贵州省17例手足口病肠道病毒71型分离株全基因组序列分析

Whole genome sequence analysis of 17 enterovirus 71 strains isolated from children with hand, foot and mouth disease in Guizhou province

目的 分析我国贵州地区17株肠道病毒71型（Enterovirus 71，EV71）分离株的基因特征及遗传进化情况，探讨本地区EV71病毒的分子流行病学特点。方法 收集2013至2015年贵州省各地区手足口病住院患儿咽拭子样本，提取其病毒核酸，利用逆转录-聚合酶链反应（reverse transcription-polymerase chain reaction，RT-PCR）分段扩增其全基因组后进行测序，测序结果利用DNAMAN8.0软件进行编辑及拼接，然后将病毒基因组序列分别与基因库中其他EV71基因组序列进行Blastn比对，应用MEGA5.2软件中的Neighbor-Joining法构建系统进化树。结果 本研究成功分离扩增出17株EV71病毒全基因组序列，其基因组全长均为7 405 bp，编码2 193个氨基酸。毒株间存在的12处氨基酸位点差异将17株分离株划分为10种氨基酸序列，不同序列与临床类型尚未表现出规律性和相关性。17株EV71分离株间VP1区、5′非翻译区（ 5′ untranslated region，5′UTR）及3′非翻译区（ 3′ untranslated region，3′UTR）核苷酸同源性均较高，其全基因组与A、B、C基因型及柯萨奇病毒A组16型（Coxsakievirus A 16，CA16）代表株进行同源性比较显示其与EV71 C4a亚型代表株同源性最高，为95.3%～98.1%，与CA16代表株同源性最低。17株分离株基于全基因组、VP1区及5′UTR核苷酸序列构建的遗传进化树显示17株分离株自成一簇，与C4a亚型代表株聚在同一大分支上，不同区域构建的进化树亲缘关系远近存在差异。结论 2013至2015年贵州地区流行的EV71毒株基因型为C4a亚型，与国内其他地区流行的EV71基因型一致，暂未出现抗原转变及新亚型毒株输入，但存在着碱基及氨基酸改变，需动态监测；17株EV71分离株氨基酸序列差异与疾病严重程度尚未表现出相关性。

Objective To inquire into the molecular epidemiology of enterovirus 71 （ EV71 ） in our region by analyzing the whole genome characteristics and genetic evolution of EV 71 strains isolated from Guizhou area. Methods The throat swabs samples of hospitalized children with hand, foot and mouth disease in Guizhou province from 2013 to 2015 were collected, the virus nucleic acid were extracted, then the whole genome of virus were piecewise amplified by reverse transcription polymerase chain reaction （RT-PCR） and sequenced. Sequencing results were edited and spliced by DNAMAN8.0 software, then the viral genome se- quences were compared with genome sequences of other EV71 strains in the genebank by Blastn, the phyloge- netic tree was constructed by the Neighbor-Joining method in MEGA5.2 software. Results The whole ge- nome sequences of 17 EV71 strains were successfully isolated and amplified,the whole genome length of 17 EV71 isolates was 7 405 base pair, encoded about 2 193 amino acids. The 17 isolates were divided into ten species of amino acid sequences by 12 differences of amino acid among the strains,different sequences and clinical types had not shown regularity and correlation. The nucleotide homology in VP1 region,5＇untranslat- ed region（5＇UTR） and 3＇ untranslated region （3＇UTR） were high among 17 EV71 isolates. The results that the whole genome of 17 EV71 isolates was compared with representative strains of EV71 A, B, C genotype and coxsakievirus A 16 （CAI6） showed that 17 EV71 isolates had higher homology with EV71 C4a sub- type,95.3%-98.1% ,and the lowest homology with CA16. The phylogenetic tree was constructed based on nucleotide sequence of the whole genome, VP1 region and 5 ＇untranslated region of 17 isolates showed that the 17 isolates were clustered into one cluster, and were clustered in the same branch with C4a isoforms, the phy- logenetic relationships among different regions were different. Conclusion The popular genotype of EV71 strains in Guizhou area for 2013-2015 was C4a subtype, consistenting with the genotype of popular EV71 in other regions of China. EV71 strains hasn＇t the antigen transformation and input of a new subtype temporari- ly, but exist nucleotide and amino acid changes, so need be chronically and dynamically monitored. There is no correlation between the amino acid sequence difference of 17 EV71 isolates and the state of an illness.