泰安市2017-2018年柯萨奇病毒A4型分离株全基因组特征

摘要

Objective To analyze the genetic characteristics of Coxsackievirus A4 isolated from Taian,2017-2018.Methods Sixty throat swab samples of the children who visited Taian Maternal and Child Health Hospital during the year 2017-2018 and were diagnosed as hand,foot and mouth disease,were collected and aseptically inoculated.Fluorescent quantitative PCR analysis was performed using the universal primer for enteroviruses.The high-throughput sequencing was applied to the enterovirus-positive samples,and the full-length genome sequences of the viruses were obtained.Phylogenetic analysis was performed using Mega5.05 and RaxML respectively,and sequence homology and amino acid mutation sites were also analyzed using Mega5.05.Results Four whole genome sequences of CV-A4 isolated from infants aged 17-19 months old were obtained.Phylogenetic analysis of the full length CV-A4 genomes showed that apart from MG550920/AA/Henan/2016,the remaining CV-A4 strains from China (97.2％),including the four strains from Taian,fell within Group 3.The VP1 genes could be classified into four genotypes and 98.5％ of the Chinese strains belonged to genotype D,and the four strains from Taian belonged to D2.It was notable that the Taian isolate A1/Taian is closely related to two strains C179 and C062 from Australia both in the complete genome and the VP1 gene,as well as one strain YT184R isolated from Yantai in 2016 by us.Compared with the prototype CV-A4 strain High Point,18 amino acid mutations were found in the P1 region.Conclusions Both phy]ogenetic trees estimated using the complete genome and the VP1 gene sequences revealed that the four CV-A4 isolates from Taian fell within the same clade with the majority of CV-A4 strains circulating in China.Compared with the prototype CV-A4 strain,several amino acid variations have occurred in the P1 region,which warrants further investigation.%目的 分析2017-2018年泰安市柯萨奇病毒A4型(Coxsackievirus A4,CV-A4)分离株的基因组特征.方法 收集2017-2018年到泰安市妇幼保健院就诊且临床表征为手足口病(hand,foot and mouth disease,HFMD)特征的患儿咽拭子样本共60份,利用肠道病毒(enterovirus,EV)通用引物,经荧光定量PCR进行鉴定.核酸阳性样本进行高通量测序.通过Mega5.05、RaxML软件进行全基因组序列及VP1基因系统发育分析,并进行同源性及氨基酸突变位点分析.结果 4条CV-A4全基因组序列,标本来自17～19月龄幼儿.系统进化分析表明,除了MG550920/AA/Henan/2016以外,包括4株泰安株以内的中国CV-A4毒株(97.2％)均位于Group 3分支.根据VP1基因可划分为4个基因型,98.5％的国内毒株均位于基因型D分支,4株泰安株位于基因型D2.值得注意的是,分离株A 1/Taian同澳大利亚株(C179、C062)的全长序列及VP1基因进化关系均较近,且均与本实验室2016年烟台分离株YT 184R高度同源.与CV-A4原型株High Point相比,4个泰安株在结构蛋白编码区即P1区共有18个位点发生氨基酸突变.结论 4株泰安CV-A4分离株同国内多数CV-A4株的全基因组及VP1基因均位于同一进化分支,为近期国内流行株,与原型株相比其P1区发生了多个氨基酸变异,值得进一步研究.