Molecular strategy for ‘serotyping’ of human enteroviruses

Valérie Caro1, Sophie Guillot1, Francis Delpeyroux1 and Radu Crainic1

Laboratoire d’Epidémiologie 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


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
To explore further the phylogenetic relationships between human enteroviruses and to develop new diagnostic approaches, we designed a pair of generic primers in order to study a 1452 bp genomic fragment (relative to the poliovirus Mahoney genome), including the 3' end of the VP1-coding region, the 2A- and 2B-coding regions, and the 5' moiety of the 2C-coding region. Fifty-nine of the 64 prototype strains and 45 field isolates of various origins, involving 21 serotypes and 6 strains untypeable by standard immunological techniques, were successfully amplified with these primers. By determining the nucleotide sequence of the genomic fragment encoding the C-terminal third of the VP1 capsid protein we developed a molecular typing method based on RT–PCR and sequencing. If field isolate sequences were compared to human enterovirus VP1 sequences available in databases, nucleotide identity score was, in each case, highest with the homotypic prototype (74.8 to 89.4%). Phylogenetic trees were generated from alignments of partial VP1 sequences with several phylogeny algorithms. In all cases, the new classification of enteroviruses into five identified species was confirmed and strains of the same serotype were always monophyletic. Analysis of the results confirmed that the 3' third of the VP1-coding sequence contains serotype-specific information and can be used as the basis of an effective and rapid molecular typing method. Furthermore, the amplification of such a long genomic fragment, including non-structural regions, is straightforward and could be used to investigate genome variability and to identify recombination breakpoints or specific attributes of pathogenicity.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Human enteroviruses (HEV) belong to the family Picornaviridae. They are major human pathogens and are associated with a broad spectrum of clinical features including acute respiratory illness, aseptic meningitis, meningoencephalitis, myocarditis, hand, foot and mouth disease, neonatal multi-organ failure and acute flaccid paralysis (Melnick, 1996 ). Outbreaks of disease associated with a single serotype of enterovirus are often reported (Gjoen et al., 1996 ; Kopecka et al., 1995 ) and represent a major public health problem. In 1998, an outbreak of enterovirus 71 infection caused hand, foot and mouth disease and herpangina in more than 100000 individuals in Taiwan, with hundreds of deaths due to complications including encephalitis, aseptic meningitis, pulmonary oedema or haemorrhagic, acute flaccid paralysis and myocarditis (Ho et al., 1999 ). HEV are currently responsible for 80 to 92% of aseptic meningitis cases with an identified aetiologic agent (Rotbart, 1995 ).

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 RT–PCR 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 VP1–2C 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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Prototype viruses.
The echoviruses, coxsackieviruses B, coxsackieviruses A and enterovirus types 68 to 71 studied in this work were the ‘prototype’ strains of each serotype (Melnick, 1996 ) and were kindly supplied by the National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands. The serotype of the reference strains was checked by seroneutralization using the Lim Benyesh-Melnick (LBM) panel of intersecting antisera pools (Melnick et al., 1973 ).

{blacksquare} 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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Table 2. Details of enteroviruses isolates used in this study

 
{blacksquare} RT–PCR.
RNA was extracted from 100 µl of infected cell culture supernatant using the Total Quick RNA Talent kit (Euromedex, France) and eluted in 70 µl of DEPC-treated water. RNA (2 µl) was used for cDNA synthesis with 10 pmol each of the antisense primers, EUC2a and EUC2b (see Table 1 for the primers). The reaction mixture (20 µl) contained 50 mM Tris–HCl pH 8·3, 75 mM KCl, 3 mM MgCl2, 10 mM dNTPs, 2 mM DTT, 20 U ribonuclease inhibitor (RNasin, Promega) and 200 U Superscript RNase H- reverse transcriptase (Life Technologies). The reaction mixture was incubated for 30 min at 42 °C and then for 5 min at 95 °C to inactivate the enzyme. The cDNA product (2 µl) was added to a PCR mixture containing 67 mM Tris–HCl pH 8·8, 16 mM (NH4)2SO4, 0·01% Tween 20, 2 mM MgCl2, 10 mM dNTPs, 1·25 U of Taq DNA polymerase (EurobioTaq, Eurobio) and 10 pmol of primers (EUC2, EUG3a, EUG3b and EUG3c). Amplification involved 29 cycles of denaturation at 95 °C for 20 s, annealing at 45 °C for 1 min and elongation at 72 °C for 1 min, followed by a final cycle of denaturation at 95 °C for 20 s, annealing at 45 °C for 1 min and elongation at 72 °C for 10 min. Amplification products (5 µl) were run on 1·5% agarose gels. The gels were stained with ethidium bromide and the DNA was viewed under UV light. RT–PCR was used to amplify a 435 bp fragment in the 5'NCR from 2 µl of purified RNA, as previously described (Balanant et al., 1991 ), using primers UC52–UG53 (Guillot et al., 1994 ).


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Table 1. Primers used for RT–PCR and sequencing of human enteroviruses

 
{blacksquare} Nucleotide sequence determination and sequence analysis.
PCR products were purified by the low-melting-point agarose gel method (Sambrook et al., 1989 ) and sequenced on an automated DNA sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer Biosystems), with the EUG3a, EUG3b and EUG3c primers. Sequences were compared with the GenBank database sequences using the program FASTA version 3.3 (Pearson & Lipman, 1988 ).

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 ).

{blacksquare} 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).


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Selection of generic primers and evaluation for amplification
Our first goal was to develop an RT–PCR assay capable of detecting all known serotypes of human enteroviruses, facilitating the identification of enteroviruses and their phylogenetic study by means of amplicon sequencing. We therefore designed a pair of generic degenerate PCR primers, binding to the sequences on either side of the sequences encoding VP1 and 2C (Table 1), based on previously published alignments of full-length prototype strain sequences (personal communication from Ann Palmenberg, http://www.bocklabs.wisc.edu/acp/picorna/aligns/). The oligonucleotides were designed to be very similar (>85% identical) to all the target enterovirus sequences already available in the database. In all cases, the three 3'-terminal nucleotides were chosen so as to be highly conserved among the available enterovirus sequences in order to avoid PCR mismatch inhibition. None of the antisense primers contained a mixed-base and none of the sense primers had more than one degenerate position. The amplicon thus obtained was 1452 bp long (relative to the PV1-Mahoney sequence) and included the 3' end of the VP1-coding sequence, the entire coding sequence of 2A and 2B, and the 5' moiety of the 2C-coding region of the enterovirus genome.

To determine specificity, an equimolar mixture of primers was first tested in an RT–PCR 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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Table 3. Correspondence between partial VP1 sequencing and seroneutralization

 
Nucleotide sequence and serotypes
We sequenced 600 to 700 nt including the 3' third of the VP1 gene and the beginning of the 2A gene for each isolate using sense EUG3a, EUG3b and EUG3c primers in combination or separately. All new sequence data presented in this work have been published in the EMBL database (Table 2). The sequences of the 2A-coding region are currently being analysed and the sequences of the 2B- and 2C-coding region have not yet been determined. The partial VP1 sequence of each of the 45 field isolates (a stretch of 365 nucleotides from 3021 to 3385, numbering according to PV1-Mahoney) was compared with all enterovirus sequences published in GenBank. The gbvrl library was used for nucleic acid analysis and the gpvrl library for protein analysis. A 100% correlation was obtained between the serotype determined from the 3' third of the VP1-coding region sequence and the serotype determined by the conventional neutralization assay for all the 45 field isolates of known serotype tested (Table 3).

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 (RT–PCR) of the 5'NCR. They were not neutralized by intersecting RIVM pools. Their genome was successfully amplified by our pair of enterovirus-specific VP1–2C 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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Fig. 1. Phylogenetic tree showing genetic relationships between the 45 enterovirus field isolates, based on the C-terminal third of the VP1-coding region sequence alignment. The prototype enterovirus representative of each species is indicated in bold. The tree was constructed by the neighbour-joining method with the maximum-likelihood method of Kishino and Hasegawa with a transition/transversion ratio of 8·0 for the distance matrix. Numbers at nodes represent the percentage of 100 bootstrap pseudoreplicates. The human enterovirus species (PV, HEV-A, HEV-B, HEV-C and HEV-D) are indicated on the right-hand side by vertical bars (distances between bars are arbitrary). Horizontal branch lengths are proportional to the number of substitutions as indicated by the scale (the scale bar represents 0·1 nucleotide substitutions per site).

 
To study more precisely the phylogenetic relationships between field isolates, we constructed phylogenetic trees within each defined species for the clinical isolates and the corresponding prototype HEV. In each tree, one sequence from each of the other species was included as an outgroup. The HEV-D species was not analysed because no appropriate isolate was available. Within each species, the same general tree topology was obtained irrespective of the method used. However, some variation in the branching order of some subgroups, in bootstrap values for certain nodes and in branch lengths was observed. Whatever the species, all homotypic strains (field and prototype) were monophyletic, as supported by parametric bootstrap analysis. Three subgroups, slightly different from the published subgroups (Oberste et al., 1999b ), were observed for HEV-A species (Fig. 2a): (1) CA2, CA6, CA10, CA4, CA8 and CA3; (2) CA7, CA14, CA5 and CA12; and (3) CA16, EV71 and the two CA16 isolates. The CA16 isolates were closely related to each other and to their homologous prototype strain. The clustering of CA16 with the prototype EV71 strain (bootstrap value of 91%), is consistent with a prior observation that CA16 has an antigen in common with EV71 (Hagiwara et al., 1978 ). The HEV-C species was divided into the same four subgroups as already published (Oberste et al., 1999b ): (1) CA1, CA19 and CA22; (2) CA21 and CA24; (3) CA11 and CA15; and (4) PV1, PV2, PV3, CA17, CA13 prototype and isolates, CA18, CA20 prototype and isolates (Fig. 2b). HEV-B species made up the largest group, with 37 isolates of the 45 analysed belonging to this phylogenetic group. By analysing 37 field strains and 44 prototype strains, including three outgroup strains (EV70, CA16 and PV1) (Fig. 2c), we found that the field isolates formed monophyletic clades with their prototype, well supported by bootstrap analysis. All the CBVs clustered together, with each isolate close to its prototype. As previously reported, CB2 and CB4 were more related to each other than to the other CB serotypes, whereas the CB1, CB3 and CB5 strains constituted a single subgroup and CB6 formed a branch of its own (Lindberg & Polacek, 2000 ). In general, the clinical isolates of a given serotype were more closely related to each other than to their homologous prototypes, which were often displayed on a different branch.




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Fig. 2. Phylogenetic trees depicting genetic relationships between field human enteroviruses and prototype HEV-A (a), HEV-C+PV (b) and HEV-B (c). Trees were based on sequence alignment of the C-terminal third of the VP1-coding region and constructed by the neighbour-joining method with the maximum-likelihood method of Kishino and Hasegawa with a transition/transversion ratio of 8·0 for the distance matrix. Numbers at nodes represent the percentage of 100 bootstrap pseudoreplicates. Branch lengths are proportional to the number of substitutions as indicated by the scale (the scale bar represents 0·1 nucleotide substitutions per site). Human enteroviruses sequenced in this work are shown in bold.

 
The two E9 prototypes were not clustered together in a single subgroup and the E9-RO-1/9/72 isolate was more closely related to the E9/Barty/57 prototype than to the other isolate, E9-RO-116/6/82. The E9-RO-1/9/72 isolate and E9/Barty/57 were very similar: E9-RO-1/9/72 was isolated from the spinal cord of a child with encephalitis and E9/Barty/57 was isolated from the cerebrospinal fluid of a child with aseptic meningitis. The two E9 field strains had an additional 10 amino acid fragment including an RGD motif in the C-terminal part of the VP1 structural protein (data not shown), which differentiated the pathogenic Barty strain from the non-pathogenic Hill strain (Zimmermann et al., 1996 ), and has been described as a probable major determinant of virulence (Nelsen-Salz et al., 1999 ; Zimmermann et al., 1997 ).


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The global eradication of poliomyelitis will soon be achieved, vaccination with live oral poliovirus vaccine will be stopped and the ecological niche taken by poliovirus will become vacant. Interest has increased in the circulation, detection, identification and evolution of non-polio enteroviruses and the emergence of new epidemic strains. The traditional classification of enteroviruses is based on antigenic specificity, as determined by serum neutralization assay, with all the disadvantages that this method implies. Genomic sequencing has made it possible to develop various molecular approaches for enterovirus identification and genealogical studies. Use of the 5'NCR or the VP2 capsid protein-coding region (Romero, 1999 ) made it possible to detect HEV, but not to identify them beyond the genus level. The discovery that the VP1 protein-coding region contains serotype-specific information in HEV (Oberste et al., 1999b ) has made possible the development of molecular methods for the identification of traditional HEV serotypes and of the newly designated HEV species (Pringle, 1999 ). We demonstrate here that a genomic segment of 364 nucleotides (relative to PV1-Mahoney) of the 3' third of the VP1-coding region of HEV genomes contains enough serotype-specific information to be used for HEV serotype identification.

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 VP1–2C 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 VP1–2A 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 RT–PCR 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 VP1–2C 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 RT–PCR 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 VP1–2C 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.


   Acknowledgments
 
We would like to thank Véronique Giacomoni-Fernandes and Ariane Dubois for expert technical assistance. We also wish to thank Dr George Anicet Dahourou, Dr Jacques Salmon, Dr Panayotis Markoulatos, Dr Mala Rakoto Andrianarivelo, Dr Albert Ras and Dr Marianna Combiescu for providing the isolates from Burkina Faso, France, Greece, Madagascar, the Netherlands and Romania, respectively.

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).


   Footnotes
 
The EMBL accession numbers of the sequences reported in this paper are AJ279151 to AJ279195.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 13 July 2000; accepted 26 September 2000.