大连市人肠道病毒71型病原学特征研究

发布时间：2018-05-30 21:28

本文选题：肠道病毒71型 + 核苷酸序列　；参考：《大连医科大学》2012年硕士论文

【摘要】：目的：了解2009～2011年大连地区EV71流行情况；对分离出的EV71毒株进行部分基因片段的核苷酸和氨基酸序列测定，构建系统发生树和同源性分析，以确定其基因型别；对与毒株毒力相关基因序列进行比较分析，探寻毒株毒力与基因序列之间的关系，为今后EV71的防治及防控提供科学依据。方法：收集大连市手足口病监测哨点医院门诊就诊病例和手足口样病例，将来自监测哨点医院的咽拭子和粪便标本用荧光RT-PCR方法检测为EV71的阳性标本接种于RD细胞，经传代培养后分离EV71病毒，，提取病毒RNA，利用RT-PCR方法分别扩增出VP1、VP4、5’UTR基因片断，对扩增产物进行核苷酸序列测定，用DNAStar软件与EV71各基因亚型代表株进行序列比较，分析其核苷酸序列和氨基酸序列的同源性，以及不同临床症状的毒株与核苷酸和氨基酸序列之间的关系。结果： 1、2009～2011年期间共检测533份样品，其中EV71阳性333份，阳性率为62.48%。533份病例中，男317例，女216例，男女比例为1.47:1。其中男性患者EV71阳性率为61.51%，女型患者EV71阳性率为63.89%。0～5岁为手足口病高发年龄，以托幼儿童为主。 2、VP1区序列分析表明：选取9株RD细胞分离培养的EV71毒株进行VP1序列扩增并测序，其结果用DNAStar软件分析，这9株EV71毒株均属于C4a亚型，其核苷酸之间的同源性为88.7%～99.9%；氨基酸之间的同源性为96.7%～100%。与BRCR-CA-70的核苷酸同源性为79.4%～81.5%；与B型核苷酸同源性为：81.8%～84.7%；与C亚型核苷酸同源性为85.2%～99.2%,而与C4同源性高达90.0%～99.2%，其中与C4a的同源性最高为93.7%～99.2%，与C4b的同源性为90.0%～94.1%。对重症来源的毒株，在第227位核苷酸发生了T→C的碱基置换，而轻症来源的毒株均未发生改变。对于氨基酸序列比较上未发现一致性改变。 3、VP4区序列分析表明：9株毒株的VP4氨基酸序列高度一致，只是在第52位dalian1-09株发生了K→R的改变，第62位氨基酸dalian4-10株发生了T→S的改变，其余各株氨基酸序列均一致。没有发现重症来源的毒株和轻症来源的毒株在氨基酸或核苷酸序列上相应固定的变异。 4、5’UTR区序列分析表明：9株毒株在第208位核苷酸,重症来源的样品发生了碱基置换（G→A）；在第254位核苷酸，重症来源的样品发生了碱基置换（A→G）。氨基酸序列未发现一致性改变。结论： 1、2009年-2011年大连地区手足口病的主要病原体为EV71，其基因型别为C4a亚型，这与中国大陆的大部分地区流行株相一致。 2、基于VP4的基因分型与VP1的基因分型相一致，均为C4a亚型，且VP4序列高度保守。 3、在毒株的毒力分析上，重症来源的毒株，在VP1的第227位核苷酸发生了T→C的碱基置换，而轻症来源的毒株均未发生改变。VP4的核苷酸序列和氨基酸序列均未发现与临床症状相对应的改变，而在5’UTR序列上发现了第208位核苷酸的碱基置换（G→A），这一结果还有待于进行下一步的研究。
[Abstract]:Objective: to investigate the prevalence of EV71 in Dalian from 2009 to 2011, to determine the nucleotide and amino acid sequences of partial gene fragments of isolated EV71 strains, and to construct phylogenetic tree and homology analysis to determine their genotypes. In order to find out the relationship between virulence and gene sequence of virulence of virus Methods: the outpatient and hand foot mouth like cases of surveillance sentinel hospital of hand, foot and mouth in Dalian were collected. Throat swabs and stool samples from sentinel hospital were inoculated into Rd cells by fluorescence RT-PCR method. After passage culture, EV71 virus was isolated, virus RNAs were extracted, and VP1- VP4- 5 UTR gene fragments were amplified by RT-PCR method. The amplified products were sequenced by nucleotide sequencing and compared with the representative strains of EV71 gene subtypes by DNAStar software. The homology of nucleotide sequence and amino acid sequence, and the relationship between nucleotide sequence and amino acid sequence of virus strains with different clinical symptoms were analyzed. Results: From 2009 to 2011, a total of 533 samples were detected, of which 333 were positive for EV71, the positive rate was 62.48.533 cases, 317 males and 216 females, the ratio of male to female was 1.47: 1. The positive rate of EV71 was 61.51% in male patients and 63.89% in female patients. 2 sequence analysis of VP1 region showed that 9 EV71 strains isolated from Rd cells were amplified by VP1 sequence and sequenced. The results showed that the 9 EV71 strains belonged to C4a subtype by DNAStar software. The homology between nucleotides and amino acids was 88.7 and 99.9, respectively. The nucleotide homology with BRCR-CA-70 was 79.4%; with B type was 84.8%; with C subtype was 99.2; with C subtype was 99.2, and with C4 was 90.099.2. The highest homology with C4a was 93.799.2and 90.0799.2 with C4a, 90.0799.2with C4b and 90.094.1 with C4b. The base replacement of T-C was observed in the 227 nucleotides of the strains from the severe disease, while none of the strains from the mild disease changed. For amino acid sequence comparison, no change of consistency was found. The sequence analysis of VP4 region showed that the amino acid sequence of VP4 was highly consistent among the 9 strains, except for the change of K-R in the 52nd dalian1-09 strain, the change of T-S in the 62nd amino acid dalian4-10 strain, and the identical amino acid sequence of the other strains. No variations in amino acid or nucleotide sequences were found in severe and mild source strains. The sequence analysis of the UTR region showed that at the 208 nucleotides, the nucleotide substitution of G ~ (+) was found in the samples from severe source, and at the 254 nucleotides, the nucleotide substitutions of A ~ (1 +) were found in the samples from the severe source. The amino acid sequence showed no change in consistency. Conclusion: The main pathogen of HFMD in Dalian from 2009 to 2011 is EV71.The genotype of EV71is C4a subtype, which is consistent with the epidemic strains in most areas of mainland China. 2. The genotyping based on VP4 was consistent with that of VP1, all of them were C4a subtypes, and the VP4 sequence was highly conserved. 3. In the virulence analysis of the virulence of the virus strain, the base substitution of T-C was observed in the 227th nucleotide of VP1. The nucleotide sequence and amino acid sequence of VP4 did not change with clinical symptoms. On the 5'UTR sequence, the sequence of the 208th nucleotide was found, and the result is still to be studied in the next step.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373.2

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