华东和华南地区商品猪戊型肝炎病毒携带率和基因分型分析

目的 了解华东和华南地区商品猪戊型肝炎病毒(HEV)的携带率和基因分型.方法 2007-2009年收集华东和华南地区5家屠宰场商品猪胆汁600份,应用巢式RT-PCR方法检测HEVRNA,测序后做系统进化分析.结果 600份猪胆汁中有47份(7.83%)分离出HEVRNA.基于HEV ORF2区域内150-nt序列的系统进化分析结果,分离的47份HEV病毒株均属于Ⅳ型,与Ⅰ、Ⅱ、Ⅲ和Ⅳ型参考病毒株的核苷酸同源性分别为75.0%～83.4%、75.0%～84.6%、71.9%～80.7%和88.1%～91.5%,且大部分病毒株按采样地区来源表现出地区聚集性.结论 HEV在华东和华南地区的商品猪中广泛存在,应加强对商品猪食用安全问题的关注.     

中国南方某农村地区人与猪戊型肝炎病毒的检测和部分序列分析

目的调查中国南方某农村地区戊型肝炎病毒（HEV）人株与猪株的相关性。方法应用逆转录-巢式聚合酶链法（RT—nPCR）对一般人群中HEV-IgM阳性者、急性戊型肝炎患者和当地某养猪场的猪进行HEV RNA检测，并对HEV RNA阳性标本进行克隆测序和序列分析。结果132份猪粪便标本中13份为HEV RNA阳性；26份IgM阳性一般人群血清标本中有4份HEV RNA阳性；4例急性戊型肝炎患者中有1例的血清和粪便标本为阳性。序列分析发现该地区HEV人株与猪株在ORF2部分区域的核苷酸序列同源性为89．3％～100．0％，这10株HEV序列与HEVⅠ型、Ⅱ型、Ⅲ型和Ⅳ型在同一区域的核苷酸序列的同源性分别为78．7％～84．7％、83．3％～85．3％、76．0％～80．0％和84．7％～95．3％。结论该地区人群及猪群中流行的HEV同属基因Ⅳ型。     

中国发现基因3型戊型肝炎病毒

目的通过对中国华东地区分离出的2株基因3型戊型肝炎病毒株进行同源性分析，探讨其分子起源和传播问题。方法收集华东某地区13个养猪场的133份猪粪便标本，利用巢式RT—PCR技术扩增戊肝病毒基因组的开放读码区2（ORF2）和RNA依赖的RNA聚合酶区（RdRp，ORF1），并进行基因同源性分析。结果戊肝病毒RNA检出率为43．61％（58／133）。其中，在同一个养猪场检出的2份阳性标本与23条戊肝病毒基因3型全长序列在ORF2和ORF1区的同源性分别为77．10％～92．64％和77．49％～91．14％，被划分为基因3型。另外12个养猪场的56份阳性标本均为基因4型。同时发现，某些日本分离的基因3型病毒株比其他基因3型株更接近本次分离的病毒株。结论这是在中国大陆地区首次发现基因3型戊肝病毒。同时证明在一个较为局限的群体中基因3型和4型病毒可以共存。     

上海地区猪戊型肝炎感染状况及病毒序列分析

[目的] 调查上海地区猪戊型肝炎感染现况,掌握上海市猪感染戊型肝炎病毒的型别,以进一步探讨猪戊型肝炎感染与病人感染的可能关系。[方法]采集上海市3个区的不同季度的3月龄猪血样,用ELISA法检测抗-HEV 特异性抗体水平,并用RT-nPCR方法检测猪粪便中HEV病毒,进行RNA核酸序列分析和基因进化树分析。 [结果] 共检测猪血清标本1 798份,HEV抗体阳性率为89．38％;44份猪粪便样品中17份RT、-nPCR为阳性,HEV RNA阳性率为38．64％,病毒序列经同源性分析,与戊型肝炎病毒I、Ⅱ、Ⅲ、Ⅳ型的同源性分别为78．5％-84．0％、76．5％-80．7％、 77．3％-82．7％、84．6％-90．7％,基因进化树的分析显示,病毒序列与HEVⅣ型的ⅣA形成同一分支。[结论] 上海地区的猪戊型肝炎感染率较高,戊型肝炎病毒型别属基因型Ⅳ型。     

杭州市散发性戊型肝炎基因型别分析研究

目的了解杭州地区急性散发性戊型病毒性肝炎患者基因型特点。方法采用病毒核酸提取方法进行基因提取,荧光定量PCR方法筛选,选取代表株进行克隆测序,利用生物信息学软件进行多序列比对排列及构建进化树。结果对78份标本进行检测,经荧光PCR检测16份阳性,阳性率为20.51%。3株代表株经克隆测序,并进行BLAST比较和进化树分析,均属HEVⅣ型,与国内其他地区分离的Ⅳ型毒株核苷酸同源性为84.6%~96.7%。结论杭州地区的急性散发性戊型病毒性肝炎病毒属于Ⅳ型。     

马鞍山地区不同人群戊型肝炎病毒基因型初步分析

目的了解马鞍山地区不同人群中戊型肝炎病毒（HEV）流行株基因型的分布状况。方法对来自一般人群、有偿献血人群、吸毒人群的血清标本进行HEV IgG、IgM抗体检测并将HEV IgM阳性的血清标本进行RT-PCR扩增,PCR阳性标本进行基因测序及序列分析。结果 HEV PCR扩增成功16份,其中HEV IgM抗体阳性病人和正常人群中10份标本阳性,吸毒人群和有偿献血人群中各3份标本PCR阳性,测序结果显示均为HEVⅣ型毒株感染。结论马鞍山地区不同人群中HEV流行株均为基因Ⅳ型,但不同人群内部的流行株存在较大变异。     

北京市2008年肠道病毒71型VP1编码区基因特征分析

目的了解北京市2008年手足口病（Hand,Foot and Mouth Disease,HFMD）流行期间,人肠道病毒71型（Human Enterovirus71,HEV71）分离株的VP1编码区基因特征。方法采集发病1～3d的咽拭子标本33份,采用人横纹肌肉瘤（Human Rhabdomyosarcoma,RD）细胞进行EV分离,用特异引物通过逆转录-聚合酶链反应扩增进行毒株鉴定。对分离到的HEV71进行VP1编码区全长扩增,并对扩增产物采用双脱氧链终止法进行序列测定,Bioedit7.0.5和Mega3.1软件进行序列比对和系统进化树分析。结果从33份咽拭子标本中共分离到16株病毒,经鉴定：HEV7114株,柯萨奇病毒（Coxsackie Virus,CV）A组16型（CVA16）2株,其中1例为HEV71和CVA16混合感染。对10株HEV71进行了VP1编码区全序列测定,10株病毒的核苷酸和氨基酸序列同源性分别为95.5%～100%和98.9%～100%;与安徽省阜阳市2008年流行株的核苷酸和氨基酸序列同源性分别为95.4%～99.1%;与C4基因型代表株的核苷酸序列同源性〉92%。基于HEV71的VP1编码区核苷酸序列构建亲缘性关系树,北京市2008年流行株与C4亚型代表株聚为一簇,属于C4基因亚型中的C4a分支;且10株病毒处于四个相对独立的进化分支。结论北京市2008年从HFMD患儿分离的HEV71均为C4亚型,且属于2004年以来我国的优势分支——C4a。亲缘性分析提示,此次流行中至少存在4个HEV71传播链。由于近年来HEV71在中国持续大规模流行,迫切需要对HEV71进行连续的分子流行病学监测,及时阐明现阶段流行毒株的基因特征,为疾病预防控制提供病毒学依据。     

上海市部分地区散发性戊型肝炎病毒基因型别和发病危险因素研究

目的探讨上海市部分地区急性散发性戊型肝炎（戊肝）病毒（HEV）型别和发病危险因素.方法用巢式RT-PCR方法检测HEV序列,并进行序列的同源性比较分析;同时采取1：2病例对照研究的方法,选取上海市3个区2003-2004年急性散发性确诊戊肝86例住院病例,分别配以本区和其他区健康人群对照组,用单因素和多因素logistlc回归模型进行分析.结果病毒序列分析表明戊肝病例中的HEV病毒序列属于Ⅳ型;单因素分析结果显示居住条件、外出就餐、有海鲜河鲜食用史等均为戊肝发病的危险因素;多因素分析结果显示海鲜（生、炝）食用史（OR=7.048）是戊肝感染 的危险因素.结论上海市部分地区散发性HEV流行株以Ⅳ型病毒株为主,海鲜（生、炝）食用史等是戊肝发病的危险因素.     

华东地区猪基因4型戊肝病毒的季节变化及与人同源性的分析

目的 戊型肝炎病毒的人兽共患特征被认可,本研究检测华东地区猪群在不同季节中基因4型戊肝病毒(HEV)的感染情况,了解猪病毒株之间以及与人病毒株的同源性关系.方法2007年9月-2008年6月在华东地区浙江、安徽和江苏的3家屠宰场中共采集猪胆汁标本1200份,应用巢式RT-PCR方法检测HEV RNA,对阳性标本测序并结合同一地区人HEV序列进行同源性分析.结果猪胆汁标本中HEV RNA总检出率为4.5%,各季节猪群的检出率为9-10月平均检出率6%,12-1月4.33%,3-4月4.33%,5-6月3.33%,各地区的平均检出率为江苏6%、安徽5%、浙江2.5%.不同地区猪与猪、猪与人基因4型HEV病毒株的同源性都较高(猪株之间核苷酸序列约为80%～100%,氨基酸序列约为96%～100%;猪与人株之间核苷酸序列约为76%～99%,氨基酸序列约为95%～100%).部分猪HEV与人HEV构成独立的同源性分支,进化起源关系密切.结论 基因4型HEV在华东地区猪群中常年广泛流行,且可能拥有共同的进化与传播起源.猪群携带HEV可能会对人群戊肝的流行趋势产生影响.     

新疆猪戊型肝炎病毒株CHN-XJ-SW33全基因组序列分析

目的测定从新疆南部地区分离的猪戊型肝炎病毒(HEV)株全基因组序列,并在此基础上分析猪HEV与人源HEV的关系。方法设计HEV基因4型通用PCR引物,用逆转录巢式聚合酶链反应法(RT-nPCR)分段扩增猪HEV株CHN-XJ-SW33的全基因组序列;用cDNA末端快速扩增法(RACE)扩增其末端序列;对扩增的目的片段进行克隆测序,并对拼接后的基因组进行序列比对和进化分析。结果除3′poly(A)尾外,CHN-XJ-SW33基因组全长为7 238 nt,由3个开放读码框(ORF1-3)组成,分别编码1 706、674和114个氨基酸。CHN-XJ-SW33全基因组序列与HEV基因1-3型病毒株同源性仅为72.1%~74.9%,而与HEV基因4型病毒株同源性高达82.8%~95.5%,其中与日本人源中国输入型HEV株JKO-ChiSai98C同源性最高,为95.5%。基因进化分析显示,CHN-XJ-SW33属于HEV4a基因亚型。结论猪HEV与人HEV在全基因组核苷酸序列上高度同源,提示基因4型HEV可能由猪传染给人。     

深圳地区HEV流行基因型及动物宿主带毒状况分析

目的了解深圳地区戊型肝炎病毒(HEV)流行基因型及动物宿主带毒状况。方法通过同源比对HEV四种基因型的戊型肝炎病毒序列,选取其保守序列部分设计两对巢式聚合酶链式反应的简并引物,建立一种可同时扩增戊型肝炎四个基因型病毒RNA的PCR快速检测方法。对深圳市采集的80份猪粪便标本和6份戊型肝炎病人血清,提取病毒RNA,然后进行PCR检测,阳性时对扩增片段进行基因序列测定,并进行基因型分析。结果80份猪粪便标本5份HEV—RNA阳性,6份病人血清样本中4份阳性。序列分析显示全部为HEV基因IV型,5份猪标本中的HEV基因序列与GenBank中人HEV的基因序列最相似,同源性为88%～94%。结论深圳地区HEV流行株为基因IV型,而且猪HEV基因序列与人HEV之间有着高度的相似性,证实猪是HEV的宿主,戊型肝炎是一种人兽共患传染病。     

Phylogenetic analysis of 626 hepatitis E virus (HEV) isolates from humans and animals in China (1986-2011) showing genotype diversity and zoonotic transmission

Hepatitis E is considered as a public health problem in China. To determine the overall molecular epidemiology of hepatitis E virus (HEV) and analyze the situation of cross-species transmission between humans and swine in China over the last 25 years (1986-2011), 626 HEV complete and partial sequences (89 isolates identified by our group) isolated from humans and animals in China were retrieved from GenBank and subjected to phylogenetic analysis. There were three genotypes and 11 sub-genotypes of HEV prevailing in China. Furthermore, rabbit HEVs, of which the genotype is controversial, are also widespread in China. Genotype 1 was the most isolated genotype prior to 2000 and mainly detected in Xinjiang, Beijing and East China. However, genotype 4, which was identified in most regions of China during the last 10 years, has overtaken genotype 1 in frequency of isolation nationwide. Genotype 3 HEV strains have been found only in eastern China and were thought to be imported from Japan. Both genotypes 3 and 4 were found in humans and swine and cross-species transmission from pigs to humans of the two genotypes may have occurred in Northeast, Northwest, North, East and South China. These results indicate that HEV strains with considerable genetic diversity are widespread and the zoonotic transmission between swine and humans appears ubiquitous in China.     

Comparison of hepatitis E virus genotypes from rabbits and pigs in the same geographic area: No evidence of natural cross-species transmission between the two animals

Domesticated pigs have been shown to be a reservoir of genotypes 3 and 4 hepatitis E virus (HEV). Farmed rabbits were recently recognized as the host of a novel virus, rabbit HEV. In order to determine whether HEV is transmitted naturally between rabbits and pigs, a survey on HEV infections was conducted in rabbits and pigs aged 2–4 months from rabbit and pig farms located near to each other in nine villages in three counties of Hebei Province, China. The overall anti-HEV antibody positivity rates in serum samples of swine and rabbits were 61.7% (58/94) and 23.2% (67/289), and the positive rates for HEV RNA were 23.4% (22/94) and 10% (29/289), respectively. In addition, 37 of 125 swine fecal samples (29.6%) were HEV RNA positive. The nucleotide sequences of a 304 bp region within HEV ORF2 have identity ranging from 84.5% to 100% among the rabbit isolates and from 82.3% to 100% among the swine isolates. In contrast, the nucleotide identity between the two species groups was only 72–76.6%. Consequently, the two groups were clearly separated in the phylogenetic tree that showed all of the rabbit isolates are closely related to the rabbit HEV reported recently and the swine isolates belong to genotype 4, including subgenotypes 4a, 4c and 4d. The results showed that HEV is highly prevalent in farmed rabbits and pigs in these areas. However, genotype 4 HEV and rabbit HEV are circulating separately in pigs and rabbits in the same area. In conclusion, there was no evidence of cross-species transmission of HEV between pigs and rabbits. The frequency of HEV transmission events between these two animal species is likely low in commercial farms.     

Genotype analysis of hepatitis E virus from sporadic hepatitis E cases in northern China

Hepatitis E is an important public health problem in many countries. However, there is no definite conclusion about the zoonotic reservoir, transmission patterns and risk factors of hepatitis E in the human population. The aim of this study was to analyze the epidemiological and viral genotype characteristics of hepatitis E cases in northern China. Surveillance was conducted in two hospitals in Liaoning and Hebei province from July 2010 to June 2012. Out of a total of 116 diagnosed patients, 88 (75.9%) were male and 28 (24.1%) were female and most (73%) were in the age group 40–70 years. In both hospitals, cases were diagnosed more frequently in March than in other months. HEV RNA was amplified from 41 patients and characterized by nucleotide sequencing and phylogenetic analysis. Most of the isolates (37 strains, 90.3%) were genotype 4, including subgenotypes 4a, 4b, 4d, 4h, 4i and a new subgenotype. One subgenotype 3a strain was isolated from Baoding, Hebei province. Three genotype 1b strains were found from patients in Jinzhou, Liaoning province. Most of the genotype 4 strains and the genotype 3 strains were phylogenetically related to known swine isolates. In conclusion, the finding that HEV infects mostly middle-aged and elderly men and that the incidence spiked in March may reflect the zoonotic transmission characteristics of HEV infection. Pigs, but not rabbits, were the important reservoirs in this area, because genotype 4 HEV was found to be responsible for the majority hepatitis E cases. However, genotype 1 is still present in northern China. Also, the first isolation of genotype 3 HEV in this area indicates that alternative routes of HEV transmission might exist.