Genome Analysis of Coxsackievirus A4 Isolates From Hand, Foot, and Mouth Disease Cases in Shandong, China

Abstract

Coxsackievirus A4 (CVA4) is one of the most prevalent pathogens associated with hand, foot and mouth disease (HFMD), an acute febrile illness in children, and is also associated with acute localized exanthema, myocarditis, hepatitis and pancreatitis. Despite this, limited CVA4 genome sequences are currently available. Herein, complete genome sequences from CVA4 strains (n = 21), isolated from patients with HFMD in Shandong province, China between 2014 and 2016, were determined and phylogenetically characterized. Phylogenetic analysis of the VP1 gene from a larger CVA4 collection (n = 175) showed that CVA4 has evolved into four separable genotypes: A, B, C, and D; and genotype D could be further classified in to two sub-genotypes: D1 and D2. Each of the 21 newly described genomes derived from isolates that segregated with sub-genotype D2. The CVA4 genomes displayed significant intra-genotypic genetic diversity with frequent synonymous substitutions occurring at the third codon positions, particularly within the P2 region. However, VP1 was relatively stable and therefore represents a potential target for molecular diagnostics assays and also for the rational design of vaccine epitopes. The substitution rate of VP1 was estimated to be 5.12 × 10^-3 substitutions/site/year, indicative of ongoing CVA4 evolution. Mutations at amino acid residue 169 in VP1 gene may be responsible for differing virulence of CVA4 strains. Bayesian skyline plot analysis showed that the population size of CVA4 has experienced several dynamic fluctuations since 1948. In summary, we describe the phylogenetic and molecular characterization of 21 complete genomes from CVA4 isolates which greatly enriches the known genomic diversity of CVA4 and underscores the need for further surveillance of CVA4 in China.

Keywords: Coxsackievirus A4; VP1; and mouth disease; foot; genotypes; hand; phylogenetic analysis.

FIGURE 1

The maximum likelihood phylogenetic tree of the CVA4 whole genome sequences (n = 31). The 21 CVA4 isolates described in this study are colored in rose and the nodes of the reference stains downloaded from GenBank are colored in lavender. The three clades (I, II, and III) are colored in blue, purple and light green, and the prototype strain CVA4 High Point (AY421762) is colored in red.

FIGURE 2

Genetic diversity of the CVA4 polyprotein genes (n = 31). (A) Average pairwise genetic diversity of all CVA4 polyprotein genes was calculated using a sliding window of 300 nts with a step size of 50 nts. (B) Positional entropy values and the Sn values at partitions 1, 2, and 3 of each codon were estimated, respectively. ^∗∗∗p-value <0.001 in the Wilcoxon rank-sum test. (C) The Sn values of nucleotides at positions three of the codons were estimated in VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C, and 3D genes, respectively. ^∗∗∗p-value <0.001 in the Wilcoxon rank-sum test between 2B, 2C, 3A, 3C, 3D genes and VP4, VP2, VP3, VP1 genes, respectively.

FIGURE 3

Bayesian phylogenetic analysis and demographic reconstruction of the CVA4 VP1 gene sequences (915 nts). (A) Bayesian phylogeny was conducted by BEAST with a Bayesian skyline tree prior and a relaxed molecular clock model. (B) The x-axis is the time scale (years) and the y-axis is the effective population size in logarithmic Neτ scale. The thick solid line indicates the median estimates and the shaded area indicates the 95% highest posterior density.