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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 understand the VP1 coding region of human Enterovirus 71 (HEV71) isolates during the epidemic of Hand, Foot and Mouth Disease (HFMD) in Beijing in 2008. Methods Thirty-three throat swabs were collected from 1 to 3 days after the onset of infection. Human Rhabdomyosarcoma (RD) cells were used for EV isolation and specific primers were used to identify the strains by reverse transcription-polymerase chain reaction amplification. The full length of VP1 coding region of HEV71 was amplified, and the amplified product was sequenced by dideoxy chain termination method. Bioedit7.0.5 and Mega3.1 software were used for sequence alignment and phylogenetic tree analysis. Results Totally 16 viruses were isolated from 33 throat swab specimens. HEV7114 strain and 2 strains of CVA16 strain of Coxsackie virus (CVA16) were identified. One of them was a mixed infection of HEV71 and CVA16 . The complete VP1 coding sequence of 10 HEV71 strains was determined. The nucleotide and amino acid sequence homologies of 10 isolates were 95.5% -100% and 98.9% -100%, respectively. Compared with the strains of epidemic strains of Fuyang in 2008 The nucleotide and amino acid sequence identities were 95.4% -99.1%, respectively. The homologies with nucleotide sequences of C4 genotypes were more than 92%. Based on the nucleotide sequence of the VP1 coding region of HEV71, the phylogenetic tree was constructed. In 2008, the epidemic strains and C4 subtypes of Beijing epidemic strains clustered into clusters, belonging to the C4a branch in the C4 gene subtype; and 10 viruses were in four Relatively independent evolutionary branch. Conclusions The HEV71 isolated from children with HFMD in Beijing in 2008 are all C4 subtypes, belonging to the dominant branch in China since 2004 - C4a. Affinity analysis suggested there were at least 4 HEV71 transmission chains in this epidemic. Due to the continuous epidemic of HEV71 in China in recent years, there is an urgent need for continuous molecular epidemiological surveillance of HEV71, to clarify the gene characteristics of the epidemic strains in time and provide virological basis for disease prevention and control.