Molecular Evolutionary Dynamics of Coxsackievirus A6 Causing Hand, Foot, and Mouth Disease From 2021 to 2023 in China: Genomic Epidemiology Study

Abstract

Background: Hand, foot, and mouth disease (HFMD) is a global public health concern, notably within the Asia-Pacific region. Recently, the primary pathogen causing HFMD outbreaks across numerous countries, including China, is coxsackievirus (CV) A6, one of the most prevalent enteroviruses in the world. It is a new variant that has undergone genetic recombination and evolution, which might not only induce modifications in the clinical manifestations of HFMD but also heighten its pathogenicity because of nucleotide mutation accumulation.

Objective: The study assessed the epidemiological characteristics of HFMD in China and characterized the molecular epidemiology of the major pathogen (CV-A6) causing HFMD. We attempted to establish the association between disease progression and viral genetic evolution through a molecular epidemiological study.

Methods: Surveillance data from the Chinese Center for Disease Control and Prevention from 2021 to 2023 were used to analyze the epidemiological seasons and peaks of HFMD in Henan, China, and capture the results of HFMD pathogen typing. We analyzed the evolutionary characteristics of all full-length CV-A6 sequences in the NCBI database and the isolated sequences in Henan. To characterize the molecular evolution of CV-A6, time-scaled tree and historical population dynamics regarding CV-A6 sequences were estimated. Additionally, we analyzed the isolated strains for mutated or missing amino acid sites compared to the prototype CV-A6 strain.

Results: The 2021-2023 epidemic seasons for HFMD in Henan usually lasted from June to August, with peaks around June and July. The monthly case reporting rate during the peak period ranged from 20.7% (4854/23,440) to 35% (12,135/34,706) of the total annual number of cases. Analysis of the pathogen composition of 2850 laboratory-confirmed cases identified 8 enterovirus serotypes, among which CV-A6 accounted for the highest proportion (652/2850, 22.88%). CV-A6 emerged as the major pathogen for HFMD in 2022 (203/732, 27.73%) and 2023 (262/708, 37.01%). We analyzed all CV-A6 full-length sequences in the NCBI database and the evolutionary features of viruses isolated in Henan. In China, the D3 subtype gradually appeared from 2011, and by 2019, all CV-A6 virus strains belonged to the D3 subtype. The VP1 sequences analyzed in Henan showed that its subtypes were consistent with the national subtypes. Furthermore, we analyzed the molecular evolutionary features of CV-A6 using Bayesian phylogeny and found that the most recent common ancestor of CV-A6 D3 dates back to 2006 in China, earlier than the 2011 HFMD outbreak. Moreover, the strains isolated in 2023 had mutations at several amino acid sites compared to the original strain.

Conclusions: The CV-A6 virus may have been introduced and circulating covertly within China prior to the large-scale HFMD outbreak. Our laboratory testing data confirmed the fluctuation and periodic patterns of CV-A6 prevalence. Our study provides valuable insights into understanding the evolutionary dynamics of CV-A6.

Keywords: CV-A6; China; HFMD; coxsackievirus A6; evolution; hand, foot, and mouth disease; molecular epidemiology.

Figure 1.. Pathogen spectrum of HFMD in Henan, China, from 2021 to 2023: (A) the monthly incidence of HFMD and (B) composition ratio of various EVs in Henan, from 2021 to 2023. CV: coxsackievirus; EV: enterovirus; HFMD: hand, foot, and mouth disease.

Figure 2.. Subtyping analysis of CV-A6. (A) Genetic evolutionary characteristics of enteroviruses isolated in Henan, China. Maximum likelihood phylogenetic trees for enteroviruses were constructed with CV-A6 full-length sequences. The red box marked the strains that have been isolated this study. The colors of the branches represent countries, and the colors on the strains represent subtypes. (B) Annual percentage distribution of subgenotypes D1, D2, and D3 associated with HFMD from 2009 to 2023 in mainland China. CV: coxsackievirus; HFMD: hand, foot, and mouth disease.

Figure 3.. The temporal and geographical distribution characteristics of CV-A6 in China. (A) Annual and regional distribution of 514 CV-A6 strains in China. (B) The distribution of CV-A6 strains associated to HFMD in 7 significant regions of China, where both region and sequence quantity are denoted and differentiated by the provided color key. The colors on the map represent different geographical areas, and the colors on the pie charts represent the pathogens of HFMD. CV: coxsackievirus; HFMD: hand, foot, and mouth disease.

Figure 4.. Molecular evolution analysis of CV-A6. (A) Temporal signal analysis of root-to-tip divergence regression versus date (R²=1). (B) The phylogenetic tree was constructed with maximum clade credibility for 313 VP1 sequences of CV-A6 strains. The purple bars at the nodes indicate the 95% HPDs of tMRCAs. Red vertical lines represent the 34 CV-A6 strains isolated in this study. CV: coxsackievirus; HPD: highest posterior density; tMRCA: time of the most recent common ancestor.

Figure 5.. Amino acid mutation sites and genetic differences analysis of the VP1 gene. (A) Analysis of amino acid mutations in the VP1 gene of the isolated strains of CV-A6. (B) Estimates of evolutionary divergence between sequences of isolated CV-A6 strains and the prototype CV-A6 strain (AY421764.1). Values are indicated by color shading. Analyses were conducted using the maximum composite likelihood model. The analysis involved 35 nucleotide sequences. CV: coxsackievirus.