河南省2008年手足口病病原体（EV71）分离株全基因组序列

发布时间：2018-01-15 23:10

本文关键词：河南省2008年手足口病病原体（EV71）分离株全基因组序列　出处：《郑州大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的了解河南省2008年手足口病病原体流行特征,获得一株EV71分离株的全基因组核苷酸序列并对其进行同源性分析,构建系统发生树,获得该EV71毒株的基因型。材料与方法收集2008年河南省18个地市在门诊就诊和住院的手足口临床诊断病例的血清和/或粪便(咽拭子、疱疹液、脑脊液)标本,运用国家CDC推荐的分子生物学方法对其进行鉴定；同时选取部分标本接种人横纹肌肉瘤细胞(RD细胞)进行病毒分离。提取其中一株EV71分离毒株的RNA,设计八对引物,用反转录聚合酶链式反应(reverse transcription PCR即RT-PCR)扩增其全基因组序列,产物纯化后另外设计八对引物对其进行双向测序；用Clustal (1.8) X、GeneDoc、DNAStar、MEGA3等软件进行核苷酸整理、拼接组装、校对、同源性比较、氨基酸序列分析、基因进化树构建等分子变异研究；与GenBank中EV71毒株的各基因亚型代表株的VP1段进行同源性分析并对该株毒株定型。结果 1样本检测结果共检测435份手足口病临床诊断病例的不同标本,EV71检出率为16.78%,Cox.A16检出率为1.38%,经检验差异具有统计学意义(P0.01)。检出阳性病例中,男、女性别比为2.3:1(54/23),但差异无统计学意义。对69份标本进行了细胞培养和病毒分离,7份标本出现明显致细胞病变效应(CPE),其中EV71占85.7%(6/7),Cox.A16占14.3%(1/7)。对分离株再次用RT-PCR检测,阳性率达100%。 2一株EV71分离株的全基因组序列分析 EV71 HENAN08分离株全基因组序列长度为7405bp(未包括多聚腺苷酸尾),其中腺嘌呤核苷酸(A)占27.01%,鸟嘌呤核苷酸(G)占24.00%,胸腺嘧啶核苷酸(T)占24.92%,胞嘧啶核苷酸(C)占24.07%。A和T丰富。从基因组的5'末端开始有742个碱基的5'非编码区(5'UTR),与SHZH98株、BrCr株、Zhejiang08株和TW2086株(743个碱基)不同。5'UTR之后为6579bp的编码区,编码含2193个氨基酸的多聚蛋白,其后为84个碱基的3′非编码区(3′UTR)。与其它EV71毒株相比,HENAN08株在编码区没有核苷酸的缺失和插入,但在5′UTR和3′UTR区存在个别核苷酸的缺失和插入。核苷酸同源性比较：在整个基因组,包括5′UTR区、编码区(P1、P2和P3)和3′UTR区,和AnhuiFY08株以及Zhejiang08株的同源性均高于95%；在P1区,HENAN08与AnhuiFY08、SHZH98、Zhejiang08、SHZH03株的同源性均90%,与BrCr和TW2086的同源性均80%,而与Cox.A16的同源性最低,70%；在3′UTR区,与标准株BrCr以及TW2086的同源性最低。氨基酸同源性比较：在整个编码区,EV71 HENAN08株与其它国内、外EV71株的同源性均较高,与Cox.A16同源性较低；在P2和P3区,HENAN08与其它国内EV71株的同源性均较高,而与标准株BrCr以及TW2086的同源性低于Cox.A16;结构蛋白VP1区,EV71 HENAN08株与AnhuiFY08、SHZH98、标准株BrCr、Zhejiang08、SHZH03、TW2086株的同源性均95%,与Cox.A16的同源性最低,为72.7%。结构蛋白VP4区的同源性比较都为100%。结论 1 EV71是引起2008年河南省手足口病的优势病原体。 2河南省首次成功提取EV71病毒株的全部基因片段,基因序列号为GU366191。 3本次分析的EV71分离株同安徽省分离的毒株在基因亲缘关系和流行时间关系上最接近,同属于一种基因型——C基因型C4亚型。
[Abstract]:objective
To understand the epidemiological characteristics of HFMD pathogens in Henan Province in 2008, we obtained a complete nucleotide sequence of a EV71 strain and analyzed its homology. The phylogenetic tree was constructed to obtain the genotype of the EV71 strain.
Materials and methods
18 cities in Henan province in 2008 were collected and serum in outpatient and hospitalized HFMD cases and / or feces (swab, herpes fluid, cerebrospinal fluid specimens), using molecular biology method recommended by national CDC to identify them at the same time; selected specimens were inoculated human rhabdomyosarcoma cells (RD cells) virus the extraction separation. One strain EV71 isolated strains of RNA, eight pairs of primers were designed by reverse transcription polymerase chain reaction (reverse transcription PCR RT-PCR) to amplify the whole genome sequence of the purified product, also designed eight pairs of primers were sequenced to Clustal; (1.8) X, GeneDoc, DNAStar, MEGA3 and other software nucleotide finishing, assembly, proofreading, homology, amino acid sequence analysis, gene molecular phylogenetic tree constructed with GenBank mutation; EV71 strain subtypes strains VP1 The segment was analyzed by homology and the strain of the strain was stereotyped.
Result
1 sample test results
A total of 435 test samples of different specimens of clinically diagnosed cases of HFMD, the detection rate of EV71 was 16.78%, Cox.A16 positive rate was 1.38%, the difference was statistically significant (P0.01). The positive cases, male and female sex ratio was 2.3:1 (54/23), but the difference was not statistically significant. The 69 specimens were cell culture and virus isolation, 7 specimens appeared obvious cytopathic effect (CPE), which accounted for 85.7% of EV71 (6/7), Cox.A16 (1/7) accounted for 14.3%. Of the isolates again with RT-PCR detection, the positive rate was 100%.
Whole genome sequence analysis of 2 EV71 isolates
EV71 HENAN08 isolates complete genome sequence of length 7405bp (not including Polya tail), which accounted for 27.01% of adenine nucleotides (A), guanine nucleotide (G) accounted for 24%, accounted for 24.92% of thymine nucleotide (T), cytosine nucleotide (C) for 24.07%.A and T rich. From the 5'end of the genome has 742 base 5' (5'UTR), and the encoding region of SHZH98 strain, BrCr strain, Zhejiang08 strain and TW2086 strain (743 BP) in different.5'UTR 6579bp encoding region, encoding 2193 amino acids of the polyprotein, followed by a 84 BP 3 'non encoding region (3' UTR). Compared with other EV71 strains, HENAN08 strains in the encoding region without nucleotide deletion and insertion, but in the 5 'UTR and 3' UTR region deletion and insertion of individual nucleotides.
The nucleotide homology in the whole genome, including the 5 'UTR region, encoding region (P1, P2 and P3) and 3' UTR region, and AnhuiFY08 strain and Zhejiang08 strain homology were higher than 95%; in P1, HENAN08 and AnhuiFY08, SHZH98, Zhejiang08, SHZH03 strain homology was 90%, and the BrCr and TW2086 homology 80% with Cox.A16, and the lowest homology, 70%; in 3 'UTR region, and the standard strain BrCr and TW2086 the lowest homology.
The homology of amino acid encoding: in the whole area, EV71 HENAN08 and other domestic and foreign strains, EV71 strains are of high homology, the homology with Cox.A16 is low; in P2 and P3, HENAN08 and other domestic EV71 strains are of high homology, and standard strains BrCr and TW2086 homology is lower than Cox.A16; VP1 structural protein HENAN08 strain and AnhuiFY08, EV71, SHZH98, Zhejiang08, SHZH03 standard strain BrCr, strain TW2086, the homology was 95% Cox.A16, and the lowest homology, VP4 72.7%. structural protein homology is 100%.
conclusion
1 EV71 is the dominant pathogen of hand foot and mouth disease in Henan Province in 2008.
2 the whole gene fragment of EV71 virus strain was successfully extracted in Henan province for the first time. The sequence number of the gene was GU366191.
3 the EV71 isolates from this analysis were closest to the Anhui isolates, and they belonged to a genotype C genotype C4 subtype.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373.2

【引证文献】