1 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
2 Institute of Public Health, Dhaka, Bangladesh
Correspondence
M. Steven Oberste
soberste{at}cdc.gov
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY697458AY697507.
Supplementary tables are available in JGV Online.
Present address: VaxGen Inc., 347 Oyster Point Blvd, Suite 102, South San Francisco, CA 94080, USA.
Present address: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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INTRODUCTION |
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In previous studies, we used analysis of partial or complete VP1 nucleotide sequences as a surrogate for antigenic typing to distinguish enterovirus serotypes (Oberste et al., 1999a, 2000
, 2003
). This method was used to identify a new enterovirus serotype, EV73, from among enterovirus isolates that were deemed untypable by classical identification methods (Oberste et al., 2001
). Four additional new types, EV74, EV75, EV77 and EV78, have also been identified by similar methods (Norder et al., 2003
; Oberste et al., 2004c
). The identification of these new types suggested that there may be many other additional enterovirus serotypes awaiting identification.
In this study, 19 enterovirus isolates are characterized as members of four new types within HEV-A. Isolates within each of these four groups are significantly different from all known enterovirus serotypes, as determined on the basis of sequences from multiple genome regions. Sequence comparisons identified all four groups as members of the species HEV-A, but analysis of partial 3D sequences showed that they also form a distinct subgroup within HEV-A. We propose that these isolates be classified as members of four new human enterovirus types in the species HEV-A.
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METHODS |
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Isolates were further characterized by sequencing of the complete capsid (P1) region. RT-PCR primers were designed to anneal to sites encoding amino acid motifs that are highly conserved among members of the species HEV-A (Oberste et al., 2004d). Specific, non-degenerate primers were designed from preliminary sequences to close gaps between the original PCR products. The complete genome sequences of each of the proposed new prototype strains were also determined using a similar strategy, with additional primer-walking to close large gaps as necessary. For the non-prototype strains, a portion of the 3D region was amplified and sequenced using primers 782 (5'-TAGYCTTTCTCCYGCYTGG-3', EV76 nt 66726690) and 783 (5'-GAYCGCACRTGRTCTTGIGT-3', EV76 nt 72057186). For comparison with isolates that are members of previously recognized HEV-A serotypes, the partial 3D sequences of 16 contemporary Bangladeshi HEV-A isolates were determined using primers 233 (5'-TTGAYTACWCWGGNTATGATGC-3', PV1 nt 66816702) and 130 (5'-WGSRTTCTTKGTCCATC-3', PV1 nt 72097191) (Oberste et al., 2004e
). For all sequence determinations, the PCR products were purified for sequencing by using a High-Pure PCR product purification kit (Roche Molecular Biochemicals), and both strands were sequenced by automated methods, using fluorescent dideoxy-chain terminators (Applied Biosystems).
Sequence analysis.
The nucleotide and deduced amino acid sequences of the isolates were compared to one another and to those of other enteroviruses by using the programs Gap and Distances (Wisconsin Package, version 10.3). Nucleotide sequences were aligned using the PILEUP program (Wisconsin Package) and adjusted manually to conform to the optimized alignment of deduced amino acid sequences. Phylogenetic relationships were inferred from the aligned nucleic acid sequences by the neighbour-joining method implemented in MEGA, version 2.1 (Kumar et al., 2001), using the Kimura two-parameter substitution model (Kimura, 1980
) and a transition-transversion ratio of 10. Regions containing alignment gaps were omitted from the analysis. Support for specific tree topologies was estimated by bootstrap analysis with 1000 pseudo-replicate datasets.
Nucleotide sequence accession numbers.
The sequences described here have been deposited in the GenBank sequence database, accession numbers AY697458 to AY697507; the accession numbers are listed in Table S1 (JGV Online) by strain and region sequenced. Other enterovirus sequences used in comparisons are listed in Table S2 (JGV Online).
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RESULTS |
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To confirm the typing identifications that were based on the partial VP1 sequences, complete VP1 sequences were determined for each of the isolates. The sequences were compared with each other and with the complete VP1 sequences of all known enterovirus serotypes. Like the partial VP1 sequences, the complete VP1 nucleotide sequences of all 19 isolates are less than 70 % identical to those of the established enterovirus serotypes, and they are most closely related to those of members of HEV-A (54·969·4 % nucleotide sequence identity) (Table 2); members of other HEV-A serotypes are 54·573·2 % identical to one another (Oberste et al., 1999b
, 2004d
). We have previously shown that strains that are at least 75 % identical in VP1 sequence belong to the same serotype, whereas those that are less than 70 % identical to one another belong to different serotypes (Oberste et al., 1999a
, b
, 2000
, 2001
, 2003
). In almost all cases, the VP1 sequence of each of the 19 isolates is most closely related to that of one of the simian enteroviruses in HEV-A, A13, SV19/SV26/SV35 (a single serotype), SV43 or SV46 (Fig. 1
a). Comparison of the complete VP1 sequences with one another confirmed that they formed four distinct groups (provisional new types). Within each of the four new types, the VP1 sequences are at least 78·1 % identical to one another (93·2 % amino acid identity), but members of the different types differ from one another by at least 29·7 % (Table 2
). These new types have been provisionally named enteroviruses 76, 89, 90 and 91. EV77 and EV78, both members of HEV-B, have been recently described (Bailly et al., 2004
; Norder et al., 2003
); enteroviruses numbered from 79 to 88 are also members of HEV-B and will be described elsewhere (M. S. Oberste, unpublished data).
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P1 sequences
As in VP1, all of the complete capsid sequences are monophyletic, both by type and as a group (Fig. 1b), and the capsid sequence clustering is more firmly supported by bootstrap analysis (97 %). Complete capsid sequences are not yet available for the simian viruses in HEV-A. The 11 EV76 capsid sequences remain clustered in the same three subgroups, with 100 % bootstrap support for each subgroup. The deduced capsid protein sequences are highly conserved within a type (94100 % identity), whereas capsid sequence identity between the four types ranges from 79 to 82 % (Table 3
). However, they are no more than 76 % identical to those of any other enterovirus serotype. By comparison, other members of HEV-A are 6685 % identical to one another in deduced capsid protein sequence (Oberste et al., 2004d
). The capsid protein sequences of the EV76 candidates are 94·3100 % identical to one another, and at least 99 % identical within each subgroup (Table 3
and data not shown). Sites at which the amino acid sequences vary among the EV76 isolates are distributed throughout the three major capsid proteins, 16 in VP2, 13 in VP3 and 20 in VP1; the VP4 sequences are identical in all isolates (data not shown). Eight of 16 variable sites in VP2 are in the puff region and 10 of 13 variable sites in VP3 are in the knob region. In enteroviruses of known 3D structure, the puff and knob are predominant surface protrusions and contribute to neutralizing epitopes on the capsid surface (Muckelbauer et al., 1995
). In VP1, most of the variable sites are near the ends of the mature protein: eight in the first 27 residues at the amino terminus and six in the last 26 aa at the carboxyl terminus. The capsid sequences of the EV89, EV90 and EV91 isolates are very highly conserved within type (99·4, 98·299·2 and 99·0 % identity, respectively) (Table 3
).
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DISCUSSION |
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Analysis of enterovirus prototype strains has suggested that recombination is a frequent event in enterovirus evolution and that recombination only occurs between viruses of the same species (Andersson et al., 2002; Brown et al., 2003
; Oberste et al., 2004a
, d
; Santti et al., 1999
). Similar results have been obtained by analysis of more recent clinical isolates (Lindberg et al., 2003
; Lukashev et al., 2003
, 2004
; Oberste et al., 2004e
). Phylogenetic analysis of partial 3D sequences suggests that EV76, EV89, EV90 and EV91 have recombined with one another but not with viruses of other HEV-A serotypes (Fig. 3
). Similarly, the analysis of 3D sequences shows that the Bangladesh enteroviruses of other types are also recombinant with respect to each other and to the HEV-A prototype strains, as the recent clinical isolates do not necessarily cluster by type in the 3D tree. The biological factors underlying the apparent restriction of recombination within a species remain unknown.
Four serotypes of simian enteroviruses, A13, SV19/SV26/SV35, SV43 and SV46 are provisionally classified as members of HEV-A (Oberste et al., 2002). Other than A13, all of these viruses were originally isolated from Asian primates of the genus Macaca, most commonly Macaca mulatta (rhesus macaque) and Macaca fascicularis (cynomolgous macaque) (Heberling & Cheever, 1965
; Hoffert et al., 1958
; Hull et al., 1958
). While complete capsid sequences are not yet available for A13, SV19, SV43 or SV46, the VP1 sequence phylogeny suggests that EV76, EV89, EV90 and EV91 may be most closely related to the simian enterovirus serotypes in HEV-A (Fig. 1a
). In the partial 3D phylogeny, EV76 and EV89-91 form a distinct group within HEV-A, separate from both the human and simian prototype strains of other serotypes, suggesting that these viruses may occupy a unique ecological niche (Fig. 3
). Rhesus macaques are indigenous to Bangladesh and elsewhere in South Asia. Isolation of enteroviruses from wild primates species in South Asia may yield additional clues to the genetic relationships between the human and simian viruses in HEV-A.
EV76, EV89, EV90 and EV91 represent the first new HEV-A types identified since the isolation of EV71 in 1972 (Kennett et al., 1974; Schmidt et al., 1974
). Whereas EV71 is closely related to the previously identified members of HEV-A, particularly to coxsackievirus A16 (Brown & Pallansch, 1995
), the four newest members of the species are clearly distinct from other HEV-A viruses throughout their genomes. It will be interesting to see whether additional HEV-A types will be discovered in the future and whether these new types more closely resemble the conventional HEV-A viruses or the members of the EV76-EV89-EV90-EV91 group.
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ACKNOWLEDGEMENTS |
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Received 27 July 2004;
accepted 19 October 2004.