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.     

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。     

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.     

淮安市戊型病毒性肝炎人畜共患性及病毒基因型研究

目的探讨淮安市戊型病毒性肝炎病例与猪之间的联系,为控制HEV在人畜间的传播提供依据。方法在淮安市收集急性戊肝、肝炎未分型病例血液标本193份,同期在屠宰场收集生猪胆囊标本510份,采用Real-time RT-PCR和RT-PCR提取目标核苷酸,测定序列后进行分析。结果所有标本共分离出HEV-4型毒株20株,分4个亚型,HEV-4d型为优势流行毒株亚型;人源与猪源HEV毒株间的同源性为:80.3%～97.7%。结论淮安市戊肝病例与生猪感染的HEV高度同源,生猪是戊肝传播过程中一种重要的动物宿主。     

Close similarity between sequences of hepatitis E virus recovered from humans and swine, France, 2008-2009

Frequent zoonotic transmission of hepatitis E virus (HEV) has been suspected, but data supporting the animal origin of autochthonous cases are still sparse. We assessed the genetic identity of HEV strains found in humans and swine during an 18-month period in France. HEV sequences identified in patients with autochthonous hepatitis E infection (n = 106) were compared with sequences amplified from swine livers collected in slaughterhouses (n = 43). Phylogenetic analysis showed the same proportions of subtypes 3f (73.8%), 3c (13.4%), and 3e (4.7%) in human and swine populations. Furthermore, similarity of >99% was found between HEV sequences of human and swine origins. These results indicate that consumption of some pork products, such as raw liver, is a major source of exposure for autochthonous HEV infection.     

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.     

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。     

山东省某地屠宰场猪肝内戊型肝炎病毒感染状况研究

目的研究商品猪肝脏内戊型肝炎（戊肝）病毒（HEV）的检出状况及其基因分型。方法采集35份山东省某个人屠宰场和某城镇大型屠宰场内的猪肝，处理后采用逆转录．巢式聚合酶链反应（nested RT·PCR）方法检测HEV RNA。并与GenBank中登录的HEV全长序列进行同源性分析。结果35份猪肝标本中共有3份检出HEVRNA，阳性率为8．57％。同源性分析显示这3株病毒均为Ⅳ型HEV。结论即将上市的商品猪肝脏内存在HEV感染，其基因型与中国大陆HEV流行株相同，均为基因Ⅳ型。     

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.     

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.     

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.     

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。     

中国部分地区猪胆汁戊型肝炎病毒基因型和中和抗原位点变异研究

目的调查猪胆汁戊型肝炎病毒(HEV)携带率、基因型和中和抗原位点变异。方法在中国4个地区屠宰场采集成年商品猪的胆汁标本,采用real-time RT-PCR检测HEV RNA;采用巢式RT-PCR扩增HEV ORF2区部分基因片段,进行基因分型和中和抗原位点氨基酸序列分析。结果猪胆汁标本HEV RNA总阳性率为8.6%(46/532),4个地区阳性率在3.0%-12.5%之间。19株HEV基因分型成功,均为基因4型,其中11株HEV基因亚型分型成功,均为4a亚型。在该11株HEV的21个中和抗原位点中,2株HEV在ORF2区分别发生第487位氨基酸突变(Ser487Tyr)和第564位氨基酸突变(Thr564Pro)。结论中国部分地区猪胆汁HEV携带率较高,为中国人群中优势流行的基因4型,且部分中和抗原位点发生突变;应加强猪群HEV的持续监测。     

Seroepidemiology and genetic characterization of hepatitis E virus in the northeast of china

The aim of this study was to analyze the prevalence of infection and genotype of hepatitis E virus (HEV) in people and animals in the northeast of China (Heilongjiang, Jilin and Liaoning provinces). This seroepidemiological study was conducted using enzyme immunoassays and human sera positive for HEV antigen or anti-HEV IgM, and animal sera positive for HEV antigen or with an S/CO ≤10 for anti-HEV were tested for HEV RNA using real-time RT-PCR and nested RT-PCR. In humans, the overall prevalence of anti-HEV IgG was 31.6% (311/985), 28.6% (147/514) and 21.1% (841/3994) in individuals frequent, infrequent, and very rare contact with swine, respectively. The overall prevalence of anti-HEV was 81.6% (1737/2127) in pigs above 3 months of age, 66.4% (1644/2473) in pigs below 3 months of age, 18.7% (301/1612) in cattle and 12.4% (162/1302) in sheep. 1211 samples were tested for HEV RNA using real-time RT-PCR and 71 were positive. 30 of the 71 samples also were positive for HEV RNA using nested RT-PCR. These 30 isolates shared 81.2–100% sequence identity with each other at the nucleotide level and belonged to HEV genotype 4, regardless whether from human or animals. The results indicate that HEV infection is widely spread in the northeast of China. The prevalence of anti-HEV in individuals with frequent contact with pigs was significantly higher than those without and the HEV sequences isolated from such individuals were related more closely to isolates from pigs. These support strongly the hypothesis of a zoonotic origin of hepatitis E.     

Sources of Hepatitis E Virus Genotype 3 in the Netherlands

Non–travel-related hepatitis E virus (HEV) genotype 3 infections in persons in the Netherlands may have a zoonotic, foodborne, or water-borne origin. Possible reservoirs for HEV transmission by water, food, and animals were studied. HEV genotype 3/open reading frame 2 sequences were detected in 53% of pig farms, 4% of wild boar feces, and 17% of surface water samples. HEV sequences grouped within 4 genotype 3 clusters, of which 1 is so far unique to the Netherlands. The 2 largest clusters contained 35% and 43% of the animal and environmental sequences and 75% and 6%, respectively, of human HEV sequences obtained from a study on Dutch hepatitis E patients. This finding suggests that infection risk may be also dependent on transmission routes other than the ones currently studied. Besides the route of exposure, virus characteristics may be an important determinant for HEV disease in humans.     

Phylogenetic analysis of Hepatitis E virus strains isolated from slaughter-age pigs in Colombia

Hepatitis E virus (HEV) is a zoonotic virus that causes acute hepatitis in humans, and can be transmitted via the fecal–oral route. Pigs are considered to be a reservoir for this infection—mainly where the disease is not endemic. In a previous study conducted in Antioquia, which is a region in Colombia where the production and consumption of pork meat is higher than in the rest of the country, the presence of anti-HEV IgG-type antibodies was reported in slaughter-age pigs. Aiming to identify the HEV genotype circulating in swine, animal liver, and feces samples from five swine cattle slaughterhouses located in six different sub-regions of Antioquia were collected. A nested RT-PCR (nRT-PCR) was used in order to amplify the HEV ORF-1 (170 bp) and ORF-2 (348, and 958 bp). The amplicons yielded in this study were sequenced, and a molecular phylogeny analysis based on the maximum likelihood method, including HEV sequences reported in several countries, was performed. Phylogeny analysis revealed that HEV amplification fragments from Antioquia's pigs were grouped in three clades within the sub-genotype 3a without a specific geographical structure, and were also genetically related to Japanese and American HEV sequences. This analysis provides the first approach on the genetic diversity and circulation dynamics of HEV in Colombian herds.     

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。     

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。