Genotyping and phylogeographic dynamics of coxsackievirus A16

Figures

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  Fig. 1. The ML phylogenetic trees based on complete genome sequence (A), VP4 ORF sequence (B), or VP1 ORF sequence (C) of 287 CV-A16 strains. The trees were constructed using the best-fit model GTR + F + I + G4 for complete genome sequence, TNe + G4 for VP4 ORF sequence, and SYM + G4 for VP1 ORF sequence in IQ-TREE version 1.6.12 with 1000 bootstraps. The tree was visualized and modified using FigTree v1.4. Strains isolated in China are indicated with red color. The blue star, red star, and black star indicate the previous A type (GenBank ID: JQ746659.1), C type (GenBank ID: JX481738.1) [56], and D type (GenBank ID: MG957117.1) of CV-A16 strains [57], respectively. One D type strain (MG957117.1) is also the virus identified in a CV-A16 outbreak in Shanghai, China, in 2016. All remaining virus strains belong to B type of previous VP1-based classification. The genotypes were defined according to complete genome sequence-based ML tree in (A) and indicated with symbols in different colors. The scale bar represents nucleotide substitutions per site. The information on each virus in (A), (B), and (C) is detailed in Supplementary Figs. S1, S2, and S3, respectively. (D) The global longitudinal…

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  Fig. 2. Pairwise distance and root-to-tip divergence of CV-A16. (A) The pairwise distance among all the full-length genomes indicates the G-a to G-d groups are genetically closer to each other, while the G-e appears as distanced strains. (B) The root-to-tip diversion analysis of all the full-length genomes of CV-A16, showing the departure of G-e strains from the remaining CV-A16 strains. The pairwise distance was calculated using MEGA11 software, while the root-to-tip diversion was determined using the TempEst v1.5.3. The green lines represent the ancestor traces.

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  Fig. 3. Phylogeographic network of 287 full-length CV-A16 sequences worldwide from 1994 to 2019. Phylogenetic networks were inferred using MSN networks implemented with PopArt v1.7. G-a strains dominated the network and formed several major branches. G-c virus strain PM-14660-97 isolated in Malaysia in 1997 (GenBank ID: JQ746665.1) is genetically connected with G-a, G-b, and G-d genotypes through varied mutation steps. Viruses identified in different countries are indicated with different colors.

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  Fig. 4. Genomic recombination events of CV-A16 strains. (A) Diagram showing the full-length genome of CV-A16 and the corresponding regions encoding P1, P2 and P3 domains. The P1 gene encodes four subdomains: VP4, VP2, VP3, and VP1; P2 gene encodes three subdomains: 2A, 2B, and 2C; P3 gene encodes 3A, 3C, and RdRp. (B) Schematic representation of the potential recombination events listed in Supplement Table S2. Recombination event serial numbers and the description of potential recombinants (GenBank ID: virus name/country-province-year of collection) are shown on the left. The orange and brown blocks represent the DNA regions from representative minor or major parent viruses, respectively. The numbers on the top of filled orange blocks indicate the nucleotide positions of breakpoints relative to the corresponding recombinant viruses on the left.

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  Fig. 5. Amino acid variability landscape of full-length CV-A16 polyproteins. The plot represents amino acid variations in aa 1–750 position (A), aa 751–1501 (B), and aa 1501–2193 (C). The ORF nucleotide sequences were used to acquire their consensus amino acid sequence using the Wu-Kabat variability coefficient implemented by PVS. Y-axes represent the Wu-Kabat variability coefficient values, where the estimation limit is 1. Above the limit of 1 represents variations. X-axes represent the amino acid positions. The variability coefficient was calculated as following equation: variability = n∗k/N. The n denotes the number of sequences in the alignment, k denotes the number of different amino acids at a given position, and N is the time available for the most frequently identified amino acid.