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目的对陕西省2009~2012年肠道病毒71型(Enterovirus Type 71,EV71)的分子流行病学特征进行研究。方法采用体外逆转录-聚合酶链反应(Reverse Transcriptase-Polymerase Chain Reaction,RT-PCR),对陕西省2009~2012年分离自手足口病(Hand,Foot and Mouth Disease,HFMD)病例的38株EV71进行衣壳蛋白(Capsid Protein)VP1编码区基因序列扩增。通过双脱氧末端终止法(Sanger法)测序,获得VP1编码区核苷酸序列。采用软件Sequencher 5.0和MEGA 5.0对序列进行生物信息学分析。结果陕西省2009~2012年流行的EV71与C4基因型C4a分支具有最高的核苷酸序列同源性,为93.4%~99.4%;与C4a分支代表株在亲缘性关系树上具有最近的进化关系。陕西省EV71具有较高核苷酸和氨基酸序列同源性,分别为93.3%~100%(平均97.3%)和98.3%~100%(平均99.4%)。但相同年代和不同年代EV71毒株间的核苷酸序列平均差异随年代逐渐增大。结论陕西省2009~2012年流行的EV71均属于C4基因亚型C4a分支,为中国近年来流行的优势基因亚型。陕西省EV71具有较高的基因同源性,但基因多态性随着时间逐渐增大。进一步阐明EV71在陕西省的变异变迁规律,需继续开展并加强EV71的分子流行病学监测。
Objective To study the molecular epidemiology of Enterovirus Type 71 (EV71) from 2009 to 2012 in Shaanxi Province. Methods Thirty-eight EV71 isolates from Hand, Foot and Mouth Disease (HFMD) cases from 2009 to 2012 in Shaanxi Province were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) The capsid protein VP1 coding region gene sequence was amplified. The VP1 coding region nucleotide sequence was obtained by sequencing by the dideoxy terminator method (Sanger method). The sequences were analyzed by bioinformatics software Sequencher 5.0 and MEGA 5.0. Results The highest nucleotide sequence homology of EV71 and C4 genotype C4a in Shaanxi province from 2009 to 2012 was 93.4% -99.4%, which had the closest evolutionary relationship with the C4a branch in phylogenetic tree . Shaanxi Province EV71 has high nucleotide and amino acid sequence homologies of 93.3% -100% (average 97.3%) and 98.3% -100% (average 99.4%), respectively. However, the average difference of nucleotide sequences between EV71 strains of the same age and different ages gradually increased with age. Conclusion The EV71 strains that belong to C4 subtype C4a in Shaanxi province during 2009 ~ 2012 are the dominant subtypes prevailing in China in recent years. Shaanxi Province EV71 has a high genetic homology, but genetic polymorphisms gradually increased over time. Further clarify the EV71 mutation in Shaanxi Province, the need to continue and strengthen EV71 molecular epidemiological surveillance.