猪戊型肝炎病毒荧光定量PCR检测方法的建立及其分子流行病学

对已发表的戊型肝炎病毒(HEV)不同基因型毒株全序列进行分析,针对ORF3的保守区设计引物和探针优化建立了HEV荧光RT-PCR检测体系。分析表明该反应体系具有良好的扩增效率、特异性和稳定性,且检测灵敏度比普通RT-PCR方法高10～100倍。利用所建立的荧光定量PCR方法对采集自浙江及其周边上海、江苏等地区规模化猪场的样品以及HEV抗体阳性人血清样品进行了HEV核酸检测。其中,77份人血清样本仅有2份为HEV核酸阳性(2.6%)。而猪粪便样品阳性率则高达20.5%(56/273),且所调查的猪场均存在HEV感染。进一步的分子流行特征分析表明,浙江及周边地区的猪HEV毒株多为基因Ⅳ型,与其他地区的Ⅳ型HEV毒株同源性较高(83.4%～89.5%),但它们之间也存在较多的变异位点。猪源HEV毒株PJ-1则与多数Ⅲ型HEV毒株同源性较高(88%),进化分析也表明其为基因Ⅲ型。人血清样品中检测到的2份HEV毒株Human-1与Human-2与本地区Ⅳ型猪源毒株在分子进化关系上具有很高的亲源性,它们分布于同一分支内,而不构成独立分支。以上分析结果表明浙江及其周边地区猪HEV感染较为广泛,且以基因Ⅳ型毒株为主,但也存在...     

我国鸡群禽戊型肝炎病毒RT-PCR检测及部分ORF2基因序列分析

为从核酸水平证实我国鸡群中禽戊型肝炎病毒(HEV)的存在,本实验从山东省某鸡场患有肝脾肿大综合征的病鸡中采集10份粪便和8份胆汁样品,利用RT-PCR方法检测其中禽HEV ORF2基因片段,并将阳性PCR产物克隆测序。结果显示:18份粪便和胆汁样品中,13份为禽HEV RNA阳性;其序列间的同源性为97%～99%,与GenBank中登录的参考序列同源性为76.6%～98.1%;进化树显示与欧洲地区的禽HEV在同一分支,属于禽HEV基因3型。禽HEV ORF2基因的检出为进一步了解禽HEV对我国家禽养殖业的危害以及在我国的流行情况奠定了基础。     

四川地区猪源戊型肝炎病毒感染的分子流行病学调查

为了解四川地区猪源戊型肝炎病毒(hepatitis E virus, HEV)的感染情况,采集四川13个地区屠宰场的358份生猪组织样品,采用荧光RT-PCR进行HEV检测,对阳性样品进行开放阅读框(open reading frame, ORF)两部分序列测序、同源性及系统进化分析。结果显示:猪感染HEV的阳性率为4.75%,17个毒株ORF2部分序列之间同源率为85.4%～99.4%,均属于基因4型,其中4a亚型8株、4b亚型3株、4d亚型6株。与人、猪等不同宿主的HEV参考毒株比较,四川猪源HEV与我国主要流行的人源HEV遗传关系密切,其中10株与人源毒株同源率超过95%。结果表明:四川地区生猪HEV流行毒株以基因4型为主,具有较大的人畜共患风险,应有针对性、系统性地对其进行预防和控制。     

2015年-2016年度南宁部分地区猪戊型肝炎病毒基因型分析

为了解广西猪群中戊型肝炎病毒(HEV)基因型,利用套式RT-PCR用对南宁周边猪场采集到的104份新鲜猪粪便进行HEV检测、分离并成功扩增和克隆了11株阳性毒株的ORF2基因部分保守片段.序列测定结果表明,11株HEV ORF2序列自身核苷酸同源性为97.7%~100%,推导编码的氨基酸同源性为97.9%~100%;与4型HEV代表毒株Ch-S-1、Ch-T11、swGX40等的核苷酸同源性为84.6%~90.6%,而与其他各型之间的同源性较低.系统发育进化树结果表明,11株HEV广西株为基因4型,其中,10株HEV为4a亚型,1株毒株为4e亚型.结果表明,广西猪群中流行的HEV均为基因4型,且以4a亚型为优势流行毒株.     

河南省猪场戊型肝炎病毒流行病学调查

为了解河南地区猪戊型肝炎感染情况,同时掌握该地区猪感染戊型肝炎病毒的基因型,对来自河南新乡、平顶山、南阳等地区不同商品猪场血清、肝脏、粪样品,采用ELISA技术检测猪血清中抗HEV特异性抗体水平,并使用套式RT-PCR检测肝脏和粪便中猪戊型肝炎病毒核酸。结果显示530份样品中HEV抗体阳性率0.6%,HEV RNA均为阴性,推测目前河南省猪戊型肝炎病毒感染率较低。     

东北边境地区野猪及放养杂交野猪戊型肝炎流行病学调查

为了解东北边境地区野猪及放养杂交野猪群体猪戊型肝炎病毒(HEV)感染情况,于2015—2018年在吉林省、黑龙江省的中朝、中俄边境和内蒙古自治区境内加格达奇周边地区采集6月龄以上杂交野猪血清、粪便或肛拭子样品共520份,采集野猪血清和粪便样品共248份。ELISA检测、RT-nPCR检测、全基因组测序、同源性及进化分析结果显示,杂交野猪和野猪感染HEV的血清抗体总阳性率为34.1%(136/399);核酸总阳性率为1.56%(12/771),12份核酸阳性样品均来自杂交野猪,病毒基因组ORF2部分核苷酸序列同源性为85.4%～100.0%,属于基因4型,4a、4b亚型。对4a亚型的1份阳性样品(LJG-18)进行病毒全基因组扩增测序,其核苷酸序列与日本的人源毒株JKO-ChiSai98C同源性最高,为94.9%,与吉林省猪源毒株Ch-S-1同源性为90.2%。结果表明:东北边境地区放养杂交野猪群具有较高的HEV血清抗体阳性率,HEV流行毒株以4a亚型为主。本试验针对我国野猪及放养杂交野猪群体开展猪戊型肝炎流行病学调查,为该病的流行情况提供了新的科学数据,对我国养猪业健康发展和公共卫生安全具有重要意义。     

四川省甘孜州藏猪戊型肝炎病毒分子流行病学调查

为了解甘孜州藏猪戊型肝炎病毒(HEV)的感染情况，分析其相似性和遗传进化关系。本试验用反转录套式聚合酶链反应(RT-nPCR)方法对采集于2018—2019年甘孜藏族自治州藏猪主要养殖区域4个县10个藏猪养殖场的229份藏猪粪便样本进行核酸检测，并对全部阳性样本进行ORF2基因部分片段扩增测序，遗传进化分析。结果显示：猪粪便HEV核酸阳性率为16.59%(38/229,95%CI=12.00%～22.10%),猪场HEV阳性率为70%(7/10,95%CI=34.8%～93.3%);38个ORF2基因片段彼此之间核苷酸序列相似性为76.1%～100.0%,推导的氨基酸序列相似性为79.6%～100.0%,系统进化树分析显示所有阳性毒株均为基因4型，且与人源HEV毒株亲缘关系近。结果表明甘孜州藏猪中存在一定程度的HEV感染，与人源HEV毒株遗传关系近，相关部门采取防控措施以降低HEV感染的公共卫生风险。     

湖南地区猪戊型肝炎流行病学调查研究

为了解湖南地区猪戊型肝炎感染情况,同时掌握该地区猪感染戊型肝炎病毒的基因型,以便进一步探讨猪戊型肝炎病毒在猪不同组织中的含量分布及组织损伤,采用ELISA技术检测猪血中抗-HEV特异性抗体水平,并用RT—nPCR检测猪粪便中HEVRNA和进行RNA核苷酸序列分析。结果表明,猪血中HEV抗体阳性率为84.3％;猪粪便样品中HEVRNA阳性率为28．9％。所得5株HEV的ORF2部分核苷酸序列与戊型肝炎病毒Ⅰ、Ⅱ、Ⅲ、Ⅳ型的同源性分别为80．7％～84．O％、78．7％。80．7％、77．3％～82．7％和86．7％～90．7％,其中与Chi-xinjiang株同源性为83.3％～84．0％,与Chi—ⅣT1亚型的同源性为87．7％～90．7％。湖南地区猪戊型肝炎感染率比较高,且戊型肝炎病毒的基因型属Ⅳ型。     

禽戊型肝炎病毒人兽共感染的可能性研究

戊型肝炎是由戊型肝炎病毒(Hepatitis E Virus,HEV)引起的急性病毒性肝炎,主要经肠道传播,临床表现与甲型肝炎类似,主要流行于亚洲、非洲和中关洲的发展中国家.急性肝损坏和散发急性肝炎病例中50%以上由戊型肝炎引起.这使戊型肝炎成为一个重要的公共卫生问题.随着猪HEV的发现,禽HEV于2001年由美国的Meng博士领导的研究室发现和鉴定.在过去的几年里,我们与Meng博士研究室合作对禽HEV结构蛋白(ORF2)的B细胞抗原表位做了较系统的研究,鉴定出两个中和抗原表位,证明了禽HEV ORF2基因重组抗原免疫后能够产生中和抗体并可以防止禽HEV的感染.我们同时阐明了在禽HEV ORF2抗原内至少有一个禽HEV特异的抗原表位,一个人、猪和禽HEV三种病毒共有的抗原表位,至少有一个禽、人HEV共有的抗原表位.这些研究结果表明禽HEV ORF2蛋白的抗原表位可以作为有效的免疫诊断抗原,用来鉴别人和禽HEV感染.我们最近对健康人群HEV血清抗体检测结果表明抗禽HEV ORF2抗体阳性率约为1%,其中约4.5%的人群血清中含抗人HEV抗体.在对鸭群的血清学检测证明健康鸭群的抗禽HEV ORF2抗体阳性率约为20%.这些研究结果提示人可能被禽HEV感染.     

陕西西安蛋鸡禽肝炎脾大综合征HEV基因3型病毒分离与鉴定

2019年9月,陕西西安市某海兰褐蛋鸡场场出现产蛋率下降,死淘率升高,剖检部分死亡鸡只表现肝脾肿大且易脆破裂,为弄清该鸡场疫情的病因。我们采集了鸡血清32份,胆汁13份,粪便、肝脏和脾脏各11份,然后利用间接ELISA检测血清中是否存在禽HEV抗体,巢式RT-PCR检测粪便、肝脏、脾脏和胆汁样品中是否存在禽HEV核酸。间接ELISA检测结果发现,28份鸡血清禽HEV抗体阳性,阳性率为87.5%;巢式RT-PCR检测结果发现,6份胆汁,5份粪便,3份肝脏和脾脏样品为禽HEV RNA阳性。阳性PCR产物测序后基因序列同源性分析发现,获得的禽HEV ORF2部分基因序列相互之间同源性为99%～100%,与GenBank已知的禽HEV序列同源性在78%～99%,进化树分析表明分离的禽HEV与国内多数禽HEV分离株以及欧洲部分分离株在同一分支上,属于基因3型。上述临床剖检病变和实验室检测技术说明,该鸡场存在禽HEV的感染,此次疫情可能主要是由该病毒感染引起的。     

基因4型HEV上海株ORF_2编码蛋白表达及其抗原性分析

为了表达基因4型戊型肝炎病毒(HEV)上海株ORF2编码蛋白并分析其抗原性,采用套式RT-PCR方法扩增上海猪基因4型HEV代表株结构蛋白基因ORF2部分片段,构建含有该基因片段的pET30a(+)重组表达质粒,命名为pET-E;将重组表达质粒转化大肠杆菌BL21,经1mmol/L IPTG诱导得到表达,进行SDS-PAGE分析,然后用Western blot鉴定其抗原性,最后纯化蛋白。结果:SDS-PAGE证明该片段得到融合表达,分子量为33 ku。Western blot分析表明,重组蛋白可以与HEV阳性血清反应,表明该蛋白具有良好的抗原性;该融合蛋白含有组氨酸标签,可以用MagExtractor-His-tag-kit进行纯化。用纯化的重组蛋白作为包被抗原建立的间接ELISA检测89份猪血清样品,阳性率达80.4%,与已有的试剂盒检测结果相比,阳性符合率达95%。     

猪戊型肝炎病毒大庆株DQ1全基因序列分析

本试验采用RT．-PCR的方法对戊型肝炎病毒大庆株（DQ1HEV株）的全基因进行分片段扩增，并对其两个末端采用末端快速扩增法（RACE）进行扩增、克隆，测序。与已报道的人的14株HEV的4个基因型的核苷酸和氨基酸进行比较，与IV型HEV的同源性最高。ORF1区与IV型HEV核苷酸的同源性为82．6％～83．6％．氨基酸同源性为93．5％，ORF2区与IV型HEV核苷酸的同源性为87．0％～88.4％，氨基酸同源性为95．8％～97．4％。ORF3区与IV型HEV核苷酸的同源性为94．4％～96．5％，氨基酸同源性为90.3％～96．5％。其结果表明DQ1 HEV株为IV型HEv。     

猪群HEV血清抗体调查及一株新的猪HEV ORF2部分基因序列分析

戊型肝炎病毒(Hepatitis E virus,HEV)主要引起人的戊型肝炎,新近研究发现猪在病毒传播中可能发挥重要作用.本研究对我国部分省区HEV感染血清学调查,在被检的1 138份血清中,有666份(57.5%)为HEV抗体阳性,猪群抗体阳性率随着月龄增长而升高.通过RT-PCR方法从一份猪粪中扩增并克隆了HEVORF2 N端主要抗原决定区339bp基因片段,序列分析显示,该段基因与我国人群HEV基因4型毒株核苷酸序列同源性为85.9%,但氨基酸序列完全一致.这一结果提示我国猪群存在广泛的HEV感染,并在一定程度上与人群HEV毒株密切相关.     

Hepatitis E virus: Animal reservoirs and zoonotic risk

Hepatitis E virus (HEV) is a small, non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2 kb in size. HEV is classified in the family Hepeviridae consisting of four recognized major genotypes that infect humans and other animals. Genotypes 1 and 2 HEV are restricted to humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 HEV infect humans, pigs and other animal species and are responsible for sporadic cases of hepatitis E in both developing and industrialized countries. The avian HEV associated with Hepatitis-Splenomegaly syndrome in chickens is genetically and antigenically related to mammalian HEV, and likely represents a new genus in the family. There exist three open reading frames in HEV genome: ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein, and the ORF3 encodes a small phosphoprotein. ORF2 and ORF3 are translated from a single bicistronic mRNA, and overlap each other but neither overlaps ORF1. Due to the lack of an efficient cell culture system and a practical animal model for HEV, the mechanisms of HEV replication and pathogenesis are poorly understood. The recent identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by these animal strains raise potential public health concerns for zoonotic HEV transmission. It has been shown that the genotypes 3 and 4 HEV strains from pigs can infect humans, and vice versa. Accumulating evidence indicated that hepatitis E is a zoonotic disease, and swine and perhaps other animal species are reservoirs for HEV. A vaccine against HEV is not yet available.     

应用HEV ORF2蛋白McAb间接免疫荧光法检测HEV的研究

应用抗猪戊型肝炎病毒（HEV）ORF2蛋白单克隆抗体和猪戊型肝炎病毒多克隆抗血清对感染猪戊型肝炎病毒的A549细胞培养物进行了间接免疫荧光检测病原的比较研究。单克隆抗体（McAb）进行的间接免疫荧光染色后,HEV感染细胞培养物中多数细胞在其胞浆和细胞膜上出现黄绿色荧光,未接种病毒的细胞培养物被伊文思蓝染成红色,未见有黄绿色荧光染色的细胞存在;多克隆抗血清对接种病毒的细胞培养物染色后,荧光强度高,呈现亮绿色,但未接种HEV的细胞培养物亦有一定的非特异性荧光反应。结果表明,利用所研制的抗HEVORF2蛋白McAb进行间接免疫荧光法对病原检测具有很高的特异性。     

Hepatitis E virus infection of slaughtered healthy pigs in Brazil

Hepatitis E virus (HEV) is an emerging pathogen that can be transmitted through contaminated raw or undercooked meat derived from domestic pigs. HEV infections have been documented among pig herds, pig products and environmental samples raising concern about the spread of the virus. HEV genotypes 3 and 4 are considered zoonotic and have been linked to human cases. HEV was detected in 51 of 335 bile samples (15.2%) from healthy pigs in Minas Gerais, Brazil. Phylogenetic analysis of partial sequences from ORF1 and ORF2 regions yielded discordant results, assigning isolates to subtypes 3c and 3i, respectively, suggesting intragenotypic HEV recombination.     

Use of heteroduplex mobility assays (HMA) for pre-sequencing screening and identification of variant strains of swine and avian hepatitis E viruses

Hepatitis E virus (HEV), the causative agent of human hepatitis E, is an important public health problem in many developing countries and is also endemic in many industrialized countries including the US. The discoveries of avian and swine HEVs by our group from chickens and pigs, respectively, suggest that hepatitis E may be a zoonosis. Current methods for molecular epidemiological studies of HEV require PCR amplification of field strains of HEV followed by DNA sequencing and sequence analyses, which are laborious and expensive. As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. In this study, we developed two heteroduplex mobility assays (HMA) (one for swine HEV based on the ORF2 region, and the other for avian HEV based on the ORF1 region) to genetically differentiate field strains of avian and swine HEVs from known reference strains. The ORF2 regions of 22 swine HEV isolates and the ORF1 regions of 13 avian HEV isolates were amplified by PCR, sequenced and analyzed by HMA against reference prototype swine HEV strain and reference prototype avian HEV strain, respectively. We showed that, in general, the HMA profiles correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Field isolates with similar HMA patterns generally showed similar sequence identities with the reference strains and clustered together in the phylogenetic trees. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies.     

Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb

Strains of hepatitis E virus (HEV) genotypes 1 and 4 have been detected on the Chinese mainland although there have been no previous reports of zoonotic genotype 3 HEV. In the present study, 65 swine fecal specimens were collected from five pig farms located in different Shanghai suburbs. RT-PCR and nested PCR were undertaken using partial nucleotide sequences of Open Reading Frame 2 (ORF2) of HEV to detect HEV RNA. Genetic analysis was based on alignments of an amplified 150-nt ORF2 sequence. RT-PCR revealed 15 HEV positive samples among 65-pig fecal specimens examined. Phylogenetic analysis of the amplified sequences indicated seven HEV strains belonged to genotype 3 and eight strains to genotype 4. This is the first time that genotype 3 hepatitis E virus has been identified on the Chinese mainland.     

猪戊型肝炎病毒ORF2片段克隆及原核表达

设计套式引物，内套引物含有BamHⅠ／XhoⅡ酶切位点，采用RT-PCR技术扩增猪戊型肝炎病毒（UEV）结构蛋白基因ORF2部分片段，长度为634bp，将其克隆于PGEX-T载体，测序结果表明插入的片段属于戊型肝炎病毒ORF2部分，将该片段插入PproEXHTb表达载体，经酶切鉴定证实获得了重组表达质粒。将重组质粒转化大肠杆菌BL21，经1mmol／L IPTG诱导，得到表达，表达蛋白分子量为27Ku，以包涵体形式存在。Western blot分析表明，该蛋白可以与猪戊型肝炎阳性血清反应，表明该蛋白具有良好的反应原性。