2012-2023年882例重症手足口病病例病原谱及CV-A6基因特征分析

Pathogenic Spectrum and CVA6 Gene Characteristics of 882 Severe Hand,Foot,and Mouth Disease from 2012 to 2023

为了解重症手足口病(Hand,foot,and mouth disease,HFMD)病原谱变化情况以及重症HFMD相关柯萨奇病毒A组6型(Coxsackievirus A6, CVA6)的基因特征,本研究收集了2012-2023年各省级HFMD监测网络实验室送检的882份重症HFMD病例病毒分离物,通过扩增病毒的VP1编码区进行分子分型以确定肠道病毒的血清型,并对其中的CVA6进行全长VP1区扩增和核苷酸序列测定分析,进行最大似然法系统进化树和氨基酸置换熵值等生物信息学分析。结果发现,2018年开始,以CVA6和CVA10为主的肠道病毒逐渐取代EV-A71和CV-A16成为导致重症HFMD的主要病原体,在每年检出的病原体中的比例均超过50.0%。除2013年重症HFMD中分离的CVA6属于D2基因亚型外,2013年后重症HFMD中分离的CVA6均属于D3a基因亚型,但2019年以后分离的CVA6序列与以往分离株序列的进化距离明显增大。此外,针对氨基酸位点的置换熵值研究表明,参与构成构象表位的VP1-283易发生突变,该位点从2017年开始由亲水的苏氨酸突变成疏水的丙氨酸,导致其可能分别与VP3-57N、VP3-66R之间形成氢键,这可能会影响到病毒的感染过程。本研究明确了2012-2023年中国重症HFMD病原谱的变化特征,探究了导致重症HFMD的CVA6的进化特点以及重要氨基酸变异特征,为重症HFMD的预防控制策略的制定提供了理论依据。

In order to understand the changes in the pathogenic spectrum of severe hand,foot,and mouth disease(HFMD)and the genetic characterization of coxsackievirus A6(CVA6)associated with severe HFMD,this study collected 882 virus isolates from severe HFMD cases isolated by provincial HFMD surveillance network laboratories from 2012 to 2023.Molecular typing of enterovirus was determined by amplifying the VP1 coding region of the virus,and full-length VP1 region amplification and nucleotide sequencing analysis were performed on CVA6,followed by bioinformatics analysis such as maximum likelihood phylogenetic tree and amino acid substitution entropy.The results showed that since 2018,enteroviruses,mainly CVA6 and CVA10,had gradually replaced EV-A71 and CVA16 as the main pathogens causing severe HFMD,accounting for over 50.0%of detected pathogens each year.Except for CVA6 isolated from severe HFMD in 2013,which belonged to the D2 sub-genotype,all CVA6 isolated from severe HFMD after 2013 belonged to the D3a sub-genotype.However,the evolutionary distance between CVA6 sequences isolated in 2019 and those previous isolates were significantly increased,indicating that the VP1 of CVA66was continuously evolved.In addition,studies on the substitution entropy of amino acid sites showed that VP1-283,which participated in the formation of conformational epitopes,was susceptible to mutation.And this site was mutated from a hydrophilic threonine to a hydrophobic alanine since 2017,leading to the formation of a hydrogen bond between it and VP3-57N as well as VP3-66R,respectively,which may affect the viral infection process.This study clarified the changes in the pathogenic spectrum of severe HFMD in China from 2012 to 2023,exploring the evolutionary characteristics of CVA6 and important amino acid variation characteristics,providing a theoretical basis for the formulation to prevent and control severe HFMD.