Molecular Evolutionary Dynamics of Coxsackievirus A6Causing Hand, Foot, and Mouth Disease From 2021 to 2023in China: Genomic Epidemiology Study

Background: Hand, foot, and mouth disease (HFMD) is a global public health concern, notably within the Asia-Pacificregion. Recently, the primary pathogen causing HFMD outbreaks across numerous countries, including China, is coxsackievi-rus (CV) A6, one of the most prevalent enteroviruses in the world. It is a new variant that has undergone genetic recombinationand evolution, which might not only induce modifications in the clinical manifestations of HFMD but also heighten itspathogenicity because of nucleotide mutation accumulation. Objective: The study assessed the epidemiological characteristics of HFMD in China and characterized the molecularepidemiology of the major pathogen (CV-A6) causing HFMD. We attempted to establish the association between diseaseprogression 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 usedto analyze the epidemiological seasons and peaks of HFMD in Henan, China, and capture the results of HFMD pathogentyping. We analyzed the evolutionary characteristics of all full-length CV-A6 sequences in the NCBI database and the isolatedsequences in Henan. To characterize the molecular evolution of CV-A6, time-scaled tree and historical population dynamicsregarding CV-A6 sequences were estimated. Additionally, we analyzed the isolated strains for mutated or missing amino acidsites 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 Juneand 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 8enterovirus serotypes, among which CV-A6 accounted for the highest proportion (652/2850, 22.88%). CV-A6 emerged asthe major pathogen for HFMD in 2022 (203/732, 27.73%) and 2023 (262/708, 37.01%). We analyzed all CV-A6 full-lengthsequences in the NCBI database and the evolutionary features of viruses isolated in Henan. In China, the D3 subtype graduallyappeared from 2011, and by 2019, all CV-A6 virus strains belonged to the D3 subtype. The VP1 sequences analyzed in Henanshowed that its subtypes were consistent with the national subtypes. Furthermore, we analyzed the molecular evolutionaryfeatures of CV-A6 using Bayesian phylogeny and found that the most recent common ancestor of CV-A6 D3 dates back to2006 in China, earlier than the 2011 HFMD outbreak. Moreover, the strains isolated in 2023 had mutations at several aminoacid sites compared to the original strain Conclusions: The CV-A6 virus may have been introduced and circulating covertly within China prior to the large-scaleHFMD outbreak. Our laboratory testing data confirmed the fluctuation and periodic patterns of CV-A6 prevalence. Our studyprovides valuable insights into understanding the evolutionary dynamics of CV-A6