阜阳市手足口病柯萨奇病毒A10型VP4区基因特征分析

Gene characteristics analysis for CoxA10 VP4 region of hand-foot-mouth disease in Fuyang

目的分析2011-2012年阜阳市手足口病(hand,foot and mouth disease,HFMD)患儿咽拭子标本分离的柯萨奇病毒A10型(coxsackievirus A10,CoxA10)VP4区的基因特征,初步了解该病毒的变异情况。方法对49份HFMD患儿的咽拭子标本进行病毒分离,出现人肠道病毒(human enterovirus,HEV)特征性病毒致细胞病变效应(cytopathic effect,CPE)后提取病毒RNA,用VP4区分型引物进行半巢式反转录-PCR,再通过测序和GenBank Blast比对确定病毒型别,并对其VP4区进行序列进化分析。结果 49份咽拭子标本有16份出现了特征性CPE,病毒分离阳性率32.7%。其中三株经鉴定为CoxA10,阳性率18.8%。它们的VP4区核苷酸序列同源性为95%～99%,与GenBank中参考毒株VP4区核苷酸序列同源性为77%～97%。其中与中国株HQOO5431同源性为96%～97%,与中国株JN639887同源性为96%～97%,与中国株FJ598089同源性为95%～96%,与日本株AB282808同源性为85%～86%,与日本株AB162743同源性为87%～89%,与CoxA10原型株Kowalik(AF081300)同源性为77%～78%。进化分析表明这三株分离株与Kowalik株聚成一簇,均属于CoxA10,与日本各地分离的CoxA10的病毒核苷酸差异较大,但与我国本土分离株自成一簇。结论 2011-2012年阜阳地区手足口病病原体CoxA10 VP4与中国地区分离株在进化上密切相关,其序列未发生明显变异。

Objective To understand the mutation of Coxsackie virus A10（CoxA 10） through the analysis of VP4 of CoxA10 isolated from the throat swab specimens of the patients with hand,foot and mouth disease（HFMD） in Fuyang in 2011-2012.Methods The virus was isolated from the samples.After the appearance of cytopathic effect（CPE）,VP4 genotyping primers was used to conduct half nested RT-PCR,then the virus type and the sequence of its VP4 region was determined.Results 49 specimens were seeded cells,16 specimens appeared CPE.The positive rate was 32.7%.Three was identified as CoxA10.The nucleotide homology was 95%-99%.The homology between them and the reference strain of GenBank was 77%-97%.The homology among them and HQOO5431 was 96%-97%.The homology among them and JN639887 was 96%-97%.The homology among them FJ598089 was 95%-96%.The homology among them and AB282808 was 85%-86%.The homology among them and AB162743 was 87%-89%.The homology of CoxA10 and Kowalik was 77%-78%.Evolution analysis showed that this three and Kowalik got together into a cluster,and they all belonged to CoxA10.They were very different from the virus in Japan.But they became to a cluster with Chinese local separation.Conclusions The CoxA10 strains isolated in Fuyang in 2011-2012 had no significant variation in the VP4 region.