Molecular evolution of the human enteroviruses: correlation of serotype with VP1 sequence and application to picornavirus classification

Abstract

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.

FIG. 1

Consensus phylogenetic tree for representative human picornaviruses and nonhuman enteroviruses constructed by the neighbor-joining method (17) with a Ts/Tv ratio of 8.0. Numbers at nodes represent the percentage of 100 bootstrap pseudoreplicates that contained the cluster distal to the node. Major clusters supported by bootstrap values of at least 67% are enclosed by circles. For clarity, branch lengths are not drawn to scale. PV, poliovirus.

FIG. 2

Consensus phylogenetic trees for human enterovirus clusters A, B, and C, constructed by the neighbor-joining method (17) with a Ts/Tv ratio of 8.0. Branch lengths are proportional to genetic distances calculated by the maximum-likelihood method implemented in Puzzle (64). Numbers at nodes represent the percentage of 100 bootstrap pseudoreplicates that contained the cluster distal to the node. Genetic distance is indicated by the bar at lower left in each panel. (A) CA16-like viruses (cluster A). (B) CB-like viruses (cluster B). (C) Poliovirus-like viruses (cluster C).

FIG. 3

Frequency distribution of pairwise identity scores for comparison of VP1 nucleotide (A) and deduced amino acid (B) sequences.