【作者】 朱永豹；

【导师】 黄元成；

【作者基本信息】 华中科技大学 ， 内科学， 2011， 硕士

【摘要】 背景戊型肝炎是由戊型肝炎病毒(hepatitis E virus, HEV)引起一种急性自限性疾病,其主要经粪-口传播,呈散发和爆发流行两种形式。患者临床表现类似于甲型肝炎,但病死率较其他各型肝炎高,尤其孕妇感染后病死率高达30%～100%。中国是戊型肝炎高发地区之一,其中新疆维吾尔族自治区在1986年～1988年期间发生了迄今为止世界上规模最大的一次戊型肝炎流行,累计有119 280人发病,707人死亡,给社会及经济造成了极大的危害。近年来,我国散发性急性戊型肝炎发病率呈持续上升趋势。HEV是一单股正链RNA病毒,无包膜,基因组全长约7.3kb,含有三个开放读码框(ORFs),其中ORF1位于基因组的5′端,编码与RNA复制有关的非结构蛋白, ORF2位于基因组的3′端,编码病毒衣壳蛋白,ORF3同样位于基因组的3′端,编码一种磷蛋白,其功能尚未完全阐明。Ⅰ、Ⅱ和Ⅲ型HEV ORF3基因片段长372 bp,编码蛋白由123个氨基酸组成,而基因Ⅳ型HEV由于在其ORF3插入一个核苷酸(U),从而导致阅读框移位,ORF3编码的蛋白N末端较其他基因型缺失9个氨基酸,含有114个氨基酸。本课题对武汉同济医院感染科门诊和住院部急性戊型肝炎患者的一般情况(如性别和年龄等)进行调查及HEV基因分型,并对基因Ⅳ型HEV ORF3基因及其编码蛋白变异情况进行检测和分析。目的1.探讨武汉地区散发性急性戊型肝炎流行特点和病毒基因型,为明确武汉地区HEV基因型分布提供一定的线索。2.研究武汉地区基因Ⅳ型HEV ORF3基因及其编码蛋白变异情况,为进一步探讨基因Ⅳ型HEV ORF3基因变异的临床意义奠定基础。方法1.收集2009年4月～2010年6月我院感染科门诊和住院部急性戊型肝炎患者血清162份,分装后-80℃保存待检。调查患者一般情况(如性别和年龄等)。2.采用逆转录-巢式聚合酶链反应(RT-nPCR)对162份急性戊型肝炎患者血清HEV RNA进行检测,并对PCR产物进行测序,然后根据测序结果进行HEV同源进化分析。3.采用逆转录-巢式聚合酶链反应(RT-nPCR)对41株基因Ⅳ型HEV RNA两个基因片段(5020～5392nt和5347～5956nt, EF570133)进行扩增,并对PCR产物进行测序,然后使用ContigExpress将测序结果拼接(含有HEV ORF3全序列),使用MegAlign(DNAStar软件包)对HEV ORF3进行比较,查看HEV ORF3基因及其编码蛋白变异情况。结果1.武汉地区急性戊型肝炎患者男女比例为4.40:1,30～59岁年龄段占71.60%,162份急性戊型肝炎患者血清中,有41份血清HEV RNA阳性,阳性率为25.31%,同源进化分析表明41株HEV均为基因Ⅳ型,各毒株间的核苷酸同源性为84.7%～100%,基因Ⅳ型又可进一步分为5种亚型,即Ⅳ-A、Ⅳ-B、Ⅳ-C、Ⅳ-D和Ⅳ-E亚型,各亚型内部的核苷酸同源性分别为91.3%～100%、98.0%、92.0%～100%、98.0%～100%、100%,其中Ⅳ-A亚型与广西株、上海株和西安株的核苷酸同源性分别为91.3%～96.0%、92.0%～97.3%和94.7%～99.3%,Ⅳ-D亚型与北京株的核苷酸同源性为95.3%～96.7%。2. 41株基因Ⅳ型HEV RNA两个基因片段均扩增出来的毒株为18株,18株HEV ORF3共检测出47个位点存在核苷酸替换,其中21个替换发生在密码子第一、二位核苷酸,26个替换发生在密码子第三位核苷酸;其编码蛋白存在19个位点氨基酸变异。结论1.武汉地区急性戊型肝炎以男性多见,好发于30～59年龄段,且均由基因Ⅳ型HEV引起,基因Ⅳ型又可进一步分为Ⅳ-A、Ⅳ-B、Ⅳ-C、Ⅳ-D和Ⅳ-E 5种亚型,其中Ⅳ-A亚型与广西株、上海株和西安株亲缘关系较近,Ⅳ-D亚型与北京株亲缘关系较近。2.武汉地区基因Ⅳ型HEV ORF3存在多个位点核苷酸替换,这些位点变异在急性戊型肝炎病情进展中有何意义,有待于进一步研究。更多还原

【Abstract】 BackgroundHepatitis E virus(HEV) is a major cause of acute, self-limiting disease. It is primarily transmitted by the fecal–oral route and frequently results in sporadic,as well as epidemic, hepatitis. The clinical manifestations of acute hepatitis E is similar to those of hepatitis A, but the mortality rate of hepatitis E is higher than that of other hepatitides, especially up to 30%–100% in infected pregnant women. China is one of the high incidence areas of hepatitis E.So far, the world’s largest hepatasit E outbreak had occurred in Xinjiang Uighur Autonomous Region between1986 and1988 with about 12,000 individuals infected and 707 people died,which did great harm to society and economic. In recent years, the incidence of acute sporadic hepatitis E has been rising in our country.HEV is a a positive sense, single-stranded RNA virus without envelope.The genome of is approximately 7.3 kb and contains three open reading frames (ORFs).ORF1 locates in the 5 ’end of the genome and encodes non-structural protein which is essential for HEV replication.ORF2 locates in the 3 ’end of the genome and encodes the viral capsid protein.ORF3 also locates in the 3’ end of the genome and encodes a small phosphoprotein whose function(s) has not been fully defined. ORF3 of genotypeⅠ,ⅡandⅢwas 372 bp and encodes a protein of 123 amino acids.Compared to ORF3 of genotypesⅠ,ⅡandⅢ, ORF3 of genotype 4 contains a nucleotide insertion(U) which changes the downstream reading frames and this frame shift is predicted to shorten the ORF3 protein by 9 amino acids,including 114 amino acids.For these reasons,in our study,we firstiy investigate general situation(such as gender and age) and performed genotyping of HEV isolates among out-and in-patients with acute hepatitis E in the department of Infectious Diseases of Tongji Hospital in Wuhan.Then we detect and analyze the mutation of typeⅣHEV ORF3 and the encoded protein.Objective1. To investigate the epidemiological characteristics and genotype of hepatitis E virus (HEV) in Wuhan,which will provide some clues to determine genotype distribution of HEV in Wuhan.2. To detect and analyze the gene mutation of GenotypeⅣHEV ORF3 in Wuhan,which will further lay the foundation forinvestigating clinical significance of GenotypeⅣHEV ORF3 gene mutation.Methods1. 162 serum samples were collected from out-and in-patients with acute hepatitis E in the department of Infectious Diseases of our hospital between April 2009 and June 2010 and they were saved in -80℃after new package to test for HEV RNA. General situations(such as gender and age) of patients were surveyed.2. 162 serum samples collected from patients with acute hepatitis E were tested for HEV RNA by reverse transcription-nested polymerase chain reaction (RT-nPCR). The PCR products were sequenced. Then the sequencing results were used for phylogenetic analysis of HEV3. Two gene fragments of 41 GenotypeⅣHEV RNA(5020～5392nt and 5347～5956nt,EF570133) were amplified by reverse transcription-nested polymerase chain reaction (RT-nPCR).The PCR products were sequenced.Then these sequences were stitched(containing complete HEV ORF3) with ContigExpress and HEV ORF3s were aligned with MegAlign(DNAStar software package).The mutations of HEV ORF3 gene and ORF3 protein were analysed.Results1. The ratio of male and female patients with acute hepatitis E was 4.40:1 .The patients aged from 30 to 59 years accounted for 71.60%.41 out of 162 serum samples were positive for HEV RNA.The positive rate was 25.31%.Phylogenetic analysis showed that the 41 isolates belonged to genotypeⅣHEV. They shared 84.7.0%～100% identity with each other at the nucleotide level.The genotypeⅣisolates could be further divided into 5 subtypes, designating as A-E. The nucleotide sequence homologies within the five subtypes were 91.3%～100%、98.0%、92.0%～100%、98.0%～100% and 100%,respectively.SubtypeⅣ-A had 91.3%～96.0%、92.0%～97.3% and 94.7%～99.3% nucleotide sequence homology with the HEV isolates from Guangxi、Shanghai and Xian.SubtypeⅣ-D had 95.3%～96.7% nucleotide sequence homology with the HEV isolate from the Beijing.2. Both of HEV RNA gene fragments were amplified in 18 HEV isolates.There were 47 nucleotide substitutions of ORF3 gene in 18 HEV isolates.Among them ,21 nucleotide substitutions were found in the first or second nucleotide of codon and 26 nucleotide substitutions were found in the third nucleotide of codon.There were 19 amino acid substitutions of ORF3 protein in 18 HEV isolates.Conclusion1. AHE mostly occurs in male.People aged from 30 to 59 years are more susceptible to infection.The pathogen of acute hepatitis E is genotypeⅣHEV in Wuhan.The genotypeⅣisolates could be further divided into 5 subtype, designating asⅣ-A toⅣ-E. There is a close relationship between subtypeⅣ-A and Guangxi、Shanghai and Xian strains.There is a close relationship between subtypeⅣ-D and Beijing strain.2. There are a large number of nucleotide substitutions in GenotypeⅣHEV ORF3 gene in Wuhan. The significance of these gene mutations further needs to be studied in the progression of acute hepatitis E.更多还原