北京市2008年肠道病毒71型VP_1编码区基因特征分析

Genetic Analysis of VP_1 Sequences of Enterovirus 71 Isolated from Patients of Hand,Foot and Mouth Disease in Beijing,2008

目的了解北京市2008年手足口病(Hand,Foot and Mouth Disease,HFMD)流行期间,人肠道病毒71型(Human Enterovirus71,HEV71)分离株的VP1编码区基因特征。方法采集发病1～3d的咽拭子标本33份,采用人横纹肌肉瘤(Human Rhabdomyosarcoma,RD)细胞进行EV分离,用特异引物通过逆转录-聚合酶链反应扩增进行毒株鉴定。对分离到的HEV71进行VP1编码区全长扩增,并对扩增产物采用双脱氧链终止法进行序列测定,Bioedit7.0.5和Mega3.1软件进行序列比对和系统进化树分析。结果从33份咽拭子标本中共分离到16株病毒,经鉴定:HEV7114株,柯萨奇病毒(Coxsackie Virus,CV)A组16型(CVA16)2株,其中1例为HEV71和CVA16混合感染。对10株HEV71进行了VP1编码区全序列测定,10株病毒的核苷酸和氨基酸序列同源性分别为95.5%～100%和98.9%～100%;与安徽省阜阳市2008年流行株的核苷酸和氨基酸序列同源性分别为95.4%～99.1%;与C4基因型代表株的核苷酸序列同源性>92%。基于HEV71的VP1编码区核苷酸序列构建亲缘性关系树,北京市2008年流行株与C4亚型代表株聚为一簇,属于C4基因亚型中的C4a分支;且10株病毒处于四个相对独立的进化分支。结论北京市2008年从HFMD患儿分离的HEV71均为C4亚型,且属于2004年以来我国的优势分支——C4a。亲缘性分析提示,此次流行中至少存在4个HEV71传播链。由于近年来HEV71在中国持续大规模流行,迫切需要对HEV71进行连续的分子流行病学监测,及时阐明现阶段流行毒株的基因特征,为疾病预防控制提供病毒学依据。

Objective To get genetic information of VP1 coding region of HEV71 in Beijing in 2008. Methods Enteroviruses were isolated from samples of throat swabs collected from 33 HFMD patients within 3 days after onset by rhabdomyosarcoma（RD） cells and identified by RT-PCR method with specific primers to human enteroviruses, then VP1 coding region was amplified and sequenced by Sanger dideoxy. Bioedit 7.0.5 and MEGA3.1 were used for the nucleotide sequence alignment and phylogenetic analysis. Results 16 virus strains were isolated from 33 samples, of which 14 strains were identified as HEV71 and 2 were CVA16, and 1 was co-infected with CVA16 and HEV71. Sequence analysis of VP1 nucleotide sequences of 10 HEV71 isolates showed that homologous analysis of nucleotide identity amino acid 95.5%-100% and 98.9%-100% respectively; representative strains of Fuyang in 2008, nucleotide identity was 95.4%-99.1%; with representative strain of C4 nucleotide identity was over 92%. Phylogenetic analysis of HEV71 strains for the nucleotide sequence of VP1 coding region clarified that the HEV71 isolates in Beijing belonged to C4a cluster of C4 sub-genotype and 10 strains formed four relatively separated clusters. Conclusions The HEV71 viruses isolated from children of HFMD in Beijing belonged to C4 sub-genotype, and C4a cluster which was the predominant in China since 2004. According to phylogenetic analysis, HEV71 which belonged to more than 4 different clusters were circulating in Beijing in 2008. More virological suggestion for disease control and prevention, and information of HEV71 molecular epidemiology need to be collected urgently due to the successive large epidemic of HEV71 in China.