Journal of General Virology (1990), 71, 2141-2147.
Printed in Great Britain
Detection and differentiation of picornaviruses in clinical samples
following genomic amplification
D. Michael Olive, l,2* t Siham Al-Mufti, 2 Wahiba Al-Mnlla, 1 M. A. Khan, 3 Alexander Pasca, 1
Glyn Stanway 3 and Widad AI-Nakib 1,2
~Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, 13110 Kuwait, ZPublic Health
Virology Laboratory, 5540 Shaab, 13056 Kuwait and 3Department of Biology, University of Essex, Colchester C04 3SQ,
U.K.
A polymerase chain reaction (PCR) assay was used to
detect and differentiate picornaviruses (PVs), using
primers homologous to the 5' non-coding and VP2
regions of the PV genome. The P C R resulted in a
530 bp PCR product for human rhinoviruses (HRVs)
and a 650 bp product for polioviruses, coxsaCkieviruses
(CV) or echoviruses. The P C R assay could detect as
little as 1 p.f.u, of virus in either cerebrospinal fluid
(CSF) or stool, using ethidium bromide-stained gels.
Standard strains of poliovirus, CV, echovirus and
H R V were detected, with the exception of echovirus
type 22. In contrast, heterologous viruses, such as
herpes simplex virus, human cytomegalovirus, adeno-
Introduction
Picornaviruses (PVs) are a diverse group of human
pathogens which cause a wide variety of disease
syndromes. In terms of clinical significance, entero-
viruses (EVs) and human rhinoviruses (HRVs) are the
most clinically important genera of PVs, causing millions
of infections each year. Infections due to EVs range in
severity from asymptomatic to aseptic meningitis,
encephalitis, paralysis, pneumonia and myocarditis
(Bowles et al., 1986, 1987; Easton & Eglin, 1988; Leslie et
al., 1989). Although infections of the central nervous
system and heart are of greatest concern, these viruses
are multi-systemic in their tissue tropisms and virulence.
For this reason, the clinical diagnosis of EV infection can
be complicated by the non-specific nature of many of the
syndromes, which often makes them indistinguishable
from diseases caused by bacteria and other viruses.
HRVs are the major causative agents of the common
cold (Couch, 1985). Although infection with HRVs is
usually limited to the upper respiratory tract, they can
t Present address: GENE-TRAK Systems, 31 New York Avenue,
Framingham, Massachusetts 01701, U.S.A.
0000-9581 © 1990 SGM
2141
virus, influenza virus and rotavirus, as well as human
and monkey cell DNA, were not amplified. In nasal
swabs taken from patients with respiratory infections,
the PCR detected 27 of 28 H R V isolation-positive
specimens. All specimens from which viruses other
than HRVS were isolated were negative by PCR. The
P C R definitively identified poliovirus and CVs from
the CSF or stool of patients with aseptic meningitis, as
well as CV in the pericardial fluid of a patient who had
suffered a myocardial infarction. Specimens taken
from patients with similar pathologies, and from which
heterologous viruses were isolated, were uniformly
negative by PCR.
infect the lower respiratory tract (Krilov et al., 1986;
Monto et al., 1987). In one study, 6 5 ~ of infected
individuals who were 40 or more years of age had lower
respiratory tract infections due to HRVs (Monto et al.,
1987). HRVs have also been isolated from a high
percentage of children and infants hospitalized with
lower respiratory tract infections (Krilov et al., 1986).
Finally, HRVs are known to exacerbate episodes of
bronchitis and asthma (Gregg, 1983). With the develop-
ment of drugs such as R61837 which have been recently
shown to suppress colds in vivo yet are highly specific for
HRVs (A1-Nakib et al., 1989), it is imperative to be able
to diagnose HRV infections rapidly.
The broad antigenic diversity among the nearly 70
different serotypes of EVs and over 100 different
serotypes of HRVs has made immunoassays impractical
(Yolken & Torsch, 1980, 1981; Dearden & A1-Nakib,
1987). Nucleic acid hybridization assays have been used
but they lack sensitivity (Rotbart et al., 1984; A1-Nakib
et al., 1986; Bruce et al., 1989). Despite its suboptimal
sensitivity, tissue culture remains the mainstay of the
laboratory diagnosis of EVs and HRVs.
The polymerase chain reaction (PCR; Saiki et al.,
1988) has been used to detect pathogen-specific nucleic
2142 D. M. Olive and others
acids from viruses such as human immunodeficiency
virus type 1 (Kwok et al., 1987), human T-cell lympho-
tropic virus type 1 (Duggan et al., 1988), human cyto-
megalovirus (HCMV) (Demmler et al., 1988; Shibata et
al., 1988; Olive et al., 1989a, b) and PVs (Gama et al.,
1988, 1989; Torgersen et al., 1989; Hyypifi et at., 1989).
However, the PCR assays which have been described for
PVs have either not successfully differentiated EVs and
HRVs (Gama et aL, 1989; Hyypi/i et al., 1989) or have
suffered from a lack of specificity (Hyypi/i et al., 1989).
The assays described thus far have not been tested
directly on clinical specimens containing wild-type virus.
We have therefore developed a PCR assay, using
primers which detect and differentiate between EVs and
HRVs in clinical specimens on the basis of the size of the
PCR product. In this report, we demonstrate the
specificity of the assay for this group of viruses, as well as
its ability to detect wild-type clinical isolates of EVs and
HRVs from a variety of clinical specimens. The use of
this PCR assay for detection and differentiation of PVs
should greatly aid in the clinical management of disease
due to PVs.
Methods
Clinical specimens. Either nasal swabs or throat washings were taken
from patients presenting with respiratory infections. Clinical
specimens were processed immediately by inoculation onto Ohio HeLa
or human C16 cells (Phillpotts, 1983) for isolation of HRVs and
coronaviruses respectively. HRVs were also examined for acid lability
after isolation. Influenza virus isolates were obtained following growth
in embryonated eggs (Kendal et al., 1985). Cerebrospinal fluids (CSF)
and stool swabs were taken from patients presenting with various
symptoms possibly due to enterovirus infection. Stool swabs were
placed in 2 ml of tissue culture medium and, along with CSF
specimens, processed for isolation of virus by inoculation onto Vero,
HEp-2 or HeLa cells. EVs were typed using intersecting hyperimmune
serum pools A to H obtained from the World Health Organization
(WHO). Standard strains of EVs and HRVs were obtained from
Dr D. A. J. Tyrrell at the MRC Common Cold Unit, Salisbury, U.K.
S y n t h e t i c oligonucleotide primers. The mixed primer OL68-1
(5' GGTAAQTTCCACCACCANCC 3') is complementary to the
genomic sense RNA at positions 1058 to 1077 (Hughes et al., 1988) in
the VP2 region of HRV-1B. The OL68-1 primer contains degeneracies
at positions 6 and 18 (Q is either T or C; N is A, G, C or T). The primer
OL24 (5' CTACTTTGGGTGTCCG 3') is complementary to anti-
genomic sense RNA at positions 542 to 567 (Hughes et al., 1988) in the
5'-non-coding region of HRV-1B. The degree of homology exhibited
by the primers to known EV and HRV sequences is shown in Fig. 1
(M. A. Khan & G. Stanway, unpublished results). The alkaline
phosphatase-labelled oligonucleotide probe, PV. 1 (5' AGATTGT-
TATCATAAAGCGA 3'), for detection of poliovirus PCR products,
was homologous to nucleotides 607 to 626 of poliovirus type I (Toyoda
et al., 1984). All oligonucleotides were purchased from Genetic
Designs.
E x t r a c t i o n o f viral nucleic acids. A 100 ~tl portion of either a nasal
swab in transport medium, CSF or stool suspension was used for
DNAsynthesis-~ ~---DNAsynthesis
OL24: $'CTACTTTGG6TGTeCG-3" 3"-CCI,IACCACCACCTT0/~TGG-5" :OL68-1
HRV1B . . . . . *****~***** ********************
HRVI4 **************** ********c***********
HRV89 *** . . . . . *** . . . . * ****** . . . . . . *****c**
CAV9 *** ..... *** .... . *****************c**
CAV21 **************** **************cG****
CBV] **************** ********************
CBV3 **************** ***************c.G**
Poliol **************** *****************G**
Polio3 **************** ********'***'*******
Fig. 1. The sequences of the OL24 and OL68-1 primers used for
amplification are shown. The complementary sequences of known EVs
and HRVs are shown to illustrate the homology. In representing the
primer sequences, N is any nucleotide (A, G, C or T) and Q is either T
or C. The sequences were derived as follows: HRV1B, Hughes et al.
(1988); HRV14, Stanway et al. (1984); HRV89, Deuchler et al. (1987);
CAV9, Chang et al. (1989); CAV21, Hughes et al. (1989); CBV1,
Iizuka et al. (1987); CBV3, Lindberg et al. (1987), Jenkins et al. (1987);
Polioviruses 1, 2 and 3, Toyoda et al. (1984). Polio 1, 2 and 3,
polioviruses 1, 2 and 3.
nucleic acid extraction. Vanadyl ribonucleoside complex was added to
a final concentration of 10 mM. As a carrier, 1.0 ~tg of phenol-extracted
yeast tRNA was added. The samples were extracted with an equal
volume of phenol saturated with TE buffer (10 mM-Tris-HC1 pH 7-5,
1 mM-EDTA). The phenol phase was back-extracted with 100 ~tl of
sterile water and the aqueous phases were combined. The aqueous
nucleic acid was extracted three more times with phenol-TE, followed
by two extractions with equal volumes of chloroform. The nucleic acid
was precipitated by addition of 0.1 volume of sterile 3 M-sodium acetate
(pH 6.5) and 2.5 volumes of 95 ~ ethanol, followed by refrigeration at
- 70 °C for 15 min. The nucleic acid was pelleted by centrifugation in a
microfuge for 5 min and the pellet washed once with 70~ ethanol. The
pellet was dried under vacuum and dissolved in 50 ktl of sterile water.
The nucleic acid from each sample was extracted in triplicate and
processed for cDNA synthesis and amplification.
R e v e r s e transcription. Synthesis of cDNA was performed in a
reaction mixture containing 25 Ftl of viral nucleic acid, 8 p.l of 5 x
reverse transcriptase buffer (200 mM-Tris-HCI pH 8.3, 375 mM-KC1,
50 mM-dithiothreitol, 15 m~-MgC12), each dNTP at 0.5 mM, 40 units
RNAguard (Pharmacia), 1 pl of OL68-1 primer stock (1-0 A260
units/ml) and 200 units of Moloney murine leukaemia virus reverse
transcriptase (Bethesda Research Laboratories) in a total volume of
40 ~tl. The reaction was incubated at 37 °C for 1 h and was terminated
by the addition of 4 I-tlof 3 ~-sodium acetate pH 6.5 and 100 I.d of 95
ethanol. The mixtures were refrigerated at - 7 0 °C for 15 min, pelleted
and the cDNA was dissolved in 50 I.d of TE buffer.
P o l y m e r a s e chain reaction. For PCR, 15 p.1 of the eDNA was
combined with 10 I.tlof 10 x Taq salts (500 mM-KC1, 100 mM-Tris-HC1
pH 8.3, 25 mM-MgC12, 0.1 ~ gelatin), 10 ~tl of 8 mM-dNTP stock (2 mM
each dNTP), 2 ~tl each of OL24 and OL68-1 primer stocks (1.0 A z 6 o
units/ml each), 60 lal of water and 2.5 units of T h e r m u s a q u a t i c u s DNA
polymerase (Perkin-Elmer Cetus). The mixture was amplified by 30
successive cycles of heating at 94 °C for 2 min, renaturing at 50 °C for
2 min and polymerizing at 72 °C for 3 min. A further unit of enzyme
was added after 15 cycles. The PCR products were analysed by
 Genomic amplification o f picornaviruses 2143

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11

738 b p - -
615 b p - -

738 b p - -
615 bp--

492 b p - -

Fig. 3. The PCR reaction was tested for cross-reactivity with a variety
of heterologous viruses and cell types. Lane 1, marker DNA; lane 2,
poliovirus type 1; lane 3, HSV; lane 4, HCMV; lane 5, Ad2; lane 6,
Ad5; lane 7, rotavirus; lane 8, HeLa cell DNA; lane 9, HEp-2 cell
DNA; lane 10, Vero cell DNA; lane 11, marker DNA.

electrophoresis on composite agarose gels (3% Nusieve agarose, 1%

HGT agarose). DNA bands were visualized following staining with
ethidium bromide and exposure to u.v. light. Positive controls for the
PCR reaction consisted of cloned cDNAs from poliovirus type 1 or
HRV-14.
Hybridization with an alkaline phosphatase-labelled probe. Portions
(50 ~tl) of the products of the PCR reactions were combined with 50 pl
of 0-6 M-NaOH and incubated at room temperature for 15 min. The
Fig. 2. The PCR products resulting from amplification of some of the denatured DNA was applied to a nylon filter using a filtration manifold
clinical specimens in Tables 2 and 3 are shown. Lane 1, marker DNA; and the samples were neutralized by washing each well with 100 pl of
lane 2, CSF containing vaccine strain poliovirus type 2 (meningitis); 2 M-ammonium aceta~.e. The filters were heated in a vacuum oven for
lane 3, CSF containing vaccine strain poliovirus type 2 (meningitis); 2 h. The filters were prehybridized for 15 min at 45 °C in buffer
lane 4, CSF containing CBV3 (meningitis); lane 5, stool containing containing 5 x SSC (1 × is 0.15 M-NaC1, 0-015 M-sodium citrate), 1%
CBV5; lane 6, pericardial fluid containing CBV2; lane 7, nasal swab SDS and 0-5% bovine serum albumin. The filters were hybridized for
containing wild-type HRV; lane 8, nasal swab containing wild-type 15 min at 45 °C in the same buffer containing 50 pl of a 0.4 ~tMstock of
HRV; lane 9, marker DNA. probe. The filters were washed twice with 100 ml of 1% SDS in 1 x SSC
T a b l e 1. Amplification of standard virus strains at 45 °C, followed by two washes in 100 ml each of 1% Triton X-100 in
1 x SSC at 45 °C. Finally, the filters were washed in 1 × SSC at room
Virus PCR Virus PCR temperature.
strain* result strain* result The washed filters were immersed in 7.5 ml of alkaline phosphatase
buffer (100 mM-Tris HC1 pH 8.5, 100 mM-NaC1, 100 mM-ZnCI2)
HRV2 + Echo2 + containing 33pl of Nitro Blue tetrazolium solution (75mg/ml
HRV9 + Echo3 + in dimethylformamide) and 25~tl of 5-bromo-4-chloro-3-indolyl
HRV14 + Echo4 + phosphate solution (50 mg/ml in dimethylformamide). The colour
Polio 1 + Echo6 + reaction was allowed to proceed for 4 h and terminated by washing in
Polio2 + Echo8 + distilled water.
Polio3 + Echo9 +
CAV1 + Echol l +
CAV9 + Echol6 +
CAV10 + Echol9 + Results
CAV 16 + Echo22 -
CAV21 + Echo25 + Detection o f standard virus strains
CAV24 + HSV -
CBV1 + HCMV - Standard strains of EVs and HRVs were amplified, and
CBV2 + Ad2 - t h e P C R p r o d u c t s w e r e d e t e c t e d b y gel e l e c t r o p h o r e s i s .
CBV3 + Ad5 -
CBV4 + Rotavirus - A s s h o w n i n F i g . 2, t h e E V s t e s t e d g a v e r i s e t o a
CBV5 + HeLa - characteristic PCR product approximately 650 bp in
CBV6 + HEp-2 - length. In contrast, HRV strains gave rise to a PCR
Echol + Vero -
p r o d u c t o f a p p r o x i m a t e l y 530 b p , u s i n g t h e O L 2 4 / 6 8 - 1
* Abbreviations: Poliol, poliovirus type 1 ; Polio2, poliovirus type 2; primers. Table 1 summarizes the results of the PCR assay
Polio3, poliovirus type 3; CAV, coxsackievirus A; Echo, echovirus. on standard strains of HRVs, polioviruses, coxsackie-
2144 D. M. Olive and others
1 2 3 4 5 6 7 8 9 10
738
615
Fig. 4. The sensitivityof the PCR was determined by detecting known
amounts of poliovirusin either stool suspensionor CSF. Seriallydiluted
virus was added to 100 ~tlof either stool or CSF and processed for virus
detection. Lanes 2 to 5 show the results of the titration in stool, whereas
lanes 7 to 10 show the resultsof the CSF titration. The samplesare: lane
1, marker DNA; lane 2, 125 p.f.u.; lane 3, 25 p.f.u.; lane 4, 5 p.f.u.;
lane 5, 1 p.f.u.; lane 6, marker DNA; lane 7, 125 p.f.u.; lane 8,
25 p.f.u.; lane 9, 5 p.f.u.; lane 10, 1 p.f.u.
viruses (CV) and echoviruses, as well as several unrelated
viruses and cell lines. With one exception, the character-
istic P C R product was seen for all of the PVs tested.
Echovirus 22, however, did not amplify using this assay.
In contrast to the P C R assay previously reported by
Hyypi~i et al. (1989), the OL24 and OL68-1 primers did
not give rise to spurious amplification products when
tested on herpes simplex virus (HSV), H C M V , adeno-
virus (Ad), rotavirus, H e L a cell D N A , HEp-2 cell D N A
or Vero cell D N A (Fig. 3). Only PVs were amplified and
detected.
Detection limits of the PCR assay
In order to determine the amount of virus which could be
detected in clinical samples, a titrated stock of poliovirus
was serially diluted and added to a negative stool or CSF
specimen. The viral nucleic acid was then extracted,
reverse-transcribed and amplified. Fig. 4 shows that
1 p.f.u, of virus could be detected on ethidium bromide-
stained agarose gels regardless of the nature of the
clinical specimen. Previous studies have reported com-
parable sensitivities using titrated virus stocks ( G a m a et
al., 1989; Torgersen et al., 1989).
Detection of HR V in patients with respiratory infections
Nucleic acids from either nasal swabs immersed in tissue
culture medium or nasal washings were extracted and
subjected to P C R analysis. As shown in Fig. 2, the
characteristic 530 bp P C R product was detected from
patients infected with wild-type HRVs. The P C R assay
Table 2. Detection of HR Vs from nasal swabs taken from
patients with respiratory infections
Virus No. of No. positive
isolated specimens by PCR
HRV 28 27
CV229E* 7 0
Influenza A 4 0
RSVp" 2 0
* CV229E, Coronavirus 229E.
t The diagnosisof RSV infection was based on a positive result in an
enzyme immunoassay rather than virus isolation.
was applied to 28 specimens taken from patients with
respiratory infections from whom H R V s had been
isolated (Table 2). H R V s were detected in 27 of these
patients by PCR. In cases where similar respiratory
infections were caused by coronaviruses, influenza virus
type A or respiratory syncytial virus (RSV), the P C R
assay was uniformly negative.
Detection of enteroviruses from clinical specimens
EVs are known to be causative agents of aseptic
meningitis and encephalitis (Melnick, 1985). During the
early acute phase of infection, infectious virus can
sometimes be isolated from the CSF whereas, in the later
stages of infection, virus can also be isolated from stool.
In cases of myocarditis, however, virus is rarely isolated,
presumably due to low numbers of infectious particles
(Leslie et al., 1989). Specimens taken from patients
hospitalized for confirmed EV infections were analysed
by P C R along with specimens taken from patients with
similar pathologies which were either verified or
suspected of being caused by infection with viruses other
than PVs (Table 3). In all cases, the characteristic 650 bp
P C R product was detected after amplification of
patients suffering from EV-mediated disease. The
procedure used in this study was suitable for a variety of
clinical specimens, including stool, CSF and pericardial
fluid. Cases of aseptic meningitis caused by either CVs or
the vaccine strain of poliovirus 2 were clearly detected
(Fig. 2). In two other patients with aseptic meningitis,
CVs were detected in stool cultures (Fig. 2). Significant-
ly, coxsackievirus B2 (CBV2) isolated from the pericard-
ial fluid of a patient who had suffered a myocardial
infarction was also detected by P C R assay (Fig. 2). The
P C R was unreactive with specimens taken from patients
with similar pathologies containing non-PV viral agents.
Hybridization with a poliovirus-specific probe
The identity of poliovirus P C R products was verified by
hybridization to an alkaline phosphatase-labelled oligo-
 Genomie amplification o f picornaviruses 2145

Table 3. Detection of enteroviruses from clinical specimens 1 2 3 4 5

Virus PCR
Specimen Patient diagnosis isolated result

CSF Meningitis CBV3 + [

CSF Meningitis CBV3 +
PCF* MI~ CBV2 +
Stool Meningitis CBV5 + Fig. 6. Identification of poliovirus PCR products by hybridization
Stool Meningitis CBV 1 + with the PV. 1 probe. The samples are derived from amplification of:
CSF Meningitis CBV5 + 1, poliovirus type 2, aseptic meningitis; 2, CBV3, aseptic meningitis; 3,
CSF Meningitis Polio2:~ +
CSF Meningitis Polio2:~ + poliovirus type 1; 4, poliovirus type 2; 5, poliovirus type 3. The PCR
CSF Encephalitis HSV - products for the aseptic meningitis samples are shown in Fig. 2, lanes 3
CSF Encephalitis HSV - and 4.
CSF SSPE§ None -
CSF SSPE None - shows considerable m i s m a t c h with the sequences o f
CSF SSPE None - other characterized EVs. The identity of the P C R
CSF Multiple sclerosis Nonell -
product obtained by amplification of the C S F material
* Pericardial fluid. from a patient with aseptic meningitis due to poliovirus
~"Myocardial infarction. type 2, was confirmed with the P V . 1 probe (Fig. 6). I n
Vaccine strain. contrast, a similar case of meningitis caused by CBV3
§ The diagnosis of subacute sclerosing panencephalitis (SSPE) was
based on high titres of anti-measles antibody in both serum and CSF. was unreactive with the P V . 1 probe (Fig. 6). The P V . 1
IIThe patient specimen was sent for investigation into possible viral probe did not detect any o f the other EVs tested,
causes. presumably owing to the heterogeneity o f this region
a m o n g EVs (unpublished data).

607 626
Discussion

5" - A C - , ~ T T G T T A T C A T ~ G C ~ - 3 " Efforts to detect and differentiate PVs directly f r o m

clinical samples by using nucleic acid hybridization
Poliol ~ ~ ~ ~ techniques have been largely unsuccessful. Bruce et al.
(1989) have shown that, although radioactively labelled
Polio2 ~ ~ ~ ~ synthetic oligonucleotide probes homologous to the 5'
untranslated region of H R V s can be used to detect most
Polio3 ~ ~ ~ ~ k n o w n serotypes, the sensitivity can vary significantly
depending on the viral isolate. In addition, the radioac-
cBvl **G******C****T**TT* tive label used is unsuitable for routine usage. Alkaline
phosphatase-labelled probes have been used as an
CBV3 -~ -~ ~- * -~ -x.~ .w--~ C -I~-~ -I( -~ -~ ~ ,I~-~ w-T alternative but are even less sensitive (Rotbart et al.,
1988). More recently, Petitjean et al. (1990) have used
CBV4 * * * * * * * * * C * * * * T * * * T * radioactively labelled riboprobes homologous to the 5'
non-coding region o f EVs. A l t h o u g h virus could be
CAV9 * * * * * * * * * C * * * * T * * * T * detected in most stool specimens, the detection o f EVs in
C S F again showed low sensitivity.
CAV21 C-¢~******* C C * * * T * * * T * The P C R assays described thus far have been unable
to give a simple, clear differentiation between EVs and
Fig. 5. The DNA sequence of the poliovirus detection probe, PV. 1.
The sequences of known EVs are shown to illustrale homology. The H R V s , nor have they been applied directly to clinical
sources of the sequence information are those mentioned in Fig. 1. samples ( G a m a et al., 1989; Torgersen et al., 1989;
Nucleotide numbers are indicated above the sequence. Hyypi~i et al., 1989). The specificity of antiviral drugs for
treatment of respiratory disease makes it imperative to
nucleotide probe ( P V . 1 ) specific for a conserved be able to detect and differentiate infections caused by
sequence o f the 5'-non-coding region o f polioviruses (Fig. H R V s and EVs directly on clinical specimens since
5). This sequence is not found in H R V s . The P V . 1 probe severe respiratory infections can be caused by both viral
hybridized specifically to P C R products obtained by groups. In addition, acute diseases such as aseptic
amplification of poliovirus types 1, 2 and 3 (Fig. 6). As meningitis or encephalitis require rapid diagnosis for
can be seen in Fig. 5, however, the P V . 1 probe sequence effective clinical m a n a g e m e n t .
2146 D. M. Olive and others
Therefore, we have developed a PCR assay which
detects and differentiates PVs directly from clinical
specimens. The OL24 primer sequence is derived from a
highly conserved region of the 5' non-coding region of the
PV genome. The OL68-1 primer is built around a highly
conserved stretch of three tryptophan codons (UGG)
found in the VP2 region of the PV genome. Using these
primers, we have been able to exploit the observation
that the 5' non-coding regions of EVs are approximately
120 bp longer than that of HRVs.
The PCR assay detected and differentiated all
serologically identified EVs or HRVs tested with the
exception of echovirus type 22. The unreactivity of
echovirus type 22 in our assay is not surprising, however,
since other studies which have tested probes for PV
detection have been uniformly unreactive with echovirus
type 22 (Auvinen et al., 1989; Hyypi~i et al., 1989).
Auvinen et al. (1989) have shown that the nucleotide
sequence of echovirus type 22 diverges significantlyfrom
that of other PVs. Presumably this is the cause of the
unreactivity of echovirus type 22 in our and other PCR
and hybridization assays.
Previously, PCR sensitivity has been determined on
viral stocks rather than clinical samples. We have shown
here, however, that as little as 1 p.f.u, of virus could be
detected in CSF or stool samples. This level of sensitivity
is sufficient for clinical application. The conditions
described here for nucleic acid extraction, reverse
transcription and amplification were suitable for all
types of clinical samples tested.
For detection of PV-mediated infections, we have
relied primarily on the electrophoretic detection of
specific sizes of PCR products without further identifi-
cation by hybridization, restriction enzyme digestion or
DNA sequencing. Although we have been able to use a
probe for identity confirmation of poliovirus PCR
products, this technique is not generally applicable to PV
PCR confirmation as much of the region of the viral
genome flanked by the OL24/OL68-1 primers shows
significant variability among PVs. Torgersen et al.
(1989) have used restriction enzyme digestion patterns of
PCR products obtained after amplification as a means of
serotyping HRVs. Owing to the large number of PVs, as
well as the high degree of sequence variability in the
region amplified in our assay, mapping with restriction
enzymes would be impractical on unknown virus
isolates. Furthermore, owing to the limited amount of
sequence information available for the more than 170
serotypes of PVs, it is likely that not even DNA
sequencing would definitively identify the PCR products
as PV-derived. Despite this limitation, we have not
observed spurious amplification of any of the heter-
ologous samples examined and the PCR assay resulted in
products of the expected size for almost every HRV or
EV tested.
By applying the PCR assay to respiratory illness, 27 of
28 wild-type HRVs isolated from nasal swabs were
correctly identified. The single HRV not identified
probably had a variant nucleic acid sequence at the site
of one of the primers. Similarly, EV isolation-positive
CSF fluids and stool specimens taken from patients with
aseptic meningitis gave rise to the EV-specific 650 bp
PCR product. None of the pathologically similar cases
from which heterologous viruses had been isolated were
amplified.
Significantly, CBV2 was detected in the pericardial
fluid of a patient who had suffered a myocardial
infarction. CVs have long been suspected of playing a
significant role in myocarditis, but definitive data have
been lacking due to the low recovery rates of virus from
pericardial fluids (Melnick, 1985; Leslie et al., 1989). The
ability to detect virus in pericardial fluid using the PCR
assay should facilitate a better definition of the role
played by these viruses in cardiac disease.
In summary, even in the absence of sequence
confirmation, we conclude that the assay is specific for
PVs and, in addition, will differentiate infection by EVs
and HRVs. The PCR assay described here is also rapid
and can be completed in a single day. Indeed, the rapid
detection and differentiation of PVs using the PCR assay
should allow better clinical management of PV-mediated
disease.
This work was supported by Kuwait University grant number
MI063.
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(Received 20 March 1990; Accepted 4 June 1990)