Molecular evolution of the major capsid protein VP1 of enterovirus 70

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.