贵州省2019-2021年人源和猪源戊型肝炎病毒基因特征

目的 分析贵州省部分地区人源和猪源戊型肝炎病毒(Hepatitis E virus,HEV)流行株基因特征。方法 2019-2021年在贵州省医疗机构收集急性HEV感染者(抗-HEV IgM阳性)血清标本,在屠宰场收集猪胆汁和血清标本,提取HEV核酸,对HEV ORF2区基因进行扩增和测序,构建系统进化树,分析基因型、基因亚型及其核苷酸和氨基酸同源性。结果 从450例急性HEV感染者标本和196份猪标本中共获得53条人源和2条猪源HEV序列。人源序列的核苷酸和氨基酸同源性分别为81.39%-100%和95.92%-100%,猪源序列分别为87.40%和100%;人源和猪源HEV均与HEV基因4型参考株的核苷酸同源性最高,分别为89.93%和89.83%。55条HEV序列位于系统进化树的3个分支,其中24条与4b亚型、20条与4a亚型、11条与4d亚型参考株处于同一分支,且核苷酸同源性最高,分别为95.54%、93.10%、95.72%。2条人源序列各发生1个位点的氨基酸替换。结论 2019-2021年贵州省部分地区人群和猪群中HEV优势流行株为基因4型,包括4b、4a和4d亚型,氨基酸...     

广西地区动物HEV基因型及亚型分布

目的 了解广西地区猪、鼠、狗、猕猴和鱼5种动物戌型肝炎病毒(HEV)基因型和亚型分布.方法 对广西地区猪、鼠、狗HEV抗体阳性血清170份,鼠肝组织150份,3月龄以下狗粪便标本80份,鱼胆汁标本120份,3月龄以下猪粪便标本129份,龙虎山自然景区猴粪便标本130份,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(HEV RNA),对RT-nPCR阳性扩增产物进行克隆测序,用Vector NTI Suite 9.0和Treeview软件与目前新分型方法所选参考序列进行核苷酸序列相似性比较和生物进化树分析.结果 广西地区170份猪、鼠和狗血清HEV RNA检测均为阴性.在150份鼠肝组织,120份鱼胆汁标本,80份狗和130份猕猴粪便标本中均未检出HEV RNA;3月龄以下猪粪便标本HEV RNA阳性率10.08(13/129).对13株猪HEV开放读码框架(ORF)1区部分核酸序列进行分析,其与Ⅰ、Ⅱ、Ⅲ和Ⅳ型HEV的相似性分别为73%～77%,76%～79%,76%～81%,82%～97%;与6个亚型的相似分别为4a:82%～94%;4b:82%～98%;4c:82%～87%;4d:81%～87%;4f:84%～86%;4g:81%～84%,其中2株与N a亚型相似性是高为82%～94%,11株与Ⅳ b亚型相似性最高为82%～98%.结论 广西地区猪HEV均为基因型4,2株属于基因型Ⅳ a,Ⅱ株为Ⅳb.HEV在猪、鼠、狗血清、鼠肝组织、鱼胆汁、狗和猕猴粪便标本中均未检出.     

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.     

北京市郊区八种动物戊型肝炎病毒抗体流行率调查及病毒基因型研究

目的 调查北京地区与人密切接触的猪、牛、羊、马、驴、犬、鸡、鸭8种动物血清中戊型肝炎病毒(HEV)抗体流行率及该地区猪HEV基因型和亚型.方法 收集8种动物的血清标本及幼猪粪便标本,用双抗原夹心ELISA法检测血清中抗-HEV;用巢式反转录聚合酶链反应法(RT-nPCR)检测HEV RNA;对部分PCR产物进行克隆和测序,并对测序结果进行基因分型.结果 在8种动物中抗-HEV阳性率分别为猪80.43%(481/598),其中成猪(>6月龄)为87.86%(369/420),幼猪(2～3月龄)为62.92%(112/178);牛15.02%(52/346);马14.29%(40/280);驴0(0/26);羊9.88%(33/334);犬0(0/21);鸭3.03%(7/231);鸡2.53%(8/316).RT-nPCR检测3月龄以下猪粪便标本HEVRNA阳性率为66.67%(74/111).HEV RNA阳性的标本测序后可归为6株(分别命名为bjsw1、bjsw2、bjsw3、bjsw4、bjsw5和bjsw6),6株HEV开放读码框2(ORF2)部分核苷酸序列的相似性为94.5%～99.6%,与1、2、3、4型HEV的相似性分别为75.6%～78.6%、75.6%～76.2%、77.1%～80.7%和83.7%～94.5%.6株HEV与人HEV 4d亚型的同源性最高,为90.0%～94.5%.结论 北京市郊区猪、牛、马、羊、鸭、鸡中均存在HEV感染,其中猪抗-HEV流行率最高,驴、犬抗-HEV的流行率最低;6株猪HEV属于基因4型、4d亚型.     

两种核酸检测方法在猪源与人源戊型肝炎病毒监测中的应用

目的评价两种核酸检测方法在猪源与人源的戊型肝炎病毒(HEV)监测应用的可行性。方法收集江苏地区急性戊肝病例血液标本和屠宰场生猪胆囊标本,采用Real-time RT-PCR法和RT-PCR法进行RNA检测,对阳性检出情况和核酸序列比对结果进行分析。结果两种方法监测急性戊肝病例和猪胆囊标本的结果完全一致,患者血清HEV RNA检出阳性率为38.94%,猪胆汁标本HEV RNA检出阳性率为4.67%。HEV-4型为2014年本省主要人源和猪源毒株,且病毒核苷酸序列同源性为83%~97%。结论Real-time RT-PCR法快捷、简便性和RT-PCR的分型、序列分析作用相结合,可在HEV感染和传染源监测中发挥重要作用。     

西安市家畜戊型肝炎病毒携带情况调查

目的 探讨西安市家畜戊型肝炎(戊肝)病毒(HEV)携带情况.方法 收集猪、犬等常见家畜的胆汁标本,采用逆转录巢式聚合酶链反应(RT-nPCR)技术扩增HEV基因组开放读码2(ORF2)区域内150 nt片段;阳性标本PCR产物直接测序,并与GenBank数据库进行比对,构建进化树分析遗传特征.结果 共收集猪胆汁标本194份,犬胆汁标本178份,牛胆汁标本79份,羊胆汁标本191份.RT-nPCR检测结果表明,四种家畜胆汁标本HEV RNA阳性率分别为4.10%、0%、0%、0%;遗传距离分析发现本次得到的HEV与Ⅳ型HEV距离最近,其次为Ⅰ型、Ⅱ型和Ⅲ型;氨基酸水平也显示了同样的规律.结论 猪是HEV家畜宿主,Ⅳ型是主要流行型.     

兰州市猪戊型肝炎血清学及病毒基因型分析

目的 调查甘肃省兰州市猪戊型肝炎病毒(HEV)感染情况以及基因型.方法 收集兰州市部分地区猪血清,用ELISA诊断试剂盒分别检测HEV抗原和抗体;其中抗原阳性的血清,采用HEV ORF2引物进行套式逆转录聚合酶联反应(RT-PCR)扩增,对阳性扩增产物进行克隆和测序,并对序列进行分析,确定其基因型.结果 3月以下猪戊型肝炎病毒抗原、抗体阳性率分别为1.56%和90.27%,3个月以上分别为2.35%和86.99%,抗原、抗体水平在3月龄以上和以下二者中差异无统计学意义,抗原阳性血清共有22份,其中7份为HEV核酸阳性,其核苷酸序列与HEV 4型的同源性为82.2%～99.3%,分别为4a,4d和4f亚型.结论 兰州市猪群存在HEV感染,且为基因4型.     

铜川市2013-2015年A型流感病毒HA基因特性研究

目的 应用生物信息学数据库和工具,了解2013-2014年铜川市A型H1N1和2013-2015年A型H3N2流感病毒血凝素(HA)基因特性及其抗原变异情况。 方法 收集铜川市哨点医院流感样病例标本,采用MDCK细胞分离流感病毒。用RT-PCR对部分A型H1N1、H3N2流感病毒进行HA基因序列扩增和纯化,序列测定,并用MEGA软件构建种系发生树分析。 结果 2013-2015年铜川市流感核酸检测标本共2 333份,流感阳性476份,阳性率20.40%。4株A型H1N1亚型流感病毒与WHO推荐A/California/07/2009(H1N1)疫苗株氨基酸序列比对,共发生11次氨基酸突变,其中3个氨基酸位点突变发生在抗原决定簇。3株A型H3N2亚型与WHO推荐的北半球2013-2014年A/Victoria/361/2011(H3N2)疫苗株比对共有9个氨基酸位点发生了突变,其中3个氨基酸位点发生在抗原决定簇;与WHO推荐的北半球2014-2015年A/Texas/50/2012(H3N2)疫苗株比对共有10个氨基酸位点发生了突变,有5个氨基酸位点发生在抗原决定簇。 结论 2013-2015年铜川市流行的A型H1N1、H3N2亚型流感病毒氨基酸发生突变导致抗原漂移。     

猪粪便中戊肝病毒实时荧光RT-PCR定性检测方法的建立及其分子流行病学特征

目的 建立以TaqMan探针为基础的戊肝病毒(HEV)实时荧光RT-PCR检测方法和构建系统发育树,对戊肝病毒进行基因分型,检测中国部分省市猪场的猪粪便样本HEV污染水平。方法 参照GenBank HEV基因型序列,针对HEV保守区设计引物和探针,优化反应体系,建立实时荧光RT-PCR和巢式RT-PCR检测体系。结果 建立的HEV实时荧光RT-PCR检测方法的灵敏度为19.9拷贝/μL,扩增效率92.9%～109.1%,与札如病毒(Sa)、诺如病毒(Nov)、甲肝病毒(HAV)均不发生交叉反应。荧光RT-PCR检测猪粪便样本342份,其中HEV阳性样本210份,阳性率61.4%,育肥前阳性率56.6%,育肥后阳性率66.9%,育肥前后样本阳性率差异具有统计学意义(χ~2=24.8,P<0.05);经基因分型鉴定体系测定阳性毒株基因型均为HEV-4型,且存在4b、4d、4h三种基因亚型。结论 中国部分省市猪场中HEV感染普遍,基因型均为HEV-4型,各省市猪场感染率和基因亚型存在差异。     

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。     

Isolation, detection and characterization of swine hepatitis E virus from herds in Costa Rica

Although swine HEV isolates from North America, Europe, and Asia have been genetically characterized, little is known about the strains presumed to be circulating in Latin America. In this study, seven commercial swine production sites in Costa Rica were surveyed for HEV. Using RT-PCR, with primers located in ORF2, 19/52 fecal samples produced a product of the expected size following two rounds of amplification. Most positive samples were from swine between the ages of 1.5 and 4 months. This study provides documented evidence for the endemicity of HE infections in swine residing in Central America. Through nucleic acid sequencing, isolates were found to be genetically similar, if not identical, with no amino acid substitutions. By comparison of swine and human HEV strains representing all four genotypes and phylogenetic analysis, our isolates closely resembled the US swine and human and other Genotype III strains, with 85-93% nucleic acid identity.     

The genetic divergences of codon usage shed new lights on transmission of hepatitis E virus from swine to human

Hepatitis E virus (HEV) is an important pathogen causing public health burden. Swine has been recognized as a main reservoir. Interestingly, genotype 1 HEV only infects human; whereas genotype 3 and 4 are zoonotic. However, there is a lack of in-depth understanding in respect to the transmission from swine to human. Codon usage patterns generally participate in viral survival and fitness towards its hosts. We have analyzed codon usage patterns of the three open reading frames (ORFs) for 243 full-length genomes of HEV genotypes 1, 3 and 4. The divergence of synonymous codon usage patterns is different in each ORF for genotypes 1, 3 and 4, but the genotype-specific codon usage bias in genotype 1 is stronger than those of genotypes 3 and 4. In respect to genotypes 3 and 4, compared with strains isolated from human, HEV isolated from swine shows appreciable variation in adaptation of codon usages to human or swine. These results may help to understand the transmission and host adaptation of HEV genotypes 3 and 4 from swine to human.     

Detection and molecular characterization of swine hepatitis E virus in North Carolina swine herds and their faecal wastes

Recent findings of almost genetically indistinguishable swine and human strains, have suggested swine play a role in the transmission of hepatitis E virus (HEV). The extent to which HEV may be present and persist in the faecal waste generated from intensive swine operations is largely unknown. The fate of swine waste liquid is often land application, possibly resulting in unintentional seepage into groundwater or run-off into surface waters, hence validating concerns of human exposure risks. Freshly passed swine faeces, barn flush liquid waste, and lagoon liquid from production sites in North Carolina were surveyed periodically for HEV using RT-PCR primers located in ORF2. On three farms where HEV RNA was detected in swine faeces, it was also found in stored liquid waste on several occasions. HEV presence was related to swine age but not to animal management and waste management procedures, which varied amongst the farms. Seasonal patterns of HEV prevalence could not be established as viral RNA was isolated at all time points from two farms. Phylogenetic analysis of 212 bases of the genomic RNA indicated that isolates resembled the known US swine and human strains (percentage nucleic acid homology 91 to 94%), with one amino acid substitution.     

Analysis of the complete genome sequences of one swine and two human hepatitis E virus genotype 4 strains isolated in Beijing,China

Full-length sequences were determined and analyzed for two human (MO and W3) and one swine (W2–5) hepatitis E virus (HEV) isolates from Beijing, China. The genomes of the three strains were composed of 7242, 7239, 7239 nucleotides, respectively, excluding the poly (A) tails, and were 84% identical to each other. All were classified into genotype 4. Sequence analysis shows that the 2 human isolates have up to 91–94% nucleotide identity in full length genome with swine strains isolated in China, while the swine isolate share 92% identity with the human strain T1 from Beijing. At the amino acid level, the three strains share 94%, 97% and 89–92% identity in the ORF1, ORF2 and ORF3, proteins respectively. The human strains MO and W3 have the highest identity, 97%, 98–99% and 96–98% in ORFs 1–3, respectively, to swine strains CHN-XJ-SW13 and CHN-XJ-SW33 from Xinjiang, China, while swine strain W2–5 has highest identity with the human strain HE-JA2, 96%, 99% and 91% in ORFs 1–3, respectively. Genotype specific amino acid substitutions were found at a single site in all three ORFs by sequences alignment, and genotype specific short sequences (5–10aa in length) were found in ORF1 and the C-terminus of ORF3. However, no difference was found at any amino acid position that discriminates between human and swine HEVs within genotype 4 for any of the three ORFs. These results indicated that the genotype 4 HEV strains from humans and pigs in China may evolve from the common ancestor.     

Assessment of the cross-protective capability of recombinant capsid proteins derived from pig, rat, and avian hepatitis E viruses (HEV) against challenge with a genotype 3 HEV in pigs

Hepatitis E virus (HEV), the causative agent of hepatitis E, is primarily transmitted via the fecal-oral route through contaminated water supplies, although many sporadic cases of hepatitis E are transmitted zoonotically via direct contact with infected animals or consumption of contaminated animal meats. Genotypes 3 and 4 HEV are zoonotic and infect humans and other animal species, whereas genotypes 1 and 2 HEV are restricted to humans. There exists a single serotype of HEV, although the cross-protective ability among the animal HEV strains is unknown. Thus, in this study we expressed and characterized N-terminal truncated ORF2 capsid antigens derived from swine, rat, and avian HEV strains and evaluated their cross-protective ability in a pig challenge model. Thirty, specific-pathogen-free, pigs were divided into 5 groups of 6 pigs each, and each group of pigs were vaccinated with 200μg of swine HEV, rat HEV, or avian HEV ORF2 antigen or PBS buffer (2 groups) as positive and negative control groups. After a booster dose immunization at 2 weeks post-vaccination, the vaccinated animals all seroconverted to IgG anti-HEV. At 4 weeks post-vaccination, the animals were intravenously challenged with a genotype 3 mammalian HEV, and necropsied at 4 weeks post-challenge. Viremia, fecal virus shedding, and liver histological lesions were compared to assess the protective and cross-protective abilities of these antigens against HEV challenge in pigs. The results indicated that pigs vaccinated with truncated recombinant capsid antigens derived from three animal strains of HEV induced a strong IgG anti-HEV response in vaccinated pigs, but these antigens confer only partial cross-protection against a genotype 3 mammalian HEV. The results have important implications for the efficacy of current vaccines and for future vaccine development, especially against the novel zoonotic animal strains of HEV.