Complete genome sequences of all members of the species Human enterovirus A

M. Steven Oberste, Silvia Peñaranda, Kaija Maher and Mark A. Pallansch

Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-17, Atlanta, GA 30333, USA

Correspondence
M. Steven Oberste
soberste{at}cdc.gov


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The species Human enterovirus A (HEV-A) in the family Picornaviridae consists of coxsackieviruses (CV) A2–A8, A10, A12, A14 and A16 and enterovirus 71. Complete genome sequences for the prototype strains of the 10 serotypes whose sequences were not represented in public databases have been determined and analysed in conjunction with previously available complete sequences in GenBank. Members of HEV-A are monophyletic relative to all other human enterovirus species in all regions of the genome except in the 5' non-translated region (NTR), where they are known to cluster with members of HEV-B. The HEV-A prototype strains were about 66 to 86 % identical to one another in deduced capsid amino acid sequence. Antigenic cross-reactivity has been reported between CVA3-Olson and CVA8-Donovan, between CVA5-Swartz and CVA12-Texas-12 and between CVA16-G-10 and EV71-BrCr. Similarity plots, individual sequence comparisons and phylogenetic analyses demonstrate a high degree of capsid sequence similarity within each of these three pairs of prototype strains, providing a molecular basis for the observed antigenic relationships. In several cases, phylogenies constructed from the structural (P1) and non-structural regions of the genome (P2 and P3) are incongruent. The incongruent phylogenies and the similarity plot analyses imply that recombination has played a role in the evolution of the HEV-A prototype strains. CVA6-Gdula clearly contains sequences that are also present in CVA10-Kowalik and CVA12-Texas-12, suggesting that these three strains have a shared evolutionary history despite their lack of similarity in the capsid region.

Supplementary table of primers used to amplify and sequence the genomes of the HEV-A prototype strains in JGV Online.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The human enteroviruses include the polioviruses (PV), coxsackie A viruses (CVA), coxsackie B viruses (CVB), echoviruses (E) and numbered enteroviruses (EV). More than 60 human enterovirus serotypes are divided among five species within the genus Enterovirus (family Picornaviridae): Poliovirus (PV1–3), Human enterovirus (HEV) A (CVA2–8, 10, 12, 14, 16 and EV71), HEV-B (CVA9, CVB1–6, E1–7, 9, 11–21, 24–27, 29–33 and EV69 and 73), HEV-C (CVA1, 11, 13, 15, 17–22 and 24) and HEV-D (EV68 and 70) (King et al., 2000). The close genetic relationship between the polioviruses and members of HEV-C suggests that they should be considered a single species (Brown et al., 2003; Hyypiä et al., 1997; Pöyry et al., 1996). Although most infections are asymptomatic, enteroviruses may be associated with a wide range of diseases, including mild upper-respiratory illness, febrile rash (hand, foot and mouth disease), herpangina, aseptic meningitis, encephalitis, myocarditis and acute flaccid paralysis (Pallansch & Roos, 2001). The viruses in HEV-A, particularly CVA16 and EV71, are often associated with hand, foot and mouth disease, a relatively common febrile rash illness in children.

With the exception of EV71, the prototype strains of the HEV-A serotypes were isolated between 1947 and 1951 (Pallansch & Roos, 2001); the EV71 prototype strain was isolated in 1970 (Schmidt et al., 1974). Most of the viruses in HEV-A have not been examined in detail, but antigenic relationships (weak or non-reciprocal cross-reactivity) have been observed for some serotypes (Beeman et al., 1952; Contreras et al., 1952; Dalldorf & Sickles, 1956; Hagiwara et al., 1978). EV71 has been studied more extensively due to the occurrence of severe, sometimes fatal disease during outbreaks of hand, foot and mouth disease in Malaysia and Taiwan in the late 1990s (Chan et al., 2000; Shimizu et al., 1999) and its association with severe neurological disease in earlier outbreaks (Alexander et al., 1994; Melnick, 1984). While EV71 circulated widely in the affected communities during the Malaysia and Taiwan outbreaks, EV71 has not been unequivocally established as the only factor related to the fatal outcome in some patients during these outbreaks.

The enterovirus genome is a single-stranded, polyadenylated, positive-sense RNA of approximately 7·4 kb, with a 22 aa virus-encoded protein (3BVPg) covalently linked to the 5' end. Flanked by 5'- and 3' non-translated regions (NTRs), the single long open reading frame encodes a polyprotein of approximately 2200 aa that is processed during and following translation by viral proteases to yield the mature viral polypeptides. The P1 region encodes the capsid proteins 1A–1D (VP4, VP2, VP3 and VP1, respectively). The P2 and P3 regions encode proteins involved in polyprotein processing, RNA replication and shut-down of host-cell protein synthesis. Complete genome sequences were previously available for CVA16 and EV71, but only partial sequences (generally less than one-third of the genome) were available for the remaining 10 members of HEV-A. Phylogenetic analyses of available sequences have shown that the members of HEV-A are closely related to one another in multiple regions of the genome (Hyypiä et al., 1997; Oberste et al., 1998, 1999b; Pöyry et al., 1996), but the full extent and details of their genetic relationships have not been described.

We present here the first analysis of the complete genome sequences of the prototype strains of all serotypes in HEV-A. Individual sequence comparisons and phylogenetic analyses explain, at least partially, some of the observed antigenic relationships between these serotypes. Furthermore, these studies suggest that certain members of HEV-A have recombined with one another at some point in their evolutionary history.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viruses.
The prototype strains of coxsackieviruses A2–A8, A10, A12 and A14 were obtained as National Institutes of Health research reference reagents from the National Institute of Allergy and Infectious Diseases (Bethesda, MD) (Table 1) and propagated in cell culture by standard methods (Melnick et al., 1979). The original materials are now distributed by the American Type Culture Collection (Manassas, VA).


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Table 1. Prototype strains of Human enterovirus A serotypes

 
Nucleotide sequencing.
Complete genomic sequences were determined for each of the 10 strains indicated in Table 1. Overlapping fragments representing each complete viral genome were amplified by RT-PCR using degenerate, inosine-containing primers designed to anneal to sites encoding amino acid motifs that are highly conserved among enteroviruses. The generic primers are listed in the supplementary Online material (Table S1). Specific, non-degenerate primers were designed from preliminary sequences to close gaps between the original PCR products. The PCR products were purified for sequencing by using a High-Pure PCR product purification kit (Roche Molecular Biochemicals). Both strands were sequenced by automated methods, using fluorescent dideoxy-chain terminators (Applied Biosystems) with a mean of 4·6-fold redundancy. The complete genome sequences for CVA16-G-10 and EV71-BrCr were obtained from GenBank.

Sequence analysis.
The pairwise sequence identities among the nucleotide and deduced amino acid sequences of all of the HEV-A serotypes were calculated by use of the programs Gap and Distances (Wisconsin Sequence Analysis Package, version 10.2, Accelrys, Inc.). Nucleotide sequences were aligned using the PILEUP program (Wisconsin Package) and adjusted manually to conform to the optimized alignment of deduced amino acid sequences. Regions of the alignment containing gaps were excluded from the phylogenetic analyses. Phylogenetic relationships were inferred from the aligned nucleic acid sequences by the neighbour-joining method implemented in the programs DNADist and Neighbor (PHYLIP: Phylogeny Inference Package, version 3.57, http://evolution.genetics.washington.edu/phylip.html), using the Kimura two-parameter substitution model (Kimura, 1980) and a transition–transversion ratio of 10. The transition–transversion ratio was chosen based on that used for poliovirus type 1 (Liu et al., 2000); ratios in the range of 8 to 10 produce trees with essentially the same topology (data not shown). Support for specific tree topologies was estimated by bootstrap analysis with 1000 pseudo-replicate datasets. Branch lengths in consensus trees were calculated by the maximum-likelihood quartet-puzzling method, using the nucleotide substitution model of Tamura & Nei (1993) as implemented in TreePuzzle 5.0 (Strimmer & von Haeseler, 1996). Similarity plots depicting the relationships among the aligned nucleotide sequences were generated using SimPlot, version 3.2 beta (Lole et al., 1999). Similarity was calculated in each window of 200 nt by the Kimura two-parameter method (Kimura, 1980) with a transition–transversion ratio of 10. The window was successively advanced along the genome alignment in 20 nt increments. Simplot was also used to analyse the relationships among the HEV-A polyprotein sequences by using the Jukes–Cantor distance method (Jukes & Cantor, 1969) to calculate similarity in a window of 200 residues with a step of 20 residues.

Nucleotide sequence accession numbers.
The sequences reported here were deposited in the GenBank sequence database, accession nos. AY421760–AY421769.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Non-coding regions
Phylogenetic trees were constructed for each of the major functional units in the genome, including the 5' and 3' NTRs, the capsid coding region (P1) and the regions encoding the non-structural proteins (P2 and P3) (Fig. 1). Each of the trees includes a representative of each of the other three human enterovirus species as reference points. The 5' NTR sequences of all members of HEV-A are closely related to one another and to the 5' NTR of CVB1, the representative of HEV-B (Fig. 1A). In the 5' NTR, the viruses in HEV-A and HEV-B are all closely related to one another and intermix without regard to species, forming enterovirus 5' NTR group II, whereas HEV-C (including the polioviruses) and HEV-D form group I (Brown et al., 2003; Hyypiä et al., 1997; Oberste et al., 2004; Santti et al., 1999). The 3' NTR sequences of HEV-A viruses are similar to one another (77·1 to 98·8 % identity), but they are quite distinct from members of the other HEV species, both in pairwise sequence identity (<61 % identity) and in phylogeny (Fig. 1B). Most of the HEV-A 3' NTRs are 83 nt long, but those of CVA4, CVA14 and CVA16 are only 81 nt in length, sharing a 2 nt deletion near their 5' ends. These three serotypes also clustered together phylogenetically (Fig. 1B).



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Fig. 1. Unrooted phylogenetic trees based on HEV-A virus nucleotide sequences. Each of the major functional regions of the genome was analysed independently. Bootstrap values (percentage of 1000 pseudoreplicate datasets) supporting each cluster are shown at the nodes; for clarity, only values over 70 % are shown. For ease of interpretation, the unrooted trees were similarly oriented relative to the outgroup taxa, CVB1, PV1 and EV70, which are representatives of HEV-B, HEV-C and HEV-D, respectively. All trees are plotted to the same scale except panel (B); the scale bars indicate genetic distance (substitutions per nucleotide). (A) 5' NTR; (B) 3' NTR; (C) complete P1 region; (D) complete P2 region; (E) complete P3 region.

 
Capsid region
The HEV-A deduced capsid protein sequences vary in length from 858 to 870 aa. The length differences tend to accumulate in regions of known diversity among the enterovirus capsid proteins (data not shown). For example, CVA2, CVA3, CVA5, CVA8, CVA10 and CVA12 have a gap of 1 aa in the VP2 ‘puff’ region relative to the longest VP2 sequences (CVA4 and CVA6), while CVA7, CVA14, CVA16 and EV71 have a 2 aa gap at the same position. The same four serotypes (CVA7, CVA14, CVA16 and EV71) have a 2 aa insertion in the VP3 ‘knob’ relative to all other serotypes. Length differences in VP1 cluster in the amino-terminal domain (1–5 aa gaps), the B-C loop (1–2 aa gaps) and in the carboxyl-terminal domain (gaps of 1–14 aa).

The HEV-A prototype strain capsid-coding sequences (P1) are monophyletic relative to members of the other human enterovirus species, with strong bootstrap support (Fig. 1C). The observed antigenic cross-reactions in complement fixation or neutralization tests had suggested a relationship between CVA3 and CVA8 (Beeman et al., 1952; Contreras et al., 1952; Dalldorf & Sickles, 1956), between CVA5 and CVA12 (Contreras et al., 1952; Dalldorf & Sickles, 1956) and between CVA16 and EV71 (Hagiwara et al., 1978). As reported previously, these relationships correlate with similarities in complete VP1 sequence (Oberste et al., 1999b). To investigate further the genetic basis for these relationships, pairwise sequence identities were calculated for the deduced complete capsid protein sequences of all 12 HEV-A viruses and for the sequences of each of the mature capsid proteins, VP1–VP4. The complete capsid sequences are 66·2 to 85·4 % identical to one another (Table 2). CVA3 and CVA8 are the most closely related (85·4 % identity), followed by CVA5-CVA12 (82·9 % identity) and CVA16-EV71 (79·3 % identity). Within the capsid region, the VP1 region is the most divergent (55·4 to 83·3 % sequence identity). In each of the mature capsid proteins, CVA3-CVA8, CVA5-CVA12 and CVA16-EV71 are consistently among the most closely related sequence pairs (Table 2). The CVA3-CVA8 and CVA5-CVA12 nucleotide sequences also cluster phylogenetically in the VP2, VP3 and VP1 trees, but by contrast, CVA16 and EV71 cluster together only in VP4 and VP1 (Fig. 2A–D); in most cases, however, the bootstrap support is relatively low.


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Table 2. Pairwise amino acid sequence differences among the capsid proteins of HEV-A serotypes

 


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Fig. 2. Unrooted phylogenetic trees based on HEV-A virus nucleotide sequences. The regions encoding each of the mature capsid proteins and the mature proteins derived from the P2 and P3 regions were analysed independently. Bootstrap values (percentage of 1000 pseudoreplicate datasets) supporting each cluster are shown at the nodes; for clarity, only values over 70 % are shown. For ease of interpretation, the unrooted trees were similarly oriented relative to the outgroup taxa, CVB1, PV1 and EV70, as in Fig. 1. The outgroup taxa are represented schematically by the branch at the left of each tree. Trees in panels (A)–(D) are plotted to the same scale, as are those in panels (E)–(J) (see scale bars). (A) 1A (VP4); (B) 1B (VP2); (C) 1C (VP3); (D) 1D (VP1); (E) 2A; (F) 2B; (G) 2C; (H) 3AB; (I) 3C; and (J) 3D.

 
The deduced amino acid sequences for the complete polyproteins of all 12 serotypes were also analysed with Simplot, using each serotype in turn as the query sequence, with a sliding window of 200 residues and a step of 20 residues (Fig. 3). In some of the comparisons, the query sequence was approximately equidistant from all other serotypes. For example, there were no obvious pairing partners for CVA2, CVA4 and CVA6 (Fig. 3A, C and E). In other cases, specific relationships were readily apparent. The most striking examples are the capsid region comparisons for CVA3-CVA8 and CVA5-CVA12 (Fig. 3B, G, D and I, respectively), which agree well with the pairwise amino acid sequence identities (Table 2) and the nucleotide phylogenies (Fig. 1).



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Fig. 3. Similarity plots of HEV-A virus amino acid (aa) sequences calculated by SimPlot 3.2 beta (Lole et al., 1999). Each point represents the similarity between the query sequence and a given heterologous sequence, within a sliding window of 200 aa centred on the position plotted, with a step of 20 residues between points. Positions containing gaps were excluded from the analysis. The enterovirus genetic map is shown at the top of each column of panels (A–L). For each plot, the identity of the query sequence is indicated in the upper left corner. (A) CVA2; (B) CVA3; (C) CVA4; (D) CVA5; (E) CVA6; (F) CVA7; (G) CVA8; (H) CVA10; (I) CVA12; (J) CVA14; (K) CVA16; and (L) EV71.

 
Non-structural region
The P2 and P3 regions are collinear for all members of HEV-A. The P2 region is 1734 nt long (578 aa) and P3 is 2259 nt long (753 aa). As in the capsid-coding region, the members of HEV-A are monophyletic in both P2 and P3 with respect to the other species of HEV (Fig. 1D and E), as well as in each of the individual mature proteins derived from P2 and P3 (Fig. 2E–J and data not shown). CVA4, CVA14 and CVA16 cluster together throughout P2 and P3 (Figs 1D, E and 2E–J), but the clustering relationships of the other prototype strains change depending upon genome position. For example, CVA5 clusters with CVA10 in 2A, with CVA4 in 2B, on an independent branch near CVA4-CVA14-CVA16 in 2C, 3AB and 3C, and with CVA7 and CVA8 (and distinct from CVA4-CVA14-CVA16) in 3D.

Within the P2 and P3 regions, conservation of amino acid sequence is much higher than seen in the capsid region, >96 and >92 % identity, respectively. For this reason, the capsid sequence similarities observed in the protein Simplots are not as prominent in the nucleotide Simplots (Fig. 4). The CVA16-EV71 capsid relationship is barely discernible in the nucleotide sequence comparison due to a comparatively higher proportion of synonymous differences, which lowers the overall nucleotide similarity (Fig. 4K and L). In contrast, however, the P3 similarities between CVA4, CVA14 and CVA16 are more readily apparent in the nucleotide analysis (Fig. 4C and J, K) than in the amino acid comparison (Fig. 3C and J, K), probably because of the overall high degree of amino acid conservation in P2 and P3 among all members of HEV-A. As described for HEV-B (Oberste et al., 2004), there are four basic patterns of similarity in the HEV-A nucleotide Simplots: (i) relatively low similarity (about 65 to 85 %) to all other prototype strains in P2 and P3; (ii) a high degree of similarity (80 to 95 %) to many other strains, throughout most of P2 and P3; (iii) high similarity to many other strains, with peaks in discrete regions of P2 and P3; and (iv) low similarity to most prototype strains but with a high degree of similarity to one or a few other strains in discrete regions of P2 and P3. CVA7 is an example of the first pattern, with no significant peaks of similarity above the mean (Fig. 4F), and CVA2 is an example of the second case, having 85 to 90 % similarity to several other strains throughout P2 and P3 (Fig. 4A). CVA6, CVA10 and CVA12 are examples of the third pattern, displaying a complex pattern of relationships to one another in 2C (CVA6–CVA10) and 3D (CVA6 and CVA12), as well as somewhat weaker relationships to numerous other strains throughout P2 and P3 (Fig. 4E and H, I). The fourth pattern type is best exemplified by CVA4, CVA14 and CVA16, which are much more closely related to one another from 2C to the C terminus of the polyprotein than they are to any other prototype strain (Fig. 4C and J, K).



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Fig. 4. Similarity plots of HEV-A virus nucleotide (nt) sequences calculated by SimPlot 3.2 beta (Lole et al., 1999). Each point represents the similarity between the query sequence and a given heterologous sequence, within a sliding window of 200 nt centred on the position plotted, with a step of 20 residues between points. Positions containing gaps were excluded from the analysis. The enterovirus genetic map is shown at the top of each column of panels (A–L). For each plot, the identity of the query sequence is indicated in the upper left corner. (A) CVA2; (B) CVA3; (C) CVA4; (D) CVA5; (E) CVA6; (F) CVA7; (G) CVA8; (H) CVA10; (I) CVA12; (J) CVA14; (K) CVA16; and (L) EV71.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
During the initial characterization of the enteroviruses in the early 1950s, individual strains were classified as ‘coxsackie A’ or ‘coxsackie B’ on the basis of their pathogenesis in intracranially inoculated newborn mice, with group A producing flaccid paralysis and group B leading to spastic paralysis (polioviruses and echoviruses are typically avirulent in newborn mice). Strains were further classified into serotypes by cross-complement fixation or cross-neutralization, using antisera raised against specific strains (Dalldorf & Sickles, 1956). Strains that cross-reacted with one another to a similar titre were considered to belong to the same serotype. Weak or non-reciprocal cross-reactivities were often observed, leading to difficulties in assay interpretation. Despite these limitations, the neutralization assay served as the reference standard for enterovirus identification for nearly 50 years, until the recent introduction of molecular typing methods (Oberste et al., 1999a, 2000, 2003). Recent molecular data have confirmed many of the cross-reactivity observations and provided a framework to explain the molecular basis for the observed antigenic relationships. For example, as previously reported for HEV-C, reciprocal cross-reactivity was observed for CVA11 and CVA15 and for CVA13 and CVA18 (Dalldorf & Sickles, 1956). A high degree of capsid sequence conservation (96 % aa identity) has confirmed these relationships and suggests that CAV15 and CAV18 should be classified as strains of CAV11 and CAV13, respectively (Brown et al., 2003). Similarly, cross-reactivity has been observed between CVA3-Olson and CVA8-Donovan in both neutralization and complement fixation tests (Contreras et al., 1952; Dalldorf & Sickles, 1956). Our data show that the capsid sequences of CVA3-Olson and CVA8-Donovan are also related to one another, but to a lesser degree (85·4 % identity) than observed in the examples cited from HEV-C, whereas the mean identity among HEV-A prototype strains is only 71·5 % (Table 2). Thus, CVA3 and CVA8 probably represent related, but distinct, serotypes that relatively recently derived from a common ancestor. The specific epitopes responsible for their antigenic cross-reactivity remain unknown.

The incongruent phylogenies and the Simplot analyses imply that recombination has played an evident role in the evolution of some HEV-A prototype strains. CVA6-Gdula clearly contains sequences in the non-capsid region that are also present in CVA10-Kowalik and CVA12-Texas-12, suggesting that these three strains have a shared evolutionary history, despite their lack of similarity in the capsid region. Likewise, CVA4-High Point, CVA14-G-14 and CVA16-G-10 are clearly related to one another in P2 and P3, despite there being little similarity in P1. This suggests that two or more recombination events have coupled related P2 and P3 sequences to the three heterologous and unrelated capsid sequences.

The HEV-A Simplots and deduced patterns of recombination are not as numerous or dramatic as those generated for members of HEV-B (Oberste et al., 2004). This may simply reflect the fact that there are three times as many serotypes in HEV-B or the apparent abundance of infections with viruses in this species. The smaller number of evident recombination events could also be influenced by the lack of temporal and geographical heterogeneity in HEV-A relative to HEV-B. Eight of the 12 HEV-A prototype strains were isolated in states on the United States' Atlantic coast, and only two are from outside the United States (both are from South Africa), whereas the HEV-B prototype strains are from five countries on three continents. Similarly, 11 of the 12 HEV-A prototype strains were isolated over a 4 year period, from 1947 to 1951, while isolation of the HEV-B prototypes was distributed across a 14 year period, from 1947 to 1961 (Pallansch & Roos, 2001). Analysis of clinical isolates from a wider geographical area and over a longer period of time might help resolve the relative importance of these factors as they contribute to the observed genetic diversity within HEV-A.

As with HEV-B and HEV-C (Brown et al., 2003; Oberste et al., 2004), the relatively high nucleotide sequence diversity among the capsid-coding sequences of HEV-A serotypes suggests that nucleotide substitution is the dominant evolutionary force in this region of the genome. Closely related sequence pairs, such as CVA3-Olson and CVA8-Donovan, probably derived relatively recently from a common ancestor. This divergence was most likely the result of amino acid substitutions, rather than by sharing sequences through recombination, because their divergence across the capsid follows the same pattern as the mean similarity among strains and there are no detectable recombination junctions in the capsid sequences of CVA3-Olson and CVA8-Donovan.

Regions of amino acid conservation within the capsid probably reflect a restriction due to functional constraints, such as maintaining the {beta}-barrel structural elements or conservation of receptor-binding domains; however, receptor usage has not been reported for any of the members of HEV-A. The availability of complete genome sequences for all serotypes within HEV-A will provide a context for future genetic studies and support investigations of HEV-A structure and receptor utilization.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 7 November 2003; accepted 16 January 2004.