人、猪、禽戊型肝炎病毒血清学关系的研究

目的研究人、猪、禽戊型肝炎病毒（HEV）的血清学关系。方法应用ELISA分别以人、猪、禽HEVORF2重组蛋白p166human、p166swine、p268avian检测人、猪、鸡血清及其他标本中抗．HEVIs,G，用SAS软件进行统计学分析，同时进行序列同源性比较。结果p166human和p166swine对HEV实验感染动物血清、HEVORF2重组蛋白免疫血清和单克隆抗体均呈阳性反应，而p268avian均呈阴性反应。以p166human、p166swine、p268avian检测人、猪、鸡血清抗．HEVIgG，戊型肝炎患者血清检出率分别为98．5％、97．7％和1．5％，正常人血清为10．0％、10．0％和4．0％，猪血清为26．9％、25．6％和1．3％，鸡血清为4．3％、2．2％和33．3％。p268avian与p166human或p166swine的检出率差异有统计学意义（P〈0．001）。相关性分析表明，p166human和p166swine对不同样本的检测均呈直线正相关，而p268avitm与p166human或p166swine无直线相关性。人和猪HEVpORt2的序列同源性在88．2％。99．2％，而禽HEV与人、猪HEVpORt2的同源性仅为45．5％～46．1％，其中含有多个插入和缺失突变。结论人和猪HEV血清学关系密切，而禽HEV与人、猪HEV抗原性差异有统计学意义，无血清学相关性。因此禽HEV与人、猪HEV的关系应予进一步考证。     

戊型肝炎病毒Ⅳ型全基因序列的分析

目的 分析戊型肝炎病毒（HEV）Ⅳ型的全基因序列以比较与其他基因的差异。方法 设计PCR引物对全基因进行分自然扩增,并两个末端采用末端快速扩增方法（RACE）进行扩增,对扩增产物进行克隆及测序。结果 HEV－T1全序与Ⅰ型、Ⅱ型和Ⅲ型的核苷酸同源性分别为（74．8－75．5）％、74．5％和（75．3－76．3）％。ORF1与已报道的Ⅰ型、Ⅱ型和Ⅲ型氨基酸同源性分别为（85．0－86．7）％、85．0％和（88．5－88．7）％;ORF2与已报道的Ⅰ型、Ⅱ型和Ⅲ型的氨基酸同源性分别为（91．6－92．4）％、90．1％和（91．9－93．0）％。ORF3与已报道的Ⅰ型、Ⅱ和Ⅲ型的氨基酸同源性分别为（75．9－77．8）％、75．0％和（79．6－83．3）％。结论 这一研究为今后发展戊型料诊断试剂及戊型肝炎疫苗提供了新的、重要的分子生物学基础。     

戊型肝炎病毒与戊型肝炎

病毒性肝炎是我国最重要的传染病之一，国内外对其病原学研究进展甚快，至少已确认有五种肝炎病毒，其中戊肚的散发流行。为使从事传染病防治方面的专业人员病房了解本病毒的特征，进一步完善肝炎的病原学诊断及流行病学调查结果，本刊编辑部特约请第二军医大学微生物教研室戚中田教授撰写了有关戊肚用其戊肝病毒的综述；　　本综述简要报道戊型肝炎病毒的生物学及分子生物学特征，戊型肝炎的临订特征及特异性诊断，并对戊肝免疫预防、基因疫苗的展望作了介绍，对从事病毒学及传染病学的及传染病学的工作人员具有重要的参考价值。     

散发性戊型肝炎病毒部分核苷酸序列分析

目的为阐明戊型肝炎病毒（ＨＥＶ）毒株的地理分布、流行特征以及研制血清学诊断试剂和基因工程疫苗提供资料。方法选择１例浙江仙居山区散发性ＨＥ患者，从其血清中分离ＨＥＶＲＮＡ，通过逆转录－套式聚合酶链反应法（ＲＴ－ｎＰＣＲ），扩增该散发性ＨＥＶ（Ｚ－３３株）ＯＲＦ２区部分ｃＤＮＡ片段（４９７ｂｐ），然后进行直接序列分析，并与ＨＥＶ各主要代表株序列作比较。结果Ｚ－３３株与新疆流行株ＣＨ１．１的核苷酸及氨基酸序列同源性分别为９６．７％及９８．２％；与缅甸流行株的同源性分别为９４．２％及９９．１％；与缅甸散发株的同源性分别为９５．２％及９９．１％，与墨西哥株的同源性分别为８１．８％及９４．５％。结论浙江仙居散发性ＨＥＶＺ－３３株和新疆流行株ＣＨ１．１一样，与ＨＥＶ缅甸株可能为同一亚型。     

猪HEV基因型和亚型的研究

目的调查猪HEV的基因型和亚型。方法收集新疆南部和广西柳州地区3月龄以下猪粪便标本134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（HEVRNA），对RT-nPCR阳性扩增产物进行克隆测序，用VectorNTISuite9．0和TreeView软件与目前新的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。结果新疆和广西猪粪便标本的HEVRNA阳性率分别为3．64％（2／55）和3．80％（3／79），命名为XJ3-2、XJ3-6、GX3-1、GX5．2和GX6-3。5株猪HEV的开放阅读框架1（ORFI）的部分核苷酸序列与Ⅰ、Ⅱ、Ⅲ和Ⅳ型HEV的相似性分别为74％-77％、76％～77％、73％-81％和82％一98％。同时对开放阅读框架2（ORF2）部分核苷酸序列进行了分析，GX3．1和GX6-3与1Vb亚型相似性最高，为88％～95％。GX5．2、XJ3．2和XJ3．6与Ⅳa亚型相似性最高，为90％-96％。结论新疆和广西5株猪HEV均属于基因型Ⅳ，新疆2株为IVa亚型，广西1株为IVa，另2株为IVb亚型。未检测到HEV基因Ⅰ型和Ⅲ型。     

云南大理猪戊型肝炎病毒分子流行病学调查

目的：了解云南大理农村的猪群中戊型肝炎病毒（ hepatitis E virus, HEV）感染情况,为HEV的防控提供理论依据。方法利用逆转录巢式聚合酶链反应（ RT?nested PCR）技术,对所采集大理41份猪粪便样品进行HEVORF2基因部分片段扩增,经琼脂糖凝胶电泳后对目的条带进行回收纯化及克隆测序,序列利用DNAStar和MEGA4．0软件进行同源性比较和进化树构建。结果 RT?nested PCR方法扩增出348 bp目的基因序列,系统进化树显示该病毒属于基因4型h亚型HEV。此次实验共检测41份猪粪便样品,其中23份为阳性,阳性率为56．10％。结论大理猪群存在较高的HEV感染率,存在HEV跨种间感染人的风险,应该加强防控以免HEV爆发流行。     

戊型肝炎病毒基因型地域分布特征的演变

自Reyes1990年首次克隆出缅甸株HEV基因序列以来，国内外不同基因型的HEV毒株陆续被发现。科学家们对已分离的HEV毒株进行了全基因及部分基因的克隆及测序，借助于系统软件的分析对HEV进行了基因分型，发现在世界范围内HEV基因型地域分布特征发生了很多变化。本文回顾了HEV基因型地域分布演变的过程，概括了HEV基因型新的地域分布特征。     

戊型肝炎病毒摩洛哥株非编码区基因序列鉴定及基因型分型

目的　检测戊型肝炎病毒 (HEV)摩洛哥株非编码区 (UTR)序列 ,探讨HEV非编码区序列能否作为其基因型分型的依据。方法　使用基于RNA连接酶的cDNA末端快速扩增法 (RLM RACE)扩增HEV摩洛哥株 5′和 3′端片段并测序。所得序列使用LASERGENE和PHYLIP软件包与其他 2 9株HEV序列比较。结果　只有基于甲基化帽子结构的RLM RACE扩增出了 5′端片段。HEV摩洛哥株 5′端UTR有 2 6个核苷酸 ,3′端polyA之前有 6 5个核苷酸。基于 3′端UTR序列的进化树与基于全基因序列的进化树不全相同。HEV 3′端至少需要 10 0个左右核苷酸长的序列才可进行HEV基因型分型。结论　HEV摩洛哥株 5′端有甲基化帽子结构。HEV 3′端UTR序列不能完全代替全基因组序列进行基因型分型。部分序列替代全基因组进行基因型分型时需要考虑其部位和长度因素。     

戊型肝炎病毒研究进展

本文重点介绍了近几年来戊型肝炎病毒的分子生物学研究进展。以生物学特性、分子生物学特性及诊断方面概述了近年的研究成果，并提出了尚需解决的问题和今后的研究方向。     

Full genomic sequence analysis of swine genotype 3 hepatitis E virus isolated from Shanghai

The full genomic nucleotide sequence of a previously identified genotype 3 hepatitis E virus (HEV), strain SAAS-JDY5, was obtained using RT-PCR and rapid amplification of cDNA ends (RACE). The genome consisted of 7225 nucleotides, excluding a poly-A tail at the 3′ terminus, and contained three open reading frames (ORFs), ORF-1, ORF-2 and ORF-3, encoding 1702, 660 and 113 amino acids, respectively. Phylogenetic analysis confirmed that SAAS-JDY5 belonged to genotype 3 HEV and was most closely related to the Japanese isolate wbJYG1 (AB222184). SAAS-JDY5 shared approximately 87% nucleotide similarity to human and swine strains from the United States, compared with 74–75% similarity to Asian (genotype 4) and Mexican strains (genotype 2). Alignment of the SAAS-JDY5 genomic sequence with reference sequences of the same genotype revealed one nucleotide substitution and one deletion at positions 5145 and 7189 (3′ UTR), respectively. Moreover, SAAS-JDY5 contained two additional nucleotides (AC) at the very end of the 3′-terminus preceding the poly-A tail of the genome. Comparison of the putative amino acid sequence encoded by the SAAS-JDY5 genome with sequences of other genotype 3 isolates revealed 15 unique amino acid substitutions and one deletion in ORF-1, and three substitutions in ORF-2.     

Development and validation of a genotype 3 recombinant protein-based immunoassay for hepatitis E virus serology in swine

Hepatitis E virus (HEV) is classified within the family Hepeviridae, genusHepevirus. HEV genotype 3 (Gt3) infections are endemic in pigs inWestern Europe and in North and South America and cause zoonotic infections inhumans. Several serological assays to detect HEV antibodies in pigs have beendeveloped, at first mainly based on HEV genotype 1 (Gt1) antigens. To develop asensitive HEV Gt3 ELISA, a recombinant baculovirus expression product of HEV Gt3 openreading frame-2 was produced and coated onto polystyrene ELISA plates. Afterincubation of porcine sera, bound HEV antibodies were detected with anti-porcineanti-IgG and anti-IgM conjugates. For primary estimation of sensitivity andspecificity of the assay, sets of sera were used from pigs experimentally infectedwith HEV Gt3. For further validation of the assay and to set the cutoff value, abatch of 1100 pig sera was used. All pig sera were tested using the developed HEV Gt3assay and two other serologic assays based on HEV Gt1 antigens. Since there is nogold standard available for HEV antibody testing, further validation and a definitesetting of the cutoff of the developed HEV Gt3 assay were performed using astatistical approach based on Bayes'' theorem. The developed and validated HEVantibody assay showed effective detection of HEV-specific antibodies. This assay cancontribute to an improved detection of HEV antibodies and enable more reliableestimates of the prevalence of HEV Gt3 in swine in different regions.     

Molecular characterization and phylogenetic analysis of the complete genome of a hepatitis E virus from European swine

Hepatitis E virus (HEV) has been detected in humans and in a broad range of animals, including pigs. For the first time the full-length genomic sequence of a HEV of European porcine origin, termed swX07-E1, was determined. Comparative analysis of 76 complete or nearly complete nucleotide sequences showed that swX07-E1 shares the highest nucleotide identity with Japanese swine HEV swJ8-5 and swJ12-4. The whole-genome phylogenetic analysis showed that swX07-E1 from Europe belongs to genotype-3 HEV, clusters with variants from Japan, Mongolia and Kyrgyzstan in subgroup 3c, but it is divergent from the prototype US HEV. Our analysis indicates that swX07-E1 represents a new subgroup of genotype-3 and that analysis of full-length sequences is necessary to discover new subgroups of HEV. According to our knowledge, swX07-E1 is the first full-length genome sequence of HEV from European swine. Knowledge about the full length HEV sequence from European swine is very important for understanding the HEV evolutionary events and the molecular mechanism of infection in human and in animals.     

烟台沿海地区人源和猪源戊型肝炎病毒基因型别的研究

目的 调查烟台市沿海地区人源与猪源戊型肝炎病毒(HEV)基因型别的相关性.方法 应用逆转录一巢式聚合酶联反应( RT-nPCR)方法对当地急性散发戊型肝炎患者、正常人群中抗HEV-IgM阳性者和当地养猪场的猪进行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 16例急性散发戊型肝炎患者中有7例粪便标本HEV RNA阳性;51份lgM阳性正常人群血清标本中有1份HEV RNA阳性;34份猪胆汁标本中有1份HEV RNA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87％～ 98.1％.7株患者的戊肝病毒基因型和1株猪的戊肝病毒基因型均为Ⅳ型,基因序列同源性在87％ ～ 98.1％之间;其中有6例患者和猪的基因序列同源性在93.9％ ～98.1％之间,为Ⅳ型a亚型;1例患者和猪的基因序列同源性为87％,为Ⅳ型d亚型.正常人群的1例戊肝病毒基因型为Ⅰ型d亚型.该地区人与猪HEV的ORF2的部分基因片段与HEV Ⅰ～Ⅳ型的代表株进行比较,核苷酸序列同源性分别是82.5％～100％,81.7％ ～92.9％,81.4％ ～93.9％,84.9％ ～ 100％.结论 该地区人群中流行的HEV存在2个基因型3个亚型,主要以基因Ⅳa型为主,与猪群中流行的HEV基因Ⅳa型同源性较高;HEV Ⅰ型在人群中散在存在.     

烟台沿海地区人源和猪源戊型肝炎病毒基因型别的研究

目的调查烟台市沿海地区人源与猪源戊型肝炎病毒（HEV）基因型别的相关性。方法应用逆转录一巢式聚合酶联反应（RT—nPcR）方法对当地急性散发戊型肝炎患者、正常人群中抗HEV-IgM阳性者和当地养猪场的猪进行HEVRNA检测,并对HEVRNA阳性标本进行克隆测序和序列分析。结果16例急性散发戊型肝炎患者中有7例粪便标本HEVRNA阳性;51份IgM阳性正常人群血清标本中有1份HEVRNA阳性;34份猪胆汁标本中有1份HEVRNA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87％～98．1％。7株患者的戊肝病毒基因型和1株猪的戊肝病毒基因型均为Ⅳ型,基因序列同源性在87％～98．1％之间;其中有6例患者和猪的基因序列同源性在93．9％～98．1％之间,为Ⅳ型a亚型;1例患者和猪的基因序列同源性为87％,为Ⅳ型d亚型。正常人群的1例戊肝病毒基因型为Ⅰ型d亚型。该地区人与猪HEV的ORF2的部分基因片段与HEVⅠ～Ⅳ型的代表株进行比较,核苷酸序列同源性分别是82．5％～100％,81．7％～92．9％,81．4％～93．9％,84．9％～100％。结论该地区人群中流行的HEV存在2个基因型3个亚型,主要以基因Ⅳa型为主,与猪群中流行的HEV基因Ⅳa型同源性较高;HEVI型在人群中散在存在。     

Identifications of polyphyletic variants in acute hepatitis suggest an underdiagnosed circulation of hepatitis E virus in Argentina

Background

In recent years, an increasing number of infections with genotype 3 hepatitis E virus (HEV) have been reported in western countries. Data in South America, however, are still scarce. Swine and human variants previously described in Argentina are closely related to a human Austrian one.

Objective

To identify whether HEV is still circulating in Argentina.

Study design

Sera and stool samples from adults and children with unexplained acute liver disease referred to our center during the last six years were prospectively studied. Dual infection with hepatitis A was retrospectively studied in a group of children with fulminant hepatic failure.

Results

Fifteen new cases (13 adults and 2 children), seven of whom required hospitalization, were diagnosed. Nine had detectable HEV RNA, and one had imported genotype 1. Subgenotype 3i HEV-related variants are still circulating. Five autochthonous sequences, related to European, American and Japanese ones, grouped in subgenotype 3a. One case had a subgenotype 3b variant.

Discussion

The polyphyletic variants widespread in Argentina suggest multiple sources of infection. Whether or not their reservoir is swine merits further investigation. Since hepatitis E is still considered rare, differential laboratory testing in unexplained acute liver disease is not routinely performed in Argentina. Broadening awareness of this disease is important in light of the decrease in hepatitis A incidence since universal vaccination was implemented in 2005. The diagnosis of hepatitis E with a combination of serological and molecular tools is needed to better understand its epidemiology and impact on the clinical management of patients with unexplained increased transaminases.     

PCR结合单酶切对戊型肝炎病毒进行基因分型的研究

目的　建立PCR结合单酶切对戊型肝炎病毒(HEV)进行基因分型的方法,并将其应用于HEV基因型分布的研究。方法　采用简并引物扩增1～4型HEVORF1片段,1、2型HEVPCR产物为2 75、2 6 9bp ,3、4型PCR产物为317、314bp ,分别应用NaeⅠ、NotⅠ消化两种长度的PCR产物,根据酶切结果区分4种基因型。结果　采用此方法对4种基因型的HEV标准株分型,结果与预期一致;4 3份HEVIgM阳性的临床标本中,19份PCR阳性,分型结果均为4型HEV。结论　此方法可以快速简便地区分HEV 4种基因型。南京地区散发戊型肝炎大多由4型HEV所致。     

Complete or near-complete nucleotide sequences of hepatitis E virus genome recovered from a wild boar, a deer, and four patients who ate the deer

Zoonosis has been implicated in hepatitis E virus (HEV) transmission. We examined wild boar living in a forest of Hyogo prefecture, Japan, and found HEV RNA in three of seven boars. A full-genome HEV isolate from one of them was revealed to be 99.7% identical to a previous isolate from a wild deer hunted in the same forest and to those from four patients who contracted hepatitis E after eating raw meat of the deer. These findings suggest an interspecies HEV transmission between boar and deer in their wild life, and that both animals might serve as an infection source for human beings as suggested previously.