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
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ABSTRACT |
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Supplementary table of primers used to amplify and sequence the genomes of the HEV-A prototype strains in JGV Online.
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INTRODUCTION |
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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 1A1D (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.
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METHODS |
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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 transitiontransversion ratio of 10. The transitiontransversion 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 transitiontransversion 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 JukesCantor 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. AY421760AY421769.
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RESULTS |
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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, VP1VP4. 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. 2
AD); in most cases, however, the bootstrap support is relatively low.
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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 (CVA6CVA10) 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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DISCUSSION |
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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 -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.
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Received 7 November 2003;
accepted 16 January 2004.