Using Statistical Phylogenetics for Investigation of Enterovirus 71 Genotype A Reintroduction into Circulation

Figures

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  Figure 1. (a) Maximum likelihood (ML) phylogenetic tree used for TempEst analysis (panel (a)) inferred under a Kimura 2-parameter model with invariable sites. Stars at tree nodes indicate nodes with bootstrap values greater than 80%. Letters to the right and branch colors indicate enterovirus 71 (EV71) genotypes. TempEst analysis of the temporal population structure in the total EV-A71 alignment with 16 BrCr-like Chinese sequences, midpoint tree root (b), the sole genotype A (16 Chinese sequences and 4 prototype BrCr sequences), best-fitting tree root (c), and the sole genotype C, represented by 55 sequences (d). White circles in panel (b) correspond to genotype A sequences (prototype strain sequence JN874547 and Chinese sequences), and grey circles correspond to the sequences of other genotypes. The correlation coefficient, R2, and substitution rate (regression line slope) are indicated in each plot.

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  Figure 2. (a) Maximum clade credibility (MCC) tree for EV-A71 sequences of different subtypes and 16 BrCr-like Chinese isolates. The scale bar below the tree corresponds to 10 years. Subtypes other than A are collapsed for graphical reasons. The asterisks indicate node posterior probabilities above 0.85. (b) Frequency distribution of branch substitution rate logarithms in the MCC tree shown in panel (a). The solid red line indicates the mean, and the dashed lines indicate standard deviations from the mean. (c) MCC tree for 16 BrCr-like Chinese sequences. The scale bar below the tree corresponds to one year. Abbreviations in Chinese sequences’ names correspond to the cities or provinces of isolation: BJ—Beijing, HB—Hubei province, LN—Luan, WH—Wuhan, YN—Yunnan, NA—unknown.

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  Figure 3. Possible pattern of EV-A71 genotype A release and spread in China. Hypothetical intermediate ancestors of present viruses are indicated in dashed squares. Suggestive source strain is 0B (Table 1). Isolates are labeled with Arabic numbers, hypothetical intermediate viruses with N1–N3. Arrows mark likely transmission events and dashed arrows indicate alternative transmission events. Isolation year and substitutions in the VP1 region are provided. Synonymous nucleotide mutations are given in small letters and marked with asterisks and amino acid substitutions are in capital letters; positions are given for VP1-encoding sequence or the protein, respectively. Key mutations mentioned in the text are shown in color. Polymorphisms that likely originated from the heterogeneity observed among source BrCr variants are underlined. Full information about sequences is provided in Table 1. Provinces where BrCr-like Chinese viruses were isolated are colored in grey. The MCC phylogenetic tree was built using Chinese GtA sequences and BrCr sequence JN874547, which was used to root the tree, but then omitted. Tips are numbered according to the map. Nodes with posterior probabilities above 0.9 are…

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  Figure 4. Mapping of 14 amino acid substitutions found in Chinese BrCr-like isolates. The residues are colored according to the structural region of the capsid: the canyon northern rim (red), canyon floor and its proximity (green), canyon southern rim (cyan), and other regions (orange), based on the EV-A71 structure 4CDQ [36]. Residues that belong to the major epitopes of VP1 [34] are colored in dark blue. (a) Side views of a cartoon representation of the EV-A71 icosahedral asymmetric unit; (b) surface residues mapped to the molecular surface of EV-A71 pentamer formed by five icosahedral asymmetric units (left) and a single icosahedral asymmetric unit in cartoon representation (right) viewed down a five-fold axis. The capsid VP1 protein is colored in black, VP2 colored in grey, VP3 colored in white, and VP4 colored in wheat. Positions of the icosahedral symmetry elements are indicated. The visualization of EV-A71 crystal structure was performed in PyMOL [37].