一起由肠道病毒CoxB4引起的手足口病聚集性疫情的病原学鉴定及其基因型分析

Pathogenic identification and genotype analysis of an hand-foot-mouth disease epidemic caused by coxsackievirus B4

目的:对引起一起手足口病聚集性疫情的未分型肠道病毒进行病原学鉴定,并分析其基因型。方法:对采集的标本用Real-time PCR方法检测肠道病毒核酸,并选取其中通用阳性,EV71和CoxA16阴性的标本用自行设计长距离PCR通用引物进行两步法RT-PCR,将得到的PCR产物进行测序,将所得序列在GenBank中进行同源性搜索以确定是何种血清型肠道病毒,同时选取部分标本进行全序列的测定并构建分子系统树,从而确定引起本次手足口肠道病毒为何种基因型。结果:34份标本经Real-time PCR检测,19份标本肠道病毒核酸通用阳性,EV71阴性和COX16阴性,选取其中的10份测定其VP1区序列,经同源性搜索确定为肠道病毒CoxB4,且10标本的序列同源性均在99%以上。全序列测定结果与其他CoxB4进行系统发生分析显示,本次分离到的CoxB4属于第VIII类基因型。结论:引起本次手足口病聚集性病例的病原为肠道病毒CoxB4,第VIII类基因型。

Objective：To identify the pathogen that caused the outbreak of HFMD epidemic,and to find out the genetic character of the pathogen.Methods： After pan-enterovirus PCR primers positive-detected but EV71 and CoxA16 negative-detected specimens were screened by Real-time PCR kit,ten positive specimens were subject to two-step RT-PCR using our designed long range PCR universal primers.After sequencing of the PCR products,the sequence homology search was done on GenBank to determine which serotype they belong,and further the molecular phylogeny tree was constructed to determine which genotype the enterovirus belong.Results： 19 of the total 34 specimens were pan-enterovirus positive-detected but EV71 and CoxA16 negative-detected by realtime PCR.The 10 positive specimens were subject to VP1 sequencing,and classified to coxsackievirus B4 by homology search.And the phylogeny analysis with other coxsackievirus B4 indicated that the isolated coxsackievirus B4 should be classified to the VIII genotype of Coxsackievirus B4.Conclusion： The pathogen of this epidemic of Hand-Foot-Mouth Disease were determined as the VIII genotype of Coxsackievirus B4.