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Previous Article | Next Article 
Journal of Virology, March 1999, p. 1941-1948, Vol. 73, No. 3
Respiratory and Enteric Viruses Branch,
Division of Viral and Rickettsial Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia 30333
Received 3 September 1998/Accepted 30 November 1998
Sixty-six human enterovirus serotypes have been identified by serum
neutralization, but the molecular determinants of the serotypes are
unknown. Since the picornavirus VP1 protein contains a number of
neutralization domains, we hypothesized that the VP1 sequence should
correspond with neutralization (serotype) and, hence, with phylogenetic
lineage. To test this hypothesis and to analyze the phylogenetic
relationships among the human enteroviruses, we determined the complete
VP1 sequences of the prototype strains of 47 human enterovirus
serotypes and 10 antigenic variants. Our sequences, together with those
available from GenBank, comprise a database of complete VP1 sequences
for all 66 human enterovirus serotypes plus additional strains of seven
serotypes. Phylogenetic trees constructed from complete VP1 sequences
produced the same four major clusters as published trees based on
partial VP2 sequences; in contrast to the VP2 trees, however, in the
VP1 trees strains of the same serotype were always monophyletic. In
pairwise comparisons of complete VP1 sequences, enteroviruses of the
same serotype were clearly distinguished from those of heterologous
serotypes, and the limits of intraserotypic divergence appeared to be
about 25% nucleotide sequence difference or 12% amino acid sequence difference. Pairwise comparisons suggested that coxsackie A11 and A15
viruses should be classified as strains of the same serotype, as should
coxsackie A13 and A18 viruses. Pairwise identity scores also
distinguished between enteroviruses of different clusters and
enteroviruses from picornaviruses of different genera. The data suggest
that VP1 sequence comparisons may be valuable in enterovirus typing and
in picornavirus taxonomy by assisting in the genus assignment of
unclassified picornaviruses.
Human enteroviruses (family
Picornaviridae) infect millions of people worldwide each
year, resulting in a wide range of clinical outcomes ranging from
inapparent infection to mild respiratory illness (common cold),
hand-foot-and-mouth disease, acute hemorrhagic conjunctivitis, aseptic
meningitis, myocarditis, severe neonatal sepsis-like disease, and acute
flaccid paralysis (reviewed in references 43 and
45). In the United States, enteroviruses are
responsible for 30,000 to 50,000 meningitis hospitalizations per year
as a result of 30 million to 50 million infections. Serologic studies
have distinguished 66 human enterovirus serotypes on the basis of an
antibody neutralization test (43), and additional antigenic
variants have been defined within several of the serotypes on the basis
of reduced or nonreciprocal cross-neutralization between prototype and
variant strains (6, 8, 68, 71, 72). On the basis of their
pathogenesis in humans and experimental animals, the enteroviruses were
originally classified into four groups, polioviruses, coxsackie A
viruses (CA), coxsackie B viruses (CB), and echoviruses, but it was
quickly realized that there were significant overlaps in the biological
properties of viruses in the different groups (8). The more
recently isolated enteroviruses have been named with a system of
consecutive numbers: EV68, EV69, EV70, and EV71 (42).
A comparison of nucleotide and deduced amino acid sequences at the 5'
end of VP2 has identified four major phylogenetic groups within the
Enterovirus genus: CA16-like viruses (cluster A), a CB-like
group containing all CB and echoviruses as well as CA9 and EV69
(cluster B), poliovirus-like viruses (cluster C), and EV68 and EV70
(cluster D) (23, 24, 49, 53, 54, 73). However, pairwise
alignments and phylogenetic analyses within these groups demonstrated
that the VP2 sequence does not fully correlate with serotype, as
viruses known to belong to the same serotype often failed to cluster
together (2, 49). (E22 and E23 are genetically distinct from
enteroviruses [24], and their reclassification into a
separate genus has been proposed [45]).
VP1 is the most external and immunodominant of the picornavirus capsid
proteins (58). A number of major neutralization sites reside
in the VP1 proteins of many picornaviruses (reviewed in references
40 and 44), but the specific
epitopes responsible for serotype specificity and intratypic variation
have not been identified. Similarly, the genetic correlates of serotype
identity remain unknown. If the important serotype-specific
neutralization sites reside in VP1, then the VP1 sequence or some
portion thereof would be predicted to correlate with serotype. Studies
on the three serotypes of poliovirus have shown that a partial VP1
sequence correlates well with serotype (32). In addition,
genetic lineages based on the VP1 sequence can be used to define
poliovirus reservoirs and chains of transmission (reviewed in reference
30). To test whether the VP1 sequence might be
applied to the classification of nonpolio enteroviruses and to the
analysis of the phylogenetic relationships among the human
enteroviruses, we determined the complete VP1 nucleotide sequences for
47 human enterovirus prototypes and 10 well-characterized antigenic
variants. These data, together with previously available sequences,
comprise a database of complete VP1 sequences for all known human
enterovirus serotypes and 12 natural antigenic variants. This database
will be useful for molecular epidemiologic studies of enteroviral
disease outbreaks, to obtain a better understanding of the genetic
correlates of serotype, and for the development of enteroviral
molecular diagnostic reagents.
Viruses, RNA extraction, and reverse transcription-PCR.
The
viruses used for sequence analysis and phylogenetic reconstruction are
listed in Table 1. RNA
isolation and reverse transcription-PCR were carried out as described
previously (48). Briefly, viral RNA was extracted from
infected cell culture supernatant or 10% infected mouse brain
homogenate with Trizol LS (Life Technologies, Inc., Gaithersburg, Md.),
and cDNA was synthesized by use of a random hexamer primer and a
SuperScript preamplification kit (Life Technologies, Inc.). From each
viral cDNA, an amplicon of approximately 900 to 950 bp, encompassing
the 3' end of VP3, all of VP1, and the 5' end of 2A, was amplified by
PCR with primers for VP3 and 2A (Table
2). For some viruses, VP1 was amplified
as two overlapping fragments with internal VP1 primers as well as the
VP3 and 2A primers. The PCR products were gel isolated and purified for
sequencing with a QIAquick gel extraction kit (Qiagen, Inc., Santa
Clarita, Calif.) and sequenced on an automated DNA sequencer with
fluorescent dideoxy chain terminators (PE-Applied Biosystems, Foster
City, Calif.). Complete VP1 PCR products of viruses for which VP1
primers were not available were cloned into pGEM-T (Promega Corp.,
Madison, Wis.), and nested-deletion subclones were constructed with an Erase-a-Base kit (Promega). For each virus, at least two independent clones were sequenced by automated methods as described above.
0022-538X/99/$00.00+0
Molecular Evolution of the Human Enteroviruses:
Correlation of Serotype with VP1 Sequence and Application to
Picornavirus Classification
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
TABLE 1.
Enterovirus VP1 sequences used in sequence comparisons
and phylogenetic analyses
TABLE 2.
Primers used for PCR amplification of the VP1 region
of enteroviruses
Sequence analysis. Pairwise nucleotide and amino acid sequence identities were calculated by alignment of all possible sequence pairs with the program Gap (Wisconsin Sequence Analysis Package, version 9.1; Genetics Computer Group, Inc., Madison, Wis.). For phylogenetic reconstructions, groups of nucleotide sequences were aligned with Pileup (Genetics Computer Group). The alignment was manually adjusted to account for codon boundaries, optimal alignment among closely related sequences, and optimal alignment of highly conserved amino acid motifs. Phylogenetic relationships were inferred with the programs Neighbor and DNApars (PHYLIP version 3.57 [17]) and Puzzle (version 4.0 [64]). The maximum-likelihood method of Kishino and Hasegawa (33), with a transition/transversion (Ts/Tv) ratio of 8.0, was used to construct a distance matrix for neighbor-joining analysis. The statistical significance of phylogenies constructed with Neighbor and DNApars was estimated by bootstrap analysis with 100 pseudoreplicate data sets. Puzzle was executed by use of the distance method of Kishino and Hasegawa (33), with a Ts/Tv ratio of 8.0, and the reliability of phylogenetic reconstructions was estimated by use of 1,000 puzzling steps. Branch lengths of the neighbor-joining trees were calculated by the maximum-likelihood method with Puzzle. The robustness of subtrees within cluster B (CB-like group) was confirmed by construction of a Puzzle tree containing each of the subtree taxa, as well as their nearest sibling taxa, for a total of 14 sequences. The sibling taxa were those with the highest amino acid identity scores compared with the taxa in the subtree of interest. For subtree analysis, Puzzle was executed with Ts/Tv ratios of 1.0, 6.0, and 10.0, and well-supported Puzzle trees were taken as evidence in support of the subtree in the original phylogram.
Nucleotide sequence accession numbers. The sequences reported here were deposited in the GenBank sequence database under accession no. AF081293 to AF081349.
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Complete VP1 nucleotide sequences were determined for 57 human enterovirus strains for which VP1 sequences were not previously available (Table 1). Forty-seven of the strains were prototype strains for recognized human enterovirus serotypes (43). The other 10 sequenced strains were well-characterized antigenic variants which, while antigenically distinct from their respective prototype strains, were similar enough to the prototype strains to have been considered of the same serotype (8, 43). Combined with the 21 previously available complete enterovirus VP1 sequences, the 57 sequences reported here comprise the first collection of complete gene sequences representing each of the 66 human enterovirus serotypes.
The boundaries of the newly sequenced VP1 genes were predicted by comparison of the nucleotide and deduced amino acid sequences with those of previously characterized enteroviruses. Human enterovirus VP1 sequences varied in length from 834 to 951 nucleotides (278 to 317 amino acids). The CB had the shortest predicted VP1 amino acid sequences (278 to 298 amino acids), while EV68 and EV70 had the longest ones (312 and 317 amino acids). The newly determined enterovirus sequences were compared with previously available human enterovirus VP1 sequences and with the sequences of other closely related picornaviruses, including E22 and E23, provisionally reclassified as the only members of the genus Parechovirus (45); porcine enterovirus 9; bovine enterovirus types 1 and 2; human rhinovirus types 2 and 14; and human hepatitis A virus.
To assess the broad relationships among the enteroviruses and other human picornaviruses, a phylogenetic tree was constructed with representatives of each of the four human enterovirus clusters (A: CA2, CA12, and CA16; B: CA9, CB1, E26, and EV69; C: PV1, CA19, and CA24; D: EV68 and EV70), the nonhuman enteroviruses (BEV1, BEV2a, BEV2b, and PEV9), and other human picornaviruses (E22, E23, HAV, HRV2, and HRV14). As expected, the human enteroviruses clustered into four major groups (Fig. 1), consistent with published enterovirus phylogenies (23, 49, 53, 54, 73). Picornaviruses from the genera Parechovirus and Hepatovirus were distinct from the enterovirus clusters. The human rhinoviruses HRV2 and HRV14 clustered among the human enteroviruses, consistent with previous phylogenies, but the precise position of HRV2 was poorly supported (bootstrap value, 37%). The nonhuman enteroviruses, PEV9, BEV1, BEV2a, and BEV2b, formed a monophyletic group distinct from but related to cluster A. The human enterovirus clusters were very strongly supported, with bootstrap values of 100%, and the relationship between clusters B and C was also well supported (80%). The relationship of the nonhuman enteroviruses to cluster A was supported by a bootstrap value of 62%. In some cases, PEV9 fell outside this group, resulting in the low bootstrap value.
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To determine the phylogenetic relationships among individual prototype
viruses and, where available, prototypes and their antigenic variants,
intracluster phylogenetic trees were constructed with several phylogeny
reconstruction algorithms (neighbor-joining, maximum parsimony, and
maximum likelihood) and included one sequence from each of the
heterologous human enterovirus clusters as an outgroup. Within each of
the four major clusters, trees constructed by different methods were
congruent in overall structure and in general clustering patterns but
often differed slightly in the order of one or more distal branches or
in the bootstrap support for specific nodes. Viruses in cluster A
segregated into three distinct subgroups
(i) CA7, CA14, CA16, and
EV71; (ii) CA3, CA4, CA6, CA8, and CA10; and (iii) CA5 and CA12with
59 to 99% bootstrap support (Fig. 2A).
CA2 appeared to be distinct from the other viruses in cluster A. Cluster C viruses segregated into four subgroups(i) CA1, CA19, and
CA22; (ii) CA21, CA24, CA24v, and E34; (iii) CA11 and CA15; and (iv)
CA13, CA17, CA18, CA20, PV1, PV2, and PV3with bootstrap support of 67 to 100% (Fig. 2C). Within cluster B, some of the viruses clustered
into subgroups, but few of the subgroups were well supported by
bootstrap analysis (Fig. 2B). Stable subgroups included (i) E3 and E12;
(ii) E11, E11', and E19; (iii) E2 and E15; (iv) E13 and EV69; (v) the
six CB serotypes; (vi) E1, E8, E4-Pesacek, E4-DuToit, and
E4-Shropshire; (vii) E6-D'Amori, E6-Charles, E6'-Cox, and E6"-Burgess;
and (viii) E21, E25, E29, E30-Bastianni, E30-Frater, E30-Giles, and
E30-PR-17. The other viruses could not be reliably subgrouped, as the
bootstrap values were extremely low. For every cluster, all serotypes
which were represented by more than one isolate were monophyletic. In
most cases, bootstrap support was strong (60 to 98%), but the value
for CA24 strains was only 43%.
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Sequence relationships within a serotype, within a cluster, between
clusters, and between human enteroviruses and other picornaviruses were
analyzed by comparison of the nucleotide and deduced amino acid
sequences of all possible sequence pairs. The relationships were
visualized by plotting the frequency of pairwise identity scores versus
percent identity, rounded down to the nearest integer, as a histogram
(Fig. 3). For both the nucleotide (Fig.
3A) and amino acid (Fig. 3B) pairwise identity distributions, the
scores fell into four categories. The highest scores (nucleotide
identity, 
75%; amino acid identity 88%) depicted relationships
among viruses of the same serotype (e.g., the four E30 strains) or
among prototype viruses that have been proposed to be homologous based
on antigenic relatedness (e.g., CA13 and CA18). Nucleotide identity
scores for pairwise comparisons within a major cluster ranged from 48.9 to 73.2% and defined a peak that was clearly delineated from that of
the homologous pairs and from the peak of scores comparing viruses of
different phylogenetic clusters (Fig. 3A). Cluster A scores ranged from
58.5 to 73.2%, while cluster C scores ranged from 55.9 to 70.6%.
Viruses in cluster B appeared to be somewhat more heterogeneous, with
scores ranging from 48.9 to 71.8%. Scores for the heterologous
comparison peak ranged from 42.1 to 64.5% nucleotide identity. The
final peak, containing the lowest scores, represented comparisons of
viruses of different genera within the family
Picornaviridae. In the amino acid identity distribution (Fig. 3B), the heterologous cluster peak appeared to be composed of two
overlapping peaks. The peak with higher scores represented comparisons
of viruses from phylogenetically related clusters (e.g., clusters B and
C), whereas the peak with lower scores represented comparisons of
viruses from more distant clusters (e.g., clusters A and B).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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VP1 is the major surface-accessible protein in the mature
picornavirus virion; it is arrayed around the fivefold axis of symmetry of the icosahedral virion (1, 21, 39, 58). VP2 and VP3 comprise the remainder of the virion surface. Each of the capsid proteins is composed of conserved elements that form the 
-barrel structural elements of the capsid, with variable loops between the
-barrel structures (reviewed in reference 44).
Many of the loops are exposed on the virion surface, and studies of
monoclonal antibody-resistant mutants have shown that a number of the
loops contribute to specific antigenic neutralization sites. VP1
contributes to all three of the major neutralization sites that have
been identified on the poliovirus surface, whereas VP2 and VP3
contribute to two and one of the sites, respectively. For example, the
B-C loop is one of five VP1 loops forming VP1 antigenic site 1. Replacement of the VP1 B-C loop of CB3 by that of CB4 through
site-directed mutagenesis produced a viable virus with a mixed
neutralization phenotype, demonstrating the presence of a
serotype-specific antigenic neutralization site in the B-C loop
(57). Our sequencing studies confirmed the presence of
sequence domains that are conserved among all members of the
Enterovirus genus, as well as intervening domains that vary
in sequence between strains of different serotypes and in some cases
within a serotype. Due to the complexity of the three-dimensional
structure of the enterovirus capsid and the fact that most of the
neutralization sites are discontinuous, it is not possible to correlate
specific VP1 residues with antigenic sites responsible for serotype
specificity. Sequence comparisons and phylogenetic reconstructions
suggest that VP1 contains serotype-specific information that can be
used for virus identification and evolutionary studies. For the
polioviruses, serotype-specific sequences in VP1 have been exploited to
produce type-specific molecular diagnostic reagents (see below).
Pairwise sequence identity has been applied to the taxonomy of plant viruses in the family Potyviridae, with clear distinction of viruses of the same strain, viruses of different strains within a genus, and viruses of different genera (67). Our data suggest that a similar system based on the VP1 sequence can be used to classify viruses within the family Picornaviridae (Fig. 3). Enteroviruses of the same serotype were clearly distinguished from those of heterologous serotypes, and the limits of intraserotypic divergence appeared to be about 25% nucleotide sequence difference or 12% amino acid sequence difference. Likewise, strains of a homologous serotype clustered together in phylogenetic analyses (Fig. 2). Sequencing of additional strains within several different serotypes will provide more extensive data to determine whether this distinction is valid and will provide a more accurate measure of the serotype boundaries. Since enterovirus serotypic differentiation is based on neutralization and the VP1 sequence correlates with neutralization type, it is logical to assume that molecular diagnostics targeted to the VP1 coding region should give typing results that also correlate with the serotype determined by neutralization with type-specific antisera. The molecular distinction between serotypes has obvious applications to the typing of enterovirus isolates in the clinical laboratory. Molecular assays directed to specific sequences in VP1 have already been applied to the serotyping, genotyping, and group identification of polioviruses (12, 13, 30-32, 69, 70). Pairwise identity scores have also placed viruses of the same cluster in a single frequency peak, demonstrating that gross intercluster sequence differences exist, whereas differences within a cluster occur on a smaller scale. The molecular distinction between clusters suggests that it may be useful to extend the taxonomic classification of enteroviruses to include each of the clusters as a subgenus. Similarly, VP1 sequence comparisons may prove valuable in the assignment of unclassified picornaviruses to one of the existing genera or point out the need for the introduction of new genera.
The CB were originally distinguished from the CA and polioviruses on the basis of differences in pathogenesis when inoculated intracerebrally into newborn mice. The echoviruses were, by definition, apathogenic in newborn mice; however, there are examples of echovirus isolates that are mouse virulent. In phylogenetic trees based on the partial sequence of VP2 (or VP4-VP2), the CB failed to cluster together, being interspersed among the echoviruses in a large CB-echovirus group (23, 49). In addition, analysis of 5' nontranslated region sequence of seven CB5 clinical isolates showed that there is little or no genetic linkage between the 5' nontranslated region sequences and serotype, probably due to a high frequency of recombination, whereas VP1-VP2A junction sequences are monophyletic and are correlated with serotype (35). In the present work, VP1 trees contained a monophyletic group consisting only of CB, suggesting that biological properties exclusive to the CB may be partially or completely encoded within the VP1 gene. Although bootstrap support for this subgroup was relatively low in the overall cluster B tree (Fig. 2B), additional tree reconstruction with only sequences most closely related to the CB supported the existence of a distinct CB subgroup within cluster B (data not shown). The nature of the CB-specific determinants remains unknown but could include receptor specificity and, hence, host range, cell or tissue tropism, and pathogenesis, as structural studies have shown that VP1 forms part of the picornavirus receptor-binding pocket (58).
CA1, CA19, and CA22 are the only enterovirus serotypes that have never been successfully adapted to growth in cell cultures, instead requiring passage by intracranial inoculation of suckling mice (18, 29, 43). In parsimony and neighbor-joining analyses with partial VP2 sequences, these three viruses formed a monophyletic cluster within the poliovirus-like group, but with low bootstrap support (49). The maximum-likelihood method produced a VP2 tree with a star topology within the poliovirus-like cluster and failed to show specific clustering of CA1, CA19, and CA22. When complete VP1 sequences were used, the same algorithms consistently produced trees in which CA1, CA19, and CA22 formed a well-resolved monophyletic cluster within the poliovirus-like group, with bootstrap support of 77 to 100%, suggesting that VP1 may play a role in the common host range and/or cell tropism of these three viruses.
E8 (strain Bryson) was isolated in 1953 and initially classified as a separate enterovirus serotype (8, 56). Later serologic studies demonstrated the antigenic relatedness between E8 and E1 (9, 20), and E8 has been designated a strain of the E1 serotype (45). Our genetic comparison of E1 and E8 agrees with the previous antigenic studies, with E1 and E8 being 76.0% identical in nucleotide sequence and 93.2% identical in amino acid sequence, and supports the reclassification of E8 as a variant of E1, based on the homologous serotype boundary described above. The VP1 nucleotide sequences of CA11 (Belgium-1) and CA15 (G-9) were 80.1% identical to one another (96.7% amino acid identity). Partial VP2 sequences had shown that the two viruses were 100% identical in the 50 amino acids at the amino terminus of VP2 (49). Similarly, the complete VP1 nucleotide sequences of CA13 (Flores) and CA18 (G-13) were 77.2% identical to one another (95.1% amino acid identity). Antigenic cross-reactivity has been noted between CA11 and CA15 and between CA13 and CA18 (9). Taken together, the serologic and genetic relationships suggest that CA15 may be a strain of CA11 and that CA18 may be a strain of CA13. In contrast, antigenic cross-reactivity has been reported for CA3 and CA8 (10), but their VP1 amino acid sequences were only 83.3% identical, suggesting that they are genuinely distinct serotypes that share a common epitope(s).
We have demonstrated the general utility of the enterovirus VP1 sequence database by its application to basic problems in enterovirus classification, phylogeny, and evolution. Based on the success of poliovirus molecular diagnostics targeted to VP1 (12, 13, 31, 32, 69, 70) and the shortcomings of current molecular methods for identifying nonpoliovirus enteroviruses (2), we conclude that future enterovirus molecular diagnostic development efforts should be targeted to the genomic region encoding VP1. Hybridization, PCR, and sequencing assays targeted to other regions of the genome may still be useful in identifying and characterizing intertypic enterovirus recombinants. The existence of a complete VP1 sequence database that includes representatives of all human enterovirus serotypes will facilitate the development of generic and serotype-specific molecular reagents for the rapid diagnosis of enterovirus infections, for the detailed molecular epidemiologic study of enterovirus disease outbreaks, for the characterization of newly discovered enteroviruses, and for the study of enterovirus evolution within and among the 66 human enterovirus serotypes.
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FOOTNOTES |
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* Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Rd. NE, Mailstop G-17, Atlanta, GA 30333. Phone: (404) 639-2751. Fax: (404) 639-4011. E-mail: mbo2{at}cdc.gov.
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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