2016年昆明市肠道病毒EV71分离株全基因序列分析

发布时间：2018-06-22 00:30

本文选题：EV71 + 全基因组　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]分析2016年昆明市手足口病(HFMD)患儿肠道病毒EV71分离株的全基因组序列特性(不包含多聚腺苷尾),以阐明当年昆明流行EV71毒株的分子生物学特点,通过与标准株比较,了解病毒的生物学性状是否发生改变,进一步了解其进化的规律和发病特点。[方法]将昆明市妇幼保健院送检的20个EV71咽拭子阳性样本提取液接种到非洲绿猴肾细胞(Vero-E6)进行扩增,从接种后生长较好的细胞中随机选取10株(KM01-KM10)的标本用于后续实验。利用RT-PCR扩增覆盖基因组全长的片段并测序,采用Over-lap extension PCR获得10个分离株的基因组序列;从GenBank中选取来自不同地区、时期及不同类型的26个EV71毒株及1株CA16毒株的全基因组序列,与KM01-KM10进行同源性、进化、重组等分析。[结果]10株EV71昆明株基因组全部符合肠道病毒基因组的基本特征,获得的基因组全长均为7405nt,其开放阅读框为6582nt,编码一个含2194个氨基酸的多聚蛋白,10株EV71全长序列与挑选的标准菌株比较显示,其编码区内未见有核苷酸的插入或缺失;10株EV71毒株的VP1、VP4、P1、P2、P3及全长的核苷酸和氨基酸同源性分析显示KM01-KM10与阜阳EU703812菌株的相似度最高;VP1、P1、P2+P3与全长区段的进化分析显示KM01-KM10处于C4亚型分支内部的最末端,与阜阳EU703812菌株处于同一簇,进化关系很近。[结论]1.本研究获得昆明10株EV71病毒(KM01-KM10)的全基因组序列。2.通过核苷酸及氨基酸序列的相似性比对和遗传进化分析显示KM01-KM10病毒属于C4亚型,它们与2008年分离的C4亚型EV71毒株亲缘关系很近,可能起源于同一种系。3.昆明KM01-KM09等9株EV71病毒的全基因组序列没有发生型内重组现象,而KM10的可能发生重组。
[Abstract]:[objective] to analyze the whole genome sequence characteristics (excluding polyadenosine tail) of enterovirus EV71 isolated from children with hand, foot and mouth disease (HFMD) in Kunming in 2016, in order to elucidate the molecular biological characteristics of EV71 strain in Kunming, and compare it with the standard strain. To understand whether the biological characters of the virus have changed, to further understand the evolution of the law and the characteristics of the disease. [methods] Twenty EV71 pharyngeal swabs positive samples from Kunming Maternal and Child Health Hospital were inoculated into the kidney cells of African green monkey (Vero-E6). Ten samples (KM01-KM10) were randomly selected from the cells growing well after inoculation. The genomic sequences of 10 isolates were amplified by RT-PCR and sequenced by overlap extension, 26 EV71 strains from different regions, different periods and different types and 1 CA16 strain were selected from GenBank. Analysis of homology, evolution and recombination with KM01-KM10. [results] the genomes of 10 EV71 Kunming strains all accord with the basic characteristics of enterovirus genome. The total length of genome was 7405nt.The open reading frame was 6582nt.The full-length sequence of 10 EV71 strains encoding 2194 amino acids was compared with the selected standard strain. No nucleotide insertion or deletion was found in the coding region. Analysis of nucleotide and amino acid homology showed that KM01-KM10 had the highest similarity with Fuyang EU703812 strain. Evolution analysis of KM01-KM10 showed that KM01-KM10 had the highest similarity with the full-length region. Evolutionary analysis showed that KM01-KM10 had the highest similarity with the full-length region. The phylogenetic analysis of KM01-KM10 showed that KM01-KM10 had the highest similarity with that of Fuyang EU703812 strain. At the end of the subtype C4 branch, The strain EU703812 is in the same cluster as Fuyang strain, and the evolutionary relationship is very close. [conclusion] 1. In this study, the whole genome sequence of 10 strains of Kunming EV71 virus (KM01-KM10) was obtained. The similarity analysis of nucleotide and amino acid sequence and genetic evolution analysis showed that KM01-KM10 virus belongs to C4 subtype, which is closely related to the strain of C4 subtype EV71 isolated in 2008, and probably originated from the same strain. 3. The whole genome sequence of 9 strains of KM01-KM09 et al., Kunming KM01-KM09, etc., did not recombine in the whole genome, but the recombination of KM10 was possible.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.1

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