2016年盐城地区非EV71非CVA16其他肠道病毒分子分型及肠道病毒CVA6型VP1基因特征分析

Molecular genotyping of the enterovirus for non-enterovirus 71 and non-coxsackievirus A16 in Yancheng area in 2016 and analysis of the characteristics of VP1 gene of coxsackievirus A6

目的了解2016年盐城地区手足口病非肠道病毒71型(enterovirus,EV71)非柯萨奇病毒A组16型(coxsackievirus A16,CVA16)其他肠道病毒病原谱分布情况,并对鉴定为肠道病毒CVA6型VP1基因进行分子流行病学研究。方法对分子分型鉴定为CVA6型的肠道病毒,通过反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)的方法扩增VP1基因全长编码区,扩增产物经纯化测序,采用相应的生物信息软件进行核苷酸及氨基酸序列比对及基因种系进化特征分析。结果 EV71、CVA16与非EV71非CVA16三者核酸阳性率差异有统计学意义(x^2=87.72,P<0.001)。非EV71非CVA16其他肠道病毒基因定型结果显示分属7个基因型CVA4、CVA6、CVA10、埃可病毒3(echovirus 3,E-3)、E-9、E-13、CVB4,分属肠道病毒A、B两组。遗传进化树分析显示,盐城56株CVA6型肠道病毒分属D大分支D3、D4进化分支。其中,54株毒株属D4分支,为国内主流优势流行分支,在D4分支内又聚类成众多细小分支,远离原型株Gdula。结论 2016年盐城市手足病流行的病原谱呈现多样性,有多种基因型的肠道病毒共同流行,CVA6是优势病原体,D4分支的毒株为优势进化分支,形成多条传播链。

Objective To study the pathogenic spectrum of the enterovirus for non-enterovirus 71 （ non-EV71 ） and non-coxsackievirus A16（non-CVA16） for hand, foot and mouth disease （HFMD）in the city of Yancheng in 2016, and characterize the molecular epidemiology of the VP1 gene of coxsackievirus A6 strains. Methods The serotype of coxsackievirus A6 strains were identified by molecular subtyping. The sequences of the VP1 gene of coxsackievirus A6 strains were amplified through one step reverse transcription polymerose chain reaction （RT-PCR） method and the PCR products were sequenced. The sequences of nucleotide and amino of the VP1 gene of coxsackievirus A6 were analyzed and the evolutional tree was generated using bioinformatics software. Results There was significant difference among the three positive rates in EV71 ,CVA16 and non-EV71, non-CVA16（ %2 = 87.72 ,P 〈 0. 001 ）. The strains of the enterovirus for non-EV71 and non-CVA16 were classified into seven genotypes （ CVA4, CVA6, CVA10, Echovirus 3 （ E-3）, E-9, E-13, Coxsackievirus B4（CVB4） ） and belonged to human enterovirus A group and human enterovirus B group. Phylogenetic tree analysis showed that 56 strains of enterovirus coxsackievirus A6 in Yancheng area belonged to D3, D4 evolutionary sub-branches. A- mong them, 54 strains belonged to D4 evolutionary sub-branch, which was the mainstream sub-branch in china. Within the D4 sub-branch, 54 strains were clustered into numerous small sub-branches and away from the prototype Gdula strain. Conclusions The spectrum of pathogens causing HFMD epidemics in Yancheng area in 2016 was multifarious and the genotype of coxsackievirus A6 was the prevalent pathogen. D4 sub-branch of strains was the dominant evolutionary branch and formed multiple transmission chains.