Evolutionary genetics of human enterovirus 71: origin, population dynamics, natural selection, and seasonal periodicity of the VP1 gene

Abstract

Human enterovirus 71 (EV-71) is one of the major etiologic causes of hand, foot, and mouth disease (HFMD) among young children worldwide, with fatal instances of neurological complications becoming increasingly common. Global VP1 capsid sequences (n = 628) sampled over 4 decades were collected and subjected to comprehensive evolutionary analysis using a suite of phylogenetic and population genetic methods. We estimated that the common ancestor of human EV-71 likely emerged around 1941 (95% confidence interval [CI], 1929 to 1952), subsequently diverging into three genogroups: B, C, and the now extinct genogroup A. Genealogical analysis revealed that diverse lineages of genogroup B and C (subgenogroups B1 to B5 and C1 to C5) have each circulated cryptically in the human population for up to 5 years before causing large HFMD outbreaks, indicating the quiescent persistence of EV-71 in human populations. Estimated phylogenies showed a complex pattern of spatial structure within well-sampled subgenogroups, suggesting endemicity with occasional lineage migration among locations, such that past HFMD epidemics are unlikely to be linked to continuous transmission of a single strain of virus. In addition, rises in genetic diversity are correlated with the onset of epidemics, driven in part by the emergence of novel EV-71 subgenogroups. Using subgenogroup C1 as a model, we observe temporal strain replacement through time, and we investigate the evidence for positive selection at VP1 immunogenic sites. We discuss the consequences of the evolutionary dynamics of EV-71 for vaccine design and compare its phylodynamic behavior with that of influenza virus.

FIG. 1.

Phylogenies of human enterovirus 71 (EV-71) VP1 genes. The maximum-likelihood phylogenetic trees of EV-71 VP1 genes from genogroup B and C are shown. Complete VP1 genes of genogroup B (n = 283) and C (n = 344) with known sampling dates (1972 to 2008) were used. Each genogroup is classified into five subgenogroups, denoted B1 to B5 and C1 to C5, respectively. The trees are midpoint rooted, and significant bootstrap support values (≥80%; 1,000 bootstrap replicates) are indicated by asterisks at major nodes. Scale bars signify a genetic distance of 0.02 nucleotide substitutions per site. For clarity, the year of isolation of each sequence is not shown in this figure but is listed in Fig. S1 and S2 in the supplemental material. Several C3- and C4-like isolates are also shown at the base of the C3 and C4 clusters, respectively.

FIG. 2.

Origin of human enterovirus 71 (EV-71). (A) Phylogenetic relationships of EV-71 genogroups with coxsackievirus A16. Coxsackievirus A16 sequences shown in the cladogram were previously reported by Perera et al. (56). (B) The date of the MRCA of EV-71 was estimated to be 1941.0 (95% CR, 1928.8 to 1952.2) using a relaxed molecular clock and an exponential population growth model (22), as implemented in BEAST (24).

FIG. 3.

The genetic diversity dynamics of EV-71 genogroups B and C. (A) Bayesian skyline plot estimates depicting the past genetic diversity dynamics through time of EV-71 genogroups B and C. The plot for genogroup B shows a sharp rise in relative genetic diversity in the late 1990s. Other increases can be seen in the early 1970s and mid-1980s. The putative EV-71 subgenogroups that are proposed to be implicated in escalating genetic diversity are indicated by arrows. Similarly, the plot for genogroup C shows sharp rises in genetic diversity in the mid-to-late 1980s and late 1990s. (B) The global reporting of EV-71 infections from the 1970s to 2008, compiled from the published literature (see Table S4 in the supplemental material), suggests that at least three independent waves of major outbreaks—one in each decade—have occurred worldwide since 1970. These episodes of outbreaks are indicated by gray shading in all plots. Country names are abbreviated as follows: US, United States; AU, Australia; JP, Japan; BU, Bulgaria; FR, France; HK, Hong Kong; TW, Taiwan; MY, Malaysia; SG, Singapore; CN, China; KR, Republic of Korea; VN, Viet Nam; HU, Hungary; MN, Mongolia; BN, Brunei.

FIG. 4.

Evolutionary dynamics of EV-71 subgenogroup C1. (A) Molecular clock phylogeny of EV-71 subgenogroup C1, longitudinally sampled between 1986 and 2006. The phylogeny branch lengths are in units of time. The trunk lineages (which include both core and secondary trunk lineages) are drawn thicker than the terminal lineages; all tip branches are colored according to their countries of isolation. Clusters of secondary trunk and terminal lineages are annotated by dashed horizontal lines, and the ranges of sampling time for these isolates are shown, below which the duration in years of the secondary trunk lineage is shown in parentheses. The subgenogroup C1 phylogeny shows temporal strain replacement (a ladder-like topology; also seen in the ML phylogeny). (B) An illustration of the temporal amino acid evolution of the VP1 gene for subgenogroup C1. Observed amino acid substitutions are plotted against their estimated times of occurrence, as obtained from the molecular clock phylogeny in panel A using the joint ML method implemented in HyPhy.

FIG. 5.

Adaptive evolution and periodicity of EV-71. (A) Phylogeny of the longitudinally sampled EV-71 subgenogroup C1, together with a comparable phylogeny of the human influenza A virus. Population sequences from 73 individuals of the human influenza A virus (subtype H3N2) hemagglutinin (HA) gene (∼1.7 kb) isolated from six countries across Asia and sampled between 1985 and 2007 were downloaded from the NCBI's Influenza Virus Resource (7). Trees were plotted using PAUP*, version 4.0 beta (78), and scale bars represent 0.02 nucleotide substitutions per site. (B) The annual incidence of EV-71 infections in Japan from 1982 to 2008. EV-71 isolations/detections are reported by public health institutes and compiled by the Infectious Disease Surveillance Center (IDSC) of the National Institute of Infectious Diseases, Tokyo (idsc.nih.go.jp/iasr/index).