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。     

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。     

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.     

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。     

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。     

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.     

Virulent Strain of Hepatitis E Virus Genotype 3, Japan

Hepatitis E virus (HEV) genotype 3, which usually causes asymptomatic infection in Japan, induced severe hepatitis in 8 patients. To better understand genetic features of HEV associated with increased virulence, we determined the complete or near-complete nucleotide sequences of HEV from these 8 patients and from 5 swine infected with genotype 3 strain swJ19. Phylogenetic analysis showed that the isolates from the 8 patients and the 5 swine grouped separately from the other genotype 3 isolates to create a unique cluster, designated JIO. The human JIO-related viruses encoded 18 amino acids different from those of the other HEV genotype 3 strains. One substitution common to almost all human HEV strains in the JIO cluster was located in the helicase domain (V239A) and may be associated with increased virulence. A zoonotic origin of JIO-related viruses is suspected because the isolates from the 5 swine also possessed the signature V239A substitution in helicase.     

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.     

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。     

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.     

Hepatitis E virus in domestic pigs and surface waters in Slovenia: Prevalence and molecular characterization of a novel genotype 3 lineage

A novel hepatitis E virus (HEV) genotype 3 lineage was identified in Slovenian pig herds. Stool samples from six Slovenian pig farms were collected and tested for the presence of HEV RNA. Of 85 individual samples 15 (20.3%) were positive for HEV RNA of which 2/38 (5.3%), 6/21 (28.6%) and 7/26 (26.9%) were from suckling, weanling and fattening pigs, respectively. Additionally, 51 pooled porcine stool samples were tested in one of the biggest pig farm and the estimated infection rate of individual pig was calculated, resulting in 7.8%, 10.6% and 24.2% for suckling, weanling and fattening pigs, respectively. The majority of HEV positive porcine samples were from the same pig farm. Out of 17 Slovenian patients with confirmed recent hepatitis E in the period 1999–2011, the serum samples of 10 patients were tested and 3 samples turned out to be HEV RNA positive. Furthermore, 60 surface water samples were tested throughout the country, of which 2 (3.3%) were positive for HEV RNA, one of them in the near vicinity of a pig farm. All HEV strains were analysed at 5’ ORF1 and 5’ ORF2 regions and both genome regions confirmed that Slovenian HEV strains represent a distinct genotype 3 lineage, diverse from all other genotype 3 lineages available in GenBank and described in the literature to date. All but one HEV strains detected in pigs in Slovenia represent a monophyletic branch in phylogenetic trees, with a high degree of sequence identity. One human HEV strain belonged to genotype 1 and two to genotype 3 but did not match the new genotype 3 lineage detected in Slovenian pig herds.     

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.     

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。     

广西地区猪、鼠、狗戊型肝炎病毒感染血清学分析

目的了解广西地区猪、鼠和狗血清中抗．戊型肝炎病毒（HEV）IgG抗体流行情况。方法应用HEV基因1型开放读码框（ORF）2和ORF3多肽包被酶标板，辣根过氧化物酶（HRP）标记的羊抗猪、羊抗鼠和羊抗狗建立的酶联免疫吸附试验（ELISA）法检测猪、鼠和狗血清中抗．HEV IgG。结果总抗．HEV IgG阳性率为31．84％（170／534），其中猪血清抗．HEV IgG阳性率为26．40％（66／250），鼠血清抗．HEV IgG阳性率为43．02％（77／179），狗血清抗．HEV IgG阳性率为25．71％（27／105）。结论猪、鼠和狗血清中存在HEV感染，鼠抗．HEV IgG阳性率最高。     

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.     

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.     

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.