戊型肝炎病毒ORF2基因分段克隆与序列分析
摘要
戊型肝炎（Hepatitis E）是由戊型肝炎病毒（Hepatitis E virus HEV）引起的急性自限性疾病。其临床特征为通常表现是发热、全身疲乏、食欲不振、厌油腻、恶心呕吐、尿色深黄如浓茶、眼睛和皮肤发黄，肝功能检查出现转氨酶迅速升高到几百甚至几千单位，黄疸较常见，并可持续一周。在亚洲、非洲和美洲等发展中国家人群中发生比较普遍，在一些地区可占到急性病毒性肝炎的50％，是导致发病和病死的重要因素，尤其对孕妇可造成有20％的病死率。对人类健康具有严重危害性。
HEV是一种无包膜的正义单链RNA病毒，目前归于嵌杯病毒科，直径为27－32nm，基因组全长大约为7.5kb，具有3个互相重叠的开放阅读框架即ORF1、ORF2和ORF3，并且5’－端有27个核苷酸的非编码区，3’－端为非结构区，ORF1长约5kb，它主要编码与HEV复制相关的非结构蛋白；ORF2长约2kb，编码病毒衣壳蛋白，为主要的结构基因编码区。ORF3含有369个核苷酸，编码产物为磷蛋白，与细胞的支架及HEV特异性免疫活性有关。
不同地区来源的HEV基因序列有一定的差异，但是同一地区来源的HEV的基因序列保持相对稳定。随着HEV不断被分离，克隆和鉴定，根据各毒株核苷酸，氨基酸的同源性的大小以及系统进化树的分析，全球的HEV至少有8个基因型。我国主要为1和4型，而且散发性戊型肝炎中以4型为主。在发展中国家主要引起病毒性肝炎的戊型肝炎病毒（HEV），已经在北美和亚洲猪体内检测出，关于是否能在人畜之间传播引起了人们极大的关心。
本研究对黑龙江省一株猪粪便中的HEV的部分特性进行研究，根据已知序列设计了5组兼并引物，采用逆转录聚合酶链式反应（RT-PCR）检测猪粪便中的HEV RNA,分段的将PCR阳性产物的ORF2片断基因进行了克隆测序，并与已知人的HEV和猪的HEV进行了序列比较分析。结果该株序列ORF2区与已知人的1，2，3，4型HEV ORF2区的同源率为59.6％、56.8%、59.6％、74.4％。与美国猪和日本猪的HEV的同源率只用有57.7％。说明该株猪源的HEV与中国人戊型肝炎病毒基因IV型的关系比较近，但是有着较高的基因异质性，是一株比较独特的HEV株。
综上所述，本实验对黑龙江省一株猪源的HEV病毒的部分特性进行研究，并对其ORF2区基因进行了克隆比较分析，对于更好地了解HEV变异性和流行病学特征有重要意义；同时，可以为寻找HEV共同抗原和通用性PCR引物，研制更加敏感、特异的HEV诊断试剂盒，研制行之有效的HEV基因工程疫苗及为HEV感染的预防和临床治疗提供资料。
Hepatitis E is a self-limited acute illness which caused by Hepatitis E virus (HEV). Acute icteric hepatitis, the commonest recognizable form of illness, is usually insidious in onset and has an initial prodromal phase lasting a week, with a variable combination of flu-like symptoms, fever, mild chills, abdominal pain, anorexia, nausea, aversion to smoking, vomiting, clay-coloured stools, darkor tea-coloured urine, diarrhoea, arthralgia, asthenia and a transient macular skin rash. In serum alanine aminotaansferasa elevate quickly to hundreds Unit. The illness may be particularly severe among pregnant women, with mortality rates reaching as high as 20%.Human healthy will be severe harmed.
Hepatitis E virus (HEV) is a non-enveloped RNA (7.5 kb) virus, 27-32nm diameter particle with icosahedral symmetry. The viral genome, approximately 7.5kb in length, is a single-stranded, positive-sense, polyadenylated RNA molecule that contains short 5,and 3, non-coding regions of 27 and 68 nucleotides, respectively. It contains three overlapping open reading frames (ORF).ORF1 (5.0kb) is predicted to code for amino acid polyprotein consisting of non-structural proteins that are involved in viral genome replication and viral protein processing. ORF2 (2.0kb) encodes for the viral capsid protein. It is a major coding region of structural protains.ORF3 (369bp) is a phosphoprotein, which appears to associate with the cytoskeleton and immunocompetence of HEV.
HEV gene has some difference with different areas. It retains relative stable sequence which stemmed from same regions. With HEV isolated、cloned、identified, eight genotypes of HEV were divided in world. Isolates 1 and 4 are found in China, and the major is isolate 4 in dispersed Hepatitis E.HEV, formerly, a major cause of viral hepatitis in much of the developing world, has recently been detected in swine in North America and Asia, raising concern about potential for zoonotic transmission
To investigate if HEV is commonly present in swine in the province of Heilongjiang, pooled stool samples from swine farms were assayed by reverse transcription-polymerase chain reaction (RT-PCR) amplification. We designed 5 group primers, HEV ORF2 gene was detected by RT-PCR and was sequenced, and the results were compared with published sequences of HEV genotypes from humans and swine. HEV strains from swine in Heilongjiang has 59.6%、56.8％、59.6％ and 74.4％ identity to genotype I、II、III and IV in ORF2 regions. They had 57.7% identity to swine HEV from America and Japan. Our data show that HEV in swine in Heilongjiang are genetically closely related to HEV isolates from humans HEV type IV in ORF2 regions, but there were a lot of differences.
In short, Characters of HEV strains from swine in Heilongjiang were examined, and ORF2
regions were cloned and sequenced, which is important to understand variation and epidemiology of hepatitis E; the same time, the study provided much useful statistics for seeking common antigenic determinants and PCR primer which could be used for making more sensitive specific diagnostic kits and vaccine for HEV.
Candidate：Yu Min Major：Preventive Veterinary Science
Supervisor:Prof. Li Jingpeng
HEV是一种无包膜的正义单链RNA病毒，目前归于嵌杯病毒科，直径为27－32nm，基因组全长大约为7.5kb，具有3个互相重叠的开放阅读框架即ORF1、ORF2和ORF3，并且5’－端有27个核苷酸的非编码区，3’－端为非结构区，ORF1长约5kb，它主要编码与HEV复制相关的非结构蛋白；ORF2长约2kb，编码病毒衣壳蛋白，为主要的结构基因编码区。ORF3含有369个核苷酸，编码产物为磷蛋白，与细胞的支架及HEV特异性免疫活性有关。
不同地区来源的HEV基因序列有一定的差异，但是同一地区来源的HEV的基因序列保持相对稳定。随着HEV不断被分离，克隆和鉴定，根据各毒株核苷酸，氨基酸的同源性的大小以及系统进化树的分析，全球的HEV至少有8个基因型。我国主要为1和4型，而且散发性戊型肝炎中以4型为主。在发展中国家主要引起病毒性肝炎的戊型肝炎病毒（HEV），已经在北美和亚洲猪体内检测出，关于是否能在人畜之间传播引起了人们极大的关心。
本研究对黑龙江省一株猪粪便中的HEV的部分特性进行研究，根据已知序列设计了5组兼并引物，采用逆转录聚合酶链式反应（RT-PCR）检测猪粪便中的HEV RNA,分段的将PCR阳性产物的ORF2片断基因进行了克隆测序，并与已知人的HEV和猪的HEV进行了序列比较分析。结果该株序列ORF2区与已知人的1，2，3，4型HEV ORF2区的同源率为59.6％、56.8%、59.6％、74.4％。与美国猪和日本猪的HEV的同源率只用有57.7％。说明该株猪源的HEV与中国人戊型肝炎病毒基因IV型的关系比较近，但是有着较高的基因异质性，是一株比较独特的HEV株。
综上所述，本实验对黑龙江省一株猪源的HEV病毒的部分特性进行研究，并对其ORF2区基因进行了克隆比较分析，对于更好地了解HEV变异性和流行病学特征有重要意义；同时，可以为寻找HEV共同抗原和通用性PCR引物，研制更加敏感、特异的HEV诊断试剂盒，研制行之有效的HEV基因工程疫苗及为HEV感染的预防和临床治疗提供资料。
Hepatitis E is a self-limited acute illness which caused by Hepatitis E virus (HEV). Acute icteric hepatitis, the commonest recognizable form of illness, is usually insidious in onset and has an initial prodromal phase lasting a week, with a variable combination of flu-like symptoms, fever, mild chills, abdominal pain, anorexia, nausea, aversion to smoking, vomiting, clay-coloured stools, darkor tea-coloured urine, diarrhoea, arthralgia, asthenia and a transient macular skin rash. In serum alanine aminotaansferasa elevate quickly to hundreds Unit. The illness may be particularly severe among pregnant women, with mortality rates reaching as high as 20%.Human healthy will be severe harmed.
Hepatitis E virus (HEV) is a non-enveloped RNA (7.5 kb) virus, 27-32nm diameter particle with icosahedral symmetry. The viral genome, approximately 7.5kb in length, is a single-stranded, positive-sense, polyadenylated RNA molecule that contains short 5,and 3, non-coding regions of 27 and 68 nucleotides, respectively. It contains three overlapping open reading frames (ORF).ORF1 (5.0kb) is predicted to code for amino acid polyprotein consisting of non-structural proteins that are involved in viral genome replication and viral protein processing. ORF2 (2.0kb) encodes for the viral capsid protein. It is a major coding region of structural protains.ORF3 (369bp) is a phosphoprotein, which appears to associate with the cytoskeleton and immunocompetence of HEV.
HEV gene has some difference with different areas. It retains relative stable sequence which stemmed from same regions. With HEV isolated、cloned、identified, eight genotypes of HEV were divided in world. Isolates 1 and 4 are found in China, and the major is isolate 4 in dispersed Hepatitis E.HEV, formerly, a major cause of viral hepatitis in much of the developing world, has recently been detected in swine in North America and Asia, raising concern about potential for zoonotic transmission
To investigate if HEV is commonly present in swine in the province of Heilongjiang, pooled stool samples from swine farms were assayed by reverse transcription-polymerase chain reaction (RT-PCR) amplification. We designed 5 group primers, HEV ORF2 gene was detected by RT-PCR and was sequenced, and the results were compared with published sequences of HEV genotypes from humans and swine. HEV strains from swine in Heilongjiang has 59.6%、56.8％、59.6％ and 74.4％ identity to genotype I、II、III and IV in ORF2 regions. They had 57.7% identity to swine HEV from America and Japan. Our data show that HEV in swine in Heilongjiang are genetically closely related to HEV isolates from humans HEV type IV in ORF2 regions, but there were a lot of differences.
In short, Characters of HEV strains from swine in Heilongjiang were examined, and ORF2
regions were cloned and sequenced, which is important to understand variation and epidemiology of hepatitis E; the same time, the study provided much useful statistics for seeking common antigenic determinants and PCR primer which could be used for making more sensitive specific diagnostic kits and vaccine for HEV.
Candidate：Yu Min Major：Preventive Veterinary Science
Supervisor:Prof. Li Jingpeng
引文
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Berke T, Matson DO Reclassification of the Caliciviridae into distinct genera and exclusion of hepatitis E virus from the family on the basis of comparative phylogenetic analysis Arch Virol 2000:145:1421-36.
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Jameel S, et al. Virolgy 1995;47:299-302.
Zafrullah, M., M. H. Ozdener, S. K. Panda, and S. Jameel. 1997. The ORF3 protein of hepatitis E virus is a phosphoprotein that associated with the cytoskeleton. J. Virol. 71:9045-9053.
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Erker J C et al. A hepatitis E virus variant from the United States :molecular characterization and transmission in cynomolgus macaques J Gen Virol 1999,80：681－690.
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Zhang JZ et al Conformational antigentic determinants generated by interactions between a barterially expressed recombinant peptide of the hepatitis E virus structural protein J Med Virol 2001, 64: 125~132.
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Reyes G R,et al. Isolation of a Cadan from the virus responsible for enterically transmitted non-A non-B hepatitis. Science ,1990,247:1335.
Datta R et al.Acute sporadic non-A non-B viral hepatitis of adults in India:epidemiological and immunological studies[J].J Gastroenterol Hepatol,2:333-345.
Zhuang H ,Cao X Y,et al. Epidemiology of hepatitis E in China[J]. Gastroenterologia Japonica ,1991,36(supp13):135-138.
Van Cugch-Gendre H ,et al .J Med Virol, 1998;55:197－202[28].
Panda，S.K.and Jameel，S.(1997)Hepatitis E virus：from epidemiology to molecular biology.Vir Hep Rev 3,227-251.
王宇明等，戊型肝炎的研究进展 中华传染病杂志 1993 ，11（3）：158.
Berke T, Matson DO Reclassification of the Caliciviridae into distinct genera and exclusion of hepatitis E virus from the family on the basis of comparative phylogenetic analysis Arch Virol 2000:145:1421-36.
Nanda SK et al.Protranteed viremia during acute sporadic hepatitis E virus infection .Gastroenterology 1995;108:225-30.
Khudyakov, Y. E., E. N. Lopareva, D. L. Jue, T. K. Crews, S. P. Thyagarajan, and H. A. Fields. 1999. Antigenic domains of the open reading frame 2-encoded protein of hepatitis E virus. J. Clin. Microbiol. 37:2863-2871.
Jameel S, et al. Virolgy 1995;47:299-302.
Zafrullah, M., M. H. Ozdener, S. K. Panda, and S. Jameel. 1997. The ORF3 protein of hepatitis E virus is a phosphoprotein that associated with the cytoskeleton. J. Virol. 71:9045-9053.
Meng J ,et al . J Med Virol 1999 ;57:126-133.
Erker J C et al. A hepatitis E virus variant from the United States :molecular characterization and transmission in cynomolgus macaques J Gen Virol 1999,80：681－690.
Schlauder G G et al.The sequence and phylogentic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States J Gen Virol 1998,79;447-456.
Hsieh S Y et al. Indentification of a novel strain of hepatitis E virus responsible for sporadic acute hepatitis in Taiwan J Med Vorol 1998,53: 300-304.
Zanetti A R et al. Indentification of a novel variant of hepatitis E virus in Italy[J] J Med Virology 1999, 57 : 294-297.
Karetnyi Y V,et al. J M icrobiol Epidemiol Immunol ,1993,4:52-56.
Meng XJ et al Genetic and experimental evidence for cross-species infection by the swine hepatitis E virus J virol 1998 ,72 : 9714~9721.
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