猪戊型肝炎病毒IgG抗体检测和部分核酸序列分析

目的 了解猪戊型肝炎病毒(HEV)感染情况,探讨猪HEV基因型与人HEV的关系。方法用酶联免疫吸附法(ELISA)和逆转录巢式聚合酶链反应法(RT-nPCR)分别检测猪血清抗-HEV IgG抗体和猪HEV RNA,并用Vector NTI Suite 7和TreeView软件进行人与猪HEV核酸序列和基因进化树分析。结果 猪抗-HEV抗体阳性率为16.67%(18/108),并从18份抗-HEV IgG阳性的猪血清中扩增到2株(11.11%,分别命名为S18和S43)HEV 开放读码框(ORF)1片段(102～387bp),两者核苷酸序列同源性为99%,与HEV基因型1～8的核苷酸序列同源性分别为76%～77%、78%、76%～79%、85%～86%、77%、80%、79%和75%～79%。其中1株(S18)还扩增出HEV ORF2片段(5994～6297bp),与HEV基因型1～4的核苷酸序列同源性分别为76%～78%、74%、74%～77%、85%～94%。结论 分离的2株猪HEV属于HEV基因型4。     

武汉地区戊型肝炎病毒开放读码框3的基因序列分析

目的 对武汉地区戊型肝炎病毒(HEV)开放读码框(ORF)3基因序列进行分析,并确定病毒基因型. 方法 收集103份抗-HEV IgM阳性血清,采用逆转录-巢式聚合酶链反应扩增HEV RNA两个基因片段(5020 ～ 5392nt和5347 ～ 5956 nt,EF570133);对PCR产物进行测序,并用ContigExpress将测序结果拼接(含有ORF3基因),对ORF3基因序列进行分析.结果 103份抗-HEV IgM阳性血清中HEV RNA两个基因片段均扩增出来的样本为18份,18株HEV ORF3基因全长均为345 bp,编码114个氨基酸.各毒株间的核苷酸同源性为92.5％ ～ 99.4％,与基因Ⅰ型、Ⅱ型、Ⅲ型、Ⅳ型HEV的核苷酸同源性分别为83.5％ ～ 86.7％、83.2％ ～ 85.2％、84.6％ ～ 87.2％、92.0％ ～ 96.5％;系统进化分析表明该18株HEV均为基因Ⅳ型. 结论 武汉地区HEV主要为基因Ⅳ型,ORF3基因序列可用于同源进化分析.     

武汉地区散发性急性戊型肝炎流行特点和病毒基因型分析

目的：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。方法：采用逆转录-巢式聚合酶链反应（RT-nPCR）检测162份急性戊型肝炎患者血清HEV RNA,并对PCR产物进行测序及同源进化分析。结果：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有41份HEVRNA阳性,阳性率为25.31%,同源进化分析表明41株HEV毒株均属于基因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,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与上海株、西安株和广西株的核苷酸同源性分别为92.0%～97.3%、94.7%～99.3%和91.3%～96.0%,Ⅳ-D与北京株的核苷酸同源性为95.3%～96.7%。结论：武汉地区急性戊型肝炎以男性多见,好发于30～59岁年龄段,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与上海株、西安株和广西株亲缘关系较近,Ⅳ-D与北京株亲缘关系较近。     

长春地区人与猪戊型肝炎病毒分子流行病学及部分基因序列分析

目的分析长春地区猪和人群流行的戊型肝炎病毒的系统进化关系。方法参照文献设计引物,对长春地区猪和人群流行的8株HEV(其中3株来源于人,5株来源于猪)进行RT-PCR,并将RT-PCR产物克隆到p MD18-T载体,筛选阳性重组质粒测序,测序结果采用Clustal X v.1.8进行多重比对分析,并与基因1~4型HEV代表株的核苷酸及氨基酸进行同源性比较,绘制遗传进化树。结果本研究获得的8株HEV核苷酸同源性在91.2%~99.1%,推导氨基酸同源性在97.4%~100%之间;基因1~4型内各株序列间同源性分别为:87.9%~100%,100%,85.9%~96.6%,84.8%~100%。结论研究表明获得的长春各株病毒均属基因4型,由进化关系看长春地区猪HEV与人HEV可能由同一毒株进化而来。     

烟台市2015-2018年人感染戊型肝炎病毒基因型分析北大核心CSCD

目的分析2015-2018年烟台市人感染戊型肝炎病毒(HEV)基因型别变化,探讨分子流行病特征。方法选取烟台市2015-2018年国家法定传染病报告系统上报的戊型肝炎病例为研究对象,采集血清标本检测HEV-IgM抗体,对血清HEV-IgM阳性病例的血清和粪便标本提取HEV核酸,采用逆转录PCR方法对HEV开放式阅读框架2区域内的核苷酸片段(644 bp)进行扩增,测序后进行系统进化分析。结果共采集360份血清样本,198份(55%)样本HEV-IgM阳性。HEV-IgM阳性的血清标本,HEV核酸检出率27.78%(55/198);粪便标本HEV核酸检测阳性率39.58%(19/48)。总阳性病例数72例,男性48例,女性24例。本次检测的72例HEV病毒株基因型全部是Ⅳ型,以4d亚型为主,占77.78%(56株);其次是4b亚型,占19.44%(14株);4a亚型占2.78%(2株)。72株HEV核苷酸序列的遗传距离为0.002~0.303。56株4d亚型HEV序列与山东地区猪源(KF176351)、羊源(KU904267)、牛源(KU904278)HEV序列的遗传距离为0.021~0.160、0.010~0.178、0.008~0.176。结论基因Ⅳ型HEV在2015-2018年烟台地区占主导趋势,4d亚型为主要流行株。本地人感染HEV可能与进食动物制品或密切接触动物相关。     

猪源戊型肝炎病毒上海株全基因组序列分析

目的分析猪源戊型肝炎病毒(HEV)上海株swChinash与已知其它HEV基因型的差异和人源HEV的关系。方法设计22对PCR引物,分片段进行扩增,两末端采用快速扩增方法(RACE)扩增,扩增产物克隆到pMD18-T载体并测序,用DNASTAR软件与53株人源和猪源HEV的基因组进行同源性分析,用Clustalx和Mega3.0软件绘制基因进化树。结果swChinash全序列除去poly(A)尾,由7235个核苷酸组成,ORF1～3分别编码1707,660,122个氨基酸。全基因序列与Ⅳ型的核苷酸同源性为83.4%～84.7%,与其他三型为73.3%～76.3%,其中与中国人源长春株最高,为84.7%。结论基于ORF1～3和全长基因组的进化树,猪源HEV上海株swChinash属于基因Ⅳ型,与其他Ⅳ型HEV亲缘关系较远,单独在一个进化分支上。     

云南某农村猪戊型肝炎病毒分子流行病学调查

目的 了解云南农村散养猪群HEV感染情况,为HEV防控提供理论依据。方法 利用反转录套式聚合酶链反应（RT-nested PCR）技术,对所采集云南猪群78份粪便样品进行HEV ORF2基因部分片段扩增,经琼脂糖凝胶电泳后对目的条带进行回收纯化及克隆测序。序列利用DNAStar和MEGA4.0软件进行同源性比较和系统进化树构建。结果 RT-nested PCR方法扩增出了350bp目的基因序列,该序列与GenBank中HEV各型的同源性在77.4~97.4%之间,与基因4型同源性最高（97.4%）,与3型同源性最低（77.4%）。系统进化树显示测定的序列与基因4型聚为一支,表明该序列属于基因4型。本次实验共检测了78份猪粪便样品,其中8份为阳性,阳性率为10.26%。结论 云南农村人群存在感染HEV的风险,应该加以防控以免HEV暴发流行。     

武汉地区散发性戊型肝炎流行病学及病毒基因型

为了解武汉地区戊型肝炎病毒(HEV)流行病学特点及基因型。回顾性分析2000年至2004年同济医院就诊急性肝炎916例流行病学特点,应用逆转录-套式聚合酶链反应法(RT-nested-PCR),扩增HEV开放读码框架2(ORF2)的部分序列120份,用Clustal X和Treeview软件比较武汉地区HEV序列与4个HEV主要代表株序列。发现所扩增的HEV,核苷酸同源性为(82.61~98.55)%,与Ⅰ型(缅甸株)、Ⅱ型(墨西哥株)、Ⅲ型(美国株)、Ⅳ型(中国/台湾株)核苷酸同源性分别为(76.52~81.74)%、(70.43~73.04)%、(76.52~81.16)%和(84.35~88.70)%。故认为武汉地区散发性戊型肝炎患者以HEVⅣ型感染为主,其发病率呈上升趋势,发病年龄以30~59岁为主,全年均可发病,3~6月为高峰季节。     

戊型肝炎

实验感染HEVⅣ型后恒河猴HEVRNA及抗HEVIgM（IgG）动态变化研究——杨志国等（江苏南京中国人民解放军第81医院南京军区肝病研究所210002）；《中国现代医学杂志》，2006，16（12）：1828-1830[目的：观察实验感染HEV（Hepatitis E Virus）Ⅳ型后恒河猴HEVRNA及抗HEV—IgM（IgG）的动态变化，探索HEVⅣ型感染的规律。方法：用逆转录PCR检测实验动物接种前后血清及粪便HEVRNA，用酶联免疫法（ELISA）检测血清抗HEV-IgM及抗HEV-IgG，并同步观察其血生化及肝脏组织学的变化。结果：感染后的恒河猴均发生了典型的急性肝炎，血清HEVRNA首先呈现阳性，相继出现谷丙转氨酶（ALT）升高、抗HEV-IgM阳性及抗HEV—IgG阳性。     

广西地区5种动物戊型肝炎病毒RNA的检测

目的了解广西地区与人关系密切的猪、鼠、狗、猕猴和鱼等5种动物戊型肝炎病毒（HEV）。方法对广西地区3月龄以下狗、猪粪便标本及龙虎山自然景区猴粪便标本，鱼胆汁标本，鼠肝组织，猪、鼠、狗HEV抗体阳性血清，用巢式逆转录聚合酶链反应法（RT—nPCR）检测戊型肝炎病毒核糖核酸（HEVRNA）。结果在3月龄以下猪粪便标本检测出HEVRNA，阳性率为10．08％（13／129），其余各种动物粪便、肝组织、胆汁、HEV抗体阳性血清标本均未检测出HEVRNA。结论猪粪便标本存在HEVRNA，该地区应加强动物粪便特别是猪粪便的管理，以预防戊型肝炎病毒的感染。     

Swine hepatitis E virus in rural southern China: genetic characterization and experimental infection in rhesus monkeys (Macaca mulatta)

BACKGROUND: In rural areas of southern China, where hepatitis E is endemic, residents generally rear pigs in pigsties near their houses. The study was conducted to assess the possibility that hepatitis E virus (HEV) infections in this region are acquired primarily through contact with swine. METHODS: One hundred twenty swine fecal samples collected from pigsties located in eight rural communities of southern China were tested for HEV RNA. The swine HEV isolates were analyzed genetically and were experimentally inoculated into rhesus monkeys to determine the potential risk of cross-species infection. RESULTS: Twenty-nine of the 120 swine fecal samples were positive for HEV RNA. The nucleotide sequences of these swine HEV strains shared 85%-99% identities with the local human genotype 4 isolates and belonged to two subgroups of genotype 4. Importantly, swine HEV strains representing both subgroups induced hepatitis in rhesus monkeys by inoculation with the virus, evidenced by elevated serum alanine transaminase (ALT), viremia, fecal viral shedding, anti-HEV seroconversion, and liver histopathological changes. CONCLUSIONS: Swine may be the principal reservoir for human HEV infection in rural southern China.     

rSj26-Sj32-HRP-IgG-Dot-ELISA用于急性日本血吸虫病患者的诊断价值

目的探讨rSj 26-Sj 32-HRP-IgG-Dot-ELISA用于急性日本血吸虫病患者的诊断价值。方法利用纯化的rSj26-Sj32融合蛋白和日本血吸虫成虫抗原(SjAWA)建立HRP-IgG-Dot-ELISA检测急性日本血吸虫病患者血清,并以华支睾吸虫病、卫氏并殖吸虫病、棘球蚴病、乙型肝炎、肺结核病患者和健康人血清作对照,观察二者的检测结果。结果 rSj 26-Sj 32和SjAWA检测急性日本血吸虫病患者阳性检出率均为100%,特异性分别为95.35%和93.02%;二者均与华支睾吸虫病、卫氏并殖吸虫病和泡型棘球蚴病患者血清存在不同程度的交叉反应。结论纯化的rSj26-Sj32融合蛋白是一种较好的免疫诊断抗原。     

Features of hepatitis E virus infection in Japan

Hepatitis E virus (HEV) is a major cause of acute hepatitis in many developing countries. HEV is transmitted principally by the fecal-oral route, and water-borne epidemics are characteristic of hepatitis E. Recently, there is growing consensus that HEV-associated hepatitis also occurs among individuals in industrialized nations who had no history of travel to endemic areas. Zoonotic spread of HEV has been suggested as human and swine HEV strains are closely related genetically and experimental cross-species infection of swine HEV to a chimpanzee and that of human HEV to swine have been demonstrated. This review describes the clinical, epidemiological and virological characteristics of domestic HEV infection in Japan, the genetic relatedness of Japanese human and swine HEV strains, and possible modes of HEV transmission, emphasizing that HEV should be considered in the diagnosis of acute or fulminant hepatitis of non-A, non-B, non-C etiology, even in patients who have not traveled abroad.     

Human and swine hepatitis E viruses from Western India belong to different genotypes

BACKGROUND/AIMS: Hepatitis E is endemic in India. Earlier, we showed prevalence of IgG antibodies to hepatitis E virus (IgG-anti-HEV) in different animal species and inability of at least one human hepatitis E virus (HEV) strain to infect pigs. In the US where hepatitis E is not endemic in humans, zoonotic spread of HEV was suspected as swine and human HEV were closely related and cross-species infection was documented. The present study attempts to identify and partially characterize swine HEV from India. METHODS: Serum samples from 284 pigs were screened for the presence of HEV-RNA (nested polymerase chain reaction; PCR) and IgG-anti-HEV (enzyme-linked immunosorbent assay; ELISA). PCR products (Open Reading Frame-2 region) were sequenced and subjected to phylogenetic analysis. Two sero-negative pigs were inoculated with swine HEV-positive serum pool. RESULTS: ELISA and PCR positivity were 42.9 and 4.6%, respectively. All Indian swine HEV sequences clustered with genotype IV. Pigs could be experimentally infected with swine HEV. CONCLUSIONS: Swine HEV circulates in Indian pigs. In contrast to US and Taiwan wherein both human and swine HEV isolates belong to same genotype, Indian human HEV isolates belong to genotype I whereas genotype IV circulates in swine. Though experimental infection with Indian swine HEV was possible, at least one human HEV strain could not infect pigs.     

Swine as a principal reservoir of hepatitis E virus that infects humans in eastern China

BACKGROUND AND METHODS: Genotype IV hepatitis E virus (HEV) has been isolated from humans and swine. To study the relationship between the human and swine reservoirs, we estimated their respective viral burden, analyzed the genetic makeup of the virus populations, and assessed the risk of infection associated with swine farming. RESULTS: In 2 swine-farming districts of eastern China, 9.6% of swine and 0.3% of healthy human subjects excreted HEV in stool, as did 68.8% of patients with confirmed HEV infection. The virus population circulating in humans consisted of genotype I and at least 4 phylogenetically distinct subgroups of genotype IV viruses, 2 of which concurrently circulated among swine. Persons engaged in occupations related to swine farming were found to have a 74% higher risk of infection than those engaged in other occupations, and persons living in communities downstream of the swine farms were found to have a 29% higher risk of infection than persons living in communities upstream. CONCLUSIONS: Genotype IV HEV is freely transmitted between humans and swine. Because the size of the swine population and its viral burden are much larger than those of humans, transmission of the virus most likely is directed from swine to humans. Infection can be acquired through contact with swine and their waste.     

糖尿病足感染中耐甲氧西林葡萄球菌的脉冲场凝胶电泳分型研究

目的研究分离白糖尿病足感染溃疡创面的耐甲氧西林金黄色葡萄球菌（MRSA）和耐甲氧西林表皮葡萄球菌（MRSE）的分子流行病学特点。方法收集2008年1月至2010年6月从天津医科大学代谢病医院388例糖尿病足溃疡住院患者中分离的24株MRSA和18株MRSE菌株,应用脉冲场凝胶电泳方法对菌株进行同源性分析。结果MRSA菌株相似系数为42％～97％,分为11型（A—K）,12株为A型克隆株（其中11株分离于2008年的1至5月）,包含12个亚型;B型2株;c型2株;其余8型各1株。MRSE菌株相似系数为34％～100％,分14型（A—N）,5株A型克隆株中3株A1亚型,A2、A3亚型各1株;其余13型各1株。结论本院糖尿病足病科2008年1至5月分离的MRSA菌株很可能有A型MRSA克隆株的暴发流行,2008年6月至2010年6月呈散发感染;2008年1月至2010年6月分离的MRSE菌株呈散发性,未发现有克隆株暴发流行。     

浙江省猪血清中乙脑病毒的分离鉴定及抗体的测定

目的对浙江嘉兴地区采集的猪血清进行乙型脑炎病毒分离鉴定以及了解该地区猪感染乙型脑炎病毒的状况。方法在浙江省嘉兴地区采集猪血清标本,利用病毒分离方法检测猪血清是否携带乙脑病毒,利用间接免疫荧光法对猪血清进行抗体检测。应用荧光定量RT PCR方法对新分离病毒进行鉴定;设计特异性引物,利用RT PCR方法扩增新分离乙脑病毒PrM-E基因,并对新分离病毒进行基因分型和E基因的分析。结果共采集240份猪血清标本,抗体检测阳性率为61.6%。其中6月中旬50%以上的猪乙脑病毒抗体呈阳性。分离出3株病毒。经荧光定量RT PCR鉴定3株病毒均为乙脑病毒,基因分析表明3株病毒均属于基因I型乙脑病毒。对这2株乙脑病毒的E基因区段进行分析,它们之间核苷酸和氨基酸的同源性均为100%。与疫苗株SA14 14 2相比,核苷酸同源性在87.7%,氨基酸同源在97.0%以上。结论浙江省嘉兴地区猪群中存着乙脑病毒的隐性感染或曾经感染过,提示在该地区自然界中存在着乙脑病毒。