culture isolation. The test is based on the synthesis of cDNA, the polymerase chain reaction and the use of oligonucleotide probes. The primers were selected ...
J. gen. Virol. (1989), 70, 3261-3268. Printed in Great Britain
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Key words: PCRihuman picornaviruses/hybridization
Polymerase Chain Reaction for Human Picornaviruses By T I M O H Y Y P I A , * P E T R I A U V I N E N
AND M A R I T A
MAARONEN
Department of Virology, University of Turku, Kiinamyllynkatu 13, SF-20520 Turku, Finland (Accepted 29 August 1989) SUMMARY
We have used enzymic amplification of specific nucleic acid sequences followed by hybridization, for the rapid detection and typing of human picornaviruses after cell culture isolation. The test is based on the synthesis of cDNA, the polymerase chain reaction and the use of oligonucleotide probes. The primers were selected from the 5' non-coding region of the genome representing highly conserved regions. Sequences specific to enteroviruses and rhinoviruses were used as probes. The assay was able to identify all the picornavirus reference strains analysed and it was also possible to discriminate between enteroviruses and rhinoviruses by the hybridization procedure. When 29 picornavirus clinical isolates were analysed, all except one were detected by gel electrophoresis and a specific hybridization signal was obtained with all except three strains using the oligonucleotide probes. INTRODUCTION Human picornaviruses consist of approximately 200 serologically distinct members which are important pathogens. Human rhinoviruses (HRVs) are the most frequent cause of the common cold whereas members of enterovirus subgroups (coxsackie-, echo- and polioviruses) are responsible for e.g. meningitis, paralysis, myositis and myocarditis. Because of the large number of serotypes, specific virological diagnosis of these infections is problematic. Enteroviruses are currently detected using isolation in cell culture followed by neutralization typing with antiserum pools. Rhinoviruses are identified after isolation on the basis of their characteristic acid lability. These methods are laborious and time-consuming because at least two cycles in cell culture are needed for specific recognition of the virus. Since these viruses are common causes of human disease there is an obvious need for new rapid and reliable diagnostic procedures. The nucleotide sequences of approximately a dozen human picornaviruses are already known and these data can be used for the selection of nucleic acid probes for diagnostic purposes. The 5'-terminal non-coding part and regions of the ssRNA genome encoding non-structural proteins are highly conserved among human picornaviruses. Both cloned c D N A (Hyypi~t et al., 1984; Rotbart et al., 1984; A1-Nakib et al., 1986) and synthetic oligonucleotides (Rotbart et al., 1988) have been used successfully as broadly reacting probes in hybridization tests. However, these assays require a passage of the virus in cell culture for amplification of the target. Because it would be desirable to have an assay that could directly demonstrate the presence of virus in clinical samples, we have studied the possibility of using a combination of the polymerase chain reaction (PCR) (Saiki et al., 1988) and oligonucleotide hybridization, for human picornavirus detection. With this aim, we have first considered an assay system and reagents which can be used for identification of picornaviruses after a passage in cell culture. Our results indicate that the system based on these methods can be used for broad detection and typing of enteroviruses and rhinoviruses and that it could replace cell culture amplification. METHODS
Virus propagation and preparation of nucleic acids. Picornavirus standard strains included coxsackievirusesA2, A16, A21, B3, B4 and B6, echoviruses 11, 18 and 22, polioviruses 1 and 3, and rhinoviruses 1B, 2, 9, 14, 22 and 89. 0000-9167 © 1989 SGM
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T. HYYPI.~, P. AUVINEN AND M. MAARONEN
All the strains were from the American Type Culture Collection. Clinical virus strains were from the collection of routine isolation samples at the Department of Virology, University of Turku and they had been stored at - 70 °C for 2 to 2.5 years prior to re-inoculation. The enterovirus isolates had been typed previously using WHO serum pools A to H (WHO, Copenhagen) and rhinoviruses had been identified on the basis of their acid lability. Standard serotypes and clinical virus isolates were propagated in LLC (enteroviruses) and HeLa (rhinoviruses) cell cultures. When the c.p.e, was complete the cells were centrifuged and total nucleic acids were isolated by proteinase K-SDS treatment (at concentrations of 100 ktg/ml and 0.5 ~, respectively) for 30 min, at 37 °C, followed by phenol extraction and ethanol precipitation (Hyypi~i et al., 1984). cDNA synthesis. Prior to the PCR, cDNA synthesis was carried out using nucleic acid extracts from approximately 50000 cells. In addition to the sample the reaction mixture (40 ~1) contained 50 mM-Tris HCI pH 8-3, 75 mM-KC1, 10 mM-DTT, 3 mi-MgCl2, 0.5 mM-dNTPs (Pharmacia), 1 ~tl human placental ribonuclease inhibitor (Amersham), 0.1 nmol of the cDNA ( - ) primer (Table 1) and 1 ~tl of Moloney murine leukaemia virus reverse transcriptase (Bethesda Research Laboratories, BRL). The reaction was carried out at 37 °C for 1 h. Polymerase chain reaction. Five ~tl of the cDNA reaction mixture was further amplified in 40 cycles of the PCR using Taq DNA polymerase (New England Biolabs). Temperatures and times for denaturation, annealing and enzyme reaction were 95 °C (2 min), 40 °C (2 min) and 70 °C (4 min), respectively. The amplification was carried out in 16-6 mM-(NH~)2SO4,67 mM-Tris-HC1 pH 8.8, 6.7 mM-MgC12, 1 mM-2-mercaptoethanol, 170 ktg/ml bovine serum albumin and 0.1 nmol of the cDNA ( - ) and PCR (+) oligonucleotide primers. The reaction (total volume of 100 ~tl)was started by adding 2-5 units of Taq polymerase, another 2.5 units was added after 15 to 20 cycles and the procedure was then continued for an additional 25 to 20 cycles. A DNA thermal cycler (Perkin-Elmer Cetus) was used to perform the amplification. Ten ~tl of the final reaction product was analysed in a 1% agarose gel containing 1 ~tg/ml of ethidium bromide. Hybridization procedure. Ten ktl of the amplification reaction products was applied to GeneScreen Plus (New England Nuclear) after denaturation for 10 min at 95 °C. Oligonucleotide probes were 5'-end-labelled (BRL) using [~-32p]ATP (Amersham) as the precursor, to a specific activity of 2 x 10s to 10 x l0 s c.p.m./pmol. After labelling the probes were run through a Sephadex G-25 (Pharmacia) spin column. The filters containing the samples were prehybridized for 15 min in 6 x SSC, 5 × Denhardt's solution, 1~ SDS and 100 ktg/ml of single-stranded herring sperm DNA at a melting temperature of - 6 °C to - 1 0 °C. The labelled probe was added and after a 2 h hybridization the membranes were washed twice at the hybridization temperatme for 20 min each, with 2 x SSC containing 1~ SDS and then autoradiographed on an X-ray film (Trimax XD, 3M). Computer analysis. The selection of the primers and probes was based on alignments of poliovirus 1 and 2, coxsackievirus B4 and A21, and rhinovirus i B, 2, 14 and 89 sequences (Kitamura et at., 1981 ; Toyoda et al., 1984; Jenkins et at., 1987; Hughes et al., 1988, 1989; Skern et al., 1985; Stanway et al., 1984; Duechler et al., 1987) by a recently developed program (Vihinen, 1988) on a VAX 8800 computer. For use as primers, regions of approximately 20 nucleotides exhibiting the highest percentage of homology at the 5' end of the genome were selected (Table 1). Sequences between these primers which had the maximum difference between enteroviruses and rhinoviruses in addition to intra-genus conservation were used as probes. Echovirus 22 primers and probe were selected from a preliminary 3' end nucleotide sequence (our unpublished results).
RESULTS Detection o f standard virus strain T h e principle o f the c o m b i n e d a m p l i f i c a t i o n and oligonucleotide h y b r i d i z a t i o n assay is s h o w n in Fig. 1. C o x s a c k i e v i r u s A2, A16, A21, B3, B4 and B6, e c h o v i r u s 11, 18 and 22, poliovirus 1 and 3, and H R V 1B, 2, 9, 14, 22 and 89 reference strains were first tested by the c D N A ( - ) and P C R ( + ) p r i m e r s (Table 1) in o r d e r to e v a l u a t e the cross-reactivity o f the oligonucleotides a m o n g m e m b e r s o f h u m a n picornaviruses. Analysis by gel electrophoresis r e v e a l e d t h a t all the strains e x c e p t e c h o v i r u s 22 had r e a c t e d specifically; D N A f r a g m e n t s o f the e x p e c t e d size were seen in the samples and no reaction was o b s e r v e d w i t h the u n i n f e c t e d control cells (Fig. 2a). In the case o f c o x s a c k i e v i r u s B4 and e c h o v i r u s 18 the b a n d was rather w e a k but clearly visible in the original gels. W h e n the e c h o v i r u s 22 p r i m e r s w e r e used, faint bands o f various sizes were o b s e r v e d for all the e n t e r o v i r u s e s (data not shown). T h e amplification p r o d u c t s were tested further by using oligonucleotide probes, selected on the basis o f specificity for enteroviruses and rhinoviruses. All the e n t e r o v i r u s strains e x c e p t e c h o v i r u s 22 were d e t e c t e d by the e n t e r o v i r u s p r o b e w h e r e a s no reaction was seen w h e n the r h i n o v i r u s oligonucleotide was used in h y b r i d i z a t i o n (Fig. 2b). O n the o t h e r h a n d , the r h i n o v i r u s
P C R for human picornaviruses
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cDNA synthesis
5'.
M
1
Denaturation Binding of the primer
Repeat n times
°oo.
j
\
Poylmerae reaction
m m m
c
2
3
Fig. 1. The principle of the amplification reaction for picornavirus detection. cDNA is first generated in a reverse transcriptase-catalysed reaction using a primer complementary to the viral RNA genome. The product is then amplified in a Taq DNA polymerase-catalysed reaction with repeated cycles using an additional primer which represents the opposite polarity. Oligonucleotide probes are used 2° copies of target DNAdetected by a for specific identification of the amplified sequences. specific probe
Table 1. Enterovirus and rhinovirus primers and probes used in the study Similarity Reagent
Sequence
Map position*
Enteroviruses
cDNA ( - ) primer PCR (+) primer t Enterovirus ( - ) probe Rhinovirus ( - ) probe
5' CATTCAGGGGCCGGAGGA 5' AAGCACTTCTGTTTCC 5' GGCCGCCAACGCAGCC 5' GGCAGCCACGCAGGCT
443-468 163-178 357-372 349-364
18/18 14-16/16 16/16 8-12/16
Rhinoviruses 18/18 16/16 8-11/16 15-16/16
* The map positions refer to poliovirus 1 (Kitamura et al., 1981; cDNA and enterovirus) and HRV-14 (Stanway et al., 1984; PCR and rhinovirus) sequences. ~"Gama et al. (1988).
probe recognized all the H R V strains and HRV-14 gave a signal with the enterovirus probe (Fig. 2c). The oligonucleotide derived from the echovirus 22 sequence did not react with the amplification products of other strains (Fig. 2b and c). Detection o f clinical isolates To determine whether the combined amplification and hybridization system would be applicable for routine detection of human picornaviruses after cell culture isolation, 29 coded samples representing randomly selected, previously tested rhinovirus and enterovirus specimens were analysed. The original clinical specimens were inoculated into cultured cells and at advanced c.p.e, nucleic acid extracts were prepared for the amplification test. Twenty-eight (97 ~ ) of the samples were reactive as determined by gel electrophoresis (Fig. 3 a and b). The only exception was echovirus 27 (sample no. 31). In all cases the D N A fragment of specific size could be differentiated with a high probability, on the basis of its size being distinct from the other bands observed in a few samples. However, some of the non-related viruses tested in parallel, especially adenovirus and cytomegalovirus (CMV), showed reaction products which were close in size to the appropriate band.
T. HYYPI.~., P. AUVINEN AND M. MAARONEN
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(a)
I m IA21A~61A~IB3IB~IB6IE~IEmlE~P~IP3I~B[2 ]9 I ~
[28[891
_m ~ I W ~
(b)
m
D
....
ILtCIA21A'OIA2 I831B IBoIE"IE'81E221P'IP31
H toI Blz i9
I zzleg]
Fig. 2. Detection of standard picornavirus strains by in vitro amplification and oligonucleotide hybridization after a passage in cell culture. (a) Agarose gel electrophoresis of the amplification products. (b) Identification of amplified enteroviruses sequences by enterovirus (1) rhinovirus (2) and echovirus 22 (3) oligonucleotide probes. (c) Identification of rhinoviruses by the probes. Lane m, Mr markers; A, coxsackievirus A; B, coxsackievirus B; E, echovirus; P, poliovirus; 1B, 2, 9, 14, 22 and 89 are rhinovirus strains; LLC and HeLa are cell lines which were used to propagate the viruses.
The products of the amplification reaction were then tested by using both enterovirus and rhinovirus hybridization probes (Fig. 3 c, d and e). The results of these experiments are summarized in Table 2. All except two of the 14 HRV strains were detected by the rhinovirus probe. In addition five specimens reacted with the enterovirus oligonucleotide which detected all but one of the different members of coxsackie-, echo- and polioviruses. None of these strains were reactive with the rhinovirus probe. The hybridization reaction was also highly specific for human picornaviruses; no signal was observed with any of the control viruses (Fig. 3).
PCR for human picorna~iruses
~°)
3265
L.' I E I R 1~21221 ~ 18 12812ol ~ 1~ol3ol ~ I g 12gl3 L~3L~I2sl
~b) 1713312~ 1,713, I~s118 I,, 1261~ 121,91, 1,21271. I A I, I c I H I
(c) E
R
32
22
5
8
28
20
6
10
30
1
9
29
3
13
1/,
25
7
33
21
17
31
15
18
11
26
16
2
19
&
12
27
M
A
I
C
H
(a)
(e)
Fig. 3. Detection of clinical picornavirus isolates by gel electrophoresis and oligonucleotide hybridization after amplification. Coded enterovirus and rhinovirus specimens were inoculated into cell cultures and at complete c.p.e., nucleic acids were extracted. The extract was amplified using common primers and the product was analysed by agarose gel electrophoresis (a and b) and by hybridization using enterovirus (d) and rhinovirus (e) probes. The codes of the virus isolates (a, b and c) refer to Table 2 where the results are summarized. Lane m, Mr markers; E, enterovirus standard; R, rhinovirus standard; M, mumps virus; A, adenovirus; I, influenza A virus; C, cytomegalovirus; H, herpes simplex virus type 1.
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T.
HYYPI.A.,
P.
AUVINEN
AND
M.
MAARONEN
Table 2. Summaryof the results of coded clinical isolates and virus strains presented in Fig. 3 Probe Specimen no.
•
Virus
~
-~
GE*
Rhinovirus
Enterovirus + + + + +
1 2 3 4 5 6 7 8 9 10 11 12
HRV HRV HRV HRV HRV HRV HRV HRV HRV HRV HRV HRV
+ + + + + + + + + + + +
+ + + + + + + + + +
13 14
HRV
+
+
-
HRV H e L a cells Echo 3 CA9 Echo 9 E c h o 17 E c h o 23 CA9 CB4 CB2 Echo 6 CB5 Polio 3 E c h o 11 CA16 E c h o 27 E c h o 21 LLC cells Mumps Adeno Influenza A CMV HSV-1;~
+ rt + + + + + + + + + + + + + r + r r r r r r
+ -
+ + + + + + + + + + + + + + -
15 16 17 18 19 20 21 22 25 26 27 28 29 30 31 32 33
* G E , Gel electrophoresis. t r, Reactive. Indicates that the :~ Herpes simplex virus type 1.
fragment length differs from the controls.
DISCUSSION
The current methods used for human picornavirus identification are cumbersome and do not provide a specific diagnosis at the acute phase of illness. Also, owing to the large number of serologically distinct viruses, the use of immunological methods for direct detection of enteroviruses in clinical samples is difficult. Nucleic acid probes offer a possibility of taking advantage of the highly conserved regions at the 5' end of the genome which is not known to code for proteins. As the sensitivity of the hybridization assays is not sufficient for direct detection of the virus in clinical material a passage in cell culture is needed. Therefore these tests have only limited use in diagnostic laboratories. The use of the PCR could replace cell culture amplification by a biochemical method provided that the problems concerning the test system and treatment of the specimens can be solved. Our aim was to study the reagents needed for such an assay system and to develop a test which could detect human picornaviruses rapidly and also differentiate between enteroviruses and rhinoviruses. The selection of reagents was carried out using a computer program which can align several nucleotide sequences for analysis of common or specific regions in the genome (Vihinen, 1988).
P C R for human picornaviruses
3267
By using this system an oligonucleotide representing a highly conserved region was selected for cDNA ( - ) primer, and a primer described earlier (Gama et al., 1988) was used for the initiation of the synthesis of the complementary ( + ) strand. Probes which had maximum specificity for enteroviruses and rhinoviruses between the primer sequence regions were chosen. Broad reactivity of the primers was observed in the gel electrophoresis and the specificity of the probes was evident in hybridization with the exception that HRV-14 reacted also with the enterovirus probe although the computer analysis of the probe region in HRV-14 did not show any exceptional homology with this sequence when compared to other HRVs. Therefore the most obvious reason for this result is a genetic difference between the sequenced virus and the strain used here. Echovirus 22 failed to react in this assay system but was detected by primers and probe derived from a preliminary 3' end sequence. The difference can be understood easily: this virus type does not react with probes from any major enterovirus subgroup (Auvinen et al., 1989). When coded clinical isolates were tested in this combined assay, all except one of the specimens reacted when analysed by gel electrophoresis. This was an echovirus 27 strain which did not show progressive c.p.e, in cell cultures after re-inoculation. This serotype is known to exhibit a hybridization pattern characteristic for echoviruses (Auvinen et al., 1989). Hybridization with the rhinovirus probe failed to detect two of the HRV specimens whereas all the enteroviruses were detected by the specific probe with the exception of the sample (no. 31, echovirus 27) which also gave a negative result by gel electrophoresis. In addition to enterovirus samples, this probe gave a signal with five HRV samples. This is an interesting finding as some of the rhinoviruses are known to resemble polioviruses closely. Although these viruses were not typed they are known to fall into a 'HRV-14-1ike' category in hybridization analysis by cDNA probes (P. Auvinen et al., unpublished results). Combination of the two probes makes it possible to discriminate between enteroviruses and rhinoviruses because the HRV probe had no reactivity with samples representing the enterovirus group. This assay system can be applied directly for routine laboratory diagnosis; rapid and non-radioactive confirmation of picornavirus growth in cell culture isolation is possible by gel electrophoresis and the same material can be used in typing by oligonucleotide probes. The approach is of practical advantage as chemically synthesized reagents and commercial kits can be used instead of serum pools of limited availability. The greatest improvement in the diagnosis of picornavirus infections would be the replacement of cell culture isolation by the in vitro amplification method. According to our experience some nasopharyngeal samples can be directly detected as positive by this assay system, as already reported by Gama et al. (1988). The optimization of the procedure for clinical material, including stool specimens, cerebrospinal fluid, muscle biopsies etc., needs further development of the technique to obtain maximum sensitivity, to remove inhibitors of the reaction in samples and to minimize contamination problems during the procedure. We thank Dr Glyn Stanway for providing us with the unpublished coxsackievirus A21 nucleotide sequence and Dr M a u n o Vihinen for advice in the computer analysis.
REFERENCES AL-NAK1B,W., STANWAY,G., FORSYTH, M., HUGHES, P. J., ALMOND,J. W. & TYRRELL, D. A. J. (1986). Detection of h u m a n rhinoviruses and their molecular relationship using c D N A probes. Journal of Medical Virology 20, 289-296. AOVINEN, P., STANWAY,G. & HYYPI.~.,T. (1989). Genetic diversity of enterovirus subgroups. Archives of Virology 104, 175-186. DUECHLER, M., SKERN, T., SOMMERGRUBER,W., NEUBAUER,C., GRUENDLER, P., FOGY, I., BLAAS,D. & KUECI-ILER,E. (1987). Evolutionary relationships within the h u m a n rhinovirus genus: comparison of serotypes 89, 2, and 14. Proceedings of the National Academy of Sciences, U.S.A. 84, 2605-2609. GAMA, R. E., HUGHES, P. J., BRUCE, C. B. & STANWAY, G. (1988). Polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material. Nucleic Acids Research 16, 9346. HUGHES, P. J., NORTH, C., JELLIS, C. H., MINOR, P. D. & STANWAY,G. (1988). The nucleotide sequence of h u m a n rhinovirus 1B: molecular relationships within the rhinovirus genus. Journal of General Virology 69, 49-58.
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HUGHES, P. J., NORTH, C., MINOR, P. D. & STANWAY,G. (1989). The complete nucleotide sequence of coxsackievirus A21. Journal of General Virology 70, 2943-2952. HYYPIA., T., ST.~LLHANDSKE,P., VAINIONP.~, R. & PETTERSSON, U. (1984). Detection of cnteroviruses by spot hybridization. Journal of Clinical Microbiology 19, 436-438. JENKINS, O., BOOTH,J. D., MINOR, P. D. & ALMOND,J. W. (1987). The complete nucleotide sequence of coxsackievirus B4 and its comparison to other m e m b e r s of the Picornaviridae. Journal of General Virology 68, 1835-1848. KITAMURA,N., SEMLER, B. L., ROTHBERG, P. G., LARSEN, G. R., ADLER, C. J., DORNER, A. J., EMINI, E. A., HANECAK,R., LEE, J. J., VAN DER WERF, S., ANDERSON,C. W. & WIMMER, E. (1981). Primary structure, gene organization and potypeptide expression of poliovirus R N A . Nature, London 291, 547-553. ROTBART,H. A., LEVIN,M. J. a VlLLARREAL,L. P. (1984). Usc of subgenomic poliovirus D N A hybridization probes to detect the major subgroups of enteroviruses. Journal of Clinical Microbiology 20, 1105-1108. ROTBARr, n. A., EASrrC~N, P. S., RUTH, J. I., hqRATA, K. K. & LEVIN, M. J. (1988). Non-isotopic oligomeric probes for the h u m a n enteroviruses. Journal of Clinical Microbiology 26, 2669-2671. SA1KI,R. K., GELFAND, D. H., STOFFEL,S., SCHARF,S. J., HIGUCHI, R., HORN, G. T., MULLIS,K. B. & ERLICH, H. A. (1988). Primer-directed enzymatic amplification of D N A with thermostable D N A polymerase. Science 239, 487491. SKERN, T., SOMMERGRUBER,W., BLAAS,D., GRUENDLER, P., FRAUNDORFER,F., PIELER, C., FOGY, I. & KUECHLER, E. (1985). H u m a n rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region. Nucleic Acids Research 13, 2111-2126. STANWAY, G., HUGHES, P. J., MOUNTFORD, R. C., MINOR, P. D. & ALMOND, J. W. (1984). The complete nucleotide sequence of a c o m m o n cold virus: h u m a n rhinovirus 14. Nucleic Acids Research 12, 7859-7875. TOYODA, H., KOH~A, M., KATAOKA,Y., SUGANUMA,T., OMATA, T., IMU~,A, N. & NOMOTO, A. (1984). Complete nucleotide sequences of all three poliovirus serotype genomes. Journal of Molecular Biology 174, 561-585. vn-n/~N, M. (1988). A n algorithm for simultaneous comparison of several sequences. Cabios 4, 89-92.
(Received 11 July 1989)
