constructed using wire) were erected
other diseases, such as African swine fever (ASF) in South Africa to prevent the spread of rinderpest
and FMD, that approach is not currently recom- in the late nineteenth Century. Until quite recently,
mended for reasons which are not apparent. Even it was implicitly accepted that livestock farming
for ASF there is seemingly a suggestion – in the was so fundamentally vital to rural development of
form of a draft chapter for possible future incorpo- the region that collateral damage caused by VCFs
ration into the TAHC – that this approach should could be disregarded. However, a large body of
be adopted for ASF as well. If such an approach evidence has accumulated over several decades that
were adopted for FMD, especially if accompanied provides a basis for questioning that presumption
by more flexible standards based on management (e.g. Osofsky et al., 2008; a collection of contribu-
of sanitary risks along value chains, it would have tions edited by Ferguson and Hanks, 2010; Gadd,
major benefits for countries and zones in sub- 2012; Woodroffe et al., 2014; Cumming et al., 2015;
Saharan Africa. Durant et al., 2015). This evidence has largely con-
It is perhaps time that it is frankly admitted cerned the environmental effects of such fences but
that in most extensive systems, especially those the socio-economic impacts of zonation, enabled
where wildlife maintain SAT-type FMD, eradica- by veterinary cordon fences, have also been shown
tion or regional elimination of the infection is to be potentially detrimental (Barnes, 2013; Cas-
not currently possible (Thomson et al., 2015). In sidy et al., 2013). Of crucial importance is the fact
such cases the emphasis arguably needs to be on that current approaches to FMD management are
clearly far less effective than they should be (Thom- region into consideration, but this is not always
son et al., 2013a). practically possible, as vaccine manufactures have
The net result seems to be acceptance that fences to carry the cost of development, and cost recovery
can have both beneficial and/or detrimental effects. is uncertain in regions of the world where vaccine
So, as has been suggested by others, individual purchase is erratic. FMD vaccines are expensive and
fences and their alignments need more careful few countries in Africa have the resources to pay for
evaluation than has been the case in the past. Only development of vaccine strains that are specific for
in that way can it be ensured that benefits of FMD their needs. Vaccine strains with wide antigenic
management outweigh the costs (Woodroffe et al., cover are therefore an imperative, whether they
2014). be naturally occurring strains or genetically engi-
neered.
Vaccination of free-living wildlife as part of
Vaccination FMD control is not a viable option due to the
Routine vaccination of cattle against FMD in logistical and financial difficulties of vaccinating
areas adjacent or close to wildlife areas has been large numbers of free-living animals in areas that are
practised in southern Africa since the mid-1970s. frequently inaccessible. However, the use of pro-
However, because even the best FMD vaccines are jectile syringes incorporating marker devices fired
relatively inefficient, it has been shown by experi- from helicopters could be used on a small scale.
ence in southern Africa that reliance on vaccines In an experiment where buffalo, impala, eland and
exclusively is unreliable. The reasons for this are cattle were vaccinated using an inactivated trivalent
two-fold. Firstly, immunity following a primary vaccine with alhydrogel/saponin as the adjuvant, it
inoculation is ephemeral (<2–3 months only in was found that all animals developed serum neu-
cattle when the vaccine contains alhydrogel/sapo- tralizing antibody responses of a lower order than
nin as the adjuvant; oil-adjuvanted vaccines may be those of cattle and there was considerable variation
more effective in this respect). In addition, due to in the responses of individual animals (Hedger et
constraints, very few countries apply the primary al., 1980). Therefore, it was recommended that a
immunization as prescribed by the manufacturers, double dose of vaccine should be used for wildlife.
i.e. two doses of vaccine administered 1–2 months Correlation between serum neutralization antibody
apart. Following that, only when individual cattle levels and resistance to challenge have not yet been
have received several inoculations does the level established for wildlife species, but convalescent
of immunity engendered remain high against antibody levels following experimental or contact
challenge with the homologous virus (Barteling, infection have been recorded (Hedger et al., 1972;
2002). Thereafter annual vaccination is required to Anderson et al., 1975). Convalescent antibody
keep levels of immunity at a satisfactory level. The responses in buffalo and eland were of a similar
second problem is that of antigenic variation. It is order to those of cattle and it may be that protec-
well established that animals recovered from infec- tive antibody levels are likely to be correspondingly
tion with one FMD virus serotype are susceptible similar. In contrast, convalescent antibody levels of
to re-infection with any of the other 6 virus sero- impala were found to be lower than those of cattle
types (Doel et al., 1994). (Hedger et al., 1980).
Due to the large number of topotypes identified Oil adjuvanted vaccines have not been tested in
for most serotypes prevalent in sub-Saharan Africa wildlife except in a single pilot study that was con-
(Vosloo et al., 1995; Bastos, 1998, 2001; Bastos et ducted into the possibility of vaccinating buffalo
al., 2001, 2003; Maree et al., 2014), and the fact calves within breeding herds in a wildlife reserve
that different genotypes within a virus serotype in South Africa with high antigen-payload vaccines
are sometimes antigenically diverse enough to (P. Hunter, personal communication, 1997). The
require different vaccine strains to protect against results were inconclusive. For domestic animals it
disease caused by the respective topotypes, control has been shown that such adjuvants provide longer
through vaccination alone may not be sufficient. lasting immunity (Barteling, 2002) and the success
Ideally, vaccine strains should be chosen for differ- of improved control of FMD in South America
ent regions taking the topotypes occurring in that was, among other factors, attributed to the use
of oil-adjuvanted vaccines (Naranjo and Cosivi, and so address the problem of antigenic variation
2013). observed with the SAT serotypes more effectively.
Although vaccines have been shown to be effec- That would enable the manufacture of vaccines that
tive in protecting domestic livestock from clinical stimulate broader immune responses.
disease caused by FMD viruses, protection against
infection is less efficient resulting, in ruminants, in a
proportion of animals that are persistently infected, Establishment of disease-free
i.e. so-called carriers (De Leeuw et al., 1979; Don- buffalo herds
aldson et al., 1987; Donaldson and Kitching, 1989; A solution to the dilemma posed by the widespread
Salt et al., 1996). Because of this, trade restrictions desire to maintain African buffalo within recognized
are frequently imposed on countries where there is FMD-free areas lies in the establishment of buffalo
a possibility that such animals exist. The argument herds free of FMD and other diseases (Condy and
that ‘carrier’ animals transmit FMD virus so infre- Hedger 1978, Vosloo et al., 2001). Several protocols
quently as to be epidemiologically insignificant and have been followed to ensure that calves born to
that this possibility can be safely ignored (Barteling, infected mothers were reared without becoming
2002; Sutmoller and Casas, 2002), is possibly infected with FMD virus (Laubscher and Hoff-
dangerous in view of the enormous economic man, 2012). In South Africa at least, a protocol was
repercussions that could result if such an unlikely established to ensure that breeding of these calves
possibility came to pass (Muckspreader, 2001). by private enterprises occurred under strict control
Developments in vaccine preparation (purifica- of veterinary services and this practice was permit-
tion of viral antigens to the extent that NSPs are ted between 1996 and 2011.
excluded from the final product, with appropriate These animals, as well as buffalo from popula-
certification) and companion serological tests, have tions historically free from FMD in South Africa,
enabled animals, including wildlife, that have been have been used to breed significant numbers of
vaccinated and subsequently infected, to be dif- buffalo free from FMD. To date this approach has
ferentiated from animals that have been vaccinated delivered disease-free buffalo that now populate
but not infected. In addition, new technologies areas of South Africa where buffalo have not
such as viral-vectored FMD vaccine platforms occurred for many decades. It has also enabled
(reviewed in Rodriguez and Gay, 2011) that offer the preservation of genetic material and boosted
improved opportunities for differentiating infected eco-tourism, hunting and thereby the broader
from vaccinated animals, should enable vaccina- economy. However, the relative scarcity of so-called
tion of wildlife without subsequent restrictions for FMD-free buffalo has resulted in a large price dif-
international trade. While the current technology is ferential between these animals and similar animals
based on the expression of FMD virus antigens via in FMD-infected areas. This provides an obvious
a replication defective vector (Mayr et al., 1999), incentive for smuggling infected animals and selling
it is not impossible to envisage a vector that could them as animals free of FMD (Vosloo et al., 2001).
spread naturally between wildlife and so overcome Such cases have been proven (Vosloo et al., 2001)
the problem of vaccinating large numbers of free- and threaten the status of the zone recognized as
living animals. The development of more stable free of FMD in southern Africa. Vigilance on the
vaccines (Porta et al., 2013) also offer benefits that part of veterinary services is therefore essential to
could be applied to wildlife where novel routes of ensure that buffalo are not moved illegally. In South
administration could become reality, such as oral Africa at least, it is now required that all buffalo
vaccines distributed in the field for animals to con- movements may only occur after the animals have
sume. Reverse genetics provide opportunities to been subjected to appropriate testing.
engineer attenuated viruses, but at present deficien- Novel fields of development, such as treatment
cies in understanding virus–host interaction and with antiviral drugs (reviewed in Goris et al., 2008)
mechanisms of pathogenesis, even in domestic ani- may, in future, be used to eliminate infection from
mals, makes this a challenging field. Taken together, persistently infected buffalo. Research into the
these technologies could improve the antigenicity efficacy of antiviral inhibitors and mutagenic agents
of the vaccine by incorporating engineered epitopes that target viral replication in persistently infected
buffalo specifically, could assist in eliminating virus Caprivi, Namibia, with potential wider implications
for regional transfrontier conservation areas. Technical
from animals and so establish infection-free herds. report to the Wildlife Conservation Society’s AHEAD
Such treatment would also be valuable should Program and the World Wildlife Fund. 84pp.
infection-free herds inadvertently become infected Barteling, S.J. (2002). Development and performance of
(as has occurred in Zimbabwe in recent years) by inactivated vaccines against foot-and-mouth disease.
Rev. Off. Int. Epizoot. 21, 577–588.
providing a mechanism for recovery of infection- Bastos, A.D.S., Boshoff, C.I., Keet, D.F., Bengis, R.G.,
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and FMD in livestock better controlled, holds the foot-and-mouth disease virus between African buffalo
potential for transforming sub-Saharan Africa’s (Syncerus caffer) and impala (Aepyceros melampus) in the
Kruger National Park, South Africa. Epid. Infect. 124,
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immunomodulation, which can lead to severe (Summerfield et al., 2001). Certainly, FMDV was
immunopathology. not found in either lymphocytes or DCs following
FMDV infection will cause local cell damage, infection (Nfon et al., 2010).
which together with virus infection-related prod- Both lymphocyte and DC subsets are also
ucts provide ‘danger’ signals recognized by the involved in down-regulation of inflammatory
host immune defences. Local tissue MΦ (his- response to a resting state once a pathogenic
tiocytes) will attempt to control the infection and threat has been removed. Monocytotropic viruses
remove potentially harmful material arising from induce aberrant control of inflammatory responses,
the infection, including virus. These responses causing immunomodulation in terms of excessive
self-amplifying through initiation of local inflam- inflammatory reaction and maintenance of blood
matory reactions – recruit blood monocytic and leukopenia (Summerfield et al., 1998, 2000). The
granulocytic cells, as well as blood DCs, together transient leukopenia observed with FMDV is
enhancing local tissue MΦ and DC activity. Inflam- more typical of immunoregulated inflammatory
matory response-induced recruitment of blood responses. Nonetheless, an immune system under
leucocytes also ultimately involves lymphocytes, pressure to control an FMDV infection does not
hence the transient leukopenia forming an impor- have the same capacities as under homeostasis;
tant characteristic of the local immune defence if the balance between initial host immune activ-
against infection. ity and virus replication does not control of the
Local immune response have been identified virus infection, disproportionate inflammatory
with skin DCs producing type I interferon (IFN) responses can ensue.
in response to live FMDV (Bautista et al., 2005). Efficient development of immune defence
FMDV infection of pigs can induce increased against FMDV requires detection of the virus and its
plasmacytoid DC (pDC) numbers and activ- infection-products as antigenic and ‘danger’ entities.
ity – serum IFN-α (Nfon et al., 2010). Thereafter, Innate immune cells again play a critical role, pro-
pDC numbers declined transiently, reflecting more cessing virus antigen to activate adaptive immune
an inflammatory response rather than infection responses into antibody- and T-lymphocyte-
as typified by classical swine fever virus (CSFV) mediated defence (Fig. 10.1). Following control of
Figure 10.1 Generalized overview showing the salient points involved in APC interaction with B and
T-lymphocytes, leading to antibody production.
any virus infection, the adaptive immune responses molecular patterns’ (PAMPs), as well as ‘danger-
provide the core elements for protective and durable associated molecular patterns’ (DAMPs) found
defence. Vaccination seeks to imitate this scenario with tissue and cell damage. Both processes involve
by inducing the protective immune defence with- pattern recognition receptors (PRRs) associated
out recourse to virus infection. Analyses of the with innate immune cells, which can be restricted
different immune compartments, and how FMDV to particular cell subsets (Gallucci and Matzinger,
interacts with the cells therein, provide the essential 2001; Medzhitov and Janeway, 2000a, b; Medzhi-
knowledge defining protective immunity and its tov and Janeway, 2000c; Pulendran and Ahmed,
induction. 2006).
Innate defences actually begin with the physical
Innate and adaptive immune and secretory barriers of the body. For example,
defences pertinent to FMDV the skin and mucosal epithelial cell layers, as well
as secretions at mucosal surfaces and the pH in the
Distinguishing characteristics of innate stomach and intestine, provide physical/chemical
and adaptive immune defences barriers as first lines of innate defences (Bals, 2000;
As already mentioned, induction of adaptive Fellermann and Stange, 2001). Once a pathogen
immune defence requires innate immune cells – or other foreign antigenic entity passes such bar-
antigen-presenting cells (APC) such as DCs – to riers, cellular elements of innate immune defences
present antigen in combination with co-stimulatory – histiocytes and other monocytic cells within
molecules and the appropriate balance of cytokines tissues, tonsils and mucosal-associated lymphoid
(Fig. 10.1) (Banchereau and Steinman, 1998; Car- tissues (MALT) – provide the first encounter.
roll and Janeway, 1999; McCullough et al., 2012; These cells include DCs, MΦ, neutrophils, natural
McCullough and Summerfield, 2005; Medzhitov killer (NK) cells, mast cells, and probably γδ-T-cells
and Janeway, 2000a; Mellman, 2013; Mellman and (Banchereau and Steinman, 1998; Biron et al.,
Steinman, 2001; Steinman and Hemmi, 2006). In 1999; Boismenu and Havran, 1997; Caux et al.,
turn, lymphocytes of the activated adaptive immune 2000; Feger et al., 2002; Mellman, 2013; Mell-
compartment influence the activation status within man and Steinman, 2001; Steinman and Hemmi,
the innate immune compartment. One major dis- 2006; Steinman and Pope, 2002). Mo also play
tinction between innate and adaptive responses is a role, often observed as differentiation of these
the kinetics of response development. Another lies predominantly blood-associated cells into inflam-
in the specificity of the response for the antigen in matory DCs and MΦ upon migration into tissues
question, as well as the expansion of that recogni- and organs following inflammatory signalling. The
tion capacity. Nonetheless, the distinctive roles pDCs, representing a specialized subset of the DC
played by the different immune compartments and family, play a prominent role early during immune
their co-operative functionality define the power of defence development. This cell type is by far the
developing effector immune defence processes. strongest producer of Type I interferon (IFN), rep-
resenting both a potent antiviral factor and potent
General characteristics of innate signalling component for driving forward induc-
immune defences tion of adaptive immune responses (Liu, 2005).
Innate defences are more rapidly activated after Endothelial cells are also important in innate
infection, but display lower specificity and shorter defences, through cytokine production and
duration than adaptive responses (Carroll and responses to cytokines from inflammatory innate
Janeway, 1999; Hoffmann et al., 1999; Medzhitov immune cells in the adjacent tissues. The latter leads
and Janeway, 2000a). The innate immune cells are to upregulated adhesion molecules on the endothe-
relatively non-specific for antigenic entities within a lium for enhanced adhesion of blood DCs, Mo,
foreign body such as virus or vaccine; they also lack neutrophils, NK cells and even lymphocytes – initi-
the genetic re-assortment found with lymphocytes ates their extravasation into the tissues. Interactions
of adaptive immunity to ‘fine-tune’ antigen recog- of the induced cytokines – include interleukin (IL)
nition. There is a form of specificity with innate 1β, IL-6, IL-10, IL-12, IL-15, tumour necrosis
immune cells –recognition of ‘pathogen-associated factor (TNF) α, transforming growth factor (TGF)
β, and IFN-α/β/γ – and chemokines direct both the ‘fine-tuning’ of antigen recognition – and there-
the activation and migratory behaviour of the fore specificity and affinity of reaction – brought
developing immune defence (innate and adaptive) about by genetic rearrangements of the antigen
and inflammatory responses. Accordingly, these responsive genes defining antigen recognition
responses will be defined by the cell populations receptors. Only antigen-specific lymphocytes
involved, and therefore their environment – for display ultimate expansion of clones specifically
example, responses differ between the dermis and recognizing particular antigenic determinants
the mucosal surfaces (see ‘MΦ and DC responses With FMDV, antibodies of high affinity and
to FMDV infection and vaccination’ and ‘Innate quantity have been implicated in protective
immune cell regulation of adaptive defence devel- immune defence (McCullough et al., 1986, 1998,
opment’). 1992b,c; Mulcahy et al., 1990, 1992; Pay and Hing-
Serum proteins – such as acute phase pro- ley, 1987; Steward et al., 1991). This characteristic
teins, complement, natural antibodies and other is dependent on the efficiency with which immune
opsonins (including lipopolysaccharide (LPS)- responses are stimulated and the ensuing matura-
binding protein and mannan-binding protein) tion of adaptive immune compartments. While
– enhance innate defences through opsonization of antibody production requires B-lymphocytes,
the antigen/virus characteristics (Agah et al., 2001; Th-lymphocyte activity is also critical (Fig. 10.2).
Barrington et al., 2001; Biron et al., 1999; Matsu- Effector antibody-based immunity is dependent
shita et al., 1998; Rossi and Zlotnik, 2000; Zlotnick on the correct activation of Th-lymphocytes –
et al., 2000); opsonization is also an important through humoral factors (cytokines) (Fig. 10.2)
function with antigen-specific antibody (see ‘Effec- and intercellular communications (cell surface
tor humoral immune defence’). Nonetheless, these co-stimulatory molecules) (Fig. 10.1) – to drive
innate defences are limited in their efficacy. Whilst antigen-stimulated B-cells towards antibody-
capable of removing a viral threat when the level of producing plasma cells.
virus activity is relatively low, their lack of specific- B-lymphocytes respond directly with antigen,
ity for the antigen in question can lead to a more but T-lymphocytes require antigen processing into
prominent virus infection overcoming the innate small peptides for presentation in association with
defences. Therein lies the particular power of adap- MHC molecules (Fig. 10.1). This antigen presenta-
tive immune defence, providing specificity, avidity tion is a core contribution from APC, of which DCs
and rapidity of recall (see ‘Effector lymphocyte play major roles due to their status as ‘professional
responses against FMDV’). antigen-presenting cells’ (Banchereau and Stein-
man, 1998; McCullough et al., 2012; Mellman,
General characteristics of adaptive 2013; Mellman and Steinman, 2001; Steinman
immune responses and Hemmi, 2006; Steinman and Pope, 2002). The
Initiation of innate immune response activity is T-lymphocyte receptor for this processed antigen
also ultimately responsible for promoting adap- – the ‘T-cell receptor’ (TCR) – can only recognize
tive immune defence development. Of particular epitopes within processed peptides presented in the
importance are the DCs. Their transport of anti- context of the MHC. In contrast, the ‘B-cell recep-
gen to lymphoid organs and tissues, as well as the tor’ (BCR) does not require antigen to be processed
processing into peptides for MHC molecule asso- or presented. This BCR is an immunoglobulin
ciation, is critical for adaptive immunity to develop. molecule with the same antigen recognition char-
The hallmark of an effective immune response acteristics as the antibody that will eventually be
following infection or vaccination is the induction secreted – by the plasma cells differentiating from
of specific immunity. Durable immunity – immu- activated antigen-specific B-lymphocytes. This
nological memory and rapid recall responses to delivery of antigen to B-lymphocytes also involves
prevent subsequent infection – is only associated DCs (Fig. 10.1), but without the antigen processing
with the adaptive immune compartment, relating (Le Roux et al., 2011; Wykes et al., 1998).
to the specificity of foreign body recognition by the DC interaction with FMDV (virus or vaccine)
two compartments. Upon response to antigen, the is essential for adaptive immune defence induc-
adaptive immunity expands in both numbers and tion. Endocytosis of virus or vaccine (Fig. 10.3)
Figure 10.2 A primary function of DC: to degrade antigen and present the derived peptides to antigen-specific
Th lymphocytes, which in turn provide the cytokines (following stimulation by the peptide–MHC complexes) to
promote and control the developing immune response. *Endocytosis: the process referred to in this image is
likely to be clathrin-independent endocytosis – macropinocytosis or phagocytosis. This process is particularly
important in dendritic cells, macrophages, monocytes, and granulocytes with respect to the uptake of
particulate antigenic material, although clathrin-dependent processes are also operative. In the case of FMDV,
it appears that either macropinocytosis or phagocytosis dominates with macrophage and dendritic cell uptake
of the virus, and probably differs from the entry of virus into epithelial cell lines.
allows for DC delivery of antigen to B-lymphocytes viral RNA genome and dsRNA replicative form,
or processing into peptides for presentation to or adjuvant components in formulated vaccines.
T-lymphocytes (Fig. 10.1). Critically important Appropriate maturation of the DCs upregulated
for the latter is that the DCs mature – ensured by the chemokine receptor CCR7, promoting DC
their response to PAMPS and DAMPs such as the migration into lymphoid tissue for interaction
Figure 10.3 Interaction of FMDV (O1 Lausanne) (green) with porcine monocyte-derived dendritic cells (DCs)
and macrophages (MΦ). (a) FMDV binding to the surface of DCs held on ice, 30 min. (b) Endocytosis of FMDV
by DCs after 30 minutes at 39°C. (c) Rapid interaction of DMFV with MΦ at 39°C, showing binding to the MΦ
surface and initial endocytic events with the more active cells (displaying lamellipodia and filipodia). (d) FMDV
internalized by MΦ within 30minutes at 39°C. In all images, FMDV was detected using antibody against a
conformational neutralisable antigenic site (4C9), and Leica TCS SL confocal laser scanning microscopy with
a Nomarski Interference Contrast attachment (Leica AG). Analysis of the images employed Imaris 8.0 (Bitplane
AG).
with the lymphocytes. Enhanced trafficking of follicles and extra-follicular areas (see ‘Immu-
leucocytes has been reported early after vaccina- nological Memory Induction: in balance with
tion of pigs against FMDV (Rigden et al., 2003). effector immunity’) for appropriate signalling to
In the lymphoid tissues and organs, DCs deliver T-lymphocytes recognizing MHC-presented pep-
antigen to the B-lymphocytes (Le Roux et al., tides (see ‘Antigen-presenting cell interaction with
2011; Wykes et al., 1998), and present antigenic T-lymphocytes’ and ‘T-lymphocyte Induction’).
peptides associated with MHC molecules to acti- Without ligation of these co-stimulatory mol-
vate antigen-specific T-lymphocytes (Fig. 10.1) ecules, the responding T-lymphocytes are induced
(Banchereau et al., 2000; Banchereau and Stein- into anergy or apoptosis (immunoregulation and
man, 1998; Steinman and Hemmi, 2006; Steinman tolerance). Accordingly, appropriate lymphocyte
and Pope, 2002). Critical for the latter, DC matura- receptor and accessory molecule ligation controls
tion also ensures up-regulation of co-stimulatory the nature and intensity of antigen-specific adaptive
molecules such as CD80/86 –essential in lymphoid immune responses.
accepted that RIG-I ligands include ssRNA bearing preventing endosomal acidification such as chlo-
a 5′-triphosphate and a blunt-ended region at the 5′ roquine (Guzylack-Piriou et al., 2006a), and by
end, as well as short dsRNA, while MDA5 binding suppression of IFN-α responses using specific
is associated to long dsRNA (Berke et al., 2013; oligonucleotide inhibitors of TLR7 (Lannes et al.,
Schlee, 2013). While RIG-I is required for sensing, 2012). More details on the interaction of FMDV
among others, paramyxo-viruses, influenza virus, with pDC will be given in ‘FMDV and mononuclear
Japanese encephalitis virus and hepatitis C virus, innate immune cells’ and ‘Consequences of FMDV
MDA5 is involved in recognizing picornaviruses, interaction with mononuclear innate immune cells’.
mouse norovirus, mouse hepatitis virus and defec-
tive interfering particles of paramyxoviruses (Feng Viral immune evasion strategies
et al., 2014). During the long co-evolution with their hosts,
During picornavirus replication, a fully comple- viruses have acquired strategies to actively counter-
mentary dsRNA product is synthesized that has act host antiviral responses, and picornaviruses are
recently been identified as the ligand that activates no exception. In addition to their function in poly-
MDA5 (Feng et al., 2012). In that work, RNA from protein processing, viral proteases target a variety
several picornaviruses, including equine rhinitis of host proteins for efficient virus replication. These
A virus (ERAV), a member of the Aphthovirus include proteins involved in translation, transcrip-
genus, were analysed. Replication of all viruses in tion, immune signalling, or nucleocytoplasmic
mouse embryonic fibroblasts transfected with the traffic. Some viral proteases can inhibit type I
corresponding viral RNAs induced an MDA5- and IFN production and NF-κB signalling through
MAVS-dependent, but RIG-I-independent, IFN-β the cleavage of receptors, adaptors, and regulators
response providing experimental evidence that a participating in these pathways. A wide variety of
broad spectrum of picornaviruses is indeed recog- strategies to evade the RLR-mediated type I IFN
nized by MDA5. response have been reported for picornaviruses,
In 2011, using lentivirus-driven RNA interfer- including FMDV (Dotzauer and Kraemer, 2012;
ence, Husser et al. (2011) reported that FMDV Feng et al., 2014).
was sensed by MDA5 but not by RIG-I or TLR3 in Aphthovirus Lpro impairs cap-dependent trans-
porcine epithelial cells. Six hours after FMDV infec- lation through cleavage of initiation factor eIF4G,
tion, IFN-β mRNA induction was only reduced leading to a translational host shut-off (Devaney et
significantly in MDA5 silenced PK-15 cells. al., 1988; Medina et al., 1993). Cleavage of eIF4AI
In pDCs it appears that FMDV is sensed by by FMDV 3Cpro also contributes to the host shut-
TLR7 inside the endosomal compartment (Fig. off (Li et al., 2001). Picornavirus-induced host
10.4). This is supported by sensitivity to drugs gene expression shut-off is known to contribute to
IFN-α/β suppression though the primary blockade research over the last few years. FMDV has been
of this pathway lies upstream of IFN production. shown to use a repertoire of strategies to coun-
MDA5 and MAVS cleavage during enteroviral teract the host innate immune response. The viral
infections has been associated mainly to 2Apro, proteases Lpro and 3Cpro have been found to
though cleavage of overexpressed MAVS by 3Cpro antagonize the host type I IFN response. Using a
has also been shown (Feng et al., 2014). Interest- genetically engineered FMDV lacking the Lpro
ingly, cleavage of RIG-I by 3Cpro has also been coding region (A12-LLV2), de los Santos et al.
reported for various enteroviruses (Barral et al., (2006) demonstrated that Lb, the most abundant
2009; Papon et al., 2009). However, it remains to be form of Lpro in cells as the result of translation
elucidated why these viruses target an RNA sensor started at the second AUG, inhibits the induc-
that does not participate in their recognition. It tion of IFN-β mRNA and the expression of
has been proposed that RIG-I may directly turn IFN-α/β-stimulated genes in IBRS-2 swine cells.
on ISG transcription via STAT1 activation in an This inhibitory effect was further associated with
IFN-α/β-independent fashion ( Jiang et al., 2011). Lpro translocation to the nucleus in correlation
Cardioviruses do not target MDA5 or MAVS with a decrease in the amount of nuclear p65/RelA,
though effectively prevent IRF-3 phosphorylation. a subunit of NF-κB (de Los Santos et al., 2007),
The cardiovirus leader proteins have long been then inhibiting NF-κB dependent gene expres-
established as the main IFN antagonists. These sion (Zhu et al., 2010). A putative SAP (SAF-A/B,
multifunctional proteins have neither enzymatic Acinus, and PIAS) domain between amino acids 47
activity, nor known homologues but interfere with and 83 of Lb was predicted and disruption of the
critical cellular processes such as IFN production, Lpro SAP domain selectively prevented p65/RelA
nucleo-cytoplasmic trafficking, apoptosis, stress processing without affecting the ability of Lpro to
granule formation or MAP kinase activity. Theiler’s cleave eIF4G (de los Santos et al., 2009). A different
murine encephalomyelitis virus (TMEV) L has report showed that both forms of Lpro (Lab and
been shown to form a tight complex with the small Lb) suppress dsRNA-induced IFN-β mRNA tran-
GTPase Ran, disrupting the RanGDP/GTP gradi- scription in PK-15 porcine cells through decreasing
ent across the nuclear pore, a crucial regulatory IRF-3/7 expression. The Lpro ability to process
mechanism for nuclear import and export (Porter eIF4G was not required for the inhibitory effect
et al., 2006). The induced disorder may interfere (Wang et al., 2010).
with IRF-3 activation by preventing its shuttling Lpro has been associated with regulation of the
and/or nuclear translocation upon activation inflammatory and antiviral chemokine RANTES
by TBK1. TMEV L* inhibits the activity of the (regulated upon activation, normal T-cells
IFN-induced RNase L, by direct protein–protein expressed and secreted, also known as CCL5),
interaction (Sorgeloos et al., 2013). suggesting another strategy by which FMDV
MAVS cleavage activity has been reported for counteracts the cellular inflammatory responses to
3ABC, a precursor of hepatitis A virus (HAV, genus viral infection. Lpro limits RANTES and TNF-α
Hepatovirus) 3Cpro. The transmembrane domain transcription by degradation of p65/RelA (de
in 3A targets 3ABC to the mitochondria outer Los Santos et al., 2007). In the case of RANTES,
membrane where MAVS is localized and cleaved suppression via ISRE by interfering with IRF-3/7
(Yang et al., 2007). activation has also been reported (Wang et al.,
Coxsackievirus B, enterovirus 71 (EV71) and 2011a,b,c). Catalytic activity and the SAP domain
HAV are also known to cleave the TLR3 adaptor of Lpro seem to be necessary for suppressing
TRIF in infected cells and 3Cpro has been associ- dsRNA-induced RANTES expression.
ated with those cleavage events (Lei et al., 2011; Sequence alignment and structural bioinfor-
Mukherjee et al., 2011; Qu et al., 2011). matics analyses suggested similarities between
the topology of FMDV Lbpro and that of some
FMDV interference with antiviral cellular and viral deubiquitylation enzymes
functionality (DUBs). Experimental evidence confirmed that
The study of FMDV mechanisms interfering with the papain-like protease of FMDV, Lbpro, is also
immune functions has been a very active area of a novel viral DUB that cleaves ubiquitin moieties
from critical signalling proteins of the type I IFN also shown in PK-15 cells after FMDV infection (Du
signalling pathway, such as RIG-I, TBK1, TNF et al., 2014). The KPNA1 degradation by 3Cpro is
receptor-associated factor 3 (TRAF3), and TRAF6 the first example of a viral immune evasion mecha-
(Wang et al., 2011a,b,c). Mutations that ablate the nism that involves direct degradation of KPNA1 by
catalytic activity or disrupt the SAP domain of a viral protease, not by a proteasome- and caspase-
Lbpro abrogate the DUB activity and also the abil- dependent pathway.
ity of Lbpro to block IFN-β induction. The cleavage of TRAF family member-associated
FMDV Lpro has been found to be an IFN-III NF-κB activator (TANK) by viral proteases
antagonist in PK-15 porcine cells. Recombinant encoded by several positive RNA viruses has just
porcine IFN-λ1 exhibited significant antiviral activ- been reported (Huang et al., 2015). The cleavage
ity against FMDV but IFN- λ1 induction could not of TANK by encephalomyocarditis virus (EMCV)
be detected in infected cells. Expression of Lpro 3C impairs the ability of TANK to inhibit TRAF6-
inhibited poly(I:C)-induced IFN-λ1 promoter mediated NF-κB signalling. FMDV 3C also cleaves
activity (Wang et al., 2011a; Wang et al., 2011b; TANK although the cleavage sites are different and
Wang et al., 2011c). In this case, the catalytic activ- a 15 kDa N-terminal fragment is produced. The
ity and the SAP domain of Lpro were required for abundance of TANK was reduced when FMDV
suppression. Lpro was co-expressed with TANK, but no cleaved
Evidence that FMDV 3Cpro is another virus- fragment was detected. TANK cleavage by FMDV
encoded IFN antagonist was firstly provided in 3C is dependent on its protease activity. Addition-
2012. Proteolytical cleavage of the NF-κB essential ally, TANK is cleaved by porcine reproductive and
modulator (NEMO, also known as Ikk-Ɣ), an respiratory syndrome virus (PRRSV) nsp4 and
adaptor protein essential for viral activation of equine arteritis virus (EAV) nsp4, which are the
IRFs and NF-κB (see above), was reported (Wang main proteases required for viral polyprotein cleav-
et al., 2012). NEMO cleavage impairs its ability to age. However, enterovirus 71 (EV71) 3C protease
activate downstream IFN production in the RLR did not cleave TANK (Huang et al., 2015). The
and TLR pathways as the fragments resulting from cleavage of TANK may represent a common mech-
3Cpro cleavage were either severely impaired for anism adopted by several positive RNA viruses to
or completely deficient in activation of NF-κB, regulate NF-κB signalling and interfere with host
IRF, and IFN induction. The cleavage of porcine innate immune and inflammatory responses. How-
NEMO in infected cells was also demonstrated. ever, the importance of viral protease–mediated
3Cpro specifically targeted the Gln 383 residue cleavage of TANK and the functions of the cleaved
and catalytically inactive mutants (H46Y, C163G, fragments under normal physiological conditions
and H181Y) failed to cleave NEMO (Wang et al., remain unknown.
2012). All the above information illustrates the wide
FMDV has been recently shown to inhibit the variety of strategies evolved by FMDV for coun-
type I IFN signalling pathway (Du et al., 2014). teracting the host defence using the viral-encoded
Expression of 3Cpro significantly reduced the tran- proteases.
script levels of ISGs and ISRE promoter activity. New insight into understanding how viral
3Cpro inhibits the JAK/STAT signalling pathway inhibitors have been selected and optimized during
by blocking the nuclear translocation of STAT1/ evolution will help in the design of more effective
STAT2. Phosphorylated STAT1 accumulates in the measurements for disease control, and elucidating
nucleus via interaction with a specific nuclear local- the intricate pathways that guide immune responses.
ization signal receptor, karyopherin α1 (KPNA1).
KPNA1 degradation was specifically attributable to
the protease activity of FMDV 3Cpro. The catalytic FMDV and mononuclear innate
triad residues H46, D84, and C163 of 3Cpro are immune cells
essential for FMDV 3Cpro to suppress ISRE pro- Innate mononuclear cells are divided into ontoge-
moter activities as the catalytically inactive 3Cpro netically distinct populations derived from bone
mutants were unable to induce KPNA1 degrada- marrow precursors (for reviews see (Haniffa
tion. The degradation of endogenous KPNA1 was et al., 2013; Merad et al., 2013)). MΦ colonize
tissues during embryonic development and can leucocytes at the site of virus entry/vaccina-
self-renew locally independent of bone-marrow- tion modulate MΦ (and DC) activities – these
derived immigration. Tissue DCs also arise from a include resident and recruited effector Th/Tc/Treg
common bone marrow precursor, but are of a dif- lymphocytes, NK cells and γ/δ T-lymphocytes.
ferent bone marrow lineage to the MΦ (and Mo). Recruitment is an effect of the initial activation
Mo are more dominant in the blood, but are par- of resident MΦ – initiation of the inflammatory
ticularly important as Mo-derived MΦ and DCs. response – and also involves blood DCs and Mo
Under inflammatory conditions, Mo differentiate differentiating into DCs and MΦ. The recruit-
into cells functionally related to the tissue cells. ment is effected through chemokine production,
Blood DCs appear to be a mixture of bone marrow with inflammatory cytokines modifying the local
derived and Mo-derived cells, although the origin endothelium to enhance leucocyte adherence lead-
of the SLAN-Tip-DCs in the blood – and tissues ing to tethering, rolling and extravasation into the
under inflammatory conditions – is still uncertain. inflammatory tissues.
The distinct lineage of bone-marrow-derived DCs Interaction of virus or vaccine with MΦ and
is composed of two major subsets: Conventional DCs will have major consequences on the outcome
(or classical) DC (cDCs) with antigen presentation of infection and the ability of the host to mount
and induction of naive T-cell responses as their a its immune defence. The rapidity with which
main function; the pDCs specialized in sensing MΦ innate defences respond places them at the
nucleic acids and producing large quantities of type forefront of a pathogen attack, with the potential
I IFN in response to TLR and helicase stimulation to destroy that pathogen. While many pathogens,
– hence the term ‘interferon-producing cells’ (IPC) including most viruses, have evolved to circumvent
or ‘natural interferon producing cells’ (NIPC) (Liu, this destructive capacity of MΦ, FMDV does not
2005). The identity of these population has been fit into these scenarios (McCullough et al., 1992b,
described in pigs, cattle and sheep (for review see 2009; Summerfield et al., 2009; Summerfield and
Summerfield et al., 2015). McCullough, 2009a). MΦ and DCs are important
elements of early immune defence following both
Monocytes and macrophages infection and emergency vaccination (Barnett et al.,
Mo and MΦ, together with Mo-derived MΦs 2002; Rigden et al., 2003), but the control of infec-
(MdMs) and DCs (MoDCs), are important actors tion is predominantly dependent on their activities
in effector innate immune defence. Together with in promoting and controlling adaptive immune
neutrophils, they are central in acute inflammatory defence development. Of particular pertinence to
reactions. Parenteral vaccination involves initially the MΦ is the capacity for rapidly detecting virus–
tissue histiocytes and dermal DCs, while mucosal antibody complexes (Fig. 10.4), leading to their
infection or vaccination involves local mucosal MΦ removal and destruction (see ‘Effector humoral
and DCs (for DCs, see next subsection). Upon immune defence’). Yet, MΦ (Fig. 10.5) and DCs
activation, MΦ initiate an inflammatory response (Fig. 10.3) can both endocytose FMDV in the
defined by their profile of cytokine and other absence of specific antibody.
inflammatory mediators. Parenteral vaccination
also causes local tissue damage due to the injec- Conventional and monocyte-derived
tion, against which an inflammatory response by dendritic cells
the resident and then recruited MΦ will develop; As with MΦ, the local DCs are first to encounter
of course the adjuvant in a vaccine formulation and respond against FMDV at mucosal surfaces or
also provides signalling to enhance inflammatory within tissues following parenteral introduction.
responses. When FMDV disseminates from its pri- Both local DCs and MΦ initiate the inflammatory
mary site of infection, local MΦ in the secondary response recruiting cells to augment DC (and
tissues and organs respond in a manner similar to MΦ) activity. Although DCs and MΦ have many
those encountering parenteral vaccine delivery. similarities, there are major distinctions between
Migratory leucocytes tend to be dominated them. MΦ recruit lysosomal proteases to phago-
by neutrophils and Mo, typifying innate defence somes, and probably most endocytic pathways,
response (Murdoch and Finn, 2000). Other earlier and at higher levels than DCs (Amigorena
Figure 10.5 Confocal microscopy image of FMDV virion antigen (green) in pulmonary macrophages, detected
with 4C9 antibody at 6 hours post-infection with FMDV O1 Lausanne. Actin microfilaments were stained red
using Alexa 546 phalloidin. The main picture is an x-y image through a single plane of the cell. The small
pictures to the left, right and below the main picture are z-stack sectional views of the left-hand and right-hand
cells shown in the main picture, to demonstrate that the virus antigen has been internalized (on the cytoplasmic
side of the microfilament cytoskeleton. Adapted from Rigden et al. (2002).
and Savina, 2010). Consequently, DCs degrade Following virus dissemination from the primary
endocytosed material at slower rates, with an site of infection, resident tissue DCs such as dermal
overall less acidic phagosomal/endosomal pH DC subsets become involved, relating to the situ-
than MΦ. These DC characteristics relate to their ation with parenteral vaccination (McCullough et
biological roles – processing and delivering antigen al., 2012). Common characteristics with tissue and
to activate lymphocyte development in second- mucosal DCs are the control of local inflammatory
ary lymphoid organs. MΦ characteristics provide responses and ultimate interaction with the adap-
a cellular defence more pertinent for removing tive immune system. Initially, local DCs are the
infectious threats to the host, as well as damaged major players, ultimately involving DCs recruited
or dying cells. Nonetheless, these prominent roles by the inflammatory response. The latter are derived
are neither absolute nor mutually exclusive, and from blood cells such as blood Mo, but also include
both DCs and MΦ interact during inflammatory blood DCs such as the SLAN + TIP-type of DC, by
responses and recruitment of additional DCs, MΦ, inference to the murine and human DC responses.
T-lymphocytes and NK cells. These SLAN+ TIP DCs are important murine and
At mucosal surfaces, DCs underlying the human subsets for processing antigen and promot-
mucosal epithelia interact indirectly with antigen ing maturation of DCs for antigen presentation (see
following virus/vaccine transport across or through below). This function of DCs will be discussed in
the mucosal epithelium (transcytosis). Importantly, the context of adaptive immune defences in ‘Innate
DCs mucosal surfaces can also open tight junctions to adaptive: inducing adaptive immune defence
between epithelial cells to access the mucosal sur- against FMDV’.
face, while ensuring integrity of these gap junctions
(Chieppa et al., 2006; Rescigno, 2010; Rescigno Plasmacytoid dendritic cells or
and Di Sabatino, 2009; Rescigno et al., 2001, 2008). natural interferon-producing cells
By such means, DCs send cellular protrusions into As mentioned above, porcine pDCs can sense
the lumen at the mucosal surfaces, thus capturing FMDV through TLR7 and produce IFN-α in
foreign material. response to infection (Fig. 10.4). Nevertheless, the
levels of IFN-α release are relatively modest when humoral immune defence’, subsection ‘The role of
compared with other viruses such as influenza virus innate effector plasmacytoid DCs’).
or porcine respiratory reproductive syndrome virus
(Baumann et al., 2013; Bel et al., 2011; Guzylack- MΦ and DC receptors for
Piriou et al., 2006a; Lannes and Summerfield, endocytosis of FMDV
2013; Reid et al., 2011; Summerfield et al., 2009).
Considering that FMDV has efficient mechanisms General appreciation of innate cell
for controlling such responses in non-innate cells receptors
– ultimately including its cytopathogenicity for Innate immune defence cell receptors are involved
epithelial cells – it is unclear if these mechanisms in many aspects of host environment surveillance,
operate in pDC. The pDC responses to FMDV including pathogen uptake and promoting inflam-
differ from those against influenza virus, being matory reactions (Gallucci and Matzinger, 2001;
observed only with live virus. In fact, FMDV initi- Medzhitov and Janeway, 2000a; Wagner and Roth,
ates replication in pDCs in terms of non-structural 2000). Receptors such as C-type lectins, integrins
protein expression. However, this replication is and CD44 can enhance binding to and uptake
abortive and does not result in release of progeny by the cells, leading into endocytic processing.
virus (Guzylack-Piriou et al., 2006a), similar to the PRRs such as Toll-like receptors (TLRs), as well
situation in MΦ (see ‘Consequences of FMDV as complement receptors and mannose-binding
interaction with mononuclear innate immune receptors including CD206, are important for
cells’). inflammatory responses (McCullough et al., 2012;
The pDC responses against FMDV can be Medzhitov and Janeway, 2000a,b; Pulendran and
enhanced by the Th1 cytokine IFN-γ, as well as Ahmed, 2006; Rossi and Zlotnik, 2000). While
certain haematopoietic cytokines enhancing pDC ligation of receptors such as TLRs, SigLecs, Galec-
survival. Type I IFN itself also enhances pDC tins and CD14 activate innate defence processes,
responses in a positive amplification loop (Lannes they can also promote antigen uptake, either alone
and Summerfield, 2013). Despite the evasion or in co-operation with C-type lectins or integrins
strategies described in ‘Interaction of FMDV (Dzopalic et al., 2012; McCullough et al., 2012;
with innate viral sensors’, infected pigs and cattle Ouasti et al., 2011). Fc-receptors (FcαR/FcγRI/
can mount a short, systemically detectable IFN-α FcγRII/FcγRIII) are also involved in endocytosis,
response (Nfon et al., 2010; Reid et al., 2011). The when pathogen is opsonized by antibody; similarly
cattle studies employed anti-CD4 monoclonal anti- with complement receptors (CR1/CR2/CR3/
body treatment to deplete CD4+ Th-lymphocytes. CR4/C3aR/C5aR) when appropriate complement
Bovine pDC relate to porcine pDCs, which components are associated with the antigen–anti-
are CD4+ (Guzylack-Piriou et al., 2004, 2006a; body complexes. This latter area is pertinent to the
Summerfield et al., 2003; Summerfield and early studies on MΦ and DCs uptake of FMDV
McCullough, 2009b); anti-CD4 treatment may complexed with antibody (Baxt and Mason, 1995;
well deplete pDCs. McCullough et al., 1986, 1988, 1992b; Rigden et al.,
During in the early phase of a naive FMDV 2002). This aspect of immune defence is dependent
infection, induction of pDC IFN-α responses on adaptive immune responses, and will be con-
could benefit the host in two ways: A direct anti- sidered in ‘Effector lymphocyte responses against
viral effect limiting FMDV replication; Type I IFN FMDV’. Direct interaction of FMDV with MΦ
system-dependent alarm leading adaptive immune and DCs in the absence of specific antibody does
responses (Summerfield et al., 2009; Summerfield occur (Harwood et al., 2008; Rigden et al., 2002).
and McCullough, 2009a). Following develop- The resultant endocytosis leads to retention of virus
ment of adaptive immunity, the pDCs would play antigen for prolonged periods, clearly distinct from
important roles. The cells are strongly activated the rapid uncoating of virus and replication (within
by FMDV complexed with antibodies, resulting a few hours) to produce progeny virus during a
in secretion of high IFN-α levels (Guzylack-Piriou single-cycle infection in permissive epithelial cells.
et al., 2006a). In this way, pDCs also contribute to FMDV interaction with MΦ and DCs via
antibody-mediated effector functions (see ‘Effector integrin-binding is likely distinctive to the
integrin-binding on permissive epithelial cells, same integrins as epithelial cells (Harwood et al.,
further complicated by the adaptation of vaccine 2008). The binding to MΦ and DCs compared
viruses upon cell culture passage for binding with with epithelial cells may require different accessory
proteoglycans of heparan sulphate (HS)-containing receptors – tetraspanins are well documented for
glycosaminoglycans. It is important to consider this type of activity (Berditchevski, 2001; Charrin
how interaction with MΦ and DCs relates to bind- et al., 2014; Dahmane et al., 2014; Scheffer et al.,
ing with epithelial cells, particularly considering the 2014). These molecules have been implicated in
importance of HS-independent to the handling of several cellular activities, including interactions
vaccine virus. with viruses, due to their ability in organizing
membrane microdomains and thus modulating the
Involvement of integrins function of associated molecules and subsequent
FMDV employs the Arg-Gly-Asp (RGD) sequence signal transduction. Importantly for virus entry,
in the G-H loop of VP1 capsid protein for binding this includes the outcome of initial interaction
with integrin receptors on susceptible cells (Baxt between other receptors including integrins and
and Becker, 1990; Berinstein et al., 1995; Fox et their ligands (Berditchevski, 2001; Charrin et al.,
al., 1989; Jackson et al., 1997, 2000a,b; Leippert et 2014). Tetraspanins are also important accessory
al., 1997; Liebermann et al., 1991; Neff and Baxt, molecules in organizing a number of immune cell
2001; O’Donnell et al., 2005). MΦ and DCs also cognate interactions (Levy and Shoham, 2005).
express integrins capable of interacting with RGD
motifs, which should provide potential for FMDV Involvement of proteoglycans
interaction. Importantly, the integrins reported as It appears that the cell culture passage selects for
main receptors for FMDV infection leading to rep- FMDV variants with positively charged amino
lication in epithelial cells are αvβ1, αvβ3, αvβ6 and acids on their capsid surface, capable of interact-
αvβ8 (Berinstein et al., 1995; Berryman et al., 2005; ing with heparan sulphate-containing structures
Jackson et al., 1997, 2000a,b, 2002, 2004; Johns et (Baranowski et al., 2000; Jackson et al., 1996; Neff
al., 2009; O’Donnell et al., 2005). Human DCs do et al., 1998; Sa-Carvalho et al., 1997). Cell-passaged
express β1 and β3 integrins (D’Amico et al., 1998; FMDV, as used for vaccine production, employs
Jancic et al., 1998), but not β6 integrins; in sheep, HS-containing proteoglycan receptors ( Jackson et
only epithelial tissues carry αvβ6 (Brown et al., al., 1996; Zhao et al., 2003). These are glycosami-
2006). Porcine MΦ and DCs poorly express these noglycan polymers of highly sulphated, negatively
epithelia cell integrin receptors for FMDV, if at all charged, disaccharide repeats – hence their involve-
(Harwood et al., 2008). The β1 and β3 chains were ment with cell culture-adapted virus carrying
detected, but the presence of β chains does not positively charged surface amino acids. Although
ensure association with appropriate α chain to form selection of HS-binding virus should dispense with
receptors for FMDV productive infection. Indeed, an obligation for integrin receptors, the situation
antibodies more specific for αvβ3, αvβ5 and αvβ8 may be more elaborate.
gave only weak reactions with porcine MΦ and DCs While membrane proteoglycans act as endocytic
(Harwood et al., 2008). Moreover, detection of receptors, they can also function as co-receptors,
integrin-binding virus interaction with the porcine particularly with a number of tyrosine-kinase type
DCs was more difficult compared with detection of growth factors. Membrane proteoglycans also
HS-binding virus. Nonetheless, peptide antigens cooperate with integrins (and other cell adhesion
carrying the B-cell epitope – and RGD motif – of molecules) for promoting cell–cell and cell–extra-
antigenic site A contained within the G-H loop cellular matrix interactions (Esko et al., 2009).
interacted most efficiently with porcine DCs, and Accordingly, HS-containing proteoglycans could
were internalized by various endocytic routes strengthen relatively weak interactions with the
(K.C. McCullough, D. Andreu and F. Sobrino, integrins on MΦ and DCs. Alternatively, soluble
unpublished data). This would suggest that FMDV proteoglycans could bind with FMDV to form
could indeed bind to integrins expressed by DCs. complexes more readily interactive with the MΦ/
Certainly, it has been proposed that FMDV inter- DC integrins. Either of these scenarios may equally
action with MΦ and DCs would not employ the occur with permissive epithelial cells, but the αvβ1,
αvβ3, αvβ6 or αvβ8 do not display an apparent obli- response function. Comparison with epithelia cell
gation for proteoglycan involvement. endocytosis has helped broaden our understand-
Using cloned viruses possessing no binding ing of virus/antigen interaction with MΦ and DCs
to HS-containing glycosaminoglycan structures, (Ewers and Helenius, 2011; McCullough et al.,
proteoglycan-independent binding with MΦ 2012; Pelkmans et al., 2001; Vazquez-Calvo et al.,
and DCs was clearly less efficient compared with 2012).
HS-proteoglycan-enhanced binding (Fig. 10.3)
(Harwood et al., 2008; Rigden et al., 2002). This Endocytosis of FMDV by epithelial cells
is not a failing of the virus G-H loop carrying the FMDV entry via integrin receptors expressed by
RGD motif, because peptides from this region epithelial cells – human MCF-10A cells express-
readily interact with DCs and are efficiently endo- ing human αvβ3 and αvβ6 integrins (O’Donnell
cytosed (K.C. McCullough, D. Andreu and F. et al., 2005), or human adenocarcinoma-derived
Sobrino, unpublished data). Clearly, the interac- SW480 cells and Chinese hamster ovary (CHO)
tion of FMDV with MΦ and DCs, and probably cells stably transfected for αvβ6 expression (Berry-
also epithelial cells, is more complex than simple man et al., 2005) – was related to clathrin-mediated
integrin-dependent of HS-proteoglycan dependent endocytosis. Apparent co-localization of FMDV
binding, and accessory interactions such as those with clathrin was reported (O’Donnell et al., 2005),
proposed for tetraspanins are likely to be influential although no co-localization studies were per-
(Berditchevski, 2001; Charrin et al., 2014; Dah- formed. Importantly, the majority of virus particles
mane et al., 2014; Scheffer et al., 2014). were not associated with clathrin, a characteristic
retained during the 30-minute period reported.
Consideration of vaccine virus Inhibition of FMDV uptake by MCF-10A cells
With DCs being critical to vaccination and effi- using chlorpromazine (O’Donnell et al., 2005) – a
cient induction of immunity, HS-proteoglycans cationic amphiphilic drug interfering with clathrin
become pertinent for vaccine virus interaction with AP-2 subunit, preventing clathrin binding to and
DCs and MΦ (Fig. 10.3). Vaccine virus binding loss of clathrin coated pits at the plasma membrane
to porcine DCs via HS-containing proteoglycans – was taken to show clathrin-dependency. However,
(Harwood et al., 2008) relates to observations detection was assessed after 4 hours, which does not
on human papillomavirus 16 binding to human relate to the rapid (minutes) kinetics of clathrin-
DCs (Bousarghin et al., 2005). The dominance of mediated processes. Moreover, such a duration
HS-proteoglycans on cell surfaces and as secreted would inhibit the sourcing of endosomal structures
proteins provides a powerful mechanism for DC and contents from the trans-Golgi (Gallon and
interaction, particularly in the context of vaccine Cullen, 2015), and eventually retromer-based
delivery targeting DCs (McCullough et al., 2012, recycling and trans-Golgi communications would
2014b). With cell culture adaptation of FMDV for be disrupted, inhibiting most endocytic pathways.
vaccine production enhancing DC interaction (Fig. Additional analysis relating to AP180 – a mono-
10.3), the positively charged amino acids involved meric assembly protein stimulating clathrin latex
in reaction with HS-containing proteoglycans may formation on membranes to generate the clathrin
also influence the DC endocytic route in the direc- cages for clathrin-mediated endocytosis – employed
tion of cytosolic translocation (McCullough et al., AP180 C-terminus expression as a double-negative
2012). While this can facilitate various antigen inhibitor of clathrin-dependent endocytosis. In
processing/delivery pathways, a particular applica- SW480-αvβ6 cells and IBRS-2 cells, this inhibited
bility is cross-presentation (see ‘Antigen-presenting FMDV uptake (Berryman et al., 2005; Johns et
cell interaction with T-lymphocytes’). al., 2009). Nonetheless, the approach would also
impact on the aforementioned endosomal sourcing
Endocytic routes employed by MΦ from the trans-Golgi and retromer-based recycling
and DCs with FMDV (Gallon and Cullen, 2015).
MΦ and DCs employ their variety of endocytic With SW480-αvβ6 cells and CHO-αvβ6 cells,
routes for processing and degradation of internal- rapid (5 minutes) co-localization of endocytosed
ized material, thus influencing innate immune FMDV was noted with early endosomal antigen
positive (EEA-1) and transferrin, but not with clathrin-coated vesicle formation, and the intracel-
lysosomal antigen LAMP-2 up to 30 minutes post lular movement after clathrin uncoating along actin
infection (Berryman et al., 2005). Once again, the microfilaments. This involves primarily short-term
majority of virus antigen+ vesicles were not associ- peripheral events in mammalian cells, moving endo-
ated with these endosomal markers, suggesting cytic vesicles from the actin-rich periphery to early
either a particularly asynchronous procedure, or endosomes. Nonetheless, early endosomes leading
the involvement of the different endocytic path- into endocytic processing routes is dependent on
ways known to be operative (Kumari et al., 2010; kinensin and dynein motors for microtubules; the
McCullough et al., 2012; Sandvig et al., 2011; initial role for microfilament transport is to delivery
Sorkin and von Zastrow, 2009). Nocodazole (depo- endocytic vesicles into the microtubule transport
lymerizes microtubules) and wortmannin (inhibits system.
PI3 kinase Type I and macropinocytosis) could By 30 minutes, the amount of detectable virus
not prevent FMDV infection in epithelial cells was reduced, associated neither with EEA-1+
(Berryman et al., 2005) – clearly divergent from structures nor with late endosomes/lysosomes
observations in DCs (see ‘Endocytosis of FMDV (Berryman et al., 2005). This is expected due
by MΦ and DCs’, and Figs. 10.5 and 10.6) – nor did to the conditions in maturing endolysosomes
the virus clearly associate with caveolin-1 (caveolar degrading virus beyond recognition by antibod-
endocytosis) (Berryman et al., 2005; O’Donnell et ies. With degradative clathrin-mediated endocytic
al., 2005). Of course, inhibition of one endocytic pathways occurring rapidly, virus-positive inclu-
route would still allow other routes to operate, both sions remaining at 30 minutes likely reflected
clathrin dependent and independent. The apparent slower clathrin-independent or late-endosome-
independence from microtubule function (unaf- independent routes. The former would explain
fected by nocodazole) is surprising considering the the virus+ vesicles not co-localizing with EEA-1+
role of microtubules in the transport and matura- structures. Association with transferrin and trans-
tion of endosomal structures. Actin-dependent ferrin receptor at 15–30 minutes would suggest
transport does exist – myosin VI is involved in retrograde transport associated with recycling/
Figure 10.6 Heparan sulphate-binding FMDV O1 Lausanne internalized by porcine MoDC showing antigen
accumulation during the initial four hours 4 h. (a) Vesicular inclusions of endocytosed FMDV (red) both peripheral
and deeper in the cells, in juxtaposition to microfilaments stained with phalloidin (green). (b) Vesicular inclusions
of endocytosed FMDV (green) peripherally in the cells, attached to the ends of microtubules, visualized through
inhibiting endocytic progression with the vacuolar-H+-ATPase inhibitor bafilomycin. Adapted from Harwood et
al. (2008).
sorting endosomes. Indeed, at 15 minutes post and is not particular to one endocytic route. In con-
infection, some virus+ vesicles co-localized with trast to Rab5, the small GTPase Rab11 (Golgi and
transferrin receptor, indicative of recycling/sorting recycling endosomes) had only a minor influence,
endosomes, presenting the possibility for recycling while Rab4 (endosomal recycling to the plasma
of virus to the plasma membrane or retrograde membrane) had no influence ( Johns et al., 2009).
transport into the Golgi/ER (Lu and Hong, 2014; This suggests that the majority of virus has contact
Mallard et al., 1998) – but see ‘FMDV uptake by with early endosomes, although a small portion
epithelial cells suggestive of multiple endocytic may pass through the retrograde pathway into the
routes’ and ‘Endocytosis of FMDV by MΦ and Golgi, but not recycling back to the plasma mem-
DCs’. brane (Lu and Hong, 2014; Mallard et al., 1998;
Overall, the results with epithelial cells implicate Sandvig and van Deurs, 2005; Scott et al., 2014).
different endocytic routes operative with FMDV Some of the above characteristics from FMDV
uptake. The dominance of clathrin-mediated interaction with epithelial cells (O’Donnell et
endocytosis leading into the maturing endolyso- al., 2005) may be indicative of macropinocyto-
somal system is typical of most cells. By providing sis (McCullough et al., 2012; Vazquez-Calvo et
a rapid passage through the maturing endosomal al., 2012). Certainly, HS-binding virus uptake
system, this is potentially problematic for viral by MCF-10A epithelial cells (O’Donnell et al.,
genome cytosolic translocation – leading more 2008) was reported as dominated by caveolar-
to degradation than initiation of virus replication like endocytosis. This relates to HS-mediated
(McCullough et al., 2012; Pelkmans et al., 2001; internalization of ligands employing caveolin-
Vazquez-Calvo et al., 2012). Such a scenario has dependent pathways (Reilly et al., 2004). Caveolar
been described with murine polyoma virus, which endocytosis was slower than clathrin-mediated
uses integrin α4β1 for binding (Mannova and uptake – co-localization with EEA-1+ structures
Forstova, 2003; Richterova et al., 2001). High num- was still detectable after 1 hours (O’Donnell et al.,
bers of virions or pseudocapsids were required for 2008), although many virus+ structures were not
productive infection, the majority of virus particles EEA-1+ at this time. Of course, the slower kinet-
apparently being degraded following uptake by the ics would show more readily the asynchrony of
cells. Accordingly, successful interaction of FMDV the process – both material interacting with early
with epithelial cells leading to virus replication endosomes and caveolar endocytic vesicles not
may involve more than a single endocytic route, to yet interacting. HS-binding virus was also noted
ensure virus genome escape from the rapid degra- to associate with transferrin+ and transferrin
dation offered by the dominant clathrin-dependent receptor+ structures, implying the involvement
endocytosis. of both clathrin-independent and -dependent
endocytosis.
FMDV uptake by epithelial cells
suggestive of multiple endocytic routes Endocytosis of FMDV by MΦ and DCs
Using double negative mutants of the small The above observations that the receptor employed
GTPase Rab5 – associates with EEA-1+ early (proteoglycans compared with integrins) can
endosomes – inhibited the FMDV infectious pro- influence the endocytic route of uptake are impor-
cess ( Johns et al., 2009), intimating a requirement tant when considering MΦ and DCs, wherein
for clathrin-mediated endocytosis. However, Rab5 macropinocytosis is a prominent endocytic route
functionality will impact on all endocytic routes (Basta et al., 1999, 2010; Harwood et al., 2008;
dependent on early endosomes, including macro- McCullough et al., 2012; Natale and McCullough,
pinocytosis and caveolar endocytosis (Ewers and 1998; Rigden et al., 2002). Both MΦ and DCs
Helenius, 2011; Kumari et al., 2010; Pelkmans and display similar dominance of the different endo-
Helenius, 2002; Sandvig et al., 2011; Sorkin and cytic routes (Kumari et al., 2010; McCullough et
von Zastrow, 2009). This small GTPase functions al., 2012; Pelkmans et al., 2001; Vazquez-Calvo
more generally for early endosome fusion with et al., 2012), but do not express the integrins
endocytic vesicles following endocytic internaliza- associated with FMDV infection of epithelial
tion (Somsel Rodman and Wandinger-Ness, 2000), cells. Infection of MΦ (Fig. 10.5) and DCs (Fig.
10.6) via proteoglycans does occur (Harwood et DCs (Fig. 10.6), as well as infectious virus (Fig
al., 2008; Rigden et al., 2002), which is important 10.7a and b) (Harwood et al., 2008; Rigden et al.,
for vaccine interaction with the cells. Integrin- 2002). Integrin-binding virus was not so easily
binding FMDV also interacts with MΦ and DCs, detected (Fig 10.7c and d) (Harwood et al., 2008)
to lower levels compared with HS-binding FMDV (F. Caridi, F. Sobrino and K.C. McCullough,
(Harwood et al., 2008). Internalized HS-binding unpublished data).
virus was readily detectable in terms of antigen Endocytosis of FMDV by MΦ and DCs nota-
associated with microfilaments in MΦ (Fig. 10.5) bly required microfilaments (Fig. 10.7e; inhibition
and both microfilaments and microtubules in by cytochalasin D), and close association with
Figure 10.7 Interaction of heparan sulphate-binding FMDV O1 with porcine MoDC. (a) pH resistance of
DC-associated FMDV was measured by infecting 4-day-old MoDC with FMDV O1 at an MOI of 1 TCID50/cell
for 1 hours at 4°C. Cells were then washed eight times to remove unbound virus followed by the addition of
prewarmed medium, and the temperature was then shifted to 39°C. At the indicated time points, half the cells
were treated with 0.05 M sodium hydrogen phosphate (pH 6) to destroy virus on the cell surface. Cells were
then lysed to measure the titres of cell-associated virus by titration on BHK-21 cells. (b) Replication of FMDV
O1 was determined by pretreating cells with cycloheximide and measuring infectious cell-associated virus
by titration on BHK-21 cells. (c) Comparison of heparan sulphate-binding and non-heparan sulphate-binding
FMDV interaction with MoDC in terms of infectious virus. Four-day-old MoDC were infected with FMDV O1
and FMDV C-S8c1 at an MOI of 1 TCID50/cell for 1 hours at 4°C. Cells were then shifted to 39°C for the
duration of the experiment. At each of the time points shown on the x axis, the cells were washed eight times
to remove any unbound virus. The cells were then lysed after the final centrifugation to release cell-associated
virus, and the infectious virus titres measured by titration on BHK-21 cells. (d) Following transfection of MoDC
with O1K/C-S8c1 infectious RNA, measurement of ECV and cell-associated virus (CAV) at different times
post-transfection by titrating on BHK-21 cells demonstrated that the transfection results in abortive replication.
(e) The influence of metabolic inhibitors on the binding (30min p.i.; pronase-sensitive) and internalization (4
hours p.i.; pronase-resistant) of FMDV by MoDC, measured as the percentage of bound virus in a non-pronase
treated control. Adapted from Harwood et al. (2008).
Date: 10:24 Monday 12 September 2016
UNCORRECTED PROOF File: FMDV 4P
Immune Cell Interaction with FMD | 229
microfilaments and microtubules (Figs. 10.5 and by sensitivity to wortmannin, the influence of
10.6). The involvement of multiple endocytic filipin (cholesterol and caveolar endocytosis
routes was witnessed by the inhibitory influence inhibitor) and chlorpromazine suggests additional
of chlorpromazine, filipin and wortmannin, none involvement of lipid rafts, caveolar endocytosis
of which alone could abrogate uptake of the virus and clathrin-dependent processes (Fig 10.7e)
by DCs (Harwood et al., 2008). The acidifying (Harwood et al., 2008). Concerning clathrin-
machinery of the endosomal system – identified dependent endocytosis of HS-binding virus by
by sensitivity to bafilomycin and chloroquine – DCs, chlorpromazine modified antigen distri-
was also important, more notable at the later 4 bution in the cell to be punctate without the
hours post infection than the earlier 30 minutes perinuclear localization observed in the absence
(Fig 10.7e) (Harwood et al., 2008). This would of metabolic inhibitors (Harwood et al., 2008).
reflect processing via early endosomal maturation, Together with being influential only at the later 4
rather than retrograde processes via the Golgi hours rather than 30 minutes post infection (Fig
into the ER (see (Lu and Hong, 2014; Scott et al., 10.7e) suggests more an influence on delivery of
2014; Vazquez-Calvo et al., 2012). Interaction endosomal material from the Golgi.
with DCs in particular led to de novo production Overall, DC and MΦ endocytosis of FMDV
of both virion antigen (Fig. 10.7b) and infectious leading to genome activity and antigen processing/
virus (Fig. 10.7c). Although the observed virus presentation involves a dominance of macropino-
replication was ultimately abortive (Fig 10.7c cytosis. This is likely similar for DC endocytosis
and d) (Harwood et al., 2008), the translation of HS-binding and HS-independent FMDV. The
of antigen and its retention for at least 8 hours abortive replication of the virus and retention
requires cytosolic translocation of the genome of antigen would be important for induction of
from the endocytosed FMDV. This relates to the adaptive immunity. Indeed, DC endocytosis of
influence of bafilomycin/chloroquine inhibition HS-binding and HS-independent FMDV did pro-
of the endosomal acidification machinery (Fig. vide conditions for re-stimulating FMDV-specific
10.7e), potentially involving pore formation or lymphocytes (Fig. 10.8) (Harwood et al., 2008).
endosomal membrane polarization as reported Relating to the differences in endocytosis activ-
with rhinoviruses (Brabec et al., 2003; Fuchs and ity for the two forms of FMDV, re-stimulation
Blaas, 2010; Konecsni et al., 2009) – also employ with HS-independent virus was 40–50% of that
both clathrin-dependent (Snyers et al., 2003) and obtained with HS-binding FMDV. This immune
clathrin-independent (Bayer et al., 2001) routes of response was related to genomic activity of the
endocytosis. virus, determined by transfecting DCs with
Macropinocytosis – a more dominant endo- viral RNA (Fig. 10.7d) (Harwood et al., 2008).
cytic process with MΦ and DCs compared to The greater capacity for DCs to endocytose HS-
epithelial cells – is a major force in the internaliza- binding virus is an important characteristic for
tion of FMDV (Fig 10.7e; wortmannin-sensitive) vaccines. For field viruses, as during an infection,
(Harwood et al., 2008; Rigden et al., 2002). This interaction with the DCs may provide less antigen
was observed with both HS-binding virus and initially for the cell to process, but the DCs only
peptides carrying the epitopes from the site A of require a small amount of antigen to be efficient
the integrin-binding G-H loop (McCullough, at promoting adaptive immune defence develop-
Andreu and Sobrino, unpublished data). Moreo- ment. The capacity of these HS-independent
ver, DC endocytosis of lipopeptides carrying these viruses to translate in DCs, albeit with an abortive
FMDV epitopes – self-assembly into synthetic infection (see ‘FMDV within MΦ and DCs’ and
virus-like particles (SVLPs) – employs primarily ‘Effector humoral immune defence’), will increase
macropinocytosis (Sharma et al., 2012, 2016), the antigen content therein. The observed acti-
defined by co-localization with high molecular vation of immune and naive T-lymphocytes
weight dextran accumulating in macropinosomes. demonstrates the importance of this interaction
Although the importance of macropinocytosis in with DCs for initiating adaptive immune defence
DC endocytosis of HS-binding virus is witnessed (Harwood et al., 2008).
Figure 10.8 MoDC from a vaccinated pig were infected for 4 hours with heparan sulphate-binding FMDV (C1,
MARLS, and O1K/C-S8c1/p5) and non- heparan sulphate-binding FMDV (C-S8c1 and O1K/C-S8c1/p2), after
which the cells were extensively washed before coculturing with CD172a– sorted lymphcoytes. The anti-FMDV
IgG production was measured by ELISA. Adapted from Harwood et al. (2008).
Figure 10.9 FMDV interaction with pulmonary macrophages (MØ). (a, b) Presence of virion antigen at: between
30 min and 48 hours post-infection detected using the 4C9 antibody against a conformational epitope. The
grey histogram (Neg.) shows the staining of cells treated with mock antigen. (c, d) Presence of infectious FMDV
associated with the FMDV-treated MØ culture (filled circles), in comparison with infected BHK-21 cells (unfilled
circles), measured in terms of cell-free virus (c) and cell-associated virus (d) released by sonication of the cells.
Adapted from Rigden et al. (2002).
Date: 10:24 Monday 12 September 2016
UNCORRECTED PROOF File: FMDV 4P
Immune Cell Interaction with FMD | 231
FMDV, only HS-binding virus was reported to cell response against FMDV not only enhances
replicate; integrin-binding virus had to be immune endocytic uptake, but can also activate the cells
complexed (Robinson et al., 2011). Although a pro- (McCullough et al., 2012; Medzhitov and Janeway,
ductive infection was linked to cytopathogenicity, 2000a,b; Pulendran and Ahmed, 2006; Rossi and
this is in contradiction to the majority of analyses Zlotnik, 2000). Vaccination or infection expands
on FMDV infection of DCs – and MΦ – which these outcomes through release of exogenous
clearly demonstrate a transient, ultimately abortive mediators due to local tissue damage, referred to as
replication without notable cell death (Bautista et ‘danger signals’ or DAMPs (Gallucci and Matzinger,
al., 2005; Baxt and Mason, 1995; Guzylack-Piriou 2001). As with PAMPs associated with the virus or
et al., 2006a; Harwood et al., 2008; Lannes et al., its infection, DAMPs are recognized by receptors
2012; Ostrowski et al., 2005; Reid et al., 2011; on histiocytes (MΦ and neutrophils), DCs and
Rigden et al., 2002; Summerfield et al., 2009). An mast cells at the site of injury or vaccine deposition.
explanation for this discrepancy may relate to the The resultant production of exogenous mediators
high multiplicity of infection employed and/or elaborates immune defence development through
the immune complexes (see ‘Effector humoral local inflammatory reactions, recruiting additional
immune defence’). Certainly, integrin-binding innate cells and effector lymphocytes. Inflamma-
virus alone did not induce cell death in bovine tory signalling modifies endothelial cell adhesion
DCs. Importantly, infection was described in terms molecule expression in the inflammation locality,
of non-structural protein 3A detection. It was increasing local endothelium permeabilization.
unclear if this came from the original inoculum – Chemokines, released from the endothelium and
MΦ and DCs are efficient at internalizing any viral inflammatory innate immune cells, provide gra-
protein including non-structural proteins (Rigden dients for chemo-attraction of blood leucocytes
et al., 2002). The 3A staining pattern extending as well as providing receptors when present on
from peripheral to perinuclear vesicular sites is endothelial cell surfaces. Together with integ-
indicative of compartmentalization within the rins and selectins, receptors on cells of inflamed
endosomal system. These authors did not employ endothelium regulate the tethering/rolling process
cycloheximide to check if the observed staining leading to extravasation of migratory cells. Blood
was de novo, as was employed with porcine MΦ leucocytes adhere to modified endothelium in
to show internalization of non-structural protein the vicinity of the inflammation; the modulated
rather than synthesis (Rigden et al., 2002). Nor receptor expression facilitates increased binding
was infectious RNA employed to determine if cell and rolling on the endothelium, loosening intracel-
death was caused by genome replication. Certainly, lular tight junctions for extravasation into the site of
excessive endocytosis of 3A could prove detrimen- inflammation.
tal to cell survival (R. Rigden, C. Carrasco and K.C. Enhanced recruitment by extravasation pro-
McCullough, unpublished data). vides increased numbers of MΦ and DCs, with
The majority of studies on FMDV interaction increased capacity for endocytosing the virus.
with MΦ and DCs describe abortive virus replica- The abortive replication observed in DCs (see
tion without notable cytopathogenicity. The cycles ‘FMDV within MΦ and DCs’) provides PAMPs
of antigen production are important, particularly associated with the viral RNA and dsRNA replica-
with viable DCs, leading as they do to efficient tive intermediates for further promoting innate
induction of both naive and effector/memory lym- responses (Guzylack-Piriou et al., 2006a; Harwood
phocyte responses (Harwood et al., 2008). et al., 2008). Antibody against dsRNA confirmed
the abortive replication in porcine cDC subsets
MΦ and DC responses to FMDV (Harwood et al., 2008; Rigden et al., 2002), also
infection and vaccination observed in murine DCs (Ostrowski et al., 2005)
and porcine pDCs (Guzylack-Piriou et al., 2006a).
Danger recognition The latter was particularly evident after endocytosis
As mentioned in ‘Innate and adaptive immune of FMDV–antibody complexes, demonstrating
defences pertinent to FMDV’, the initial employ- another advantage of specific antibody against the
ment of cell surface receptors by innate immune virus – facilitating uptake by pDCs leading to
enhance innate signalling required by cDCs and CD80/86 (Bautista et al., 2005). FMDV-infected
adaptive immune responses. Thus, DCs endocy- porcine MoDCs also remain immunocompetent,
tosing FMDV would respond to the presence of readily stimulating FMDV-specific Th-lymphocytes
dsRNA intermediates, as well as to double stranded (Harwood et al., 2008).
secondary structures formed by hairpin structures Innate immune responses are important in vivo
in the genomic RNA, as identified using mRNA early post-vaccination, which may be enhanced by
molecules with DCs (Ceppi et al., 2005). the subsequently developing antibody responses.
Early innate immune responses were observed
Innate immune cytokine responses in terms of increased chemotactic activity in sera
In the context of the pDCs, a particularly impor- from vaccinated pigs and increased blood leucocyte
tant factor with respect to viral infections is Type migration (Rigden et al., 2003). Innate cytokines
I IFN (see ‘Interaction of FMDV with innate viral IL-6, IL-8 and IL-12 were also detected early post
sensors’) (Fig. 10.4). Within innate defences, the vaccination (Barnett et al., 2002), before anti-
most potent producer of IFN-α is the pDC (natural FMDV antibody, and would relate to the enhanced
interferon-producing cell, NIPC) (Nowacki and migration by the blood leucocytes, particularly
Charley, 1993; Riffault et al., 1997; Summerfield with the IL-8.
et al., 2003, 2015; Summerfield and McCullough,
2009b). FMDV can induce IFN-α production
by pDCs in vitro (see ‘The role of innate effector Ranking innate immune
plasmacytoid DCs’) (Guzylack-Piriou et al., 2006a; defences
Lannes et al., 2012; Reid et al., 2011; Summerfield The host possesses additional innate defences
et al., 2003, 2009). Serum IFN-α and pDC activity against virus infection, including NK cells and γ/δ
is also observed to increase early after infection of T-lymphocytes (Golde et al., 2008; Toka et al.,
pigs with FMDV (Nfon et al., 2010). Using adeno- 2009a, 2011). NK cell activity is likely to be impor-
virus vectors encoding FMDV antigens and IFN-α, tant in the innate defence against FMDV. At rest,
vaccinated pigs showed increases in DC and natural these cells show little cytolytic activity; upon acti-
killer (NK) cells levels in the skin and lymph nodes, vation by pro-inflammatory cytokines, which could
as well as delayed or decreased clinical signs follow- be derived from MΦ and DCs as well as lympho-
ing FMDV challenge infection (Diaz-San Segundo cytes, they efficiently lyse FMDV-infected targets
et al., 2010). (Toka et al., 2009b). NK-like cells from vaccinated
The immune system does not always show a cattle displayed a non MHC-restricted cytolytic
positive antiviral direction. Acute infection of activity against FMDV-infected cells (Amadori et
pigs with FMDV was noted to result in increased al., 1992). Shortly after FMDV infection in pigs, a
IL-10 production by DCs, which in turn had a transient decrease in NK cells was noted (Toka et
negative influence on antigen presentation and al., 2009a). As with peripheral lymphocyte and DC
T-lymphocyte activation (Diaz-San Segundo et al., numbers, such a transient effect is more typical of
2009). The FMDV infection of DC precursors may an inflammatory response against the virus rather
have caused the interference with both DC matura- than the immunomodulation observed with mono-
tion and antigen presentation, possibly linked to cytotropic viruses such as CSFV (Summerfield et
the observed induction of IL-10. In mice, abortive al., 1998, 2001). Moreover, the NK cell function-
infection of FMDV in DCs led to down-regulation ality remained intact following FMDV infection
of MHC Class II and CD40 (Ostrowski et al., 2005). (Golde et al., 2008; Toka et al., 2009a).
Taken together with the IL-10 secretin by these As for γ/δ T-lymphocytes, their situation in the
cells could explain the observed failure to stimu- defence against FMDV remains unclear. Following
late T-lymphocytes in vitro. Nonetheless, this may infection of cattle, an early and transient modula-
reflect particular conditions and not hold true for tion of their surface receptors has been noted, again
all situations. Porcine skin DCs respond to FMDV more likely reflecting immune response against
by producing Type I IFN, without detectable detri- virus presence. Indeed, the altered functionality
mental effect on cell viability, antigen processing/ of the γ/δ T-lymphocytes was in terms of perforin
presentation, or expression of MHC Class II and production and killing of xenogeneic targets (Toka
et al., 2011). This implies that the transient activity lymphoid tissue and maturation of the DCs to
noted for γ/δ T-lymphocytes was indeed a response express the co-stimulatory molecules ensuring acti-
against the virus infection, which in turn strength- vation of T-lymphocyte immunity development
ens the argument for the reported decreases in rather than anergy. This T-lymphocyte activation in
circulating lymphocytes, NK cells and DCs being turn ensures differentiation of the B-lymphocytes
part of this immune response. into antibody-producing plasma cells. By such
Whilst MΦ- and DC-dependent innate interactions between innate and adaptive immune
defences can prevent virus infection and disease compartments, together with the cognate lympho-
development, there is clearly a limit to their han- cyte interactions, development of antigen-specific
dling of virus load, above which the virus can gain adaptive immune responses is ensured.
the upper hand. Thereby, adaptive immunity is
required to ensure ultimate protection of the host,
particularly when the virus can initiate replication Innate to adaptive: inducing
in permissive cells such as epithelial cells. The adaptive immune defence
latter is more likely at the mucosal surfaces with against FMDV
which the virus has initial contact, particularly in
the absence of mucosal immunity as in a naive Inducing adaptive immunity
animal or host receiving parenteral vaccination
with inactivated vaccine. Although the innate At the heart of the matter – antigen
immune system is still of critical importance for presentation
initialising adaptive immune defence, the latter is Endocytosis and intracellular processing of anti-
essential for protection against FMDV. Despite gen leading to ‘antigen presentation’ is at the core
this, the innate system still plays a critical role. of adaptive immune defence induction, with DCs
Both before and after specific immune defence playing the major role as sentinels of the immune
development, innate effectors will operate, effec- system in all lymphoid and non-lymphoid tis-
tively enhanced by and enhancing the capacity of sues (Banchereau and Steinman, 1998; Mellman,
the specific defences to eliminate the virus (see 2013; Mellman and Steinman, 2001; Steinman and
‘Effector lymphocyte responses against FMDV’). Hemmi, 2006; Steinman and Pope, 2002). It should
The function of innate immune responses is most be noted that DCs are actually a heterogeneous
easily observed early after infection or vaccina- population of subsets (Bekiaris et al., 2014; Haniffa
tion. In the first days post vaccination – before et al., 2015; Kopf et al., 2015; Malissen et al., 2014;
anti-FMDV antibody – IL-6, IL-8 and IL-12 Summerfield and McCullough, 2009b). The main
were detected (Barnett et al., 2002). This would antigen-processing DC subsets are collectively
influence the activity of other innate cells, includ- termed ‘conventional DCs’ (cDCs). Generally
ing DCs and NK cells. Moreover, the IL-8 at speaking, they are involved in antigen uptake and
least would influence chemotactic and migratory processing, leading to association of antigen with
activities of these cells, observed in samples from MHC molecules for presentation to the adaptive
vaccinated pigs (Rigden et al., 2003). immune compartment. The DC subset involved
Concerning induction of the adaptive immune and the manner of that ultimate presentation deter-
responses, antibodies of high affinity and quantity mines the type of adaptive immune response to be
are the major force in protective immune defence, induced (see ‘Antigen presenting cell interaction
their induction dependent on the interplay among with T-lymphocytes’ to ‘Antigen presenting cell
DCs, B-lymphocytes and T-lymphocytes (Fig. interaction with B-lymphocytes’).
10.1). DCs process antigen for presenting peptides Maturation of DCs during their antigen process-
in association with MHC to T-lymphocytes, and ing also impacts on the form of immune signalling
deliver antigen to B-lymphocytes without pro- to be delivered. This maturation phenomenon has
cessing. Following interaction with and uptake of led to the terminology of immature and mature
FMDV by DCs, additional response to DAMPS DCs (iDCs and mDCs respectively) (Steinman,
and PAMPs – either viral or adjuvant components 2007, 2008; Steinman and Hemmi, 2006). Langer-
– for DC maturation promotes migration into hans cells (LC), other skin DC and subsets of
Figure 10.10 Interactions between antigen presenting cells and lymphocytes, and infected targets and
lymphocytes, involving both MHC Class II and MHC Class I presentation of the antigen peptides, resulting in
initiation of the antigen-specific immune defences.
mucosal DCs are good examples of iDC, seeking 2000; Pulendran and Ahmed, 2006; Pulendran
out antigenic material for endocytosing and pro- et al., 2001; Steinman, 2007, 2008; Steinman and
cessing into peptide structures to be associated with Hemmi, 2006).
the MHC molecules (Figs. 10.1, 10.2 and 10.10).
Presentation of the antigen associated with MHC The requirement for DC maturation
molecules is the means by which adaptive immune Inflammatory ‘danger’ signals including pro-inflam-
defences are promoted, but the maturation process matory cytokines and factors released during tissue
determines the manner of ‘communication’ with damage can promote DC maturation, the DCs
the adaptive immune system (Banchereau et al., responding to the ‘danger’ or DAMP signals therein.
PAMPs contained in microbial products – include Kzhyshkowska and Krusell, 2009; McCullough
lipopeptides, lipopolysaccharides, peptidoglycans, et al., 2009; McCullough and Summerfield, 2005;
DNA and RNA – can also induce maturation. DCs Mercer and Greber, 2013; Schlitzer et al., 2015;
recognize different DAMPs and PAMPs through Summerfield and McCullough, 2009a,b); MΦ pro-
their pattern recognition receptors (PRR), which vide major ‘scavenger’ phagocyte functions within
vary between different DC subsets and for recog- effector immune defences. Moreover, Mo and MΦ
nition of different molecular patterns (Mellman, have been associated with regulatory activities – as
2013; Pulendran and Ahmed, 2006; Pulendran et are DC subsets (Fig. 10.2) – suppressing develop-
al., 2001). Application of adjuvants in vaccine for- ment of antigen-specific responses (Fig. 10.11)
mulation can ensure DAMP or PAMP activation of (Barnard et al., 2016; Basta et al., 2000; Piersma et
DC. al., 2006).
Maturation of antigen-processing iDC into
antigen-presenting mature DC (mDCs) ensures the From endocytosis to antigen
up-regulation of chemokine receptors to enhance presentation
migration of the cells into lymphoid organs wherein
naive T-lymphocytes reside, particularly important Antigen entry into the processing
for inducing primary responses (Cerovic et al., pathways
2014; van der Aa et al., 2015). Effector or memory The iDCs internalize antigen via the same endocytic
T-lymphocytes in tissues require interaction with pathways discussed in ‘Endocytic Routes employed
processed antigen presented locally. For both naive by MΦ and DCs with FMDV’ concerning FMDV
and effector/memory T-lymphocytes, the up- interaction with MΦ and DCs. In addition to
regulation of co-stimulatory molecules on mDCs endocytic processes ultimately degrading internal-
is critical (Figs. 10.1, 10.2 and 10.10). These ensure ized antigen within lysosomes or by proteasomes,
the correct secondary signal for antigen presentation DCs employ the endosomal system or retrograde
in association with MHC molecules, to promote transport through the ER to process antigen into
activation and expansion of the T-lymphocytes peptide fragments. These processes involve the
into effector cells (Bakdash et al., 2014; Cerovic et MIIC (Fig 10.1) or the immunoproteosomes (Fig
al., 2014; Mellman, 2013; van der Aa et al., 2015). 10.10) for ultimate association with MHC Class II
Once activated, the T-lymphocytes can bypass or Class I molecules, respectively (Amigorena and
this requirement for co-stimulatory molecules by Savina, 2010; Kumari et al., 2010; Lu and Hong,
employing CD40–CD40L interaction with DCs 2014; McCullough et al., 2012; Pelkmans et al.,
presenting antigen (Bakdash et al., 2014). As such, 2001; Sandvig et al., 2011; Sorkin and von Zastrow,
local effector T-lymphocytes – particularly when 2009; ten Broeke et al., 2013). For antigen process-
activated – do not always require the same degree ing, macropinocytosis and caveolar endocytosis
of maturation in local DCs for antigen presentation. are more often involved, although slower clathrin-
dependent endocytic routes may also permit
Antigen presentation beyond DCs antigen processing for presentation.
B-lymphocytes can also present processed antigen in Compared with MΦ, DCs are regarded as pos-
association with MHC molecules to T-lymphocytes, sessing slower kinetics of endocytosis and lower
but primarily to already educated T-lymphocytes – proteolytic activities – considered to favour antigen
memory and effector T-lymphocytes (Fig. 10.1). processing for presentation, particularly when the
Only the DCs present antigen to both effector/ antigen is held within macropinosomes or caveolar
memory and naive T-lymphocytes – hence their endocytic vesicles prior to interaction with early
description as ‘professional antigen-presenting endosomes. The latter provide the early enzymes
cells’; DCs also ‘cross-present’ exogenous antigen and facilitate initiation of the acidification via vacu-
in association with MHC Class I molecules (Fig. olar H+-ATPases, essential events for processing
10.10) (see ‘Antigen presenting cell interaction with antigen into peptides for association with MHC
T-lymphocytes’). Mo and MΦ perform pertinent Class II molecules (Fig. 10.1). Initial acidification
roles including differentiation of Mo into Mo- of endocytosed material by early endosomes is also
derived DCs and MΦ (Italiani and Boraschi, 2014; important for processing endocytosed exogenous
(a) (b)
(c) (d)
Figure 10.11 (a) Relative antigen presenting cell activity of lung macrophages (Lung-MΦ) compared with the
APC present in PBMC, with respect to presentation of FMDV antigen or mock BHK cell lysate to FMDV-specific
lymphocytes. (b) Relative antigen presenting cell activity of lung macrophages (Lung-MΦ) compared with
monocytes and the APC present in PBMC, with respect to presentation of FMDV antigen or mock BHK cell
lysate to FMDV-specific lymphocytes. (c) Efficacy of porcine monocyte-derived dendritic cells (MoDCs) at
presenting FMDV antigen to lymphocytes from an FMDV-vaccinated animal. (d) Comparison of MoDCs with
monocytes as antigen presenting cells, using the same source of lymphocytes as in (c). Adapted from Basta
et al. (2000).
antigen leading to associate with MHC Class I (Amigorena and Savina, 2010; Neefjes et al., 2011).
(Fig. 10.10) – the so-called ‘cross-presentation’ These aspects of peptide association with MHC
pathways (Amigorena and Savina, 2010; Monu and molecules will be discussed further in ‘Antigen
Trombetta, 2007). Thereby, the influx of protons presenting cell interaction with T-lymphocytes’.
leads to modification of the antigen and poten- Overall, the different endocytic routes employed
tially membrane stability of the endocytic vesicle by DCs for processing endocytosed material will
(McCullough et al., 2012; Neuberg and Kichler, depend upon the nature of that material and the
2014; Sandvig and van Deurs, 2005; Zaki and Tire- ultimate consequence of the endocytic processing
lli, 2010; Zuber et al., 2001), promoting cytosolic in terms of the type of MHC-based presentation.
translocation. An alternative route to the cytosol is
mediated from the early endosome directing mate- DC output following endocytosis of
rial into the Golgi, and by retrograde transport into FMDV
the ER, from where chaperones transfer the antigen DC processing of live FMDV showed both effi-
to the cytosol (Lu and Hong, 2014; Sandvig and ciency at inducing antigen-specific lymphocyte
van Deurs, 2005). Within the cytosol, the antigen responses and a lack of FMDV-impairment in DC
becomes poly-ubiquitinated for transport into the processing of antigen to promote adaptive immune
immunoproteasome, which generates the pep- defence development (Fig. 10.8) (Harwood et
tides for association with MHC Class I molecules al., 2008). An additional important role for DC
communication was also observed, particularly Fig. 10.2), but recent evidence shows that such
in terms of B-lymphocyte induction (Bergamin et subsets also exist in other species, at least swine
al., 2007a,b). DC production of B-cell-activating (Summerfield et al., 2015; Auray et al, submitted for
factor belonging to the TNF family (BAFF) was publication). While the cDC1 subset represents the
essential for driving B-lymphocyte responses into major MHC Class I cross-presenting DC, with the
antibody production. This DC function could cDC2 subset more involved in MHC Class II pres-
replace T-lymphocyte cognate interactions with entation, this is a relative dominance of each subset
the B-lymphocytes, although the T-lymphocyte in the two processes (Cerovic et al., 2014; Schlitzer
cytokine IL-2 was still required. In contrast, et al., 2015). There are also species differences
BAFF could not completely replace induction of between human and murine subsets, concerning the
T-lymphocytes in vivo, probably relating to this relative dominance of different activities (Schlitzer
continued retirement for IL-2, although a com- et al., 2015). Overall, greater cross-presentation is
munication influence from BAFF was apparent associated with the cDC1 subset, possibly relating
(Bergamin et al., 2007a). Interestingly, the effi- to their unique expression of Clec9A facilitating
ciency of inducing lymphocyte activities was higher endocytosis of apoptotic/necrotic cells. With virus-
with HS-proteoglycan-binding virus (Barnard et infected cells, this fosters cross-presentation of the
al., 2005; Bergamin et al., 2007b; Harwood et al., viral antigens. Human CD141+ cDC1 cells show
2008), possibly reflecting the greater endocytosis higher capacity for cross-presenting a number of
and retention of this virus/antigen in DCs; cer- viral antigens compared with human CD1c+ cDC2
tainly favourable for cell culture-passaged vaccine cells – includes cytomegalo-, hepatitis B and human
virus. immunodeficiency viruses. Yet human CD1c+
cDC2 cells can cross-present antigens from certain
Antigen-presenting cell interaction viruses, such as influenza virus (Yu et al., 2013). In
with T-lymphocytes contrast, murine CD11b+ cDC2 cells are generally
Although all subsets of DCs possess the capac- regarded as poor cross-presenters compared with
ity to promote MHC Class I and Class II antigen murine CD8α+CD103+ cDC1 cells (Schlitzer et al.,
presentation, there is a subset-based predilection 2015).
for the different processes. While this is not an Although both murine and human cDC1 cells are
absolute division of labour, it facilitates the study involved in regulatory responses, human CD141+
and understanding of the processes (Cerovic et al., cDC1 cells are relatively weak, the human intestinal
2014; Schlitzer and Ginhoux, 2014; Schlitzer et al., CD1c+ cDC2 cells being the more potent at induc-
2015). An important consideration is the interac- ing Treg-lymphocytes (see Fig. 10.2). Both human
tion with the family of Th-lymphocyte subsets. and murine cDC2 cells can produce retinoic acid
Together, they are critical for ensuring appropriate for skewing T-lymphocyte differentiation towards
development of both humoral (B-lymphocytes) tolerogenesis, and can express TGF-β – important
and cytotoxic (Tc-lymphocyte) immunity, and for controlling Treg-lymphocyte induction (Guil-
essential for expansion of naive cells upon first liams et al., 2010; Yu et al., 2013). Concerning the
encounter with virus/antigen. Following encoun- Treg/Th17 balance (see Fig. 10.2), murine responses
ter with the antigen for which they are specific, are split between the cDC1 (Treg-lymphocytes)
Th-lymphocyte subsets develop into effector cells, and cDC2 (Th17 homeostasis) subsets, whereas
which also play important roles in regulating innate human intestinal CD1c+ cDC2 cells induce Th17-
immune responses, including MΦ and DCs. This lymphocytes and Treg-lymphocyte proliferation/
forms part of a bidirectional interchange of cognate differentiation (Schlitzer et al., 2015).
interactions and cytokine-based signalling (Figs. The cDC2 subset is most often associated with
10.2 and 10.10), important when interpreting the MHC Class II presentation (Fig. 10.1), but again
functionality of DCs. only relatively. It is the functional activity of phe-
notypically defined cDC1 and cDC2 subsets that
Considerations of DC subsets determines the type of immune response induced,
The existence of DC subsets has been studied in in terms of Th1, Th2, Th17, Treg and Tc lymphocyte
more depth with human and murine cells (see induction (see Fig. 10.2) (Schlitzer et al., 2015).
Both murine and human cDC1 cells favour induc- this point, MHC Class II molecules vary in terms
tion of Th1- (and Tc-lymphocyte) lymphocyte of ‘recognizing’ a particular peptide amino acid
responses, while cDC2 cells favour Th2 and Th17 sequence, for presenting different epitopes to dif-
responses (Schlitzer et al., 2015). Although their ferent clones of antigen-specific T-lymphocytes.
relative roles may differ dependent on the antigen, Non-activated, immature DCs show a relative
and their ability to induce Treg-lymphocytes, the instable expression of the majority of their MHC
outcome of virus interaction with DCs in terms of Class II molecules at the plasma membrane, fre-
the functional T-lymphocyte activities is the deter- quently recycled and transported into lysosomes
minant factor rather than any particular DC subset. for degradation. MHC Class II molecules on non-
Therein, the manner by which the DC handles the activated cells can also be ubiquitinated via the
antigen for processing fashions the outcome of cytoplasmic domain of the MHC Class II β chain,
T-lymphocyte interaction, regardless of species. facilitating proteasomal degradation. Alternatively,
MHC Class II molecules on non-activated DCs
Considerations for MHC Class II can play an immunoregulatory role. In the absence
processing of PAMPs or DAMPs – that is under steady state
The endocytic route leading to antigen presenta- homeostasis without pathogen incursion or vac-
tion requires maturation of the endosomal system cination – the MHC Class II molecules are loaded
with which the endocytic vesicles carrying the with ‘self ’ peptides within MIICs, prior to transfer
virus or antigen have interacted (Fig. 10.2). This back to the plasma membrane. Being non-activated,
maturation involves the stages of early endosome, the absence of co-stimulatory molecules allows the
recycling/sorting endosome, multivesicular presentation of these self-peptides to induce anergy
bodies (MVBs; also known as endosomal car- or clonal deletion, thus providing self-tolerance.
rier vesicles) and late endosome. During the Similar scenarios can be found at mucosal surfaces
maturation process, the activity of the enzymes with food tolerance, where it is important to rec-
involved is altered, relating to the function of the ognize food as a ‘non-danger’ entity, as opposed to
different structures and the gradual reduction pathogens or toxins.
of the pH therein through the action of vacuolar Once DCs are activated, the scenario changes.
H+-ATPases (Amigorena and Savina, 2010; Monu Response to PAMPs or DAMPs promotes expres-
and Trombetta, 2007; Scott et al., 2014; van der sion of co-stimulatory molecules such as CD80 and
Goot and Gruenberg, 2002). When antigen is CD86. This is the situation found following endo-
retained within the maturing endosomal system, cytosis of FMDV or vaccine antigen, with either
it is gradually processed for final delivery into the virus or the adjuvant providing the PAMP-
either lysosomal structures (degradation) or signalling. The processed antigenic peptide in
the late endosome/lysosome-like MHC Class complex with MHC Class II becomes more stably
II Compartment (MIIC) (Neefjes et al., 2011). expressed at the plasma membrane, providing an
Directing antigen processing by fusing the enhanced interaction with the Th-lymphocyte
maturing endosomal structures with the special- antigen receptors (TCRs). Activation of the Th-
ized MIIC containing MHC Class II molecules lymphocytes can occur in the absence of CD80/86,
leads to antigen presentation for activating T h- but this requires that the DC present antigen to
lymphocytes – T h1, T h2 or T h17 (Neefjes et al., activated/effector Th-lymphocytes, whereby the
2011; Schlitzer et al., 2015). Within this MIIC, CD80/86 signalling is replaced by CD40/CD40L
peptides derived from the processed antigen interaction.
interact with the peptide binding cleft of the MHC
class II molecules; this is made available following Considerations for MHC Class I
degradation of the invariant chain (Ii) (ten Broeke processing
et al., 2013), which also occurs within the endoso- Dependent on the endocytosed material, and
mal system. Processing of the Ii leaves the CLIP particularly its molecular and ionic structure, early
fragment occupying what is termed the peptide- compartments of the endosomal system facilitate
binding groove or cleft. Within MIICs, the CLIP cytosolic translocation for cross-presentation
is exchanged for the antigen-derived peptides. At involving the proteasome and MHC Class I
molecules (Fig. 10.10) (Amigorena and Savina, ‘delivery’ than ‘presentation’ of antigen. Although
2010; Heath et al., 2004; Monu and Trombetta, ‘passive transfer’ of antigen between cells is a pos-
2007; Neefjes et al., 2011; van der Goot and Gru- sibility, if the antigen were retained on the surface
enberg, 2002). Cytosolic translocation may occur of DCs, it is likely that cognate interactions are
directly from early or late endosomes, or following more important, particularly when considering
retrograde transport from early or recycling/sort- the reported roles of molecules such as BAFF (see
ing endosomes to the Golgi and ER (Lu and Hong, ‘DC interaction with B-lymphocytes’). Antigen
2014; Sandvig and van Deurs, 2005). The route is delivery by DCs to B-lymphocytes following
dependent on the structure of the antigen endo- endocytosis of the antigen has been demonstrated,
cytosed, but will be influenced by any formulation both in vitro and in vivo (Le Roux et al., 2011;
employed, whether that be the adjuvant or delivery Wykes et al., 1998). Intriguingly, the DCs employ
vehicle such as lipoplexes, liposomes or other nano- a small GTPase rab7-dependent route having char-
particles (Démoulins et al., 2015; McCullough acteristics related to the late endosomal route, but
et al., 2014a,b; Thomann-Harwood et al., 2013). without processing or degradation of the antigen.
Overall, the critical determinant is escape from the Such cell-directed delivery of antigen to B-lym-
lysosomal compartment wherein rapid degradation phocytes by DCs is certainly more in keeping with
will result, impairing cross-presentation (Schuette biological operational procedure, in contrast to the
and Burgdorf, 2014; Shete et al., 2014). ‘hope’ that antigen would arrive in lymphoid fol-
Many aspects concerning MHC Class I antigen- licles by simple passage along the lymph without
presentation relate to MHC Class II presentation. direction by the immune system (McCullough et
Both exogenous antigen and endogenous antigen al., 2012). Biology does not operate by serendip-
can be processed. Differences emerge concerning ity; reliance upon uncontrolled chance would
trafficking into the final destination for presenta- render inefficient this important immune defence
tion. Cytosolic translocation for MHC Class of humoral immunity against pathogen attack.
I presentation is the frontier at which the two The high propensity of DCs for homing to lymph
processes part company. This can be due to retro- nodes, together with their presence within extra-
grade transport through the Golgi into the ER (Lu follicular and follicular sites, endows them with
and Hong, 2014; Sandvig and van Deurs, 2005), or the characteristics to provide antigen delivery to
directly from early endosomes (Sandvig and van B-lymphocytes as well as antigen-presentation to
Deurs, 2005), before the pH of the maturing endo- T-lymphocytes.
somal system becomes too acidic (Amigorena and
Savina, 2010). Direct translocation or retrograde DC interaction with B-lymphocytes
transport may also occur from later endosomal DC interaction with B-lymphocytes goes beyond
structures (Lu and Hong, 2014; Sandvig and van antigen delivery (Litinskiy et al., 2002; Schnei-
Deurs, 2005), providing that the pH-dependent der, 2005). Promoting B-lymphocyte expansion
maturation of the endosomes has not progressed also involves provision of appropriate signalling
too far, which in turn will depend on the nature to drive B-lymphocyte immunoglobulin isotype
of the antigen or vaccine delivered (Lu and Hong, switching. While this latter area is also depend-
2014; Mallard et al., 1998; McCullough et al., ent on Th-lymphocyte cognate interactions and
2012; Sandvig and van Deurs, 2005). cytokine profiles – such as combinations of
IL-2 with IL-4 and IL-5 – DC regulation of
Antigen-presenting cell interaction B-lymphocyte activity can be either dependent
with B-lymphocytes or independent of Th-lymphocytes. There is no
MHC involvement, but accessory molecules pro-
Antigen delivery to B-lymphocytes duced by the DC are important; BAFF and ‘a
In addition to driving forward T-lymphocyte proliferation-inducing ligand’ (APRIL), which can
responses, DCs also facilitate antigen encounter by be secreted or present as membrane-bound mol-
antigen-specific B-lymphocytes (Fig. 10.1) in lym- ecules, are two proven factors (Bergamin et al.,
phoid follicles and germinal centres; this is more 2007a,b; Litinskiy et al., 2002; Schneider, 2005).
Innate immune cell regulation of 10.2). DC:T–lymphocyte ratio can influence the
adaptive defence development response; lower ratios, a possible ‘default’ response
in the absence of adequate signalling and low or
Defining the T-lymphocyte response deficient IL-12 production, lead to more Th2-
Co-stimulatory molecule expression on the dominated outputs (Eisenbarth et al., 2003). Other
DCs, such as CD80/86, is essential for ensur- cytokines – particularly IL-6, possibly together
ing T-lymphocyte expansion (Figs. 10.1, 10.2 with IL-18 and IL-25 – may influence polarization
and 10.10), although this can be replaced by towards Th2-responses (Fig. 10.2). DC produc-
CD40 on DCs interacting with T-lymphocyte tion of IL–1/IL-6/IL-23 combinations, together
CD40L. Absence of both CD80/86 expression with the presence of TGFβ (from DCs and/or
and CD40–40L cognate interactions is impor- non-immune accessory cells) and IL-21 (from
tant for regulation and tolerance (Ezzelarab and γ/δ-T-lymphocytes) have been implicated in the
Thomson, 2011; Oh and Shin, 2015). Even when induction of Th17-lymphocytes (Walsh and Mills,
CD80/86 is expressed on the DC, activation of 2013). Such manipulations of cytokine profiles
the T-lymphocyte can be prevented through liga- can be controlled by the application of defined
tion of the co-stimulatory molecules by CTLA-4 adjuvants in the formulation of vaccines (Ebensen
on the lymphocyte. In addition to these cognate and Guzman, 2009; Riese et al., 2013), particularly
interactions, cytokines produced by the DCs upon critical when vaccines are being applied via the
activation by PAMP or DAMP will influence the mucosal route.
nature and characteristics of the T-lymphocyte
response (Fig. 10.2) (Eisenbarth et al., 2003; Walsh DC modulation of anti-FMDV
and Mills, 2013). This has major implications on T-lymphocyte responses
both antigen dose and adjuvant form – in terms The capacity of DCs to induce and regulate anti-
of Th1-response or Th2-response – applied during FMDV T-lymphocyte responses is well established
vaccine development. While particular DC subsets (Fig. 10.11) (Barnard et al., 2016; Bergamin et
can differentially influence induction of Th1, Th2 al., 2007a,b; Harwood et al., 2008; Piersma et al.,
and Th17-lymphocytes, both the signal received by 2006). Although porcine Mo, MΦ and DC show
the DCs and the cytokines they produce influence similar characteristics for endocytosis of FMDV
the outcome (Fig. 10.2) (Eisenbarth et al., 2003). (Harwood et al., 2008; Rigden et al., 2002), Mo and
Of course cytokine responses can be employed MΦ show more immunosuppressive characteristics
towards regulatory processes; IL-10 and TGFβ than DCs (Figs. 10.11 and 10.12) (Basta et al., 2000;
production by DCs play critical roles with induc- Carrasco et al., 2001), leading to their implication
tion of Treg-lymphocyte responses (Morel and in regulation of adaptive immune responsiveness
Turner, 2011; Walsh and Mills, 2013), which in (Barnard et al., 2005, 2016; Bergamin et al., 2007b;
turn produce more IL-10 and TGFβ to maintain Piersma et al., 2006). Not surprisingly, removal of all
the cycle. The latter situations are found at mucosal porcine blood CD172a+ cells (Mo and DCs) from
surface where tolerance to food antigens must be PBMCs abrogated lymphocyte responsiveness to
maintained FMDV antigen, which was restored by replacement
Consideration of these interactions between of the CD172a+ cells (Fig 10.12) – clearly demon-
DCs and T-lymphocytes can facilitate the for- strating the importance of DCs. Yet, CD172a+ cells
mulation of more efficacious vaccines. At low in PBMC are dominated by CD14+ Mo (Bergamin
antigen and PAMP doses, Th2-responses are noted, et al., 2007b; Piersma et al., 2006; Summerfield et
whereas Th1-responses a more readily observed al., 2003; Summerfield and McCullough, 2009b).
with higher antigen and PAMP doses. The relative Partial depletion of the CD172a+ cells to increase
dose of antigen and type/degree of PAMP signal- the representation of CD14– cells enhanced the
ling can influence the DC cytokine profile. For capacity of DCs to promote antigen-specific
example, IL-12, together with the related IL-23 T-lymphocyte responses (Barnard et al., 2016;
and IL-27 as well as IL-18, is more critical for Th1- Bergamin et al., 2007b; Piersma et al., 2006). When
responses than Th2-lymphocyte induction (Fig. the depleted cells were returned to the cultures,
Figure 10.12 (a) The influence of SWC3+ cells on SWC3– cells (containing CD14– SWC3+ cells on stimulation
of anti-FMDV lymphocyte responses. PBMC were sorted by labelling with antibody against the pan-myeloid
marker SWC3, to separate into an SWC3+ and SWC3– fractions. These fractions were tested for their response
to FMDV, and the SWC3+ fraction was back-titrated into the SWC3– fraction (x-axis; ratio SWC3+: SWC3–).
The responsiveness to FMDV stimulation was measured at different times after stimulation (y-axis; day
post-stimulation), in terms of the relative cpm (3H-TdR incorporation) (SI; z-axis)with respect to that obtained
with the SWC3– fraction alone (SWC3– only). (b) Partial removal of the SWC3+ cells from PBMC, dominated
by CD14+ cells, increases the ability of the lymphocytes in the SWC3– fraction to respond to FMDV antigen
presented by the remaining SWC3+ cells (dominated by CD14– cells) in this SWC3- fraction. Re-constitution
of the SWC3+ fraction with the SWC3– fraction reduces the lymphoproliferation. Adapted from Barnard et al.
(2003a).
Figure 10.13 Relative role of T-cells during anti-FMDV antibody responses were assessed using cells from
immune pigs cultured for 6 days with FMDV. FMDV-specific IgG was detected by ELISA. (a) Antibody responses
with T-cell-depleted PBMC (Δ T-cells) and different concentrations of IL-2 (x-axis). (b) Antibody responses
in co-cultures with different ratios of APC, T-cells and B-cells (x and z axes). The number of B-cells was
kept constant at 1.5x106. (c) BAFF as a mediator of APC help for antibody production. B-cells (1.6x106) were
cultured with FMDV in absence or presence of IL-2 and different concentrations of human recombinant BAFF.
Antigen-specific IgG secreted in the supernatants was detected by ELISA (y axis); the background cut-off
values are indicated as dashed lines. (d) BAFF as a substitute for T-cell contact in antibody response. B-cells
(1x106)were cultured for 6 days in presence of inactivated FMDV, 100 U/mL IL-2, different concentrations of
CD40L (indicated on the x axis), and either the presence (filled circles) or absence of 200 ng/mL BAFF (empty
circles). The reactivity of the supernatants in the FMDV-specific IgG ELISA is shown on the y axis. Error bars
indicate standard deviation for duplicate wells in cell culture and triplicate wells in the ELISA. The background
cut-off value in the ELISA is indicated as a dashed line.
on the APC population, described as containing adjuvant having a major impact on the characteris-
Mo and Mo-derived DC, but not pDCs (Bergamin tics of that response.
et al., 2007b). Similar results were obtained when
the T-lymphocytes were replaced with IL-2 only
(Fig 10.13). This APC-mediated help was through Adaptive immune responses
soluble factors, including BAFF (Bergamin et al., against FMDV
2007b), demonstrating that not only T-lympho-
cytes, but also APC directly modulate anti-FMDV B-lymphocyte responses
B-lymphocyte activities. Although certainly valid
for secondary B-lymphocyte responses, BAFF General considerations for
clearly plays important roles under conditions B-lymphocyte responses
physiologically relevant for secondary lymphoid While assessment of anti-FMDV responses after
organs. vaccination or infection has concentrated on anti-
The outcome from cross-linking the BCR body, recall responses analysed in vitro and in vivo
depends on the antigen load delivered by the DCs. showed the importance of T-B lymphocyte col-
Under conditions of high antigen load, T-cell- laboration (Barnard et al., 2005; Collen et al., 1984,
independent responses may show higher potential. 1989; Rodriguez et al., 1995). Activation of B-lym-
Conversely, high antigen dose may initially promote phocytes would clearly require stimulation of both
a T-lymphocyte dependent response, but the acti- the plasma membrane immunoglobulin-like BCR
vated B-lymphocytes can continue their response by antigen, and the surface cytokine receptors by
to the higher levels of antigen without obligation for factors produced by Th-lymphocytes. CD40-medi-
further T-lymphocyte help, perhaps employing the ated cognate interactions become important when
DC help through factors such as BAFF and APRIL. the T-lymphocyte CD40L plays a key role in selec-
When antigen load is below a certain threshold, tive B-lymphocyte differentiation. As discussed
T-lymphocyte help can appear more obligatory. in ‘DC control of B-lymphocyte responses’, DC
Consequently, both the B-lymphocyte responses factors such as BAFF and APRIL are also involved
and T-lymphocyte activity in terms of the Th1/ (Fig. 10.13), certainly for isotype switching (Ber-
Th2 balance show dependency on the antigen dose gamin et al., 2007a,b). Under conditions of BAFF/
reaching the DCs, which in turn have implications APRIL-induced isotype switching, a physical inter-
for vaccine design. Delivery of inactivated vaccines, action between B- and Th-lymphocytes is no longer
as commonly employed with FMDV, provides a required, and the T-lymphocyte cytokines can be
scenario more related to one of lower antigen load replaced, at least partially (Bergamin et al., 2007b;
associated with DCs, and therefore more T-cell- Litinskiy et al., 2002).
dependent. Use of self-replicating vaccines, such Both B- and T-lymphocytes respond by clonal
as those based on replicon RNA (Atkins et al., expansion, restricted to particular clones recogniz-
2008; Demoulins et al., 2015; Khromykh, 2000; ing the so-called B-cell (B-lymphocyte) and T-cell
Ljungberg and Liljestrom, 2015; McCullough et (T-lymphocyte) epitopes on the virus or antigen
al., 2012, 2014a; Pijlman et al., 2006; Rayner et (Fig. 10.14) (Blanco et al., 2013; Blanco et al., 2001;
al., 2002), can provide increasing antigen dose Blanco et al., 2000; Collen et al., 1991, 2002; Gerner
above that possible with an inactivated vaccine, et al., 2006, 2007, 2009; Haghparast et al., 2000;
due to the translation and replication of the RNA. Hohlich et al., 2003a,b; Perez Filgueira et al., 2000;
Accordingly, these vaccines could eventually pro- Rodriguez et al., 1994). Antigenic sites contain-
vide the high antigen loads whereby T-lymphocyte ing B-cell epitopes – chemically defined antigenic
dependency becomes less obligatory during later determinants – recognized by the BCR relate to the
stages of humoral immune defence development. recognition associated with the antigen-binding
Nonetheless, it is clear that the final product of an site or paratope of the anti-FMDV antibody mol-
induced humoral response is dependent upon the ecules produced by the B-lymphocytes. Effectively,
interplay between B-lymphocytes, T-lymphocytes the B-lymphocyte BCR and antibodies produced
and DC subsets, wherein the ultimate antigen load by the differentiated antibody-producing cell are
influences the type of response induced, with the immunoglobulin molecules displaying the same
B-cell sites, relating to the efficiency with which antigenic site against which it is reactive, leading to
they activate antigen-specific B-lymphocytes into increased affinity and titre. This is further enhanced
FMDV-specific antibody production. Although by antibodies of efficient ‘fit’ recognizing more than
one tends to discuss ‘neutralisable epitopes’ and one epitope on the virus, thus increasing immune
neutralizing antibodies, the important immu- defence efficacy. Certainly, high-affinity antibody
nological process is the antibody opsonization against a single epitope would prove superior to
of virus. Neutralization of virus infectivity alone low-affinity antibodies against different epitopes.
is inadequate for immune protection. Antibody Such a high affinity antibody is more likely capable
opsonization of the virus enhances uptake by MΦ of reacting with related antigenic determinants on
for destruction of the infectious threat – this is the quasispecies than would low affinity antibodies.
immunological protection (see ‘Effector humoral Thereby, protection could be afforded with opsoniz-
immune defence’). The power of this opsonization ing antibody against only one neutralisable epitope
process is dependent on the antibody specific- of appropriately high affinity to facilitate reaction
ity and affinity, as well as the number of antibody with modified antigenic determinants of the qua-
molecules interacting with the virus particle. When sispecies, promoting their destruction by MΦ.
more than one of the neutralisable sites on the virus Nonetheless, the greater the number of high affinity
for antibody opsonization is engaged by antibody, reactions with different epitopes on the virus, the
the protective force therein is enhanced. Antibody greater the efficacy of the humoral defence.
against only one of the immunodominant B-cell
sites can more readily select amino acid variation in Considering the route and form of
heterogeneous FMDV populations (quasispecies) vaccination
(Domingo et al., 1992, 2003, 2005), leading to an Following parenteral vaccination, a typical profile
increased spectrum of antigenic and immunogenic for the induction of anti-FMDV antibodies begins
variability and appearance of ‘antibody-resistant’ with IgM, the first detectable serum neutralizing
or ‘escape’ variants. The presence of antibodies antibody, appearing at 3–4 days following vaccina-
against more than one antigenic site decreases the tion. Maximum serum IgM responses are observed
likelihood of virus escape variants covering all the around 10–14 days post infection, after which the
antibody specificities. This is likely to be one of the response declines. Serum IgGs can be detected as
driving forces in FMDV evolution, and is probably early as 4–7 days post vaccination, these isotypes
involved in the origin of the seven distinct FMDV becoming the major neutralizing antibodies within
serotypes currently in existence (reviewed in two weeks of encounter with antigen.
Domingo et al., 2003, 2005; Sobrino et al., 2001). An important aspect of immune defence against
In addition to the number of antigenic sites FMDV is the induction of mucosal immunity. With
targeted by the humoral defence, the avidity of the the upper respiratory tract being an important
antibody reaction is important for weakening the portal of entry for FMDV to initiate infection,
virus escape process. This involves the affinity of oranasal vaccination would present advantages, in
each antibody interacting with a particular epitope particular induction of a local antibody response
on the virus, and the avidity of the combined action in the upper respiratory tracts. Both oranasal and
from different antibodies. More than a single anti- parenteral vaccination induce humoral immune
body specificity would increase this avidity, due to responses maturing from IgM to IgG, with con-
the observed avidity of multiple antibody interac- comitant increase in titres and affinities. Particular
tions being greater than the sum of the individual to the mucosal (local) immune response is the
antibody affinities. Accordingly, selection of virus dominance of dimeric IgA antibodies following the
escape variants is more likely with low titre and/or isotype switching early during humoral immunity
low-affinity antibody responses, as during primary maturation. This dimeric molecule is referred to
immune responses when antibody ‘fit’ for the anti- as secretory IgA, due to its secretion at mucosal
genic site is still relatively poor and not yet refined. surfaces – distinguishable from serum IgA, which is
Continued stimulation of the antigen-specific monomeric and not secreted.
lymphocytes increases ‘maturation’ within the The characteristics of vaccine-induced immune
immune response – increasing antibody ‘fit’ for the defence will be influenced by the Th1/Th2/T17
balance (see ‘Innate immune cell regulation of appropriate epitopes – on MHC molecules by APC
adaptive defence development’), relating in turn such as DCs (Figs. 10.1, 10.2 and 10.10) (see ‘From
to the DC subsets involved. This Th1/Th2/T17 endocytosis to antigen presentation’ and ‘Antigen
balance can also be modified through the use of presenting cell interaction with T-lymphocytes’).
highly defined adjuvants formulated with the vac- The number of processed peptides that can be pre-
cine (Ebensen and Guzman, 2009; Riese et al., sented by APC is limited by the alleles in the loci
2013). While dominant Th1 response are generally encoding MHC class I or class II – provides MHC
favoured for vaccination against viruses, this cannot ‘recognition’ of the peptides. Through possession
be considered as a hard and fast rule (see ‘Innate of different alleles, so-called MHC restriction
immune cell regulation of adaptive defence devel- exhibits a variation in the peptide recognition pat-
opment’). Moreover, the role of the adjuvant will terns expressed by individuals. The polymorphism
depend on the route of vaccination, particularly in MHC class II loci leads to species-specific and
mucosal compared with parenteral (Ebensen and individual-specific repertoires for efficient recogni-
Guzman, 2009; Riese et al., 2013). The influence tion of FMDV peptides by CD4+ Th lymphocytes
of different adjuvants on vaccine-induced immune (Collen et al., 1998; Collen and Doel, 1990; Garcia-
responses should be proven experimentally, and Briones et al., 2000; Gerner et al., 2006, 2007, 2009;
the influence of the Th1/Th2/T17 balance defined Glass and Millar, 1994; Glass et al., 1991; Haghpar-
for different vaccine strategies – inactivated, vector- ast et al., 2000; Van Lierop et al., 1995). MHC Class
based, replicon-based or peptide-based. The area I haplotype restriction is also an important con-
of FMDV vaccines will be covered in more dealt in sideration for Tc-lymphocyte epitopes (Liu et al.,
Chapters 12 to 14 of this book. 2011); compared with studies on Th lymphocytes,
Increased ‘fit’ or ‘tuning’ of the immune there have been fewer reports on FMDV-specific
responses comes from the number of re-encounters Tc-lymphocyte epitopes (Guzman et al., 2008; Liao
between FMDV-specific lymphocytes and antigen. et al., 2013; Liu et al., 2011; Rodriguez et al., 1996).
This ‘tuning’ of antigen recognition is observed The Th lymphocytes are essential for ensuring
in terms of specificity, affinity and titre, as well as efficient development of the majority of specific
increased numbers of responsive cells – clonal immune responses, in terms of both cognate inter-
expansion. Fine-tuning of antigen-specific response action and their cytokine profiles – termed
is equally important for the expansion of the ‘immunological help’ (Figs. 10.1 and 10.2). Acti-
FMDV-specific T-lymphocytes (see ‘T-lymphocyte vation through MHC Class II-restricted antigen
Induction’). Only when the combination of appro- presentation (‘Antigen presenting cell interaction
priate B-lymphocyte and T-lymphocyte responses with T-lymphocytes’) ensures appropriate interac-
is induced, will an efficacious immune defence tion with antigen-activated B-lymphocytes, for
arise. Inactivated, recombinant or peptide vaccines differentiation into antibody production. These
are limited in the number of rounds of stimulating activated (effector) Th lymphocytes also provide
antigen-specific lymphocytes they can provide. immunological help for development of antigen-
Self-replicating vector vaccines can increase the specific effector Tc-lymphocytes (Fig. 10.10)
antigen load, through their limited replicative char- – activated by appropriate antigen presentation in
acteristics (see Chapter 14). Recent developments association with MHC Class I (‘Antigen presenting
with self-amplifying replicon RNA vaccines are cell interaction with T-lymphocytes’). Although
providing further advance in this area (McCullough the role of Tc-lymphocytes against FMDV requires
et al., 2012, 2014a,b; Demoulins et al., 2015). further study, it is clear that they are induced
(Bucafusco et al., 2015; Guzman et al., 2008;
T-lymphocyte induction Patch et al., 2011, 2013; Rodriguez et al., 1996),
particularly after infection. Nonetheless, humoral
General considerations for T-lymphocyte immunity remains the major force in protective
responses immune defence, and current vaccines – based on
The major consideration for antigen-specific T-lym- inactivated virus, subunit or recombinant antigen
phocyte immunity is the antigen processing for – have a low capacity for inducing Tc-lymphocyte
presentation of the derived peptides – carrying the immunity.
dominating or a balance between two. Certain and T-lymphocyte-derived cytokines (Fig. 10.2),
defined adjuvants induce a dominance towards Th1 rather than a simple profile from one T-lymphocyte
or Th2, while others will induce a more balanced subset.
Th1/Th2 response (Ebensen and Guzman, 2009;
Riese et al., 2013).
Cytokines elaborate the interaction between Effector lymphocyte responses
cells and components of the immune system. It against FMDV
is not possible to generalize on these processes,
because the interactions at the cellular level will Components of effector immunity
influence the action due to cytokines, and the com- As with the induction of immune responses, the
bination of cytokines will influence the outcome effector mechanisms responsible for immune
of cellular interactions. For example, interaction of defences can be compartmentalized. The most
immature DCs (no co-stimulatory molecules) with rapidly assimilated defences are innate (see ‘Intro-
resting Th-lymphocytes (no CD40L interaction) duction’ to ‘Ranking innate immune defences’),
will result in CD152 expression and IL-10 produc- which also ensure the correct and efficient stimula-
tion, enhancing regulatory Treg-lymphocyte activity tion of adaptive responses. While specific humoral
and anergy of T-cell responsiveness (Adorini, 2003; immunity dominates immunological protection
Jonuleit et al., 2000). In contrast, interaction of against FMDV, the destruction and removal of the
mature DCs (co-stimulatory molecules expressed) virus infectious threat requires effector cells from
with Th-lymphocytes will induce production of innate defences. The necessity and functionality
IL-2 and IFN-γ along with CD70 expression, pro- of this partnership relates to the characteristics of
moting Th-lymphocyte expansion and ultimately the humoral response; one must consider how
antigen-activated B-lymphocyte differentiation into antibody actually operates, to appreciate protective
antibody-producing cells. When Treg-lymphocytes humoral immunity.
become involved, cytokine expression is observed
to shift from IFN-γ to IL-10 with down-regulation Anti-FMDV antibody
of Th-lymphocyte responses. Durable protection against FMDV is elicited by
It is often remarked that a Th1 cytokine profile humoral immune defences, particularly specific
is desirable for inducing effective immune defence anti-FMDV antibodies (Black et al., 1984; Lavo-
against virus infection, but this is misleading. As ria et al., 2012; McCullough et al., 1986, 1992a;
discussed in ‘Innate immune cell regulation of Oliveira et al., 2005; Ostrowski et al., 2007; Pay
adaptive defence development’, vaccine-induced and Hingley, 1987; Rieder et al., 1994; Scicluna
immune responses will be a balance of cytokine et al., 2001; Steward et al., 1991; Van Maanen and
profiles, influenced by the adjuvant employed. Vac- Terpstra, 1989). Experiments performed more
cination against FMDV led to a combined Th1- and than a century ago had already demonstrated this
Th2-response (Barnard et al., 2005) – Th1 cytokines importance (Loeffler and Frosch, 1897). Immuno-
IFN-γ and IL-2, as well as Th2 cytokines IL-6 and logically speaking, one cannot rationalize analyses
IL-10 were induced. IL-6, and to a lesser degree of serum antibody diluted in vitro using simple buff-
IL-8 and IL-12, can be induced in the serum of ers with events in vivo. Dilution of serum to a point
vaccinated pigs early post-vaccination (Barnett et where specific and non-specific/aspecific reac-
al., 2002). With a dose of inactivated FMDV anti- tions are separable over a long incubation period
gen that alone did not induce detectable immune is useful for the serological test, but cannot reflect
responses, combination of IL-1 (Mo/DC-derived mechanisms of immunological protection. Dilutied
cytokine) or IL-2 (Th1-derived cytokine) facilitated sera ignores the roles played by low-titre antibod-
induction of anti-FMDV antibody within 14 days ies, natural immunoglobulins and other opsonins
post vaccination (McCullough et al., 1992c). IL-1 in humoral immune defences. While these reac-
was more effective when administered 24 hours tions alone will be inefficient at protecting against
before the vaccine, while IL-2 was most effective FMDV – non-immunized animals are sensitive to
when concomitant with the antigen. Overall, effica- FMDV infection – the combination of specific and
cious vaccination relies on both Mo/DC-derived non-specific/aspecific humoral factors will provide
bidirectional influences. Individual affinities of virus infection with anti-FMDV serum antibody
each component for binding to the virus combine assessed by traditional in vitro assays shows that
to provide an avidity of reaction greater than the similar antibody titres cannot guarantee resistance
simple sum of those individual affinities. to challenge infection (McCullough et al., 1992a;
Nonetheless, humoral responses per se cannot Van Maanen and Terpstra, 1989). In reality, a ‘grey
provide clinical protection (Harwood et al., 2008; zone’ exists for estimation of antibody titres by in
McCullough et al., 1986, 1988, 1992a,b; Rigden et vitro serological assay (Fig. 10.15), within which it
al., 2002; Van Maanen and Terpstra, 1989). Anti- is not possible to distinguish protected from sus-
body–virus interaction is only a chemical reaction, ceptible animals (McCullough et al., 1992a). Such
and not a biological event. Neutralization of virus inconsistencies relate to the chemical nature of
infectivity is measured as an in vitro event, not an antibody interaction with antibody being defined
in vivo immunological process. Moreover, in vitro according to the law of mass action, whereas
assays pre-incubate diluted serum antibody with immune protection in vivo is dependent on bio-
virus prior to addition to susceptible cells, for a lim- logical events involving both humoral and cellular
ited period (often 30–60 minutes) before removal. components of immune defences (see ‘Effector
This does not occur in vivo. Protection could be humoral immune defence’).
conferred directly by antibody if the interaction dis-
rupted virion structure, but this has been reported The law of mass action
for only one antibody against a particular conforma- Following antibody interaction with virus, the
tional epitope at high concentrations (McCullough law of mass action prevails. This dictates that the
et al., 1987b). Comparison of protection against initial chemical reaction of antibody with antigen
proceeds predominantly in one direction towards
the formation of antigen–antibody complexes. As
the reaction advances, the reverse rate progressively
increases until the forward and reverse equalize. At
this point, the rate of antibody–antigen complex
formation and the dissolution rate to release free
antibody and antigen are equal; the reaction is said
to have reached its equilibrium (Fig. 10.16). With
virus, the law of mass action dictates that there will
be free virus at any point in time; due to elution of
antibody from the complexes at equilibrium, the
virus infectious threat remains, albeit impeded by
the reaction rate of complex formation.
Neutralization assays identify antibody opsoni-
zation of virus at adequate avidity to interfere with
virus infection during the time of incubation with
susceptible cells. In a natural situation, this is only
the starting point. Without removal of the com-
plexes, the virus retains potential for initiating an
infection, beyond the incubation conditions of neu-
tralization assays, wherein complexes are removed
by washing. Antibody-mediated interference with
Figure 10.15 The Grey Zone of antibody tires with
respect to protection against challenge infection
virus infection facilitates the downstream processes
by FMDV. Protected animals are shown with white involving interaction with innate immune defence
circles, and unprotected with the black circles. The cells. This second phase is essential for ensuring
white zone identifies antibody titres more often protection – removal of immune complexes and
associated with protected animals, the black zone
antibody titres associated with susceptibility to
destruction of the virus. A virus clearance mecha-
infection, and the grey zone antibody titres which do nism essential for protective immune responses
not relate to protection. Adapted from McCullough against FMDV is phagocytosis of virus–antibody
et al. (1992).
Date: 10:24 Monday 12 September 2016
UNCORRECTED PROOF File: FMDV 4P
Immune Cell Interaction with FMD | 251
Figure 10.16 Schematic of virus–antibody complex formation and relative stability, showing displacement of
the antibody with time, and freeing of virus to infect. Antibody interferes with and retards the availability of
the virus to infect. The MΦ stabilizes the complexes on its surface by interacting with the FcR and enhancing
uptake and destruction of the virus.
complexes by MΦ (Fig. 10.16) (McCullough et al., FMDV (Dar et al., 2013; Mulcahy et al., 1990;
1992b), wherein antibody opsonization of virus to Perez Filgueira et al., 1995); their induction will be
a minimum avidity is critical. influenced by the adjuvant in vaccine formulations
(Dar et al., 2013; Eble et al., 2007; Perez Filgueira
Effector humoral immune defence et al., 1995). Secretory (mucosal) IgA is clearly
important for mucosal immune defences; different
Antibody interaction with FMDV IgG isotypes have distinct influences on effector
Effector immune defence against FMDV clearly immunity, particularly in terms of interaction with
begins with antibody interaction. The kinetics phagocyte Fc receptors (FcR), and activating the
of proficient defence in vivo must function more complement cascade. These latter processes are
rapidly than the protracted times employed with activated by conformational alterations occur-
traditional in vitro methods measuring antibody ring in the antibody Fc following interaction with
interaction. Reactivity of immune serum shows antigen. FcR involvement is important for removal
>90% interaction with antigen within 10–60 of immune complexes; activation of complement
seconds (Fig. 10.17) (Scicluna et al., 2001; Sci- results in deposition of activated products on to the
cluna and McCullough, 1999). The non-specific/ antigen, which can also enhance interaction with
aspecific reactions found with non-immune sera phagocytes through specific receptors. Certain
required several minutes to show interaction with complement activation by-products are involved in
antigen. Rapid reaction with virus during seconds enhanced recruitment of phagocytes to the sites of
would have greater capacity to impede the infec- immune complex formation.
tious potential, whereas the slower reactions over
minutes found with non-immune animals would The role of innate effector MΦ
have less protective capacity. Indeed, sera from pro- Antibodies that are efficient opsonins (Crowther
tected animals showed shorter reaction times (<60 et al., 1993; McCullough et al., 1987a) are also
s) with the virus, compared with sera from unpro- capable of neutralizing virus infectivity in vitro. For
tected animals (Fig. 10.17) (Scicluna et al., 2001). protection in vivo, effector innate immune defences
Antibody isotypes also play important roles involved in phagocytosis of the virus and destruc-
in humoral immune defences, including against tion of infectivity were obligatory (McCullough
Figure 10.17 Analysis in the Rate-Limiting ELISA of serum reactivity against FMDV O1 Lausanne antigen
following vaccination. Serial bleeds from two FMDV O1 Lausanne vaccinates were analysed at different time
points after primo-vaccination and subsequent booster vaccination (arrow). The values displayed have had the
mean values obtained with non-immune sera subtracted. (a) A405 for the positive sera, 60-second reaction
time. (b) A405 for the positive sera, 10-second reaction time. (c) The relative rate of the 10 and 60-second
reactions, determined from the slope of the reactions (0 to 10 s and 0 to 60 s, respectively). Adapted from
Scicluna et al. (Vaccine 2001).
Figure 10.18 The rate of uptake (a) and destruction (b) of antibody-opsonized FMDV by macrophages. In
(a), the uptake of virus alone (unopsonized) is shown for comparison. Adapted from Rigden et al. (2002) and
McCullough et al. (1988).
et al., 1986, 1988, 1992b). An important effec- complexing FMDV’) (Figs 10.16 and 10.18).
tor therein is the MΦ (McCullough et al., 1988, Phagocytosis of virus/antibody complexes has
1992b). MΦ are designed to phagocytose entities been demonstrated to promote destruction of
recognized as ‘foreign’ or of potential ‘danger’ in FMDV infectivity (McCullough et al., 1986, 1988).
their environment, enhanced when the entities Transient, abortive replication can be observed
are complexed with antibody interacting with in cells lines transfected to express FcR (Baxt and
FcRs (see ‘Biological consequences from antibody Mason, 1995), relating to the transient, abortive
replication in MΦ and DCs with virus alone (see of antibody prior to forming complexes with the
‘FMDV within MΦ and DCs’). When virus is com- virus also impaired phagocytosis and destruction
plexed with antibody, MΦ provide the biological of the virus, but not neutralization of virus infec-
system involved in immune protection (Fig. 10.16). tivity (McCullough et al., 1986, 1988, 1992b).
Impairing MΦ phagocytosis activity, in vitro Vaccination of cattle leading to protection induced
or in vivo, weakens their ability to destroy FMDV increased numbers of MΦ and opsonization-
infectivity (Fig. 10.19). Removal of the Fc portion enhanced phagocytosis (Quattrocchi et al., 2014),
Figure 10.19 Importance of MΦ in the immune defence against FMDV. (a, b) MΦ destroy FMDV complexed
with monoclonal antibodies against different epitopes involved with virus infectivity (grey bars). In (a), removal
of the Fc portion of antibody complexed with FMDV impairs their uptake via the FcR on MΦ, and thus both the
phagocytosis and destruction of the virus. In (b), the functional activity of MΦ interacting with these infectious
complexes is impaired, through blocking of phagocytosis with silicon dioxide (white bars), which impairs their
ability to destroy FMDV infectivity. Adapted from McCullough et al. (1988). (c–f) The protective immune defence
against FMDV requires MΦ to remove the antibody/virus complexes. In (c), antibodies against different epitopes
important to virus infectivity protect against disease, even when the antibody has not neutralized the virus. In
(d), removal of the Fc portion of antibody complexed with FMDV does not influence in vitro neutralization of
virus infectivity. In (e), removal of the Fc portion of antibody complexed with FMDV prevents their uptake via
the FcR on MΦ, and thus protection against disease. In (d), impairment of MΦ function using silicon dioxide
treatment of the animals prevents antibody from protecting against disease. Adapted from McCullough et al.
(1986, 1988).
Date: 10:24 Monday 12 September 2016
UNCORRECTED PROOF File: FMDV 4P
254 | McCullough et al.
although this may have been influenced by the virus titre association with the cells. Although the
adjuvant employed. above studies in bovine DCs did not go beyond 5h,
Consequently, direct neutralization by anti- it is clear that this is only the initial phase in cell han-
body of FMDV infectivity for susceptible cells is dling of the virus. Increases in infectious titres were
incomplete for ensuring protection against disease. observed up to 8 hours p.i. with porcine MoDCs,
Antibody will provide an initial interference with after which they declined to undetectable levels by
the spread of the virus, but the main consequence 24 hours (Harwood et al., 2008). Although DCs
is increased phagocytic capacity of innate immune tend to favour this transient, abortive replicative
defence cells such as MΦ and neutrophils. Com- cycle by FMDV, contrasting with the more degra-
plexes of antibody with virus facilitate interaction dative MΦ, the ultimate outcome with the DCs is
with innate immune cell FcRs (McCullough et al., also destruction of the virus (Guzylack-Piriou et al.,
1992b). In the absence of antibody, phagocytosis 2006a; Harwood et al., 2008; Lannes et al., 2012).
and destruction of the virus would still occur,
but with reduced efficiency (Rigden et al., 2002). The role of innate effector plasmacytoid
Through these studies it has become clear that anti- DCs
body-mediated phagocytosis by MΦ, and probably Activation of pDCs has a particular importance in
neutrophils, rather than just neutralization of virus immune defence with respect to producing high
infectivity are key elements for protective immune levels of serum IFN-α, described in pigs early after
defence. infection with FMDV (Nfon et al., 2010). FMDV
infection can downregulate IFN-response genes
The role of innate effector conventional (see ‘Interaction of FMDV with innate viral sen-
DCs sors’), which may relate to the observed modulation
As with MΦ, DCs internalize FMDV in immune of immune regulation during early phases after
complexes (Baxt and Mason, 1995; Guzylack- FMDV infection in vivo (Golde et al., 2011; Golde
Piriou et al., 2006a; Harwood et al., 2008; Lannes et al., 2008). Later in infection, or after vaccination,
et al., 2012; Ostrowski et al., 2005; Reid et al., anti-FMDV specific antibody modify the manner
2011; Rigden et al., 2002; Robinson et al., 2011; by which pDCs respond, and how the virus inter-
Summerfield et al., 2009), again via FcRs (see ‘Bio- acts with these cells (Guzylack-Piriou et al., 2006a;
logical consequences from antibody complexing Lannes et al., 2012; Summerfield et al., 2009). As
FMDV’). When live virus is internalized by DCs with cDCs and MΦ, this relates to FcR function of
– cDCs and pDCs – an increase in virus titre can be the pDCs (see ‘Biological consequences from anti-
observed (Guzylack-Piriou et al., 2006a; Harwood body complexing FMDV’). When pDCs become
et al., 2008; Lannes et al., 2012; Robinson et al., involved, they employ their FcR isoform FcγRII
2011), leading to abortive replication of the virus. (CD32) to bind FMDV–antibody complexes. Inter-
Although immune complexes apparently enhanced nalization of immune complexes by pDCs induces
interaction with bovine MoDCs, in terms of infec- high levels of IFN-α (Fig. 10.4) (Guzylack-Piriou et
tious virus titres released from the cells (Robinson al., 2006a,b), but dependent on the presence of live
et al., 2011), it is unclear if this reflected replication, virus within the immune complexes – inactivated
elution from the cells as observed with epithelial virus proved ineffective. Related to FMDV interac-
cells (Baxt and Bachrach, 1980), or recycling of tion with porcine cDCs (Harwood et al., 2008),
input virus (Berryman et al., 2005; O’Donnell et al., uptake of immune complexes by pDCs resulted in
2008; O’Donnell et al., 2005). Unfortunately, these an abortive replicative cycle. This provided viral
authors did not perform the critically important RNA molecules for activating the pDCs. via a
and informative measurement of infectious titres TLR7-dependent pathway (Lannes et al., 2012).
within the cell. They also failed to report on infec- Significantly, the opsonizing capacity of antibody
tious virus titres obtained with HS-binding virus, rather than neutralization of virus infectivity was
which initiates abortive replication in porcine DCs the critically important activity. While the degree
(Harwood et al., 2008). of pDC activation varied dependent on the FMDV
An important consequence of immune complex isolate, the role of opsonizing antibodies in pDC
endocytosis by DCs is the kinetics of infectious production of IFN-α provided a broader reactivity
than the neutralizing activity of the antibodies. manner to FcγRII, and would play important roles
Uptake of virus and induction of IFN-α were also in mucosal immunity. When two FcR become
subsequently demonstrated with bovine cells cross-linked by antibody molecules within the
defined as pDCs (Reid et al., 2011). Again, only live same immune complex, conformational changes
virus in immune complexes induced pDC produc- are induced in the FcR (Leslie, 1985b,c; Ravetch,
tion of IFN-α. 1997). This results in the increased stabilization of
antibody interaction with virus, reducing antibody
Biological consequences from antibody elution from the complexes.
complexing FMDV FcR cross-linking promotes cytoplasmic sig-
nalling via immunoreceptor tyrosine activation
The role of FcRs motifs (ITAMs) in the FcR cytoplasmic tails
Biological aspects of antibody-based immune (Leslie, 1985a,b,c; Nimmerjahn and Ravetch,
defence take the process beyond the Law of Mass 2007; Ravetch, 1997; Worth et al., 2001). ITAMs
Action. Antibody binding to antigen induces con- promote protein tyrosine kinase activation, thus
formational alterations in the Fc portion of the inducing GTPase-dependent internalization of the
antibody, allowing it to interact more efficiently complexes into phagosomes. There are also FcR
with the low affinity FcRs CD16 and CD32 on isoforms carrying the more regulatory immunore-
the surface of phagocytes and DCs. Therein, ceptor tyrosine inhibitory motifs (ITIMs), but this
interaction with the innate cells is dependent on will be considered in ‘The influence of FcR signal-
the FcR-expressing cell type, and probably how ling on effector versus memory immunity’.
the immune complexes interact with the different With immune complex uptake, a major differ-
FcRs on MΦ compared with DCs. There may be ence from the entry of virus into susceptible cell
a difference between species, but it is more likely lines for replication is that the degradative process
that differences in the immune complex load and of the phagocytes becomes a dominant factor
the MΦ/DC subset involved would have a greater (Blander and Medzhitov, 2006; Canton, 2014;
bearing. Fairn and Grinstein, 2012; Nordenfelt and Tapper,
FcR binding of antibody-virus immune com- 2011; Tjelle et al., 2000). Phagocytosed virus is
plexes actually upsets the chemical equilibrium brought into contact with acidic pH and degrada-
dictated by the Law of Mass Action. The cellular tive enzymes by sequential interaction with early
FcRs can stabilize the antibody binding in favour of and late endosomes, and lysosomes. This is the
the complex side of the equilibrium, thereby reduc- process by which the virus is gradually degraded
ing or even preventing elution of antibody from the (Fig. 10.18), observable in MΦ and DCs as a
virus in the complex (Leslie, 1985a, b, c; Leslie and time-dependent destruction of FMDV infectivity
Coupland, 1985; McCullough et al., 1992b). This (Harwood et al., 2008; McCullough et al., 1988;
activity is further enhanced by the modified Fc of Rigden et al., 2002).
antibody in immune complexes activating comple-
ment components (see below). Antibody isotype The role of complement
is important in this area, influencing both the Similar to enhanced antibody binding with phago-
capacity to interact with different FcRs, and activa- cyte FcRs, activation of the complement cascade
tion of the complement cascade. Enhancement of also requires conformational alterations in anti-
antibody-mediated effector immunity is due to the body Fc. Following deposition of certain activated
means by which FcR function. MΦ possess two products from the complement cascade on to the
major classes of FcR for IgG – FcγRIII (CD16) and antigen, fragments derived from activated C3 and
FcγRII (CD32). They bind primarily IgG–antigen C4 components enhance immune complex interac-
complexes (Leslie, 1985a,b,c; Leslie and Coupland, tion with phagocytes, and ultimately destruction
1985) at a higher affinity than IgG alone (hence the of the pathogen. Phagocytes possess receptors for
term ‘low affinity FcR’ for CD16 and CD32) (Nim- these – C3b, iC3b and C4b. Similar to the role played
merjahn and Ravetch, 2007; Ravetch, 1997). An by FcRs, CR1 (CD35) binds with and responds
FcR for IgA (FcαR) also exists on eosinophils and to C3b or C4b (C3b > C4b > iC3b), promoting
mononuclear phagocytes. It functions in a similar phagocytosis. The iC3b fragment can also interact
with mononuclear phagocytes, neutrophils and NK 1994; Scicluna et al., 2001; Steward et al., 1991;
cells through CR3 – the β2 integrin CD11b/CD18, Van Maanen and Terpstra, 1989). The protective
which also binds to ICAM-1 – and CR4 – the β2 immune response involves uptake and ultimate
integrin CD11c/CD18. B-lymphocyte activity destruction of the virus by MΦ and DCs, likely
can also be enhanced by activated products from elaborated by induction of IFN-α production by
the complement cascade. Together with iC3b, the pDCs (Baxt and Mason, 1995; Guzylack-Piriou et
fragments C3d and C3dg generated from C3 can al., 2006a; Harwood et al., 2008; Lannes et al., 2012;
bind to CR2 (CD21) as co-receptors for activat- McCullough et al., 1986, 1988, 1992b; Ostrowski et
ing B-lymphocytes (C3d, C3dg > iC3b). Activated al., 2005; Rigden et al., 2002). With live virus in the
complement components also enhance leucocyte immune complexes, an abortive replication in the
chemotaxis and inflammatory responses – further DCs provides RNA for important PAMP signal-
promoting enhanced pathogen destruction; the ling to activate the innate defence mechanisms of
best-described examples are the anaphylatoxins DCs, both pDCs (Guzylack-Piriou et al., 2006a;
C3a, C4a and C5a. Lannes et al., 2012; Reid et al., 2011; Summerfield
et al., 2009), and skin DCs (Bautista et al., 2005).
The biological impact on the law of mass Importantly, porcine DCs remain immunocom-
action petent and functionally active following uptake of
Interaction of antibody with the virus must be of immune complexes carrying live FMDV (Bautista
minimum avidity for immune protection to ensue, et al., 2005), and stimulate FMDV-specific Th-
dependent on the specificity of the antibodies lymphocytes (Harwood et al., 2008).
and maturation of the adaptive immune system
responding to virus infection or vaccination. The Cytotoxic effector immune responses
avidity of that reaction ensures a relative degree of Related to the role of phagocytes in destroying
stability, within the law of mass action, inducing virus infectivity within immune complexes, the
the necessary conformational alterations in the phagocyte FcR can also engage antibody bound to
antibody Fc portions, to facilitate interaction with the surface of infected cells. This requires that the
phagocyte FcRs and complement activation. The virus infection express viral proteins on the cell sur-
complexes influence cytokine induction – particu- face, which is not as obvious with a non-enveloped
larly IFN-α – promoting immune cell functions virus as with an enveloped virus. Nonetheless,
including DC maturation. Yet, elevated levels can immune defence against a non-enveloped virus
be more regulatory, including inducing apoptosis in such as FMDV does not preclude the presence of
cells. Overall, the outcome of antibody interaction virus infection-derived proteins at the infected
with FMDV is determined by how MΦ and DCs cell surface. If these induce humoral immunity,
interact with immune complexes in vivo, and what phagocyte-dependent attack of virus-infected cells
levels of complexes likely interact with a particular through antibody-dependent cellular cytotoxicity
cell at any particular moment in time. (ADCC) becomes a reality (Biburger et al., 2014).
However, it is unlikely that inactivated or peptide/
The outcome of MΦ and DC interaction protein-based vaccines that do not replicate would
with FMDV immune complexes induce such antibody specificities. Even vector vac-
MΦ and DCs can destroy FMDV in immune com- cines are unlikely to induce such specificities, which
plexes (Baxt and Mason, 1995; Guzylack-Piriou et is more reliant on an intact virus genome.
al., 2006a; Harwood et al., 2008; Lannes et al., 2012; Phagocytes are not alone in their capacity to
McCullough et al., 1986, 1988, 1992b; Ostrowski destroy infected cells, although the other cytotoxic
et al., 2005; Rigden et al., 2002; Summerfield et immune processes are not reliant on antibody reac-
al., 2009). Such observations relate to the central tion with infected cells. Natural killer (NK) cells
importance of antibody in immunological protec- target viral antigens on infected cell surfaces, but
tion against FMDV, reported by several groups do not require involvement of MHC molecules
(Black et al., 1984; Lavoria et al., 2012; McCullough (in contrast with antigen-specific Tc-lymphocytes
et al., 1986, 1992a; Oliveira et al., 2005; Ostrowski – see below) (Lugli et al., 2014; Waggoner et
et al., 2007; Pay and Hingley, 1987; Rieder et al., al., 2015). NK cell activity does relate to that of
Th- and Tc-lymphocytes in their innate-adaptive P1 precursor polypeptide of FMDV induced cel-
communications through production of IFNγ. NK lular rather than humoral immunity (Sanz-Parra
cell activity has been identified in infected cattle et al., 1999a,b). Additional studies on adenovirus-
(Patch et al., 2014), but could not be demonstrated vectored FMDV vaccination (Patch et al., 2011), as
following vaccination – in line with NK cells attack- well as fowlpox virus-based and inactivated FMDV
ing antigens expressed on virus-infected cells. Yet, vaccines (Guzman et al., 2008), showed induction
a non-MHC restricted FMDV-specific cytolytic of Tc-lymphocyte activity. A partial protection in
activity has been identified in NK-lineage cells iso- the absence of detectable humoral immunity was
lated from vaccinated cattle (Amadori et al., 1992). also related to Tc-lymphocyte activity (Patch et al.,
NK cell responsiveness to the presence of FMDV 2013). Although only partial protection was noted,
infection of pigs was also reported (Toka et al., this would fit to the expected role of effector cellular
2009a), and activated NK cells will efficiently lyse immunity, particularly if that were due to Tc-lym-
FMDV-infected targets (Toka et al., 2009b) (see phocytes. While antibody would be most effective
‘Ranking innate immune defences’). Detection of at preventing virus spread and therefore the acute
NK cell-like activity following vaccination suggests phases of infection, cytotoxic immunity would be
a virus-derived protein as the NK cell target, rather more efficient at removing foci of infected cells
than an infected cell-derived entity. Nonetheless, and finally clearing the infection. In the absence of
the contribution of such NK activity to protection antibody, an initial infection by the virus would be
or viral clearance remains unclear. possible, but the infected cells may ultimately suc-
The other main component of antigen-specific cumb to the cytotoxic defence, although the acute
cytotoxic immune defence is associated with the phase of infection would still be observable.
Tc-lymphocytes, generated by MHC Class I pres- Overall, current evidence suggests that cytotoxic
entation of antigen (Fig. 10.10). Vaccination could immune defences are important components of
generate such cells, through cross-presentation (see effector immunity, even when humoral immunity
‘Considerations for MHC Class II processing’), is the dominant entity in the protective immune
and certainly infection should induce such immu- response, as with FMDV. A continued study of
nity. However, cellular cytotoxic responses (NK or the relative role played by cell-mediated immunity
Tc-lymphocyte) would have to target infected cells against FMDV, and the means of induction through
early after infection by FMDV, prior to virus killing vaccination should provide the necessary informa-
of the infected targets. Induction of FMDV-specific tion for designing new vaccines generating a more
effector Tc-lymphocytes has been difficult to evalu- compete immune defence.
ate, possibly due to the short life-span of infected
cells to provide appropriate targets. Virus infection Immunological memory induction: in
was also reported to induce a rapid reduction of balance with effector immunity
MHC class I expression on susceptible cells (Sanz- When effective immune defence has surmounted
Parra et al., 1998), which would impair effector the antigenic and infectious threat from FMDV,
Tc-lymphocyte responsiveness. On the contrary, the immune system enters the phase of regulation
down-regulation of MHC Class I molecules on to dampen down activities within the innate and
infected cells would facilitate detection of viral anti- adaptive compartments. Concomitantly, the adap-
gens on the cell surface by NK cells. tive immune system develops a memory immune
Despite these scenarios, anti-FMDV Tc- defence. This allows for a certain retention of the
lymphocyte activity has been identified. Peptides expanded antigen-specific lymphocyte clones,
containing T-cell epitopes have been described together with their increased reactivity derived
in terms of porcine MHC Class I-dependent from maturation – ‘fine tuning’ for recognition
(SLA-I) presentation (as well as MHC Class II of the epitopes – in response to several rounds
dependency) (Fig. 10.20), as well as their ability of antigen stimulation. By such means, there are
to activate different T-lymphocyte subsets, includ- more lymphocytes available for responding to a
ing CD4–CD8+ porcine Tc-lymphocytes (Blanco subsequent encounter with the virus, and their
et al., 2000). Vaccination of pigs and cattle with responsiveness is more specific and of higher affin-
recombinant adenovirus vaccines expressing the ity for the virus epitopes. This should be the main
Figure 10.20 (a) SLA-dependency of FMDV peptide-induced specific lymphoproliferation. Anti-SLA class I
(white bars) and -SLA class II (grey bars) MAb blocking of the lymphoproliferation obtained with peptide VP4–0
[20–34], compared with peptide in the absence of the MAb (black bars). The percentage of inhibition obtained
with each MAb is shown. (b–e) Relative responses of cytotoxic CD4–CD8+ Tc cells (b), memory/activated helper
CD4+CD8+ Th cells (c), non-Th/Tc CD4–CD8– cells (d), and naive CD4+CD8– Th cells (e), by measurement of
IL-2 receptor (CD25) expression (dark grey histograms) in cultures stimulated with a BT tandem peptide. Light
grey histograms show the CD25 expression in control unstimulated cultures. Adapted from Blanco et al. (2000).
aim of vaccination (Sallusto et al., 2010). There development is the replacement of antigen by
is both central memory – associated with lymph antigen/antibody complexes (Kurosaki et al., 2010;
nodes, spleen and bone marrow – and peripheral McHeyzer-Williams et al., 2012). This is due to the
memory – in dermal and mucosal tissues – enhanc- progression of the effector immune defences into
ing the capacity of adaptive immune defences to production of the antibody now reacting with the
respond in a rapid and specific manner for eliminat- antigen. As mentioned above in ‘Effector humoral
ing any future threat, before infection can create immune defence’, when low affinity FcγRII and
problems for the host. FcγRIII on phagocytes bind antibody-complexed
antigen, the cytoplasmic portion of the FcRs is
The influence of FcR signalling on modified, resulting in enhanced phagocytosis.
effector versus memory immunity An additional consequence of antibody/antigen
One important signal for the shift from active gen- complex formation is the interaction with the
eration of effector cells to immunological memory FcRs on B-lymphocytes (Kurosaki et al., 2010). In
addition to FcγRII and FcγRIII involved in posi- and Weisel, 2012; Takemori et al., 2014; Yoshida
tive signalling of the cell, a second isoform of the et al., 2010). Switching B-lymphocyte differentia-
FcγRII termed FcγRIIB is involved in a suppres- tion from antibody-producing, short-lived plasma
sive response (Nimmerjahn and Ravetch, 2007; cells to memory B-lymphocyte development
Ravetch, 1997). These FcRs are also present on (including long-lived plasma cells) involves modi-
DCs, whereby their relative roles in induction and fications of the surface interactions between B- and
regulation of immune responses are found (Nim- Th-lymphocytes, and the cytokines involved. Acti-
merjahn and Ravetch, 2007). B-lymphocytes also vation via FcγRIIA or FcγRIII involves IFN-γ,
carry FcαR for binding immune complexes of anti- TNFα and the complement product C5a as impor-
gen with IgA. Similar to FcγRIIB, cross-linking of tant cofactors; with FcγRIIB ligation, the cytokines
FcαR by immune complexes provides a suppressive TGF-β and IL-4 become more prominent cofactors
signal (Nimmerjahn and Ravetch, 2007; Ravetch, (Nimmerjahn and Ravetch, 2007). In addition,
1997; Tsujimura et al., 1990). immune complexes are also reported to influence
With the activating FcRs – such as FcγRIIA memory cell development. When captured on the
and FcγRIII – the outcome is ITAM activation follicular dendritic cells (FDC), germinal centre
in the cytoplasmic tail of the FcR, leading to a B-lymphocytes are observed to scan these FDC
Syk kinase- and PI3 kinase-dependent activation when ‘laden’ with immune complexes (McHeyzer-
pathways (Nimmerjahn and Ravetch, 2007). In Williams et al., 2012). Yet, this process appears
contrast, ligation of the inhibitory FcγRIIB induces to be more relevant to selection of high-affinity
ITIM activation in the cytoplasmic tail of this FcR, BCR-bearing B-lymphocytes, and thus refining
leading to an SH2-domain containing inositol 5′ the humoral immune defence. Indeed, memory
phosphate (SHIP)-dependent inhibitory signal- B-lymphocyte generation can occur in the absence
ling pathway (Nimmerjahn and Ravetch, 2007). of antigen-containing immune complexes held by
FcγRIIB is involved in deletion and/or inactivation the FDC (Anderson et al., 2006).
of self-reactive B-lymphocytes, in both the bone
marrow and the periphery. With plasma cells, the Memory B-lymphocytes
FcγRIIB triggering induces apoptosis, thus control- Memory B-lymphocyte development can be driven
ling plasma cell homeostasis. by activated Th-lymphocytes (Kurosaki et al., 2015;
An important structure on the antibody mol- McHeyzer-Williams et al., 2012; Shlomchik and
ecule influencing the selection of the FcR isoform Weisel, 2012; Takemori et al., 2014; Yoshida et al.,
for binding is the sugar moiety. For example, fucose 2010). This can lead to immunological memory
in the antibody sugar moiety at asparagine residue development, either dependent or independent of
297 influences binding to human FcγRIII but not the germinal centres; the independent processes can
FcγRII (Nimmerjahn and Ravetch, 2007). In con- actually occur prior to germinal centre formation
trast, terminal sugar residues of sialic acid rather (Takemori et al., 2014). Memory B-lymphocytes
than galactose are important for regulating anti- can also be found outside the lymphoid organs, for
body activity in vivo. When these sialic acids are at example in the bone marrow wherein long-lived
high levels, impaired binding to FcRs is noted, but ‘memory’ plasma cells have been described (Manz
terminal sialic acids are still important as noted by et al., 1998; Yoshida et al., 2010). These cells are
their reduced levels in autoimmune diseases (Nim- particularly important to effector immunity, with
merjahn and Ravetch, 2007). This is a combination their long duration of antibody production, which
of reduced binding of the sialic acid-rich antibodies may remain over a lifetime (Yoshida et al., 2010).
to FcRs together with actively promoting an anti- They are distinct from the short-lived plasma cells in
inflammatory response. extrafollicular foci of antibody production (Kuro-
It should be noted that induction of memory saki et al., 2015), seen by their elevated expression
B-lymphocytes is not separable from activation of of anti-apoptotic Bcl-2 (Yoshida et al., 2010).
the B-lymphocytes into antibody-producing plasma An active effector process does not need to be
cells. Memory development will occur during the particularly advanced before memory development
active phase of antibody induction (Kurosaki et al., is initiated. It has been suggested that within 3 days
2015; McHeyzer-Williams et al., 2012; Shlomchik of immunization/vaccination, Th-lymphocytes
interacting with processed antigen presented a major impact on germinal centre B-lymphocyte
on DCs will interact with antigen-activated proliferation and differentiation into memory cells.
B- lymphocytes via CD40–CD40L interac- An important distinction between the germinal
tion, determining the differentiation of the centre-dependent and -independent memory
B-lymphocytes into memory or germinal centre B-lymphocytes is their affinity for interacting with
cells for antibody production (Takemori et al., antigen, and therefore responsiveness upon antigen
2014). Memory B-lymphocytes can be found of re-encounter. The germinal centre-independent
IgM+, IgG+, IgA+ and IgE+ phenotype (Takemori cells have lower affinity than the germinal centre-
et al., 2014). All show the characteristics of being dependent cells (Takemori et al., 2014).
long-lived, an essential quality for ensuring durable Overall, the generation of different forms of
immunity, but it appears that IgM+ memory cells memory B-lymphocytes affords the immune
are more long-lived than the IgG+ cells (Kurosaki defences with a variety of responses upon antigen
et al., 2015). It has been suggested that this reflects re-encounter. Regardless of the differences in their
difference in their self-renewal activities. The IgG+ affinity of reaction, their relative persistence pro-
cells may require more regular BCR-mediated vides a durable response upon re-encounter with
signalling, for example by FDCs acting as a depot antigen or pathogen. The differences in affinity can
for antibody–antigen complexes. There is also facilitate broadening the responsiveness upon chal-
the reported influence of DC-derived BAFF and lenge with a modified pathogen, while the higher
APRIL, which do not affect survival of the memory affinity IgG+ memory cells would require more self
IgG+ cells, but the memory IgM+ cells may require renewal through re-encounter. Both these charac-
these cytokines similarly to naive B-lymphocytes teristic are important in the context of vaccination,
(Kurosaki et al., 2015). and should be analysed in terms of both the vaccine
A major involvement in memory cell develop- and adjuvant employed in any formulation.
ment is this CD40-CD40L cognate interaction and
its durability. While durable T–B conjugates facili- Memory T-lymphocytes
tate B lymphocyte differentiation into germinal As with the antigen-specific B-lymphocytes, clones
centre B-cells, shorter conjugate formation is more of specific T-lymphocytes can be expanded by
likely to drive the B-lymphocytes into the germinal antigen presentation of DCs into both effector cells
centre-independent memory cell pool; as for the and memory cells. The Tfh-lymphocytes mentioned
germinal centre-dependent memory cells, these above as important for germinal centre-dependent
can derive from stochastic differentiation of germi- B-lymphocyte responses, including memory, also
nal centre B-lymphocytes (Kurosaki et al., 2015). differentiate into memory cells (Hale et al., 2015).
Therein, metabolic programming and autophagy The memory Tfh-lymphocytes can develop from the
appear to be major factors for the generation and Th1, Th2 and Th17 lineages (Fig. 10.2). The cytokine
maintenance of memory B-lymphocytes; expres- profiles involved in the T-lymphocyte expansion
sion of genes regulating autophagy initiation and from naive cells will define the memory subset to
autophagosome maturation are detected in these develop. IL-12 and Type I IFN are important for
memory cells, leading to elevated levels of basal Th1 development, IL-4 for Th2 and IL-6 plus IL-1β
autophagy (Chen et al., 2014). and TGFβ for Th17. The presence of IL-1, IL-21 and
Germinal centre-dependent memory B-cell ICOS (inducible co-stimulator) will influence this
development requires immunological help from the differentiation towards the memory context (Hale
follicular Th-lymphocytes (Tfh-lymphocytes), as et al., 2015).
does germinal centre B-lymphocyte differentiation. Related to the extrafollicular B-lymphocytes,
While the germinal centre-independent memory such as the long-lived memory plasma cells, there
B-cell development also requires immunologi- are also memory T-lymphocytes found in the
cal help, this is from Th-lymphocytes that are not tissues, inducing both Th- and Tc-lymphocyte
Tfh-lymphocytes (Kurosaki et al., 2015; Takemori subsets (Schenkel and Masopust, 2014). These
et al., 2014). An additional signal involved in the tissue resident memory cells can be related to the
germinal centre-dependency of memory B-cell central memory (Tcm) and effector memory (Tem)
development is IL-21, signalling from which has cells (Sallusto et al., 2010; Sallusto et al., 1999).
generate mucosal immunity following parenteral neutrophils via interaction of the complexes
immunization, and is a major area of current with cell surface FcRs or CRs, leading to
research in vaccinology. Further studies on DCs (x) consequential signalling of the phagocytes to
with respect to their targeting through vaccina- internalize the complexes,
tion and homing to mucosal surfaces, are major (xi) promoting endosomal association of the
themes currently under scrutiny in the further- complexes together with maturation of the
ance of studies on the immunology of efficacious endosomal system to
vaccination. (xii) degrade the virus leading to destruction of its
Consequently, successful vaccination lead- infectivity and therefore
ing to induction of efficacious immune defence (xiii) immune protection, which is subsequently
provides for removal of the FMDV infectious enhanced by
threat. This requires initial interaction with specific (xiv) expansion of the antigen-reactive lymphocyte
antibody to form immune complexes, completed clones to provide
through phagocyte involvement, as well as DCs (xv) more rapidly responding memory of both
– particularly pDCs. Activating both humoral B- and T-lymphocytes, ensuring
(B-lymphocyte) and T-lymphocyte immunity (xvi) maintenance of a rapid and long-lasting
promotes effector immune defences, together with immune protection.
expansion of antigen-specific lymphocyte clones
into memory cells. Not only do memory cells reside Overall, these are the components that need
in the lymphoid organs wherein naive cells first to be considered in the context of vaccination and
responded to antigen, they also migrate into other therefore development of efficacious vaccines and
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Zaki, N.M., and Tirelli, N. (2010). Gateways for the Rev. 52, 245–253.
intracellular access of nanocarriers: a review of
include virological methods that detect virus, viral a permanent pig kidney cell line (IB-RS-2; De
antigen or genome, and are supplemented by Castro, 1964). A goat tongue cell line (ZZ-R 127;
nucleotide sequencing methods used for strain Brehm et al. 2009) has also been shown to be highly
characterization purposes. Serological approaches sensitive to infection with wild-type and cell-
detect FMDV-specific antibodies; these methods adapted strains of FMDV, and has proved useful
inform on prior infection and vaccination status for isolation from a variety of clinical samples from
and are widely used for diagnostic and surveillance animals experimentally inoculated with FMDV
purposes. (Fukai et al., 2013). Molecular approaches are now
In addition to tests that are used in centralized being explored in attempts to develop improved
laboratories, there is increasing emphasis on devel- cell lines that are suitable for FMD diagnosis.
oping diagnostic technologies that can be deployed Work in this area has recently yielded a continu-
close to the animals with suspected clinical signs. ous bovine kidney cell line which stably expresses
These assays are variously referred to as ‘point-of- both subunits of αVβ6 integrin, the principal cell
care tests’ (POCT), ‘pen-side’, ‘portable’, ‘on-site’ or receptor for FMDV (LFBK- αVβ6: LaRocco et al.
‘field’ tests, though perhaps a more accurate generic 2013). These cells were found to be more sensitive
term would be ‘point of decision’ tests. Recent to infection with FMDV material of animal origin
developments in this area include simple-to-use (vesicular fluid, tissue homogenates) than BHK,
lateral-flow devices (LFD) for the detection of IB-RS-2 and MVPK cells. Although cell cultures
FMDV, as well as new hardware PCR platforms and systems can be highly sensitive, testing of samples
disposable isothermal assays for deployment into is relatively slow (taking between 1–4 days), results
the field for use by non-specialists. can be influenced by the host-specificity of certain
FMDV strains (depending upon the particular cell
culture systems used), and are critically impacted
Virological tests by the quality of the individual specimens submit-
Virus-detection assays aim to detect FMDV ted to laboratories. Furthermore, the investment
in clinical or preclinical specimens collected in sterile facilities and technical expertise required
from animals, and include (1) virus isolation in for the maintenance of cells (and regular sourcing
cell culture systems to propagate ‘live’ FMDV of animals certified to be FMD-free in the case of
and other viruses (such as swine vesicular dis- BTy cells) makes virus isolation still inaccessible to
ease virus) causing signs of vesicular disease in the vast majority of local and smaller laboratories.
susceptible species, (2) immunoassays such as Cytopathic effect in susceptible cells is not suf-
enzyme-linked immunosorbent assays (ELISAs) ficient alone to provide a conclusive diagnosis of
to detect and characterize FMD viral antigen in FMD. An antigen detection ELISA is typically used
clinical specimens and material from cell culture to assign the serotype and verify FMDV presence.
tests, and (3) molecular tests such as real-time These assays have lower analytical sensitivity com-
reverse-transcription polymerase chain reaction pared to virus isolation when used to detect FMDV
(rRT-PCR) to detect viral genome (RNA). Biopsy in clinical epithelial samples, and are not suitable
samples (and fluid) collected from vesicular for blood, milk and swab samples. Typically these
lesions represent the ideal material for virus detec- assays utilize characterized polyclonal antisera in
tion assays and sequencing protocols, while the an antigen-capture (sandwich) format (Ferris et
utility of tests for other types of clinical specimens al. 1988); although review and production of new
such as blood/sera, milk, swabs and oesophageal– antibody reagents is required to detect new FMDV
pharyngeal fluid ‘probang’ samples is governed strains as they continuously emerge in endemic
by ‘diagnostic windows’ in the respective hosts settings. Recent advances with the antigen-ELISA
(Alexandersen et al., 2003). test have involved the use of a purified recombinant
Highly sensitive in-vitro cell cultures are used αVβ6 integrin as a trapper coupled with type-specific
to isolate and propagate FMDV, and are widely monoclonal antibodies (Ferris et al., 2005, 2011).
considered to be the ‘gold-standard’ diagnostic Amplification of specific nucleic acid sequences
test for FMD diagnosis; these include primary using reverse-transcription polymerase chain reac-
bovine thyroid cells (BTy; Snowdon, 1966) and tion (RT-PCR) is now widely used for the laboratory
detection of FMDV. Molecular assays are suitable As more viral genome data become available,
for the diverse range of different sample types that there is now focus on the development of serotype
might be submitted for laboratory investigation (or lineage)-specific rRT-PCRs to supplement
(including tissues, blood, swabs, oesophageal/phar- the pan-serotypic molecular tests used for FMD
yngeal (OP) scrapings, faecal samples and milk). diagnosis. These tools can be used in FMD-
Over the past 15 years, improvements have been endemic countries for the identification of the
made to RT-PCR protocols used for the detection serotypes of the causative virus strains, providing
of FMDV. Initially, assays that targeted conserved important information for vaccine selection, and
regions of the genome [3D: Meyer et al. 1991; for tracing the source of outbreaks. Conventional
Rodrıguez et al., 1994; and 5′ untranslated region RT-PCR procedures using primers corresponding
(5′ UTR): Reid et al. 2000] utilized agarose gel to the VP1 (1D) coding region and to the 2A/2B
electrophoresis for the detection of amplified prod- coding regions for serotyping of FMDV have been
ucts. However, these labour-intensive procedures reported (Vangrysperre and De Clercq, 1996; Cal-
carry a high risk of generating false positives due lens and De Clercq, 1997). However, subsequent
to carry-over of PCR amplicons and are therefore studies have shown that these agarose gel-based
not generally considered ideal for routine testing RT-PCR assays have relatively poor sensitivity
of large numbers of samples. Real-time RT-PCR and specificity due to the genetic diversity within
(rRT-PCR) assays have now largely replaced aga- FMDV serotypes. While these procedures can be
rose gel based assay formats. These more rapid used in conjunction with Ag-ELISA and VI to pro-
fluorescence-based approaches are highly sensitive vide additional information, they are insufficiently
enabling simultaneous amplification and quanti- sensitive to replace them for primary diagnosis of
fication of FMDV specific nucleic acid sequences. FMD (Reid et al., 1999). A conventional RT-PCR
In addition to enhanced sensitivity, the benefits procedure for differentiation of FMDV serotypes
of these closed-tube rRT-PCR assays over con- native to India using multiple primers based mostly
ventional endpoint detection methods include a on nucleotide sequences of viruses circulating in
reduced risk of cross-contamination and an ability that geographical area was described by Girid-
to be scaled up for high-throughput applications. In haran et al. (2005). These studies demonstrated
common with the approaches used in other human the potential for using tailored molecular tools to
and animal disease diagnostic laboratories, assays identify specific serotypes as an alternative or addi-
that exploit the 5′-nuclease (TaqMan®) system for tion to pan-serotypic assays for detection of FMDV.
routine diagnosis are the most widely used for FMD More recently, lineage specific RT-PCRs and rRT-
(Reid et al. 2002; Callahan et al. 2002). In order to PCR have been reported for South East Asia (Le et
increase assay throughput and minimize error asso- al., 2012), West EurAsia (Reid et al., 2014; Jamal
ciated with human operator, these assays can be and Belsham, 2015), and exotic viral incursions in
automated using robots for nucleic acid extraction North Africa (Ahmed et al., 2012; Knowles et al.,
(Reid et al. 2003). Together with the implementa- 2014). Going forward into the future, it is possible
tion of quality assurance systems (such as ISO/IEC to imagine that a ‘diagnostic tool-box’ of RT-PCR
17025), these improvements have increased the specific assays will be available for researchers and
acceptance of the rRT-PCR assays as the front-line diagnosticians in the different global FMD endemic
test for routine diagnostic purposes (Reid et al., pools.
2009). Other real-time detection chemistries have
also been explored including the use of modified
MGB probes (Moniwa et al. 2007; McKillen et al. Serological approaches
2011), hybridization probes (Moonen et al. 2003), Serological tests are widely used to monitor the
Primer-probe energy transfer (PriProET; Rasmus- immune status of animals exposed to FMDV or
sen et al. 2003) and RT-linear-after-the-exponential FMDV vaccines. Approaches used include enzyme
PCR (LATE PCR; Reid et al. 2010); however, these linked immunosorbent assays (ELISAs) and virus
formats are not widely employed since they do not neutralization tests (VNTs), although comple-
typically provide any tangible advantages over the ment fixation tests (CFT) are still used in a limited
well-established TaqMan® system. number of laboratories. One particular application
of these serological assays is to identify animals in a included in the analysis (48–89%). The specificity
vaccinated herd that have been infected with FMDV. of all these assays in vaccinated cattle exceeded
This so called DIVA (differentiating infected from 96% (Brocchi et al., 2006). Study designs usually
vaccinated animals) principle exploits differences focus on younger animals (< 18 months of age),
in the antibody (humoral) responses generated since repeated vaccination, even with high quality
in vaccinated animals compared to those animals vaccines, can generate positive signals in the NSP
naturally infected with FMDV (whether or not ELISAs that may provide a false indication of
they have been vaccinated). High-quality FMDV FMDV infection (Elnekave et al., 2015).
vaccines are purified to contain structural protein In contrast to SP tests such as the solid-phase
(SP) viral capsid components from which most of competition ELISA (SPCE), liquid-phase blocking
the viral non-structural proteins (NSP) have been ELISA (LPBE) or VNT, NSP ELISAs are not sero-
removed. In contrast, during natural infection with type specific and can therefore be used as generic
FMDV, viral NSP are expressed that elicit a cor- screening tools. NSP sero-conversion usually takes
responding immune response that can be detected 7–14 days, after which these antibodies to these
using diagnostic approaches (Fig. 11.1). proteins can be detected in serum for months, or
There are a number of commercially available even years, depending upon the amount of virus
tests, and in-house assays that detect NSP-specific replication (Parida et al., 2005; Paton et al., 2009;
antibody responses including 3ABC, 2B, 2C, Elnekave et al., 2015). NSP surveys are used in
3B, 3B2, 3D. The strength of the NSP antibody countries progressing towards OIE FMD-freedom
responses in individual vaccinated animals can (with vaccination) as they are useful to confirm the
vary according to the extent of virus replication. absence of FMDV circulation in livestock popula-
Therefore, when the comparative performance tions (Chen et al., 2011). Since these surveillance
of five 3ABC assays and one 3B tests were evalu- exercises involve the testing of relatively large
ated (Brocchi et al., 2006), the ability of these numbers of sera, it is usually important to adopt
tests to detect vaccinated animals that have been a layered testing approach to accommodate the
subsequently exposed to FMDV varied consider- inherent performance of the NSP assays (Brocchi
ably (from 38% to 74%), although these sensitivity et al., 2006) and the expected number of false posi-
values were higher when only carrier animals were tives. Tests with high diagnostic sensitivity (such
as a 3ABC ELISA) are normally used to screen
the sera, and positive results are confirmed using a
second NSP antibody assay with at least equivalent
Vaccinated
Vaccinated sensitivity and specificity (Brocchi et al., 2006;
and infected
Paton et al., 2006). Furthermore, to rule out the
false-positives, epidemiological investigations and
analysis of probang samples by real-time RT-PCR
may be recommended (Paton et al., 2006, 2009). In
Vaccine: purified this context, SP testing could be explored but would
to remove NSPs NSPs
SPs
require a detailed knowledge of typical responses
Virus against vaccines in use to identify unexpectedly
replication high titres associated with infection. It is important
to know the design prevalence of these studies
Abs against Abs against Structural since this will impact upon the interpretation of
Structural Proteins and NS Proteins
data. Rather than employing random sampling, the
Figure 11.1 The principle of using non-structural
adoption of a risk-based sampling strategy may be
proteins (NSPs) tests to differentiate between more effective to recognize rare events. Although
vaccinated and infected animals. Both structural VNTs are largely considered the ‘gold standard’ for
(SP) and NSP antigens induce the production of trading purposes and post-vaccination monitoring
antibodies in infected animals. In contrast, vaccinated
animals that have not been exposed to replicating
advances have been made in the use of ELISAs and
virus will only develop antibodies to the viral capsid there are now a number of different antibody sero-
(SP) antigens. specific ELISA kits available on the market. While
some of these commercial kits rely on polyclonal 8300 nucleotides in length. The enzyme (RNA-
sera for the detection and trapping of antigen, other dependent RNA polymerase) responsible for
kits use a monoclonal antibody-based system. replication of this genome has poor fidelity, such
Manufacturers are also promoting the supply of that changes to the nucleotide sequence frequently
pre-coated plates which has the advantage of reduc- occur and are inherited by progeny viruses. Nucleo-
ing human error as well as operator time needed to tide sequencing is an established tool that is used
perform the assay. to compare field strains with reference viruses
Serological approaches (VNT and LPBE) are and genetic characterization of FMDV routinely
also employed by FMD Reference Laboratories to uses VP1 sequence data generated by RT-PCR
assess the antigenic match between vaccine strains (Knowles and Samuel, 2003). This region of the
and circulating FMD viruses causing field out- FMDV genome is approximately 630 nucleotides in
breaks. Vaccine matching using these approaches is length and encodes a protein (1D) that comprises
used to calculate a relationship coefficient (r1-value) an important component of the FMD viral capsid
between the vaccine virus and the field virus under containing several of the antigenic determinants
investigation. Although these methods provide an for the virus (see Chapter 4). Phylogenetic analysis
indication of how the field virus and vaccine virus are of these sequences can be used to categorize field
antigenically related, due to the nature and inherent strains into discrete subgroups (or topotypes)
variation of the tests it can often be difficult to inter- which frequently show geographical clustering
pret the results especially for borderline samples. based on the historical distribution of the virus (for
Standardization and harmonization of these tests is a review see Di Nardo et al., 2011). The distribution
challenging for FMD Reference Laboratories and of these different serotypes and topotypes varies
requires access to vaccine virus strains from vaccine according to countries and continents and in some
manufacturers and suitable bovine vaccine sera. In areas, several serotypes and variants can occur at
endemic settings, where multivalent vaccines and the same time. Analysis of VP1 sequences is widely
booster vaccination regimes are usually employed, used to reconstruct the spatial and temporal spread
there is often emphasis placed on post-vaccination of different FMD lineages across international
serological studies in host species to supplement boundaries; such as the recent spread during 2013–
the results of in vitro vaccine-matching studies. As 15 of O/ME-SA/Ind-2001 lineage from the Indian
an alternative to vaccine-matching, research is now sub-continent to countries in the Middle East and
focused at developing models to improve our abil- North Africa (Knowles et al., 2014).
ity to predict the antigenic phenotype directly from Although useful for strain characterization and
nucleotide sequence data (Ludi et al., 2014; Reeve to trace regional-level spread of FMDV, outbreaks
et al., 2010; Borley et al., 2013). in the United Kingdom (during 2001 and 2007)
highlighted the limitation to which VP1 sequence
data can be used to discriminate sequences from
Sequencing field cases of disease. VP1 is relatively short (~ 8%
FMD viruses are distributed on three continents of the genome length) and as a consequence, phy-
(Asia, Africa and South America) and can be fur- logenetic trees generated for FMDV sequences
ther subdivided into seven major geographical virus recovered within outbreaks clusters are typically
pools of infection (Paton et al., 2009). Identifying flat, with poor resolution between the closely
the sources of FMD outbreaks plays an important related viruses. Furthermore, limiting sequence
role in disease control; however, this can be con- analysis to only VP1 reduces the ability to identify
founded by incomplete epidemiological evidence broad-scale recombination events that may drive
and the numerous routes that can potentially be step-changes in the generation of new genetic and
involved in the spread of the virus (such as via antigenic variants, and to fully characterize all of
aerosols, movements of infected animals or their the antigenic determinants that are encoded by
products, and spread of fomites on contaminated other components of the viral capsid (such as VP2
persons and objects). and VP3). Improvements to sequencing protocols
The genome of FMDV comprises a single- and the development of computational phyloge-
strand of positive-sense RNA of approximately netics have opened up opportunities to study the
evolution of RNA viruses in real time. Recent (1) allow more samples to be characterized within
advances in laboratory methodologies allow rapid a given time, and (2) provide an approach to
sequencing of complete FMDV genomes (Carrillo dissect the heterogeneous swarm of similar but
et al., 2005; Cottam et al. 2006; Abdul-Hamid et al., non-identical genomes viral genomes that exist as
2011). For the first time, this opens up the potential a consequence of the high replication rate and poor
for using genome sequencing to reconstruct virus proof-reading ability of the FMDV RNA-dependent
transmission trees with extremely high resolution RNA polymerase. Using NGS performed on a
and to reveal and identify the origin of unresolved Genome Analyser platform (Illumina), the viral
transmission events within discrete infection clus- populations within bovine epithelial samples (foot
ters quickly. lesions) have been determined (Wright et al., 2011;
Using full FMDV genome sequences deter- Morelli et al., 2013). This approach revealed the fine
mined from field samples collected from the 2001 polymorphic sub-structure of the viral population,
FMD outbreak in the UK, retrospective analysis from nucleotide variants present at just below 50%
demonstrated that it was possible to reconstruct frequency to those present at fractions of 1%. Some
transmission events even at the level of farm-to- of the higher frequency polymorphisms identified
farm spread (Cottam et al., 2006, 2008). Known encoded changes in the heparan sulphate receptor
patterns of spread of the virus were reproduced binding site revealing intermediate stages in the
by statistical parsimony-based analyses of these evolution of a tissue-culture adapted viral genome
data. During the 2007 FMD outbreaks in the UK, upon replication within a mammalian host. These
real-time analysis of full genomes was used to sup- technologies are still being rapidly improved, and
port epidemiological investigations (Cottam et al. newer ‘single-molecule’ sequencing methods such
2008a). Analyses of these data have revealed the as the MinION (Oxford Nanopore) may in the
most likely chain of transmission events, and pre- future even offer the possibility to deploy sophis-
dicted undisclosed infected premises prior to their ticated sequence analysis to remote settings and
discovery by serological surveillance. Thus, results poorly equipped laboratories.
demonstrate that full-genome sequencing can
be used for fine-scale epidemiology to reveal and
identify the origin of FMDV causing outbreaks, Field tests
and that the interpretation of transmission trees is Much has already been written about the poten-
statistically robust and not impacted by sampling tial of pen-side tests to provide a rapid diagnostic
biases on individual farms (Valdazo-Gonzalez et al., capability for FMD (Sammin et al., 2010). In addi-
2015). By linking the sequence data to empirical tion to the use of these tests in emergency settings
epidemiological data that is collected during FMD (such as disease incursions in FMD-free countries
outbreaks (such as date, location and lesion age), in Europe and North America), these test formats
genomics can ultimately drive the reconstruction of may be particularly suited for use in FMD-endemic
FMDV movements on multiple scales ranging from areas such as those countries within sub-Saharan
intra-host, to farm, to region, to country and also Africa, where the time taken to collect and dispatch
contribute to the wider global picture regarding the samples to a laboratory for disease investigation can
epidemiology of FMD, and providing insights into be protracted. The characteristics of an ideal test
virus transmission chain, i.e. ‘who-infected-who’. would be rapid (less than 30 min in most instances),
This knowledge directly supports decision- and inexpensive (less than 15 euros per test) and simple
policy-makers in outbreak management. to use. For FMD, disposability of the test, or the
Conventional di-deoxy ‘Sanger’ sequenc- ability to effectively decontaminate equipment to
ing methods are now being replaced by minimize the risk of fomite transmission, is also
Next-Generation Sequencing (NGS) techniques important. In terms of assay characteristics, these
that offer an unprecedented ‘step-change’ increase in tests need an appropriate level of diagnostic speci-
the amount of sequence data that can be generated ficity and sensitivity, which may not necessarily be
from a sample. Bespoke NGS (Illumina) protocols completely equivalent to laboratory-based tests.
have been recently developed for FMDV (Wright The first generation of POC tests based on LFDs is
et al., 2011; Logan et al., 2014). These methods epitomized by the success of the home pregnancy
test kit. LFDs based on ClearviewTM technology field (Callahan et al., 2002; Hearps et al., 2002; King
(Unipath) were initially developed during the et al., 2008). The focus of current work is the devel-
mid-1980s for clinical applications, and are now opment of hardware platforms (Madi et al. 2012)
most widely used as the basis for home pregnancy that incorporate a simple-to-use and robust tem-
testing kits measuring human chorionic gonadotro- plate extraction process such that all the steps of the
pin (hCG). These devices are rapid, inexpensive, assay can be performed without user intervention.
disposable and simple to use. A positive test result These steps include (i) nucleic acid extraction (ii)
is signified by the appearance of a line at the ‘test’ PCR set-up, (iii) amplification and (iv) unambigu-
position within 10 min of the addition of a sample ous calling of results. Although there is only limited
to the device, making the results easily interpretable access to these technologies outside of Europe
by a non-specialist. A number of LFDs have been and North America, there is an ongoing concerted
developed for the detection of FMDV, including effort to evaluate these platforms in field settings,
monoclonal antibody-based pan-serotypic assays particularly in endemic setting in Africa and Asia.
(Ferris et al., 2009; Oem et al., 2009; Morioka et In addition to the performance of these equipment
al., 2015; Yang et al., 2015), and tests that are able and assays, the availability and cost of consumables,
to detect specific FMDV serotypes (Ferris et al., as well as mechanisms to locally service and repair
2010), and FMDV-specific antibodies (Yang et al., the machines will be crucial factors for the routine
2010). Sample preparation in field conditions can implementation of these tests.
be achieved using simple homogenizers for prepar- The recent development of portable equipment
ing epithelial suspensions. In terms of diagnostic for PCR is an important step towards the estab-
sensitivity and specificity, it has been shown that the lishment of molecular diagnosis of FMD in the
performance of the FMDV LFD is broadly equiva- field. However, this approach relies on precision
lent to laboratory-based antigen ELISAs. Although thermocycling requiring instrumentation which
the analytical sensitivity appears to be lower than can be fragile, expensive and that will need thor-
rRT-PCR, the ability to sample multiple animals ough decontamination when transferred between
within a herd increases confidence in the result (at locations. As an alternative to PCR, isothermal
a herd level). These data illustrate the potential for (single temperature) amplification methods for
the LFD to be used next to the animal in the pen- the detection of FMDV have been developed, such
side diagnosis of FMD and for providing rapid and as reverse-transcription loop-mediated amplifica-
objective support to veterinarians in their clinical tion (RT-LAMP). RT-LAMP is an isothermal
judgement of the disease. Limited field trials of autocyling strand-displacement DNA synthesis
LFDs were undertaken during the FMD outbreaks technique which utilizes four specific primers to
in southern England during 2007: positive results recognize six regions of the target genome (Notomi
were obtained on one of the infected premises et al. 2000). Formation of loop structures enables
within 10 minutes (Ryan et al., 2008). Using LFDs explosive polymerase-based enzymatic amplifica-
for field diagnosis may also have an impact upon the tion, which generates double-stranded, multi-sized
shipment of samples to FMD Reference Laborato- amplicons. Pan-serotypic RT-LAMP assays have
ries. Recent work (Fowler et al., 2014) has shown been designed for FMDV (Dukes et al., 2006; Shao
that it is possible to recover viral RNA (suitable for et al., 2010; Yamazaki et al., 2013). Validation data
rRT-PCR and VP1 analysis), and even ‘live’ FMDV indicate that RT-LAMP has equivalent analytical
via electroporation from FMDV-positive LFDs. sensitivity to rRT-PCR and may be less sensitive
Although further evaluation, validation and a full to inhibition by problematic sample matrices such
bio-risk assessment are needed, this approach may as ‘probang’ fluids and faecal samples. RT-LAMP
provide an opportunity to use LFDs for the dry, products are generated in abundance and can be
inexpensive and less-hazardous transportation of detected using equipment to monitor turbidity,
samples from FMD endemic countries to interna- agarose gels or real-time PCR machines. However,
tional reference laboratories. the sheer amount of product generated also raises
Recent developments in the area of field tests the spectre of cross-contamination (and generation
have also explored the use of new hardware plat- of false positive results) for these assays. In response
forms to allow PCR testing to be deployed into the to these concerns, RT-LAMP assay formats have
been developed to visualize dual-labelled LAMP availability of trained field personnel and a strong
amplicons using novel lateral flow devices ( James supporting laboratory infrastructure. In addition,
et al., 2010). Together with simple methods to international cooperation, transparency between
prepare template RNA (Waters et al., 2014), and different countries, sharing of epidemiological data
stabilized reagents (Howson et al., 2015), these and ownership of disease are also key factors in the
simple readouts for RT-LAMP may provide a solu- control of important transboundary disease such as
tion to fully realize the potential of molecular assays FMD.
for rapid (and sensitive) diagnosis of FMD in field
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from vaccine production plants (Beck and Strohm- and frequently of dubious quality and not always
aier, 1987). Also in this decade methods have been available in sufficient quantity. Also there was poor
developed to concentrate and purify inactivated management on their use and way of application.
antigens including polyethylene glycol precipita- This resulted into incomplete herd protection
tion and ultrafiltration techniques (Morrow et al., allowing ongoing clinical and subclinical disease.
1974). The availability of well controlled, high-quality
In the 1990s, considerable improvements vaccines in sufficient quantity was essential for the
were established in aspects related to the quality control of outbreaks and for the eradication cam-
control (QC) of the vaccine, either during the paigns implemented in most European and South
production process or in the final product, which American countries. In fact, vaccination policies put
were accompanied by greater regulatory demands into practice within a strategic eradication plan in
imposed mainly by the increased requirements for the 1980s and 1990s in Europe and South America,
biological products destined to livestock of human respectively and during recurrence of the disease in
consumption. Also, new biosafety policies were free regions were quite successful. In Europe, up to
implemented that led to major investments to adapt 1991–1992, FMD was controlled and eradicated by
the infrastructure of the companies and staff skills. systematic vaccination of the whole cattle popula-
Another important change has been the tion. Then, after Europe had been disease free for a
increasing incorporation of the concept known as number of years, vaccination was discontinued. In
reduction, refinement and replacement (3R). To the case of South America, eradication programs
this aim, the veterinary vaccine industry and regula- implemented in 1988 through the Hemispheric
tors have been gradually replacing in vivo methods Plan for Eradication of FMD (PHEFA) were quite
by in vitro approaches. In fact, substantial efforts successful and today no outbreaks were reported
have been made to replace challenge potency since the year 2012 (Sánchez Vázquez, 2015). This
tests by serological assays (Casas Olascoaga et al., Plan included intense vaccination campaigns of
1999). Additionally, in vitro analytical parameters cattle under supervision of the national veterinary
during the vaccine manufacturing process are being service and technical capacity for diagnosis and
defined, which if implemented under strict applica- vaccine control, as well as a good management
tion of a quality system typically based on good for the implementation of vaccination programs,
manufacturing practices (GMP), will ensure the with full commitment of the farmers. By applying
effectiveness of the vaccine without the need for in this strategy, proper herd immunity was achieved.
vivo methods. It is interesting to note that this success could be
While vaccination constituted an important attained despite the fact that in some regions vac-
support for the eradication campaigns, applying cinated cattle shared pastures with non-vaccinated
vaccination generated uncertainties which pro- sheep (Sutmoller et al., 2003).
moted sanctions on trade. Main justification for Such achievements contributed towards gen-
these regulations was related to the fact that vac- erating more reliable and visible outcomes of
cinated animals can become carriers and, in spite vaccination programs, opening the way towards
of the lack of proof, the idea that FMD carriers a new policy, known as ‘vaccination to live’. This
represent a considerable risk of transmission of the policy includes the acceptance of vaccination as
disease, constituted the basis to support that vacci- general component of response to incursions in
nation may hide silent viral circulation (Sutmoller free countries with smallest consequences to the
and Barteling, 2004; Sutmoller et al., 2003). This country/region and minimizing the dependence
misconception explains the massive stamping out on large-scale culling of animals to control the dis-
policy applied during the epidemic in the United ease. A major support for this strategy has been the
Kingdom in 2001 (Thompson et al., 2002). The development and application over the past years
widespread opposition to this mass culling of ani- of methods to distinguish infection regardless of
mals encouraged intense debates on the positive vaccination, known as non-capsid protein (NCP)
and negative aspects of vaccination. In fact, appre- tests or DIVA tests (Bergmann et al., 1993, 1998;
hensions regarding vaccination originated from the Blanco et al., 2002; Chung et al., 2002; De Diego et
time when vaccines were not adequately controlled al., 1997; Malirat et al., 1998; Neitzert et al., 1991;
Shen et al., 1999). The possibility of using vaccina- processes through timely measurements of critical
tion allied to serosurveys to confirm viral clearance process parameters (CPPs) which affect critical
opened the way to demonstrate the importance of quality attributes (CQAs) (FDA, 2004).
vaccination to stop the spread of the disease and The PAT initiative is basically linked to the
also to bring to an end the misconception that vac- concept of quality-by-design (QbD), which is a
cines are not entirely effective and that vaccination scientific, risk-based and proactive approach that
masks asymptomatic viral circulation (Bergmann emphasizes the understanding of a product and
et al., 2005). This transformation in the perception its process of manufacture as well as the in-process
on the benefits of vaccination as an appropriate tool monitoring. It is based on several pillars like the
for FMD prevention and control, led to important use of qualified scale down models, application of
changes in the World Organisation for Animal risk assessment methodologies and experimental
Health (OIE) Code, such as the incorporation of planning (Craven and Whelan, 2015; Rathore and
the category of FMD-free country/zone where vac- Winkle, 2009). This approach drives to the concept
cination is practised, as well as the reduction of the of ‘the process is the product’, enhancing the role of
waiting period to regain the free status when vacci- the in-process control. In this context, it is relevant
nation is applied to control an emergency outbreak to identify CPPs in the production process and
(OIE, 2015t). accordingly monitor them in a timely manner by
This chapter describes the basis of vaccine determining the extent to which any process varia-
manufacturing and its in process and final product tion can affect the quality of the product. Successful
quality control. Evolving concepts and modern implementation of QbD concepts requires coop-
technology to characterize vaccines while in eration among multidisciplinary teams from R&D,
process are discussed. Also addressed will be the to manufacturing and to quality management.
criteria for selection of vaccine strains, and the stor- This interaction is essential to support continuous
age of concentrated and purified antigens which are improvement through the introduction of innova-
essential to establish vaccine banks for emergency tive technologies.
vaccination. A detailed description of the manufacturing
process and the key elements for current vaccine
production has been published (Barteling, 2004).
Vaccine manufacturing
In overall, vaccines used to control FMD should Manufacturing facility
comply with the general and specific international Facilities for manufacturing FMD vaccines should
standards outlined in the OIE Manual of Diagnos- be designed in various zones, according to the level
tic Tests and Vaccines for Terrestrial Animals (OIE, of biosafety risk involved in the various steps of the
2015a), as well as with national and regional production process. Those procedures that do not
requirements. Vaccine manufacturing process involve the handling of live virus, such as serum
should be carried out under GMP standards, to processing, preparation of growth media, cell cul-
ensure consistency of production, a key factor for ture, as well as vaccine formulation and filling of the
achieving a high quality product that complies with final product can be performed in so called ‘clean
safety, purity, efficacy and potency. areas’ that is outside the biosafety facility. Inacti-
Due to the increased regulatory and biosafety vated antigens whose safety has not been verified
requirements put into practice during the last 15 and confirmed, need to be processed in a ‘buffer or
years, many manufacturing plants implemented quarantine’ zone under biocontainment.
considerable investments to adequate their All stages that require handling of live virus
infrastructure and staff skills. Facilities and manu- must be carried out in a Biosafety level 4 OIE
facturing processes are under continuous audits to (BSL4 OIE) facility. Differential airflow created
monitor compliance with updated regulations. As by negative pressure gradients, ensure that the air
part of the improvements, veterinary vaccine indus- moves from low risk areas (e.g.: locker rooms, cor-
try is gradually adopting a strategic concept known ridors) to higher risk areas (e.g. infection rooms,
as process analytical technology (PAT), to design, seed preparation laboratories, etc.). Independ-
analyse and control pharmaceutical manufacturing ent air handling systems are required to prevent
cross-contamination and minimize any outward addition, the presence of antibodies to FMD virus
flow of potentially contaminated air from inside. In and to BHK21 cells needs to be avoided, in order
addition, risk assessment studies, biocontainment to prevent interference in the infection process
principles, biosafety practices and procedures, as (Barteling, 2004). In this regard, the use of bovine
well as maintaining staff with specific training in sera from FMD free zones where vaccination is
FMD virus handling are also essential components not practised is quite convenient. If such source of
of a biosafety policy. FMD-antibody-free serum is not available, immu-
A biosafety system management ensures noglobulins should be removed with methods like
continuous improvement. There are periodic self- polyethylene glycol precipitation (Abaracón and
inspection and audit programs performed by a Giacometti, 1976; Barteling, 1976).
national expert committee associated with national The scaling up of the BHK21 cell suspensions
veterinary services and international boards. is performed through an in-line process involving
a series of increasing size linked vessels, according
Starting materials and cell culture to the number of passages defined in the outline
scale-up of production. This in-line approach prevents not
In common with the prerequisites for the produc- only contaminants entering the closed system but
tion of other inactivated viral vaccines, for FMD also any chance of virus escaping from it. Cells are
vaccines the starting materials of animal origin, grown in a nutrient medium supplemented with
such as master seed viruses and master cell seed adult bovine serum which is sequentially adjusted
stocks, should also have a clear record of its origin until the culture reaches an appropriate volume, at
and be subjected to strict QC to assure its identity which point the used medium is removed and the
and purity. As far as potential contamination with cells suspended in fresh medium without serum
bovine spongiform encephalopathy is concerned, (infection media) for subsequent infection.
all components of bovine origin should originate During these steps a series of important param-
from countries with negligible risk of transmissible eters related to growth and metabolic properties
spongiform encephalopathies (TSE) and measures of the cells are monitored, leading to a detailed
should be implemented to minimize the risk of con- understanding of the process, thereby establishing
tamination with TSE risk materials (OIE, 2015a). controlled conditions to reproduce the performance
Regarding the substrate for virus infection, the from batch to batch. In fact, control of biologi-
BHK21 cell line adapted to grow in suspension is cal properties (cell concentration and viability),
the most widely used due to its capacity to reach physiological parameters (substrate, metabolite and
high virus yield, thus, facilitating the scaling up of product concentration) and physicochemical fea-
virus production. Appropriate aliquots of BHK21 tures (pH, temperature, osmolarity) characterizes
cell line can be stored over liquid nitrogen following the cell line culture and enables tracking potential
the criteria of the cell bank system. An important deviations. The availability of process information
advantage of using large-scale suspension cultures in real time is of particular value to address the
is that this system promotes uniformity, allowing variability and unpredictability of cell cultures, as
the implementation of controls to monitor and it opens the possibility of implementing advanced
guarantee identity, genetic stability and absence control strategies capable of impacting directly on
of contaminants and adventitious agents. The CPPs and CQAs (Schwamb et al., 2015).
number of serial passages from the master cell stock Regarding the vaccine viruses to be included in
necessary to obtain working stocks and for the pro- vaccine formulations, in South America, national or
duction line should be strictly limited in order to regional reference FMD laboratories provide vac-
prevent changes in the characteristics of the BHK21 cine strains to manufacturers as aliquots of bovine
cells, which could have an effect on quality and tongue epithelia or tissue culture supernatant.
productivity. The growth media required for cell Thus, sanitary authorities have complete knowl-
cultivation is generally supplemented with adult edge of the antigenic and genetic characteristics of
bovine serum of qualified sources, which should the strains incorporated in the vaccines applied in
be previously treated to inactivate potential con- the vaccination programs. In other regions, vaccine
taminants (i.e. irradiation with gamma source). In strains are considered proprietary assets limiting
somehow reference laboratories from constantly quality that may influence the vaccine characteris-
matching the existing vaccine antigens against tics, its thermal stability and also have effects on the
evolving viruses (Lombard and Fussel, 2007). local reaction at the site of inoculation and the rare
The selected viruses are passaged in BHK21 appearance of anaphylactic reactions.
cell cultures in order to prepare the master and The concentrated and purified antigens can be
working seed banks. When emergent field viruses formulated into vaccines or stored at low tempera-
are selected to integrate the vaccines, they need tures for many years for future vaccine preparation
to be previously adapted to this cell line. Master when required (Forman and Garland, 2002) (see
and working seed stocks are controlled for purity, ‘Antigen and vaccine banks’, below).
identity, infectious titre, absence of pathogens and Purification steps become particularly relevant
adventitious viral, bacterial or fungal impurities and to support claims of purity regarding appropriate
mycoplasmas (OIE, 2015a). Viral passages from removing of NCPs (Bergmann et al., 2006; Espi-
master seed virus stock to production virus suspen- noza et al., 2004; OIE, 2015a) which are pertinent
sion should be kept at a minimum number in order especially when the vaccines are to be applied in
to avoid antigenic drift. This should be studied by FMD free regions where vaccination is practised.
the manufacturer and defined in their outline of In such cases, serosurveys based on the detection
production (OIE, 2015a). The criteria for vaccine of antibodies against NCPs are required to confirm
strain selection will be addressed later. absence of viral circulation in order to obtain or
maintain their OIE free status recognition. Con-
Antigen production sequently, antibodies induced by residual NCPs
As mentioned, the steps for antigen production in the vaccines would interfere with the correct
which implicates handling of live virus must be interpretation of the serosurveys (Bergmann et al.,
carried out in the BSL4 OIE area. The infection 2003a).
process of BHK21 suspension cultures consists
in adding the virus seed at a given multiplicity Adjuvants
of infection, and incubation with stirring. Each Once the antigen is concentrated and purified, and
vaccine strain displays an individual infection QC comply with overall requirements, the antigen
kinetics which influences the incubation time at is mixed with adjuvant, usually of oil or aqueous
which optimum virus concentration is achieved. nature, to obtain the final vaccine. Aluminium
Thereafter, the virus suspension is clarified, often hydroxide/saponin aqueous adjuvant proved to be
by chloroform treatment followed by centrifuga- efficacious to control and eradicate FMD in Chile
tion or filtration, to remove proteins and cell debris. (1981) and Europe (1991) (Sutmoller et al., 2003).
Subsequently, the antigen is chemically inactivated Oil-adjuvant vaccines are usually formulated
with BEI a first-order inactivant, added in two steps as water-in-oil single emulsion (W/O) using min-
(Bahnemann, 1990). After the first addition of BEI eral oils, and emulsifiers derived from mannitol,
the virus suspension is transferred to a new vessel sorbitan and purified oleic acid of vegetable origin
to eliminate the chance of ‘dead spots’ where virus (Aucouturier et al., 2001). Some manufacturers
may not have been exposed to the inactivant. add saponin as immunomodulator (Mattion et al.,
1998). These vaccines were a key factor in achiev-
Antigen concentration/purification ing FMD free areas in South America, a region with
The inactivated virus may be purified from residues one of the highest cattle populations in the world.
of the production process such as cell and media W/O vaccines were applied in Taiwan vaccination
components and viral NCPs, and concentrated programmes in pigs with good performance in con-
by procedures such as adsorption on aluminium trolling FMD emergency outbreaks (Chen et al.,
hydroxide gel (Abaracón et al., 1982), ultrafiltration 1999) and presently they are still in use.
(Barteling, 2002), polyethylene glycol precipita- Compared with aqueous vaccines, W/O vac-
tion (Panina and de Simone, 1973) and further by cines have important advantages such as longer
methods such as chromatography (Doel, 2003). duration of immunity, requiring less frequent revac-
The purification undertaken by the different cinations, and effectiveness in inducing protective
manufacturers can result in differences in vaccine responses not only in ruminants, but also in pigs
(Augé de Mello and Gomes, 1977; Hunter, 1996; laboratories. The following assays are the most
Rivenson et al., 1982b; Sutmoller et al., 2003). critical: virus titration, in vitro safety tests (inacti-
Double oil emulsion vaccines (water-in-oil-in vation kinetics, inactivation control), sterility test,
water emulsion) have similar attributes as W/O antigen yield determination, FMD virus strain
vaccines (Cox and Barnett, 2009; Selman et al., identity and purity test. Most of these in-process
2006). However, some reports documented that analytical methods have been previously reviewed
W/O vaccines induced higher antibody responses, (Barteling, 2004; Doel, 2003; OIE, 2015a). Briefly,
and longer duration of immunity in cattle and pigs we will illustrate the tests and procedures con-
(Smitsaart et al., 2004; J. Filippi unpublished). ducted routinely in South America, to assess the
Regarding syringibility, both double oil emulsion quality of FMD oil vaccines through the produc-
and W/O vaccines prepared with newly developed tion process.
adjuvants resulted in products of low viscosity Both the inactivation kinetics (Bahnemann,
and with appropriate efficacy, safety and stability 1990) during the inactivation process and the
(Aucouturier et al., 2001). inactivation control on several steps of the manu-
A high quality and a consistent source of adju- facturing process are the most significant QC of
vant, together with adequate technical support FMD vaccine and ensure the safety of the final
from the supplier and routine testing in the plant of product (Casas Olascoaga et al., 1990).
every batch, are essential to ensure the consistency In order to assess the inactivation kinetics, the
of the manufacturing process and the final product virus titre is monitored in various samples removed
(see ‘In-process and final product controls’, below). at regular time intervals during the inactivation
New adjuvants and immunomodulators that process. The decrease in virus titre plotted loga-
enhance efficacy, short and/or long-term immu- rithmically must be linear and extrapolation should
nity, and safety are being investigated in animal indicate that there is less than 1 infectious virus
models and in target species both with inactivated unit per 104 litres of liquid preparation at the end of
whole antigen and with antigen subunits (Borrego inactivation (OIE, 2015a).
et al., 2013; Cao, 2014; Dar et al., 2013; Park et al., Regarding inactivation control, intermediate
2014; Quattrocchi et al., 2013; Quattrocchi and products such as antigen suspension prior to the
Zamorano, 2007; Saravanan et al., 2015). It should concentration step, concentrated antigens, inacti-
be noted that when assessing the performance of vated virus before formulation, and final vaccine
adjuvants in clinical trials, care should be taken are tested for residual live virus, which involves
when comparing studies performed under different three blind serial passages in susceptible BHK21
conditions, since the results could be influenced cells (usually the same cells used in antigen produc-
by the vaccine strain and antigen payload used and tion) grown in roller bottles (OIE, 2015a). The
also by the conditions under which the challenge test should demonstrate absence of active virus in
studies were conducted (e.g. route of inoculation, suspensions equivalent to 200 doses of vaccine.
virus dose, time of exposure – in case of contact Cell sensitivity should be periodically tested and
challenge – and match of challenge) (Cox and absence of interference of the sample components
Barnett, 2009). If the adjuvants are assessed for with virus detection needs to be demonstrated.
immunogenicity on target species, the ELISA for Usually such controls take place once a year or
antibody quantification showed more reproducible whenever deemed necessary.
results than virus neutralization (VN) tests (Van The traditional method to determine the con-
Maanen and Terpstra, 1989). centration of inactivated entire virus particles
during the vaccine manufacturing process and in
In process and final product controls the final vaccine (antigen yield) is the 140S quan-
The production process is thoroughly monitored titative sucrose density gradient analysis (Barteling
by numerous QC tests. Usually, independent and Meloen, 1974). A method to quantify FMD
authorities audit the manufacturing process and virus particles based on separation of components
QC procedures at suitable intervals, and are also by size‑exclusion chromatography (SEC) with UV
allowed to collect samples of the intermediate and detection has a good correlation with the 140S test
final products to run QC tests in their government (Spitteler et al., 2011). Recently, a high performance
liquid chromatography (HPLC) on SEC materi- assessment of viral particle size of the FMD virus
als was successfully developed, and validated (Fig. antigen. This combined quantitative and qualitative
12.1) (Bellinzoni et al., 2012). The SEC/HPLC approach provides a robust control for the quality
system has several advantages when compared with parameters of the antigen in both intermediate
the 140S test, since it is more precise and easy to manufacturing materials and final vaccine products
automate. Furthermore, the tandem coupling of an (Fig. 12.2) (Bellinzoni et al., 2015).
in‑line dynamic light scattering (DLS) detector to These well-defined, precise and reproducible
the HPLC equipment affords a simultaneous, direct methods can also be applied to monitor stability of
Figure 12.1 FMD virus antigen measurement by SEC/HPLC of a recovered aqueous phase of a water-in-oil
FMD vaccine. U.V. absorbance trace at 254 nm. The virus content of the sample is derived from the area under
the curve for the virus peak (RT = 15.235 min), the intrinsic U.V. absorptivity of the FMD virus and the sample
injection volume (Bellinzoni et al., 2015).
Figure 12.2 Dynamic light scattering (DLS) particle size assessment of the virus peak in the HPLC analysis.
Dots represent the particle size readings of DLS detector in nm. The continuous line is the UV trace. Arrow
indicates a constant size reading of about 30 nm for the FMD virus peak (Bellinzoni et al., 2015).
stockpiles in antigen banks (see ‘Antigen and vac- means of supporting information on the purity
cine banks’, below). related to NCPs. Also, the critical levels of NCPs
The control of identity and purity related to that avoid interference will need to be established
the presence of FMD virus strains is performed for each manufacturing procedure. This is expected
through a trapping ELISA method using strain taking into account that the induction of antibodies
specific monoclonal antibodies (MAbs) on several is highly dependent on factors including adjuvants,
materials such as master and working seed viruses, immunostimulants, scheme of virus concentration/
intermediate and final concentrates, and inactivated purification, and inherent variations of individual
virus pools before formulation (Seki et al., 2009). In animal responses, among others (Bergmann et al.,
addition, the identity of the strains present in each 2006).
monovalent or polyvalent vaccine can be clearly The monitoring of physicochemical param-
established even in the aqueous phases extracted eters through droplet test, conductivity, viscosity,
from the final oil vaccines. These strain-specific particle size and stability at various temperatures
MAbs can also be used to analyse the integrity of (usually 4°C and 20°C) allows to verify that the
viral particles belonging to each one of the indi- emulsion process is appropriate (Aucouturier et
vidual strains included in the polyvalent vaccine, by al., 2001; Bahnemann and Mesquita, 1987). Vac-
assessing MAbs reactivity in fractions correspond- cine emulsions can be further characterized by
ing to the 140S peak of a sucrose density gradient measuring distribution of droplet sizes by laser dif-
(La Torre, 2010). fraction analysis (Mie scattering) both to guide the
Further evaluation of the FMD virus antigens emulsifying process and to evaluate final product
during the process can be assessed by polyacryla- formulation attributes (Fig. 12.3).
mide gel electrophoresis to detect and quantify the In South America, final product testing is
degree of potential VP1 cleavage (Doel and Collen, usually carried out both by the manufacturer
1983). and by independent authority on every batch,
Regarding control of purity related to NCPs, the before release, for sterility, purity, safety, potency
development of methods to detect NCPs in viral and emulsion quality following the OIE Manual
suspensions destined for vaccine production as an guidelines and established national legislation.
in-process control during vaccine manufacture have Safety tests of the final product are performed in
been of great value. However, it is unlikely that any vitro (inactivation control- previously described)
purification process can completely remove NCPs, and in vivo in laboratory animals (CFR, 2012a,b).
3B and 3D are known to be part of the virion struc- Additionally, the in vivo safety control in target
ture (Newman et al., 1994), and there is currently species is carried out along with potency test by
no method for predicting the immunogenicity of examination of general or local side-effects after
any protein remaining. Therefore, demonstrating vaccination.
that the vaccine does not induce antibodies against It should be noted that the testing of residual live
NCPs in immunized animals represents the only virus, quantification of virus particles and integrity
Figure 12.3 Distribution of droplet sizes of oil vaccine by laser diffraction analysis (Mie scattering).
assessment in the final oil vaccine requires the process and ongoing stability testing is required for
breaking of the emulsion with butanol or chloro- marketed products. Antigenic stability should be
form, in order to extract the aqueous phase (Casas proven through potency tests in cattle at different
Olascoaga et al., 1999). times after vaccine manufacturing and up to three
For vaccine registration efficacy control is usu- months after the expiration date (OIE, 2015a).
ally carried out by in vivo challenge test in cattle Oil vaccine has shown antigenic stability in cattle
[protection to podal generalization (PGP), in up to at least twenty-four months post elaboration
South America or, in other regions, protective dose (A.M.Espinoza, unpublished).
50% (PD50)], which is affected by high costs and Stability testing by the use of validated analyti-
ethical issues derived from the suffering of animals cal procedures that characterize the vaccine and its
(OIE, 2015a). main active principle (entire virus particle) should
In South America, routine batch-release testing be considered for FMD vaccines to reduce the in
is performed in cattle by quantifying antibodies at vivo testing and also to minimize variation derived
28–60 days post vaccination, and by extrapolat- from biological assays (Pfannenstiel and Inman,
ing antibody titre to transform in expectancy of 2012).
protection (EPP) by using predetermined tables of
correlation between serological titres and clinical Replacement of in vivo tests
protection (by PGP), established for each vaccine FMD vaccine would provide a good opportunity
strain based on a significant number of vaccination/ to adopt the 3R concept during potency testing. It
challenge tests (Allende et al., 2003; Maradei et al., is known that the integrity of FMD virus particles
2008). These procedures require the provision of is essential to confer protection by FMD vaccines
naive cattle subjected to rigorous sanitary controls (Doel and Chong, 1982). Several dose‑response
and selected according to specific requirements. It studies that relate virus particle concentration
is worth noting that in many countries, the official with vaccine potency are documented (Black et
control involving clinical trials and the serology al., 1986; Black et al., 1984; Pay and Hingley, 1987;
assessment is public, allowing the presence of Rweyemamu et al., 1984). Moreover, the qualita-
observers of vaccine manufacturers in each step of tive and quantitative determination of the antigen
vaccine control (SENASA, 2006). content in the final vaccines (and/or its production
For purity control, in terms of no induction of intermediates), along with a detailed characteriza-
NCP antibodies after vaccination, the OIE manual tion of the formulations resulting from a defined
guidelines are followed (OIE, 2015a). For registra- and validated manufacturing process provides a
tion, manufacturers should demonstrate that the much higher degree of confidence in the product
product do not induce antibodies against NCPs quality than bioassays. As the integrity of the virus
after two revaccinations with vaccines formulated particle is needed to assure vaccine potency, this
with the highest antigen concentration declared in parameter may be also studied as indicator of vac-
their product dossier. Every batch of vaccine is usu- cine stability along with other physical–chemical
ally controlled for purity in parallel with potency properties of the vaccine.
and safety test in cattle (MAPA, 2008; SENASA, Regulatory agencies and vaccine manufactur-
2006). ers with the support of the scientific community
Regarding duration of immunity (DOI), in should encourage the replacement of the use of
South America manufacturers are required to test animals, given the availability of alternative
show data supporting that their product is able to in vitro methods which demonstrated satisfactory
induce protective antibody levels in cattle for 180 precision and good correlation with in vivo results.
days after one dose and for 1 year after revaccina- Such in vitro alternatives were already implemented
tion (performed 6 months after the first dose). This with other inactivated vaccines (Newcastle disease,
requirement is based on documented field trials canine leptospiral, feline leukaemia vaccines)
with oil emulsion vaccines (Abaracón et al., 1980; (Stokes et al., 2012).
Rivenson et al., 1982a). The set-up of a robust quality system guaran-
Antigen stability data supporting expiration teeing a consistent production of vaccine batches
date are required during the vaccine registration through the effective use of in vitro analytical tools
would highly restrict the need of animal testing on method based on PD50, recommend the use of
the final product (De Mattia et al., 2011). higher potency vaccines (> 6 PD50) during emer-
gencies, particularly in naive populations, while
standard vaccines (up to 3 PD50) would mainly
Performance in target species be recommended for use during regular vaccina-
Taking into account that the outcome of vaccine tion programs. However, this distinction could be
performance may be highly dependent on the doubtful considering the low precision of the PD50
composition and quality of the vaccine, which may method (Goris et al., 2007). Since, according to
include a vast universe of versions, this section will dose–response studies, vaccines formulated with
go over mainly on the results of clinical trials related a higher antigen concentration result in higher
to the general attributes of oil based vaccines. This potency (EMEA, 2004), this division may be more
type of oil vaccine constitutes an important support precise by determining the antigen mass.
for many FMD control programs, including the Studies in pigs demonstrated that after a single
successful eradication programs in South America, vaccination with oil based vaccine, neutralizing
as well as for most preparedness and contingency antibodies were first detected between 4 and 7 days
plans. Nevertheless, these results should be taken after vaccination (Eble et al., 2004; Salt et al., 1998;
broadly, as they may differ with different experi- J. Filippi, unpublished). Peak antibody levels were
mental designs, including vaccination approaches. reported between 21 and 60 days post vaccination,
In overall, oil based vaccines are capable of which persisted for at least five to six months (Liao
stimulating a potent and long-lasting immunity et al., 2003; Selman et al., 2006; J. Filippi, unpub-
for at least 6 months after a single vaccination lished). Protection from FMD virus challenge was
(Augé de Mello et al., 1975). In the case of cattle, observed for as long as seven months post vaccina-
controlled studies showed that after a first dose, tion (Cox et al., 2003).
detection of antibodies occurred between 4 and 7 In sheep and goats, high titres of neutralizing
days post vaccination, reaching peak antibody titres antibodies were detected after a one-dose vaccina-
between 30 and 60 days. High levels of antibodies tion with single and double oil emulsion vaccines at
were maintained for at least 6 months post vacci- 15–60 days which persisted for more than 5 months
nation (Augé de Mello et al., 1975, 1980a; Cox et (Cox et al., 2003; Hunter, 1996; Patil et al., 2002b;
al., 2005; Hunter, 1996; Patil et al., 2002a), during Selman et al., 2006; Späth et al., 2008).
which time in vivo protection against challenge was Regarding early protection and effect on virus
also demonstrated (Abaracón et al., 1980; Cox et transmission, animals vaccinated with emergency
al., 2010). A second dose applied 6 months after vaccines and exposed to FMD virus between 2 and
the first vaccination, induced a significant rise of 7 days after vaccination were protected from chal-
antibody titres 4 days after revaccination (Augé lenge, preventing virus transmission in ruminants
de Mello and Gomes, 1977), reaching a peak 2 and pigs. These responses have been extensively
days later, which were maintained at high levels for reviewed (Cox and Barnett, 2009). In addition,
more than 12 months after the second vaccine dose challenge studies in cattle performed at seven
(Augé de Mello et al., 1980b). days post vaccination with standard vaccines dem-
These data support the vaccination intervals onstrated satisfactory protection. Moreover, no
applied for systematic vaccination programmes transmission was recorded either during the conva-
in South America. Moreover, the relatively rapid lescent period or during the persistent state (Duffy
response to a first dose of vaccine makes these et al., 2012; Golde et al., 2005; Quattrocchi et al.,
standard vaccines also valuable for vaccination 2014). Similar experiments in calves demonstrated
during an emergency, to control the spread of the that vaccination seven days before FMD virus inoc-
disease. Indeed, the application in South America ulation induced protection, blocked virus shedding
of this kind of standard vaccine was quite valuable and avoided environmental contamination by
to control the disease not only during systematic FMD virus secretions and excretions, thus prevent-
vaccination, but also for emergency application. ing new infections (Bravo de Rueda et al., 2015). It
It is interesting to note that some reports which should be noted that protection outcome may be
refer to the European Pharmacopoeia potency influenced by the route of inoculation, virus dose,
time of exposure (in case of contact challenge) and potency of the vaccine and with the titre of MDA
match of challenge (Cox and Barnett, 2009). present in the piglets at the time of vaccination.
Early studies showed that this suppression was
Influence of maternally derived complete in 1-, 2- and 4-week-old piglets and par-
antibodies in the immune response tial in 8-week-old piglets (Francis and Black, 1986).
Maternally derived antibodies (MDA) play a key Later studies demonstrated that MDA did not
role in the protective mechanism against infectious entirely suppress the antibody response induced by
diseases during the early life of animals; however, vaccination of 2-week-old piglets (Chenard et al.,
they may partially suppress the response to early 2008). Additionally, vaccination at 10 days of age
active immunity induced by vaccines. The quantity and revaccination at 60 days overcame the window
of MDA transferred to individuals varies greatly of susceptibility to FMD derived from the drop in
and depends on many factors, such as the degree of MDA, recording high levels of antibodies up to 6
immunity of the mother and the quantity and time months of age (Fondevila et al., 1996). Vaccination
of uptake by the neonate after birth (EMEA, 2007). at 8 weeks induced protective levels of neutralizing
Early studies with aqueous FMD vaccines antibodies that were maintained for the productive
documented failure to induce antibody responses 24-week period, time at which protection against
in calves with MDA (Graves, 1963; Nicholls et al., challenge was also registered (Liao et al., 2003).
1984). Conversely, later reports described the ben- Vaccination of 30-day-old lambs or younger in
efits of high quality oil vaccines for immunization the presence of MDA induced an antibody response
of calves born to regularly vaccinated cows in the that persisted at protective levels for at least up to
presence of high levels of MDA. In spite of certain 100–150 days of age (Cunliffe and Graves, 1970;
suppression of the post vaccinal response due to Späth et al., 2008).
MDA, vaccination of calves at early age prevented
the decline in antibody levels. In fact, vaccina-
tion of 20- to 40-day-old calves induced antibody Performance of FMD vaccine
responses, resulting in the detection 3 months later under field conditions
of significantly higher levels than those in non- The importance of routine mass vaccination
vaccinated calves (Spath et al., 1995). programs to reduce and eradicate FMD is unques-
Other studies indicated that vaccinating 30- to tionable but its success depends on a number of
90-day-old calves born to systematically vaccinated factors (Doel, 1999; McVey and Shi, 2010) related
cows resulted in high antibody levels for two to: the quality, purity, potency and safety of the vac-
months after first vaccination (Aznar et al., 2011). cine, and its match against the circulating viruses;
Secondary responses have been shown in boosted immunity and health status of the host; the age of
calves which received their first dose in the pres- calves at first vaccination; intrinsic animal aspects,
ence of high levels of MDA (Aznar et al., 2011; which may result in significant animal to animal
Smitsaart et al., 1996). variation; the operational aspects of the vaccination
Considering these results and from a practical campaign including proper records of livestock to
point of view, the recommendation would be to be vaccinated, vaccination regime and intervals
vaccinate all the calves irrespective of age along with between vaccination events; vaccine storage and
the whole herd, particularly in the case of extensive distribution. Finally, the application of the vaccine
breeding areas. Moreover, taking into account that should follow the recommendations of the manu-
a first dose is able to prime the immune system, facturer.
even in the presence of high levels of MDA, revac- In order to ensure the successful outcome of
cination of animals before moving to fattening areas these multifaceted vaccination campaigns, the
would be highly recommended in order to ensure a national services need to substantiate their effec-
satisfactory immunity. This approach is mandatory tiveness by demonstrating absence of viral activity
in several countries in South America. (Bergmann et al., 1996, 2003c) and population
In piglets born to FMD vaccinated sows, MDA immunity (León et al., 2014). In fact, these are part
had a suppressive effect on the early vaccination of the OIE requirements in order to apply for rec-
response, which may vary with the quality and ognition of the FMD free status where vaccination
is practised or to substantiate freedom from FMD The estimation of the proportion of protected
infection after emergency vaccination (OIE, 2015t; animals and protected farms in vaccinated popula-
Paton et al., 2006). tions is based on the evaluation of antibody levels
Of major relevance for the confirmation of against specific structural proteins measured by a
absence of viral activity has been the development single dilution liquid phase competitive blocking
and application over the past years of methods ELISA (slpELISA) (Robiolo et al., 2010). As men-
to distinguish infection regardless of vaccination tioned before, predetermined correlation tables
(NCP or DIVA tests). The diagnostic strategy is associating probability of protection (referred to
based on the detection of antibodies against viral as EPP) and slpELISA titres are used to classify the
NCPs (wrongly named non structural) that take samples as belonging to protected or unprotected
part during the replication process and that, in animals, which will depend on a cut-off titre value
principle, are induced only during infection and not specific for each FMD virus strain.
after immunization with conventional inactivated Annually, the Argentine National Animal Health
vaccines (Bergmann et al., 1993, 2000; Neitzert et and Agri-food Quality Service (SENASA) evaluates
al., 1991). Due to the conserved nature of these the effectiveness of the mass vaccination campaign
proteins, infection with any serotype of FMD virus through the analysis of serum samples obtained
can be detected with a single serological test. The using a stratified random design, usually collected
NCP test system consists of a screening ELISA before the next vaccination cycle (180 days after
method that detects antibodies against the poly- vaccination). Approximately 30,000 samples of
protein 3ABC, followed by confirmation of suspect 2250 herds were collected between 2005 and 2009,
or positive samples by an enzyme linked immuno- and analysed initially by the ELISA/EITB system to
electrotransfer blot assay that detects antibodies exclude animals with antibodies induced by even-
against five NCPs (Bergmann et al., 2000; Malirat tual infection, so that only the antibodies induced
et al., 1998). by vaccination were measured. Subsequently, the
In Argentina, using this system, the testing of negative ELISA/EITB samples were tested by
more than 23,000 serum samples/year of 12- to slpELISA to detect antibodies against structural
24-month-old cattle under vaccination programme, proteins of Argentina 2001, O1 Campos, and C3
during the period 2006–2011, revealed a very Indaial strains. The results showed that vaccination
low percentage of reactive animals (Smitsaart et coverage reached during consecutive vaccination
al., 2015), which fell within the specificity of the campaigns is consistent with a level of protection
ELISA 3ABC/EITB diagnostic test system applied necessary to sustain the status of FMD-free with
(Bergmann et al., 2003a). These data demonstrated vaccination (Cosentino et al., 2013).
and confirmed previous studies that the use of
conventional purified vaccines did not interfere Safety performance
with the interpretation of the results of serologi- During vaccine development, and in order to gain
cal surveys, thus supporting the evaluation of viral regulatory approval, safety tests should be carried
circulation in the zone or country (Bergmann et al., out in the target species in order to record atypical
1996, 2000, 2003c). local or systemic adverse reactions. For the case,
In terms of herd immunity, the goal is to confer such as in South America, where all vaccine batches
immunity on a sufficient number of animals and are independently controlled in cattle by the official
farms to prevent the transmission of the virus if it veterinary service, verification of absence of such
is introduced in the population to be protected. reactions is performed in the same animals used
Consequently, monitoring the levels of protection for the vaccine potency and purity control tests.
achieved after vaccination, in terms of both indi- Also, as part of the evaluation of the vaccination
vidual animals and farms, is essential to identify programme, monitoring of post-marketing adverse
eventual failures of the campaign. Although it would reactions should be considered by the veterinary
be difficult to be prescriptive about the level of vac- services.
cination coverage required, it is generally accepted The apparent high frequency of systemic adverse
that reaching 80% herd immunity would be quite effects after FMD vaccination seems more a myth
appropriate for field protection (OIE, 2015t). than a reality. This paradigm could be explained
considering that FMD vaccine is one of the most with injections that do not meet essential hygiene
widely used biologicals in the veterinary market, requirements and aseptic practices (Rebagliati et
so that in raw terms the frequency of adverse reac- al., 2005).
tions seems to be high (Black, 1979). However, the In pigs, DOE and W/O vaccines commercial-
rate of reported adverse reactions considering the ized mostly in Asian countries have reported rare
number of vaccinations applied worldwide is neg- and insignificant side effects. In controlled studies,
ligible (Casas Olascoaga et al., 1999). In the 1970s, high quality of DOE and W/O vaccines proved
the application of aqueous vaccines produced a low to be safe in pigs with little local reactivity after
incidence of postvaccinal allergic reactions of the primary intramuscularly vaccination (Barnett et al.,
immediate and delayed type (0.12 per 1000 doses 1996; Smitsaart et al., 2004).
administered in the Federal Republic of Germany
during 1967 to 1970) (Lorenz and Straub, 1973)
cited by (Black and Pay, 1975). More recently, the Vaccine matching
rate of anaphylactic reactions for W/O vaccines was The genetic/antigenic diversity of FMD viruses is
even lower (1 per 5,000,000 doses administered in well recognized (Alonso et al., 1987; Domingo et al.,
Argentina during 2007–2009) (A. Ham, unpub- 1980; Hyslop and Fagg, 1965; Pereira, 1976; Row-
lished). lands et al., 1983; Sobrino et al., 1983) and often
The vaccine components responsible of aller- generates debates on the selection and efficacy of
genic reactions remain uncertain. Nevertheless, it inactivated vaccines containing specific viral strains
is speculated that they may be associated with anti- (Doel, 2003; Mattion et al., 2004). Infection or
biotics, BHK21 cell components of non-purified vaccination with one of the seven serotypes known
vaccines (Casas Olascoaga et al., 1999), viral pro- worldwide (Arrowsmith, 1977; Pereira, 1981) does
teins (Black, 1979) and bovine serum (Black and not confer protection against the others. Within each
Francis, 1988). The unusually high anaphylactic serotype new antigenic variants arise continuously,
reactions reported in 1984–1985 were detected due to mutation and recombination (Arrowsmith,
mostly in multivaccinated dairy cattle and in black 1977; Brooksby, 1982; Domingo et al., 1990; Kitch-
or white breeds (Casas Olascoaga et al., 1999). ing, 2005; Knowles and Samuel, 2003; Mittal et al.,
Regarding local reactions at the site of inocula- 2005; Pereira, 1981) which may affect immune
tion, oil vaccines are expected to produce a non responses, and thus the ability of vaccines to effec-
pyogenic granuloma with presence of oil vacuolas tively protect against heterologous strains of the
(Rivenson et al., 1979). Important factors to mini- same serotype (Brooksby, 1982; Cartwright et al.,
mize these effects are related to the quality of the 1982; Mattion et al., 2004; Nagendrakumar et al.,
mineral oil and surfactants, the antigen purity and 2011; Upadhyaya et al., 2014).
concentration and the volume applied per dose The emergence of variants deserves special
(Aucouturier et al., 2001). attention under subneutralizing field conditions in
In large scale studies in which cattle were vac- which antigenically distinct viruses resistant to neu-
cinated by different routes with W/O vaccines, a tralization has been previously reported (Maradei
few animals presented small local reactions at the et al., 2014; Maradei et al., 2011; Tami et al., 2003).
site of inoculation shortly after vaccination, which Such situations usually occur in endemic settings,
disappeared in the following 3–4 months (Martino either as a consequence of poor vaccination cover-
et al., 2007; Rivenson et al., 1979). age, or deficient vaccine efficacy which could be
Regarding prevention of side effects, the use of due to a poor match of the vaccine strain with the
appropriate vaccination practices together with the field virus.
incorporation of new technological developments, Although the significance of FMD virus diver-
particularly concerning adjuvants and downstream sity to escape protective immunity is not quite clear,
processing, are essential to ensure maximal vaccine the relevance of having an appropriate strain in the
performance with minimum side effects (Aguilar vaccines can be inferred from field evidence. For
et al., 2012; Cai et al., 2014; Casas Olascoaga et al., example, during the epidemic waves of serotype
1999; Martinod, 1995). The loss of meat from the C in 1984 and up to 1986 that were registered in
neck region in slaughterhouses has been associated Argentina, the incorporation in the vaccine of the
C85 strain greatly reduced the number of outbreaks a single dose. This is pertinent not only for a rapid
(Bergmann et al., 1988). Another illustration can and effective response in case of an incursion in
be drawn during the 2000–2001 emergencies of the free regions, especially when large cattle popula-
Southern Cone of South America, which affected tions are involved, but also in endemic settings. In
large cattle populations in already free regions which the latter case often a relatively high percentage of
had stopped vaccination 14 months before. A rapid young livestock, the most susceptible animals, are
control of the disease was attained in Argentina being vaccinated for the first time. Additionally,
through inclusion of the field virus (strain Argen- there may be isolated pockets of virus with the
tina 2001) in the vaccines (Mattion et al., 2004), possibility of antigenic drift and selection of new
while Uruguay, having a considerable smaller cattle variants resistant to neutralization under condi-
population, managed to compensate the antigenic tions of insufficient protection conferred by the
differences between the vaccine strain and the field vaccine strain, reinforced by the rapid waning of
virus by revaccinating 30 days after first vaccination immunity which could occur when a vaccine strain
with the prototype vaccine strain A24 Cruzeiro matches poorly with the field virus (Elnekave et al.,
(Sutmoller et al., 2003). Within the Middle East, a 2013).
high rate of evolution in FMD virus and emergence In this context, one of the main challenges for
of new sublineages of serotype A viruses during the successful application of the ‘vaccination to live’
1996–2011 has required the regular development policy is to establish when to develop a new vaccine
of new vaccine strains typically every 5–10 years, strain. To this aim it is crucial to understand the
highlighting the inadequacy of the serotype A vac- unresolved, yet central issue, of the significance of
cines used in the region (Doel, 2003; Upadhyaya FMD virus variability for heterologous protection,
et al., 2014). Epidemic waves of serotype O were both pointing to routine prophylactic vaccination
registered in Ecuador up to the year 2011, despite as well as to emergency use, scenarios for which
having reported in the previous two years the great- matching requirements are not necessarily the
est historical availability of vaccines containing the same. A more broadly reactive strain may be more
O1 Campos vaccine strain for the vaccination pro- appropriate for systematic vaccination, whereas for
gram, and over 90% coverage during the 6-month emergency use a more precise match could be more
vaccination cycles. Characterization of the acting suitable.
viruses indicated poor protection of the vaccine This understanding is rather complex because
to the field viruses which could explain the rapid of the many variables that may be involved in
appearance of new strains and the co-circulation of intratypic (viruses of the same serotype) protec-
various variants (Maradei et al., 2011, 2014). tion. The different vaccine strains (i.e. serotype and
Another interesting experience which shows variants) and the various vaccine formulations, two
how a pre-existing vaccine strain can help to con- main determinants of vaccine efficacy, can alter
trol an extensive outbreak was the use of the O1 the outcome (Doel, 2003; Paton et al., 2005). In
Campos South American vaccine strain to aid fact, vaccine potency can, in part, compensate for
in the control of a devastating epidemic in pigs relevant differences between a field and a vaccine
of serotype O in Taiwan in 1997, after 68 years strain (Brehm et al., 2008; Doel, 2003). In addition,
of being FMD free without vaccination. Vaccine the final result of a vaccination process is certainly
matching tests revealed a good match between affected by previous vaccinations, previous infec-
the O1 Campos and the acting virus O Taiwan 97, tions with the same serotype or with a different
which were reflected in the effective control of the serotype. Effectiveness can vary depending on how
epidemic (Chen et al., 1999; Yang et al., 1999). successful vaccination programs are implemented
Such examples clearly indicate the significance and on how well a vaccinated animal responds to
of optimizing the vaccine program by matching the vaccine in terms of making protective anti-
the vaccine strain to the field viruses as closely as bodies. Cattle under certain conditions may have
possible and preferably including a strain covering weakened immune systems and may have a lower
a broad antigenic spectrum, so that vaccines are antibody response. Also, responses may differ for
capable of inducing high levels of protection after the different susceptible species.
Vaccine matching tests Yang et al., 2014), but the impact of the changes for
Considering that the effectiveness of a vaccine protection was mostly not determined.
depends largely on the suitability of the chosen When the results of the genetic/antigenic char-
vaccine strain, in terms of how closely it matches acterization of the field strains reveal the emergence
those viruses circulating in the field and also on its of new variants with potential changes in immuno-
capacity to protect a wide range of variants (Doel, genic sites, additional studies need to be oriented to
2003; Paton et al., 2005), permanent awareness of further assess antigenic/immunogenic relatedness
the strains prevailing in the field, their genetic dis- with the vaccine viruses (Maradei et al., 2011; Mat-
tribution/evolution and particularly, assessment of tion et al., 2004). These second group of assays can
the probable efficacy of the vaccine strain in use to be performed with representative samples of field
control the disease is of utmost importance. isolates which can be selected, for example from a
To this aim, samples collected from different genetic analysis and if pertinent, include samples
episodes, regions and at various times of exposure from different species and locations or at different
need to be characterized. A first group of assays are times.
generally oriented to determine the genetic and Humoral immunity is known to be the most
antigenic relatedness between the field samples influential factor in preventing FMD (Ahl et al.,
with the vaccine viruses available, through sequenc- 1990; Barnett et al., 2003b; Pay and Hingley, 1986;
ing of the VP1 or P1 region and monoclonal Pay and Parker, 1977; Saiz et al., 2002; Smitsaart
antibody profiling, respectively. Although genetic et al., 1998; Van Maanen and Terpstra, 1989). In
studies are relevant to detect quantitative/qualita- fact, many studies have shown that there is a strong
tive changes of the field viruses when compared to correlation between homologous protection from
other epidemiological relevant regional and extra- virus challenge and FMD virus antibody response
regional viruses and with the vaccine strain, it is not of primo-vaccinated cattle, either measured by VN
possible at present to predict the impact of specific test (Ahl et al., 1990; Pay and Hingley, 1986, 1992;
amino acid changes on antigenicity. Consequently, Sutmoller et al., 1984; Sutmoller and Vieira, 1980)
the genetic results should be taken with caution or liquid phase blocking sandwich ELISA (LPBE),
when extrapolating between nucleotide or deduced which, in principle, is considered to be at least as
amino acid differences and antigenic homology reliable and precise as the VN test (Ahl et al., 1990;
(Ludi and Rodriguez, 2013; Paton et al., 2005). Amadori et al., 1991; Goris et al., 2008b; Periolo et
In fact, it has been reported that quite distantly al., 1993; Van Maanen and Terpstra, 1989). Thus,
related isolates may have similar immunogenic in vitro assays are widely used as relatively good pre-
characteristics (Barnett et al., 2001; Hernandez dictors of homologous protection for vaccine batch
et al., 1992; Samuel et al., 1988). Conversely, very QC and to establish population immunity (León et
close sequence homology may mask large antigenic al., 2014; Maradei et al., 2008).
differences (Crowther, 1993; Maradei et al., 2014). Regarding heterologous protection, in vitro
Recent studies indicated that when structural infor- serological methods can also be used to quantify
mation on the location of the amino acid sequence antigenic/immunogenic differences and thereby,
in the virus is added to the sequence data, a better in principle, estimate the likely cross-protection
prediction of antigenic relationships has been between a vaccine strain and a field isolate. A first
obtained (Reeve et al., 2010). Particularly molecu- step is to establish antigenic/immunogenic relat-
lar dynamics may be an important asset for these edness by comparing the antibody titres of serum
predictions (V. Malirat, unpublished). samples collected from vaccinated animals against
Regarding antigenic relatedness, profiling both the vaccine strain and field virus (Brehm et al.,
ELISA using a panel of MAbs has been reported as 2008; Paton et al., 2005). Pools of reference anti-
a rapid and sensitive way to monitor the emergence sera are prepared against each vaccine strain to be
of antigenically different strains, assessing also rel- matched, so called bovine vaccinal serum (BVS).
evant differences with the vaccine virus regarding An indirect relationship value (r1 value) is calcu-
neutralization sites (Alonso et al., 1993; Mahapatra lated for each reference BVS (Alonso et al., 1987;
et al., 2008; Samuel et al., 1991; Seki et al., 2009; OIE, 2015a; Paton et al., 2005), which is the ratio of
the reciprocal heterologous (field virus) to homolo- The mentioned limitations can be largely over-
gous (vaccine strain) serum titres quantified by come by using distinctive and well-characterized
LPBE or VN assays (Brehm et al., 2008; Jangra et cell culture systems, particularly to perform the
al., 2005; Mattion et al., 2009; OIE, 2015a). For VN tests, as well as consistent and controlled viral
either VN or LPBE tests, BVS are derived from strains and BVS. In South America, a regional
cattle 21–30 days after inoculation with the vaccine network of National Laboratories selects and
to be matched. characterizes the official vaccine strains used in
There have been controversies regarding the the region. These strains are sent to the National
interpretation of r1 data in view of the many vari- Reference Laboratories for further distribution to
ables that can affect the outcome of r1 results. For vaccine manufacturers and for use in official batch
example, information on the vaccine strains is control. In addition BVS, rabbit and guinea pig anti-
not always available, since in some cases it is con- sera are produced with unified protocols.
sidered an important proprietary asset. Different In general, values of r1 > 0.4 or > 0.3 for LPBE
manufacturers may have different versions of the assay (Ferris and Donaldson, 1992) and VN test
same vaccine strain. Indeed, differences have been (Rweyemamu, 1984), respectively, have been
observed between the cross-reactivity of vaccine suggested as indicative of significant relatedness
strains with the same name (Paton et al., 2005). In between tested strains, and thus that the vaccine is
addition, there is no standardization concerning the likely to protect the field virus (OIE, 2015a).
preparation of BVS whose cross reactivity is to be While the mechanisms behind immune protec-
measured. Different BVS preparations against the tion are certainly more complex than just humoral
same vaccine strain can lead to markedly different r1 antibody responses, as already mentioned, a good
results (Kitching et al., 1988; Paton et al., 2005). In correlation has been shown between in vivo homol-
fact, BVS preparations can vary for example, in the ogous protection and antibody titres measured by
number of vaccinated animals used, the dose and VN or LPBE assays (Maradei et al., 2008; Pay and
purity of the antigen given, the adjuvant, time after Hingley, 1987; Van Maanen and Terpstra, 1989).
vaccination at which BVS was collected, the titre However, relatively few studies assessing the
range against the homologous virus, etc. (Mattion correlation between heterologous protection and
et al., 2009; Paton et al., 2005). It has been shown r1 values are available (Aggarwal and Barnett,
that results derived from pooled sera were more 2002; Barteling and Swam, 1996; Brehm et al.,
consistent than when calculated based on the mean 2008; FMD-DISCONVAC, 2013; Maradei et al.,
reciprocal serum titres (Brehm et al., 2008; Mattion 2011, 2014; Mattion et al., 2004; Nagendraku-
et al., 2009). The OIE prescribes the pooling of at mar et al., 2011) and in overall such studies gave
least five different serum samples (OIE, 2015a). In varying results. There are documented cases where
addition, low-titre sera are less suited for r1 value cross-protection was found in spite of low r1 values
determinations and, in principle, an appropriate (Brehm et al., 2008) and vice versa (Nagendraku-
reference BVS might be a medium to high VN or mar et al., 2011). These controversies are expected
LPBE titre serum (Mattion et al., 2009). considering the many variables that can affect r1
Another source of inconsistencies for r1 value determinations added to the inherent variability of
determination is that some tests have intrinsic vari- the r1 value, which were not taken into account in
ability so that various repetitions may be needed the mentioned studies. Moreover, it is known that
for full reliability (Rweyemamu, 1984). In addition, the degree of the titre that relates with protection
different laboratories may use different cell culture is not the same for different strains, so that using r1
systems and/or various protocols with results that determinations with fixed values to predict protec-
cannot be directly compared (Ahl et al., 1990; Bar- tion for the various serotypes would not necessarily
nett et al., 2003b). Consequently, results obtained be quite appropriate.
by different laboratories may not be equivalent. In Nevertheless, when r1 values were determined
fact, cross-validation studies on r1 value determina- with standardized reagents and methods, such as is
tion between laboratories (FMD-DISCONVAC, the case in South America, they were quite valuable
2013; OIE/FAO, 2012) showed huge variation to assess the immunological relatedness between
between the laboratories and techniques used. vaccine and field strains and results were quite in
line with the observed protection. Thus, r1 values suggested that for serotype A viruses, the VN assay
when determined under consistent conditions and seemed to be the preferred test for vaccine match-
with standardized methods are quite relevant and ing purposes (Brehm et al., 2008; Jangra et al.,
give an important input together with antigenic and 2005; Mattion et al., 2009; Paton et al., 2005). A
genetic characteristics on the need or not to per- more recent study also for serotype A, showed that
form studies to further determine cross-protection when compared with the VN test, not only were the
(Maradei et al., 2011, 2013, 2014; Mattion et al., LPBE results more capable to distinguish between
2004). the suitability of various vaccines to protect field
A more direct and comprehensive estimation of viruses, they were also more reproducible, as they
the protective capacity of a vaccine against a field are not influenced by variations in tissue culture
virus, widely used in South America, establishes the susceptibility (Tekleghiorghis et al., 2014a).
suitability of a vaccine strain based on the serum Significant variation has been reported by using
titres of samples derived from animals vaccinated VN test (Rweyemamu, 1984; Rweyemamu et al.,
with a particular vaccine, against the field viruses 1978), while lower variation was registered in the
(Alonso et al., 1987). This method, known as EPP, LPBE (Amadori et al., 1991; Van Maanen and
directly relates the serological titres obtained by Terpstra, 1989). In addition, LPBE can be used
VN or LPBE assays to the likelihood that cattle with inactivated antigens outside high security
would be protected against a challenge of 10,000 laboratories, the data can be obtained earlier and
infective doses after vaccination, based on predeter- they are friendlier for validation and automation.
mined correlation tables associating antibody titres In addition, LPBE can use smaller volumes of BVS
with homologous clinical protection against the which is usually available in limited amounts. A dis-
vaccine strain. The higher the titre, the better suited advantage of the LPBE method is that it is harder
the vaccine. Although the degree of the titre that to standardize the virus antigen concentration used
relates with protection is not the same for different in the test.
strains, in general titres over 2.1 and 1.6 for LPBE When the results of the genetic/antigenic/
and VN assays, respectively, can be considered as immunogenic characterization of the field strains
protective for most vaccine strains, which in overall reveal a loss in the effectiveness of the vaccine virus
results in an EPP close to 75% which, when a group to protect the field isolates, the final evaluation
of 16 vaccinated animals are used, is an indication needs to perform the gold standard test, which is the
that the vaccines will protect against the field strain most direct method to measure cross-protection. It
(Maradei et al., 2008; PANAFTOSA, 2001). This consists in vaccinating the relevant target species
correlation may not be strictly valid under heterolo- and then to challenge them by exposure to the virus
gous conditions. However, generally a curve for the isolate against which protection is assessed (OIE,
new emerging strain would not be available and up 2015a). This will take account of both potency
to date heterologous EPP estimations seem to be in and cross-reactivity. There are two direct methods
line with homologous in vivo protection (Maradei commonly in use, the PD50 which uses groups of
et al., 2011, 2013, 2014). at least five cattle inoculated with different dose
At present no conclusive results are available volumes of vaccines (Bolwell et al., 1992; Brehm et
regarding which test should be used to test cross- al., 2008; Goris et al., 2007; Willems et al., 2012)
protection through serology. Although neutralizing and the PGP assay which employs groups of 16
antibodies are considered to better correlate with animals inoculated with undiluted vaccines, the
protection, non-neutralizing antibodies may also latter approach being mainly used in South America
be protective (Dunn et al., 1998; McCullough et al., (Goris et al., 2008a; Maradei et al., 2008; Periolo et
1992). It has been suggested that other factors than al., 1993; Vianna Filho et al., 1993).
neutralizing antibodies might have an impact on Although these in vivo methods are closely linked
protection (Cox et al., 2003; Oh et al., 2006; Parida to the FMD in-field situation, observation of PD50
et al., 2006) and might be even more relevant in results indicated a lack of dose–response relation-
case of heterologous challenge (Brehm et al., 2008). ship in a large number of tests, which complicated
Immunogenic analysis of field isolates in rela- the interpretation of the results (Goris et al., 2007;
tion to vaccine strains, based on VN or LPBE tests, Pay and Parker, 1977; Stellmann et al., 1968). In
overall the PGP method, where the vaccine is used frozen antigens for rapid formulation into vaccines
undiluted, proved to be more reliable (Vianna Filho for use in case of an emergency (i.e. antigen banks).
et al., 1993) and a good indicator of the protection In South America, vaccines are formulated with
observed in the field (Goris et al., 2008a). selected strains harmonized for use in the region
In vivo cross protection approaches have many (Allende et al., 2003), choosing those of broad
disadvantages from the standpoint of animal wel- antigenic spectrum, high stability and good adapt-
fare and biosafety, since they require the use of live ability to replicate in cell culture at an industrial
FMD virus and appropriate biosecurity procedures scale. These strains are: O1 Campos, A24 Cruzeiro
and practices. In addition, they are expensive, the and most of the Southern Cone countries comprise
number of challenge viruses that can be assessed is also virus C3 Indaial. The variant Argentina 2001 is
limited and results can only be obtained after more also included in vaccine formulations in Argentina
than a month which is a major limitation consider- (Mattion et al., 2004). The production strains are
ing that the decision to vaccinate often will have to characterized and distributed by the official control
be made within days. Consequently, in most coun- laboratory at a national level. In other regions there
tries, official animal health services as well as the is variable harmonization of vaccine strain use at
OIE experts have supported the use of alternative national and regional level. Moreover, there is a lack
in vitro testing methods. In this context, the results of consistent information on availability and use of
of the indirect in vitro assays which in overall are in different vaccine strains mainly because of eventual
line with the ones observed in the in vivo challenge conflicts of interest.
test (Maradei et al., 2011; Mattion et al., 2009; The decision whether changes in the vaccine
Robiolo et al., 2010) merit further validation and strains are pertinent is rather multifaceted and is
acceptance. an issue which involves multiple focuses to work
As stated above, another important determinant together and effectively in order to attain a suc-
of the protection that a vaccine will afford is related cessful outcome. The input of the surveillance
to potency. A highly potent vaccine that stimulates system is very important. Epidemiologists need
a strong immune response may give greater protec- to have a rather effective surveillance system in
tion against a heterologous virus than a vaccine that order to capture any viral activity and particularly
stimulates a weaker immune response (Brehm et an FMD incursion. In addition, they should have
al., 2008). Furthermore, booster doses of vaccine a rather good understanding whether the vaccine
can increase potency and the subsequent breadth is being properly applied. Field veterinarians need
of antigenic cover provided by a given vaccine, to investigate outbreaks and collect proper and
although the onset of full protection may be delayed. representative samples. As mentioned before, the
It has been shown that there is no significant effect laboratory needs to determine the characteristics
of adjuvant on the range of the antibody response, of the variant involved and perform an algorithm
neither for mixing of antigens nor for the route of of tests to infer to what extent a vaccine may pro-
administration (subcutaneous versus intradermal) tect a new field strain for the various objectives
(Tekleghiorghis et al., 2014b). In contrast, the that the vaccine will be used. In addition, it should
breadth of the antibody response depends mainly assess which strains elicit antibodies capable of
on the vaccine strain. A 10-fold higher antigen dose neutralizing a broad range of field viruses which,
resulted in approximately four times higher titres in general, will be preferred as vaccine strains to
against all tested strains. those that induce responses of narrow specificity
(it should characterize and identify new vaccine
strains to cover major antigenic variants). Vaccine
Vaccine strain selection producers need to produce and supply the vac-
As mentioned before, the importance of having cine and, if pertinent, make available the vaccine
vaccine strains that are as immunogenic and cross strain as well as the corresponding panel of BVS
reactive as possible and with proven protection in order to be able to perform the assays to evalu-
against circulating viruses is a key issue not only for ate heterologous immunity. In addition, once the
systematic vaccination programs but also for the laboratory identified a candidate field virus for
incorporation to strategic FMD virus inactivated use as a vaccine strain, the manufacturer should
how a single bank assisted an emergency involving European Union (EU) established the EU vaccine
a herd of 60 million head of cattle and how it con- Bank in 1992 (Barnett et al., 2010; Lombard and
tributed to control the outbreaks in less than a year Fussel, 2007).
(Mattion et al., 2004). In South America, Argentina was the first coun-
Recently, the concept of an international diag- try to create a National A/V Bank through signing
nostic bank to cover the demand for diagnostic a contract with a vaccine manufacturer (Smitsaart
reagents that may be rapidly needed in case of an et al., 2002). Later this manufacturer was awarded
emergency has been considered. The idea, concep- as a supplier of the NAFMDVB and, additionally
tually similar to that of vaccine banks, proposes in 2011, the USDA/APHIS issued a permit for
that several countries combine their diagnostic importation and distribution of the Argentine local
resources which would then be available to mem- FMD vaccine (Bioaftogen®) to be used in case of
bers in the case of an emergency (EU, 2010). emergency in the United States (Roth and Spickler,
Valuable information to design the requirements 2014). In addition, and within the framework of the
for A/V banks and eventually of diagnostic stocks is South American Commission for the Fight against
provided by risk assessment studies of the country Foot-and-mouth disease (COSALFA), there has
or region considering herd at risk, epidemiological been progress towards the participative establish-
situation, and disease status of neighbouring areas. ment of a regional A/V bank (COSALFA, 2015).
To this aim, FMD A/V banks are expected to have a The OIE World Fund has worldwide experi-
dynamic and active cooperation between different ence in the management of vaccine banks and the
international, regional and national veterinary ser- delivery of vaccines including vaccines for FMD. In
vices, regulatory agencies, reference laboratories, this way, the OIE supports the provision of vaccines
vaccine manufacturers, academic institutes and free of charge to developing countries destined to
with other related stakeholders. Among other ben- vaccinate target animal populations at risk and also
efits, these collaborations allow becoming aware of to progressively achieve eradication (OIE, 2015s).
emerging strains that need to be incorporated in An important progress has been the creation of a
the banks, and facilitate the harmonization among network of FMD vaccine banks (International Vac-
national regulatory agencies, of the requirements cine Strategic Reserves Network -IVSRN-(Barnett
for vaccine authorization for use during FMD et al., 2010; Palma, 2004), which was endorsed by
emergency situations, which should be established the OIE and FAO. The goal of the Network is to
at least regionally. It should be noted that in the achieve, through mutually acceptable mechanisms,
context of the risk of bio-terrorism, disease control exchange of information and materials relevant to
authorities may consider it pertinent to restrict vaccine banks. Chief Veterinary Officers of QUADs
the release of information related to the storage (Australia, Canada, USA, New Zealand) are com-
of specific stockpiles of antigens and/or vaccines. mitted to support the network (Hickey, 2015).
Financial resources are needed to support A/V
banks and strategic programs must be maintained Ready-to-use vaccines
and renewed by solid and long-lasting mechanisms. Ready-to-use vaccines have the advantage of their
The OIE Manual (OIE, 2015a) describes the rapid availability for immediate use during its life-
international standards for A/V banks along with time. In this context, and as part of the contingency
the guidelines for storage and monitoring of con- plan, veterinary services may choose to use already
centrated antigen. formulated polyvalent or monovalent vaccines as
A/V Banks are being increasingly required. a primary barrier to prevent the spread of the dis-
Therefore, many countries have access to them ease. Contracts with companies that already have
either through a single contract with a vaccine an on-going production and sales in countries that
manufacturer or as a member country of an inter- practise vaccination are highly desirable (Roth and
national bank. Thus, they can have immediate Spickler, 2014).
availability of vaccines, regardless of world demand. The application of ready-to-use vaccines was
The North American Vaccine Bank (NAFM- an important adjunct for the initial control of the
DVB) was installed in 1982 for the USA, Canada emergency outbreaks which occurred in already
and Mexico (Forman and Garland, 2002) and the free regions of the Southern Cone of South America
during 2000–2001 (Mattion et al., 2004; Sutmoller vaccine strains available, assessed by in vitro match-
et al., 2003). ing tests between them and recent field isolates
(OIE/FAO, 2013). In any case, as mentioned, each
Storage of concentrated inactivated A/V reserve defines which vaccine strains should
antigen be stockpiled as concentrated inactivated antigen
As an alternative to ready-to-use vaccines, a more on the basis of risk assessment.
commonly adopted approach consists of stockpiles Master seed-stock collections constitute an
of concentrated inactivated antigens which, in gen- important support for the inactivated antigen
eral, can be stored over liquid nitrogen for a very banks. These collections allow for more alternatives
long time for subsequent formulation into vaccine of vaccine strains to be rapidly available for use in
(Barnett and Statham, 1998). This strategy has the event of new emerging viruses, without the
economic benefits and avoids continually replacing delays resulting from the adaptation of new isolates
vaccines that go beyond their shelf life. In addition, to tissue culture (EU, 2010). In addition, they pro-
it has the advantage of allowing the adjustment vide financial benefits.
of antigen payload according to the potency and Nevertheless, regulatory and legal issues need to
amount of doses required (Lombard and Fussel, be addressed to ensure that vaccines derived from
2007). master-seed stocks are rapidly authorized in order
Quantification of entire virus particles, anti- to allow its application in case of a FMD event.
gen integrity, safety and sterility controls must In the case of an emerging strain, with relevant
be performed before freezing and some of them immunogenic differences from existing vaccine
are repeated on thawed aliquots at regular time viruses, the development of a new vaccine virus
intervals for stability testing. For the approval of from a representative field isolate could be required
the concentrated antigens, a vaccine formulated at (see ‘Vaccine strain selection’). In this regard, the
a laboratory scale with a representative fraction of A/V bank should also have the technical capacity
the antigen batch must pass final product testing. It to adapt field strains to cell cultures, and generate
is recommended to prepare a new laboratory scale master seeds according to international standards.
vaccine to verify potency every 5 years after freezing. In order to avoid delays derived from the approval
The mentioned controls are quite relevant because of vaccines produced with the new antigens, the
in special situations when the vaccine is required preparation of the vaccine using an intermediate
within a short period of time, sanitary authorities antigen batch has been considered. This prepara-
may agree to release the vaccine provided that it is tion can be used for potency testing while the whole
manufactured with the same antigen content and antigen batch is being manufactured (EMEA,
formulae as the vaccine previously produced at a 2004). In this context, the use of in vitro methods
laboratory scale, prepared for the approval of the for in process and final vaccine control can save
antigen concentrate (EMEA, 2004). In order to considerable time and provide a good approxima-
gain extra confidence, it may be desirable to verify tion on vaccine effectiveness (see ‘In process and
the quality of the final finished vaccine through final product controls’).
rapid in vitro tests, such as those described in ‘In Regarding the approval of vaccines produced
process and final product control’. with these emergent variants, the EU has proposed
Regarding stability, oil vaccines formulated with that in order to facilitate the authorization process,
concentrated antigens show similar shelf life as the concept of ‘mock up authorization’, introduced
conventional vaccines prepared with freshly manu- by human flu vaccines could be applied. This
factured antigen (18–24 months) (Barnett et al., approach consists on the approval of the vaccine
2003a; A.M. Espinoza, unpublished). formulation with a specific strain along with limited
clinical data (EU, 2010). In case of an epidemic
Viral components of A/V banks with a different strain, regulators can assess data
As for the strains to be included in the banks, it has with the new strain, resulting in a brief authoriza-
been recommended to group the vaccine strains in tion process.
three levels of priority. The classification was mainly In summary, A/V banks should fulfil at least the
based on the antigenic spectrum covered by the following conditions:
• high operational capacity with procedures and veterinary services. This would be extremely valu-
facilities that meet regulatory requirements; able to provide an updated and reliable knowledge
• high capacity for storing frozen concentrated of the circulating strains and to ensure the quality of
antigens and continuous evaluation of its stabil- the vaccines in use and their performance.
ity; Post-marketing monitoring to confirm the
• high capacity to formulate vaccines as well as vaccine safety and effectiveness, together with
flexibility to deliver in short time the finished serosurveillance to assess herd immunity and viral
products produced either from stored antigens activity, is highly desirable to give public confi-
or from freshly manufactured antigens; dence.
• supply of tools for diagnosis and for post-
outbreak serosurveillance (viral activity and Acknowledgements
herd immunity assessment) We are grateful to M. Spitteler, R Bellinzoni, A.
• technical capacity to monitor FMD virus strains Suárez, F. Barroumeres and A.M. Espinoza for help-
and vaccine strain selection; ful suggestions on the manuscript.
• cooperation and collaboration with reference
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tropism of foot-and-mouth disease virus in vivo. J. Virol. tests of foot-and-mouth disease vaccines. J. Immunol.
77, 1219–1226. Methods 124, 111–119.
Tekleghiorghis, T., Weerdmeester, K., van Hemert- Vianna Filho, Y.L., Astudillo, V., Gomes, I., Fernández,
Kluitenberg, F., Moormann, R.J., and Dekker, A. G., Rozas, C.E., Ravison, J.A., and Alonso, A. (1993).
(2014a). Comparison of test methodologies for Potency control of foot-and-mouth disease vaccine
foot-and-mouth disease virus serotype A vaccine in cattle. Comparison of the 50% protective dose and
matching. Clin. Vaccine. Immunol. 21, 674–683. the protection against generalization. Vaccine 11,
Tekleghiorghis, T., Weerdmeester, K., van 1424–1428.
Hemert-Kluitenberg, F., Moormann, R.J., and Dekker, Waldmann, O., Köbe, K., and Pyl, G. (1937). Die
A. (2014b). No significant differences in the breadth of aktive Immunisierung des Rindes gegen Maul-und
the foot-and-mouth disease serotype A vaccine induced Klauenseuche mittels Formolimpfstoff. Zent. Bakt.
antibody responses in cattle, using different adjuvants, Parasit. Infekt. 138, 401–412.
mixed antigens and different routes of administration. Willems, T., Lefebvre, D.J., Goris, N., Diev, V.I.,
Vaccine 32, 5330–5336. Kremenchugskaya, S.R., Paul, G., Haas, B., and De
Telling, R.C., and Elsworth, R. (1965). Submerged culture Clercq, K. (2012). Characteristics of serology-based
of hamster kidney cells in a stainless steel vessel. vaccine potency models for foot-and-mouth disease
Biotechnol. Bioeng. 7, 417–434. virus. Vaccine 30, 5849–5855.
Thompson, D., Muriel, P., Russell, D., Osborne, P., Bromley, Yang, M., Xu, W., Goolia, M., and Zhang, Z. (2014).
A., Rowland, M., Creigh-Tyte, S., and Brown, C. (2002). Characterization of monoclonal antibodies against
Economic costs of the foot and mouth disease outbreak foot-and-mouth disease virus serotype O and application
in the United Kingdom in 2001. Rev. Off. Int. Epizoot. in identification of antigenic variation in relation to
21, 675–687. vaccine strain selection. Virology Journal 11, 136.
Upadhyaya, S., Ayelet, G., Paul, G., King, D.P., Paton, D.J., Yang, P.C., Chu, R.M., Chung, W.B., and Sung, H.T. (1999).
and Mahapatra, M. (2014). Genetic basis of antigenic Epidemiological characteristics and financial costs of the
variation in foot-and-mouth disease serotype A viruses 1997 foot-and-mouth disease epidemic in Taiwan. Vet.
from the Middle East. Vaccine 32, 631–638. Rec. 145, 731–734.
to the immune system. In this chapter we show Inactivated whole virus vaccines
how these challenges can be quite successfully
overcome by a new generation of peptide vaccines + Good immunogenicity
that integrate B- and T-cell epitopes –the former in
multimeric presentation– into a single molecular - Risk of virus escape
platform conferring solid protection against FMDV
infection. - Low stability
(cold chain required)
- Limited immunogenicity
Limitations of classical (annual re-vaccination required)
inactivated vaccines prompted - -
Site C
Site D
Site A
Integrin-Binding
Motif (RGD)
Figure 13.2 Representation of the B-cell antigenic sites on the capsid of FMDV type C. Sites are as in (Mateu
et al., 1994). On the right: a detail of the VP1 G-H loop in which the RGD triplet that mediates virus binding to
molecules of integrins – which act as cell receptors – is depicted.
to elicit neutralizing antibodies by various immuni- Diaz-San Segundo et al., 2014; Garcia-Briones et
zation approaches such as synthetic peptides (see al., 2004; Perez-Martin et al., 2014; Sanz-Parra et
below), recombinant proteins enclosing tandem al., 1999). Specific CD8+ T-cells – stimulated upon
copies of this sequence produced in different antigen processing and presentation of viral peptide
expression systems (reviewed in Brown, 1992; epitopes by antigen presenting cells (APCs), mainly
Domingo et al., 1990), or inclusion at permissive dendritic cells (DCs), in the context of MHC class
locations within heterologous virus-like particles I molecules (Fig. 13.3) – have been reported in
such as those of the hepatitis B core (Clarke et al., host animals following infection or vaccination
1987). with inactivated virus (Guzman et al., 2010; Saiz et
For these and other reasons FMDV is regarded al., 1992). Despite these findings, the role of CTL
as an attractive, challenging model to study the responses in FMDV protection remains poorly
requirements for B and T-cell stimulation result- understood.
ing in efficient protective responses against such a Relevant for mounting efficient T-cell responses
highly variable virus (see Chapter 7). Induction of is the interaction of FMDV with APCs, in particu-
specific immune response involves recognition of lar with DCs that are key controllers of immune
B-cell epitopes inducing specific antibody produc- defence development and responsiveness, provid-
tion following antigen processing and presentation ing essential antigen presentation to T-lymphocytes
in the context of MHC class II molecules ensures and antigen delivery to B-lymphocytes (Fig. 13.3).
stimulation of helper (Th)-lymphocytes to produce Although FMDV can interact with APCs and
growth and differentiation factors necessary for DCs –in an enhanced manner when complexed
development of the immune response (Fig. 13.3). to specific antibodies– producing a sort of abor-
Protection against FMDV in natural hosts has been tive infection (see Chapter 10), a clear picture is
achieved upon immunization with different vac- not available on whether and how this interaction
cine strategies that did not elicit consistent levels influences the outcome of the protective response
of neutralizing antibodies (Borrego et al., 2006; against this virus.
IFN IL-2
IFN
MIIC: TH lymphocyte
Live FMDV MHC class II –containing Co-stimulation
or inactivated late endosomal-like structure CD4
TCR
FMDV vaccine complex
Figure 13.3 Overview of main events on the interaction of antigen-presenting cells (APCs) with B and
T-lymphocytes, leading to antibody production. Adapted from McCullough and Sobrino (2004).
S STRENGTHS W WEAKNESSES
• safety: absence of infectious material è no risk • B epitope definition not always simple: linear
of reversion to virulence, genetic integration or vs. conformational (continuous/discontinuous)
recombination • T epitopes often insufficiently specified
• total differentiation between infected and • generally low immunogenicity of peptides è
vaccinated animals (DIVA capability) adjuvants, multimerization required
• fast, affordable large scale production by
robust synthetic methods
• chemical stability (lyophilized form)
• simple handling, storage and transport (no cold
chain required)
O OPPORTUNITIES T THREATS
• flexibility: irrelevant or potentially troublesome • vaccine trial policies increasingly restrictive in
sequences can be excluded some parts of the world
• easy structural fine-tuning to modulate • vaccine trials (prototype validation) are slow
immunogenicity, stability, solubility processes
• adaptability: fast response to new strains,
emergency situations
• multivalency: various epitopes combined on a
single molecular platform
• registered as a pharmaceutical (simpler than a
biological)
Figure 13.4 A SWOT (strengths, weaknesses, opportunities, threats) analysis of peptide vaccines against
FMDV at the time of this writing.
showed a limited correlation with specific serum Regenmortel et al., 1998). A single inoculation of
neutralizing activity and a higher correlation with retro-inverso peptide corresponding to FMDV
the induction of T-cell responses. Consistently, residues VP1[141–159] induced longer-lasting
associations were observed between certain DRB and higher antibody titres in immunized animals
alleles (DRB3.2 *1, 3 and 7) and increased levels than the corresponding l-peptide. The antibodies
of protection and the presence of others (DRB3.2 cross-reacted strongly with virus particles and with
*12 and 18) and lack of protection, supporting L-peptides and conferred substantial protection
that the polymorphisms in genes of bovine class I in guinea pigs challenged with the cognate virus
and class II MHC affected recognition of the indi- (Briand et al., 1997).
vidual epitopes, resulting in the animal to animal
variation observed in both humoral and cellular
immune responses (Garcia-Briones et al., 2000). Characteristics of the regions
These results reinforced the need of adequate T-cell mimicked by FMDV peptides:
activation for efficient peptide-based protection. continuous and discontinuous
The study also underlined the limitation of vaccines B-cell epitopes
based on a single linear peptide in achieving protec- Whereas most peptide-based vaccine candidates so
tion against a highly variable RNA virus as FMDV, far reported, for either FMDV or other targets, are
as escape mutants with amino acid substitutions at designed to reproduce continuous B-cell epitopes,
the GH loop could be isolated from non-protected i.e. uninterrupted stretches of a protein sequence
challenged cattle (Taboga et al., 1997; Tami et al., recognized by antibodies, there is ample evidence
2003). that most antibody binding sites are discontinu-
An additional challenge, that of substantial ous, i.e. made up from residues distant in sequence
antigenic variability, particularly at the main B-cell –often located in different subunits of a structurally
site located on the GH loop of VP1, has also to be complex antigen, e.g. a viral particle– but brought
reckoned with. In an effort to address the limita- spatially close by the folding of the native structure.
tion posed by this problem in the design of peptide Reproduction of such discontinuous –also named
vaccines against HIV, the group of Tartar produced conformational– epitopes by chemical means is a
combinatorial peptide libraries, named mixotopes, largely unaddressed task in molecular engineering.
where the different antigenic variants were propor- Much of its challenge lies in, first, defining which
tionally represented, with the assumption that an structural components of the antigen – i.e. residues
immunogen with a high level of sequence variation or parts thereof – are relevant for antibody interac-
could improve the broadness of antibody response tion and, second, spatially arranging them so that
(Gras-Masse et al., 1992). In the case of FMDV, a native-like orientation of the critically interacting
mixotopes corresponding to the GH loop and con- moieties can be accomplished. The daunting com-
taining either ca. 103 or ca. 105 peptides each, were plexity of this task explains the low success rate of
used to immunized guinea pigs, but limited immu- such efforts, judged by the rather few examples in
nogenicity was observed (Oliveira et al., 2005). the literature (Chamorro et al., 2009; Eichler, 2004;
Induction of strong immune responses requires Franke et al., 2004; Howie et al., 1998, 1999). In
a sustained presentation of antigen in a stimula- the specific field of FMDV, only two efforts in this
tory context; hence an important goal of peptide direction have been reported. A first study (Borràs
vaccine design is overcoming degradation by extra- et al., 1999) aimed to mimic the FMDV discontinu-
cellular proteases. This goal has been addressed by ous antigenic site D, defined by mutational analysis
peptides made up of non-native, protease-resistant (Mateu et al., 1998) as consisting of five residues
D-amino acids. Peptides made with D-enantiomers situated on three external loops of capsid proteins
will adopt the mirror image conformation of the VP1 (T193), VP2 (S72, N74, H79) and VP3 (E58)
canonic, l-amino acid structures. To overcome (see Chapter 4). The three loops were spliced
this problem, the D-peptides are assembled in together by means of two interconnecting units (a
the reverse order from the natural sequence, disulfide bridge and a poly-proline segment) into a
generating ‘retro-inverso’ peptides that adopt con- single peptide construction (Fig. 13.5) that was able
formations resembling the natural antigen (Van to elicit modest levels of neutralizing antibodies.
Figure 13.5 (A) Front view of antigenic discontinuous site D of FMDV, showing the five residues defining the site
(displayed in CPK) and adjoining regions. Surface most-exposed fragments are coloured white. Also included is
VP2[132–137] loop, with a highly exposed Arg (also in CPK) presumed to interact with antibodies. (B) Elements
defined in (A) are visualized as defining a cycle (purple arrow). (C) Incorporation of viral surface elements into a
26 residue cyclic peptide (Villen et al., 2006) reproducing VP1 (193–195, TGD), VP3 (58–60, ENV; Y63 and K84)
and VP2 (72–80, PSQNFGHMHK and 133–137, SEKDR, reorganized from native SDREK for structural reasons).
(D–F) Cross-sectional view of site D showing the five key residues and other elements included in a previous,
first-generation replica of the site (Borràs et al., 1999). Antigenically relevant fragments were connected via a
poly-proline module and a disulfide bond (shown in white), all involving residues at the inner side of the capsid.
In a second, more successful attempt (Villen et al., challenge. No further efforts to replicate this dis-
2006), the approach taken was to generate a mimic continuous epitope have been reported, to the best
of the contact surface, by structure-guided rational of our knowledge. However, one can hypothesize,
assembly of the native fragments adjoining the five in view of the success of vaccines based on multi-
key residues defining site D. The result of this design meric (branched) presentation of the GH loop
was a medium-size (24-residue) quasi-circular continuous epitope (see below), that combining
array that could be rather accurately mimicked by (i.e. non-covalent admixture) the dendrimeric
a disulfide-linked cyclic peptide. That a reasonably construct with the surface contact mimic of dis-
accurate replication of site D was achieved is shown continuous site D might afford a vaccine of even
by the fact that the cyclic peptide was able to bind superior performance. This hypothesis remains for
FMDV-derived monoclonal antibodies, and that the moment untested.
amino acid residues assumed – in above-mentioned
mutational studies – to be involved in antibody
recognition were confirmed by NMR to be spa- Characteristics of the regions
tially close to antibody paratopes. In addition, the mimicked by the FMDV
cyclic peptide construct was successfully evaluated peptides: T-cell epitopes
as a vaccine candidate on a guinea pig model. The For the design of novel subunit vaccines, particu-
cyclic peptide, unconjugated to a carrier protein, larly peptide vaccines, a detailed knowledge of
generated an antibody response comparable to that the pathogen (viral) epitopes eliciting cytolytic
of virus-immunized animals in terms of FMDV (CD8+) and helper (CD4+) T-cells relevant for
neutralization in vitro, but unfortunately did not protection in the natural hosts is crucial. As men-
elicit full protection of the animals against FMDV tioned above, T-cells recognize specific antigens
in the context of MHC molecules, and the genes activated, depending on the co-stimulatory signals
coding for these molecules are highly polymorphic expressed (Weber et al., 2009). Unfortunately,
within an outbred population of a particular species incorporation of the nucleotide sequence corre-
(see Chapter 10). Therefore, the recognition of sponding to the VP4[20–34] epitope into a DNA
T-cell epitopes depends on the MHC alleles pre- vaccine turned out to be detrimental, promoting
sent in a particular animal, a phenomenon termed exacerbation of clinical signs after FMDV challenge
MHC restriction. (Ganges et al., 2011), and a fusion protein corre-
The strength of T-cell epitope binding to MHC sponding to amino acid positions VP1[133–158]
molecules is one of the critical determinants of and VP4[20–34] did not afford complete protec-
immunogenicity (Lazarski et al., 2005). Peptides tion in the guinea pig (Zhang et al., 2002).
binding with higher affinity to MHC molecules Search for FMDV heterotypic T-cell epitopes
are more likely to be displayed on the cell surface has also been focused on non-structural proteins
where they can be recognized by their correspond- (NSP), since most of these proteins exhibit low
ing T-cell receptors (TCRs) (Weber et al., 2009). sequence variability among viral isolates and sero-
Among T-cell epitopes with high affinity (immu- types, and therefore are expected to be recognized
nodominant) and recognized by different MHC in the context of heterotypic FMDV infections. In
allelic forms (promiscuous), those conserved addition, expanding T-cell epitope search to NSPs
among different viral strains (heterotypic) are pre- allows broadening the repertoire of viral epitopes
ferred for vaccine application, as they can be widely recognized by host immune defences. The T-cell
recognized by host T-cells and induce heterotypic antigenicity of FMDV NSP was first analysed using
responses. These features are particularly relevant recombinant fusion proteins expressed in E. coli,
for FMDV, a virus that exhibits high sequence vari- leading to the identification of 3AB and 3C poly-
ability (see Chapter 7) and affect different domestic peptides as main inducers of specific and consistent
and wild species (see Chapters 8 and 9). lymphoproliferative responses in all the FMDV-
infected pigs analysed (Blanco et al., 2001). Fine
T helper epitopes epitope mapping of 3AB and 3C by means of over-
Identification and characterization of FMDV- lapping peptides allowed the identification of 11
specific T-cell epitopes was initially addressed by heterotypically recognized epitopes. One of them,
using outbred animals and overlapping peptides 3A[21–35], was shown to induce lymphoprolif-
spanning VP1 capsid protein. These studies allowed eration and FMDV neutralizing antibodies in vitro
identification of T-helper epitopes recognized by when displayed in linear tandem with the VP1 GH
cattle (Collen et al., 1991) and pig (Rodriguez et loop peptide. The relevance and potential of this
al., 1994) lymphocytes. A disadvantage for vaccine 3A[21–35] T-cell epitope as a key element in novel,
improvement of the T-cell epitopes identified in fully protective peptide vaccines, has been subse-
these experiments was the high sequence variability quently confirmed (Blanco et al., 2016; Cubillos et
of VP1 among different FMDV isolates and sero- al., 2008, 2012; Monso et al., 2013) and is described
types (Carrillo et al., 2005). Interestingly, in VP4, in more detail in the next section.
another structural protein highly conserved among The presence of T-cell epitopes within NSP 3D,
FMDV serotypes, MHC-promiscuous T-cell the viral RNA polymerase, has been demonstrated
epitopes were identified for cattle (Van Lierop et in both cattle (Collen et al., 1998) and swine
al., 1995); one of them, VP4[20–34], was shown to (Foster et al., 1998). Indeed, the contribution of
bind at least four different cattle MHC haplotypes this protein to the protective immune response
and to be presented by MHC class II DQ molecules to FMDV was confirmed by the observation that
(Gerner et al., 2009). Furthermore, VP4[20–34] pigs immunized with recombinant vaccinia virus
was also recognized as a porcine T-cell epitope in expressing 3D protein were partially protected
FMDV-stimulated lymphocytes from vaccinated against viral challenge (Garcia-Briones et al., 2004).
outbred pigs, providing further evidence of the pro- In this study, 3D overlapping peptides permitted
miscuous nature of this region (Blanco et al., 2000). identification of different T-cell epitopes that were
Upon T-cell epitope recognition, effector (inflam- efficiently recognized by pig lymphocytes, upon
matory) or regulatory (suppressive) T-cells can be both primary and secondary (heterotypic) FMDV
infection. Interestingly, one of these peptides, in pigs (Wang et al., 2002). However, this vaccine
3D[346–370] was also shown to be recognized by failed to afford any protection in cattle (Rodriguez
inbred pigs (Gerner et al., 2006). In conclusion, a et al., 2003).
fairly broad set of CD4+ T-cell epitopes identified
in cattle and swine is available as candidates for
subunit and peptide vaccines improvement. Current perspectives for
improved FDMV peptide
CTL epitopes vaccines
Characterization of CTL epitopes in outbred popu- Despite the caveats already mentioned earlier in
lations is complicated by the limited availability of this chapter (Fig. 13.4), peptide-based vaccines are
inbred animals among FMDV natural hosts, and arguably one of the most advantageous alternatives
has therefore been mostly done in inbred mice. to classical vaccines (i.e. dead or attenuated virus)
However, the biological significance for natural host against FMDV, not only for the complete safety
species of murine-defined CTL epitopes cannot achieved by the exclusion of the infectious agent but
be taken for granted and requires experimental also by additional advantages such as their DIVA
confirmation. In addition, the cytopathic nature (differentiation between infected and vaccinated
of FMDV has hindered the use of FMDV-infected animals) capability, or their efficient production by
cells as targets. All these limitations underscore the chemical synthesis that facilitates characterization
scarce reports on FMDV CTL epitopes (Barfoed as pharmaceuticals, or their uncomplicated, cold
et al., 2006). So far it has been described a solely chain-free transport and storage (Brun et al., 2011;
BoLA class I-restricted CD8+ T-cell epitope in the Purcell et al., 2007). While these advantages are par-
structural protein VP1 (Guzman et al., 2010). tially offset by the recognized low immunogenicity
In an attempt to circumvent the above limita- of peptides, this limitation can be addressed in
tions, predictive approaches have been recently various ways (Rueckert and Guzman, 2012),
described to identify FMDV peptides presented particularly by multimerization, whereby several
by swine (SLA) and bovine (BoLa) MHC class I epitopes can be displayed on a single scaffold. Dif-
molecules. Based on in silico predictions and in ferent varieties of scaffolds have been described,
vitro verifications, FMDV peptides were found to including template-assisted synthetic protein
bind SLA-2*0401 and SLA-1*0401 MHC alleles (TASP) platforms (Tuchscherer and Mutter, 1995),
commonly expressed in commercial pig breeds gold nanoparticles (Chen et al., 2010), aromatic
(Pedersen et al., 2013). Analyses of antigen- hydrocarbons (Chang et al., 2012), or four-armed
specific, MHC class I-restricted T-cells using star polymers (Liu et al., 2013), in addition to the
MHC tetramers made with the peptides identified, classic lysine tree-based, multiple antigenic peptide
showed that at least one of them reacts with FMDV- (MAP) design pioneered by Tam (Tam, 1988; Tam
specific T-cells (Patch et al., 2011). A similar in silico and Spetzler, 1997). Many of these approaches rely
approach predicted the binding of several peptides on the considerable advances made in chemoselec-
from FMDV structural proteins to common Bola tive ligation since the 1990s (Lu et al., 1991; Rose et
class I molecules (Pandya et al., 2015). The CD8+ al., 1995; Tam and Spetzler, 1995).
response to such peptides has not yet been evalu- In 2008 solid protection of swine against C
ated in vaccinated/infected cattle. serotype FMDV challenge was described by means
Non FMDV-specific immunodominant T-cell of a vaccine prototype consisting of four copies of
epitopes haven also been included in the design the continuous GH loop B-cell epitope tethered
of peptide vaccines to provide T-cell help (Francis through thioether bonds to a single copy of the het-
et al., 1987b). In this approach, activated T-cells erotypic 3A[21–35] T-cell epitope elongated at its
would not be re-stimulated upon subsequent virus N-terminus by a branching core of Lys residues (Fig.
encounter, which can limit the potential stimula- 13.6). This dendrimer construct, hereafter named
tory effect. Using this strategy, a linear peptide B4T, generated high titres of FMDV-neutralizing
based on the VP1 GH sequence combined with antibodies in both pigs and outbred mice (Swiss
that of a combinatorial library used as a source of CD1 strain), activated T-cells and induced IFN-γ
T-cell epitopes was reported to confer protection release and induced full protection in swine upon
B epitope
B epitope Lys
Lys-Lys-Lys- T epitope
B epitope Lys
B epitope (S)
O
tetravalent dendrimer, B4T(thi) = CH 2
B epitope
Lys-Lys-Lys- T epitope
B epitope
(S)
O
B2T(thi) = CH 2
bivalent dendrimers O O
B2T(mal) (S) N
=
O
Figure 13.6 General structure of FMDV vaccines combining one T-cell and four/two B-cell epitopes in a branched
arrangement (Blanco et al., 2016). In all cases, the T epitope reproduces residues 21–35 (AAIEFFEGMVHDSIK)
of non-structural protein 3A, and the B epitope (acetyl-PVTNVRGDLQVLAQKAARTC) reproduces the G-H loop
of VP1, residues 140–160, serotype O-UKG 11/01. The (S) corresponds to the thiol group of the C-terminal Cys
residue. All peptides are in C-terminal carboxamide form.
infection with homologous FMDV. Most remark- epitope, and showing that bivalent (B2T; Fig. 13.6)
ably, the B4T candidate vaccine elicited high titres constructs not only improve over co-linear display
of mucosal IgAs, similar to those achieved upon of B- and T-cell epitopes but also outdo tetravalent
FMDV infection, which prevented contact controls ones in both neutralizing antibody (humoral)
to become infected when vaccinated animals were and IFN-γ (cellular) responses, particularly when
challenged with the live virus (Blanco et al., 2013; maleimide linkages are used to interconnect the
Cubillos et al., 2008). Interestingly, multimeric B- and T-cell epitopes. More recently, this unex-
display of B-cell epitopes in the dendrimer was pected superior performance of the bivalent (B2T)
necessary for solid protection, since linear pep- over the tetravalent (B4T) peptide vaccine candi-
tides including the GH loop B-cell epitope and dates has been conclusively verified in the swine, an
the 3A[21–35] T-cell epitope did not afford full FMDV natural host (Blanco et al., 2016). Similar
protection in pigs (Cubillos et al., 2012). Partial to the earlier results in mice, the bivalent maleim-
protection with linear peptides containing B and ide–linked construct [B2T(mal), Fig. 13.6] has an
T-cell epitopes has been also recently reported for edge over an also bivalent but thioether-linked ver-
cattle (Zhang et al., 2015). sion [B2T(thi); Fig. 13.6], highlighting the impact
This promising lead has been since pursued with of minute structural details on complex biological
dendrimers displaying the GH loop B-cell epitope events such as vaccination with peptides. A fur-
of more epidemiologically consequential O rather ther unexpected bonus of the maleimide–based
than C serotypes. In studies with Swiss CD1 mice construct is its extremely expedient production,
(Monso et al., 2013), the conventional view that due to the highly efficient thiol-ene conjugation
higher B epitope multiplicity enhances immuno- chemistry, in contrast with thioether and other liga-
genicity was challenged by comparing the response tion chemistries. All in all, the B2T(mal) emerges
of constructs with two or four copies of the B as an efficient FMDV immunogen in swine,
eliciting a robust immune response in both serum definition for register as pharmaceuticals. While
and mucosae, with full (100%, 6/6 animals) protec- issues such as dosage, cost efficiency, immunization
tion and with additional features (i.e. production) schedule or duration of the protection remain to be
that make it a vaccine candidate with realistic per- adequately addressed, FMDV dendrimer peptide
spectives of clinical applicability. vaccines are likely to enter the real vaccine world in
the next years.
F. (1982). Protection against foot-and-mouth disease Clarke, B.E., Newton, S.E., Carroll, A.R., Francis, M.J.,
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virus (FMDV), is the type species of the Aphtho- of FMD in a country can have severe economic
virus genus of the Picornaviridae family. The virus consequences.
is antigenically variable and includes seven sero- Currently, upon an FMD outbreak countries
types, A, O, C, Asia-1, South African Territories attempt to restrict susceptible animal movement,
1–3 (SAT1–3) and multiple subtypes within each slaughter infected animals, and decontaminate
serotype. FMDV contains a positive-sense, single- infected premises. Vaccination is an option in
stranded RNA genome which is encapsidated countries in which FMD is enzootic, but disease-
by four structural proteins, VP1–4 (Mason et al., free nations prefer not to vaccinate or vaccinate
2003). The viral RNA also codes for a number of followed by slaughter of vaccinated animals, as
non-structural (NS) proteins involved in various occurred in the 2001 outbreaks in the UK and The
phases of virus replication and host cell control Netherlands (Pluimers et al., 2002; Scudamore and
including Lpro, a 16 amino acid peptide 2A, 2B, 2C, Harris, 2002).
3A, three 21–22 amino acid 3B proteins, 3Cpro, and The current approved vaccine is a chemically
the viral RNA dependent RNA polymerase 3Dpol. inactivated whole virus preparation which is com-
Numerous reviews have described in detail the role bined with an adjuvant prior to administration (see
of many of these proteins in the virus replication Chapter 12). Production of this vaccine involves
cycle (Belsham, 2005; Mason et al., 2003) and the growth of large amounts of live virus and thus
some of the chapters in this book provide more requires expensive high-containment facilities
recent information on virus molecular biology and (Doel, 2003). While the vaccine has been success-
virus structure. fully used for many decades there are a number of
The focus of this chapter, as well as a number concerns with its use including,
of other chapters in this book, is to describe the
various approaches that have been examined • It requires passage of virus in tissue culture
to effectively and rapidly control FMD. We will which could result in selection of antigenic vari-
discuss a two-pronged strategy that we have devel- ants.
oped using a viral vector, human adenovirus type • The virus may not be properly inactivated.
5 (Ad5), to deliver an FMD empty capsid subunit • Depending on manufacturer’s protocol, the
vaccine alone or in combination with Ad5 vectored virus may not be sufficiently purified and thus
type I, II or III interferon (IFN) genes. still be contaminated with viral NS proteins
resulting in difficulty in differentiating infected
Foot-and-mouth disease from vaccinated animals (DIVA).
FMD affects numerous cloven-hoofed animals • The vaccination does not induce long-lasting
including economically important livestock immunity and protection generally requires
animals such as cattle, swine, sheep, and goats as semiannual vaccination campaigns.
well as numerous species of wildlife (Grubman • The vaccination does not induce rapid protec-
and Baxt, 2004). The disease results in a high tion – usually requires about seven days to
morbidity but generally low mortality, except in induce a protective response. This is a major
young animals that can develop cardiac complica- concern since FMDV replicates and spreads
tions. Infected animals usually develop lesions very rapidly.
on the tongue, mouth, feet and teats, and as a • The vaccine is not broadly protective and each
result, often cannot eat, produce less milk, and in serotype and often subtypes within a serotype
developing countries are unable to perform vital require a specific vaccine.
utilitarian functions such as ploughing fields. The
World Organisation for Animal Health (OIE) For these and other reasons researchers have
lists FMD as a reportable disease and therefore by been attempting for the past 40 years to develop
law, member nations are required to inform the novel vaccines which address some or all of the
organization about FMD outbreaks. OIE member above mentioned concerns (Rodriguez and Gay,
nations, in which the disease is present, are not 2011). One of the obstacles in this process is the
permitted to engage in trading of FMD-suscepti- economic reality that animal vaccines or bio-
ble animals or their products. Thus, the presence therapeutics must be inexpensive. Our approach
has been to develop a product that (1) does not 1975). Vaccine studies using numerous other viral
require infectious virus and thus can be produced systems including hepatitis B, human papilloma
in the U.S. in a BSL2 facility rather than requiring virus etc. have demonstrated that viral empty
an expensive high-containment manufacturing capsids or virus-like particles produced by recom-
facility, (2) allows differentiation of infected from binant DNA techniques are effective vaccines and
vaccinated animals (DIVA), (3) is in a DNA format some of these products are in current use (Roldão
so that generation of antigenic variants during vac- et al., 2010). Based on these results and knowledge
cine production is limited in contrast to production of the replication requirements of FMDV (Mason
of the current whole virus vaccine, and (4) can be et al., 2003), we have used recombinant DNA
used in combination with a biotherapeutic that techniques to construct a replication-defective
induces rapid protection regardless of virus sero- adenoviral vector containing the portions of the
type causing the outbreak. FMDV genome coding for the structural proteins
as well as NS protein 3Cpro, which is necessary
Adenovirus as vaccine vectors for processing of the capsid protein precursor
Adenoviruses (Ads) are non-enveloped, icosahe- (Vakharia et al., 1987; Mayr et al., 1999). In the
dral pathogens associated with most vertebrates following sections of this chapter we will go into
(Davison et al., 2003). They have a linear double- detail regarding the development and testing of
stranded DNA of about 26–45 kb and belong to Ad5 vectored FMDV constructs for types A and
the family Adenoviridae, which is divided into five O in both swine and cattle (Moraes et al., 2002,
genera based on the host species and the DNA 2011; Pacheco et al., 2005). We will also present
composition. Ads vectors have become a very several approaches we have used, including addi-
popular vehicle for gene transfer into mammalian tion of adjuvants, different routes of vaccination,
cells (Tatsis and Ertl, 2004). Human Ad are by far etc. in order to achieve vaccine dose-sparing for a
the best characterized, and the vast majority of gene more practical use in the field (Diaz-San Segundo
transfer studies involving Ad, whether for therapeu- et al., 2014; Grubman et al., 2012)
tic or vaccine purposes, have been carried out with Evidence from our lab as well as others, dem-
vectors derived from serotype 5 (Ad5) (Bradley et onstrated that FMDV replication in cell culture is
al., 2012; Tatsis and Ertl, 2004). highly susceptible to IFN treatment and that all
Different strategies have been employed in the virus serotypes are affected (Ahl and Rump, 1976;
construction of Ad vectors. The most prevalent Chinsangaram et al., 2001; Grubman et al., 2012).
strategy involves deletion of one or more of the Furthermore, IFN treatment can rapidly inhibit
early genes (e.g. E1 gene) required for virus repli- virus replication. Based on this information we
cation rendering the vector replication-defective hypothesized that a combination of IFN treatment
(Schaack, 2005). Ad viruses with multiple genomic and vaccination could both rapidly protect against
deletions have been used as delivery vectors, but in infection with any FMDV serotype and induce a
this case special cell lines, that usually have proprie- longer lasting specific immunity utilizing a vac-
tary rights, are required for growth and propagation cine against the virus strain circulating in the field
thus limiting the availability of these vectors for (Grubman, 2003). In the following sections we will
research and development. Multiple features of the also present experimental data in swine and cattle
virus such as high packaging capacity for transgene examining the effectiveness of Ad5 delivered IFNs
insertion, efficient transduction of both quiescent as well as some combination of Ad5-FMD and
and dividing cells, and ability to grow with high Ad5-IFN.
titre make it a useful vector for gene delivery (Luo
et al., 2007).
Development of Ad5 vaccines to
Adenovirus vectored FMD vaccines deliver FMDV antigens
and biotherapeutics
It has been shown that FMDV empty capsids, Ad5-FMD vaccines
e.g. virus particles lacking genomic RNA, are as The use of replication-defective human adenovirus
antigenic as infectious virions (Rowlands et al., (Ad5-) to deliver FMD vaccines has been reported
by different groups (summarized in Box 14.1 and • the absence of other genomic regions such as
Table 14.1). The initial studies using this vector the 3D coding region, which codes for one of
were performed by Sanz-Parra et al. (1999) who the most immunogenic viral proteins allowing
demonstrated that two doses of an Ad5 vector con- for differentiation of infected from vaccinated
taining the P1 coding region of FMDV serotype C, animals (DIVA);
but lacking the 3Cpro coding region, induced partial • the Ad5 vaccine is produced as a DNA molecule
protection in both cattle and swine. Mayr et al. which is genetically stable since it is not subject
used the same vector and the P1–2A coding region to the high error rate of replication of RNA poly-
of FMDV A12, but included the 3Cpro coding merases, and
region since it had been demonstrated that 3Cpro • the vaccine does not contain infectious FMDV
is required for processing of the capsid precursor and therefore can be produced in a BSL2 labora-
P1–2A, allowing for capsid assembly (Mayr et al., tory even in FMD-free countries (Grubman et
1999; Vakharia et al., 1987). The vaccine also con- al., 2010).
tained partial sequences of the FMDV NS proteins
2B and 3B. There are a number of advantages of this In their proof of concept studies Mayr et al.
vaccine platform including: (1999, 2001) demonstrated that the Ad5-FMD
vaccine induced significant levels of neutralizing
P1 2A2B3B 3C
VP0 VP3 VP1
E1 E3
LITR CMV RITR
Ad5 DNA
Pac I Promoter polyA Pac I
1
0 3
-VP0
FMDV empty capsid
-VP1
-VP3
LITR, RITR-left and right internal terminal repeats of the Ad5 genome; CMV- cytomegalovirus promoter;
PacI- restriction enzyme sites in pAd5 vector; poly A- polyadenylic acid tract; ΔE1 and E3- deletions in
Ad5 early regions 1 and 3.
antibodies and protected swine against challenge neutralizing antibody response and the vaccinated
with FMDV serotype A12. Further studies demon- animals, housed in the same room as the control
strated that the approach was also effective against animals, were protected when challenged 14 days
FMDV serotype A24, a field strain of FMDV later (Grubman, 2005). These later results suggest
(Moraes et al., 2002). One dose of an Ad5-A24 that the Ad5-FMD approach could also be useful
vector administered intramuscularly (IM) con- in vaccination campaigns in enzootic areas where
ferred protection in both swine and cattle against vaccinations are given twice annually.
direct inoculation challenge with FMDV as early FMDV strains that are circulating and causing
as seven days after vaccination and protection outbreaks in many parts of the world for the past
in swine lasted for at least 42 days (Moraes et al., number of years belong to serotype O. Outbreaks
2002; Pacheco et al., 2005). Moreover a boost of serotype O have been recurrent in many parts
given to cattle at 9 weeks after the initial inocula- of Asia including Taiwan, Japan, South Korea,
tion, significantly enhanced the FMDV-specific China, Vietnam, Russia, etc. (Nishiura and Omori,
2010; Paton et al., 2009) as well as in the Americas In addition, detection of histological changes after
(Sutmoller et al., 2003). It has been demonstrated vaccination indicated the presence of extensive
that inactivated serotype O vaccine induces a oedema and cellular infiltrates consisting of large
lower immune response compared with serotype mononuclear cells with fewer neutrophils and small
A antigen (Doel et al., 1994; Pay and Hingley, mononuclear cells (Montiel et al., 2012). Notably,
1986). As a result of its poorer immunogenicity this study showed the presence of large quantities
commercial type O vaccines usually contain 4- to of cellular markers found in antigen-presenting
five-fold more antigen that A vaccines. An Ad5- (APC) and other immune cells in tissues where
O1Campos (Ad5-O1C) vaccine was also evaluated the Ad5 transgene expression was also detected –
by scientists at Plum Island Animal Disease Center providing a clear indication of the host response to
(PIADC). Swine inoculated with 5 x 109 pfu of this Ad5-FMD vectors (Montiel et al., 2013). Examina-
vaccine developed lower levels of FMDV-specific tion of Ad5-FMD biodistribution in swine or other
neutralizing antibodies than swine inoculated with FMDV host species as well as delivery by alterna-
an equivalent dose of Ad5-A24. Furthermore, in tive routes should provide additional insights for
efficacy studies this dose of the Ad5-O1C vaccine critical events in Ad5-FMD vaccine function.
did not protect pigs against homologous challenge,
although disease signs were delayed and consider- Improvement of the efficacy of Ad5
ably less severe than controls all of which died vaccines
(Caron et al., 2005). Testing in cattle of a similar To enhance the potency and efficacy of the Ad5-
vaccine resulted in 50% protection (Moraes et al., FMD vector approach and develop a commercially
2011). However, it is important to note that in both viable FMD vaccine candidate against many circu-
experiments viraemia was eliminated or consider- lating serotypes a number of strategies have been
ably reduced and virus shedding was reduced 1–3 evaluated:
logs in vaccinated animals compared with controls.
These results are particularly important since pigs Effect of inclusion of the complete
are major amplifiers of virus and a reduction in virus FMDV 2B coding region in the
shedding can significantly reduce virus spread. Nev- Ad5-FMD vector
ertheless, it is clear that further modifications are After infection of cells with FMDV, as well as other
required to use the Ad5 approach against FMDV picornaviruses, there is a drastic rearrangement
serotype O, at least the subtype O1 Campos. of intracellular membranes which subsequently
become the sites of viral replication (Aldabe and
Ad5 biodistribution in a natural host Carrasco, 1995; Buenz and Howe, 2006; Cho et al.,
Strategies to further enhance the efficacy and 1994; de Jong et al., 2002; Monaghan et al., 2004;
potency of Ad5-FMD vaccines require detailed Suhy et al., 2000). A number of viral NS proteins have
examination of their biodistribution in the natural been implicated in the rearrangements including
host. Most of what is known about the biodistribu- NS protein 2B. To attempt to enhance the synthesis
tion of recombinant Ad5 (rAd5) vector vaccine has of FMDV capsid proteins and the efficiency and/or
been performed in mouse models, which describe stability of capsid assembly we constructed second
preferential sequestration of systemically adminis- generation Ad5-FMD vectors containing the full-
tered Ad vectors to the liver posing serious adverse length 2B coding region (Ad5-FMD-2B) (Moraes
effects (Imperiale, 2008; Kalyuzhniy et al., 2008). et al., 2011; Pena et al., 2008). Infection of cells with
However, recent studies that documented the bio- this vector induced significant rearrangement of
distribution of Ad5-A24 administered IM in cattle cytoplasmic membranes similar to what is observed
(Montiel et al., 2012, 2013) showed that despite in FMDV-infected cells (Pena et al., 2008). Swine
Ad5-A24 distribution in multiple visceral organs, inoculated with an Ad5-A24–2B vector developed
vector DNA was not found in the liver. Specifically, an enhanced FMDV-specific neutralizing antibody
elevated mRNA transcription at 24 and 48 hpi with response compared with the first generation vector
peak occurrence of transgene expression at 48 hpi which contained only a portion of the 2B coding
was observed. Abundant vector DNA was detected region and these animals showed no clinical signs
at the injected sites, in muscle and lymph nodes. of disease after FMDV challenge (Pena et al., 2008).
A similar approach was applied for the vac- (Barnett, Crews and Douglas, 2002; Hidaka et al.,
cine against FMDV O1Campos (Ad5-O1C-2B). 1999). This strategy has been particularly impor-
Although complete protection was not achieved, tant for targeting dendritic cells (DCs), which
a slightly higher percentage of cattle vaccinated are critical players for the initiation of immune
with this vaccine and challenged 21 days later with responses but lack CAR receptors (Worgall et al.,
FMDV O1 Campos were completely protected 2004). A comparison of the Ad-FMD vaccine with
from clinical disease than cattle vaccinated with the and without an extra RGD motif in the fibre knob
first generation vaccine only containing a truncated was evaluated against serotype O1Campos in cattle
version of NS viral protein 2B. Interestingly, addi- (Adt.O1C.2B.RGD versus Adt.O1C.2B) (Medina
tion of full-length 2B to Ad5-O1C resulted in an et al., 2016). Although enhanced transgene expres-
approximately 10-fold reduction in virus shedding sion was demonstrated in vitro in epithelial cells
and an enhanced specific vaccine-induced T-cell infected with the Adt.O1C.2B.RGD vector, no
response compared with the group administered significant increase in efficacy of the vaccine con-
the first generation vaccine lacking the complete 2B taining the fibre RGD compared with the vaccine
coding region (Moraes et al., 2011). without the fibre RGD was detected in cattle when
challenged with FMDV O1Campos at 21 dpv. In
Effect of route and number of sites of contrast to mice and humans, addition of an RGD
inoculation for Ad5-FMD vaccines in the Ad5 fibre did not improve transduction of
A single dose of 5 × 109 pfu of Ad5-A24 adminis- cattle immune cells enriched in APCs (Medina et
tered IM can protect swine against challenge at 7, al., 2016). Nonetheless, enhanced T-cell responses
14, or 42 days post-vaccination (dpv). The effect and presumably enhanced memory T-cell responses
of route, number and location of inoculation sites in animals vaccinated with the RGD construct may
was further evaluated for their ability to enhance play a role in improving protection from challenge
vaccine potency. By simply changing the route in the long term. Further studies with a larger sta-
of inoculation from IM to subcutaneous (SC), tistical sample number and challenge at later times
increasing the number of inoculation sites from post-vaccination (e.g. six to eight weeks), as well as
one to two or four and administering and vaccinat- development of more sensitive techniques to iden-
ing in the neck instead of the back limb of swine, tify specific subpopulations of immune cells, may
it was demonstrated that the protective vaccine contribute to a better understanding of the possible
dose could be lowered by 25-fold (Grubman et effects of RGD modification of the Ad5-FMDV
al., 2012). These results indicated that the route, fibre on humoral and cellular immunity in cattle.
number and injection location of Ad5-FMD are
important determinants to consider when evaluat- Evaluation of adjuvants for Ad5-FMD
ing potency and efficacy of the Ad5-FMD vaccine.
IFN
Effect of broadening tropism of Ad5 by Numerous studies indicate that in addition to anti-
modifying the fibre protein viral activity IFN-α/β can act as an adjuvant and
Attachment of Ad5 to the cell occurs upon inter- boost the immune response to antigens (Brassard
action between the fibre and the coxsackie and et al., 2002; Cull et al., 2002; Le Bon et al., 2001;
adenovirus receptor (CAR) (Bergelson et al., Proietti et al., 2002). The adjuvant effect of porcine
1997; Tomko et al., 1997). This is followed by the interferon α (poIFNα), also delivered by an Ad5
interaction between the RGD (Arg-Gly-Asp) motif vector (Ad5-poIFNα), was examined in swine vac-
present within the penton base and the cell surface cinated with Ad5-A24 and challenged with FMDV
integrins which mediate the internalization of the at 42 days post inoculation (de Avila Botton et al.,
virus into the cell (Bergelson et al., 1997; Greber 2006). Although strong protection was observed
et al., 1993; Wickham et al., 1993). Genetically with the vaccine alone, addition of (Ad5-poIFNα)
engineered Ads that have an RGD motif inserted in resulted in improved protection. Despite the lim-
the HI-loop of the fibre can use αvβ3 and αvβ5 inte- ited knowledge of the swine immune system, the
grins to attach to host cells (Coughlan et al., 2009; authors observed that protection correlated with
Sengupta et al., 2011) broadening their tropism increased levels of IgG1 and IgG2 as well as an
increase in the IgG1/IgG2 ratio. These results were efficacy of the combination approach correlated
consistent with previously reported studies in cattle with a stronger antigen specific cell-mediated
that suggest a role for a specific antibody isotype immune response (Diaz-San Segundo et al., 2014).
in promoting protection against FMD (Capozzo Therefore, at least in swine, polyICLC is a strong
et al., 1997; Mulcahy et al., 1990). Thus, poIFN-α adjuvant of Ad5-FMD.
enhances the long-term level of protection induced
by the Ad5-FMD vaccine. Mucosal adjuvants
More recently Su et al (Su et al., 2013) have shown The oral and respiratory mucosae are primary
that IFN-α acts as an adjuvant in the generation of sites for FMDV replication in the host (Arzt et al.,
T follicular helper (Tfh) cells and antigen-specific 2010). It is hypothesized that stimulation of local
antibody responses when it is co-expressed with immunity in these tissues may help prevent initial
FMDV VP1 proteins in an Ad5-FMD subunit vac- infection and viral spread. Several bacterial proteins
cine (Ad5-VP12A-poIFNα). Inoculation of mice including V. cholera and E. coli heat-labile entero-
with Ad5-VP12A-poIFNα enhanced the genera- toxin (LT) display adjuvant activity at mucosal
tion of Tfh, the secretion of IL-21 protein and the surfaces (Rappuoli et al., 1999). Ad5 vectors encod-
expression of Bcl-6 mRNA, compared with Ad5 ing either of two LT-based mucosal adjuvants, LTB
vectors solely expressing VP1 and substantially and LTR72 have been used together with Ad5-A24
increased the number of germinal-centre (GC) vaccine to assess their ability to augment mucosal
B-cells and formation of GCs. Furthermore, IFN-α and systemic humoral immune responses and
enhanced the antibody response as revealed by protection against challenge with FMDV (Alejo
increased production of IgG and subclasses of IgG1 et al., 2013). Adult mice receiving Ad5-A24 plus
and IgG2a. Ad5-LTR72 had higher levels of mucosal and sys-
temic neutralizing antibodies than those receiving
Poly-ICLC Ad5-A24 alone or Ad5-A24 plus Ad5-LTB. This
We also examined the inclusion of an adjuvant in group also demonstrated 100% survival after intra-
the vaccine formulation. Adjuvants can significantly dermal challenge with a lethal dose of homologous
enhance the maturation of APCs including DCs FMDV serotype A24. These results suggest that
and the antigen specific cellular response (Kool et Ad5-LTR72 could be used as an important tool to
al., 2008; Pulendran and Ahmed, 2006). Poly ICLC enhance mucosal and systemic immunity against
is a synthetic, double-stranded polyriboinosinic- FMDV and potentially other pathogens with a
polyribocytidylic acid molecule stabilized with common entry pathway.
poly-L-lysine and carboxymethyl cellulose which
has enhanced biostability in animals, compared Evaluation of Ad5-FMD empty capsid
with poly IC (Nordlund et al., 1970) and is a known vaccines for different FMDV serotypes
toll-like receptor 3 (TLR3) and MDA-5 agonist Outbreaks of serotype O have been recurring in
that can activate multiple innate immune pathways many parts of Asia including Taiwan, Japan, South
(Meylan and Tschopp, 2006; Stahl-Hennig et al., Korea, China, Vietnam, Russia, as well as the
2009). In rodents and primates, poly ICLC is a Middle East, Africa and the Americas (Nishiura
strong IFN-α inducer and provides antiviral and and Omori, 2010; Paton et al., 2009). Inactivated
adjuvant activity (Caskey et al., 2011; Harrington et serotype O vaccine induces a lower immune
al., 1979; Levy et al., 1975; Tenbusch et al., 2012). response compared with serotype A antigen and
With respect to FMD, it has been shown that poly requires more antigen than A vaccines (Doel et al.,
IC has an adjuvant effect when combined with an 1994; Pay and Hingley, 1986). These results have
FMD multiepitope protein or inactivated FMD been reproduced when evaluating an O1 Campos
vaccine (Cao et al., 2012, 2013, 2014; Zhou et Ad5-O1C-2B vaccine (Caron et al., 2005; Medina
al., 2014). Combination of poly ICLC with Ad5- et al., 2016; Moraes et al., 2011).
A24–2B vaccine delivered SC at two sites in the However, recent studies have shown that a similar
neck, reduced the Ad5-A24–2B protective dose by vaccine against serotype O1 Manisa (Ad5-O1M-
approximately 80-fold compared with administra- 2B) is very effective. Swine vaccinated with as low
tion of vaccine alone. Interestingly, the enhanced as 4 × 107 pfu of Ad5-O1M-2B, a five-fold lower
dose than that required for serotype A24, were fully days to induce complete protection from clinical
protected against challenge as early as 7 dpv (de los disease (Moraes et al., 2002; Pacheco et al., 2005;
Santos, unpublished data). Pandya et al., 2012). However, in the event of
Similar results were obtained with an Ad5-Asia an FMD outbreak in a disease-free country, the
vaccine but in a mouse model. C57BL/6 mice induction of rapid protection, prior to the develop-
immunized with a single 107 pfu dose of Ad5- ment of vaccine-stimulated adaptive immunity, is
Asia-05 were protected against challenge with 100 necessary to block or limit disease spread and thus
times the lethal dose of FMDV Asia1/YS/CHA/05 potentially reduce the number of animals that have
(Zhou et al., 2013). Additional studies with Ad5 to be slaughtered.
vectors containing the P1–2A coding region from Early protection against viral infection is
numerous other FMDV serotypes and strains have dependent on the induction and the sensitivity to
been tested in cattle (Grubman et al., 2010). IFNs and IFN-stimulated genes (ISGs) (Fensterl
These studies highlight the potential use of Ad5- and Sen, 2009; Schoggins and Rice, 2011). IFNs
FMD vaccines against current circulating FMDV are glycoproteins with strong antiviral activities
strains in Asia. that represent the first line of host defence against
invading pathogens (Ank et al., 2006; Basler and
Ad5-FMD VP1 subunit vaccines Garcia-Sastre, 2002; Frese et al., 2002; Henke et
Given the immunogenicity of FMDV VP1, several al., 2001; Samuel, 2001; Shrestha et al., 2006).
studies have focused on utilizing VP1 as a peptide These proteins are classified into three groups,
vaccine (Brown et al., 1992; Sobrino et al., 1999; type I, II and III IFNs, based on the structure of
Taboga et al., 1997). However, poor results and lim- their receptors on the cell surface (Fensterl and
ited protection in the natural host were observed. Sen, 2009). Type I IFNs (IFN-α and IFN-β) signal
Utilization of FMDV VP1 as a peptide vaccine in through a heterodimeric receptor complex formed
the context of rAd5 has been examined in combina- by IFNAR1/IFNAR2, type II IFN (IFN-γ) signals
tion with porcine IFN-α (Du et al., 2008a,b). In the through the complex IFN-γR1/IFN-γR2 and type
latter study, the rAd5 expressing VP1 from FMDV III IFNs bind the receptor complex IL-28Rα/
O/LY/2000 strain fused with IFN-α enhanced IL-10Rβ. Despite the receptor differences the three
both humoral and cell-mediated immune responses IFN families transduce signals though the JAK-
to FMDV in mice (Du et al., 2008b). Interestingly, STAT pathways and type I and type III IFNs induce
addition of multiple VP1 epitopes in rAd5 VP1 (B redundant responses which result in the inhibition
and T-cell epitopes) fused with IFN-α enhanced of viral replication. Similar to many other viruses,
immune responses in mice when compared to rAd5 FMDV is highly sensitive to the action of IFN (Ahl
VP1 or rAd5 IFN-α alone. Most importantly, this and Rump, 1976; Diaz-San Segundo et al., 2013).
approach provided protection from virulent FMDV Thus, to be a successful pathogen, the virus coun-
type O challenge in guinea pigs and swine (Du teracts the innate immune response by blocking the
et al., 2008a). Recently, Su et al. (2013) provided expression of IFN (Chinsangaram et al., 1999; de
evidence that combinatorial use of IFN-α and VP1 los Santos et al., 2006) through a combination of
could enhance the generation of specific T-cells mechanisms including, degradation of translation
(Tfh), secretion of IL-21 and Bcl-6). These studies initiation factor eIF-4G, which is required intact
summarized the importance of VP1 and its use as a for translation of capped messages (Devaney et
candidate vaccine when employed in combination al., 1988; Kirchweger et al., 1994); degradation of
with an adjuvant. nuclear factor-κappa B (NF-κB) (de los Santos et
al., 2007); inhibition of the activation of interferon
regulatory factors 3 and 7 (IRF-3; IRF-7) (de los
Development of Ad5-delivered Santos et al., 2007; Wang et al., 2010) affecting
biotherapeutics/antivirals as a adaptor molecules including TRAF-6, TBK1
control strategy for FMD (Wang et al., 2011); impairment of cellular activity
Although both commercial inactivated and newly of the cytoplasmic pathogen recognition receptor
developed Ad5 based FMD vaccines fully protect (PRR) RIG-I (Wang et al., 2012). These results sug-
animals against the disease, they require about 7 gest that use of IFN could be a successful strategy to
rapidly control FMDV in vivo. However, since IFN Lastly, an alternative anti-FMD strategy using
protein is rapidly cleared, its clinical use requires Ad vectors to deliver siRNAs have been explored.
multiple inoculations of high doses for a prolonged The sections below summarize the knowledge
time (Lukaszewski and Brooks, 2000; Qin et al., thus far reported in these fields (Box 14.2 and Table
1998; Santodonato et al., 2001) which can lead 14.2).
to adverse systemic effects (Qin et al., 1998). To
overcome this problem, bovine and porcine IFN-α, Type I IFN
IFN-β, IFN-γ and IFN-λ have been delivered with Initial studies in cell culture demonstrated that in
the same replication-defective Ad5-system used for supernatants from Ad5-poIFN-α infected IB-RS-2
the FMD vaccine, thus allowing animals to produce cells, poIFN-α was detected as early as 4 hours
the protein endogenously and spread it systemi- post infection (hpi), its expression continued for
cally. at least 30 hpi and more importantly, the protein
LITR, RITR, left and right internal terminal repeats of the Ad5 genome; CMV, cytomegalovirus promoter;
PacI- restriction enzyme sites in pAd5 vector; poly A, polyadenylic acid tract; ΔE1 and E3, deletions in
Ad5 early regions 1 and 3.
obtained had biological antiviral activity against of clinical signs, and a significant reduction in virae-
FMDV (Chinsangaram et al., 2003). Furthermore, mia when the challenge was performed 7 days post
all serotypes of FMDV were very sensitive to inoculation (dpi) or one day prior to the treatment
the supernatants of these cells (Grubman et al., (Moraes et al., 2003). Similar results were obtained
2012). Studies in swine demonstrated that after with an Ad5-vector expressing poIFN-β. In this
one IM inoculation with 1 × 109 pfu/animal of case, although the antiviral activity was lower than
Ad5-poIFN-α, relatively high levels of antiviral that induced by Ad5-poIFN-α with the same doses
activity (~800 U), starting 16 hpi to 72 hpi, were of Ad5s, animals were also completely protected
detected in plasma and, importantly, animals were against ID challenge with FMDV A24 (Dias et al.,
completely protected against FMDV A24 when 2011).
challenged intradermally (ID) 24 hours after the FMDV is an antigenically variable virus and
Ad5-poIFN-α treatment (Chinsangaram et al., a control strategy using biotherapeutics could
2003). Furthermore, complete protection induced potentially rapidly protect susceptible animals
by Ad5-poIFN-α in swine lasted for 3–5 days, and against any FMDV strain and overcome the limited
there was a delay in disease onset, reduced severity coverage of vaccines against heterologous strains
(Domingo et al., 2003; Grubman and Baxt, 2004). Segundo et al., 2010, 2013a) and in epidermal DC
Swine experiments in which animals were inocu- maturation (Fujita et al., 2005). Furthermore, using
lated with the Ad5-poIFN-α vector and challenged a mouse model for FMDV developed by Salguero
ID 24 hours later with FMDV serotypes O1 Manisa et al. (2005), IP-10 C57Bl/6 knockout mice treated
or Asia1 showed complete protection, independent with murine IFN-α (muIFN-α) prior to challenge
of the FMDV serotype used for challenge (Dias et were not protected against disease whereas WT
al., 2011). Although these proof-of-concept stud- C57Bl/6 mice were completely protected, indicat-
ies demonstrated that Ad5-poIFN-α could rapidly ing that IP-10 is directly involved in protection
protect swine against FMD, these experiments induced by IFN against FMDV (Diaz-San Segundo
were performed by direct ID challenge. However, et al., 2013b).
the natural route of FMDV infection is by aerosol Preventative therapy with Ad5-type I IFN only
(Alexandersen et al., 2003; Pacheco et al., 2010, had limited efficacy in cattle. Inoculation of bovines
2012). Therefore, the efficacy of treatment of swine with 1x1010 pfu/animal of Ad5-poIFN-α or Ad5-
with Ad5-poIFN-α was also evaluated by contact bovine IFN-α (Ad5-boIFN-α) induced a relatively
exposure challenge with animals that had been low level of systemic antiviral activity (100–200 U/
previously inoculated with FMDV. At 24 hours ml) and challenge of these animals with FMDV
post Ad5-poIFN-α or Ad5-control vaccination, A24 by ID inoculation in the tongue only resulted
swine were directly exposed for 18 hours to donor in a short delay and reduced severity of disease
animals which had been previously inoculated compared with control animals (Wu et al., 2003).
with FMDV and displayed clear clinical signs of
disease. In contrast to the Ad5-control inoculated Type II IFN
animals, all Ad5-poIFN-α-treated animals were Approximately a decade after the discovery of type
completely protected against disease, indicating I IFN, another molecule with antiviral activity was
that Ad5-poIFN-α treatment is effective against described (Wheelock, 1965). In the early 1970s
multiple routes of FMDV exposure (Dias et al., the active principle was recognized as being distinct
2011). from classical virus-induced interferons, leading
In studies to understand the mechanism by to its designation as immune IFN or type II IFN
which IFN protects swine against FMD we exam- (Falcoff et al., 1973), and eventually IFN-gamma
ined the expression of ISGs in skin, peripheral (IFN- γ). This protein is a multifunctional cytokine
blood mononuclear cells (PBMCs), and lymphoid produced by T-helper 1 (Th1) and NK cells, and
tissues and also evaluated possible immune cell its biological functions include immunoregulatory,
recruitment to the skin and lymph nodes. Pro- anti-neoplastic, and antiviral properties (Biron and
tection of swine inoculated with Ad5-poIFN-α Brossay, 2001). The signal transduction pathways
correlated with recruitment of skin DCs (Diaz- elicited by type II IFNs are different than those
San Segundo et al., 2010) that showed a partial induced by type I IFN (Levy and Darnell, 1990).
maturation phenotype with increased expression Interestingly, the combination of type I and type
of CD80/86, and decreased phagocytic activity II IFNs can synergistically induce gene expression
(Diaz-San Segundo et al., 2013a). At the same time, (Levy and Darnell, 1990; Thomas and Samuel,
an increase in the number of natural killer (NK) 1992). Following the previous work with IFN-α,
cells in draining lymph nodes was noticeable (Diaz- and to examine the potential antiviral effect of
San Segundo et al., 2010). These findings correlated IFN-γ on FMDV replication an Ad5 vector con-
with up-regulation of a number of ISGs with anti- taining the porcine IFN-γ gene (Ad5-poIFN-γ)
viral activity including PKR and OAS, which block was constructed. Ad5-poIFN-γ was able to block
FMDV replication in cell culture (Chinsangaram FMDV replication in cell culture. Furthermore, an
et al., 2001; de los Santos et al., 2006) as well as enhanced protective effect against FMDV in vitro
cytokines and chemokines, including monocyte when type II IFN was combined with type I IFN
chemotactic protein-1 (MCP-1), macrophage was observed (Moraes et al., 2007). Interestingly,
inflammatory protein (MIP)-1α, and IFN induc- the action of type II IFN in combination with type
ible protein 10 (IP-10) which are involved in I IFN could synergistically block virus replication
chemoattraction of DCs and NK cells (Diaz-San in vivo; swine inoculated with the combination of
Ad5-poIFN-γ and Ad5-poIFN-α were completely combination of Ad5 expressing type I and III IFNs.
protected against FMD challenge when used at Inoculation of cattle with 1–1.5 × 1011 pfu/animal
doses that, individually, were not effective (Moraes of Ad5-boIFN-λ3 followed by ID challenge in the
et al., 2007). More recently, a similar approach in tongue with FMDV 24 hpi, resulted in a significant
swine, using an Ad5 that expressed bicistronically delay (6 to 12 days) and reduced severity of dis-
porcine IFN-α and IFN-γ also showed an enhance- ease (Perez-Martin et al., 2012). Furthermore, the
ment of the antiviral activity compared with Ad5 delay in the appearance of disease was significantly
constructs that expressed either IFN alone in swine prolonged when treated cattle were challenged by
(Kim et al., 2014). aerosolization of FMDV, using a method that best
In cattle, similarly to treatment with Ad5-type I resembles the natural route of infection (Pacheco
IFN (Wu et al., 2003) treatment with a combina- et al., 2010). In this experiment, no clinical signs
tion of type I and II IFNs (1 × 1010 pfu of each IFN/ of FMD, viraemia or viral shedding in nasal swabs
animal) followed by challenge with FMDV A24 ID were found in the Ad5-boIFN-λ3 treated animals,
1 day later only showed partial protection, with 4 for at least 9 days post challenge and one of three
days’ delay in the onset of the disease and lower animals remained free of disease during the entire
levels of viraemia than control animals (Moraes et experiment (Perez-Martin et al., 2012). These
al., unpublished). results indicated that boIFN-λ3 plays a critical role
in the innate immune response of cattle against
Type III IFN FMDV, a species in which treatment with type I
A new family of IFNs, type III IFN or IFN lambda IFN had not been previously successful.
(IFN-λ), has been identified in several species To examine the efficacy of IFN-λ in swine,
including humans, mice, chicken, swine and cattle animals were inoculated with various doses of
(Díaz-San Segundo et al., 2011; Kotenko et al., Ad5-poIFN-λ3 and one day later exposed by
2003; Sang et al., 2010; Sheppard et al., 2003). Fur- contact to swine that had clinical signs of FMD
thermore, in vitro antiviral activity against FMDV (Perez-Martin et al., 2014). Swine administered
has been demonstrated for both, bovine IFN-λ3 the highest dose of vector were completely pro-
and porcine IFN-λ1 and –λ3 (Díaz-San Segundo tected from clinical disease, had no detectable
et al., 2011; Wang et al., 2011). Although type I viraemia or viral RNA, and no virus shedding.
and type III IFNs induce similar innate antiviral Two of three animals in each of the lower dose
responses, they signal through different receptors groups were also protected. This type of chal-
(Sheppard et al., 2003; Uzé et al., 2007). A relatively lenge resembles the natural route of infection in
higher abundance of the type III IFN receptor in a commercial production facility or a small farm,
mucosal epithelial tissues makes IFN-λ an attrac- where naive animals might be physically exposed
tive candidate for control of infections that mainly to infected animals. Interestingly, protection
initiate in these tissues (Pulverer et al., 2010; Som- was achieved even when no consistent levels of
mereyns et al., 2008). In fact, the main natural route systemic antiviral activity or up-regulation in
of FMDV infection is via aerosol through the upper the expression of ISGs in PBMCs were detected
respiratory tract (Alexandersen et al., 2003), with (Perez-Martin et al., 2014), which is consistent
the nasopharynx region as the primary site of viral with previous reports showing that the expression
replication before subsequent spread to the lungs of the IFN-λ receptor and sensitivity to this type
(Arzt et al., 2010; Pacheco et al., 2010), making this of IFN are highest in epithelial tissues and not in
type of IFN a very appealing candidate to be used leucocytes (Pulverer et al., 2010; Sommereyns et
against FMDV. al., 2008). Although more experiments collecting
Inoculation of cattle with Ad5-boIFN-λ3 several tissue samples after IFN-λ3 treatment are
resulted in low systemic antiviral activity, but needed to determine the role of specific ISGs in
the induction of several ISGs in most tissues of inducing protection in swine against FMDV at the
the upper respiratory tract which are targets of primary sites of entry, replication, and dissemina-
FMDV infection (Díaz-San Segundo et al., 2011). tion, the use of type III IFN is a viable, effective,
Interestingly, there was an enhanced effect in the and safe strategy to control FMD early after infec-
upregulated ISGs when the treatment included a tion in the field.
Ad VEE PolyIC
Endosome
cGAS RIG-I/ NOD2
MDA5 DAI
TRIF
TLR3
TLR7
TLR8
TLR9
TIK2
TRAF6 IRF-1,CXCL9
Mitochondrion
cGAMP TBK1 IFN-R JAK1 Inflammatory
STAT
STAT
IKKε response
STAT
STAT
TIK2 OAS,Mx1
NF-κB AP-1
IRF3/7/? IRF3/7/? IFNs
Type I Antiviral
Type II response
IFN
Type III
STING ER
Other antiviral
Unknown
genes
nucleus mechanism
Ad, adenovirus; AP-1, activator protein 1; cGAMP, cyclic di-GMP-AMP; cGAS, cytosolic GAMP synthase;
CXCL9 (chemokine (C-X-C-motif) ligand 9; DAI, DNA-dependent activator of IRFs; ER, endoplasmic
reticulum; GAS, γ‑activated sequence; IFN, interferon; IFN-R, IFN receptor; IRF, IFN regulatory factor;
IKKε, IκB kinase‑ε; JAK1, janus kinase 1; MAVS, mitochondrial antiviral signalling protein; MDA5,
melanoma differentiation-associated gene 5; Mx1, Myxoma resistance protein; MYD88, myeloid differen-
tiation primary response protein 88; NF-κB, nuclear factor‑κB; NOD2, NOD-containing protein 2; OAS1,
2′,5′-oligoadenylate synthetase 1; PRRs, pattern recognition receptors; poly IC, polyinosinic:polycytidylic
acid; STAT, signal transducer and activator of transcription, STING, stimulator of IFN genes; TBK1,
TANK-binding kinase 1; TRAF, TNF receptor-associated factor; TRIF, TIR domain-containing adaptor
protein inducing IFNβ; TYK2, tyrosine kinase 2; VEE, Venezuelan equine encephalitis virus.
induction of the antiviral IFN response includes with Ad5-poIFN-α (10-fold reduction compared
the activation of a series of transcription factors, with the dose of Ad5-poIFN-α necessary to
i.e. IRFs, NF-κB, etc. Activated IRFs and NF-κB protect by itself), demonstrating that the use of
are required for IFN induction as well as up- PRR agonists alone or in combination with IFNs
regulation of additional antiviral genes, some of may represent an effective and broad-spectrum
which are induced by mechanisms independent of antiviral strategy to combat FMDV infection and
type I IFN (Kawai and Akira, 2011). By directly perhaps other viral diseases of livestock species
administering IFN, the molecular interactions (Dias et al., 2012).
that normally occur during viral infection are
bypassed. It is hypothesized that treatment of VRPs
animals with both IFN and various PAMPs may Pushko et al. (1997) have constructed VRPs
result in a broader, enhanced, and prolonged consisting of VEE virus particles containing a
antiviral response than with Ad5-IFN treatment defective VEE genome, in which the structural
alone, including the activation of a number of genes downstream of a subgenomic promoter are
signalling molecules that may potentially result replaced by a heterologous gene/cassette of inter-
in a positive feedback induction of additional IFN est. Packaging of these recombinant genomes is
(Honda and Taniguchi, 2006; Marié et al., 1998). performed in helper cell lines that provide VEE
Three different strategies that could enhance the capsid and replicase activity in trans. VRPs can
antiviral effects of IFN have been tested: (i) the only undergo a single-cycle of infection when
use of double stranded RNA, poly IC, in combina- used as vaccines. VRPs have been used as vaccines
tion with IFN treatment (Dias et al., 2012); (ii) the to deliver various foreign genes (Hooper et al.,
use of viral replicon particles (VRPs) derived from 2009; Lee et al., 2003) including FMDV capsids
Venezuelan equine encephalitis (VEE) virus that (unpublished data). Konopka et al. (2007, 2009)
could act as RNA factories (Diaz-San Segundo et demonstrated that null VRPs, VRPs lacking any
al., 2013b); or (iii) expression of a constitutively foreign gene, induce an early innate immune
active transcription factor, IRF7/3(5D) fusion response in mice within 1 to 3 hpi, resulting in
protein, delivered with the Ad5 vectored platform the up-regulation of a number of ISGs and the
(Ramirez-Carvajal et al., 2014). production of type I IFN protein, and can also
protect mice against VEE challenge (Konopka
Poly-ICLC et al., 2009). In the case of FMDV, pretreatment
As mentioned above, poly-IC is a synthetic, of cells with VRPs containing green fluorescent
double-stranded RNA that binds TLR3 and protein (VRP-GFP) as well as porcine IFN-α
MDA-5 cellular receptors. Importantly, inclu- (VRP-poIFN-α) significantly reduced FMDV
sion of poly-ICLC, a stabilized version of poly replication in infected porcine or bovine cells, and
IC, acts as an adjuvant of the Ad5-FMD vaccine. inhibition lasted for at least 5 days. A number of
Treatment of specific porcine and bovine cell lines genes were upregulated after treatment of swine
with poly IC alone significantly reduces FMDV cells with either VRP-GFP or VRP-poIFN-α
replication. Interestingly, in bovine cells, the com- (Diaz-San Segundo et al., 2013). VRPs were also
bination of poly IC and boIFN-α had a synergistic very effective against FMDV in vivo in a small-
effect as compared with each treatment alone, animal model. Adult C57BL/6 mice, which can
and there was up-regulation in the expression of be lethally infected with FMDV (Salguero et al.,
a number of cytokines, chemokines, transcription 2005), survived challenge when pretreated with
factors, and genes with direct antiviral activity. VRP-GFP (Diaz-San Segundo et al., 2013b), indi-
To test the effect of this molecule in vivo in com- cating that VRP treatment is an effective approach
bination with type I IFN, swine were inoculated in vivo to stimulate a strong innate IFN dependent
using 5- to 15-fold reduced Ad5-poIFN-α doses immune response. Using the VRPs to express IFN
combined with different doses of poly ICLC. The in the natural host is a very promising strategy to
results from this experiment indicated that 8 mg of initiate the IFN pathway at various levels, there-
poly ICLC is able to protect swine against FMDV fore inducing a robust innate immune response
A24 ID challenge when used alone or combined able to protect against FMD.
and biotherapeutics to induce either, a robust long (USDA Product Code 1FM1.R0; Colby et al.,
lasting adaptive and cellular immune response with 2013), a number of improvements, as well as field
strong neutralizing antibody levels or to induce testing, are still necessary for using this approach
a rapid innate immune response to control FMD as a viable alternative to the current inactivated
have been discussed. However, a complete control whole virus vaccine. Perhaps the major obstacle is
strategy would ideally include both, a rapid control the economics of Ad vaccine production based on
of disease spread as well as long-lasting protection the vaccine dose required to induce protection. As
that would ultimately cover livestock from being discussed in this article, we have shown that the
infected with FMDV. Therefore, it would be rea- dose can be considerably reduced by (a) including
sonable to combine both treatments as the next the coding region for the FMDV NS protein 2B
step in designing the best control strategy against in the construct; (b) altering the site and route of
FMD. In a proof of concept study in swine, Moraes vaccination; and (c) including an adjuvant in the
et al. (2003) treated animals with a combination vaccine formulation.
of Ad5-poIFN-α and Ad5-A24 subunit vaccine Approaches to further improve the efficacy of
challenging at 5 dpi with FMDV A24. Treated ani- the vaccine by broadening vector tropism to target
mals were completely protected against FMD, and DCs in cattle, have not resulted thus far in enhanced
developed a significant adaptive immune response protection (Medina et al., 2016). However, addi-
(Moraes et al., 2003). More recently, similar results tional experiments are needed to determine if
were described in cattle treated with a combination similar approaches such as substituting the fibre of
of Ad5-boIFN-λ3 and Ad5-O1Manisa (Ad5-O1M) Ad5 by the fibre of other Ad serotype could target
and challenged by aerosol exposure (Diaz-San Seg- bovine DCs or other APCs resulting in a beneficial
undo et al., submitted). As expected, animals treated effect. In addition, similar studies are needed in
with Ad5-boIFN-λ3 alone or in combination with other livestock species of interest, including swine
Ad5-O1M were protected when challenged at and ovine.
24 hpi while animals treated with Ad5-O1M alone Further studies are also required to determine
were not. Interestingly cattle treated with the com- the optimal adjuvant/s that should be included in
bination of Ad5-boIFN-λ3 and Ad5-O1M were the vaccine formulation. For example, adjuvants
protected when challenged 3 dpi while animals which act as PAMPs and can initiate an innate
treated with Ad5-boIFN-λ3 or Ad5-O1M alone immune response resembling natural virus infec-
showed either no or partial protection. Remarkably, tion as well as initiating/enhancing antibody and
protection of animals treated with the combination cell-mediated immune responses, have shown
occurred despite the absence of detectable neutral- advantages over some traditional adjuvants (Goulet
izing antibodies or antiviral activity in serum at the et al., 2013). As mentioned in this chapter, poly
time of challenge. ICLC is effective and results in Ad5 vaccine dose
These two studies demonstrated that with a care- sparing, but only after 21 dpv (Diaz-San Segundo
ful design strategy, it is possible to control FMD in et al., 2014). Additionally, studies on duration of
the event of outbreaks in disease-free or enzootic immunity conferred by Ad5-FMD should be inves-
countries. If successful, this approach could signifi- tigated. In a preliminary study we demonstrated that
cantly reduce disease spread and ultimately lead to cattle given an Ad5-A24 boost at 9 weeks developed
eradication. a significant increase in FMDV-specific neutralizing
antibodies and were protected when challenged 2
weeks post-boost (Grubman, 2005). This result is
Future directions of particular importance since it demonstrated that
Although significant progress has been made in the the Ad5-FMD vaccine approach could be used in
development of Ad5 delivered FMD vaccines, as vaccination campaigns without concern that anti-
demonstrated by the granting in 2012 of a condi- bodies against the Ad5 vector would block vector
tional licence for inclusion of an Ad5-A24 vaccine delivery and transgene expression.
in the US National Veterinary Vaccine Stockpile by Development of more broadly protective FMD
the Center for Veterinary Biologics of the Animal vaccines is also an area that requires more research.
Plant and Health Inspection Service of the USDA Chimeric viruses containing epitopes from more
than one FMDV serotype can protect swine against foot-and-mouth disease virus subunit vaccine. Vaccine
31, 2302–2309.
challenge with either serotype (Rieder et al., 1994), Alexandersen, S., Zhang, Z., Donaldson, A.I., and Garland,
while an Ad5 vector containing more than one A.J. (2003). The pathogenesis and diagnosis of
capsid precursor can induce an immune response foot-and-mouth disease. J Comp Pathol 129, 1–36.
to each capsid (Wu et al., 2003). Designing Ad5 Ank, N., West, H., and Paludan, S.R. (2006). IFN-lambda:
novel antiviral cytokines. J. Interferon Cytokine Res. 26,
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types are approaches that also need to be examined. Arzt, J., Pacheco, J.M., and Rodriguez, L.L. (2010). The early
The combination of an Ad5 delivered IFN or pathogenesis of foot-and-mouth disease in cattle after
transcription factor vector and Ad5-FMD vaccine aerosol inoculation. Identification of the nasopharynx as
the primary site of infection. Vet. Pathol. 47, 1048–1063.
can induce both rapid protection and hopefully De Avila Botton, S., Brum, M.C.S., Bautista, E., Koster, M.,
long lasting vaccine specific protection, but more Weiblen, R., Golde, W.T., and Grubman, M.J. (2006).
research is required to understand which approach Immunopotentiation of a foot-and-mouth disease
is appropriate for cattle and swine. Likewise virus subunit vaccine by interferon alpha. Vaccine 24,
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combination approach should be performed in adenoviral vectors. Biochim. Biophys. Acta - Gene
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subgroup C adenoviruses and group B coxsackieviruses. Wu, Q., Brum, M.C.S., Caron, L., Koster, M., and Grubman,
Proc. Natl. Acad. Sci. U.S.A. 94, 3352–3356. M.J. (2003). Adenovirus-mediated type I interferon
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foot-and-mouth disease virus polyproteins expressed in Adenovirus-vectored shRNAs targeted to the highly
a cell-free system from clone-derived transcripts. J. Virol. conserved regions of VP1 and 2B in tandem inhibits
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and Xiao, S. (2010). Foot-and-mouth disease virus leader Youngner, J.S., and Salvin, S.B. (1973). Production and
proteinase inhibits dsRNA-induced type I interferon properties of migration inhibitory factor and interferon
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399, 72–78. Zhou, C.X., Li, D., Chen, Y.L., Lu, Z.J., Sun, P., Cao, Y.M.,
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R., Liu, X., Li, K., Chen, H., et al. (2011). The leader Resiquimod and polyinosinic-polycytidylic acid
proteinase of foot-and-mouth disease virus negatively formulation with aluminum hydroxide as an adjuvant for
regulates the type I interferon pathway by acting as a foot-and-mouth disease vaccine. BMC Vet. Res. 10, 2.
viral deubiquitinase. J. Virol. 85, 3758–3766. Zhou, G., Wang, H., Wang, F., and Yu, L. (2013).
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Zhang, H., Duan, E., Luo, R., Zhang, Z., et al. (2012). foot-and-mouth disease virus capsid proteins induces
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with regards to animal welfare but is also a huge mostly empiric and dosage, route and interval of
waste of resources. This is in sheer contrast with administration are often extrapolated from data in
the twenty-first century vision of a highly produc- humans and laboratory animals. As these antiviral
tive yet sustainable and animal-friendly agriculture drugs were not specifically developed for a certain
(EU, 2013; FAO, 2014). viral infection in a certain animal species, these
Despite the disadvantages of pre-emptive culling treatments often lack effectivity due to poor bio-
and despite the fact that vaccination is a very power- availability and in some cases even can be toxic at
ful tool to prevent the spread of FMD, vaccination therapeutic doses. Treatment regimens and success
does not offer the Holy Grail. The successful eradi- rates of antiviral therapy in companion animals
cation of smallpox (1980) and rinderpest (2011) are poorly documented in scientific literature (Dal
can at least partially be attributed to the fact that Pozzo and Thiry, 2014; Hartmann, 2012).
these viruses existed in a single serotype and that The commercial availability of antiviral drugs
vaccination offered protection for at least several for large animals would have numerous advantages
decades in humans (smallpox) (Taub et al., 2008) in particular for viral diseases for which a good
or life-long in cattle (rinderpest) (Taylor et al., vaccine is not yet available, such as African swine
2006). Foot-and-mouth disease virus (FMDV), fever, or diseases for which vaccine strategies need
however, exists in seven different serotypes and a to be extensively tailored, such as FMD. In the case
variety of antigenic subtypes. Vaccine strains need of FMD manufacturers envisage an antiviral drug
to be antigenically ‘matched’ to the circulating field that is active against several serotypes, preferably all
strains and protective immune responses induced seven, and that exerts strong inhibition of the viral
by vaccination usually do not last longer than 6 replication from the first administration onwards.
months (see Chapter 12 and Domenech et al., This drug must be active at low compound con-
2010). After formulation – for emergency vaccines centrations and have a favourable Absorption,
this is usually from strategic frozen antigen stocks Distribution, Metabolism, Excretion – Toxicity
– FMD vaccines easily degrade if the stringent (ADME-Tox) profile, a high therapeutic index and
requirements for maintenance of the cold chain a high barrier towards the selection of resistant viral
are not respected. As FMD vaccines usually do not mutants. This drug should be stable at ambient tem-
contain preservatives, it is also essential to vaccinate peratures, have a long shelf life and should be easily
in a hygienic manner, according to good veterinary palatable, soluble in drinking water or formulated
practices. Once a vaccine flask has been punctured, into a bolus. With regards to food safety and the
the vaccine should be administered without any selection of resistant viral mutants in humans this
delay (Philippe Dubourget, personal communica- drug should preferably not be active against human
tion). Highly potent FMD vaccines rapidly induce viruses related to FMD, such as human entero-
a strong immune response but animals can still viruses or rhinoviruses, or have at least a short
develop clinical disease if the infection with FMDV withdrawal period. With regards to environmental
takes place within the first 4–7 days following vacci- safety it is preferred that this drug is not toxic for
nation (Golde et al., 2005). Emergency vaccination animal species after accidental uptake of medicated
is also ineffective if the animals are newly infected feed or drinking water. A persistent or ecotoxic
with FMDV before the time of vaccination. effect of the drug or its metabolites excreted by
Antiviral drugs are available for a number of treated animals in the environment is undesired.
human diseases such as HIV infection, hepatitis B In FMD-enzootic settings where vaccination
and C, influenza and various types of herpes and is not routinely applied ‒ usually in developing
provide not only a very efficient but also a very countries ‒ antiviral drugs might not only prevent
attractive way of medicinal treatment. There is a livestock to become infected with FMDV it might
significant interest in the commercial develop- also relieve disease and accelerate recovery in
ment of antiviral drugs for animals, but in Europe infected livestock and reduce mortality in young
only recombinant feline interferon omega is com- animals. In this way antiviral drugs would reduce
mercially available. Up-to-date companion animals losses of milk, meat and draught power and reduce
are increasingly treated with antiviral drugs that decreased fertility, enhancing the food security and
were originally developed for humans. Therapy is contributing to an improved household income
and livelihood of smallholders (Bayissa et al., 2011; from natural products. Induction of type I IFN
Jemberu et al., 2014). We are however well aware using strategies based on the administration of
that food safety is a major concern when antiviral pathogen-associated molecular patterns (PAMP)
drugs would be administered to smallholders’ live- and the type I IFN delivery (biotherapeutics) are
stock of which the products are intended for daily discussed in Chapters 10 and 14, respectively.
consumption (milk) or have a high value (meat).
In settings where vaccination against FMD is
routinely applied, antiviral drugs might be a com- Nucleoside analogues
plementary tool. High potency vaccines that match The RNA-dependent RNA polymerase protein
the FMD outbreak strain are not always timely (3D) is the key enzyme in the genome replica-
available in sufficient numbers or vaccination might tion of FMDV and is one of the most conserved
be difficult to deploy in more remote areas. In such FMDV proteins (Carrillo et al., 2005). This makes
cases the administration of antiviral drugs might it an interesting target for broad spectrum anti-
immediately reduce viral replication, excretion and viral drug development. In general, nucleoside
transmission, irrespective of the serotype of the analogues need to be phosphorylated to their
FMD outbreak strain and independent of technical 5′-triphosphate metabolite to inhibit the function
requirements such as maintenance of the cold chain. of the polymerase at the active sites. Triphosphates
In zones or countries that were previously free from can be incorporated as building blocks in the viral
FMD, antiviral drugs might as well bridge the time nucleic acid chain in competition with their normal
gap between the moment of vaccination and the counterparts inducing lethal mutagenesis or chain
moment that vaccinated animals are effectively termination.
protected (‘immunity gap’). Moreover, simulation The guanosine analogue ribavirin (1-β-d-r
exercises with classical swine fever in densely popu- ibofuranosyl-1H-1,2,4-triazole-3-carboxamide)
lated pig areas have shown that antiviral drugs might was discovered in 1972 and exhibits a wide antiviral
constitute a cost-effective, stand-alone alternative activity against numerous DNA and RNA viruses
for emergency vaccination (Ribbens et al., 2012; both in vitro and in vivo, including several members
Backer et al., 2013). Ideally, antiviral drugs can be of the Picornavirus family (Andersen et al., 1992;
quickly and safely administered in the feed or drink- Carasco, 1994). Known mechanisms by which
ing water or as a bolus and are an easy and efficient ribavirin exerts its antiviral action are (1) inhibition
way to treat large herds. In case of a large outbreak, of the viral RNA polymerase; (2) lethal mutagen-
this requires that antiviral drugs are sufficiently esis; (3) inhibition of the cellular enzyme inosine
available from stockpiles. In small herds (<30 ani- monophosphate dehydrogenase (IMD) which
mals) antiviral drugs might be a good alternative induces a depletion of the synthesis of guanosine
for emergency vaccination as the currently available triphosphate (GTP); (4) inhibition of mRNA cap
ELISAs that differentiate infected from vaccinated formation and (5) immunomodulation by induc-
animals are not sufficiently capable of detecting a tion of a helper-T-cell type 1 cytokine response and
single FMDV-infected animal in a small vaccinated suppression of the helper-T-cell type 2 response. In
herd (Paton et al., 2006). Antiviral drugs might also humans ribavirin is mainly used in the treatment of
be a valuable alternative for (pre-emptive) culling chronic hepatitis C virus infection (in combination
of endangered species and rare breeding stocks. with interferon alpha), respiratory syncytial virus
Antiviral drugs might also protect young animals and Lassa fever virus. Ribavirin showed some in
from disease if the presence of maternally derived vitro activity against veterinary pathogens of domes-
immunity in young animals hampers sufficient tic animals like canine parvovirus, feline calicivirus,
immunization through vaccination. feline coronavirus, but data on in vivo efficacy are
In this chapter we will systematically review the often lacking and the safety margin is usually very
history and the state-of-the-art of antiviral drugs narrow (Povey, 1998; Weis et al., 1993; Radford et
against FMD, from nucleoside analogues, small al., 2007; Hartmann and Ritz, 2008). Also against
chemical molecules and oligonucleotides over new FMDV, ribavirin showed antiviral action in vitro.
molecular biological techniques such as RNA inter- Already in 1987, de la Torre and co-workers demon-
ference and single-domain antibodies to derivatives strated that treatment of baby hamster kidney cells
(BHK-21) persistently infected with a serotype C reported an EC50 of 27 µM against the same dose of
FMDV (C1-BHK-Rcl) with 150 µg/ml ribavirin for FMDV strain O/SKR/2002 in IBRS-2 cells (Kim
72 hours resulted in undetectable levels of FMDV et al., 2012a). In that study, it was also shown that
genome and viral proteins with maintenance of the the replication of FMDV O/SKR/2002 was more
cell-viability. Cessation of the treatment did not potently reduced in IBRS-2 cells that were treated
result in re-emergence of the FMDV replication with a combination of ribavirin and a recombinant
in over 20 cell passages. The persistent infection adenovirus expressing porcine interferon-α than in
was 10 times more sensitive to ribavirin than an cells treated with one of these agents only (Kim et
acute infection of BHK-21 cells with concentra- al., 2012a).
tions that reduced the virus yield to 50% (effective Mutagenesis leading to virus extinction through
concentration EC50) of 3–6 µg/ml and 30–50 µg/ error catastrophe when the mutation rate exceeds
ml, respectively. In addition, ribavirin treatment a certain threshold value is an extensively studied
was demonstrated to delay and decrease death rates antiviral mechanism. The quasispecies dynamics
in mice infected with FMDV C (de la Torre et al., and the influence on the structure of the 3D viral
1987). Further, a 3-day ribavirin treatment (100– protein are further elaborated on in Chapters 7 and
500 µM) of BHK-21 cells persistently infected with 6. Here the focus will be on the effects of ribavirin
a European serotype C FMDV (C-S81c1) resulted on FMDV. Serial passages of the C-S81c1 strain on
in a ~ 4 log10 reduction in virus titre. This effect cells pre-incubated with increasing concentrations
was counteracted by the simultaneous addition of of ribavirin (up to 800 µM) resulted after 25 pas-
guanosine indicating that the inhibition of IMD is sages in the selection of FMDV with a decreased
involved in the mechanism of action. Importantly, sensitivity to ribavirin and an 7.6-fold increase in
the mutation frequency in the VP1 and 3D genome viral fitness in the presence of ribavirin (Sierra et
region of FMDV treated with ribavirin increased al., 2007). The specificity of the selective advantage
up to 10-fold when compared to untreated controls to grow in the presence of ribavirin of the viruses
(Airaksinen et al., 2003). A similar increase in muta- with a M296I substitution in the 3D protein,
tion rate (up to 11%) was found in the VP1 and 3D which was reported in the viruses passaged in the
genome region of FMDV serotype O passaged one presence of ribavirin compared to the controls
time in the presence of ribavirin (1000 µM) (Gu et passaged in the absence of it, was confirmed using
al., 2006) indicating the mutagenic effect of ribavi- chimeric plasmids. The mutant viruses showed
rin on the FMDV genome. Ribavirin inhibited the a decreased capacity to incorporate ribavirine
in vitro replication of other FMDV serotypes as well. triphospate instead of guanosine triphosphate.
Upon passages of FMDV serotype O in the in pres- Importantly, the M296I mutation hardly affected
ence of ribavirin (400 µM) a gradual decline in viral the viral replication in vitro in the absence of riba-
RNA production up to 100% in the fifth passage virin (Sierra et al., 2007; Ferrer-Orta et al., 2010).
was reported. The viral replication had decreased in Similarly, 18 passages of FMDV serotype Asia1
such a way that infectious virus and viral proteins in the presence of 50 µM ribavirin on BHK-21
could no longer be detected in the fourth passage cells pre-incubated with ribavirin generated virus
(Gu et al., 2006). Using a multi-cycle growth assay variants with decreased sensitivity to ribavirin that
in BHK-21 cells ribavirin was shown to inhibit remained stable upon six additional passages in the
virus-induced cytopathogenic effect (CPE) of six presence of 50 µM ribavirin. Genetic analysis of the
FMDV serotypes (O, A C, Asia1, SAT1 and SAT3) 3D gene revealed that a single D5N amino acid (aa)
with serotypes O and SAT1 being most sensitive substitution conferred for the decreased sensitivity
(EC50 of 350 and 526 µM, respectively) and sero- to ribavirin (Zeng et al., 2014).
types C and SAT3 being least sensitive (EC50 of The agent 5-fluorouracil (FU) is a base analogue
1867 and 1095 µM, respectively) (Goris et al., 2007, that mimics both uracil and thymine, 5-azacytidine
Osiceanu et al., 2014). On the other hand, the EC50 (AZC) is a chemical analogue of cytidine and
of ribavirin against 100 tissue culture infectious 6-azauridine (AZA) is an analogue of uridine.
dose (TCID)50 of FMDV O/SKR/2010 in swine These molecules are mutagenic agents developed
kidney (IBRS-2) cells was reported to be 1367 µM for and used mainly as chemotherapeutic agents
( Jeong et al., 2015) whereas earlier this group had against cancer. Serial passage of FMDV C-S8c1
in the presence of FU (0.769–7688 mM) or AZC valopicitabine (NM283) which was initially devel-
(41 µM) resulted in a 50- to 100-fold reduction oped as an inhibitor of the hepatitis C virus (Clark
of virus progeny compared to untreated controls. et al., 2005). Treatment of BHK-21 cells with
Viral extinctions only occurred when virus progeny 2′-CMC 30 min after virus inoculation resulted
from one passage was diluted at least 1000-fold in inhibition of CPE formation for seven FMDV
before the next passage. When the virus was less serotypes at EC50 values ranging from 1.4 to 10 µM
diluted, it survived and re-emerged in high titres as determined by a colorimetric cell-viability assay.
upon passage in the absence of FU or AZC (Sierra The potency of 2′-CMC to reduce virus-induced
et al., 2000). Treatment of IBRS-2 cells with CPE was 100–140 times higher than that of ribavi-
0.2 µM of AZA starting 24 hours prior to infection rin. The outcome of time-of-drug-addition studies
inhibited the replication of 100 TCID50 of FMDV indicated that the antiviral effect of 2′-CMC was
O/SKR/2002 resulting in a 3 log10 reduction of exerted through inhibition of the viral RNA replica-
viral RNA load at 48 hours post infection (hpi) tion suggesting a possible involvement of the viral
compared to untreated cells. Unlike with ribavirin, 3D polymerase (Goris et al., 2007; Osiceanu et al.,
the antiviral activity of AZA was not enhanced 2014). In vivo experiments in an FMDV-infection
when combined with porcine interferon-α, nor model in severe combined immunodeficient
when combined with ribavirin (Kim et al., 2012a). (SCID) mice (Lefebvre et al., 2010) showed that a
Guanidine hydrochloride (G) is a selective inhibi- 5-day treatment with 2′-CMC conferred complete
tor of picornaviruses and inhibits the replication of protection against disease in SCID mice infected
polioviruses, several coxsackieviruses, echoviruses, with A22 Iraq 24/64 compared to untreated mice
but not the hepatitis A virus. G also inhibits the rep- which all developed severe general symptoms and
lication of FMDV by action on the nonstructural had to be euthanized by 4 days post infection (dpi)
polypeptide P34 and inhibition of the initiation of (Lefebvre et al., 2014a). The clinical protection
the viral RNA synthesis (Saunders and King, 1982; was a reflection of the protection against viral rep-
Saunders et al., 1985). The virus progeny titre from lication. At 2 dpi the quantities of viral RNA in the
48 out of 49 FMDV strains including all 7 serotypes serum of the 2′-CMC-treated animals were on aver-
was reduced up to 5.55 log10 plaque-forming units age 1300-fold lower than in the serum of untreated
(PFU)/ml when grown in BHK-21 cells in the animals and at 14 dpi low levels of viral RNA were
presence of 5.2mM G. Based on their sensitivity detected in the serum, lungs, heart, spleen and liver
the FMDV serotypes could be divided into three of 2 out of 15 animals only (Lefebvre et al., 2014a).
groups (I) C, (II) O, A and Asia1 and (III) Sat1, Although several of the above-described mol-
2 and 3 which were most sensitive (Nettleton et ecules may seem prominent inhibitors of FMDV
al., 1982). Combined administration of FU and replication in vitro, at the present time several points
G reduced the viral progeny by 100 to 1000 times hinder the practical application to treat FMDV
compared to treatment with FU or G alone and infection under field conditions. The main concerns
resulted in virus extinction after two passages (Pari- at present are first, that suppression of the FMDV
ente et al., 2001). The mutation frequency in the 3D replication with these molecules requires high con-
coding region of FMDV treated with FU was up to centrations (e.g. for ribavirin the lowest reported
6.4 times higher than that of untreated virus (Sierra EC50 value was 122 µM, but for some serotypes it
et al., 2000, and reviewed in Pariente et al., 2005, exceeded 1000 µM). This would not only promote
and Agudo et al., 2009). Earlier it had already been adverse effects in terms of toxicity and an increased
shown that the growth of FMD strain O1 Pacheco chance of residues in products from food produc-
on BHK-21 cells in the presence of FU (200 µg/ ing animals but also implicate serious production
ml) led to the emergence of temperature sensitive and logistical costs. Second the mutagenic effect
variant viruses resistant to G at a concentration of exerted by the majority of these molecules on the
650 µg/ml (Lake et al., 1975; Saunders and King FMDV genome, a virus notorious for its extremely
1982; Saunders et al., 1985). error-prone genome replication, does not work in
Another ribonucleoside analogue with their advantage. Virus variants with reduced sensi-
reported antiviral activity against FMDV is tivity were readily selected upon and e.g. in the case
2′-C-methylcytidine (2′-CMC) or the oral prodrug of ribavirin did not have a reduced fitness to grow in
the absence of the antiviral compound. Combined inhibitor-treated cells the virus-induced degrada-
exposition of FMDV to antiviral agents with differ- tion of p220 and shut-off of host protein synthesis
ent mechanisms of action may in this sense be more is delayed (Kleina and Grubman, 1992).
advantageous than single-drug treatments. Third, The 3D gene of FMDV encodes the viral RNA-
very few in vivo data are available. dependent RNA polymerase which is the key
protein in the viral genome replication. With 74%
aa identities in the protein sequence among differ-
Small chemical molecules ent serotypes it is one of the most conserved FMDV
Small chemical molecules are designed to bind proteins (Carillo et al., 2005), making it an attrac-
with high selectivity to the active site of a critical tive target for the development of antiviral drugs.
viral enzyme or viral protein aiming at the irrevers- Screening of 2000 drug-like compounds of the May-
ible inhibition of the viral replication. Methods for bridge Hit Finder Library using a luciferase-based
antiviral drug discovery include high-throughput assay identified seven compounds that inhibited the
screening of compound libraries for in vitro activ- enzymatic activity of the FMDV (strain C-S8c1)
ity against the virus of interest and the design of 3D with EC50 values between 2.0 and 17.8 µM
molecules based on the knowledge of the potential (Ferrer-Orta et al., 2004; Durk et al., 2010). The
target sites combined with structural biology and compound 5D9 (4-chloro-N′-thieno, [2,3-d]
computational modelling. The extensive process pyrimidin-4-ylbenzenesulfonohydrazide) inhibited
from the identification of primary hit compounds the replication of FMDV A24 Cruzeiro/Brazil/55
over structure–activity-based optimization of (A24 Cruzeiro) on pre-treated BHK-cells in a
the antiviral activity in vitro to the clinical devel- dose-dependent manner. At a concentration of
opment of an effective and safe antiviral drug is 20 µM, 90% inhibition was achieved as determined
challenging. by plaque assay, in absence of apparent cytotoxic
The Leader protease (L) of FMDV is a thiol effects. Inhibition of the viral replication was
protease from the papain family that excises itself observed with several other compounds at a mul-
from the FMDV polyprotein and cleaves the cellu- tiplicity of infection (moi) of 0.01, but not at a moi
lar translation initiation factor eIF4G that interacts of 0.1. Binding of the compounds to a pocket in
with the internal ribosome entry site (IRES) to the 3D protein close to the NTP binding site and
initiate translation of the viral genome (López de the resulting non-competitive inhibition of 3D was
Quinto and Martínez-Salas, 2000). Kleina and shown to be responsible for the observed antiviral
Grubman (1992) demonstrated that the thiol activity (Durk et al., 2010). Further study using
protease inhibitor E-64 (l-trans-epoxysuccinyl- similar methodologies confirmed the antiviral
leucylamido(4-guanidino)butane) and the activity of 5D9 against FMDV A24 Cruzeiro. The
analogue E-64c completely inhibit the autocata- replication of FMDV A24 Cruzeiro inoculated at
lytic cleavage of the L protease of FMDV strains a moi of 0.01 was inhibited with 90% at 25 µM of
A12, O1 Caseros, A5, C3 Resende, SAT2, SAT3 5D9 and with 50% at 10 µM of 5D9. There was no
and Asia1 in an in vitro rabbit reticulocyte lysate significant cytotoxic effect on BHK cells that were
translation system at a concentration of 0.5 mg/ incubated with concentrations up to 200 µM for 24
ml. Upon infection of LF-BK cells with FMDV A12 hours as determined by a cell proliferation assay
or O1 Campos the more membrane-permeable (Rai et al., 2013). Serial passage of FMDV A24
analogue E-64d inhibited processing of L from P1 Cruzeiro in the presence of a gradually increasing
at concentrations of 200 or 500 µg/ml. Addition of concentration of 5D9 resulted in the absence of
the compound 1 hour before or after viral inocula- detectable virus after nine passages, by virus titra-
tion had no influence on its activity. A reduction tion and RT-PCR. Sequencing of virus after the
of viral protein synthesis and CPE formation and fourth and the eighth passages revealed single or
a decrease in virus yield up to 1000-fold were multiple mutations (P169Q, P44R, E259V, S335F
observed in treated cells compared with untreated and L376F) in the 3D polymerase of some of the
infected cells at 6 hpi. The cell monolayers were still randomly selected clones, but none of these variant
intact at 24 hpi, although there was a 50- to 100-fold clones became predominant in subsequent pas-
increase in virus yield over that at 6 hpi. In infected, sages (Rai et al., 2013).
Also with the aim of finding compounds aldehyde (GC373), α-ketoamide (GC375), and a
inhibiting the 3D polymerase of FMDV, Jeong dipeptidyl bisulfite adduct salt (GC376) with EC50
et al. (2015) screened 50,000 compounds of the values of 0.61, 0.55 and 1.16 µM, respectively. The
chemical library of the BIO-Centre in the GSTEP inhibitory effect on viral replication was confirmed
by analysis of dynamic interactions of the com- in cell-based assays for several picornaviruses but
pounds with the 3D polymerase and a polymerase FMDV was not tested. The compounds did not
elongation template. Among 150 derivatives of show any cytotoxicity at concentrations up to
the hit-compound 2-amino-4-arylthiazole were 500 µM (Kim et al., 2012b). To specifically target
designed, of which the most potent compound the FMDV 3Cpro, Roqué-Rosell et al. (2014)
exerted an inhibitory effect on the 3D polymerase modified a peptide corresponding to the optimal
activity with an EC50 value of 0.39 µM. However, in substrate at the C-terminus of this protease to
IBRS-2 cells infected with 100 TCID50 of FMDV produce inhibitors that react as Michael acceptors
O/SKR/2010 the EC50 value of this compound to with the active enzymatic site. The mechanism of
inhibit the viral RNA replication as determined by action involves the formation of a covalent complex
RT-PCR was 144 µM, and the lowest EC50 value with the 3Cpro. N-acetylated inhibitors containing
of 13 µM was achieved with another derivative. a peptide of 4 or 5 aa length potently reduced the
Important for the potency of these compounds was recombinant 3Cpro of FMDV with EC50 values of
the presence of a 3-CO2H-group and a 3-chloro/ 0.64–0.68 µM. When peptides were shorter the
fluoro-4-hydroxy-group indicating an electrostatic efficacy of the inhibitors significantly decreased and
effect related to the inhibition of the 3D polymerase it was nullified at a length of 2 aa. Of the different
( Jeong et al., 2015). analogues with modifications at the N-terminal side
Further, GPC-N114 (2,2′-[(4-chloro-1,2- that were generated, the analogue that incorporated
phenylene)bis(oxy)]bis(5-nitro-benzonitrile)), a benzoyl cap on the tetrapeptide was found to be
a broad-spectrum inhibitor of the 3D protein of most effective with an EC50 value of 0.43 µM. The
enteroviruses and cardioviruses, did not inhibit the presence of an unsaturated ester moiety showed
viral replication of FMDV serotypes O and A or of to be essential for efficient protease inhibition
equine rhinitis A virus (ERAV), another aphthovi- (Roqué-Rosell et al., 2014).
rus (van der Linden et al., 2015). The small molecule CHI-83 (1,2,4,5-
The 3C protease (3Cpro) of the Picornaviridae tetrahydro-[1,4]thiazepino[4,5-a]benzimidazole)
is another attractive target for potent inhibitors. inhibited the replication of FMDV serotypes O,
The 3Cpro of FMDV is involved in processing of the A, Asia1 and C in swine kidney (SK-6) cells with
FMDV polyprotein. This member of the trypsin EC50 values ranging between 8.0 µM and 52.4 µM,
family of serine proteinases also contributes to the but no inhibition was seen with FMDV serotypes
translational host shut-off by cleavage of eIF4AI. SAT1, SAT2 and SAT3 even at a concentration of
The aa sequence identity of 3C has been reported 100 µM CHI-83 (Lefebvre et al., 2014b). Time-of-
to be 76% across all FMDV subtypes (Carrillo et al., drug-addition assays with duration of one single
2005). In 1999, Patick et al. showed that AG7088 replication cycle of the FMDV strain O1 Manisa
(rupintrivir) irreversibly inhibits the 3Cpro of the (which was determined to be 6 hours (Goris et al.,
human rhinovirus. More recently, it was shown by 2007)) showed that addition of CHI-83 within
van der Linden et al. (2014) that AG7088 also inhib- 2 hours following infection resulted in a complete
its the 3Cpro of FMDV. Reported values for antiviral reduction of viral RNA synthesis. Addition of the
activity against FMDV are an EC50 of 22.4 µM in a compound at later time points (3 to 6 hpi) resulted
multicycle CPE reduction assay with O1 Manisa, in a gradual loss of the antiviral activity. When
an EC50 of 13.9 µM in a cell-based protease assay FMDV strains O and A were serially passaged in
and an EC50 of 4.21 µM in a FRET (fluorescence suboptimal concentrations of CHI-83 increasing
resonance energy transfer) protease assay. The same from 5 to 100 µM variant viruses which replicate
EC50 value of 4.21 µM in a FRET protease assay without apparent hindrance in the presence of
was observed by Kim et al. (2012b). In this FRET 100µM were obtained after four passages. Full
assay these authors demonstrated inhibition of the genome sequence analysis of these variant viruses
FMDV 3Cpro for protease inhibitors dipeptidyl revealed single aa mutations in the non-structural
protein 2C of the variant viruses compared with the development of fever, vesicular FMD lesions and
wild type viruses and viruses passaged four times lameness compared to untreated infected pigs. The
in the absence of the compound. This was in line T-1105 treatment also proved effective in reducing
with earlier findings with a precursor molecule of viraemia and nasal virus excretion in treated pigs, as
CHI-83 and suggests that CHI-83 interferes at a viral RNA was barely detectable in the their serum
stage that coincides with the onset of the viral RNA and no virus could be detected by plaque assay
replication, presumably by interacting with the viral from their nasal swabs from 1 to 6 dpi, whereas the
2C protein, a large membrane-associated RNA untreated infected pigs had high levels of viraemia
binding protein with ATP-ase activity (Lefebvre et on 1 to 3 dpi and excreted virus from 2 to 5 dpi. Fur-
al., 2014b). ther, the treated animals showed a lower anti-FMDV
The pyrazinecarboxamide derivative T-1105 antibody response than the untreated animals, sug-
(3-oxo-3,4-dihydro-2-pyrazinecarboxamide) was gesting a reduction of viral replication in the treated
first discovered as an influenza A virus inhibitor animal (Sakamoto et al., 2006, Furuta et al., 2009).
by the screening of a chemical library of Toyama Oral administration of T-1105 to guinea pigs at
Chemical Co., Ltd (Furuta et al., 2009). Another the same dose as administered by Sakamoto et al.
pyrazinecarboxamide derivative T-705 (favipira- (2006) to pigs, for five consecutive days starting
vir) has completed phase III clinical evaluation for one hour before viral challenge with the FMDV
influenza therapy in humans in Japan and phase II strain O1 Manisa adapted to guinea pigs resulted in
studies have been completed in the United States a significant reduction of FMDV-induced lesions
(Furuta et al., 2013). Knowledge about the mecha- and disease severity. At 2 and 4 dpi, viral RNA was
nism of antiviral action of the pyrazinecarboxamide only detected in a minority of the treated animals
derivatives results mainly from studies with influ- and viral RNA levels were significantly lower com-
enza viruses. T-705 was shown to be a prodrug that pared to untreated animals. Protection offered to
is phosphoribosylated intracellularly into the active guinea pigs by the oral administration of T-1105
form favipiravir-ribofuranosyl-50-triphosphate was comparable to protection offered by vaccina-
(RTP). Time-of-drug-addition studies indicate tion (De Vleeschauwer et al., 2014).
T-705 acts as a pseudo purine which may be mis- High-throughput screening of more than 70,000
incorporated in the viral RNA leading to lethal small chemical molecules from different compound
mutagenesis or it may act by binding to conserved libraries, including that of the Centre for Drug
polymerase domains preventing incorporation Design and Discovery (CD3) from KU Leuven
of nucleotides for viral RNA replication and tran- in Belgium resulted in the identification of several
scription. These pyrazinecarboxamide derivatives classes of antiviral compounds that selectively
were shown to exhibit antiviral activity in vitro inhibit the replication of FMDV in vitro and that
against members of several virus families including are currently being optimized to meet the require-
Bunyaviridae, Arenaviridae, Flaviviridae, Picorna- ments of an ‘ideal’ antiviral drug against FMDV as
viridae, Alphaviruses and Noroviruses (Furuta et described above (unpublished data). As an exam-
al., 2002, 2005, 2009, 2013; Gowen et al., 2007). ple, compound A inhibited the viral replication, as
Plaque reduction assay with FMDV O/JPN/2000 determined by the inhibition of CPE formation,
in IBRS-2 cells revealed that T-1105, T-1106 and the reduction of infectious virus progeny and viral
T-705 inhibited the viral replication with T-1105 RNA yield, of 6 out of 7 FMDV serotypes with EC50
being 10 times more potent than T-1106 and T-705 values between 0.89 µM and 29 µM on SK-6 cells.
(EC50 values of 1.6, 17 and 14 µg/ml, respectively) Only for SAT1 the EC50 was > 50 µM (Osiceanu
(Sakamoto et al., 2006; Furuta et al., 2009). The in et al., 2014). Data look promising but much more
vitro EC50 of T-1105 against FMDV O/SKR/2010 research needs to be done. None of the presently
in bovine kidney (IBRS-2) cells was 349 µM ( Jeong reported small chemical molecules is sufficiently
et al., 2015). Oral administration of T-1105 through potent for practical application to prevent or treat
the feed to pigs at a dose of 200 mg/kg twice daily, FMDV infections in the field. Compounds need to
starting 1 hour before the viral challenge with be optimized and EC50 values in the low nanomolar
106 TCID50 of FMDV O/JPN/2000 until 6 days region are desirable. In addition, the evaluation of
after infection protected the treated pigs from the the anti-FMDV activity is mainly based on in vitro
experiments and experiments in rodents and the VP1 protein was transiently inhibited (35–52%
question remains whether optimized molecules inhibition at 5 hpi) by the ODN targeted at the
will be effective to block FMDV replication in AUG2 sequence at concentrations of 125–250 µM
cloven-hooved animals. Other issues that remain (not 20 µM). However, no inhibitory effect on virus
to be explored include the pharmacological and yield was detected on BHK-21 covered with ‘AUG2’
toxicological profile, the stability and solubility, ODN containing medium 2 hours prior to and after
the genetic barrier to resistance of FMDV towards infection with O/Kaufbeuren at a moi of 0.001.
these compounds and the feasibility of large scale The optimized form of the ‘AUG2’ ODN which
production and administration to livestock. contained a phosphorothioate linkage reduced the
virus yield by ≥ 40% up to 19 hpi. Combined admin-
istration of the ‘AUG2’ ODN with other ODN did
Nucleic acid-based strategies not increase the viral inhibition (Gutiérrez et al.,
The study area of antiviral approaches against 1993). In addition, this group tested sense (s) and
FMDV of which the majority of reports originate antisense (as) RNA molecules that target the 3′ ter-
is the one using nucleic acid-based strategies. The minal region of FMDV serotype C (C-S8) (p-3′D
study of sense and antisense oligonucleotides to s and as, p-3′1 s and as, p-3′2 s and as) and the 5′
block FMD viral replication started in the early non-coding region including the proximal element
nineties. Studies on RNA interference (RNAi) of the IRES site and the two functional initiation
against FMDV first emerged at the beginning of AUGs (p-5′1 s and as) for their inhibitory effect on
this century. Antisense oligonucleotides and RNAi the translation of purified FMDV RNA in an in vitro
approaches act on the same principle: the binding rabbit reticulocyte lysate system. Of all molecules
of an oligonucleotide to a target RNA through tested, only the p-5′1 as molecule induced a signifi-
Watson–Crick base pairing. Sense sequences, on cant inhibition (68%) of the translation and protein
the other hand, may interact indirectly through synthesis compared to the untreated control. When
competition with viral sequences for cellular cofac- co-transfected with infectious homologous FMDV
tors required for viral RNA replication or more RNA all but the p-5′1 s molecule resulted in marked
likely through functional distortion of a structural reduction of the VP1 expression (~ 35–75% inhibi-
viral genome motif required for RNA replication tion, > 60% for p-5′1as) at 5 hpi and inhibition of
(Bigeriero et al., 1999). plaque formation (~ 25–85% inhibition, > 60% for
p-5′1as) at 36 hpi on BHK-21 cells. The degree of
Sense and antisense inhibition was inversely related to the length of the
oligonucleotides molecules. Injection of the p-5′1 as molecule in
Antisense oligonucleotides (ASO) are single- BHK-21 1 hour prior to infection with C-S8 (moi
stranded molecules of about 20 nucleotides of 0.01) inhibited plaque formation by 30–40%
designed to be complementary to target mRNA. (Gutiérrez et al., 1994). Combination of p-5′1 as
Base-pairing between the ASO and its target results with p-3′2 s or p-3′2 as increased the inhibitory
in RNase H-dependent degradation of the target effect compared to the single molecules (Bigeriero
RNA or direct inhibition of translation (reviewed et al., 1999). Inhibition percentages of heterolo-
in Cohen, 1991; Spurgers et al., 2008). gous FMDV serotypes O (O/Kaufbeuren) and A
The group of Professor Dr. Sobrino was the (A5Ww), with 79–91% sequence identity to the
first that described the anti-FMDV effect of ASO. homologous C-S8 strain in the target regions, were
Six antisense oligodeoxyribonucleotide (ODN) similar as those of C-S8 (Bigeriero et al., 1999).
sequences corresponding to the IRES (IRES1 The inhibition was specific for FMDV as the plaque
and IRES2), the functional translation initiation formation of EMCV or SVDV was not affected.
regions (AUG1 and AUG 2), the 2A gene and the The observed effects were dose-dependent and an
beginning of the 3′ NCR sequence of FMDV O/ excess (200- to 20,000-fold) of RNA molecules
Kaufbeuren were designed and micro-injected compared to FMDV RNA was needed to obtain the
either alone either as a mixture of two in BHK-21 reported antiviral effects. Efficient hybridization
cells that were immediately thereafter infected with between viral RNA and the transcripts was crucial
O/Kaufbeuren at a moi of 2. The expression of the for the inhibition of the PFU as PFU inhibition
percentages were markedly higher (up to 90% when PPMO targeting AUG1 reduced viral replication
p-5′1 as and p-3′2 s or p-3′2 as were combined) when of FMDV strain A12 to a similar extent as the A24
viral RNA and the oligonucleotides were allowed to but FMDV serotypes Asia1 and C to a lesser extent
anneal under renaturing conditions prior to trans- (~ 2 log10), and the PPMO targeting 5D resulted
fection into cells (Gutiérrez et al., 1994; Bigeriego in titre reductions from 2 log10 to 7 log10 of FMDV
et al., 1999). In further research, Rosas et al. (2003) serotypes O, Asia1, C, SAT1 and SAT2 at a PPMO
demonstrated that constitutive expression of p-3′1 concentration of 25 µM. Serial passage of FMDV
as, p-5′1 as separately or together in BHK-21 cells A24 Cruzeiro in the presence of concentrations
reduced the plaque formation (37–90%) and virus of ‘AUG1’ PPMO increasing from 0.5 µM to 1 µM
yield (37–99%) at 24 hpi and delayed CPE forma- resulted after three passages in the absence of CPE,
tion by ≥ 4 days after infection with 50–100 PFU undetectable viral RNA and the inability to isolate
of the homologous C-S8 strain, as well as with the infectious virus upon three blind passages. On
heterologous O/Kaufbeuren strain. C-S8 virus the other hand, five serial passages of FMDV A24
recovered from p3′1 as and p-5′1 expressing cells Cruzeiro in the presence of concentrations of ‘5D’
did not show a decrease in infectivity upon six fur- or ‘AUG2’ PPMO increasing from 0.5 µM to 2.5 µM
ther passages on these cells, nor were mutations in resulted in variant viruses that had advantages in
the target sequences found in the virus recovered growth in the presence of the PPMO compared
after the sixth passage (Rosas et al., 2003). with the wild type virus. Comparative sequence
analysis of the ‘5D’ and the ‘AUG2’ PPMO variant
Morpholino oligomers viruses, the wild type virus and the virus passaged
Phosphorodiamidate morpholino oligomers five times in the absence of PPMO revealed non-
(PMOs) are ASO in which the ribose ring of the synonymous nt substitutions in the PPMO target
nucleotide (nt) backbone is replaced with a mor- site (Vagnozzi et al., 2007).
pholine ring leading to an increased stability and
solubility and decreased off-target toxicity. Unlike RNA interference
ASO, PMO do not mediate their inhibitory effect via RNA interference (RNAi) is a cellular pathway
RNase-mediated degradation of their RNA target that plays a role in cellular gene regulation and may
but by interference with the mRNA processing and act as a cellular antiviral mechanism especially in
translation since they are typically designed against plants and invertebrates but the RNA silencing
IRES or AUG start codons (Spurgers et al., 2008). machinery is conserved in vertebrates (reviewed
Six peptide-conjugated PMO (PPMO) comple- in Gitlin and Andino, 2003; van Rij and Andino,
mentary to sequences in the 5′ and 3′ untranslated 2006; Arbuthnot, 2011). Although no direct
regions of the FMDV strain A24 Cruzeiro genome evidence of the function of RNAi as a natural anti-
were designed by Vagnozzi et al. (2007). Peptide viral mechanism in mammals has been reported
conjugation increases cell-uptake and persistence (Cullen, 2014), Elbashir et al. demonstrated in
of the PMO. The PPMOs targeting the 3′ side of 2001 that introduction small interfering (si) RNA
the IRES (5D) and the AUG translation initiation into mammalian cells could silence target genes
regions (AUG1 and AUG 2) induced a reduction in by RNAi. Since then, this mechanism has been
viral RNA production, viral protein expression and widely exploited as the subject of research aiming
viral titres up to 6 log10 in BHK-21 cells treated with at the development gene-specific therapeutics for
5 µM PPMO before and after infection with A24 viral diseases, including FMDV. Several RNAi
Cruzeiro at a moi of 0.5, whereas the activity of the approaches including plasmid- or vector-encoded
PPMO directed against the 5′ side of the S-fragment, short hairpin (sh) RNA and endogenous micro
the 5′ side of the IRES and the 3′UTR region was (mi) RNA complementary to the FMDV genome
less outspoken with reductions in virus titres up to have been studied for their antiviral effect against
2 log10. Cell viability staining indicated 5 µM as the FMDV in vitro and some in vivo and are discussed
maximum tolerable concentration of PPMO with- below (overview of in vivo studies in Table 15.1).
out apparent cytotoxic effects. In accordance with Some studies even aimed at the development of
the number of sequence mismatches between the transgenic animals naturally resistant to FMDV
designed PPMO and other FMDV serotypes, the infection. Some promising data have been
achieved, but generally these techniques, like s and cleavage of target transcripts through the activation
as oligonucleotides, have to overcome some obsta- of the RNAi pathway. Short hairpin (sh) RNA are
cles before practical and widespread use in the field plasmid- or vector-expressed and are intracellularly
of livestock diseases, and especially FMDV, may be processed into siRNA.
envisaged (Paroo and Corey, 2004; Grubman and Much research on this topic was led by Professor
de los Santos, 2005; Bayry and Tough, 2005). Ide- Dr Zheng at Fudan University in China. His group
ally for an effective and broad panserotype antiviral was the first to show a 80–90% sequence specific
activity against FMDV the target region in the viral reduction of the plasmid expressed VP1 region
genome is essential during the replication cycle of of the HKN/2002 FMDV serotype O genome
the virus and is highly conserved between different (pVP-EGFP-N1) in BHK-21 cells at 24 hours after
serotypes of FMDV. Moreover, the use of siRNA simultaneous transfection of these cells with plas-
as an antiviral agent could lead to a selective pres- mids expressing the inverted repeat of either a 21
sure on the siRNA target that might result in the or 63 nt sequence (shRNA) of this VP1(pNT21,
appearance of escape variants due to changes in pNT63). Reduction was determined by immu-
the target sequence, therefore high sequence iden- nofluorescence microscopy and by fluorescence
tity between the interfering RNA and the target activated cell sorting based on the expression of
sequence is warranted and simultaneous targeting the enhanced green fluorescent protein p-EGFP-
multiple genomic regions may be useful. N1 by the reporter plasmid containing the VP1
target sequences and co-transfected with the
SiRNA and shRNA shRNA-expressing plasmid and RT-PCR analysis.
Introduction of exogenous double-stranded (ds) In addition, transfection of BHK-21 cells with
RNA into cells results in the sequence specific either the pNT21 or pNT63 plasmid 24 hours
post-transcriptional silencing of complementary prior to infection with 100 TCID50 of HKN/2002
mRNA. Small interfering (si) RNAs are generally delayed CPE formation up to 24 hpi, but not up
21 to 23 nucleotide ds RNA molecules that mediate to 48 hpi. The virus yield at 12 hpi in transfected
cells was reduced by 1000-fold compared to mock- serotype Asia1 (YNBS/58) 10-fold reduction was
transfected control cells, but this effect gradually seen with the 5′NCR, Pol and 3′UTR siRNA only
waned and no difference was seen at 72 hpi. The and the effect lasted up to 48 hours but not 60 hpi.
inhibition of viral replication was demonstrated to Despite the significant reduction seen with siRNA
be sequence specific as it was not seen when cells treatment, homologous HKN/2002 virus titres still
were infected with another serotype O FMDV reached up to > 4 log10 TCID50 per 100 µl superna-
strain (CHA/99) or a pseudorabies virus. In tant at 48 hpi. Infection of BHK-21 cells 1 hours
suckling mice, subcutaneous (SC) administration before transfection resulted in an increased RNAi
of either pNT21or pNT63 plasmid at 6 hours effect (delayed CPE formation and lower virus
prior to challenge with 20 lethal dose (LD)50 of titres) and the suppression of the viral replication
HKN/2002, resulted in a survival rate of 75% of the was prolonged until 198 hpi as compared with pre-
mice at 5 dpi. No or borderline VP1 mRNA could treated cells with all but the 3′NCR specific shRNA
be detected in the internal organs of the surviving (150 hpi) (Liu et al., 2005).
treated mice at 5 dpi, whereas mock-treated animals To address difficulties that might be associated
all died within 36 hpi with high amounts of visceral with the in vitro and in vivo delivery of RNAi, this
VP1 mRNA. The protective effect of the pNT21or research group used similar plasmid construc-
pNT63 plasmids was less outspoken when admin- tion techniques (Chen et al., 2004) to examine
istered simultaneously with virus challenge or when the efficacy of shRNA directed against the VP1
the viral challenge dose was higher (100 LD50). (pAd5-NT21) and 3D (pAd5-Pol) region of
Simultaneous administration of the VP1 expression FMDV HKN/2002 delivered by a recombinant
plasmid together with the VP1 specific shRNA 6 replication-defective human adenovirus type 5
hours prior to challenge increased the survival rate (rAd5). Treatment of IBRS-2 cells at a moi of 5 and
of mice compared with that in mice challenged after 10 with pAd5-NT21 or pAd5- Pol 12 hours before
only being given the VP1 specific shRNA (Chen et infection with 100 TCID50 of HKN/2002 inhibited
al., 2004). the formation of virus-induced CPE until 72 hpi,
This research group further explored the use reduced the viral RNA yield at 36 hpi and the
of siRNA targeted to various more conserved virus yield in the supernatant at 72 hours by 100%
regions of the FMDV genome which were selected compared with the untreated control. When chal-
based on sequence similarity (85–98%) of FMDV lenged with CHA/99 only pAd5-Pol inhibited the
strains of serotype O, A, C and Asia1 in the NCBI CPE formation and the virus yield. Typical CPE,
database with the HKN/2002 strain. The selected high viral RNA and virus yield were obtained in
genome regions included the 5′ non-coding region untreated FMDV-infected and treated PRV infected
(5′NCR), the 3′ non coding region (3′NCR), VPg, control cells from 24 hpi on. Intramuscular (IM)
VP4 and 3D (Pol). ShRNA expressing plasmids injection of 106 PFU of pAd5-Pol in guinea pigs 24
and p-EGFP-N1 reporter plasmids containing the hours prior to intradermal challenge with 50 (infec-
target sequences were constructed using similar tious dose) ID50 of HKN/2002 mitigated clinical
techniques as Chen et al. (2004). In BHK-21 cells signs and protected three out of five animals com-
transfected with these shRNA plasmids, the expres- pletely from vesicular lesions and fever that were
sion of EGFP and the target RNA yield at 24 hours present in all untreated control animals. However,
post transfection (hpt) was significantly reduced when challenged with 200 LD50 only one out of five
compared to mock controls, and this was similar for treated animals was protected. Treatment results
all target sequences. Infection of BHK-21 cells with were also less favourable for pAd5-NT21. Treat-
100 TCID50 of HKN/2002 5 hours after transfec- ment of guinea pigs with a mixture of pAd5-NT21
tion and titration of the supernatant at 24 and and pAd5-Pol and viral challenge 24 hours later, or
48 hpi revealed a 10- to 1000-fold reduction in virus twice with this mixture with a 24 hour interval and
titres compared with mock controls. The antiviral viral challenge at the time of the second treatment
effect was extended to 6 dpi. When challenged with did not increase clinical protection. Large white
a heterologous serotype O (CHA/99) a similar pigs inoculated IM with a ‘low’ dose of 2 × 109 PFU
reduction in virus yield was observed at 48 hpi with for each of a mixture of pAd5-NT21 and pAd5- Pol
all siRNAs, whereas upon challenge with FMDV and challenged 24 hours later with 100 ID50 of
HKN/2002 were markedly protected against the resulted in a survival rate of 20% with 20 LD50 of
development of virus induced vesicular lesions and HKN/2002 at 5 dpi and up to 40% with YNBS/58,
fever that were present in all control pigs from 5 dpi whereas 100% of the mock-treated mice had died
on. Only one out of three treated pigs showed mild within 60 hpi. IM administration of 109 CFU
vesicles at 15 dpi. When pretreated with a double of the recombinant rS. cho vector carrying the
dose (4 x 109 PFU each) only one out of three ani- p3D-NT56 shRNA (p3D-NT56/S. cho) to guinea
mals was completely protected against clinical signs, pigs 36 hours prior to intradermal challenge with
but in the two other pigs disease was delayed (6 and 50 ID50 of HKN/2002 protected four out of five
10 dpi) and milder compared with control-treated animals from vesicular lesions and fever that were
animals. Upon infection, control-treated animals seen in all mock-treated animals. However only one
showed high degree of viraemia at 6 dpi but not at out of five animals was protected when pre-treated
14 dpi and virus neutralizing antibodies at 14 and with 108 or 1010 CFU of this recombinant. Com-
21 dpi. None of the pigs treated with the low dose bined administration of the 106 CFU of pAd5-Pol
had detectable viral RNA in their serum at 6 dpi, (Chen et al., 2006) and 109 CFU of p3D-NT56/S.
and only the one pig showing delayed clinical signs cho did not increase the level of protection as two
was viraemic at 14 dpi, but cross-contamination out of five animals developed clinical FMDV signs.
from infected animals could not be excluded. Virae- In swine IM injected in the neck with 5 × 109 CFU
mia was not examined in the pigs treated with the of p3D-NT56/S. cho and IM challenged in the
double dose. All pigs that showed clinical disease neck with 100 ID50 of HKN/2002 24 hours later
developed significantly higher antibodies against the disease onset was delayed to 10 dpi and disease
3ABC polyprotein of FMDV than animals that severity was much lower than that of mock-treated
were protected from clinical symptoms (Chen et control animals. The latter displayed severe disease
al., 2006). from 3 dpi onwards. Pigs treated with 5 × 109 CFU
To further meet the challenges of the high of p3D-NT56/rS. cho also developed lower virus
genetic variability of FMDV and in search of an neutralizing and 3ABC antibodies suggesting
optimal vector for delivery of siRNA, the group of the amount of virus in these animals was reduced
Professor Dr Zheng constructed plasmids express- compared with control animals. Increase of the
ing shRNA targeted at conserved genome regions dose of from 5 × 109 CFU to 5 × 1010 CFU, how-
that corresponded to 3D gene sequence fragments ever, decreased the level of protection (Cong et al.,
of O/HKN/2002 (P3D-NT19 and p3D-NT56), 2010).
VP4 gene sequence fragments of Asia1/YNBS/58 Several other research groups used RNAi tech-
(pVP4-NT19 and pVP4-NT65) or a 2B gene nologies to target structural and/or non-structural
sequence fragments of O/CHA/99 (p2B-NT25) viral targets. A chronologic review of these studies
and introduced those in a recombinant attenu- is presented. Kahana et al. (2004) aligned all FMDV
ated Salmonella choleraesuis delivery vector (rS. sequences available in GenBank at that time to iden-
cho). This vector was chosen because unlike the tify three regions with sequences of at least 22 bp
rAd5-vector the rS. cho vector could be more with 100% identity in all FMDV sequences, leading
often recovered from the lymph node, tonsils, lung to the design and synthesis of one shRNA target-
and alimentary tract of treated pigs, sites that cor- ing the 2B region and two targeting the 3D region.
respond more with the infection routes of FMDV In BHK-21 cells transfected with shRNA using an
than the liver which was reported to be the main site RNAi shuttle and immediately thereafter infected
of rAd5-vector recovery. The virus titres of FMDV with 103 PFU of FMDV serotype O1 Geshur, viral
serotype O strains HKN/2002, CHA/99 and sero- replication at 24 hpi was inhibited by 80–97% as
type Asia1 strains YNBS/58 and Jiangsu/2005 was determined by viral RNA synthesis when transfec-
30–300-fold reduced at 48 hpi with 100 TCID50 of tion was done with each of the shRNA separately
BHK-21 cells transfected with the p3D-NT56 or and > 98% when a mixture of all three shRNA was
the p2B-NT25 shRNA compared with the controls, used. Virus yield at that time was inhibited > 90%
whereas the other shRNA induced cross-serotype and 100%, respectively. The amount of negative-
inhibition to a lesser extent. SC injection of suck- sense RNA, reported as an essential element of the
ling mice with p3D-NT56 or p2B-NT25 shRNA viral replication, was inhibited in a similar order of
magnitude. At 30 hpt the cellular mRNA amount in 48 hpi a 100- and a 50-fold, respectively, but further
transfected cells was similar to that of untransfected experiments with the 2B1 shRNA showed that
cells suggesting no adverse effect of transfection on virus titres at 72 hpi reached similar levels as con-
the cell-viability (Kahana et al., 2004). trols (> 106 PFU/ml). The inhibitory effect of 2B1
Mohapatra et al. (2005) infected BHK-21 cells was shown not to be mediated by the activation of
transfected with a mixture of 12–30 base pair (bp) the IFN pathway. Double transfection of IBRS-2
long siRNA generated by in vitro transcription cells with 2B1 shRNA with a 20 hour interval and
against the genome regions VP3-VP1, 2A-2C or challenge 24 hours after the second transfection
3D-3′UTR of FMDV Asia1 (IND 63/72) with resulted at 6 hpi in a decrease in total viral RNA
100 TCID50 of this FMDV strain. The virus yield production of 70% and a >80% decrease in viral
of IND 63/72 in the transfected cells was > 90% proteins 2B, VP0, VP1 and VP3 as determined by
reduced in the cell supernatant at 24 hpi, compared Western and Northern Blot analyses compared to
with controls. The greatest effect (120-fold reduc- mock-transfected control cells. In addition, infec-
tion in virus titre) was seen with the siRNA mixture tion with different FMDV serotypes of 2B1 shRNA
directed against the 2A–2C region at 24 hpi. At transfected IBRS-2 cells revealed reduction of virus
48 hpi the antiviral effect had nullified. In addition, titres at 24 hpi compared to controls of 97% for the
these authors constructed 21 bp siRNAs against homologous A12-IC strain and 92% for the heter-
the L-5′UTR and the 2B–2C junction regions, as ologous strain O1 Campos, between 74 and 88% for
alignment of FMDV serotype O, A, C and Asia1 Asia1, O/Taiwan/2/99 and SAT2, and only 2% for
had revealed these regions as highly conserved C3 Resende, indicating the 2B region – being one
between the serotypes. In BHK-21 cells transfected of the most conserved sequences across all FMDV
with these siRNA a > 99% inhibition of the repli- serotypes – as another potential target for silencing
cation of FMDV serotypes O (O/IND/R2/75), of multiple FMDV serotypes (de los Santos et al.,
A (A/IND/17/77), C (C/BOM/64) and Asia1 2005).
(IND 63/72) was observed at 24 hpi. At 48 hpi, the Transient siRNA expression in BHK-21 cells
inhibition was > 87% against all four serotypes with transfected with plasmids bearing 21–23 nt of the
the most potent siRNA directed against the 2B–2C 3D (pSiFMD2) or 2B1 (pSiFMD3) sequence of
junction. Despite the high percentages of inhibition FMDV 24 hours prior to infection with FMDV
of the virus replication by these 2 siRNA, virus serotype O, A and Asia1 (5 × 10³ TCID50/cell)
titres in the supernatant still reached up to 4.5 and barely reduced the replication of FMDV as indi-
5.6 log 10 TCID50/ml at 24 hpi and 48 hpi, respec- cated by the formation of CPE at 24 hpi and the
tively. Serial passage of the supernatant collected at virus yield in the supernatants at 18 to 48 hpi,
24 hpi from siRNA transfected cells did not induce which was only slightly lower than that observed in
the emergence of mutant viruses at passage level 2 mock-transfected and untransfected controls. SC
(Mohapatra et al., 2005). administration of 5–100 µg plasmids to suckling
De los Santos et al. (2005) selected seven con- mice 6 hours before challenge with 5 LD50 FMDV
served target regions (CRE, IRES1, IRES2, 2B1, serotype O and Asia1 resulted in a survival rate of
2B2, 2C and 3D) from FMDV A12-IC based on the 30 to 40% at 5 dpi whereas mock-treated mice all
alignment of sequences of the seven FMDV sero- had died within 3 dpi. When challenged with 20
types to construct shRNA containing plasmids and LD50, the survival rate at 5 dpi decreased to 10%
corresponding firefly luciferase reporter plasmids. (Pengyan et al., 2008).
Upon transfection of human embryonic kidney Joyappa et al. (2009) identified three highly con-
(HEK)-293T-cells specific silencing of luciferase served sequences in the 3D genomic region and one
activity of 90% was obtained with the 2B1 shRNA in the IRES region by sequence alignment of strains
and 50% with the IRES1, IRES2 and 3D shRNA of three serotypes of Indian origin. Corresponding
at 24 hpt, but not with the other shRNA. Similar shRNA were tested for their inhibitory effect of
findings were done when IBRS-2 cells were trans- viral replication in BHK-21 cells transfected with
fected with the shRNA and infected 48 hours later one of these shRNAs and infected with 100 TCID50
with 100 PFU of FMDV A12-IC, i.e. 2B1 shRNA of FMDV O/R2/75 18 hours later. The shRNA
and IRES1 shRNA inhibited the virus replication at targeting the IRES region did not exhibit any
inhibitory effect on viral replication in vitro. Two lower using the Ad–2B construct alone with 70%
shRNA targeting 3D decreased viral RNA yield and reduction in viral RNA, ~ 100-fold reduction in
virus titres in supernatant at 24 hpi ~ 10,000-fold virus yield and ~ 45% in viral protein load. Cells
compared with the control. Upon IM administra- pre-treated, pre- and post-treated at 6 hpi or post-
tion of either of these two shRNA targeting the 3D treated at 6 hpi with a combination of Ad-3C1 and
region ≥ 80% of guinea pigs was protected from Ad–2B construct showed a similar ~ 1,000-fold
vesicular lesion development when challenged with inhibition of viral RNA at 48 hpi. At 72 hpi a simi-
103 ID50 of O/R2/75 24 hours later ( Joyappa et al., lar high degree of inhibition was only seen in the
2009). pre- and post-treated cells and the inhibitory effect
The VP1 gene of FMDV was also the target had declined most in the pre-treated cells. Post-
for shRNAs developed by Lv et al. (2009). Five treatment with the Ad-3C1 and Ad–2B constructs
out of 24 designed shRNAs silenced ≥ 60% of at 1 hpi or 12 hpi inhibited the RNA yield most
the expression of EGFP from reporter plasmids effectively (~ 1000-fold at 72 hpi), whereas this
containing the VP1 gene of FMDV O (O/NY00) effect was abolished when cells were post-treated
in a sequence specific manner in HEK-293T-cells. at 24 hpi. Intraperitoneal (IP) infection of mock-
A reduction of 76–97.5% in viral RNA yield was treated suckling mice with 125 LD50 of FMDV
obtained with four shRNAs in BHK-cells and three O/SKR/2002 resulted in a survival rate of 20% at
of these delayed CPE development by 6–12 hours 3 dpi and 0% at 6 dpi. When suckling mice were
and reduced the level of viral progeny ~ 100-fold 24 administered Ad-3C1, Ad-2B or a combination IP
hours after infection with O/NY00, but the differ- at both 24 hours and 6 hours prior to viral challenge
ence with mock-transfected control cells gradually the survival rate increased to 58% at 7 dpi. Further-
waned at 36–48 hpi. Mixing the shRNAs did not more, an additional injection with Ad-construct at
increase the inhibitory efficacy. One extra passage 3 dpi enhanced the survival rate up to ~ 80% at 7 dpi
of virus recovered from shRNA treated cells on (Kim et al., 2008).
cells expressing the same shRNA, did not induce Having in mind the variable nature of the FMDV
reduced susceptibility of the virus to the shRNA. and the low fidelity of the viral RNA polymerase,
On the other hand, a single point mutation in the it is not inconceivable that a selective pressure is
target region of a shRNA, which was naturally exerted on the viral target by siRNA. This might
present in the O/NY00 virus stock, reduced the give rise to the emergence of variant viruses with
silencing effect of this shRNA by 3.7-fold (Lv et al., reduced sensitivity to this siRNA. The approach of
2009). simultaneously targeting several FMDV genomic
The majority of results have been achieved by regions to counteract the viral mechanism to escape
transfection of siRNA into cells, and this may be the RNAi effect was examined. Kim et al. (2010)
problematic for practical applicability of RNAi. In studied the effect of co-expression of multiple
attempts to get around in vitro and in vivo problems shRNAs from a single vector against FMDV. There-
associated with the delivery of RNAi different fore, additional recombinant adenovirus constructs
promoter systems and delivery vectors have been simultaneously expressing shRNA targeting the 2B
studied. Recombinant adenovirus constructs and 1 or 2 3C sequences of the FMDV serotype O
expressing shRNA targeting the 3C (Ad-3C1) or (O/SKR/2000 and O/SKR/2002) under control
2B (Ad-2B) region of the FMDV serotype O (O/ of three U6 promotors (Ad-U63B-U63C1and
SKR/2000 or O/SKR/2002) were used to infect Ad-U63B-U63C1-U63C2) or U6 and CMV
IBRS-2 cells prior to and at various time points promotors (Ad-U62B-U63C1-CMV3C2) were
after infection with 100 TCID50 of FMDV strain O/ generated. The inhibitory effect of these shRNA on
SKR/2002. Pre-treatment of cells with the Ad-3C1 FMDV O/SKR/2002 was determined by RT-pPCR
and a combination of Ad-3C1 and Ad–2B construct and virus titration at 24 and 48 hpi. To this end,
prior to virus challenge resulted in a > 90% reduc- IBRS-2 cells were treated 12 hours before infec-
tion of the RNA yield, a respective ~ 10,000- or tion with 100 TCID50 of FMDV O/SKR/2002.
~ 1,000-fold reduction in virus titre and a reduction The inhibitory effect on RNA and virus yield of
of viral protein as determined by antigen ELISA of the Ad–U63B-U63C1 construct was ~100-fold
~ 90% and ~ 65%, respectively. The inhibition was higher than that of the single Ad-3C1 and Ad-2B
constructs at 48 hpi. The antiviral effect of single with A22/IRQ 24/64 or Asia1/MOG/05, was less
constructs Ad-U63C2 or the Ad-CMV3C2 was pronounced, with survival rates of ~ 50–70% at
in the same order of magnitude as that of Ad-3C1 7 dpi (Kim et al., 2010).
and Ad-2B, but the combined administration of Targeting more than one viral gene was also
Ad-U62B-U63C1 together with one of the Ad- studied by Xu et al. (2012) through the devel-
U63C2 or the Ad–CMV3C2 constructs decreased opment of an Ad-5 vector carrying VP1 and
the effect of the former. In contrast, when the three 2B shRNAs against the genome of FMDV O/
different shRNAs were simultaneously expressed HK/2002 expressed alone or in tandem (Ad-VP1,
in one construct (Ad-U63B-U63C1-U63C2 Ad-2B and Ad-VP1–2B). In IBRS-2 cells pretreated
and Ad-U62B-U63C1-CMV3C2) the anti- with Ad-VP1–2B at a moi 10 and infected with 100
viral effect was ~ 100-fold enhanced at 54 hpi PFU 12 hours later, the titres of O/HK/2002 were
with Ad-U63B-U63C1-U63C2 being most reduced by a ~1000-fold at 48 hpi compared with
effective. The effect seemed to be dose depend- the controls, and this reduction was superior to
ent, as increasing the Ad-construct dose from that of treatment with the single shRNAs or a com-
3 x 106 TCID50 to 1 x 107 TCID50 increased bination of them. Marked reduction of viral RNA
the observed antiviral effect. Treatment of the production (>99%) was obtained when IBRS-2
cells with Ad-U63B-U63C1-U63C2 showed cells were treated with Ad-VP1–2B 12 hours before
toxic to IBRS-2 cells as the cell viability was or at the time of viral infection with O/HK/2001
reduced by ~ 47% compared with untreated but reduction decreased or absent when treated at
control cells. This toxic effect was not seen with 12 or 24 hpi. Pre-and post-treatment resulted in a
Ad-U63B-U63C1-CMV3C2. Cross-inhibition of complete absence of viral RNA until 48 hpi and a
FMDV serotype O, A, Asia1 and C occurred in > 99% reduction at 72 hpi. IM injection of guinea
IBRS-2 cells with Ad-U63B-U63C1-U63C2 or pigs with a high dose (108 PFU) of Ad-VP1–2B
Ad-U62B-U63C1-CMV3C2, but was lower against and intradermal challenge with 100 ID50 of O/
FMDV serotype SAT1, SAT2 and SAT3. These HK/2002 24 hours later resulted in a protection
observations corresponded to a complete sequence against virus induced lesions and mortality of 60%
identity of the 2B target region between serotype of the animals until 7 dpi which was higher than
O and A, and of the 3C1 and 3C2 regions between the protection conferred by the single or combined
serotype O, A, Asia1 and C, whereas the SAT administration of the Ad-shRNAs. Treatment at
serotypes each showed seven differences in the 2B the time of viral challenge or double treatment
region and two to four differences in the 3C1 and with the second treatment at the time of challenge
3C2 regions. Intraperitoneal infection of mock- did not increase the protection rate. However,
treated suckling mice with 125 LD50 of FMDV O/ triple treatment before, at the time of challenge
SKR/2002 resulted in survival rate of 20% at 4 dpi and 48 hours thereafter increased the protection
and 0% at 6 dpi. When suckling mice were adminis- rate up to 80%. Viral challenge 48 or 72 hours after
tered Ad-U63C2 or Ad-CMV3C2 IP, survival rates administration of Ad-VP1–2B decreased the rate
were similar to those reported earlier with Ad-3C1, of protection to 40% and 20% respectively at 7 dpi
Ad-2B and Ad-U62B-U63C1 (Kim et al., 2008). (Xu et al., 2012).
Treatment with a mixture of Ad-U62B-U63C1 and Another approach addressing the issue of the
Ad-U63C2 slightly enhanced survival rate but this highly genetic variability and high mutation rate of
difference was not statistically significant. However, FMDV is to target a host cell sequences instead of a
mice treated with Ad-U62B-U63C1-U63C2 or virus sequence. Using a lenti-viral vector expressed
Ad-U62B-U63C1-CMV3C2 had a survival rate RNAi targeted to the porcine integrin αv subunit of
of 90% at 7 dpi, which was higher than obtained PK-15 cells, Luo et al. (2011) established a stable
with any other Ad-construct treatment. Ninety iαv-PK-15 cell line in which the expression of the
per cent of Ad-U62B-U63C1-U63C2-treated mice integrin αv subunit of the FMDV receptor was
also survived challenge with A22/IRQ 24/64 or 89.5% downregulated as determined by qRT-PCR
Asia1/MOG/05 until 7 dpi, whereas all control analysis of the αv mRNA. Infection of these cells
mice had succumbed by that time. The effect of with 100 TCID50 of FMDV O/CHA/99 resulted
Ad-U62B-U63C1-CMV3C2 against challenge in a > 1000-fold reduction of virus titre in the
supernatant at 48 hpi compared with controls (Luo At 4 months of pregnancy fetuses were surgically
et al., 2011). recovered and after confirmation of the integration
and expression of the VP4 shRNA, tongue epi-
miRNA thelium was collected. Both primary VP4 shRNA
Unlike siRNA or shRNA which are of exogenous transgenic and non-transgenic tongue epithelium
nature, micro (mi)RNA are dsRNA molecules cells were infected with 100 TCID50 of FMDV. At
encoded in endogenous genes. Generally, miRNA 24 and 48 hpi an inhibition in viral RNA yield and
does not require full complementarity to bind with virus yield of > 91% was obtained in the transgenic
target mRNA, e.g. one type of miRNA may regulate cells compared to the non-transgenic cells (Wang
many genes, as well as one gene can be regulated et al., 2012).
by several miRNAs. Transgene expressed shRNA in Jiao et al. (2013) constructed plasmids con-
transgenic cell lines or animals may in some way be taining shRNAs targeting the 3D and 2B genome
considered as artificial miRNA (amiRNA). region of FMDV HKN/2002 (PB-EN3D2B) that
Pengyan et al. (2010) used the SIFMD2 and induced a reduction of 77.7% in EGFP expression
pSiFMD3 plasmids (Penyan et al., 2008) to gen- in BHK cells co-transfected with an EGFP reporter
erate transgenic mice through microinjection of plasmid, following similar methodology as Chen et
the siRNA in the male pronucleus of the zygote al. (2004).These PB-EN3D2B plasmids were used
and embryo transplantation. Upon IP infection to establish a transgenic IBRS-2 cell lines and trans-
with 100 LD50 of FMDV Asia1 and euthanasia at genic mice. Infection of six lines of transfected cells
72 hpi, non-transgenic mice had detectable FMDV with 20 TCID50 of FMDV O/HKN/2002 resulted
antigen in their liver, kidneys and spleen as dem- in complete abrogation of viral replication in two
onstrated by immunohistochemistry, whereas cell lines (no CPE and no detectable virus in the cell
FMDV antigen was only detected in the spleen of supernatants at 72 hpi) and a delay of CPE forma-
the transgenic mice, and the number of positive tion until 48 hpi and a 94% reduction in virus titre at
cells in their spleen was reduced compared with the 48hpi in another cell line, compared with controls.
non-transgenic controls. Pathological examination In the three remaining cell lines, the replication of
revealed necrosis in the cardiac muscle and conges- FMDV was not inhibited. Similar findings were
tion in the liver of non-transfected infected control done upon challenge of these cell lines with the
mice, but not in the transfected infected mice and a FMDV serotype Asia1 ( Jiansu/2005). The shRNA
significant increase of splenic corpuscles and mac- copy number integrated in the cellular genome was
rophage epitheloid cell nodes in the junction of the shown not to be correlated with the magnitude of
white pulp and the red pulp in the transgenic mice the observed antiviral effect. Transgenic suckling
compared with non-transgenic mice (Pengyan et mice containing 1 or 2 shRNA integrations in their
al., 2010). Unfortunately, no additional virological genome did not exhibit significant resistance to
examinations were performed in this study. FMDV infection upon challenge with 10 LD50 of
Co-transfection of HEK-293T-cells with one of HKN/2002 and all died. When the challenge dose
three lentiviral shRNAs targeting the VP2, VP3 or was lowered to 3 LD50, 27–41% of the transgenic
VP4 region of FMDV and their respective FLAG- mice survived, but also 22% of control mice did
tagged viral gene sequence induced a significant ( Jiao et al., 2013).
inhibition of viral gene expression determined by Four miRNA expression plasmids targeting
Western Blot analysis as compared with mock- conserved regions of the 3D gene sequence of
transfected cells. Viral replication as expressed by different FMDV isolates significantly silenced the
TCID50 of FMDV Asia1 (Asia1/Ys/CHA/05) in EGFP reporter plasmid containing the O/CHA/99
BHK-21 cells stably expressing one of the three 3D sequence in BHK-21 cells. In BHK-21 cells
shRNAs infected with 100 TCID50 of this strain transfected with these miRNAs a 6.3- to 400-fold
was inhibited for 91–98% relative to the controls. reduction in virus titre and a 41.5–82.1% reduction
Transgenic bovine embryos were generated by in viral RNA load compared with mock-transfected
transfer of transgenic bovine fetal fibroblasts control cells was observed at 24 hpi, but the effect had
expressing VP4 shRNA into enucleated oocytes. waned at 48 hpi (Du et al., 2011). Co-transfection
The resulting embryos were transferred into cows. of one of four miRNAs targeting the IRES of
FMDV O/HN/CHA/93 and corresponding 24 hpt was abolished in both tested 3D1 transgenic
homologous EGFP reporter plasmids in BHK-21 cell lines, but was less outspoken in 3D2 transgenic
cells resulted in 44.3–81.4% EGFP silencing at 48 cells (silencing of about 50% in only one out of
hours compared with mock-transfected controls. two examined 3D2 transgenic cell lines). No cross-
When co-transfected with EGFP reporter plasmids silencing was observed when reporter plasmids
containing IRES sequences of heterologous FMDV encoding the sequence of O1 Campos were used.
strains A/AF/72 or Asia1/Jiangsu/China/2005, Degradation of the reporter RLuc mRNA was
the miRNA resulted in 38.7–71.4% EGFP silenc- shown to be involved in the observed silencing of
ing at 48 hours compared to mock-transfected A/Arg/01 reporter plasmids. However, upon infec-
controls. Transfection with a mixture of the two tion of the 3D1 transgenic cell lines with FMDV
most potent miRNAs or with a plasmid express- A/Arg/01 (moi of 0.01 and 5) the virus replica-
ing these two miRNA structures (dual miRNA) tion as evaluated by viral titres and viral RNA was
enhanced the homologous EGFP silencing up to not impaired. Sequence analysis of the 3D1 target
84.7% and 95% respectively and the heterologous region in virus that replicated in these cells did not
EGFP silencing 88.3 and 96.6% respectively. Trans- reveal any mutation that could explain reduced
genic BHK-21 cells lines with stable integration of susceptibility to the amiRNA. In addition, com-
the dual miRNA in their genome were established parison of the predicted structural organization of
and infected with one of the FMDV strains O/HN/ the amiRNA target sequence in the reporter plas-
CHA/93, A/AF/72, Asia1/Jiangsu/China/2005, mid and that of the full-length FMDV sequence,
O/Tibet/China/1/99 or O/CHN/Mya98/33-P hybridization reactions with a fluorescent DNA
at a moi of 5–50. Up to 72 hpi the virus replication oligonucleotide resembling the 3D1 amiRNA and
of all but the O/CHN/Mya98/33-P was markedly transfection with nude viral RNA showed that the
reduced as determined by qRT-PCR. Simultaneous lack of silencing of the viral replication in cell cul-
SC administration of the dual miRNA and 50 to ture as opposed to the strong silencing of reporter
100 LD50 of each of the viruses mentioned above gene expression in the 3D1 transgenic cells could
in the neck of suckling mice resulted in 100% mor- not entirely be explained by structural differences
tality with FMDV O/HN/CHA/93, AF/72 and in the target sequence between the plasmid and the
Asia1/Jiangsu/China/2005 but the time of death virus sequence (Gismondi et al., 2014).
was delayed by 6 hours compared with control mice Hu et al. (2015) aligned FMDV strains of sero-
that all had died at 42 hpi, whereas 75–100% of the type O, A and Asia1 to select conserved sequences
mice infected with O/Tibet/China/1/99 or O/ of the VP1 gene for the design of shRNA expression
CHN/Mya98/33-P survived up to 7 dpi (Chang et vectors. Upon infection with 100 TCID50 of FMDV
al., 2013). serotype O strain OS/99 of shRNA-transfected
Gismondi and co-workers (2014) established BHK-21 cells, the most potent shRNA reduced the
transgenic BHK-21 cell lines constitutively express- viral RNA yield by 96.8% in comparison to untrans-
ing amiRNA targeted against one of two sites in the fected control cells. This shRNA was selected
3D region (3D1 and 3D2) of the FMDV genome, to generate transgenic pigs through somatic cell
based on sequence homology between FMDV nuclear transfer using shRNA transfected primary
strains A/Arg/01, O1 Campos and C3 Indaial. porcine fibroblasts. Based on the expression level
Similarly, BHK-21 transgenic cells containing a of the amiRNA in their fibroblasts, the transgenic
transgene directed against the 3′UTR region of the pigs could be divided into a ‘high’ and a ‘low’
FMDV genome were established, but no expres- expression group. When fibroblasts isolated from
sion of 3′UTR amiRNA was detected. Silencing the transgenic pigs were infected with 100 TCID50
activity of the transgenic cells was evaluated by of OS/99 the viral RNA yield was reduced up to
co-transfection with reporter plasmids encoding 30-fold at 36 hpi in the ‘high’ expression group
the Renilla luciferase (RLuc) gene fused to FMDV compared with untransfected controls and the cor-
fragments enclosing the amiRNA target sequences relation between the ami RNA expression and the
of A/Arg/01 and a control plasmid encoding the inhibition of viral RNA expression was positive.
firefly luciferase. The RLuc activity, as determined Challenge of pigs with 100 LD50 of OS/99, revealed
by quantification of the total RNA by RT-PCR, at a marked protection against FMDV induced fever
and vesicular lesions in transgenic pigs compared to siRNA in the cell and an overdose of siRNA rela-
non-transgenic pigs. Whereas the latter all showed tive to target RNA is needed to obtain an inhibitory
severe disease from 3 dpi on, fever was absent in effect. This is difficult to achieve in vivo. Progress
the ‘high’ expression transgenic pigs and only one in this field has been made by vector-based deliv-
small vesicular lesion was present at 9 dpi. Lesion ery like replication-defective human adenovirus
development was also mild and delayed until 7 dpi (Chen et al., 2006) or recombinant S. cho (Cong et
in the ‘low’ expression transgenic pigs. Consistent al., 2010). Also, the emergence of FMDV genome
with the clinical protection, the viral RNA load in resistant to the siRNA seems to be easily induced
the serum of transgenic pigs was strongly reduced in siRNA treated cells. Combined administration of
at 1, 3, 5, 7 and 10 dpi and different internal organs siRNA targeting different sequences of the FMDV
at 10 dpi compared with non-transgenic pigs (Hu et has been reported as an approach to overcome
al., 2015). this hurdle. Off-target effects due to recognition
The seemingly unlimited target range for nucleic of other genes with a similar sequence should be
acid based methods implies a promising broad avoided, taking into account the different natural
therapeutic potential. In human medicine several host species of FMDV. The use of RNAi methods to
therapeutic candidates for metabolic, hereditary, generate genetically engineered transgenic cell lines
degenerative, neurological or tumoral diseases are and animals with reduced susceptibility to FMDV
in clinical development. Antiviral RNAi-based is challenging. Discussion about the potential
drugs against infections with hepatitis B and C use of amiRNA to generate animals with reduced
viruses, respiratory syncytial virus, the human susceptibility to FMDV falls beyond the scope of
immunodeficiency virus or the Ebola virus are in this chapter and for the advances in farm animal
the developmental pipeline and some have entered transgenesis the reader is referred to review articles
phase I or II clinical studies. The present overview like Kues and Niemann (2011) and other.
shows that a lot of effort has been done to study the
potential of RNAi-based strategies to inhibit the
replication of FMDV. Depending on the construct Passive immunization with llama
design, RNAi seems an efficient method to inhibit antibodies (Nanobodies®)
the replication of FMDV in vitro but in vivo experi- Passive transfer of (hyper)-immune serum may
ments yield more varying degrees of success and offer rapid protection against FMDV infection.
effects are usually short-lived. RNA-based strate- In the beginning of the twentieth century it was a
gies hold some inherent challenges and additional frequent practice in Europe to administer immune
research is needed to address these, especially serum or blood from animals recovering from
when bearing the highly contagious and genetically FMDV infection to other animals to protect or cure
diverse nature of the FMDV and the treatment of them from the disease. However, this method was
livestock in mind. A first challenge is the seemingly quickly abandoned because of the risk for iatrog-
suboptimal efficacy when it comes to reduction of enous transmission of FMDV and of inadequate or
the infectious viral load. Although virus titres in too short protection (Blancou, 2002).
infected cells are often significantly reduced when Unlike in other mammalian species, 45–75%
expressed as a percentage, the actual virus titres of of the antibodies of the members of the family of
infectious virus that can be recovered from treated- the Camelidae only contain heavy chains. The
infected cells are often quite high. In extension, the isolated variable antigen-binding single domains
transition from in vitro activity to in vivo shows that of these antibodies (VHH fragment), known and
he latter is often inferior to what would be expected patented as Nanobodies®, have been explored and
based on the in vitro findings. Target site accessibil- developed as therapeutic agents (Reviewed in Van-
ity during viral replication and the conformation landschoot et al., 2011). The passive immunization
of the target RNA are important to consider when with recombinant llama single-domain antibody
developing an RNAi strategy (Gismondi et al., fragments against FMDV was mainly described at
2014). Another challenge is the in vivo delivery, sys- the Central Institute for Animal Disease Control
temic spread and in vivo stability of the siRNA. In in The Netherlands. Upon immunization of llamas
in vitro studies transfection is used to introduce the with a mixture of four FMDV strains O1 Manisa,
A22 Turkey/98, A24 Cruzeiro and Asia1 Shamir immunoglobulin (pIg) (Harmsen et al., 2005) –
24 VHH that neutralized O1 Manisa in vitro were using three of the above described VHH against O1
selected. Mapping of the antigenic sites to which Manisa. This fusion resulted in a 100-fold increase
the VHH bind by competition ELISA revealed that in serum half-life compared to monovalent VHH.
the VHH recognized four (I, II, III and IV) func- The in vitro affinity and FMDV neutralizing capac-
tionally independent antigenic sites more or less ity of the VHH2s was comparable to that of the
corresponding to the described antigenic sites of FMDV VHH. In vivo, all but three of the 16 large
the VP1 protein of FMDV serotype O. In ELISA, all white pigs administered with one of the VHH2 or
VHH recognizing antigenic sites I, II and IV bound a mixture of them at a dose of 3 mg/kg and chal-
to homologous and heterologous FMDV strains of lenged 24 hours later with 10³ PFU of O1 Manisa
serotype O (O1 Manisa, O Taiwan/97, O1 British developed generalized FMD at 2 to 3 dpi compa-
Field Strain 1860 (BFS)/67A), A (A22 Turkey/98, rable to mock-treated control pigs. Three pigs did
A22 Iraq 24/64, A24 Cruzeiro/Brazil/55), Asia1 not develop FMD and no infectious virus could
(Asia1 Shamir) and C (C1 Detmold), although be detected in their blood or oropharyngeal fluids
for some high concentrations (up to 10 mg/ml) (OPF) up until 14 dpi when the experiment ended.
of VHH were required. VHH recognizing anti- The transmission of FMDV from VHH2 treated
genic site III on the other hand only reacted with and FMDV-infected pigs to untreated control pigs
the FMDV serotype O strains. Fifteen VHH were at 24 hpi was not prevented, despite the lower viral
able to neutralize O1 Manisa in vitro at concentra- load in the serum and OPF of the treated pigs com-
tions of 0.15–3.3 mg/ml, one at a concentration of pared with infected control pigs (Harmsen et al.,
10 mg/ml and five not at concentrations below 10 2008). To improve the in vivo activity, other FMDV
mg/ml. Some combinations of VHH resulted in a binding VHHs with higher neutralizing capacity
synergistic effect lowering the total needed VHH were selected, two FMDV VHHs were fused into
concentration by 3- to 8-fold. The four most potent a bivalent FMDV VHH2. This VHH2 was in its
VHH were PEGylated to increase their serum half- turn fused with a pIg VHH resulting in a so-called
life and administered IM at a dose of 4 mg/kg to VHH3. Three out of five pigs administered intrave-
guinea pigs 24 hours prior to intradermal challenge nously with 50mg/kg of an equimolar mixture of 2
with 10³ TCID50 of O1 Manisa. Despite the pres- VHH3s and infected intradermally 24 hours later
ence of a virus neutralizing titre in their serum at 0 with 104 TCID50 of O1 Manisa did not develop
and 3 dpi, the guinea pigs that received single VHH FMD and had reduced and variable amounts of viral
were not protected and all developed generalized RNA in their serum and OPF between 1 and 17 dpi
FMDV lesions at 3 dpi. Combination of two VHH compared with control pigs. These three treated
resulted in a 1 day delay of lesion development and pigs did not transmit FMDV to untreated control
protected three out of six animals against lesions up pigs. In the other two treated pigs both the devel-
to 5 dpi when the experiment was stopped. Guinea opment of FMDV (5–6 dpi) as the transmission
pigs that received immune serum of guinea pigs to control pigs was delayed (6 dpi) compared with
previously immunized with FMDV O1 Manisa did untreated control pigs (2 dpi). IM administration
not develop generalized FMDV until 5 dpi, despite in the hind leg of 1g of the above VHH3 mixture
the fact that this serum neutralized less efficiently at the time of IM injection in the neck with a full
in vitro and these guinea pigs had lower neutral- dose of conventional FMDV O1 Manisa double-oil
izing titres in their serum at 0 and 3 dpi compared emulsion vaccine in pigs resulted in a significant
to the VHH immunized guinea pigs. These find- decrease in FMDV virus neutralizing antibodies
ings illustrate the crucial role antibody-mediated compared with pigs that were solely vaccinated, the
opsonophagocytosis plays in in vivo protection ELISA antibody response on the other hand was
against FMDV which cannot be achieved by VHH not affected (Harmsen et al., 2009).
since they lack the Fc receptor (Harmsen et al., Similarly, two VHH with high affinity to FMDV
2007). In search to overcome this hurdle Harmsen serotype O were isolated from the immune library
et al. (2008) generated three VHH2 – that consist from two camels immunized with an inactivated
of a VHH binding to FMDV genetically fused FMDV serotype O vaccine. These VHH showed
to a VHH binding with high affinity to porcine high affinity to FMDV serotype O but did not
cross-react with FMDV serotype A and Asia1 limit- a dose dependent way as determined by a plaque
ing their practical applicability against FMDV in the reduction assay on the supernatant collected at
field. Conjugation with fluorescent semi-conductor 18 hpi. The EC50 – the concentration that reduced
nanocrystals turned these VHH into an interesting plaque counts by 50% in treated cultures compared
tool for tracing FMDV type O virions in laboratory to untreated ones – was determined at 0.5 µg/ml
studies with FMDV (Wang et al., 2015). with no signs of cytotoxicity up to 100 µg/ml. How-
The present data provide an interesting ever although the virus yield was reduced by 2 logs
proof-of concept for the use of passive FMDV in cells treated with 50 µg/ml, the virus yield still
immuno-prophylaxis using VHH but there is a reached up to 7 log PFU/ml at 18 hpi. Addition of
need for improvement with regard to potency, MA before, during or maximum 1 hour after virus
dosage, route of administration (individual intra- adsorption was necessary for a strong inhibition
venous) and panserotype activity before practical of the viral replication (PFU). Pre-incubation of
applicability against FMDV in the field would be high concentrations of MA with 106 PFU of virus
possible. It also remains to be determined whether for 1 hour before incubation on BHK-21 cells did
FMDV variant viruses with reduced sensitivity to not result in a decrease in virus titre compared to
VHH would be readily selected on, especially since untreated virus, suggesting MA did not have a
the VHH are directed towards the highly variable virucidal effect. The reducing effect of MA on the
surface proteins of FMDV. production of photoresistant neutral-red labelled
FMDV and on the staining of intracellular acidic
compartments suggested that MA inhibits the
Antiviral properties of natural process of uncoating of FMDV in BHK-21 cells
products through reduction of the acidification of intracel-
In earlier times several treatments based on natural lular acidic vesicles (Wachsman et al., 1998).
products like herbs and plants (pine tar, pomegran- Flavonoids are ubiquitous in photosynthesiz-
ate, garlic, turnip, hyssop, root of mallow) have ing cells and therefore present in most plants. The
been proposed to treat lesions associated with molecular structure of the flavonoid compounds
FMDV infections (Reviewed by Blancou, 2002), backbone is 2-phenyl-1,4-benzopyrone, which con-
however the majority if not all of them without sists of two phenyl rings and a heterocyclic ring. The
demonstrated efficacy nor scientific background. flavonoid apigenin with three –OH substitutions
Nowadays traditional medicine including herbal at carbon positions 5, 7 and 4′ is present in many
treatment is still often empirically applied to soothe fruits and vegetables (e.g. parsley, celery, celeriac)
FMDV lesion and promote their healing in coun- and has been reported to have inhibiting effects
tries endemic with FMDV. Medicinal plants, their on HIV activation and to enhance the antiviral
derivatives or animal products have been and are activity of acyclovir against human and veterinary
more and more explored as alternative sources of herpes viruses (reviewed in Cushnie and Lamb,
novel drug discovery however few have been exam- 2005). A dose-dependent effect of the flavonoid
ined for their activity against FMDV. apigenin (4′,5,7-trihydroxyflavone) on the replica-
Meliacine (MA) is a cyclic peptide isolated tion of FMDV strain O/ES/2001 in BHK-21 cells
from the leaves of the plant Melia azedarach L that was reported, whereas six other flavonoids did not
contains aliphatic amino acids and a single glucose exhibit anti-FMDV activity (Qiang et al., 2015).
unit. It was reported to inhibit the replication of Addition of cell-culture medium containing 20 µg/
FMDV strains of serotype O (O1 Campos, O 69) ml apigenin after 1 hour adsorption of the FMDV
and A (A24, A87) with 52–90% in BHK-21 cells strain O/ES/2001 at a moi of 0.1 on BHK-21 cells
infected at a moi of 1 and over 99% when infected at strongly reduced the VP1 expression at 20 hpi and
a moi of 0.001, whereas the strains O1 Caseros and the CPE formation and virus titre obtained in the
C3 Resende were not sensitive (Wachsman et al., supernatant at 24 hpi, respectively when compared
1995). In BHK-21 cells covered with medium con- to mock-treated control. The EC50 in BHK cells
taining 0.5, 5, 50, 100 or 500 µg/ml of purified MA was determined at 8.593 µg/mL and the cytotoxic
after 1 hour virus adsorption of FMDV serotype O concentration (CC)50 at 31.43 µg/mL. Time-
(O1 Campos), the virus replication was inhibited in of-drug-addition studies revealed that apigenin
(20 µg/ml) exerted its anti- FMDV effect after virus the antiviral product in all target species and the
entry in the cell. The viral replication was not inhib- pharmacological and possible toxicological effects
ited when apigenin was added before or during in humans. Second, the scale of treatment in the
adsorption of the virus, nor when the virus and event of an outbreak of an epizootic viral disease
the apigenin were pre-incubated before addition to like FMD in livestock exceeds many times that in
the cells. Intracellular viral RNA yield was nearly companion animals. The massive administration
undetectable in treated cells up until 22 hpi. Trans- of antiviral drugs yields certain risks including the
fection of BHK-21 cells with a plasmid containing possibly rapid generation of virus variants resist-
the IRES of FMDV fused to EGFP and treatment ant to the antiviral drug. To manage this risk, a
of these cells with apigenin revealed an inhibition of thorough, fundamental scientific knowledge of
the EGFP expression in a dose dependent-manner. the barrier to resistance, the fitness and transmis-
These data indicated IRES as a target of apigenin sion efficiency of any resistant variants is needed.
and the suppression of the IRES-mediated transla- Another important factor is the cost of treatment
tional activity of FMDV as the mechanism of action of livestock which is in first instance determined by
(Qiang et al., 2015). the product and the required dosage. Therefore, a
Chitosan is a linear polysaccharide com- simple and inexpensive synthetic process which is
posed of randomly distributed β-(1–4)-linked suitable for production of large quantities and a low
d-glucosamine and N-acetyl-d-glucosamine. It is effective dose is desirable. The cost of veterinary
produced from chitin derived from the cell walls interventions, withdrawal periods and residue anal-
of fungi and shells of insects and crustaceans like yses should also be considered. For the treatment
shrimp. Administration of 0.5 mg chitosan granules of large numbers of animals on short time notice
and O/CHN/99 to suckling mice resulted in an under field circumstances, administration through
average delay of death time of 0.2–2.1 hours in the the feed seems a practical approach. However, any
chitosan-treated compared to untreated animals method for in vivo administration poses specific
and little difference was seen when the chitosan demands and challenges including bio-availability,
was administered simultaneously with, or 3 or 7 systemic spread and stability. These factors are
days prior to viral challenge. Similar results were among others important to warrant administra-
seen with the Asia1/JS/05 FMDV strain (Li et al., tion of the optimal dose of the antiviral agent and
2010). by doing so to limit to a minimum the probability
Although at present the above-described prod- that variant viruses with reduced susceptibility to
ucts are far from ready to be used for the practical the antiviral agent emerge. Given the highly infec-
control of FMDV, futher investigations into this tious nature of FMDV, the short duration of the
kind of compound may allow the development of replication cycle, the large number of virus particles
efficient and pharmacologically appropriate drugs excreted by infected animals and the small number
against FMDV. of virus particles required for infection FMDV is
readily transmitted to susceptible animals often
resulting in an explosive epidemic. To be able to nip
Summary an FMDV outbreak in the bud, an antiviral agent
While to date the use of antiviral drugs in veterinary should be very effective at potently inhibiting the
medicine remains limited to companion animals, virus replication shortly after administration. Vari-
there is an increasing interest for their therapeutic ous approaches and many research efforts have been
potential to control of livestock diseases including and are pursued in search of an adequate antiviral
FMDV. The development of antiviral drugs for agent to combat FMDV and although the majority
livestock is still at an early stage and requires a very of studies remain limited to in vitro experiments
specific approach. Drug administration to food- and/or preliminary efficacy studies in laboratory
producing animals implies specific requirements animal species, some encouraging achievements
to safeguard food safety including among others have been reported. It remains to be answered
the determination of maximum residue levels in whether the antiviral activity observed in these
products of treated animals. This requires a thor- models can be translated into FMDV target species
ough knowledge of the pharmacokinetic profile of as very few studies have been performed in these
species. Every approach meets specific challenges, Comparative genomics of foot-and-mouth disease virus.
J. Virol. 79, 6487–6504.
strengths and weaknesses and these have been sum- Chang, Y., Dou, Y., Bao, H., Luo, X., Liu, X., Mu, K., Liu,
marized at the end of the corresponding sections. So Z., Liu, X., and Cai, X. (2014). Multiple microRNAs
far, the majority of agents encounter a lack in one or targeted to internal ribosome entry site against
more aspects that are vital for appropriate use in the foot-and-mouth disease virus infection in vitro and in
vivo. Virol. J. 11, 1.
control of FMD outbreaks. For each approach more Chen, W., Yan, W., Du, Q., Fei, L., Liu, M., Ni, Z., Sheng,
research is needed to improve the antiviral potency, Z., and Zheng, Z. (2004). RNA interference targeting
to gain insight into mechanisms of action and the VP1 inhibits foot-and-mouth disease virus replication in
barriers to resistance and to explore the efficacy, BHK-21 cells and suckling mice. J. Virol. 78, 6900–6907.
Chen, W., Liu, M., Jiao, Y., Yan, W., Wei, X., Chen,
pharmacokinetic behaviour and safety in FMDV J., Fei, L., Liu, Y., Zuo, X., Yang, F., et al. (2006).
target species and to examine practical synthesis Adenovirus-mediated RNA interference against
and applicability. The large number of recent stud- foot-and-mouth disease virus infection both in vitro and
ies on this topic illustrate that the field of research in vivo. J. Virol. 80, 3559–3566.
Clark, J.L., Hollecker, L., Mason, J.C., Stuyver, L.J.,
on specific antivirals against FMDV is rather young Tharnish, P.M., Lostia, S., McBrayer, T.R., Schinazi,
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positive outlook for future progress. synthesis, and antiviral activity of 2’-deoxy-2’-fluoro-2’-
C-methylcytidine, a potent inhibitor of hepatitis C virus
replication. J. Med. Chem. 48, 5504–5508.
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heterogeneity, how they can be applied to specific outbreak and how modelling of livestock disease
FMD outbreaks or epidemics, and how they can is likely to develop in the future. The emphasis
be used to explore the impact of control measures, throughout is on ‘good practice’ (Woolhouse et
proceeding to a detailed description of the applica- al. 2011), especially how theoretical models relate
tion of a particular model, the ‘Keeling’ model, of to biological data and how models can sensibly be
the spread of FMD between farms in the UK, and used to inform decisions about disease control. A
concluding with a discussion of how mathematical glossary provides explanations of technical terms
modelling of FMD has developed since the 2001 (Box 16.1).
Network. In this context, the numbers and distri- SEIR. An abbreviation for a commonly used
bution of links between hosts, where ‘link’ implies mathematical model with four compartments:
an opportunity to transmit infection. An example susceptible, exposed (latently infected), infectious
is the network of farms linked by movements of and recovered/removed.
livestock.
Stochastic. The output is not fully determined by
Noise. Inherent variability and/or imprecision in the inputs. Chance is incorporated in the process
data which makes it difficult to obtain precise and every realization of that process can produce
parameter estimates. a different outcome. Stochastic effects are par-
ticularly important where the numbers involved are
Non-independence. Correlation between two or
small, e.g. at the start or during the ‘tail’ of an epi-
more parameter estimates such that there is a
demic when there are few infectious individuals.
range of combinations of parameter values which
are consistent with the data. Transmission kernel. A mathematical function
describing the relationship between transmission
Non-linear. Here, used in the mathematical sense
rate and distance.
that the rates of change between different com-
partments in a model (e.g. susceptible to infected) Transmission rate. The average rate at which a
are not simply proportional to the value of a single single infectious individual infects susceptible
variable. This can lead to complex relationships individuals.
between the inputs and outputs of a model.
Validation. The process where the outputs of a
Non-stationary. Meaning that parameter values are model are compared with a fully independent data
not constant through time. set, i.e. one which was not used to provide esti-
mates of any of the model’s parameters.
Parameter. A constant (i.e. a fixed number) in a
mathematical term which determines how the Variable. A quantity whose value is tracked in a
value of a variable in a model changes through mathematical model. An example is the number
time. (or fraction) of individuals in one of the compart-
ments of a SEIR model.
Sensitivity analysis. A generic term for various
methods of exploring how model outputs are Well-mixed. This implies that every individual in
related to model inputs in order to determine a population is equally likely to infect every other
which of the model’s assumptions and/or param- individual. Although clearly a simplification the
eter values are most important in determining its assumption that a population is well-mixed is often
behaviour. One method for carrying out sensitivity used as a starting point in developing a model of
analysis is Latin hypercube sampling. the spread of infection.
S f (•)
E f (•)
I R
1 2
f (•)
3
(B)
Figure 16.1 Compartments, in theory and practice. (A) A diagrammatic representation of a simple SEIR model
showing the flow of hosts between susceptible, latently infected, infectious and removed compartments. The
number of hosts (or fraction or density) in these compartments is represented by the variables S, E, I and R
respectively. The rate of flow is specified by three expressions: f1(·), the rate at which susceptible hosts become
infected; f2(·), the rate at which exposed hosts become infectious; and f3(·), the rate at which infectious hosts
are removed. Different models use different mathematical expressions, representing different levels of detail
and incorporating different numbers of parameters. (B) A diagrammatic representation of the course of an FMD
infection in a single host (or single farm). The top panel illustrates that the transition between exposed and
infectious does not correspond to the appearance of clinical signs: animals may be infectious before clinical
signs appear. In practice, there is inevitably a further delay before clinical signs are observed and reported.
The bottom panel, however, illustrates how these states can be represented as distinct compartments in a
mathematical model.
dose to which it is exposed (Hughes et al., 2002c), are useful refinements which merit further develop-
which itself may be highly variable in practice. ment in future, linking mathematical analyses to
Mathematical models attempt to take account of experimental studies of the dynamics of infections
this variability within compartments in various within individual hosts (Chis Ster et al., 2012).
ways. One solution is to define more compartments A third problem is that the unit of epidemiolog-
(e.g. subclinically infected or partially immune) ical interest is not necessarily an individual host;
(Hutber and Kitching, 1996). A more sophisticated many applications of mathematical models con-
but computationally demanding alternative is to sider populations of livestock farms rather than
represent the ‘state’ of a host quantitatively rather of individual animals (e.g. Haydon et al., 1997;
than qualitatively (e.g. levels of viraemia rather than Durand and Mahul, 2000; Howard and Donnelly,
infected or not infected; antibody titre rather than 2000; Ferguson et al., 2001a; Keeling et al., 2001;
infected or recovered) (Woolhouse et al., 1996b; Tildesley et al., 2006). There is no conceptual
White and Medley, 1998; Stringer et al., 1998). This difficulty with classifying individual farms as
requires functional relationships, e.g. between anti- susceptible, exposed, infectious or removed, but
body titre and susceptibility, to be defined. These these compartments are unlikely to be equivalent
to those for individual hosts. Considerable care individuals recover or are removed. There may also
must be exercised in ‘scaling up’ from the animal be terms specifying, for example, the rate at which
level to the farm level – the time scales and nature susceptibles are vaccinated and the rate at which
of the epidemiological processes involved are vaccinated individuals lose their protection. Each
likely to be distinctly different. As an example, of these terms incorporates one or more parameters
consider how a farm makes the transition from (mathematical constants which determine how
exposed to infectious. If this occurs when the first the values of the variables change), such as the
infected animal becomes infectious then the farm- transmission rate (see below). The exact functional
level latent period will be of the same order as the form depends on the underlying biology and the
individual-level latent period. But if considerably type of mathematical model being used. To take a
more virus is required for transmission between simple example, the term σE is often used in dif-
farms than for transmission between individual ferential equation models to represent the rate at
animals, as might be expected given that differ- which exposed and infected individuals become
ent transmission routes may be involved, then the infectious. Expressed thus, the implication is that
farm-level latent period may be longer, possibly exposed and infected individuals become infec-
much longer. The situation is further complicated tious at a constant per capita rate represented by
in that the dynamics of FMDV outbreaks on the parameter σ, that the mean latent period is 1/σ,
individual farms are likely to be highly variable but that the modal latent period is extremely short.
(Woolhouse et al., 1996b), just as are the dynam- This last aspect is often regarded as unsatisfactory,
ics of infections of individual animals. This is and an alternative is to make the latent period itself
particularly evident in countries such as the USA, a fixed constant. The implication, when expressed
where there is a significant variation in farming this way, is that all exposed and infected individuals
practices in different parts of the country. For become infectious at a fixed time after they were
example, in states such as California, large dairy first infected, with no variation. The biological real-
farms can have herds in excess of 15,000 animals ity is likely to lie between these extremes. There are
and therefore it may not be reasonable to assume many potentially suitable functions to describe that
that the disease progresses through all animals on situation, e.g. the gamma distribution, but these are
the farm at the same rate (Carpenter et al., 2011). inevitably more complex (i.e. they incorporate more
Whilst its limitations must be borne in mind, it parameters) and are more problematic to compute
should be recognized that the SEIR approach has (Stringer et al., 1998). Consequently, constant rates
proved extremely useful for developing a quan- or constant periods are frequently assumed in prac-
titative understanding of the epidemiology and tice, although it is often straightforward to compare
control of a wide variety of microparasite infections models of both types to see if there are significant
(Anderson and May, 1991; Keeling and Rohani, differences in their outputs (Woolhouse et al.,
2008) and is likely to remain a standard format for 1997b; Keeling and Grenfell, 2002).
epidemiological models for some time to come. The number of parameters in a mathematical
model is limited only by the level of biological detail
Parameters and variables which is to be represented (though an increase in
An SEIR model describes infection dynamics in the number of parameters will result in an increase
terms of changes in the numbers (or densities or in computational cost to carry out the simulations).
fractions) of hosts in the compartments suscep- For example, it is possible to represent different
tible, exposed, infectious and removed. These transmission routes separately and so have one
numbers are represented by the variables S, E, I or more parameters relating to each of airborne
and R respectively. Movements between these spread, direct contact, livestock movements, vehi-
compartments occur at rates specified by a set of cle movements and so on. One microsimulation
mathematical terms (see Fig. 16.1A). In the sim- model for FMD which takes this approach has over
plest formulation, one term specifies the rate at 50 different parameters (Morris et al., 2001). This
which susceptibles become infected, another the allows the model to be more realistic, but does
rate at which exposed individuals become infec- create problems with parameter estimation and
tious, and another the rate at which infectious model validation.
Which of these paradigms is correct depends on case–control studies (see Dohoo et al., 2009),
the details of the biology of transmission and the although it can also be attempted by fitting appro-
nature of ‘contacts’ between hosts. This has been the priate mathematical models to data (Ferguson et al.,
subject of detailed experimental investigations for 2001b; Keeling et al., 2001; Tildesley et al., 2008;
various infectious diseases of livestock (e.g. Bouma Jewell et al., 2009a). There is, however, a crucial dif-
et al., 1995). The issue is particularly problematic ference between these: case–control studies usually
for FMDV because the virus can be transmitted by estimate the relative risk of infection among farms
many different routes and the relationship between of different types; whereas, fitting models allows
transmission rates and host numbers or density is estimation of both the relative risk of infection
not the same for all routes. Within a confined area and the relative risk of transmitting infection once
with intense mixing and/or aerosol spread, infec- infected (Keeling et al., 2001). Many different fac-
tion may be able to reach all susceptible hosts: here, tors might be involved: livestock species (especially
transmission is density dependent. Over a more cattle farms versus sheep farms versus pig farms);
extensive area, or between farms, infection may livestock numbers or densities; farm type (e.g.
spread through a limited number of contacts with dairy versus beef cattle farms); degree of fragmen-
susceptible hosts: here, transmission is frequency- tation (i.e. whether the farm is a single parcel of
dependent. In practice, transmission of FMDV has land, or multiple parcels); level of biosecurity; and
been modelled as both density dependent (Pech any others identified by epidemiological studies.
and Hone, 1988; Woolhouse et al., 1996b; Haydon Models that allow for these heterogeneities must
et al., 1997; Keeling et al., 2001; Tildesley et al., distinguish different types of farm. Instead of single
2006; Buhnerkempe et al., 2014) and frequency variables and single parameters, the SEIR model
dependent (Howard and Donnelly, 2000; Bouma et now has sets of variables, e.g. Sx which represents
al., 2001). In general, it is clearly important when subpopulations of susceptibles of type x, and sets
developing models of the transmission of FMDV of parameters, e.g. βxy which represents the trans-
that particular attention is paid to the nature of mission rate from infectious farms of type y to
virus transmission and contacts between hosts in susceptible farms of type x.
the context of the system being modelled.
Spatial effects
Complications also arise if the probability of
Modelling heterogeneities transmission between farms is a function of the
distance between them, i.e. the system is not well
Different host types mixed. Intuitively, this ought to be an important
The simplest way to think about the spread of any consideration: it seems unlikely that a farm is
infectious disease is to consider a population of equally likely to transmit infection to a farm many
identical hosts, each of whom is equally likely to kilometres away as it is to the farm next door.
infect any one of the others. This leads to the simple However, the importance of distance is related to
SEIR model described above, which can be thought the mechanism of spread: transmission by aerosol
of as representing a ‘well-mixed’ system. In practice, is related to distance (Donaldson et al., 2001),
this will not always be appropriate, especially when whereas transmission by movement of livestock
considering transmission between livestock farms from farm to farm need not be (e.g. within the
which differ in their composition and in their local catchment of a livestock market; Lindström et
density and distribution (Carpenter et al., 2011; al., 2013; Buhnerkempe et al., 2014). This point
Buhnerkempe et al., 2014). Similar issues may arise is important because it has been shown that a
when considering transmission within a farm with surprisingly small number of well connected
a mixture of livestock species, ages, vaccination his- ‘nodes’ (e.g. livestock markets) can have a major
tories, or housing locations (Hutber et al., 1998). impact on the dynamics of the system (Watts and
Here, however, we will focus on transmission Strogatz, 1998; Green et al., 2006; Dawson et al.,
between farms. 2015). In practice, given that livestock movement
Identifying epidemiologically significant restrictions are usually implemented during FMD
heterogeneities is usually achieved by the use of epidemics, much of the spread of infection is
localized (Gibbens et al., 2001) and spatial consid- mathematical models. One of these is percolation
erations are extremely important. theory, a stochastic approach to modelling how an
There are various ways in which spatial structure infection spreads through a geometric lattice which
can be represented in mathematical models. The has been used to explore, in general terms, how the
simplest and most transparent is to represent the nature of the farming landscape might influence
map location of each farm individually, an approach FMD dynamics (Kao, 2001; Tildesley et al., 2010).
which favours a microsimulation model. A key issue Another approach is to extend the deterministic
here is to define a function which relates the prob- SEIR framework using a technique known as
ability of transmission to distance, referred to as the moment closure. This approximates local spread by
transmission kernel. For the UK 2001 epidemic considering the status not just of individuals but of
it was possible to define this function empirically pairs, triplets etc. (Ferguson et al., 2001a). A third
based on the results of contact tracings (Gibbens technique, small world network modelling, is based
and Wilesmith, 2002) and this was a key input into on an SEIR framework but distinguishes between
microsimulation models of that epidemic (Keeling local spread and long-distance transmission events
et al., 2001). More recent mathematical models have (Ahmed et al., 2002). Percolation theory, moment
used exponential or power law functional forms to closure and network models are, at best, relatively
capture the shape of the dispersal kernel, with the crude approximations to what may in practice be
precise form of the kernel being determined by quite complex and variable spatial arrangements
fitting to the outbreak data ( Jewell et al., 2009b; of farms. Owing to this, there are many instances
Deardon et al., 2010; Gubbins et al., 2010; Hayama where it will be preferable to use spatially explicit
et al., 2013). For spatially explicit microsimulation microsimulation models despite their greater com-
models the parameter β is replaced by individual putational complexity.
values for the transmission rate between a given
pair of farms. These rates may be weighted by other
risk factors for susceptibility and infectiousness. If Model application
epidemics in large populations of farms (the UK has
over 100,000 livestock farms) are being modelled, Parameter estimation
this approach can become very computationally A major challenge for developing a mathematical
intensive. model for FMD (or any other infectious disease)
A refinement to modelling spatial transmission is how to obtain estimates of the model’s param-
is to represent different routes of transmission eters. In general, these will come from two sources:
explicitly, and to calculate probabilities of trans- (i) prior knowledge, whether from experimental
mission for each of these (e.g. Morris et al., 2001; studies or from analyses of earlier epidemics; and
Bates et al., 2003; Harvey et al., 2007). Such models (ii) estimation from the epidemic being modelled,
require even more detailed inputs – e.g. road net- whether directly from inspection of the available
works, milk tanker routes, footpaths, wind speeds data or indirectly from fitting the model to those
and direction – and so are often coupled to the data.
development of geographical information system In theory, it is desirable to obtain parameter
(GIS) representations of the landscape (Sanson, estimates from prior knowledge – the model is then
1999). At the same time, many more transmission statistically independent of the data. In practice,
parameters must be estimated and validated, and each epidemic is likely to have its own unique
the overall transmission rate between a pair of farms features and some parameter estimates will have to
is obtained by summing the rates of transmission by be obtained using data from that same epidemic.
different routes, again weighting by known risk fac- The balance between these will depend on the
tors. These models rely heavily upon expert opinion availability and quality of data from independent
to ascertain parameter values and this can present sources and the biological question of whether it is
problems regarding the transparency of the process reasonable to generalize across different epidemics.
(Woolhouse, 2011). For example, the incubation period can often be
Alternatively, there are more abstract ways specified independently (mindful of differences
in which spatial structure can be represented in between virus strains and livestock species and
the difficulty of scaling up from individual animals by considering each one separately. Two examples
to entire farms) whereas the transmission rate come from the estimation of R0 from data on the
(whether within or between farms) may often be UK 2001 epidemic: various combinations of R0 and
highly variable (depending on farm management generation time (the average time between a farm
practices, weather conditions, effectiveness of con- being infected and new farms being infected from
trol measures and many other factors) and usually it) are consistent with the initial rise in numbers of
has to be re-estimated each time a model is applied. cases per day; similarly, various combinations of
The mechanics of estimating parameters directly R0 and the number of ‘at-risk’ neighbouring farms
have been described elsewhere (e.g. Richter and are consistent with observed rates of local spread
Sondgerath, 1990; Jewell et al., 2009b). In general, (Kao, 2001; Tildesley and Keeling 2009). Non-
there are three main concerns: noise, bias and non- independence is most straightforwardly resolved
independence. Noise simply means that biological by obtaining independent estimates of one of the
data tend to be inherently variable (and sometimes parameters involved.
difficult to measure precisely): the noisier the data, Methods for estimating parameters indirectly
the less precise the parameter estimate derived from by fitting a mathematical model to data are the sub-
the data. An example of noise comes from fluctua- ject of a large literature (see Becker, 1989) and are
tions of numbers of cases reported during the UK becoming increasingly more sophisticated. Deter-
2001 epidemic. Superimposed on the main trends, ministic models can be fitted by least squares (e.g.
there was considerable day-to-day variation in num- Woolhouse et al., 1996b) or maximum likelihood
bers of cases reported per day (partly explained by methods (Anderson et al., 1996). Stochastic models
day-of-the-week effects), which is especially impor- can be fitted by a variety of techniques including
tant when numbers of cases are small, as during the martingale estimation (Becker, 1993), maximum
epidemic’s tail. This means that estimates of CRR likelihood and Bayesian methods – the last two
values have limited precision which, for example, being increasingly implemented using Markov
makes short-term projections difficult. Noise can Chain Monte Carlo (MCMC) methods (Gamer-
sometimes be reduced by making more precise man, 1997; Jewell et al., 2009a; Deardon et al.,
measurements. Where this is not possible, the 2010). Even so, the sheer complexity of the system
implications of uncertain parameter values must be to be modelled and the structure of the data often
explored using sensitivity analysis (see below). mean that the available ‘off-the-shelf ’ model-fitting
Bias means that the available data tend to over- procedures may be impractical during the course of
estimate or underestimate the value of a parameter. an outbreak when model outputs must be rapidly
A possible example of bias comes from estimated disseminated to policy-makers. As a result, model
dates of FMD infection on UK sheep farms in fitting has been historically implemented using
2001: these were often obtained by ageing lesions, ad hoc methodologies (e.g. Keeling et al., 2001).
but lesions were not always apparent, especially However, since 2001, developments in Bayesian
in sheep, and so early infections could have been methodologies and computing power has meant
missed and the date of infection underestimated that model-fitting procedures are becoming more
(Gibbens and Wilesmith, 2002). Bias can be diffi- standardized and implementable during epidemics
cult to eliminate and its potential importance needs ( Jewell et al., 2009a,b).
to be gauged; again this can be done using sensitiv- An alternative approach to estimating Rt values
ity analysis. is to use a non-parametric method which does
Non-independence means that uncertainties in not impose a model on the data. This has been
the values of two or more parameters are related attempted for the UK 2001 epidemic using infor-
to each other, e.g. a high estimate of one parameter mation on contact tracings and estimated dates of
tends to be associated with a low value for another, infection (Woolhouse et al., 2001; Haydon et al.,
and vice versa. A consequence of this is to increase 2003). These data allow the entire history of the
the uncertainty associated with a given parameter epidemic, in terms of which farm was thought to
estimate; there may be a much wider range of com- have infected which other farm, to be described. The
binations of two or more parameter values that are number of new cases per case, for any time period
consistent with the data than would be apparent or any region, can then simply be read off directly.
Uncertainty about the source of infection can be An equally important, but often neglected,
incorporated by generating epidemic histories on consideration is the starting conditions from which
a probabilistic basis and generating confidence the model is to be run. At the simplest level, start-
limits around Rt values using bootstrapping meth- ing conditions might specify the introduction of a
ods (where the data are repeatedly sampled with single primary case into a homogeneous popula-
replacement and parameter values re-estimated tion thus: S(0) = N – 1; E(0) = 1; I(0) = 0; R(0) = 0.
each time in order to generate confidence inter- More complex models may require the subpopula-
vals) (Haydon et al., 2003). This method is likely tion or the actual individual infected to be specified.
to be much more robust than methods based on In practice, information on the early course of an
model-fitting, especially for small epidemics, but epidemic is often sketchy or incomplete and, in
does require better information on individual FMD addition, the processes important early on in the
cases than is usually available. However, the now course of an epidemic may be very different from
routinely available tool of genome sequencing pro- those important in the remainder. For example, the
vides an alternative, and potentially more reliable, UK 1967/68 epidemic involved early widespread
means of tracing the spread of infection between wind-borne dissemination of infection from a few
farms (Cottam et al., 2008). primary cases (Tinline, 1970) and the UK 2001
Estimates of transmission rates can also epidemic involved early dissemination via sheep
be obtained from experimental studies of the markets, which was subsequently halted (Gibbens
transmission of FMD (e.g. Hughes et al., 2002c; et al., 2001). As a result, it is often necessary to ini-
Charleston et al., 2011) and other infections [e.g. tialise a mathematical model based on the epidemic
Bouma et al. (1995) for pseudorabies in pigs]. Such situation some time after the first introduction of
studies are extremely valuable, but are normally infection (e.g. Woolhouse et al, 1996b; Keeling et
restricted to small numbers of animals kept in a al., 2001).
limited area under experimental conditions, so the
results cannot necessarily be applied directly to the Sensitivity analysis and validation
field situation, and considerable care is required in Once a model has been constructed it is important
extrapolating to much larger scales, e.g. transmis- that its behaviour is explored in detail. There are
sion between farms. two elements to this. One is to explore the effect
of various assumptions made in developing the
Inputs and initial conditions model. Examples would be to compare the effects
The simplest mathematical models consider only of assuming a constant latent period with a con-
intrinsic dynamics; that is, the behaviour of the stant rate at which exposed individuals become
system is fully specified by the values of the model’s infectious, or to compare the effects of assuming
parameters and the starting values for each of the density- or frequency-dependent transmission (see
model’s variables. More complex models, however, above). This is referred to as the structural sensitiv-
may attempt to take into account extrinsic variables, ity of the model. Obviously, it is vital that those
such as weather conditions or the magnitude of the assumptions which are shown to have a significant
control effort, especially if these are not constant in effect on model behaviour are tested as rigorously
time or space. These will appear as an additional set as possible.
of inputs in the model, affecting the value either of The second element of exploring model behav-
one or more parameters, e.g. transmission rates as iour is to consider the effects of different inputs:
a function of wind speed. An additional, and often parameter values; starting conditions; and any
crucial, set of inputs is the demographic and epide- extrinsic variables. This is done using sensitivity
miological characteristics of the population. The analysis. The simplest form of sensitivity analysis
level of detail required is hugely variable, ranging is to alter the value of each input separately (Wool-
from simply the initial number of susceptibles in house et al., 1996a; Hutber and Kitching, 1996;
the population (N) to comprehensive information Durand and Mahul, 2000). However, because
on geographic location of, pattern of movement by, the dynamics of infectious diseases are typically
and risk factors associated with every single indi- complex, a more robust approach is to explore the
vidual in the population. effects of changes in combinations of inputs using
a factorial design. If the value of an input has a problem is suggested by using a spatially explicit
significant effect then it is clearly important that microsimulation model (Keeling et al., 2001 – see
that value is measured or monitored as precisely below). This model provides a good description of
as possible. the UK 2001 epidemic after the imposition of the
Validation of an epidemiological model is ide- national ban on livestock movements (see below),
ally achieved by comparing model output with a but without having to invoke non-stationary
fully independent data set, i.e. testing the model’s parameter values. One interpretation of the very
predictions. In practice, it is often difficult to take marked contrast between the performance of these
a model parameterized for one epidemic and apply different types of model is that the spatial distribu-
it to another: each epidemic tends to have its own tion of farms is a key determinant of the course of
unique characteristics and history. Even so, it is an FMD epidemic and that an accurate representa-
sometimes possible to use the same modelling tion of this distribution is necessary for a model to
framework in different settings, which constitutes have any explanatory power.
partial validation even if some re-parameterization
is necessary: this has been done for BSE in cattle
(Anderson et al., 1996; Ferguson et al., 1998) and Modelling control measures
scrapie in sheep (Woolhouse et al., 1999; Matthews
et al., 2001). There are two problems with model Control options
validation. First, it is not always clear what consti- Methods for controlling FMD epidemics are
tutes a good ‘fit’ between model and data. Second, discussed in detail elsewhere in this volume. They
it is often far from clear that the suggested model include enhanced surveillance, epidemiological
offers a unique solution; in other words, there may tracing, biosecurity, movement restrictions, stamp-
be other models, making different assumptions ing out, pre-emptive culling and vaccination. FMD
and using different parameter values, which fit the control programmes often involve a combination
data as well or better. Pending objective solutions of some or all of these measures and, even given
to these issues it is important that model output the inevitable constraints on what is logistically
is evaluated critically, cautiously and, preferably, feasible, the number of possible designs of control
independently. programmes greatly exceeds the number that can
be tried out in practice. Mathematical models are
Results of model fitting a potentially useful tool for exploring the potential
Several studies have attempted to fit SEIR models impacts of different control programmes. A prob-
(or elaborations thereof) to FMD epidemic data lem with using models in this way is that not all
(Haydon et al., 1997; Howard and Donnelly, 2000; interventions are readily quantifiable: for example,
Ferguson et al., 2001b). These studies variously used there are no quantitative studies of the relation-
data from epidemics in the UK in 1967/68, Taiwan ship between the implementation of biosecurity
in 1997 and the UK in 2001 and an important aim measures and a decrease in transmission rates. On
was to quantify β, the per capita transmission rate the other hand, the impact of stamping out, pre-
between farms. However, none of these model fit- emptive culling and various uses of vaccination,
ting exercises was entirely satisfactory because it including prophylactic vaccination in advance of
proved impossible to reproduce the epidemic curve an epidemic and reactive vaccination during an epi-
without invoking substantial changes in the value demic, can be usefully explored using mathematical
of β, i.e. the parameter estimate was non-stationary. models.
This occurred even when factors such as changes in It should be emphasized that, from an epide-
the control effort (directly) and the effects of local miological perspective, a key aspect of a control
spread (indirectly, using moment closure) were programme is its impact on transmission rates.
incorporated (Ferguson et al., 2001b). Interventions which are clearly bad for the individ-
It is, of course, possible that transmission rates ual farm, e.g. pre-emptive culling, may nonetheless
did vary throughout the course of these epidemics, be good for the population by preventing onward
particularly given that control actions may also vary transmission from that farm. Conversely, interven-
with time, but a more satisfactory resolution of the tions which are better for the individual farm, e.g.
vaccination, may be less effective at the population would have reduced the final epidemic size by an
level because they have a less immediate effect on estimated 52% (95% confidence interval 37–61%)
transmission. Mathematical models can be used to (Haydon et al., 2003).
analyse trade-offs of this kind.
Stamping out
Movement restrictions Stamping out, i.e. culling of livestock on farms
A ban on the movement of livestock between farms where infection is detected, acts to decrease the
affects the transmission kernel (Fig. 16.3), both infectious period. The impact of this on farm-to-
in terms of its height (overall transmission rate) farm transmission depends on how infectiousness
and shape (the fraction of long-distance transmis- varies through time, i.e. on the dynamics of infec-
sions) (Keeling et al., 2001). It has been calculated tion within the farm. Although many different
that, in the context of the UK 2001 epidemic, the dynamics are possible in principle the average
introduction of a movement ban decreased R0 relationship, at least in the UK in 2001, seems to
from just below 3 (though with wide confidence have been approximately linear, i.e. halving the
intervals around this estimate) to approximately infectious period halves net transmission (Royal
half that value (Matthews and St Rose, personal Society, 2002). However, the relationship may
communication; Haydon et al., 2003). This single well vary from farm to farm (depending on initial
measure, therefore, would have had an enormous numbers of livestock infected, the doses they were
impact on the course of the epidemic. Indeed, a infected with, how infection was introduced, the
non-parametric analysis based on the results of livestock species involved and various other factors)
epidemiological tracing studies suggests that if a and may not apply in all epidemiological settings.
national ban on livestock movements had been Various models which have explored the impact of
imposed in the UK just 2 days earlier then this stamping out (e.g. Haydon et al., 1997; Howard and
Figure 16.3 Examples of transmission kernels, relative per capita rate of transmission as a function of distance
between farms. Empirical results (grey lines with black circles) derived using data tracing studies carried out
during the UK 2001 epidemic after the imposition of a national ban on livestock movements (Keeling et al.,
2001) are compared with two standard functions: 1) k/d2 with k = 0.41 (blue line); and 2) g*exp[-hd] with g=4.8
and h=2.4 (red line). All functions show per capita transmission rates relative to that over distances of 0 to 5 km.
The constants k, g and h were fitted using the least squares method. The empirically derived transmission
kernel equates to 80% of transmission occurring over distances up to 5km. Note that function (1) overestimates
transmission rates at longer distances, whereas function (2) underestimates these.
Figure 16.4 The relationship between the expected final size of an epidemic, Ifinal, and the basic reproduction
number, R0. The relationship has the form Ifinal = 1 – (1 – I0) exp[–R0 Ifinal] (modified from Kermack and McKendrick,
1927) and is based on a SEIR model for a well-mixed, homogenous population, with I0 as the fraction of
the population initially infected. Results are shown for I0 (labelled on the each contour) ranging from near
zero (bottom line), 1%, 2.5%, 5%, 10%, 25%, 50%, and 75% (top line). If R0 << 1 then final epidemic size is
determined largely by the fraction initially infected, and if R0 >> 1 by the size of the susceptible population.
However, when R0 ≈ 1, small increases in R0 can lead to large increases in final epidemic size. Similarly, when
R0 ≈ 1 small increases in I0 can also lead to large increases in final epidemic size.
Donnelly, 2000; Tildesley et al., 2009) conclude already infected farms (such as culling dangerous
that more rapid removal of infected livestock has contacts); and reducing the availability of at-risk
a disproportionate impact because even relatively susceptible farms for infection. So, here too, it is
small decreases in net transmission can result in therefore useful to represent within the modelling
very large decreases in the final size of an epidemic framework the fraction of culled farms which were
(Fig. 16.4). and were not infected. There is general theoretical
agreement on the potential of ring culling to reduce
Pre-emptive culling the size of an epidemic, to reduce the total numbers
There are two main forms of pre-emptive cull- of livestock lost, and to reduce economic losses,
ing: culling farms designated by epidemiological provided that there is sufficiently intense local
tracings as ‘dangerous contacts’; and ring culling spread (Durand and Mahul, 2000; Ferguson et al.,
around infected farms. The culling of dangerous 2001a,b; Morris et al., 2001; Keeling et al., 2001;
contacts in mathematical models can be crudely Royal Society, 2002). This conclusion stems from
approximated as a reduction in the transmission the non-linear relationship between epidemic
rate, but a more realistic representation requires size and transmission rates, which means that the
the removal of farms at a given rate, some of which benefits of removing livestock before they transmit
will be infected and some susceptible. Where the infection can outweigh the disadvantages of culling
latter approach has been adopted (e.g. Keeling et uninfected livestock at the same time. However, if
al., 2001; Tildesley et al., 2009), there has been the the intensity of local spread varies, e.g. reflecting
difficulty that there are no independent estimates of variation in livestock densities, then ring culling
the sensitivity of dangerous contact identification, may not be the optimal solution in every affected
i.e. the fraction actually infected. area (Garner and Lack, 1995; Durand and Mahul,
Ring culling can have two effects: removal of 2000; Matthews et al., 2003).
There has been relatively little mathematical of livestock were culled for welfare reasons as were
modelling of prophylactic vaccination against culled pre-emptively for disease control purposes,
FMD. Simple SEIR models have been applied to and indirect costs to tourism and other industries
describe outbreaks of FMD amongst prophylacti- exceeded the costs of the control effort itself
cally vaccinated dairy cattle herds in Saudi Arabia (National Audit Office, 2002).
(Woolhouse et al., 1996b), and generalizations of One point on which there is general agreement
these models have been used to explore the poten- is the importance of the early application of effec-
tial for current vaccines to prevent major FMD tive control measures (Garner and Lack, 1995;
outbreaks (Woolhouse et al., 1997b). More recent Ferguson et al., 2001b; Keeling et al., 2001; Haydon
studies have implemented more detailed analyses of et al., 2003; Tildesley et al. 2006; Ward et al., 2009).
the impact of prophylactic vaccination on a national Although it could rightly be argued that this result
scale (e.g. Keeling et al., 2003; Ringa and Bauch, is qualitatively obvious, it has a disproportionate
2014). The general lesson that emerges from these significance because of the highly non-linear rela-
studies is that current vaccines have limited capabil- tionship between initial number of cases and final
ity to provide adequate herd immunity: the fraction epidemic size (Fig. 16.4).
protected is too small (even against homologous
challenge); and the duration of protection is too
short. Even so, prophylactic vaccination could still An example: the Keeling model
be effective if transmission rates are not too high, The model developed by Keeling et al. (2001) with
especially if targeted at the most susceptible farms reference to the 2001 UK epidemic illustrates many
and when combined with other control measures of the general principles developed in the preceding
(Keeling et al., 2003). sections of this chapter. The mathematical basis of
this model is relatively straightforward. It is a sto-
Costs and benefits chastic microsimulation model of the transmission
Most mathematical models of the epidemiology of FMD between livestock farms in Britain. Each
and control of FMD consider the number of cases farm is represented as in one of four possible states:
(whether individuals or farms) as the primary S, E, I or R. Susceptible farm i becomes infected on
output. However, the goal of a control programme day t with probability Pi given by the expression:
is not necessarily to minimize the numbers of
cases. Additionally, the estimated performance of ⎡ ⎤
a control programme will depend on the output Pi =1−exp⎢− ∑ βij ⎥
measure upon which it is compared and whether ⎢⎣ j∈I(t ) ⎥⎦
control programmes are compared via expecta-
tions or via the estimated risk of an extreme event where βij is the transmission rate from farm j to farm
(Probert et al., 2016). Alternative goals, or objec- i, and the summation term Σ indicates that the βij
tives, include: minimizing the total numbers of terms are summed across all farms infectious on day
livestock lost (which is especially important where t. βij in turn is given by the expression:
pre-emptive culling is a control option); minimiz-
ing the economic value of livestock lost (which βij = −aib j K (dij )
weights different species differently); and reduc-
ing the duration of an epidemic. The importance where ai is the relative susceptibility of farm i (which
of duration is often underestimated but it matters incorporates risk factors for infection, e.g. farm
because, typically, many of the negative impacts of size and livestock composition), bj is the relative
an FMD epidemic are indirect rather than direct. infectiousness of farm j (which incorporates risk
Indirect impacts include losses of livestock culled factors for transmission, e.g. farm size and livestock
for welfare reasons (largely resulting from move- composition), and K(dij) is the transmission kernel
ment restrictions imposed while FMD is present in (which represents the relative transmission rate
a country or region), losses to export markets, and as a function of the distance between farms j and
losses through negative effects on other industries. i, dij – see Fig. 16.3; also see review of Pomeroy et
For example, in the UK in 2001 similar numbers al. (2015) for an illustration of published empirical
FMD transmission kernels). The model assumes of infected farms immediately after the imposi-
that an infected farm becomes infectious after σ tion of the national ban on livestock movements.
days (which can vary according to livestock species) Estimates of the latent period and the relation-
and that an infectious farm is removed after τ days ship between transmission rate and distance were
(which can vary according to the control effort). obtained from observations made during the early
There are two types of heterogeneity in the stages of the epidemic – the latter emphasizing the
model that was utilized during the 2001 outbreak. importance of local spread once the movement ban
First, farms differ in the number and species com- was in place (Fig. 16.3).
position of their livestock, which affects both their Just four parameters had to be estimated by fit-
susceptibility, a, and infectiousness, b. Second, spa- ting the model to data on daily numbers of reported
tial heterogeneity is represented by locating each cases: three of these refer to the relative per capita
farm according to its map reference. The rate of susceptibility and infectiousness of cattle and
transmission from infectious farm j to susceptible sheep; the other refers to the fraction of dangerous
farm i, βij, is therefore a function of the livestock contacts culled which were actually infected. Model
populations on the two farms and the distance fitting was carried out in two steps: maximum likeli-
between them, dij. hood methods provided initial parameter estimates;
The 2001 epidemic control programme, a com- least squares methods were then used to optimize
bination of stamping out and pre-emptive culling the fit to the cumulative numbers of reported cases
against a background of movement restrictions, by county. Model fitting using Bayesian approaches
biosecurity and heightened surveillance, is repre- was attempted subsequently (Deardon et al. 2010).
sented as the removal of infected farms (stamping More recent developments of the model since 2001
out) and of susceptible and latently infected farms will be described in the next section of this chapter.
(pre-emptive culling) in numbers and locations The model is able to reproduce, with consider-
corresponding to the achieved culling effort at each able accuracy, both the temporal course and spatial
stage of the epidemic. distribution of the numbers of reported cases during
Inputs are therefore a map of the UK livestock the UK 2001 epidemic (Fig. 16.5). In particular, the
farms and their species compositions (based on model reproduces the timing of the epidemic’s peak,
census data) and spatiotemporal variation in the with the decline beginning as expected shortly after
culling effort. Initial conditions are the distribution the stepping up of the pre-emptive culling effort in
(A) (B)
Model range
Model average
Observed
Figure 16.5 Comparison of the output of the model of Keeling et al. (2001) with data from the UK 2001 epidemic.
Four model parameters are fitted to data on the numbers of reported cases and recorded culling effort (see
main text). (A) Time course showing number of reported cases (black lines and symbols), average output of
best-fit model (red line), and individual results from 100 simulations (red dots). (B) Spatial distribution showing
location of reported cases (left-hand side, red dots) and culls (left-hand side, black dots) and average number
of expected cases in 10km squares from 100 model simulations (right-hand side, ranging from < 1 (grey) to > 20
(orange and red).
late March (Fig. 16.5A). The model also reproduces 2013). Some of the key conclusions that emerge
the protracted epidemic tail, which results from from these analyses are that: (i) prompt and effec-
occasional long range jumps, followed by a degree tive intervention is crucial to restricting the size
of local spread before infection is locally eliminated. of an epidemic; (ii) ‘traditional’ control measures
The distribution of cases is also well reproduced, (stamping out and culling dangerous contacts) are
with hotspots in SW Scotland, NW England, mid not adequate for controlling an epidemic of a highly
Wales and SW England and small outbreaks and transmissible strain of virus in regions of high live-
sporadic cases elsewhere (Fig. 16.5B). The fitted stock densities; (iii) small-scale ring vaccination
parameter values imply that large farms were more (up to 3km around each reported case) is unlikely
susceptible and more infectious than small farms to be an effective alternative to pre-emptive cull-
and that cattle were more susceptible and more ing; (iv) large-scale reactive vaccination targeted
infectious than sheep (both of which are consistent at cattle (hundreds of thousands of immunisations
with experimental data), and that many danger- per day) may prove an effective control option, but
ous contacts (perhaps more than half) were not will not produce such an immediate effect on the
infected, although there is considerable uncertainty course of an epidemic as pre-emptive culling (a
around this estimate. prediction confirmed by later reported events in
Uruguay and Argentina in 2001, where numbers of
cases continued to increase for several weeks after
The Keeling model post 2001 the introduction of mass reactive vaccination);
Since 2001, the Keeling model has been used to (v) reactive vaccination targeted at high risk farms
explore a number of ‘what-if ’ scenarios, including can be much more efficient; and (vi) mixed pre-
the potential effectiveness of earlier implementa- emptive culling and reactive vaccination strategies
tion of more rigorous culling policies, replacing have perhaps the greatest potential for an effective
pre-emptive culling by ring, barrier, mass or targeted control policy, with the optimal ring vaccination
reactive vaccination, and adopting mixed cull- strategy being dependent upon the total resources
ing and vaccination policies (Keeling et al., 2001, available and the speed at which vaccination can be
2003; Tildesley et al., 2006, 2009; Porphyre et al., carried out (Fig. 16.6).
Figure 16.6 Number of farms infected and culled for various vaccination radii. Points represent individual
simulations, lines give a smoothed average for different vaccination capacities with large dots marking the
optimal ring size, and the dashed black line gives the average epidemic impact with prompt contiguous
premises and dangerous contacts culling but no vaccination (reprinted from Tildesley et al., 2006).
Figure 16.7 Upper tail of the distribution (based on the 97.5th percentile of 100 simulations) for the number
of counties infected when infections are introduced to each of the 3109 counties of the continental US and
assuming a state-level movement ban (reprinted from Buhnerkempe et al. (2014)).
Modified versions of the Keeling model have event of a future outbreaks occurring. Models are
also been utilized to predict the spread of disease playing ever increasing roles in such contingency
in other countries, should an outbreak of foot-and- planning and there is therefore a need for increased
mouth disease occur. One such model for the USA dialogue between modellers and policy-makers to
highlights the fact that the risk of a large outbreak ensure that models are fit for purpose.
occurring is highly dependent upon the region of Since 2001, FMD modelling has developed to
introduction (Buhnerkempe et al., 2014) and this the extent that there are now several FMD mod-
indicates that the deployment of control must take elling groups around the world who work at the
into account the characteristics and the location of interface between science and policy. In addition to
the outbreak (Fig. 16.7). Of course, as stated above, the Keeling model, FMD models that are currently
such results must be treated with an element of cau- utilized by policy makers include the Davis Animal
tion since the model was developed for one country Disease Simulation (DADS; Bates et al., 2003), the
(the UK) and is being used to simulate the spread North American Animal Disease Spread Model
of disease in another. However, it may be possible (NAADSM; Harvey et al., 2007), InterSpread Plus
to validate such a model for the USA in the event of (Stevenson et al., 2013), AADIS (Bradhurst et al.,
an outbreak, by fitting to observed epidemic data. 2015, 2016), and AusSpread (Garner and Beckett,
As discussed below, translating model predic- 2005). Modelling work has also been carried out
tions into policy advice requires considerable care on the Japan 2010 outbreak (Nishiura and Omori,
and caution. However, the consistency of model 2010).
outputs with available data and generally good The presence of multiple models creates a
agreement with other modelling approaches (see dilemma for the policy community in determin-
previous section) increase confidence in the robust- ing which model should be used in the event of a
ness of these conclusions. novel outbreak. In 2001, several models were used
to provide policy advice, but this may present a
problem should the models provide conflicting
Development of FMD models advice regarding how an epidemic will spread
since 2001 and the impact of interventions. There may be the
The 2001 outbreak raised awareness around the temptation for a policy maker to be influenced by
world of the economic impact of FMD epidemics the model that predicts the smallest outbreak and
and the need to develop contingency plans in the the least severe intervention strategy as that would
minimize the potential impact upon the farming Future of mathematical models
community and the potential backlash associated Epidemiological modelling has made enormous
with introducing control measures. Alternatively, advances since 2001, building on an intellectual
a model may be favoured for historical reasons, foundation developed over the previous hundred
owing to the use of that model in a similar context years. Some of these advances are technological:
in the past. However, a more sensible approach may increased computing power has, for example,
be to consider the predictions of multiple models made sophisticated mathematical and statistical
and analyse the predicted effect of several different approaches much more easily implemented and
control strategies upon the epidemic, to elucidate widely accessible. Other advances are technical: for
common trends and better determine which inter- example, greatly improved methods for parameter
vention should be adopted during an outbreak. estimation and sensitivity analysis. But perhaps
The outputs of multiple models can also be con- the most important advance is that mathematical
sidered in a formal framework utilizing ensemble modelling has become much better integrated with
methodology. Ensemble modelling has been used other biomedical disciplines, which has resulted
extensively in climate and weather predictions, with in models which are more realistic, more closely
the result that forecasting has become much more connected with data and, ultimately, more useful as
accurate in recent years (Palmer et al., 2004; Gneit- practical scientific tools, although much remains to
ing and Raftery, 2005). Such an approach may be done.
become more commonplace in epidemiological Against a background of continuing
modelling in the future, with recent results suggest- improvements in computer technology, future
ing that utilizing multiple models to make a single developments are likely in several key technical
prediction regarding the future spread of a disease areas. Stochastic models, especially those based
can reduce risk and provide more accurate informa- on microsimulations, have now become the first
tion regarding the impact of intervention strategies choice format for veterinary epidemiologists in
(Lindström et al., 2015; Webb et al., submitted). most situations, allowing better understanding of
In the early stages of a new outbreak, there variability in the epidemic process, especially when
will be significant uncertainty regarding how the only small numbers of hosts are infected. However,
disease may spread. Models may be unable to pro- it is important to stress that, despite computational
vide accurate predictions until sufficient data are development, models remain a complement to, not
available to parameterize the models to the new a replacement for, experience and expertise in the
epidemic. However, policy-makers cannot afford epidemiology and control of FMD (or any other
to wait for this uncertainty to be resolved before infectious disease). The development of models
making a decision – the cost of waiting to introduce requires specialist mathematical, statistical and
control could be higher than the cost of making computational skills, but these need to be deployed
an incorrect policy decision. Utilizing an adap- in concert with those with hands-on knowledge of
tive control policy that is optimized based upon FMD biology and epidemiology. With this in mind,
the available information regarding the outbreak it is clearly important that the modelling process is
may be a potential solution to this problem. Such as transparent as possible and that the assumptions
a methodology, so-called ‘adaptive management’, underlying the models, and their impact on model
allows models to be used at epidemic onset – the output, are properly communicated. Equally, the
initial control policy that is implemented will be application of mathematical models must take
chosen taking into account the uncertainty in the account of limitations of and possible deficiencies in
disease parameters and the fact that there is the pos- the available input data. Model outputs must always
sibility to switch strategies once more information be checked to ensure that they are robust to any
is accrued. Recent work suggests that multi-phase uncertainties in the inputs and, where this is not the
adaptive control can provide a framework to accu- case, deficiencies must be clearly identified. Here
rately assess which control policy should be used at too, close collaboration with those involved in the
the start of an outbreak and reduce the costs of the collection of the data is highly desirable. Often the
epidemic (Shea et al., 2014). process of constructing a model will itself indicate
where data are currently lacking and stimulate the pig-populations is independent of the size of the
population. Prev. Vet. Med. 23, 163–172.
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The beginning of a new era of emergent and The Pan American Health Organization
re-emergent diseases, and the importance of their (PAHO), as Regional Office of the World Health
potential consequences to the public health and to Organization (WHO), was established in 1920 in
the economies of countries and their citizens, has order to control the epidemics that would affect
caused a strong reaction from the animal health the American continent. FMD was always consid-
organizations particularly in the implementation ered by PAHO as a significant disease of animals
of policies relative to animal health and the manner affecting the American continent. In 1951 the Pan
in which the prevention, control and eradication of American Food and Mouth Disease Center was
animal diseases and zoonosis are addressed. created in Rio de Janeiro (Brazil) and, although
It is within this conceptual framework where focused only in the Americas, many valuable con-
international organizations related to animal health tributions for the prevention and control of FMD
have focused their activities, specifically in recent have been accomplished up to the present.
decades, developing appropriate standards and Since then these three international organiza-
helping in defining regulatory aspects and actively tions have continuously contributed in their areas
participating, within the scope of their competen- of competence in the prevention and control of
cies, in the organization and support of operational FMD worldwide. As a result, FMD has been suc-
actions at the global, regional and country levels. cessfully controlled and eradicated in some parts
The year 2011 marked a significant worldwide of the world using recommended disease control
achievement in the field of animal health, with the measures including the vaccination of domestic
global eradication of Rinderpest (Resolution eight- animals. A detailed chronology relative to FMD,
een, OIE seventy-ninth General Session, 2011). the role of international organizations, and the
Rinderpest was considered, along with Foot-and- actions that have taken place at a worldwide level
mouth Disease (FMD), to be one of the greatest up until the year 2000, can be found in the compila-
animal health threats in the world. This motivated tion of works by Blancou (Blancou, 2002; Blancou
the creation, in 1924, of the Office International et al., 2004).
des Epizooties/World Animal Health Organization During 2001 several serious FMD outbreaks
(OIE) in Paris, France. occurred, particularly in South America and Europe.
The trans-boundary nature of FMD and other The OIE and FAO convened an international
animal diseases that impact the international trade conference on FMD that strongly recommended
of animals and products requires a focus that goes a coordinated international effort against animal
beyond individual countries. This is where inter- diseases, especially FMD. The thirty-first Session
national and regional organizations play a role of the FAO Conference urged the FAO, OIE and
of critical importance in effectively advancing in WHO to continue their joint efforts to seek an
the control and eradication of these diseases. The international solution to the control of FMD and
global perspective for the animal production sector, other trans-boundary animal diseases.
with an increase in demand and price over the next In 2004, the OIE and FAO came together with a
decades, gives a strong incentive to advance towards new initiative, ‘The Global Framework for Progres-
the control and eradication of these diseases in all sive Control of Trans-boundary Animal Diseases
regions of the world. (GF-TAD)’, which combines the strengths of both
The international community has recognized organizations to achieve agreed common objec-
the importance of the FMD within the interna- tives. GF-TAD is a facilitating mechanism, which
tional trade of animals and products and its social attempts to empower regional alliances with the
and economical impact particularly in developing participation of regional organizations in the fight
and in-transition countries. against trans-boundary animal diseases (TAD). The
The United Nations established the Food and GF-TAD provide for capacity building and assis-
Agricultural Organization (FAO) of the United tance in establishing programmes for the specific
Nations in 1946, giving particular consideration to control of certain TAD based on regional priorities,
FMD as one of the principal epizootics that affect FMD being one of them. The efforts were directed
to support specific activities against diseases such as positive RNA molecule and about 8200 nucleo-
FMD and also the strengthening of veterinary ser- tides, within an icosahedral capsid made of 60
vices including their infrastructure and activities for copies each of four proteins, VP1, VP2, VP3 and
prevention and control of TAD’s such as the quality VP4. There are seven immunologically distinct
of epidemiology surveillance and disease reporting, serotypes, O, A, C, Asia1, South African Territories
in order to facilitate the evaluation of the national (SAT) SAT 1, SAT 2, and SAT 3 in circulation
disease status and to ensure the safety of world worldwide and intratypic variants is a common
trade. Regarding FMD the prevention and control finding. Infection or vaccination with one serotype
objectives were mainly achieved through better of FMDV does not cross-protect against other sero-
FMD surveillance, diagnostic capability and report- types and may also fail to protect fully against other
ing as well as vaccination using better-standardized subtypes (variants) of the same serotype.
FMD vaccines. The control programmes have been Field occurrence of FMD is strongly associ-
adapted to the various known epidemiological fac- ated with the animal production system and with
tors associated with FMD in each particular region. the eco-systems, which implies that for an effec-
In the meantime, international organizations have tive control and prevention there is a need to take
also reinforced their technical and financial support into account the dynamics of FMD, the farming
for national FMD control programs. system and cattle movement (Rweyemamu et al.,
The world distribution of FMD clearly reflects 2002). The main target for an effective control is
the economic structure of regions and countries. to identify the source of infection (endemic areas)
In general, more developed countries are free while being the most effective and sustainable strategy
the disease is endemic in developing or in-transition for controlling the disease. Other strategies include
countries. In recent years, however, several incur- bio-security and zoning that has been successfully
sions of FMD have been recorded in developed used in some regions of the world particularly in
countries, all of which have been financially and protecting free countries or zones, as well as the use
socially extremely costly to eliminate, a reminder of vaccinations.
that every country should be fully prepared to
confront such emergencies whenever necessary. The international organization and
At the same time, FMD limits many developing the global control of FMD
and in-transition countries from participating in The OIE/FAO Global Conference on Foot-and-
formal trade at regional and global levels, which mouth Disease held in Asuncion, Paraguay, in June
consequently would lead to the improvement of 2009 urged both organizations to work together on
their economies. a program for the global control of FMD (Proceed-
FMD is a highly contagious vesicular disease of ings of the First OIE/FAO Global Conference on
cattle and other cloven-hoofed animals. Although Foot-and-mouth Disease, 2009). The seventy-ninth
mortality due to the disease is very low and mostly OIE World Assembly of Delegates supported the
restricted to young animals, a drastic decrease in preparation of the Global Control Strategy and
productivity and working capacity of the animals asked that a consultation of experts and repre-
causes losses to the livestock industry. The disease sentatives of national, regional and international
has an important socio-economic impact in coun- institutions be undertaken.
tries where it is endemic, provokes huge economic The European Commission for Foot-and-mouth
consequences when outbreaks occur in disease free Disease Control (EuFMD) advanced with the elab-
regions, and is considered one of the most impor- oration of a Progressive Control Pathway (PCP),
tant constraints to international trade of livestock which was further, finalized and co published by
and animal products. the FAO, OIE and EuFMD in 2011 (The Progres-
In addition, several characteristics related to the sive Control Pathway for FMD Control, 2011), for
nature of FMD virus infection and factors inher- countries to move towards the ultimate objective
ent to the epidemiology of the disease complicate of obtaining official freedom from FMD. The OIE
the goal of global eradication of FMD. FMD virus adopted specific standards to allow for the official
(FMDV) is a member of the Picornaviridae family, recognition of the national control programme of
genus Aphthovirus. It possesses a single stranded OIE member countries to control FMD. The text
was adopted in the seventy-ninth General Session In 2015, among the 180 countries reporting
of OIE delegates in 2011 and incorporated as a their diseases to the OIE, 66 were officially FMD
normative text in the OIE Terrestrial Code (World ‘free without vaccination’. This means that one-third
Organisation for Animal Health, 2014). of the countries in the world have been able to erad-
The PCP for FMD elaborated by Eu-FMD icate the disease using animal health measures. At
proposes a stagewise approach, which presents the same time, important progress has been made
similarities to the approach known as ‘OIE Rinder- on the control of FMD by vaccination since two
pest Pathway’ followed under the successful Global countries are officially recognized as ‘free of FMD
Rinderpest Eradication Programme (GREP) now with vaccination’. Similarly, there are 11 countries
concluded. The Global FMD control strategy with officially recognized ‘FMD free zones without
allows for a regional or eco-systems based synchro- vaccination’, and in seven countries there are ‘free
nization between countries. zones with vaccinations’, five countries had official
The PCP FMD is defined under the risk due to recognition of their national control programme
the FMD virus pools occurring in the world (Ham- (China, India, Venezuela, Morocco, and Algeria),
mond et al., 2009). It consists of six stages ranging and Namibia had recognition programme for a part
from zero, when there is continuous FMDV circu- of its territory.
lation with no reporting or control actions, to five The OIE continues as the sole international
where a country is ready to be officially recognized reference organization for animal health engaged
by the OIE as free without vaccination. Currently in the prevention and control of FMD through
the OIE considers four situations for countries the fulfillment of the tasks assigned by the 180
with regard to FMD: (a) countries not free for member countries, promoting transparency
FMD (PCP stage 0–3). Countries can present in where each member country is responsible for
principle at stage 3 or before their national control reporting to the OIE on the occurrence of animal
programme to the OIE World Assembly for official diseases and zoonosis that are detected within
endorsement, (b) countries preparing dossiers to their territories. The new occurrence of FMD
be submitted to the OIE to be recognized as FMD must be communicated immediately (24 h.) to
free countries/zones practising vaccination (PCP the OIE, who will inform the rest of the countries
stage 4), (c) FMD-free countries/zones where vac- through its disease information system and the
cination is practised (PCP stage 5) and d) FMD World Animal Health Information Database, of
free countries where vaccination is not practised the reported events.
(beyond PCP stage 5). In addition, the OIE collects, analyses and
The Global FMD Control Strategy was dis- disseminates the latest scientific information on
cussed during the FAO/OIE Global Conference on disease control and prevention. This information
FMD Control held in Bangkok as a combination of is then made available to member countries in
three inter-related components, namely the control order to help them improve the methods used in
of FMD, the strengthening of Veterinary Services in the control and eradication of FMD and other dis-
charge of the control programme implementation eases. The 301 Collaborating Centres and reference
and, in parallel the prevention and control of other laboratories recognized by the OIE throughout the
major diseases affecting livestock. world also collaborate by providing technical assis-
The Conference supported to join the FAO/ tance, reagents, and generating and disseminating
OIE Global Control Strategy and the implementa- scientific information. The 12 FMD-OIE reference
tion of a plan with its three components under the laboratories in the Americas, Europe, Asia and
GF TAD mechanism. The Conference also recom- Africa provide an important service to the field and
mended that countries use the possibility of OIE operative actions for the prevention and control of
officially endorsed PCP FMD in principle at stage 3 FMD. Most of these OIE Reference laboratories are
as a recognition by the OIE World Assembly of the also referenced by FAO.
effective management of FMD control in the coun- OIE also provides international solidarity
try by entering the official OIE recognition pathway through technical assistance missions to member
for FMD-free status whenever feasible (Sumption, countries with the best international scientists in
2012). the various fields of FMD control and prevention.
The permanent contact by the OIE to interna- 1 strong participation of farmers in the
tional, regional and national donor organizations is preparation and the implementation of the
extremely important in order to convince them to programme;
invest more and improve on the control of FMD. 2 national Veterinary Services complying with
For all these activities OIE closely collaborates OIE standards on quality.
with FAO and regional organizations on FMD like
PAHO/OMS in America, AU-IBAR in Africa and The FAO plays an important role in assisting
ASEAN in South East Asia and China through the member countries to improve surveillance and
SEAC-FMD programme and OIE SEACFMD Sub control of trans-boundary animal diseases. FMD is
Commissions. considered a priority disease under the FAO and it
Of extreme importance is the work developed continuously assists member countries by provid-
for establishing the international rules for the pre- ing experts, sponsoring and organizing meetings as
vention, control and eradication of FMD, being well as technical cooperation projects (TCP).
the OIE Terrestrial Code (World Organisation for The fight against FMD is also addressed by
Animal Health, 2014) and the Manual of Diag- FAO at the regional level through, for example, the
nostic Tests and Vaccines for Terrestrial Animals, exchange of information between countries or the
the Terrestrial Manual (World Organization for harmonization of national control strategies. FAO
Animal Health (2013), the main documents best has 10 FMD Reference Centres most of them being
known to be the ‘OIE standards’ recognized by the also OIE Reference laboratories. They participate
World Trade Organization (WTO) as the refer- to the joint OIE FAO FMD Global Network of
ence to international animal sanitary rules. These Reference Laboratories. FAO hosts the European
standards contain the latest scientific information Commission for the control of Foot-and-mouth
evaluated by experts in relation to the prevention Disease (EuFMD) and one regional commission
and control of FMD. Besides, OIE has developed on animal production and health, APHCA (Animal
a specific set of tools called PVS Pathway for the Production and Health Commission for Asia and
improvement of veterinary services, particular the Pacific).
PVS Evaluation and PVS Gap Analysis, which
helps developing and in-transition countries
to improve the necessary infrastructure, their The regional activities for the
resources and capabilities (PVS Pathway, 2013). control of FMD
These PVS tools are essential components in the In the Americas, at the regional level, the Pan
field application of the Global FMD strategy along American Health Organization (PAHO) has been
the PCP FMD. a major player in the control of FMD in South
The official recognition by the World Assembly America. Historically serotypes O, A, and C have
of the OIE of country freedom with or without been recorded in South America, and in most of the
vaccination is a key technical and political booster regions FMD was present, until a regionally coor-
to allow Member Countries to embark in national dinated eradication plan was implemented in the
control programmers. The official recognition of 1980s. The strengths of the veterinary services, the
the quality of the programme is also a key tool. improvement of the diagnosis methods, the devel-
The new concepts progressively adopted by opment and use of an oil adjuvant FMD vaccine
the OIE since 1990, such as the possibility for a and the characterization of the eco and production
country to be recognized free with vaccination, systems associated with the presence of FMD were
then the possibility to be recognized for only a part determinants in the development of an Hemi-
of the territory (zoning) and the adoption of the spheric Plan for the eradication of FMD (PHEFA).
concept of containment zone, allowed developing The strategy adapted FMD control activities to the
and emerging countries to be strongly associated to specific regions and, at the same time, addressed
the global vision of the OIE towards global control the social impact and need for local participation
of FMD. with special attention to small livestock owners.
However it is now recognized that two key con- The PHEFA target is to achieve FMD freedom in
ditions must be respected everywhere: the Americas (with and without vaccination) by
the year 2020 (Hemispheric Program for the Eradi- collaboration with EuFMD and the new OIE FMD
cation of Foot-and-mouth Disease: PHEFA Action Unit in Astana, Kazakhstan, is taking a prominent
Plan, 2011). The PHEFA, which has a component role. The objective is to achieve control of FMD by
of public/private participation, has achieved very 2020 in the 14 countries of the region.
important advances in the last decades and at pre- The OIE Sub-Commission for FMD in
sent most of the countries/zones have their status South East in Asia and China (SEACFMD Sub-
officially recognized by the OIE as FMD free either Commission) follows the implementation of the
‘with or without vaccination’. The last report of SEACFMD campaign at the service of the ASEAN
FMD (type O) to the OIE was in 2012 in Paraguay. member countries. The Campaign is guided by the
Type C virus was last recorded as appearing in the ‘SEACFMD 2020 Roadmap’, which describes strat-
north of Brazil (Amazon Region) in 2004. Type A egies to achieve FMD freedom with vaccination by
virus was last recorded in 2001. To date, more than 2020 and maintain freedom in free countries and
95% of the susceptible animal population in South zones (SEAC-FMD 2020, 2007). This regional
America is based in countries/zones free of ‘FMD program for the prevention, control and eradica-
with or without vaccination’. Veterinary services tion of FMD from the countries in South East Asia
have been strengthened during the past years and was developed as a formal OIE program in 1994.
there is a system in place of epidemiological vigi- The SEACFMD Sub-Commission is chaired by the
lance for the detection of clinical diseases and their OIE along with representatives from participating
viral circulation, which operates continually. More countries, ASEAN, FAO, AusAID and other key
than 700 million doses of FMD vaccines are used donors. At the time when the campaign began, only
annually. The public/private investments that have Indonesia was recognized by OIE as FMD free.
taken place in the region to effectively control FMD The program’ strategy is based on progressive
exceed US$1300 million annually. geographic zoning and the rapid identification of
The European Commission for Foot-and-mouth the foci of infection and elimination of FMD at
Disease control (EuFMD) was created in 1954 as a the source. The use of vaccination is considered in
FAO Commission in order to coordinate the efforts order to increase the level of immunity in the herds.
of the European countries in the prevention, control FMD is endemic in Cambodia, Lao PDR,
and eradication of FMD. This regional organization Myanmar, Vietnam and China. Peninsular Malaysia
lends it services and support to 36 member states reported sporadic outbreaks. The entire Philippines
in an action coordinated with the EU through the and Indonesia, the East Malaysia, Sabah and
DG-SANCO of the European Commission and in Sarawak are officially free of FMD. The occurrence
collaboration with OIE. of FMD is associated to FMD pool type O, A and
Following the eradication of FMD in Europe Asia 1. The Chinese FMD control programme has
during the decade of the 1990s, the EuFMD, as a been adopted at the eighty-third General Assembly
specialized regional organization, maintains col- of the OIE in May 2015.
laborative projects in liaison with OIE and FAO in In other countries in South Asia, namely India,
the European neighbouring regions such as in the Bhutan and Nepal FMDV is endemic in the sus-
Caucasian region, Turkey, Syria, Iran and Northern ceptible population and pool types O, A, and Asia
Africa. 1 occurred frequently. The Indian FMD control
The situation of FMD in the European region programme has been adopted at the eighty-third
appears stable with sporadic outbreaks in Russia General Assembly of the OIE in May 2015.
(O or A), Bulgaria (O) and Central West Eurasia. FMD remains endemic in most countries in
The implementation of the Global FMD control the Middle East and North Africa (MENA) with
strategy is strongly based on regional approaches occurrence of FMD virus pool types O, A, and
and one of the major tools in that regard is the Asia 1. The social and political situation influences
organization of regular ‘Road Map meetings’. One the effectiveness of the implementation of control
of the more elaborated regional Road Map is the measures for FMD, increasing the regional risks. Of
Central/West Eurasian region for which six annual particular interest, FMD SAT 2 present in the sub-
meetings have already taken place. OIE and FAO Saharan African region was detected for the first
are organizing these meetings under GF TADs in time in North Africa (Libya and Egypt) in 2012.
In 2012, and with the coordination and support The recent success of the program to eradicate
of OIE and FAO, an FMD Regional Road Map for Rinderpest from the world achieved in 2011 has
East Africa was established. FMD is endemic in all been an incentive to motivate international organi-
the countries with circulation of FMD virus pool zations and countries to embark in a similar road
type O, A, SAT 1, SAT 2 and SAT 3. The goal is to for the control of FMD. Controlling TAD such
arrive at 2022 with some countries reaching the as FMD at its source is a shared interest between
FMD free status and most countries with FMD infected and uninfected countries and is considered
control programmes in place, meaning that veteri- a Global Public Good.
nary services should be significantly strengthened The OIE and FAO have been leading, under their
but the FMD virus would still circulate. areas of competence, the organization of a global
A FMD Road Map for the SADC member coun- response to confront the challenge of controlling
tries in the Southern African region was established FMD worldwide under the OIE/FAO GF TAD
and regular meetings are being organized with the initiative approved by both international organiza-
support of OIE and FAO. The objective of the tions in 2004. The excellent experience gained by
SADC regional strategy is to have all countries in the PHEFA in South America and SEACFMD in
this region free of FMD for 2020. Although some Asia, and previously in Europe are relevant and
countries in the SADC region are free of FMD, indicate that the regional approach is a road for an
FMD virus remains present in several member effective global control of FMD.
countries, with sporadic occurrences in the free The results obtained presently by the OIE and
countries protected by free zones. FMD virus pool FAO coordinated action, have shown significant
type O, A, SAT 1, SAT 2 and SAT 3 circulated in advances in the organization at regional and
infected countries and the role of wildlife, particu- countrywide levels. The most important advances
larly buffaloes, makes the eradication of FMD very have been on the improvement of the veterinary
complex in this part of the world. services, the transparency in the reporting, the
efficiency on the control measures applied, the
adoption of the OIE international standards, the
Conclusions use of recommended diagnostics and vaccines
FMD is not a zoonosis, but is considered a trans- as well as the participation of major donors from
boundary animal disease and one of the most national and international organizations that invest
significant and contagious diseases in animals, in the regional and national programs.
with a devastating impact on national economies The ideal target of a world free of FMD should be
and posing a great risk to world trade. At this maintained, but the actual objective is more global
time, there are more than one hundred countries control than eradication of FMD. This objective is
throughout the world, which are not recognized very ambitious but it can be achieved with the tools
officially as free, all developing or in-transition that are available. One important component yet to
countries. be addressed is the sustainability of the control of
The challenge to effectively control FMD at a FMD, particularly for developing and in-transition
worldwide level is an enormous task, and should it countries. Reaching this objective is the primary
prove to be successful, its impact on animal health responsibility of all stakeholders. The result will be
throughout the world would be an achievement a great contribution to food security, a substantial
without precedent. improvement in the safe trade of animals and animal
The history of FMD outbreaks throughout the products and a significant reduction of poverty in
years indicate that the most common spread of many developing and in-transition countries.
FMD virus occurs across national borders. This sit-
uation presents a major challenge to the veterinary References
services worldwide and the international organiza- Blancou, J. (2002). History of the control of foot-and-mouth
disease. Comp. Immunol. Microbiol. Infect. Dis. 25,
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the move towards global control of animal diseases Blancou, J., Leforban, Y., and Pearson, J.E. (2004). Control
and safe trade in animals and animal products as of Foot-and-mouth Disease: Role of International
well as the reduction of poverty. Organizations. In Foot-and-mouth Disease. Current
Perspectives, Francisco Sobrino and Esteban Domingo,
eds. (Norfolk, UK: Caister Academic Press), pp 425– of natural resource scarcity. The role of agriculture
436. technologies. (Washington DC, USA: Peer-Reviewed
Hammond, J.M., Ferris, N.P., Yanmin Li, Knowles, Publication, International Food Policy Research
N.J., King, D.P., and Paton, D.J. (2009). The Global Institute).
Situation of Foot-and-mouth Disease Occurrence-an Rweyemamu, M.M., and Astudillo, V.M. (2002). Global
Overview. In the Proceedings of the First OIE/FAO perspective for foot-and-mouth disease control. Rev.
Global Conference on Foot-and-mouth Disease: The Off. Int. Epizoot. 21, 765–773.
way towards Global Control (Paris, France: World SEAC-FMD 2020: The road map for foot-and-mouth disease
Organization for Animal Health) pp. 11–20. freedom with vaccination by 2020 in South-East Asia
Hemispheric Program for the Eradication of Foot-and-mouth (2007). (Bangkok, Thailand: Regional Coordination
Disease. PHEFA. Action Plan 2011–2020. (2011). Unit, OIE Sub-Commission for foot-and-mouth disease
(Rio de Janeiro, Brasil: Centro Panamericano de Fiebre in South-East Asia).
Aftosa OPS/OMS). Sumption, K. (2012). The Progressive Control Pathway for
Proceedings of the First OIE/FAO Global Conference on FMD (PCP-FMD): A Tool for Developing Sustainable
Foot-and-mouth Disease: The Way Towards Global Long Term National and Regional FMD Control, FAO/
Control (2009). (Paris, France: World Organization for OIE Global Conference on Foot-and-mouth Disease
Animal Health). Control, Bangkok, Thailand.
PVS Pathway. OIE Tool for the Evaluation of Performance The Progressive Control Pathway for FMD control
of Veterinary Services. OIE PVS Tool. (2013). (Paris, (PCP-FMD), Principles, Stage description and
France: World Organization for Animal Health). Standards (2011). (EuFMD, OIE and FAO).
Resolution 18th. Declaration of Global Eradication of World Organisation for Animal Health (2013).
Rinderpest and Implementation of Follow-up measures Foot-and-mouth disease Chapter 2.1.5: Manual of
to Maintain World Freedom from Rinderpest. (2011). Diagnostic Tests and Vaccines for Terrestrial Animals
79th Session of the OIE World Assembly of Delegates, (Paris, France: World Organization for Animal Health).
Paris, 22–27 May. World Organisation for Animal Health (2014).
Rosegrant, M.W., Koo, J., Cenacchi, N., Ringler, C., Foot-and-mouth Disease Chapter 8.7: Terrestrial
Robertson, R., Fisher, M., Cox, C., Garret, K., Perez, Animal Health Code (Paris, France: World Organisation
D., and Sabbagh, P. (2010). Food security in a world for Animal Health).
considerable distances to susceptible cattle espe- cost of FMD globally is between 6 and 20 billion
cially under humid conditions (Alexandersen et al., US dollars (Knight-Jones and Rushton, 2013)
2003). Infection by the aerosol route is enhanced and indeed single countries, with intensive animal
in cattle because of their large respiratory tidal production, can experience economic losses of this
volume. In contrast, pigs are much less susceptible magnitude in a single year (as in the UK in 2001)
to infection by the aerosol route (Alexandersen through losses in trade, disease control costs and
and Donaldson, 2002), indeed even a small adverse effects on tourism, etc.
physical separation (less than 1 m) is sufficient to
prevent efficient transmission between them (van
Roermund et al., 2010). The virus can be carried Controlling FMD
mechanically by farm workers, veterinarians, motor There are essentially two means of controlling
vehicles and other fomites between infected and FMD within a country: stamping out, by killing
susceptible animals. This must always be borne all diseased and potentially infected animals, with
in mind when introducing measures to control an the subsequent destruction of their carcasses fol-
outbreak of FMD. lowed by thorough cleaning and disinfection of the
affected premises; and vaccination. The stamping-
out policy has been law in the UK since 1892, and
Global impact of FMD is applied with compensation to the farmers for loss
FMD has been present throughout most of the of livestock, resulting in freedom of the UK live-
world for many years. It continues to affect more stock industry from FMD over many years. Serious
than 100 countries around the world (several epidemics occurring in 1922–24, 1967–68, and
recent reviews cover different aspects of FMDV 2001 were all controlled without vaccination. This
distribution around the world, see: Jamal and method was also used by the USA in the last out-
Belsham, 2013; Tekleghiorghis et al., 2016; Brito et break in 1929, and in Canada in 1951–52. However,
al., 2015). However, New Zealand has never expe- the large numbers of animals slaughtered in the
rienced the disease while Australia, South Korea UK in 2001 has led to a reassessment of potential
and Japan have been free of FMD for long periods, control strategies and the use of vaccination (within
more than 65 years. It is noteworthy that Japan has vaccination-to-live scenarios) to control the disease
now had outbreaks in 2000 and 2010 while South is now a possibility. However, so far this has not yet
Korea has had multiple introductions of the disease been employed within Europe. In The Netherlands
in 2002, 2010 and 2014 (Brito et al., 2015). In (2001) and Japan (2010), vaccination during an
addition, during the last century, Central American outbreak has been used to assist in controlling
countries from Guatemala to Panama have escaped the disease but the animals were still slaughtered
the disease, despite many epidemics raging in the (Bouma et al., 2003; Muroga et al., 2012).
USA and South America over many years. The USA In the South American countries, control of
experienced nine outbreaks between 1870 and FMD has generally employed vaccination. The
1929, the most serious of which, in 1914–1916, difficulty with vaccination is that only inactivated
involved 22 States and the District of Columbia. vaccines have so far been developed; these do not
FMD was introduced into South America provide long-lasting immunity and ideally need
through the importation of breeding stock in the to be administered every 6 months. In addition,
mid-to-late nineteenth century, first in Argentina the virus strain used in the vaccine should match
(1865) then Uruguay (1870), Chile (1871) and the prevailing epidemic strain to induce efficient
southern Brazil (1895). The disease then spread protection. Since FMDV undergoes rapid antigenic
across Brazil, to Paraguay and Peru (1910), Bolivia changes, producing many subtypes within any one
(1912), then much later to Mexico (1946), Ven- of the seven recognized serotypes, then this can be a
ezuela (1950), Colombia (1950), and Ecuador significant challenge. A poor match can be expected
(1956). In recent years, the FMD situation in South to reduce the effectiveness of the vaccine and it
America has improved significantly (see below). always takes some days between vaccination and the
FMD continues to be considered to be enzootic induction of protection against infection. In earlier
in Africa and Asia. It is estimated that the annual days, incomplete inactivation of the virus present
in the vaccine has occasionally been demonstrated, commercial advantage, since the absence of vacci-
leading to spread of infection rather than control nation and outbreaks is the most practical criterion
of an epidemic (Barteling, 2002), but this should to ensure that a country is free of FMD. For this
no longer be an issue. From the point of view of reason countries make every effort to guard their
trade with other countries, vaccination of livestock ‘FMD-free’ status, since a single FMD outbreak
is viewed as an admission that FMD is present in may cost the country many millions of dollars in
the country. Such factors led the European Union lost livestock export trade.
in 1992 to abolish general vaccination of livestock In countries where FMD is enzootic, including
against FMD, so creating a large susceptible animal many African and Asian countries, there have been,
population. Since the 2001 FMD epidemic, there in some cases, very limited attempts to control the
have, fortunately, only been quite limited outbreaks disease. However, the development of the FMD
of disease in Europe. However, these have included Progressive Control Pathway (PCP), which has
another in the UK in 2007 (resulting from virus been adopted by the Food and Agriculture Organi-
escape from Pirbright that has both a research zation of the United Nations (FAO)/EuFMD and
Institute plus a FMD vaccine manufacturing facil- OIE, provides a framework for all countries to move
ity) and one in Bulgaria (in 2010–2011) involving towards improved control of FMD (Sumption et
a virus closely related to others circulating in the al., 2012). The PCP provides a series of control
Anatolian region of Turkey and other countries in activity stages (6 in number) that can be achieved
the region (Valdazo-González et al., 2012). by each country in their own way and according
In 1987, the South American countries signed a to local circumstances. The end point of the PCP
Hemispheric Plan for the Eradication of Foot-and- is ‘free without vaccination’ and many countries
mouth disease, and during the 1990s, remarkable started the process at stage 0 with little knowledge
progress was made. For example, national labora- of their disease status or the nature of the circulat-
tories diagnosed 786 cases of FMD per year in the ing viruses. Clearly there are significant cost and
region in 1990, but by the late 1990s this had fallen infrastructure implications for moving all the way
to only 130 cases of FMD in the whole of South along the pathway but a large reward for doing so.
America. By 1999, the international community
recognized Argentina, Chile, Guyana and Uruguay
as free from FMD without vaccination. However, Emergence of new serotype O
in 2001 FMD reappeared in the southern region of viruses
South America forcing the reintroduction of cattle Serotype O FMDV is the most common globally
vaccination in Argentina, southern Brazil (Rio including throughout Asia, and exists there in sev-
Grande do Sul), Paraguay and Uruguay (Correa eral distinct evolutionary genotypes, that have been
Melo et al., 2002). Paraguay last had an outbreak termed ‘topotypes’ (Samuel and Knowles, 2001).
of serotype O FMDV in 2011/12 and since then The PanAsia lineage of serotype O FMDV was first
no clinical disease has been reported anywhere isolated in 1990 from outbreaks of FMD in India
in South America. Vaccination is used in various and became widespread in many Asian countries
countries and thus virus may still be in circulation (Hemadri et al., 2002). This PanAsia strain also
but as the use of vaccination declines further then moved in a westerly direction into the Middle East.
hopefully the target status of ‘free without vaccina- During 1994, this virus appeared in Saudi Arabia
tion’ can be attained for the entire continent. and spread from there to Turkey, Greece and Bul-
The World Organisation for Animal Health, garia by 1996. Outbreaks of FMD due to this strain
administered on behalf of 162 countries by the were then reported from several other countries of
Office International des Epizooties (OIE) in Paris, the Middle East, where it came to replace all other
maintains a code to establish guarantees that coun- strains circulating in those countries.
tries importing livestock are entitled to demand of In 2000 this strain spread to Japan which had
exporting countries to ensure protection against been free of FMD since 1908, and to South Korea
the introduction of the virus without creating which had been free of FMD since 1934 (Saka-
unjustified sanitary barriers. Countries recognized moto and Yoshida, 2002). This strain of virus was
as ‘FMD-free without vaccination’ thus have a clear also responsible for outbreaks in South Africa and
for the devastating epidemic in the UK in 2001 spread across North Africa (Knowles et al., 2014);
(Knowles et al., 2001; Knowles and Samuel, 2003). it has caused outbreaks in Libya (in 2013), Saudi
It is interesting that over a 12-year period, the Arabia (2013) and then Algeria (2014) and Tunisia
viruses causing these outbreaks of FMD appear to (2014) (see http://www.wrlfmd.org/fmd_geno-
belong to a single lineage based on the nucleotide typing/2014/WRLFMD-2014-00029%20O%20
sequence of the VP1 coding region, since this Tunisia%202014.pdf). The latter two countries are
region differs by no more than 5% between the fairly close to Europe and the presence of FMD in
various isolates (note: VP1 is one of the surface these countries represents a significant threat.
exposed proteins of the virus; it is highly variable Another lineage of serotype O FMDV (termed
and thus its sequence is frequently used to differen- O/SEA/Mya-98) was responsible for the severe
tiate between strains). outbreaks of disease in Japan and South Korea in
A successor to the PanAsia lineage of serotype 2010 and has also affected other countries in East
O FMDV, now termed PanAsia-II, was present in Asia including China (Valdazo-González et al.,
Afghanistan in 2004 and in Pakistan in 2005 ( Jamal 2013).
et al., 2011a) and has been responsible for extensive
outbreaks in the Middle East and within Southern
Asia. Various strains within this lineage have now The 2007 UK outbreak in the UK
emerged. One strain within this lineage, termed After the experience of the 2001 FMD epidemic,
O-PanAsia-IIANT-10, was first identified in Pakistan the UK suffered another FMD outbreak in 2007.
in 2009 (Brito et al., 2013) and it has spread west- This time the virus responsible was a derivative of
wards and caused disease in the Middle East and the O1 BFS 1860 strain (which is used as a refer-
adjacent countries including Turkey, Israel, Libya, ence strain and for vaccine production) and had
Egypt and Bulgaria; the latter is the most recent been in use within the Pirbright Institute and the
(mainly during 2011) outbreak of FMD in Europe. Merial vaccine production facility that are located
This outbreak was unusual in that it was first identi- on the same site. The index case occurred in fairly
fied in wild boar and only later on within domestic close proximity to Pirbright and it is believed that
animals. There was extensive serological and viro- the outbreak resulted from virus escape from these
logical screening of farms and wildlife in Bulgaria. premises. The outbreak appeared to occur in two
A pathway of transmission between the affected separate clusters and in total 8 separate infected
farms could be generated from the sequences of the premises were involved and were all within a
viruses that all shared a single ancestor (Valdozo- relatively limited geographical area (Cottam et al.,
Gonzalez et al., 2012). Seropositive wild boar were 2008). The use of near full-genome sequencing of
only detected in close proximity to the infected the viruses from different cases facilitated detec-
domestic livestock (Alexandrov et al., 2013) and tion of some infected premises that linked the two
the disease has not been maintained after the cull- clusters and indicated that only a single release of
ing of the infected farms. Thus it appears that the virus has occurred. The relatively restricted size of
wild boar and deer populations, at least within that this outbreak may in part be due to the cell culture
environment, were unable to maintain the virus adaptation of the virus strain that was responsible
(see Dhollander et al., 2016). Recent sub-lineages for the infection.
within the PanAsia-II lineage, e.g. PanAsia-IIAnt-10 This outbreak was in great contrast to the epi-
and PanAsia-IIFar-09 are continuing to circulate in demic in 2001 that was caused by the Pan-Asia
the Middle East and West Eurasia (see Brito et al., strain of serotype O virus. It is likely that the initial
2015). introduction of the virus into pigs in 2001 occurred
A separate lineage of type O has been identified through the illegal feeding of untreated waste food.
within Pakistan, Afghanistan and Iran ( Jamal et al., The virus most probably spread by the airborne
2011a) and was termed PanAsia-III but so far it has route from pigs to sheep on neighbouring farms,
not migrated outside of these three countries. and then by animal movements to various parts of
In contrast, another lineage, termed O/ME-SA/ the country and also to Northern Ireland, Scotland,
Ind-2001, that was initially identified within India France and the Netherlands. Although all move-
and some neighbouring countries, has recently ment of animals within the UK were stopped after
the disease was detected, more than 70 premises 2011c). Indeed, outbreaks due to this novel strain
had already become infected by that time. This have been reported in animals given vaccine based
spread of the virus was undoubtedly increased by on the Asia-1/Shamir strain. A homologous vac-
the difficulty of recognizing the disease in sheep, cine prepared from an isolate (Asia-1/TUR/11)
which show few overt clinical symptoms, as well as responsible for a field outbreak in Turkey in 2011,
by the very large trade in sheep that had developed has been found to be moderately effective to con-
in the UK. In total over 2000 premises were affected tain the spread of these Sindh-08 (Group-VII)
and nearly 4 million animals were slaughtered, of viruses (Knight-Jones et al., 2014).
these, more than 80% were sheep, 15% cattle, and
5% pigs and other animals such as goats. The costs
of the 2001 outbreak have been estimated as 3.1 Foot-and-mouth disease in
billion pounds sterling from losses to agriculture Africa
and the food chain, and between 2.7 and 3.2 billion As with many other virus diseases, FMD is endemic
pounds sterling from losses to businesses and tour- in most countries within the African continent
ism (Thompson et al., 2002). and six of the known FMDV serotypes (all except
Asia-1) have been found there. Although the dis-
ease has been largely controlled in South Africa,
Emergence of the A-Iran-05 Botswana and Namibia by strict enforcement of
lineage of FMD viruses livestock movement control and good surveillance
Serotype A FMDVs are the second most common to detect outbreaks, most other African countries
after serotype O globally. A new lineage of viruses, lack the necessary resources and infrastructure to
termed A-Iran-05 was initially identified in samples deal properly with the disease. An additional prob-
from Iran but has spread throughout the Middle lem is caused by the reservoir of FMDV that exists
East and has become the predominant serotype A in wildlife, particularly African buffalo (Condy et
lineage in the region. The virus has been identified al., 1985).
in West Eurasia including Pakistan, Afghanistan, In September 2000, the Pan-Asian type O virus
Saudi Arabia, Jordan and Turkey (Knowles et al., caused an outbreak of FMD on a pig farm near
2009). Viruses belonging to this lineage have also Pietermaritzburg, KwaZulu-Natal Province, South
been detected in North Africa including Libya Africa. This was the first FMD recorded in the prov-
(2009) and Egypt (2010, 2013–14).Initially this ince since 1956, and the first occurrence of FMDV
lineage appeared to be well matched to the A22/ type O in South Africa. It is highly likely that the
Iraq vaccine strain but more recently multiple sub- virus was carried in food waste fed to pigs (swill)
lineages have arisen that are antigenically distinct obtained illegally from a ship visiting Durban that
(e.g. A-Iran05BAR-08; Jamal et al., 2011b). had originated in South-East Asia. However, as
indicated above, the O/ME-SA/Ind-2001 lineage
has spread extensively across North Africa recently
Emergence of a new lineage of and outbreaks of other serotype O viruses continue
serotype Asia-1 FMDV to occur in East and West Africa (see Brito et al.,
A new lineage of serotype Asia-1 has emerged in 2015).
recent years. It was first detected in Pakistan in The last known cases of FMD caused by sero-
2008 ( Jamal et al., 2011c) and subsequently spread type C FMDV within Africa occurred in Kenya,
throughout West Eurasia region. It is now termed in East Africa, during 2004 (Sangula et al., 2011);
Asia-1/Sindh-08 (or Group VII) and it has caused it was also detected in Brazil in the same year. In
significant problems since the usual vaccines against Kenya, multiple isolates of serotype C had been
Asia-1 did not provide good protection against this made over the preceding 40 years with remarkably
virus. It continues to circulate in the West Eurasian little sequence change and it seems likely that these
region and has reached Turkey. Viruses belong- outbreaks resulted from reintroduction of a vac-
ing to this lineage are not efficiently neutralized cine strain of the virus into the field (Sangula et al.,
by antisera raised against the Asia-1/Shamir and 2011). No serotype C FMDV has been detected
Asia-1/Ind/8/79 vaccine strains (see Jamal et al., anywhere in the world since 2004 and it is now
possible that this serotype is extinct (Roeder and an additional seven factors in a second Institute of
Knowles, 2009). Medicine Report published in 2003 (Smolinski et
The Southern African Territories (SAT) sero- al., 2003):
types 1–3 are normally restricted to sub-Saharan
Africa and the SAT 1 and SAT 2 serotypes are much 1 microbial adaptation and change;
more prevalent that SAT 3 (Vosloo et al., 2002). 2 human demographics and behaviour;
These serotypes can be maintained for long peri- 3 breakdown of public health measures;
ods, without apparent clinical disease, in African 4 international travel and commerce;
buffalo (Syncerus caffer). Recently (2012–2104) 5 changes in technology and industry;
severe outbreaks of FMD caused by SAT 2 virus 6 economic development and land use;
have occurred in North Africa, particularly in Egypt 7 human susceptibility to infection;
and Libya (see Ahmed et al., 2012). 8 climate and weather;
Serotype SAT 3 FMDV has been isolated from 9 changing ecosystems;.
cattle and buffalo in sub-Saharan Africa. Until 10 poverty and social inequality;
recently, the last isolate of SAT 3 FMDV was made 11 war and famine;
from samples collected from buffalo within the 12 lack of political will;
Kruger National Park, in South Africa, during 2006. 13 intent to harm.
It is likely to still be maintained within this Park.
Other isolates have been made from elsewhere, The scope of the report, however, was limited
previously, including distinct variants from buffalo to infectious diseases in humans. It organized the
within Uganda. Recently a new isolate of SAT 3 contributory factors into four interlinked domains
FMDV was made from samples obtained from sen- of the emergence of infection:
tinel Long Horned Ankole calf that were introduced
into the Queen Elizabeth National Park in Uganda 1 genetic and biological factors;
in 2013 where it had close contact with other 2 physical environmental factors;
cattle and buffalo. Although no clinical disease was 3 ecological factors;
observed, the calf was found to seroconvert against 4 social, political and economic factors.
FMDV shortly after its arrival in the National Park
and FMDV was isolated from oropharyngeal fluid. In considering FMD as a re-emergent infection
Following characterization, including complete it is useful to consider to what extent these factors
genome sequencing, it was apparent that this new apply to FMD.
isolate was most closely related (but with only
about 80% identity) to another Ugandan SAT 3 Genetic and biological factors
FMDV which had been isolated in 1997 (Dhiku- FMDV is a highly mutable virus, and within each
sooka et al., 2015). Thus, it seems that the virus of the seven recognized serotypes there are multi-
has been circulating, within Uganda, during all this ple subtypes that can be distinguished genetically
time but without detection. This may reflect a lack within the virus population (Knowles and Samuel,
of sampling and/or lack of clinical disease in the 2003). The FMDV population displays high muta-
indigenous cattle resulting from infection by this tion rates and quasispecies dynamics (Domingo
virus. The circulation of FMDVs in the absence of et al., 2003). The virus evolves at a rate of about
apparent disease clearly complicates the issue of 0.5–1% of its genome per year (ca. 40–80 nt
disease control. changed). This rate of change means that essentially,
during an outbreak, that as the virus spreads from
one premises to another that its consensus genome
FMD as a re-emerging disease sequence acquires a single nt change. This accounts
The 1992 Institute of Medicine Report (Leder- for the ability to determine the pathway of virus
berg et al., 1992) listed six principal factors that transmission during outbreaks on the basis of the
contribute to the emergence or re-emergence of an complete genome sequence (Cottam et al., 2008;
infectious disease and these were supplemented by Valdazo-González et al., 2012). There is no doubt
that within this population change, mutations will rapid and long-range movement of goods for trade
occur that can result in viruses with altered host can allow inadvertent virus transmission to occur
range or pathogenicity. over very long distances. Indeed the introduction of
A potentially important biological factor in a type O (PanAsia strain) FMDV into South Africa
FMD emergence is the persistence of FMDV in in 2000 and into the UK in 2001, both appear to
many species of wild animals, especially in Afri- be mediated through such long-distance movement
can buffalo, which makes the challenge of global of contaminated material. The virus introductions
eradication of the disease very difficult. Even in occurred when there was no virus of this serotype
European countries, there are susceptible wildlife in adjacent countries and thus they represent exam-
species, such as deer and wild boar, which may be ples of virus transfer presumably through human
capable of harbouring the virus and potentially actions.
acting as a source of transmission of infection.
However, so far it does not seem that such species Social and economic factors
within Europe are able to maintain the virus for a An important social consequence of the ‘stamping-
significant period (see Dhollander et al., 2016) and out’ policy during the UK epidemic in 2001 was
it has not been necessary to cull wildlife in Europe the adverse effects of culling livestock on the non-
to control outbreaks of the disease. agricultural community. Larger numbers of animals
were slaughtered as a means of disease control than
Physical environmental factors in any other epidemic over the last 40 years, and
Many elements of the physical environment play the funeral pyres of thousands of animals were seen
a role in the survival of viruses and may affect the regularly on television as well as by the rural com-
transmission between susceptible hosts. Humid munity. Movement restrictions were imposed for
conditions seems to favour the air-borne dissemina- more than ten months not only upon susceptible
tion of FMDV (Donaldson, 1972) and virus survival livestock, but also on persons walking in the coun-
in the environment is greatly enhanced under cool tryside for recreational purposes. This undoubtedly
conditions (Bøtner and Belsham, 2012). The air- had a damaging effect upon rural businesses as well
borne spread of FMDV over many miles, especially as tourism, contributing to the enormous economic
across water, has been documented, as occurred in cost of the epidemic (Taylor, 2002). An important
1981 when the virus spread from France to the Isle economic factor that undoubtedly played a role in
of Wight over water. the UK FMD epidemic in 2001 was the increase in
However, there are considerable species dif- sheep farming consequent upon the earlier disas-
ferences in such transmission (Donaldson et al., trous BSE epidemic that had made beef production
2001). Animals vary in their ability to shed virus unprofitable for many farmers. Sheep infected
in aerosol form and also in their susceptibility to with FMDV are much more difficult to recognize
infection by airborne virus. As indicated above, compared with cattle, and it was the virus spread,
pigs exhale large amounts of virus while cattle are due to movement of infected sheep, that greatly
highly susceptible to infection by the aerosol route, increased the magnitude of the epidemic before it
thus cattle downwind of infected pigs are at high was first recognized. Trade to other countries from
risk of infection. It is also important to realize that the UK also lead to introduction of the disease into
individual virus strains may exhibit different prop- France and The Netherlands. There is concern that
erties that can affect transmission, and this should deliberate introduction of FMDV into ‘disease-free’
be taken into account when attempting to control countries could be used as an economic weapon.
an epidemic (Sutmoller et al., 2003).
the worldwide pandemic of the PanAsian strain of outbreaks with serious social and political conse-
FMDV type O were severe and, more recently, a quences in the future.
number of other virus strains have also shown their
ability to spread rapidly on a regional basis and Acknowledgements
take over from previously circulating strains (e.g. We thank Syed M. Jamal and Anette Bøtner for
A-Iran-05). Similarly, the incursion of SAT 2 virus helpful comments on this manuscript.
into North Africa demonstrates that unexpected
changes in virus distribution can occur. These References
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1A 44 Adenovirus 75, 118, 233, 257, 333–335, 339, 346, 350,
1AB 45 360, 368, 371, 375
1C 45 Adjuvants 202, 203, 211, 215, 221, 234, 236, 239–241,
1D 45 244, 245, 247–249, 251, 254, 260–262, 287, 291,
2A 9, 28, 43, 50, 52 292, 294, 299, 302, 304, 308, 317, 321, 334, 335, 337,
2A-like sequences 52, 53 339–341, 343, 347, 349, 413
2A-mediated cleavage 52 African buffalo, infection 179–192
2A-mediated translational ‘recoding’ 51 African buffalo, population numbers 182
2B 9, 28, 43, 50, 121 Antibody complexes 5, 85, 86
2BC 44 Antibody escape 89
2C 9, 28, 72, 121, 122, 124 Antibody levels 184
3A 9, 29, 122, 124 Antibody recognition 83
3ABC 54, 121, 219, 278, 298, 369 Anti-FMD vaccines 3, 61, 67, 75, 76, 79, 80, 88, 89,
3B 9, 122, 123 96, 202, 203, 287, 289–293, 295, 297, 299, 301, 303,
3BCD 10 305–308, 333, 335, 336, 338–342, 347, 348–349, 350,
3C protease 9, 10, 28, 30, 32, 43, 45, 46, 48, 53–55, 70, 73, 358, 411–412, 413, 414, 419
119, 120, 141 Antigen presentation 211, 214, 221, 222, 231, 233–235,
3C structure 47 236, 238–240, 247, 260, 261, 319
3C, catalysis 46 Antigen processing 211, 213, 214, 225, 233, 234, 236, 239,
3CD 9, 10, 30–32, 45, 46, 48, 122, 139 247, 261, 319
3D 9, 10, 30, 31, 108, 118, 122, 123, 137–139 Antigen–antibody recognition 152
3D, complex with template-primer 138 Antigenic drift and shift 152
3D, fidelity 137 Antigenic sites 87, 88, 152, 156
3D, motifs 137–141 Antigenic variants 151, 193, 279, 299, 304, 317, 322, 327,
3D, mutants 142, 143 334, 335
3D, NTPs channel 138 Antigenic variation 149, 151, 152, 180, 186
3D, processivity mutants 143, 144 Antigenic variation, in absence of immune selection 152
3D, structure 137, 142 Antigen-presenting cell 213, 214, 216, 225, 234–238, 239,
3D, template channel 138 247, 319
3D, ternary RNA-rNTP complex 142 Antisense oligonucleotides (ASO) 365, 366
5-Fluoridine triphosphate (FUTP) 141 Antiviral agents 161, 203
5-Fluorouracil (FU) 161, 162, 360, 361 Antiviral drugs 5, 24, 47, 203, 357–359, 362, 378
Arg-Gly-Asp (RGD) motif 67, 69, 85, 90–93, 109–116,
A 153, 224, 225, 318, 320, 337, 339
A-particle 117 Assembly 71, 74, 119
Acid lability 77, 78 Attenuated 6, 16, 27, 29, 93, 122, 203, 325, 369
Acid lability, and histidine residues 77 Autophagy pathway 121
Acid lability, and viral RNA 78–79
Acid resistance 78, 82 B
Ad5-FMD (Ad5 expressing FMDV capsid) 333, 335, 336, Bacteriophage φ6 158
338–341, 343, 347, 348–349, 350 Bayesian evolutionary analysis 180
Ad5-IFN (Replication-defective human Adenovirus type B-cell epitope 84, 224, 244, 245, 317, 319, 320, 322,
5 vector expressing IFN) 333, 335, 341–342, 343, 346, 325–327, 341
347, 348–349 Beclin1 121
Adaptation 94, 113–116, 123, 137, 142, 149, 151, 153, Biocontainment 289, 290
155, 201, 224, 225, 287, 305, 307, 420, 422 Biosafety 288, 289, 290, 304, 305
Biotherapeutics 335, 341, 343, 348, 349, 359 DIVA (differentiating infected from vaccinated animals) 4,
B-lymphocytes 211, 214–216, 234, 236, 238, 240, 75, 123, 278, 288, 296, 321, 325, 333, 334–336
242–249, 256, 259–260, 261–262, 319
Bus 8 E
Echovirus 9 110
C Economic impact 194
Capsid axes 65, 74, 82, 115 EITB 298
Capsid de-stabilization 118 ELISA 3, 10, 230, 239, 243, 252, 276–278, 281, 292, 294,
Capsid structure 62–64 298, 301, 359, 371, 376
Capsid with increased thermostability 79, 80 Emergency vaccination 221, 289, 296, 357–359
Cardioviruses 16 Empty capsids 62, 70, 73, 75–79
Cell culture adaptation 115 Encephalomyocarditis virus (EMCV) 16, 20, 22, 23, 26,
Cell lines, for FMDV infection 109, 110 45, 137
Cellular receptors 107–113 Endemic 2, 3, 5, 10, 108, 172, 179, 181, 182, 194, 196,
Chemically inactivated vaccines 79, 291, 317, 334, 424 198, 199, 201, 275–277, 279–281, 299, 300, 308, 377,
Chitosan 378 409, 411, 414, 415, 417, 421
Clinical signs 171–175, 188, 192, 233, 275–276, 324, 339, Endocytosis 95, 96, 117, 118, 214–216, 223, 226–229,
343–345, 368, 369, 388, 389 231, 232, 234, 236–241, 247, 254
Coevolution of antigenicity and cell tropism 152 Enterovirus (EV71) 29, 137, 140, 141
Compartments 153, 196, 211, 213, 214, 217, 234, Enzootic 33, 337, 349, 358, 418, 419
239–240, 257, 318, 377, 386–390 Epidemics 5
Complex adaptive systems 156 Epidemiological models 385, 386, 390
Consensus sequence 148, 159, 164 Epidemiology 148, 154, 179, 182, 183, 191, 192, 280, 385,
Conventional vaccine 6, 7, 307, 318, 320 388, 390, 400, 404, 405, 411
Coxsackievirus A9 (CVA9) 110 Epitopes 7, 67, 71, 76, 83–89, 152, 186, 203, 211, 214,
Coxsackievirus B3 (CVB3) 137, 140, 141 229, 239, 244–248, 253, 257, 261, 317, 319, 321–327,
Cre 8, 15, 16, 18, 30, 124, 139 341, 349–350
Critical Process Parameters (CPPS) 289, 290 Equine rhinovirus A 7
Critical Quality Attributes (CQAs) 289, 290 Eradication 6, 183, 197, 201, 275, 288, 296, 306, 349, 358,
Cryoelectron microscopy (cryo-EM) 62 409–415, 419, 423, 424
Crystallographic protomer 64 Error catastrophe 156, 159, 161
CTL (cytotoxic T lymphocytes) 241, 319, 321, 325, 327 Error threshold 160, 161
Cytokine profile 240, 241, 247, 248, 249, 260 Error-prone replication 147
Cytokines 211, 213–215, 217, 221, 223, 233, 235, 238, European Commission for FMD Control (EuFMD) 3,
240–244, 247–249, 256, 259, 260, 261, 319, 344, 347, 359 411, 413, 414, 419
Cytotoxic 53, 152, 211, 238, 256, 321, 362, 363, 366, 377 Evolutionary rate 149, 154, 155
D F
Danger-associated molecular patterns (DAMPs) 213, 215, FAO (Food and Agriculture Organization of the United
232, 234–236, 239, 240 Nations) 3, 196–198, 201, 282, 302, 306, 307, 358, 419,
DCTN3 host factor 123 424
Deep sequencing 3, 151 Favipiravir 364
Defective-interfering genomes 53 Fencing 201
Delivery of antigen 214, 215, 240 Fitness 157–159, 161, 162
Dendrimer peptide vaccine 327 Fitness, epidemiological 154
Dendrimer/Dendrimeric 248, 323, 325–327 Fitness, fluctuations 163
Dendritic cells 4, 211, 215, 216, 218, 221, 222, 237, 259, Flavonoids 377
319, 339 FMD-free 179, 180, 182, 194, 197–199, 201, 203, 275,
Deterministic behaviour 157 276, 278, 280, 287, 289, 296, 305, 333, 336, 412, 417, 419
Diagnosis 2, 3, 157, 175, 193, 275–277, 281–283, 288, Food safety 200, 358, 359, 378, 409
308, 357, 413
Diagnostic test 5, 275, 276, 282, 289, 298, 413 G
Disease control measures 180, 199 Gemin5 23, 28, 53
Disease management 196–200 Generation time 386, 391, 394
Disease notification 195–199 Genetic variation 149, 186
Disease outbreaks 1, 2, 5, 107, 122, 149, 154, 172, 173, Genome organization 14
179, 180, 186, 188–189, 192, 194–196, 198, 199, 275, Genome segmentation 150, 162–164
277, 279–281, 287–289, 291, 300, 304, 306–308, 317, Genome-scale ordered RNA structure (GORS) 7, 8
318, 333–335, 337, 340, 341, 349, 357, 359, 378, 379, GH loop 62, 65–67, 69, 83, 84, 85, 86, 92, 94, 109, 110,
385, 386, 388, 390, 391, 393, 394, 399–404, 409–411, 112, 318, 320, 327
414, 415, 417–424 GH loop, antibodies elicited by 67
Disease, early descriptions 180 GH loop, immunodominance 86
Disease-free herds 203 GH loop, mobility 65