2021年云南省手足口病病例中柯萨奇病毒A2型的测序定型和基因特征分析

Serotype detection and phylogenetic analysis of coxsackievirus A2 causing hand,foot and mouth disease in Yunnan Province in 2021

目的对2021年云南省昆明市及曲靖市手足口病(hand,foot and mouth disease,HFMD)病例中非肠道病毒A组71型(enterovirus A71,EVA71)和非柯萨奇病毒A组16型(coxsackie-virus A16,CVA16)的其他肠道病毒进行VP4/VP2区及VP1区基因测序检测,并对检测到的CVA2 VP1区基因进行基因进化分析,为CVA2的预防及控制提供实验室证据。方法按《手足口病实验室手册(2018年版)》,对2021年昆明市和曲靖市送到云南省疾病预防控制中心HFMD实验室的标本进行前处理,提取病毒RNA后,先用MD91/OL68-1引物扩增肠道病毒VP4/VP2结合区基因并测序,编辑后的序列在GenBank上搜索确定病毒型别,再用肠道病毒A组引物分两段扩增CVA2 VP1区基因完整序列并测序,通过肠道病毒在线定型工具(Enterovirus Genotyping tool Version 0.1)进行病毒型别确认。按文献下载CVA2 VP1区基因型/亚型代表株序列,Mega5.2软件构建基因进化树,分析病毒基因特征。结果共检测749份非EVA71和非CVA16的肠道病毒标本,两地皆以A组肠道病毒为主,B组肠道病毒有少量报告,CVA16居病原谱第一位。其中CVA25份,检出率为0.67%(5/749),为HFMD的稀有病原。VP4/VP2和VP1两个基因片段的测序检测、定型结果一致。基因特征分析表明,昆明市3株为基因型A,曲靖市2株为基因型D。结论2021年云南省HFMD监测网络中昆明市和曲靖市CVA2的检出率为0.67%,CVA2在这两个市是HFMD的稀有病原。基因进化分析表明,两个基因型分别在昆明市和曲靖市传播但尚未引起流行。昆明市CVA2基因型A为国内首次报道。加强云南省CVA2的实验室监测特别是分子流行病学监测,对追踪和分析病毒传染来源具有重要意义。

Objective To analyze the non-enterovirus A71(non-EVA71)and non-coxsackievirus A16(non-CVA16)enteroviruses causing hand,foot and mouth disease(HFMD)in Kunming and Qujing of Yunnan Province in 2021 by sequencing the VP4/VP2 and VP1 genes and to analyze the phylogenetic characteristics of the VP1 gene of CVA2,aiming to provide reference for the prevention and control of CVA2.Methods The samples were made and extracted strictly according to the Laboratory Manual for Hand,Foot and Mouth Disease(China Center for Disease Control and Prevention,2018 Edition).VP4/VP2 junction regions were firstly amplified and sequenced by MD91/OL68-1 primers.These sequences were firstly edited and then"blasted"on the GenBank to determine the virus serotype.To analyze the phylogenetic characteristics of CVA2,the entire VP1 gene sequences were amplified in two segments using enterovirus species A primers.Virus serotype was again confirmed online by"Enterovirus Genotyping Tool Version 0.1".The sequences of the reference virus genotypes/sub-genotypes were downloaded according to the reference.The phylogenetic trees were constructed by Mega5.2 software and the genetic characteristics were analyzed.Results A total of 749 non-EVA71 and non-CVA16 enteroviruses were detected in the two areas in 2021.Group A enteroviruses were the main pathogens,with CVA16 as the predominant virus,and a small number of group B enteroviruses were reported.Only five strains of CVA2 were detected with a detection rate of 0.67%(5/749),indicating that CVA2 was a rare pathogen for HFMD in the two areas.The sequencing and serotyping results were consistent using the two genomic regions of VP4/VP2 junction region and VP1 region.Phylogenetic analysis showed that three Kunming strains belonged to genotype A,while two Qujing strains belonged to genotype D.Conclusions The detection rate of CVA2 in Kunming and Qujing was 0.67%in 2021.CVA2 was a rare pathogen for HFMD in the two regions.Phylogenetic analysis showed genotypes A and D spread in Kunming and Qujing,respectively,but had not caused epidemics.To our knowledge,this was the first report of genotype A of CVA2 in China.Strengthening the laboratory surveillance especially molecular epidemiological surveillance is valuable for the monitor and analysis of transmission source for CVA2.