Laboratoire dEpidémiologie Moléculaire des Entérovirus, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Valérie Caro. Fax +33 1 45 68 87 80. e-mail vcaro{at}pasteur.fr
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Abstract |
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Introduction |
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Based on their antigenic properties, the original 64 HEV serotypes were initially grouped into polioviruses (PV), coxsackieviruses A (CA), coxsackieviruses B (CB), echoviruses (E), and the more recently identified enteroviruses (EV) 68 to 71. A new HEV classification based on molecular and biological data has recently been proposed as an alternative to the antigenic classification (Hyypiä et al., 1997 ; Pöyry et al., 1996
). This classification groups enteroviruses into five species: (1) PV, including poliovirus types 1, 2 and 3; (2) HEV-A, including 11 coxsackieviruses A and EV71; (3) HEV-B, including all coxsackieviruses B, all echoviruses, EV69 and CA9; (4) HEV-C, including 11 other coxsackieviruses A; and (5) HEV-D, including EV68 and EV70 (Pringle, 1999
). The enteroviruses previously classified as E22 and E23, which were shown to group independently (Hyypiä et al., 1992
), now constitute a new genus, Parechovirus (with two serotypes), in the family Picornaviridae (Mayo & Pringle, 1998
).
Enterovirus typing is required for several main reasons: (i) to distinguish polio- from non-polio-enteroviruses in the context of poliomyelitis eradication; (ii) to determine the relationship between enterovirus type and clinical syndrome; (iii) to identify new enterovirus types or variants; (iv) to analyse enteroviruses in neonates and immunodeficient patients; and (v) to investigate enterovirus molecular epidemiology and phylogeny (reviewed in Muir et al., 1998 ). The agent responsible for HEV-induced disease is currently identified by conventional virus isolation followed by neutralization with intersecting pools of type-specific antisera. Due to the large number of antigenically distinct serotypes, serotyping procedures are time-consuming, labour-intensive and costly. Moreover, the limited supply of reference type-specific sera, the limited number of serotypes covered by the intersecting pools of sera currently available (LBM or RIVM), their static character (the inability to detect new antigenic variants or emerging serotypes) are major drawbacks of neutralization typing (el-Sageyer et al., 1998
). In addition, enteroviruses are frequently found to be untypeable.
In the light of recent developments in molecular biology, several assays based on RTPCR followed by nucleic acid hybridization or sequencing have been assessed as possible approaches for the identification of enteroviruses. The enterovirus genome is a single-stranded, positive RNA molecule, approximately 7500 nucleotides long, including a 5' and a 3' non-coding region (NCR), and encompassing a single, long open reading frame. Sets of primers specific for highly conserved sequences in the 5'NCR or VP2 capsid protein-coding regions have been used to develop efficient methods for the rapid and sensitive detection of enteroviruses (reviewed by Romero, 1999 ). However, neither the 5'NCR nor the VP2-coding region (Oberste et al., 1998
) can be used for enterovirus typing, due to a lack of correlation between the nucleotide sequence of these genomic regions and serotype. VP1 is a capsid protein located mainly at the virion surface. It makes a large contribution to the constitution of neutralization antigenic sites. For this reason, the region of the genome encoding VP1 has been used to investigate the molecular evolution of poliovirus (Kew et al., 1995
), to determine poliovirus genotypes (Balanant et al., 1991
) and to develop poliovirus serotype-specific PCR primers (Kilpatrick et al., 1998
). The nucleotide sequence of the entire VP1 coding region has recently been shown to correlate with serotype in enteroviruses (Oberste et al., 1999a
), opening up possibilities for the development of molecular diagnostic reagents for serotype-specific enterovirus identification.
We present here a new approach for the molecular serotyping of HEV, involving the amplification of a genomic fragment encompassing the VP12C coding region with a single pair of enterovirus-specific primers. Restricted analysis of the 3' third of the VP1-coding region showed a good correlation between nucleotide sequence and enterovirus serotype, for both classical reference strains and field isolates, over a 30 year period, and covering widely dispersed geographical regions. This method may improve diagnosis of the diseases caused by enterovirus infection. The amplification of this long genomic fragment may also be used as a rapid and efficient tool for studies of HEV molecular epidemiology and evolution.
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Methods |
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Field isolates.
Forty-five enterovirus isolates were selected, representing 21 serotypes throughout the genus Enterovirus. The panel included 35 virus isolates from Europe (France, Greece, The Netherlands and Romania) and 10 isolates from Africa (Burkina Faso and Madagascar) (see Table 2). They were isolated from original clinical specimens: stool (n=31), cerebrospinal fluid (n=4), blood (n=1), cortex (n=1), spinal cord (n=2), nasopharyngeal secretion (n=2), vesicle (n=2) and throat (n=2). Clinical symptoms and the specimens studied in this work are described in Table 2
. The serotypes of Romanian isolates were determined by neutralization with the LBM and home-made antisera pools and those of the Dutch, French, Greek and Madagascan isolates were determined by neutralization with the RIVM pools (Kapsenberg, 1988
). The six Madagascan enterovirus isolates which were not neutralized by any of the RIVM pools were classified as untypeable.
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Pairwise nucleotide and amino acid sequence identities were calculated by aligning each field isolate sequence with each available prototype enterovirus sequence using the multiple alignment program CLUSTAL W (Thompson et al., 1994 ). To analyse phylogenetic relationships, the partial VP1 sequences of isolates were compared with those from other human enteroviruses using CLUSTAL W. Some reference enterovirus nucleotide sequences from the GenBank database were used (Oberste et al., 1999b
). Alignments were corrected manually to maximize sequence identity, to account for codon boundaries and to ensure the alignment of conserved amino acid motifs. Phylogenetic trees were generated by inputting the aligned sequences into PHYLIP (Phylogeny Inference Package) version 3.5 (Felsenstein, 1993
) and PUZZLE version 4.0 (Strimmer & von Haeseler, 1996
). Phylogenetic trees were constructed using the neighbour-joining algorithm of Saitou & Nei (1987)
, as implemented in the program NEIGHBOR, and using the maximum parsimony method, as implemented in DNAPARS. For neighbour-joining analysis, a distance matrix was calculated using the Kishino and Hasegawa method (Kishino & Hasegawa, 1989
) with a transition/transversion ratio (k) of 8·0, using DNADIST (PHYLIP). The k parameter is an empirical ratio calculated by PUZZLE from the data set. To investigate the robustness of the phylogenies constructed with NEIGHBOR and DNAPARS, bootstrap analysis was carried out on 100 pseudo-replicate data sets with SEQBOOT. Phylogenetic trees were reconstructed by the maximum likelihood method with PUZZLE, which uses QUARTET PUZZLING as the tree search algorithm. Distances were calculated with the model of nucleotide substitutions of Kishino and Hasegawa and the transition/transversion parameter was estimated directly from the data set. The reliability of tree topology was estimated by use of 1000 puzzling steps. The trees were drawn using the program TREEVIEW (Page, 1996
).
Nucleotide sequence accession numbers.
The nucleotide sequence data reported in this paper have been deposited in the EMBL sequence database under the accession numbers AJ279151 to AJ279195 (see Table 2).
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Results |
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To determine specificity, an equimolar mixture of primers was first tested in an RTPCR assay with the RNA extracted from each of the prototype human enteroviruses. Amplicons were obtained from 59 of the 64 prototype human enteroviruses (92·2%). The viruses for which the amplification reaction was unsuccessful were CA5, CA19 and CA22, which thrive only in suckling mice, EV68 and EV70, members of the distant HEV-D species, and E22 and E23, which are now classified as a new genus (Parechovirus) of the family Picornaviridae (Hyypiä et al., 1992 ). The failure of amplification was not due to a low concentration of viral RNA in the reaction, as shown by the successful amplification of an HEV-specific genomic fragment from the 5'NCR, as for all other HEV strains tested (not shown).
Amplicons were also successfully obtained from all of the 45 clinical enterovirus isolates tested (Table 3), irrespective of their date of isolation (1970 to 1998), serotype (21 different serotypes) or the geographical region in which they were collected.
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For each field isolate tested, identity was highest with the homotypic prototype strain, for both nucleotide and amino acid sequences, with identities of 74·8 to 89·4% for nucleotide sequences and from 89·8 to 98·2% for amino acid sequences (Table 3). In all cases in which a sequence from a more recent homotypic isolate was present in the database, a better score was obtained with this strain than with the homotypic reference strain (not shown). However, in one case (CB5-RO-14/5/70), the VP1 sequence of the isolate was less similar to the homotypic CB5 prototype (78·5%) than to the prototype of swine vesicular disease virus (SVDV), a pig enterovirus (89·2%), with which the highest identity score was obtained. The deduced amino acid sequence of CB5-RO-14/5/70 was 95·5% similar to those of both SVDV and CB5. This is not surprising as it has been shown that SVDV probably arose in pigs from a single transfer of a human CB5, and may therefore be considered to be a subspecies of CB5 (Zhang et al., 1999
).
In all cases, the second-highest identity scores with respect to another serotype were 67·9 to 78·5% for nucleotide sequences and 70·4 to 95·5% for amino acid sequences. In each case the prototype strain giving the second-highest score belonged to the same species as the strain giving the highest score. The range of delta scores, representing the difference in percentage nucleotide sequence identity between the highest and second-highest scores, was from 1·4 to 19·4% (Table 3). This difference, demarcating the boundary between serologically homotypic and the closest heterotypic strains, was on average 10·0% but was very low for seven clinical isolates. Isolate E1-RO-122/1/74 had a nucleotide sequence 80·5% similar to that of E1 prototype strain and 77% similar to that of E8 prototype strain. This difference of only 3·5% is consistent with the previously demonstrated antigenic relationship between E1 and E8 (Harris et al., 1973
) and the reclassification of E8 as a variant of E1. The other six isolates with a small difference in percentage nucleotide identity between the highest and second-highest scores (1·4 to 2·9%) are discussed below.
Analysis of the untypeable field enteroviruses
Strains MG-354/94, MG-356/94, MG-404/94, MG-448/94, MG-423/94 and MG-498/94 were all isolated from the stools of healthy Malagasy children (Table 2). They were identified as enteroviruses by their cytopathic effect on an enterovirus-susceptible cell line and by the enterovirus-specific amplification (RTPCR) of the 5'NCR. They were not neutralized by intersecting RIVM pools. Their genome was successfully amplified by our pair of enterovirus-specific VP12C primers and sequenced. The highest and second-highest identity scores obtained by comparing the partial VP1 sequences of these strains with those of HEV strains in the GenBank are reported in Table 3
. Isolates MG-354/94, MG-356/94, MG-404/94 and MG-498/94 were 77·4 to 80·2% identical to CA13 (Flores) and 75·4 to 78·0% identical to CA18 (G-13) prototype strains. The high level of sequence identity between these two reference strains (76·0%), consistent with their antigenic relatedness (Committee on Enteroviruses, 1962
), may account for the small difference between the two scores (1·4 to 2·2%). By neutralization with several monospecific CA13 antisera (kindly supplied by RIVM), all the above isolates were identified as serotype CA13 (not shown). A similar problem of a small difference between the highest and second-highest scores was encountered for strains MG-448/94 and MG-423/94. Indeed, MG-448/94 was 74·8% identical to CA20 (IH-35) and 71·9% identical to CA17 (G-12) reference strains, with the CA20 and CA17 prototypes displaying only 70·0% sequence identity. Similarly, strain MG-423/94 was 75·0% identical to CA20 and 72·3% identical to CA13, with the CA20 and CA13 prototypes only 71·0% identical to one another. No antigenic cross-reactivity of CA20 with CA17 or CA13 has been reported. Both isolates were identified as CA20 isolates by neutralization with monospecific sera. However, they were strongly neutralized by both anti-CA20 (IH-35) and anti-CA20a (Tulano) antibodies, and less strongly by anti-CA20b (Cecil) antibodies (not shown), reflecting the antigenic relationship of these three variants of CA20 (personal communication from Albert Ras). Thus, in this study, isolates MG-448/94 and MG-423/94 had the lowest percentage nucleotide sequence identity with the closest reference prototype strain (74·8 and 75% respectively). These field strains have probably accumulated several mutations, causing them to drift away from their homologous prototype, which was isolated in 1955.
Phylogenetic relationships of field isolates
To determine the relationships between field and prototype HEV, the sequence of the 3' third of the VP1-coding region of each field isolate was compared with those of all prototype strains by pairwise alignments, with the program CLUSTAL W. In each case, the homologous serotype pairwise comparison scores were higher than 75% for nucleotide identity and higher than 85% for amino acid identity. Furthermore, there is no overlap between the homologous serotype pairwise comparison scores of both nucleotide and amino acid sequences and the heterologous serotype pairwise comparison scores (data not shown). This confirmed the accuracy of the molecular serotyping: the serotype of every isolate could be determined if the 3' third of the VP1 sequence displayed a minimum of 75% nucleotide identity (85% amino acid identity) with a prototype strain in the database.
To explore further the evolutionary relationships between field and prototype viruses, a general tree was generated with the 45 isolates and representatives of each of the five identified HEV species (PV: PV1; HEV-A: CA2, CA12 and CA16; HEV-B: E27, CB3, CA9 and EV69; HEV-C: CA13, CA19 and CA24; HEV-D: EV68 and EV70). The same tree topology was produced, regardless of the algorithm used. The isolates clearly segregated into five distinct major groups (Fig. 1), consistent with previously published human enterovirus phylogenies (Huttunen et al., 1996
; Oberste et al., 1998
; Pöyry et al., 1996
; Pulli et al., 1995
) and the new classification. The various groups were strongly supported by bootstrap values of 100% for HEV-B, C and D and 92% for HEV-A species, regardless of the algorithm used.
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Discussion |
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The HEV capsid has an icosahedral structure with 60 repeating protomer units, each containing one set of the structural proteins, VP1 to VP4 (Rueckert, 1996 ). Variations within capsid proteins VP1 to VP3 are responsible for the antigenic diversity of enteroviruses. Three independent neutralizing antigenic sites have been described for polioviruses (Mateu, 1995
) and the major neutralization epitope has been identified within the dominant capsid protein, VP1. A complete VP1 sequence database including sequences from all known HEV serotypes has recently been established (Oberste et al., 1999b
). The phylogenetic tree constructed here shows the same clustering into four major groups as previously reported and, in each case, the field strains of a given serotype were monophyletic.
We chose the VP12C region for amplification for several reasons: (i) the VP1 protein is the most exposed structural protein, contributing to the constitution of antigenic neutralization sites, and has been shown to be the most suitable genomic region for use in a molecular typing method; (ii) the VP12A junction has proved to be useful for poliovirus genotyping (Kew et al., 1995 ); (iii) generic antisense primers could be selected in the 2C region due to the presence of conserved regions; and (iv) a significantly longer fragment (approximately 1450 bp in size) including the 2A to 2C non-structural protein-coding region would make possible the analysis of regions of the genome other than that corresponding to VP1, thus providing information about possible viral determinants of pathogenesis and virulence, or the possible correlation between enterovirus serotype and disease syndrome.
An RTPCR amplicon including the VP1 coding region was obtained for 59 of the 64 reference and all of the 45 field isolates, using an original single pair of universal HEV-specific primers, flanking the 1452 nucleotide VP12C coding region (relative to PV1-Mahoney strain). To our knowledge, this is the first time that it has been possible to detect and to identify 92·2% of all known prototype HEV with a single pair of primers. In another study (Oberste et al., 1999a ), a set of degenerate deoxyinosine-containing PCR primers designed to amplify the region encoding VP1 was shown to be effective for only 65% of enterovirus prototype strains. The same authors (Oberste et al., 2000
) had to design five different pairs of primers to amplify 54 different field isolates. In our study, the five prototype strains not recognized by our pair of primers have characteristics unusual among HEV. Indeed, only 80% and 70% identity with the sense primers was observed for the CA5, CA19 and CA22 group and for the EV68 and EV70 groups, respectively. These strains are also known to have characteristics that differ from those of other HEV: the three CA viruses thrive only in suckling mice and the two EV constitute a separate species (HEV-D). To avoid failing to detect an enterovirus with our RTPCR assay, an alternative means of detection should be used. One possibility is to include in the diagnosis procedure the classical enterovirus-specific PCR in the 5'NCR. If detected in this way, an enterovirus which was not amplified with our pair of primers could be further genotyped with an HEV-D- or CA5-, 19- and 22-specific primers. Other specific primers should be designed in the future if new HEV genotypes arise which not detected by the presently described molecular methods. Current work in our laboratory addresses these questions.
The neutralization test, the traditional standard procedure for enterovirus identification, is generally reliable but may fail to identify isolates due to mixtures of enteroviruses, aggregation of virus particles, antigenic drift, or simply because it is impossible to identify all circulating HEV serotypes with the intersecting pools of antisera in current use. With our system, it was possible to obtain amplicons from all of the 45 field strains, randomly chosen from HEV isolates representing 21 different serotypes, from various geographical regions, spanning a 30 year period. By comparing the 3' end of the VP1 sequence of each isolate with those of all prototype enteroviruses, we were able to confirm or to identify unambiguously the serotype of all these isolates. Six of the 45 isolates were untypeable with RIVM intersecting sera, but were correctly serotyped with our molecular method, the results being confirmed by neutralization with monospecific antisera. We were also able to determine the serotypes of over 100 other strains, from 26 different serotypes (not shown).
The results reported here are consistent with previous findings (Oberste et al., 1999a ), showing a good correlation between molecular and antigenic serotyping for HEV. All available data suggest that sequencing of the VP1-coding region is likely to be a useful tool for rapid identification of enteroviruses, for the diagnosis of enterovirus infections, for determining the extent of genotype divergence among isolates of a given serotype and for phylogenetic studies of enteroviruses. Thus, our molecular strategy should improve the identification and characterization of enterovirus isolates and constitute a rational basis for replacing serotyping by easy rapid genotyping.
Our method has already proved to be useful and accurate in a molecular epidemiological study of a severe epidemic associated with E11 strains in Hungary (el-Sageyer et al., 1998 ; A. Szendroi and others, unpublished results).
As the amplicon derived from our pair of primers includes the 2A-coding region, it would be very interesting to study in detail the 2A nucleotide and amino acid sequences of the prototype and field enterovirus strains. The 2A protein is a trypsin-like protease involved in polyprotein processing and in the shut-off of host-cell macromolecule synthesis by cleavage of the eIF-4G subunit of the eIF-4F complex. A recent study demonstrated that enteroviral infection of cardiac myocytes leads to disruption of the cytoskeleton through enteroviral protease 2A-mediated cleavage of dystrophin (Badorff et al., 1999 ). Another group has shown that this picornaviral non-structural protein has motifs characteristic of the H-rev107 cellular protein family, involved in the control of cell proliferation (Hughes & Stanway, 2000
). All these results demonstrate that accurate and exhaustive 2A analysis will be useful in elucidating the molecular mechanisms underlying enteroviral pathogenesis and picornavirus genome structure and evolution.
The key advantage of our molecular strategy for the identification of HEV, based on the use of VP12C primers, is that it requires only a single pair of optimized oligonucleotides for serotyping and genotyping of almost all HEV strains, opening up new possibilities for diagnosis purposes, for studying epidemiological or pathological features and for searching for new serotypes of HEV.
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Acknowledgments |
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This study was partly financed by grants from Argene-Biosoft, Varilhes, France and by grants to R.C. from the European Commission (Copernicus CIPA CT94-0123 and Inco-Copernicus ERBIC 15 CT96-0912).
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Footnotes |
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Received 13 July 2000;
accepted 26 September 2000.