Emerging recombination of the C2 sub-genotype of HFMD-associated CV-A4 is persistently and extensively circulating in China

Abstract

Sporadic outbreaks caused by coxsackievirus A4 (CV-A4) have been reported worldwide. To further elucidate the detailed genetic characteristics and evolutionary recombination events of CV-A4, virus samples from nationwide hand, foot and mouth disease (HFMD) surveillance, encompassing 27 out of the 31 provinces in China, were investigated. Comprehensive and systematic phylogenetic analyses were performed by using 29 complete genomes, 142 complete CV-A4 VP1 sequences. Four genotypes (A, B, C and D) and five sub-genotypes (C1-C5) were re-identified based on the complete VP1 sequences. C2 is the predominant sub-genotype of CV-A4 associated with HFMD and has evolved into 3 clusters. Cluster 1 is a major cluster that has been persistently and extensively circulating in China since 2006 and has been associated with all severe cases. All the sequences showed high homology with the CV-A4 prototype in the P1 region, while higher identities with CV-A5, CV-14 and CV-16 in the P2 and P3 regions. Recombination analysis revealed that C2 had two specific genetic recombination patterns with other EV-A prototypes in the 5'-UTR and 3D region compared with C5. These recombination patterns might be associated with the increased transmissibility of C2 viruses, which were obtained due to their persistent and extensive circulation in populations.

Figure 1

A phylogenetic dendrogram (neighbor-joining method) based on the 915 nt sequences of 142 representative CV-A4 isolates collected from 1948–2017. The strains isolated in different years are represented by different colours according to the legend. A solid circle indicates severe cases. The sequences downloaded from GenBank are listed in Supplementary Table S3. The prototype CV-A5 strain (Swartz) served as an out-group reference.

Figure 2

Yearly distribution of the CV-A4 sub-genotypes in China from 1996 to 2016. Different sub-genotypes are coloured according to the legend.

Figure 3

The neighbor-joining trees constructed from CV-A4 strains and EV-A prototypes based on the whole genome (A) and the P1 (B), P2 (C), P3 (D) structural protein coding regions. Hollow circles indicate strains isolated from mild cases in this study, and rhombus indicates strains isolated from severe cases in this study.

Figure 4

Similarity plot and bootscan analysis of representative CV-A4 strains of HFMD circulating in China with the EV-A prototype strains. A sliding window of 500 nucleotides moving in 20 nucleotide increments was used in this analysis. (A) Mild case of C2, 11–60-CQ-CHN-2011. (B) Severe case of C2, 16–5-HuN-CHN-2016. (C) C5, 14–41-JX-CHN-2014. (D) C5, 16–128-JX-CHN-2016.