2014—2018年辽宁省柯萨奇病毒A6型分离株VP1区基因特征分析

Genetic characteristics of the gene of VP1 region of Coxsackievirus A6 strains in Liaoning province in 2014-2018

目的了解辽宁省手足口病(hand,foot and mouth disease,HFMD)患者中分离到的柯萨奇病毒A组6型(coxsackievirus A group 6 type,CV-A6)VP1区基因特征。方法收集2014—2018年辽宁省14个市送检的HFMD患者非肠道病毒A组71型(enterovirus A group 71 type,EV-A71)和非柯萨奇病毒A组16型(coxsackievirusA16,CV-A16)肠道病毒(enterovirus,EV)的阳性标本,通过细胞培养法分离EV,提取病毒RNA,用RT-PCR法对EV VP1区基因扩增和序列测定。利用Blast对测序结果比对后确定病毒基因型别。对鉴定为CV-A6的分离株利用软件DNAStar和MEGA5.0进行VP1区同源性分析,并构建系统进化树。结果2014—2018年辽宁省共收到非EV-A71和非CV-A16其他EV的阳性标本7379份,用人横纹肌瘤细胞(rhabdomyosarcoma cells,RD)分离出111株CV-A6。通过遗传进化分析显示,CV-A6流行株形成A、B、C、D分支,D分支分为D1~D3亚分支,2014—2018年43株辽宁省CV-A6分离株处在D2和D3亚分支,D2亚分支11株,D3亚分支32株。辽宁CV-A6分离株间的VP1区基因核苷酸同源性和氨基酸同源性分别为91%~100%和95.5%~100%;与处在A分支的原型株Gdula的核苷酸同源性为79%~82%,氨基酸同源性为90.9%~92.4%,亲缘关系较远;与2008年芬兰变异株KM114057共同处在D分支,亲缘关系近,核苷酸同源性为93%~96%,氨基酸同源性为93.9%~98%。结论辽宁省CV-A6分离株有D2和D3分支共同流行传播,其中D3分支是优势流行株。

Objective To understand the genetic characteristics of the VP1 region of coxsackievirus A group 6 type(CV-A6)isolated from patients with hand,foot and mouth disease(HFMD)in Liaoning province.Methods Non-enterovirus A group 71 type(EV-A71)and non-coxsackievirus A group 16 type(CV-A16)and enterovirus(EV)-positive specimens of patients with HFMD from 14 cities in Liaoning province from 2014 to 2018 were collected.EV strains were isolated by cell culture,viral RNA was extracted,amplification and sequencing of the gene of VP1 region of EV were performed.The Blast was used to compare the sequencing result to determine the virus genotype.The isolates identified as CV-A6 were analyzed the homology of VP1 region using DNAStar and MEGA5.0 software,and a phylogenetic tree was constructed.Results In 2014-2018,Liaoning province received 7379 positive samples of other EV(non-EV-A71 and non-CV-A16).Total 111 strains of CV-A6 were isolated on rhabdomyosarcoma cells(RD).According to genetic evolution analysis,CV-A6 epidemic strains formed A,B,C and D branches,and D branch was divided into D1-D3 sub-branches.From 2014 to 2018,43 CV-A6 isolates in Liaoning province were in D2 and D3 sub-branches.Eleven strains belonged to D2 sub-branch,and 32 strains belonged to D3 sub-branch.The homologies of nucleotide and amino acid of the VP1 region of Liaoning CV-A6 isolates are 91%-100%and 95.5%-100%,respectively.The homologies of nucleotide and amino acid of the VP1 region of Liaoning CV-A6 isolates and the prototype strain Gdula in the branch A are 79%-82%and 90.9%-92.4%,the genetic relationship is far.Liaoning CV-A6 isolates are in the branch D with the Finnish mutant strain KM114057 in 2008,the genetic relationship is close,the nucleotide acid homology is 93%-96%,the amino acid homology is 93.9%-98%.Conclusions CV-A6 isolates in Liaoning province belong to D2 and D3 branches,and D3 branch is the dominant epidemic strain.