猪戊型肝炎病毒分子流行病学分析及基因3型猪戊型肝炎病毒感染性克隆的构建
|
摘要
戊型肝炎(Hepatitis E, HE)以前称非甲-非乙型肝炎,是由戊型肝炎病毒(Hepatitis E virus, HEV)感染引起的一种经粪口途径传播的疾病,呈全球分布。该病属于人畜共患病,对人类健康具有很大的危害,已引起亚洲、非洲等许多发展中国家暴发急性肝炎。研究证实HEV可感染猪、野猪、牛、山羊、绵羊、鹿、灵长类、鸡、犬、贝类、啮齿类等多种动物,在这些动物宿主中以猪的感染率最高,病毒载量最大,而且从中分离到的病毒和人源毒株也有较高的同源性。因此,调查清楚猪群中HEV的存在状况,对防控人戊型肝炎具有重要的公共卫生学意义。
1上海地区戊型肝炎病毒分子流行病学研究
为了能够准确的了解上海地区戊型肝炎在猪群中的流行情况,从上海郊区的39个猪场中采集1487份粪便样品,通过RT-nPCR(反转录套式PCR)的方法,扩增HEV ORF2的部分片断,通过测序和遗传进化分析来研究病毒的存在状况。试验结果表明,1487份样品中的297份样品为HEV RNA阳性,阳性率20%。297份阳性样品中158份为基因3型阳性,139份为基因4型阳性,分别占总阳性率的10.6%和9.30%,基因3型HEV在感染中占主导优势。各个猪场HEV阳性率在12.1%-36%之间,分型结果显示上海郊区猪场HEV基因型为基因3型和4型,从中选取的12株代表性毒株中8株3型HEV属于3b亚型。4株4型HEV分属于4b、4d和一个新的基因亚型。
对不同猪场基因3型和4型HEV的流行情况进行相关性分析后发现,上海郊区猪场基因3型和4型HEV的感染率呈负相关,提示两个基因型在生存和进化过程中可能存在相互竞争的关系。本次调查所关注的3个群体中,2-6月龄的猪群中HEV的感染率最高,为29.7%,其次为1-8周龄的猪群,感染率为6.9%,母猪的感染率仅为3.1%,在调查的群体中为最低。
遗传进化分析表明,发现两株新的HEV基因4型亚型病毒株。此外,基因3型HEV在进化树中和一株日本分离株的同源性最高。这说明基因3型HEV在上海分布已经较为广泛,但以前的研究表明,中国境内猪群中只存在基因4型HEV,因此其来源还有待进一步研究。
2猪基因3型戊型肝炎病毒上海分离株全基因组序列的测定及遗传进化分析
为了进一步分析上海地区猪群中基因3型HEV与人源HEV的关系,本研究采用RT-nPCR方法对前面分子流行病学研究中从猪粪便样品中分离到的一株基因3型HEV(命名为SAAS-JDY5)的全基因组序列进行分片段扩增,并对其5’端和3’端两个末端的序列采用cDNA末端快速扩增技术(Rapid Amplification of cDNA Ends, RACE)进行扩增、克隆和测序。
测序结果表明SAAS-JDY5去除3’端poly(A)尾全长为7225bp,5’-非编码区(NCR)为26bp,3'-NCR为73bp。5’和3’非编码区之间有3个开放阅读框(ORF),ORF1从27到5135位核苷酸,全长5109bp,编码1702个氨基酸。ORF2从5170-7152位核苷酸,全长1983bp,编码660个氨基酸。ORF3从5159到5500位核苷酸,全长342bp,编码113个氨基酸,此序列已经登陆GenBank,序列号为FJ527832。通过生物学软件,将SAAS-JDY5与其它已报道的88个HEV病毒株核苷酸和氨基酸序列比较后发现,SAAS-JDY5HEV全序列与HEV Ⅰ、Ⅱ、Ⅳ型的同源性在74-87%之间,而与3型HEV的同源性最高为90.8%,其中ORF1与3型HEV核苷酸的同源性为86.1-88.7%,氨基酸同源性为96.3-96.9%；ORF2与3型HEV核苷酸的同源性为88.7-90.8%,氨基酸同源性为97.6-98.9%。ORF3与3型HEV核苷酸的同源性为93.8-98.1%,氨基酸同源性为92.6-98.4%。
遗传进化分析表明,核苷酸的进化与基因3型HEV在一个分枝上,表明SAAS-JDY5病毒株为基因3型HEV。本研究首次报道了在中国上海地区分离到的基因3型HEV的全基因组序列。3重组HEV衣壳蛋白的原核表达、鉴定及多克隆抗体的制备
由于HEV缺乏成熟的细胞培养模型,目前一般采用ORF2编码的蛋白作为表达抗原,以此来检测HEV特异性抗体。本研究选取猪基因3型HEVSAAS-JDY5ORF21141-1842区域(a.a381-614)基因,在构建重组表达载体pET-32a-p234的基础上,成功地表达了SAAS-JDY5株的p234蛋白,通过进一步的研究证实该重组蛋白具有较好的免疫原性,能够与猪源HEV阳性血清发生免疫学反应。用该蛋白免疫新西兰大白兔,获得了较高的抗体效价,为进行下一步的研究奠定了基础。
4猪基因3型HEV上海分离株感染性克隆的构建及体外转录活性的研究
由于缺乏合适的体外细胞培养系统,影响了HEV的进一步研究。通过反向遗传学手段获得HEV的感染性克隆后,就可在DNA水平上通过突变、缺失、插入等手段来研究HEV的复制、表达、病毒的重组、病毒与宿主的相互作用等,也可进行抗病毒策略研究,还可以构建新的病毒载体。本研究在已知猪HEVSAAS-JDY5株全基因组序列的基础上,根据基因组和克隆载体的酶切位点,将已经克隆到的9段3型猪HEV克隆片段经过一系列的克隆和亚克隆后,构建了含有基因组全长cDNA克隆的pGEM4z-HEV重组质粒。在pGEM4z-HEV质粒中,cDNA克隆的5’端上游引入了T7启动子序列,并且在T7启动子的上游引入了单一酶切位点Xba I,3'端引入单一酶切位点Hind Ⅲ.基因组进行全长测序后发现了14个核苷酸的突变。不改变其中的沉默突变序列以作为遗传标志,对于其中的错义突变运用大引物PCR技术(Megaprimer PCR)进行定点突变修补突变的碱基,最终获得了含有遗传标志的基因3型HEV SAAS-JDY5林基因组全长cDNA的重组质粒pGEM4z-HEV.
为了验证所构建感染性克隆的感染性,首先在Huh7细胞中进行了细胞试验。将含有猪基因3型戊型肝炎病毒SAAS-JDY5株全基因组的以T7启动子启动转录的重组质粒pGEM4z-HEV进行体外转录,成功获得纯化的HEV基因组RNA。将其转染Huh7细胞后连续培养6天,对转染的细胞通过RT-nPCR和间接免疫荧光等方法检测,证明病毒蛋白在转染的细胞中成功进行了表达。结果表明所构建的感染性克隆pGEM4z-HEV具有感染性,为下一步的体内试验提供了可行性参考。
5基因3型HEV人工感染SD大鼠的初步研究
体外转录后的HEV RNA转染Huh7细胞,通过间接免疫荧光试验和RT-nPCR初步检测为阳性后,进行了动物试验。pGEM4z-HEV感染性克隆线性化后作为模板用体外转录试剂盒进行体外转录,转录后的RNA肝内注射SD大鼠(SPF级),同时设立阴性和阳性对照。所有动物接种后连续观察55天,分别于注射后的0、3、7、14、21、28、35、45、50、55天分别采集大鼠的粪便和血清样品,粪便样品用RT-nPCR检测HEV RNA,血清样品用北京万泰生产的HEV总抗体检测试剂盒检测血清中的抗体,用RT-nPCR检测HEV RNA.检测结果表明大鼠在感染后7天左右在粪便中检测到HEV RNA;感染后14天左右开始出现病毒血症,一直持续到第28天还能在血清中检测到HEV RNA;感染后14天左右血清anti-HEV抗体阳性,而阴性对照中均没检测到HEV RNA和特异性抗体。为了检测是否自然感染的可能,采用两套引物对遗传标志部位进行测序验证,结果证实能够检测到遗传标志。通过本研究证实猪基因3型HEV可以跨种传播给SD大鼠,提示该动物可以作为研究HEV复制及致病机理方面一种理想的动物模型。
以上体外体内试验结果证明,含有HEV SAAS-JDY5病毒株基因组全长cDNA的pGEM4z-HEV重组质粒具有感染性。本研究首次在我国构建出了猪HEV基因3型的感染性克隆。
综上所述,本研究在充分普查上海市猪群中HEV的流行分布及分析猪群中流行的HEV两个基因型之间相互关系的基础上,对来源于猪粪便中的SAAS-JDY5HEV进行了全基因组序列分析,确定了该地区猪群中流行的HEV的基因型,有利于更深入分析猪源HEV与人源HEV的关系。而且在获得全基因组序列的基础上构建了含有T7启动子的感染性克隆,并通过体外及体内试验证实所获得的转录本具有感染性,成功拯救出了基因3型猪HEV,为HEV的分子生物学研究及基因工程疫苗研究打下基础,填补了国内空白。此外,通过本研究证实了猪基因3型HEV可以经人工感染的方式跨种传播给SD大鼠,提示该动物可以作为研究HEV复制及致病机理方面一种可选的动物模型。
Hepatitis E (HE), formerly known as non A, non B hepatitis, is a fecal-oral transmission disease caused by hepatitis E virus (HEV) and showing the global distribution. It is a zoonotic disease that has significant harm to human health, and has caused the outbreak of acute hepatitis in many developing countries. Studies have confirmed that HEV can infect pigs, cattle, goats, sheep, deer, primates, chickens, dogs, shellfish, rodents and other animals. Pigs have the highest infection rate and maximum viral load in these animal hosts, and the viruses isolated from pigs have high homology with human strains. Therefore, the existence status of the disease in pig herds has important public health significance on preventing and controlling the incidence of human hepatitis E.
1Molecular epidemiological study of Hepatitis E Virus in Shanghai
In order to accurately understand the prevalence of hepatitis E in shanghai,1487stool samples were collected from39pig farms in the suburbs of Shanghai. The partial sequence of HEV ORF2region was amplified by RT-nPCR method, and then sequenced and phylogenetic analyzed for studying the existing state of the virus. The results showed that297of1487samples were positive for HEV RNA (positive rate was20%). In the297samples,158samples were positive for the genotype3HEV,139were positive for the genotype4HEV, the total positive rate of each was10.6%and9.3%, respectively. Genotype3HEV accounted for the dominance in the infection. The HEV positive rate of all tested farms was from12.1%to36%, and the classification results showed that the genotypes of the HEV infection in the suburbs of Shanghai was genotype3and genotype4, among the12selected representative strains,8strains belonged to3b sub-genotype,4strains belonged to4b,4d and a new sub-genotype.
We found that there was a middle scale negative relevance between positivity rates of genotype3and4HEVs in the different farms, which suggested a competition for niches between the two genotypes. In addition, the2-6months pigs had the highest infection rate in the three concerned groups (29.7%), the infection rate of1-8weeks pigs took second place in the survey, i.e.6.9%, the sows were only3.1%, which was the lowest among the three groups.
Phylogenetic analysis showed that two new strains of subtype4HEV were identified. In addition, the genotype3HEV had the highest homology with a Japanese strain in the phylogenetic tree. The results showed that genotype3HEV was more widely distributed in Shanghai. However, previous studies had shown that there was only genotype4HEV in mainland China, thus its origin required further study.
2The complete genome sequence determination and phylogenetic analysis of the SAAS-JDY5genotype3HEV
To further analyze the genotype3HEV infection in Shanghai suburb, the complete sequence of the genotype3HEV strain (SAAS-JDY5) was amplified by RT-nPCR and Overlap PCR method, and the both ends of5'and3'end sequences were amplified by the RACE (rapid amplification of cDNA ends) method.
The sequencing results showed that the full-length genome of SAAS-JDY5strain was7225bp (excluding the3'poly (A) tail) with26bp of5'-NCR and73bp of3'-NCR. There were3ORFs between the5'-NCR and3'-NCR, ORF1, from nt27to5135, has5109bp by encoding1702amino acids; ORF2, from nt5170to7152, has1983bp by encoding660amino acids; and ORF3, from nt5159to5500, has342bp by encoding113amino acids. The obtained sequence was submitted to the GenBank database and the accession number was FJ527832. The obtained sequence of SAAS-JDY5was compared with other88reported HEV sequences at nucleotide and amino acid level, and we found that the SAAS-JDY5complete sequences had74-87%nucleotide homology with the genotype Ⅰ, Ⅱ, Ⅳ HEV; However, it had the highest homology (90.8%) with genotype3HEV. In the ORF1region, the SAAS-JDY5strain shared86.1%to88.7%and96.3%to96.9%sequence identities with other genotype3HEV at the nucleotide and amino acid levels, respectively. The sequence and amino acid similarities in the ORF2region were88.7%to90.8%and97.6%to98.9%, respectively. In the ORF3region, they were93.8%to98.1%and92.6%to98.4%, respectively.
Phylogenetic analysis showed that the nucleotide of SAAS-JDY5was in the same branch with genotype3HEV. Our study firstly report the complete genome sequence of genotype3HEV isolated from swine in Shanghai, China.
3The expression, identification of the recombinant HEV capsid protein and preparation of polyclonal antibodies
Since there is a lack of an efficient in vitro cell culture system and a practical animal model for HEV study, HEV-specific antibodies are detected by the ORF2antigen at present.
In this study, the ORF2region (a.a381-614) of SAAS-JDY5was expressed by constructing a expression vector pET-32a-p234, which was designated p234protein. It was confirmed that the recombinant protein (p234) had a good immunogenicity and the ability to response to swine anti-HEV positive serum. High titer antiserum was obtained by immunization of rabbits by the purified protein, which laid a solid foundation for our further research.
4Construction the swine genotype3HEV infectious cDNA clone of a Shanghai strain and the research on the in vitro transcription activity of the clone
Due to the lack of a suitable in vitro cell culture system, it was diffcult to proceed the futher study of HEV, thus greatly hampered the progress of HEV research. Reverse genetic system can be a valuable tool for studying the molecular biology of HEV. In this study, based on the obtained complete sequence of SAAS-JDY5strain, we cloned9overlapping DNA fragments through a series of DNA cloning and subcloning, and then constructed the recombinant plasmid designated pGEM4z-HEV which containing the full-length genome. In the pGEM4z-HEV plasmid, we introduced a unique XbaI restriction site and a T7RNA polymerase core promoter at the extreme5'terminus, and short stretch of15adenosines (A) and HindⅢ site at the3'end of the HEV cDNA clone. The plasmid pGEM4z-HEV was subjected to complete sequencing and14nucleotides mutations were identified. Silent mutations were kept as genetic markers and non-silent mutations were corrected by megaprimer PCR mutagenesis.
In order to verify the infectivity of the constructed infectious cDNA clone, we firstly carried out cell transfection test with huh7cells. Swine genotype3HEV complete genomic RNA was synthesized by in vitro transcription with pGEM4z-HEV as template. Transfected huh7cells were cultured for6days and tested by RT-nPCR and fluorescence assay (IFA). Results proved that the viral protein synthesis was successfully expressed in the infected cells, which indicated that the infectious HEV cDNA clone was successfully constructed.
5The preliminary study of artifical infect SD rats by genotype3HEV
Following the successful test on Huh7cells, we conducted animal experiments to check infection nature of the HEV cDNA clone. The recombinant plasmid pGEM4z-HEV were linearized and used as a template for in vitro RNA transcription, and SD rats (SPF) was injected with the in vitro synthesized RNA intrahepaticly. Feces and serum samples were collected from the animals at0,3,7,14,21,28,35,45,50,55days post the injection, then the fecal samples were conducted RT-nPCR detection of HEV RNA. The serum samples were conducted double antigen sandwich ELISA and RT-nPCR for the detection of anti-HEV specific antibody and HEV RNA. Our results showed that HEV RNA was detected in the stool samples at7days post infection; The viremia began at about14days post infection and existed till28days post infection; Rats had their sero conversion at about14days post inoculation, however, the animals in the negative group remained sero negative throughout the study. In order to detect the possibility of natural infection, two sets of primers were used for the detection of the genetic markers. The results identified the genetic markers in the extracted HEV RNA. Our research confirmed that the genotype3swine HEV can infect SD rats, which suggested that the SD rat can be used as an animal model for the research of HEV replication and pathogenesis.
In summary, a detailed epidemiological investigation of hepatitis E prevalence in Shanghai revealed a middle scale negative relationship between genotype3and4HEV. The complete sequence of the SAAS-JDY5strain which was isolated from our previous investigation was obtained and a complete sequence analysis was carried out. In addition, an infectious HEV cDNA clone was constructed and confirmed to be able to infect cultured cells and SD rats. The research laid the foundation for more advanced research on HEV molecular biology. The research also suggested the possibility that SD rats be used as an animal model of HEV replication and pathogenesis study.
1上海地区戊型肝炎病毒分子流行病学研究
为了能够准确的了解上海地区戊型肝炎在猪群中的流行情况,从上海郊区的39个猪场中采集1487份粪便样品,通过RT-nPCR(反转录套式PCR)的方法,扩增HEV ORF2的部分片断,通过测序和遗传进化分析来研究病毒的存在状况。试验结果表明,1487份样品中的297份样品为HEV RNA阳性,阳性率20%。297份阳性样品中158份为基因3型阳性,139份为基因4型阳性,分别占总阳性率的10.6%和9.30%,基因3型HEV在感染中占主导优势。各个猪场HEV阳性率在12.1%-36%之间,分型结果显示上海郊区猪场HEV基因型为基因3型和4型,从中选取的12株代表性毒株中8株3型HEV属于3b亚型。4株4型HEV分属于4b、4d和一个新的基因亚型。
对不同猪场基因3型和4型HEV的流行情况进行相关性分析后发现,上海郊区猪场基因3型和4型HEV的感染率呈负相关,提示两个基因型在生存和进化过程中可能存在相互竞争的关系。本次调查所关注的3个群体中,2-6月龄的猪群中HEV的感染率最高,为29.7%,其次为1-8周龄的猪群,感染率为6.9%,母猪的感染率仅为3.1%,在调查的群体中为最低。
遗传进化分析表明,发现两株新的HEV基因4型亚型病毒株。此外,基因3型HEV在进化树中和一株日本分离株的同源性最高。这说明基因3型HEV在上海分布已经较为广泛,但以前的研究表明,中国境内猪群中只存在基因4型HEV,因此其来源还有待进一步研究。
2猪基因3型戊型肝炎病毒上海分离株全基因组序列的测定及遗传进化分析
为了进一步分析上海地区猪群中基因3型HEV与人源HEV的关系,本研究采用RT-nPCR方法对前面分子流行病学研究中从猪粪便样品中分离到的一株基因3型HEV(命名为SAAS-JDY5)的全基因组序列进行分片段扩增,并对其5’端和3’端两个末端的序列采用cDNA末端快速扩增技术(Rapid Amplification of cDNA Ends, RACE)进行扩增、克隆和测序。
测序结果表明SAAS-JDY5去除3’端poly(A)尾全长为7225bp,5’-非编码区(NCR)为26bp,3'-NCR为73bp。5’和3’非编码区之间有3个开放阅读框(ORF),ORF1从27到5135位核苷酸,全长5109bp,编码1702个氨基酸。ORF2从5170-7152位核苷酸,全长1983bp,编码660个氨基酸。ORF3从5159到5500位核苷酸,全长342bp,编码113个氨基酸,此序列已经登陆GenBank,序列号为FJ527832。通过生物学软件,将SAAS-JDY5与其它已报道的88个HEV病毒株核苷酸和氨基酸序列比较后发现,SAAS-JDY5HEV全序列与HEV Ⅰ、Ⅱ、Ⅳ型的同源性在74-87%之间,而与3型HEV的同源性最高为90.8%,其中ORF1与3型HEV核苷酸的同源性为86.1-88.7%,氨基酸同源性为96.3-96.9%；ORF2与3型HEV核苷酸的同源性为88.7-90.8%,氨基酸同源性为97.6-98.9%。ORF3与3型HEV核苷酸的同源性为93.8-98.1%,氨基酸同源性为92.6-98.4%。
遗传进化分析表明,核苷酸的进化与基因3型HEV在一个分枝上,表明SAAS-JDY5病毒株为基因3型HEV。本研究首次报道了在中国上海地区分离到的基因3型HEV的全基因组序列。3重组HEV衣壳蛋白的原核表达、鉴定及多克隆抗体的制备
由于HEV缺乏成熟的细胞培养模型,目前一般采用ORF2编码的蛋白作为表达抗原,以此来检测HEV特异性抗体。本研究选取猪基因3型HEVSAAS-JDY5ORF21141-1842区域(a.a381-614)基因,在构建重组表达载体pET-32a-p234的基础上,成功地表达了SAAS-JDY5株的p234蛋白,通过进一步的研究证实该重组蛋白具有较好的免疫原性,能够与猪源HEV阳性血清发生免疫学反应。用该蛋白免疫新西兰大白兔,获得了较高的抗体效价,为进行下一步的研究奠定了基础。
4猪基因3型HEV上海分离株感染性克隆的构建及体外转录活性的研究
由于缺乏合适的体外细胞培养系统,影响了HEV的进一步研究。通过反向遗传学手段获得HEV的感染性克隆后,就可在DNA水平上通过突变、缺失、插入等手段来研究HEV的复制、表达、病毒的重组、病毒与宿主的相互作用等,也可进行抗病毒策略研究,还可以构建新的病毒载体。本研究在已知猪HEVSAAS-JDY5株全基因组序列的基础上,根据基因组和克隆载体的酶切位点,将已经克隆到的9段3型猪HEV克隆片段经过一系列的克隆和亚克隆后,构建了含有基因组全长cDNA克隆的pGEM4z-HEV重组质粒。在pGEM4z-HEV质粒中,cDNA克隆的5’端上游引入了T7启动子序列,并且在T7启动子的上游引入了单一酶切位点Xba I,3'端引入单一酶切位点Hind Ⅲ.基因组进行全长测序后发现了14个核苷酸的突变。不改变其中的沉默突变序列以作为遗传标志,对于其中的错义突变运用大引物PCR技术(Megaprimer PCR)进行定点突变修补突变的碱基,最终获得了含有遗传标志的基因3型HEV SAAS-JDY5林基因组全长cDNA的重组质粒pGEM4z-HEV.
为了验证所构建感染性克隆的感染性,首先在Huh7细胞中进行了细胞试验。将含有猪基因3型戊型肝炎病毒SAAS-JDY5株全基因组的以T7启动子启动转录的重组质粒pGEM4z-HEV进行体外转录,成功获得纯化的HEV基因组RNA。将其转染Huh7细胞后连续培养6天,对转染的细胞通过RT-nPCR和间接免疫荧光等方法检测,证明病毒蛋白在转染的细胞中成功进行了表达。结果表明所构建的感染性克隆pGEM4z-HEV具有感染性,为下一步的体内试验提供了可行性参考。
5基因3型HEV人工感染SD大鼠的初步研究
体外转录后的HEV RNA转染Huh7细胞,通过间接免疫荧光试验和RT-nPCR初步检测为阳性后,进行了动物试验。pGEM4z-HEV感染性克隆线性化后作为模板用体外转录试剂盒进行体外转录,转录后的RNA肝内注射SD大鼠(SPF级),同时设立阴性和阳性对照。所有动物接种后连续观察55天,分别于注射后的0、3、7、14、21、28、35、45、50、55天分别采集大鼠的粪便和血清样品,粪便样品用RT-nPCR检测HEV RNA,血清样品用北京万泰生产的HEV总抗体检测试剂盒检测血清中的抗体,用RT-nPCR检测HEV RNA.检测结果表明大鼠在感染后7天左右在粪便中检测到HEV RNA;感染后14天左右开始出现病毒血症,一直持续到第28天还能在血清中检测到HEV RNA;感染后14天左右血清anti-HEV抗体阳性,而阴性对照中均没检测到HEV RNA和特异性抗体。为了检测是否自然感染的可能,采用两套引物对遗传标志部位进行测序验证,结果证实能够检测到遗传标志。通过本研究证实猪基因3型HEV可以跨种传播给SD大鼠,提示该动物可以作为研究HEV复制及致病机理方面一种理想的动物模型。
以上体外体内试验结果证明,含有HEV SAAS-JDY5病毒株基因组全长cDNA的pGEM4z-HEV重组质粒具有感染性。本研究首次在我国构建出了猪HEV基因3型的感染性克隆。
综上所述,本研究在充分普查上海市猪群中HEV的流行分布及分析猪群中流行的HEV两个基因型之间相互关系的基础上,对来源于猪粪便中的SAAS-JDY5HEV进行了全基因组序列分析,确定了该地区猪群中流行的HEV的基因型,有利于更深入分析猪源HEV与人源HEV的关系。而且在获得全基因组序列的基础上构建了含有T7启动子的感染性克隆,并通过体外及体内试验证实所获得的转录本具有感染性,成功拯救出了基因3型猪HEV,为HEV的分子生物学研究及基因工程疫苗研究打下基础,填补了国内空白。此外,通过本研究证实了猪基因3型HEV可以经人工感染的方式跨种传播给SD大鼠,提示该动物可以作为研究HEV复制及致病机理方面一种可选的动物模型。
Hepatitis E (HE), formerly known as non A, non B hepatitis, is a fecal-oral transmission disease caused by hepatitis E virus (HEV) and showing the global distribution. It is a zoonotic disease that has significant harm to human health, and has caused the outbreak of acute hepatitis in many developing countries. Studies have confirmed that HEV can infect pigs, cattle, goats, sheep, deer, primates, chickens, dogs, shellfish, rodents and other animals. Pigs have the highest infection rate and maximum viral load in these animal hosts, and the viruses isolated from pigs have high homology with human strains. Therefore, the existence status of the disease in pig herds has important public health significance on preventing and controlling the incidence of human hepatitis E.
1Molecular epidemiological study of Hepatitis E Virus in Shanghai
In order to accurately understand the prevalence of hepatitis E in shanghai,1487stool samples were collected from39pig farms in the suburbs of Shanghai. The partial sequence of HEV ORF2region was amplified by RT-nPCR method, and then sequenced and phylogenetic analyzed for studying the existing state of the virus. The results showed that297of1487samples were positive for HEV RNA (positive rate was20%). In the297samples,158samples were positive for the genotype3HEV,139were positive for the genotype4HEV, the total positive rate of each was10.6%and9.3%, respectively. Genotype3HEV accounted for the dominance in the infection. The HEV positive rate of all tested farms was from12.1%to36%, and the classification results showed that the genotypes of the HEV infection in the suburbs of Shanghai was genotype3and genotype4, among the12selected representative strains,8strains belonged to3b sub-genotype,4strains belonged to4b,4d and a new sub-genotype.
We found that there was a middle scale negative relevance between positivity rates of genotype3and4HEVs in the different farms, which suggested a competition for niches between the two genotypes. In addition, the2-6months pigs had the highest infection rate in the three concerned groups (29.7%), the infection rate of1-8weeks pigs took second place in the survey, i.e.6.9%, the sows were only3.1%, which was the lowest among the three groups.
Phylogenetic analysis showed that two new strains of subtype4HEV were identified. In addition, the genotype3HEV had the highest homology with a Japanese strain in the phylogenetic tree. The results showed that genotype3HEV was more widely distributed in Shanghai. However, previous studies had shown that there was only genotype4HEV in mainland China, thus its origin required further study.
2The complete genome sequence determination and phylogenetic analysis of the SAAS-JDY5genotype3HEV
To further analyze the genotype3HEV infection in Shanghai suburb, the complete sequence of the genotype3HEV strain (SAAS-JDY5) was amplified by RT-nPCR and Overlap PCR method, and the both ends of5'and3'end sequences were amplified by the RACE (rapid amplification of cDNA ends) method.
The sequencing results showed that the full-length genome of SAAS-JDY5strain was7225bp (excluding the3'poly (A) tail) with26bp of5'-NCR and73bp of3'-NCR. There were3ORFs between the5'-NCR and3'-NCR, ORF1, from nt27to5135, has5109bp by encoding1702amino acids; ORF2, from nt5170to7152, has1983bp by encoding660amino acids; and ORF3, from nt5159to5500, has342bp by encoding113amino acids. The obtained sequence was submitted to the GenBank database and the accession number was FJ527832. The obtained sequence of SAAS-JDY5was compared with other88reported HEV sequences at nucleotide and amino acid level, and we found that the SAAS-JDY5complete sequences had74-87%nucleotide homology with the genotype Ⅰ, Ⅱ, Ⅳ HEV; However, it had the highest homology (90.8%) with genotype3HEV. In the ORF1region, the SAAS-JDY5strain shared86.1%to88.7%and96.3%to96.9%sequence identities with other genotype3HEV at the nucleotide and amino acid levels, respectively. The sequence and amino acid similarities in the ORF2region were88.7%to90.8%and97.6%to98.9%, respectively. In the ORF3region, they were93.8%to98.1%and92.6%to98.4%, respectively.
Phylogenetic analysis showed that the nucleotide of SAAS-JDY5was in the same branch with genotype3HEV. Our study firstly report the complete genome sequence of genotype3HEV isolated from swine in Shanghai, China.
3The expression, identification of the recombinant HEV capsid protein and preparation of polyclonal antibodies
Since there is a lack of an efficient in vitro cell culture system and a practical animal model for HEV study, HEV-specific antibodies are detected by the ORF2antigen at present.
In this study, the ORF2region (a.a381-614) of SAAS-JDY5was expressed by constructing a expression vector pET-32a-p234, which was designated p234protein. It was confirmed that the recombinant protein (p234) had a good immunogenicity and the ability to response to swine anti-HEV positive serum. High titer antiserum was obtained by immunization of rabbits by the purified protein, which laid a solid foundation for our further research.
4Construction the swine genotype3HEV infectious cDNA clone of a Shanghai strain and the research on the in vitro transcription activity of the clone
Due to the lack of a suitable in vitro cell culture system, it was diffcult to proceed the futher study of HEV, thus greatly hampered the progress of HEV research. Reverse genetic system can be a valuable tool for studying the molecular biology of HEV. In this study, based on the obtained complete sequence of SAAS-JDY5strain, we cloned9overlapping DNA fragments through a series of DNA cloning and subcloning, and then constructed the recombinant plasmid designated pGEM4z-HEV which containing the full-length genome. In the pGEM4z-HEV plasmid, we introduced a unique XbaI restriction site and a T7RNA polymerase core promoter at the extreme5'terminus, and short stretch of15adenosines (A) and HindⅢ site at the3'end of the HEV cDNA clone. The plasmid pGEM4z-HEV was subjected to complete sequencing and14nucleotides mutations were identified. Silent mutations were kept as genetic markers and non-silent mutations were corrected by megaprimer PCR mutagenesis.
In order to verify the infectivity of the constructed infectious cDNA clone, we firstly carried out cell transfection test with huh7cells. Swine genotype3HEV complete genomic RNA was synthesized by in vitro transcription with pGEM4z-HEV as template. Transfected huh7cells were cultured for6days and tested by RT-nPCR and fluorescence assay (IFA). Results proved that the viral protein synthesis was successfully expressed in the infected cells, which indicated that the infectious HEV cDNA clone was successfully constructed.
5The preliminary study of artifical infect SD rats by genotype3HEV
Following the successful test on Huh7cells, we conducted animal experiments to check infection nature of the HEV cDNA clone. The recombinant plasmid pGEM4z-HEV were linearized and used as a template for in vitro RNA transcription, and SD rats (SPF) was injected with the in vitro synthesized RNA intrahepaticly. Feces and serum samples were collected from the animals at0,3,7,14,21,28,35,45,50,55days post the injection, then the fecal samples were conducted RT-nPCR detection of HEV RNA. The serum samples were conducted double antigen sandwich ELISA and RT-nPCR for the detection of anti-HEV specific antibody and HEV RNA. Our results showed that HEV RNA was detected in the stool samples at7days post infection; The viremia began at about14days post infection and existed till28days post infection; Rats had their sero conversion at about14days post inoculation, however, the animals in the negative group remained sero negative throughout the study. In order to detect the possibility of natural infection, two sets of primers were used for the detection of the genetic markers. The results identified the genetic markers in the extracted HEV RNA. Our research confirmed that the genotype3swine HEV can infect SD rats, which suggested that the SD rat can be used as an animal model for the research of HEV replication and pathogenesis.
In summary, a detailed epidemiological investigation of hepatitis E prevalence in Shanghai revealed a middle scale negative relationship between genotype3and4HEV. The complete sequence of the SAAS-JDY5strain which was isolated from our previous investigation was obtained and a complete sequence analysis was carried out. In addition, an infectious HEV cDNA clone was constructed and confirmed to be able to infect cultured cells and SD rats. The research laid the foundation for more advanced research on HEV molecular biology. The research also suggested the possibility that SD rats be used as an animal model of HEV replication and pathogenesis study.
引文
[1]Zhuang, H., X.Y. Cao, C.B. Liu, et al., Epidemiology of hepatitis E in China. Gastroenterologia Japonica[J],1991,26 Suppl 3:135-138.
[2]Irshad, M., Hepatitis E virus:an update on its molecular, clinical and epidemiological characteristics. Intervirology[J],1999,42 (4):252-262.
[3]Balayan, M.S., A.G. Andjaparidze, S.S. Savinskaya, et al., Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology[J],1983,20 (1):23-31.
[4]Reyes, G.R., M.A. Purdy, J.P. Kim, et al., Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science[J],1990,247 (4948): 1335-1339.
[5]Purcell, R.H. and S.U. Emerson, Hepatitis E:an emerging awareness of an old disease. Journal of Hepatology[J],2008,48 (3):494-503.
[6]Navaneethan, U., M. Al Mohajer, and M. Shata, Hepatitis E and pregnancy: understanding the pathogenesis. Liver International[J],2008,28 (9):1190-1199.
[7]Vitral, C.L., M.A. Pinto, L.L. Lewis-Ximenez, et al., Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil. Memorias do Institute Oswaldo Cruz[J],2005,100 (2):117-122.
[8]Ellis, J., E. Clark, D. Haines, et al., Porcine circovirus-2 and concurrent infections in the field. Veterinary Microbiology[J],2004,98 (2):159-163.
[9]Tan, D., S.W. Im, J.L. Yao, et al., Acute sporadic hepatitis E virus infection in southern China. Journal of Hepatology[J],1995,23 (3):239-245.
[10]Tam, A.W., M.M. Smith, M.E. Guerra, et al., Hepatitis E virus (HEV):molecular cloning and sequencing of the full-length viral genome. Virology[J],1991,185 (1):120-131.
[11]Balayan, M.S., R.K. Usmanov, N.A. Zamyatina, et al., Brief report:experimental hepatitis E infection in domestic pigs. Journal of Medical Virology[J],1990,32 (1):58-59.
[12]Lu, L., J. Drobeniuc, N. Kobylnikov, et al., Complete sequence of a Kyrgyzstan swine hepatitis E virus (HEV) isolated from a piglet thought to be experimentally infected with human HEV. Journal of Medical Virology[J],2004,74 (4):556-562.
[13]Meng, X.J., Swine hepatitis E virus:cross-species infection and risk in xenotransplantation. Current Topics in Microbiology and Immunology[J],2003,278: 185-216.
[14]Meng, X.J., R.H. Purcell, P.G Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[15]Payne, C.J., T.M. Ellis, S.L. Plant, et al., Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus. Veterinary Microbiology[J],1999,68 (1-2):119-125.
[16]Haqshenas, G., H.L. Shivaprasad, P.R. Woolcock, et al., Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis-splenomegaly syndrome in the United States. Journal of General Virology[J], 2001,82 (Pt 10):2449-2462.
[17]Erker, J.C., S.M. Desai, G.G Schlauder, et al., A hepatitis E virus variant from the United States:molecular characterization and transmission in cynomolgus macaques. Journal of General Virology[J],1999,80 (Pt 3):681-690.
[18]Gouvea, V., C.H. Hoke, Jr., and B.L. Innis, Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis. Journal of Virological Methods[J],1998, 70 (1):71-78.
[19]Schlauder, G.G. and I.K. Mushahwar, Genetic heterogeneity of hepatitis E virus. Journal of Medical Virology[J],2001,65 (2):282-292.
[20]Schlauder, G.G, S.M. Desai, A.R. Zanetti, et al., Novel hepatitis E virus (HEV) isolates from Europe:evidence for additional genotypes of HEV. Journal of Medical Virology[J], 1999,57 (3):243-251.
[21]Schlauder, G.G., B. Frider, S. Sookoian, et al., Identification of 2 novel isolates of hepatitis E virus in Argentina. The Journal of infectious diseases[J],2000,182 (1): 294-297.
[22]Tsarev, S.A., L.N. Binn, P.J. Gomatos, et al., Phylogenetic analysis of hepatitis E virus isolates from Egypt. Journal of Medical Virology[J],1999,57 (1):68-74.
[23]van Cuyck-Gandre, H., H.Y. Zhang, S.A. Tsarev, et al., Short report:phylogenetically distinct hepatitis E viruses in Pakistan. American Journal of Tropical Medicine and Hygiene[J],2000,62 (2):187-189.
[24]Wang, Y, R. Ling, J.C. Erker, et al., A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. Journal of General Virology[J],1999,80 (Pt 1):169-177.
[25]Zhai, L., X. Dai, and J. Meng, Hepatitis E virus genotyping based on full-length genome and partial genomic regions. Virus Research[J],2006,120 (1-2):57-69.
[26]Lu, L., C. Li, and C.H. Hagedorn, Phylogenetic analysis of global hepatitis E virus sequences:genetic diversity, subtypes and zoonosis. Reviews in Medical Virology[J], 2006,16 (1):5-36.
[27]Jameel, S., M. Zafrullah, Y.K. Chawla, et al., Reevaluation of a North India isolate of hepatitis E virus based on the full-length genomic sequence obtained following long RT-PCR. Virus Research[J],2002,86 (1-2):53-58.
[28]Aggarwal, R., K. McCaustland, J. Dilawari, et al., Genetic variability of hepatitis E virus within and between three epidemics in India. Virus Research[J],1999,59 (1):35-48.
[29]Arankalle, V.A., S. Paranjape, S.U. Emerson, et al., Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993). Journal of General Virology[J],1999,80 (Pt 7): 1691-1700.
[30]Vaidya, S.R., S.D. Chitambar, and V.A. Arankalle, Polymerase chain reaction-based prevalence of hepatitis A, hepatitis E and TT viruses in sewage from an endemic area. Journal of Hepatology[J],2002,37 (1):131-136.
[31]Tsarev, S.A., S.U. Emerson, G.R. Reyes, et al., Characterization of a prototype strain of hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1992,89 (2):559-563.
[32]Shrestha, S.M., S. Shrestha, F. Tsuda, et al., Molecular investigation of hepatitis E virus infection in patients with acute hepatitis in Kathmandu, Nepal. Journal of Medical Virology[J],2003,69 (2):207-214.
[33]Shrestha, S.M., S. Shrestha, F. Tsuda, et al., Genetic changes in hepatitis E virus of subtype 1a in patients with sporadic acute hepatitis E in Kathmandu, Nepal, from 1997 to 2002. Journal of General Virology[J],2004,85 (Pt 1):97-104.
[34]Drabick, J., J. Gambel, V. Gouvea, et al., A cluster of acute hepatitis E infection in United Nations Bangladeshi peacekeepers in Haiti. The American journal of tropical medicine and hygiene[J],1997,57 (4):449.
[35]Bi, S.L., M.A. Purdy, K.A. McCaustland, et al., The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China. Virus Research[J],1993,28 (3): 233-247.
[36]Wang, Y., H. Zhang, Z. Li, et al., Detection of sporadic cases of hepatitis E virus (HEV) infection in China using immunoassays based on recombinant open reading frame 2 and 3 polypeptides from HEV genotype 4. Journal of Clinical Microbiology[J],2001,39 (12): 4370-4379.
[37]Yin, S., R.H. Purcell, and S.U. Emerson, A new Chinese isolate of hepatitis E virus: comparison with strains recovered from different geographical regions. Virus Genes[J], 1994,9(1):23-32.
[38]Chatterjee, R., S. Tsarev, J. Pillot, et al., African strains of hepatitis E virus that are distinct from Asian strains. Journal of Medical Virology[J],1997,53 (2):139-144.
[39]Grandadam, M., S. Tebbal, M. Caron, et al., Evidence for hepatitis E virus quasispecies. Journal of General Virology[J],2004,85 (Pt 11):3189-3194.
[40]He, J., L.N. Binn, S.A. Tsarev, et al., Molecular characterization of a hepatitis E virus isolate from Namibia. Journal of Biomedical Science[J],2000,7 (4):334-338.
[41]Nicand, E., GL. Armstrong, V. Enouf, et al., Genetic heterogeneity of hepatitis E virus in Darfur, Sudan, and neighboring Chad. Journal of Medical Virology[J],2005,77 (4): 519-521.
[42]van Cuyck, H., F. Juge, and P. Roques, Phylogenetic analysis of the first complete hepatitis E virus (HEV) genome from Africa. FEMS Immunology and Medical Microbiology[J],2003,39 (2):133-139.
[43]Huang, C.C., D. Nguyen, J. Fernandez, et al., Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology[J],1992,191 (2):550-558.
[44]Buisson, Y, M. Grandadam, E. Nicand, et al., Identification of a novel hepatitis E virus in Nigeria. Journal of General Virology[J],2000,81 (Pt 4):903-909.
[45]Maila, H.T., S.M. Bowyer, and R. Swanepoel, Identification of a new strain of hepatitis E virus from an outbreak in Namibia in 1995. Journal of General Virology [J],2004,85 (Pt 1):89-95.
[46]Worm, H.C., H. Wurzer, and G Frosner, Sporadic hepatitis E in Austria. New England Journal of Medicine[J],1998,339 (21):1554-1555.
[47]Mansuy, J.M., J.M. Peron, F. Abravanel, et al., Hepatitis E in the south west of France in individuals who have never visited an endemic area. Journal of Medical Virology[J],2004, 74 (3):419-424.
[48]Dalton, H.R., H.J. Fellows, E.J. Gane, et al., Hepatitis e in new zealand. Journal of Gastroenterology and Hepatology[J],2007,22 (8):1236-1240.
[49]Schlauder, GG, GJ. Dawson, J.C. Erker, et al., The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. Journal of General Virology[J],1998,79 (Pt 3):447-456.
[50]Zanetti, A.R., GG Schlauder, L. Romano, et al., Identification of a novel variant of hepatitis E virus in Italy. Journal of Medical Virology[J],1999,57 (4):356-360.
[51]Ijaz, S., E. Arnold, M. Banks, et al., Non-travel-associated hepatitis E in England and Wales:demographic, clinical, and molecular epidemiological characteristics. The Journal of infectious diseases[J],2005,192 (7):1166-1172.
[52]Dalton, H.R., P.H. Thurairajah, H.J. Fellows, et al., Autochthonous hepatitis E in southwest England. Journal of Viral Hepatitis[J],2007,14 (5):304-309.
[53]Buti, M., P. Clemente-Casares, R. Jardi, et al., Sporadic cases of acute autochthonous hepatitis E in Spain. Journal of Hepatology[J],2004,41 (1):126-131.
[54]McCrudden, R., S. O'Connell, T. Farrant, et al., Sporadic acute hepatitis E in the united kingdom:an underdiagnosed phenomenon? Gut[J],2000,46 (5):732-733.
[55]Kwo, P.Y., G.G. Schlauder, H.A. Carpenter, et al., Acute hepatitis E by a new isolate acquired in the United States. Mayo Clinic Proceedings[J],1997,72 (12):1133-1136.
[56]Herremans, M., H. Vennema, J. Bakker, et al., Swine-like hepatitis E viruses are a cause of unexplained hepatitis in the Netherlands. Journal of Viral Hepatitis[J],2007,14 (2): 140-146.
[57]Munne, M.S., S. Vladimirsky, L. Otegui, et al., Identification of the first strain of swine hepatitis E virus in South America and prevalence of anti-HEV antibodies in swine in Argentina. Journal of Medical Virology[J],2006,78 (12):1579-1583.
[58]Cooper, K., F.F. Huang, L. Batista, et al., Identification of genotype 3 hepatitis E virus (HEV) in serum and fecal samples from pigs in Thailand and Mexico, where genotype 1 and 2 HEV strains are prevalent in the respective human populations. Journal of Clinical Microbiology[J],2005,43 (4):1684-1688.
[59]Banks, M., GS. Heath, S.S. Grierson, et al., Evidence for the presence of hepatitis E virus in pigs in the United Kingdom. Veterinary Record[J],2004,154 (8):223-227.
[60]van der Poel, W.H., F. Verschoor, R. van der Heide, et al., Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerging Infectious Diseases[J], 2001,7 (6):970-976.
[61]Tei, S., N. Kitajima, K. Takahashi, et al., Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet[J],2003,362 (9381):371-373.
[62]Lewis, H., D. Morgan, S. Ijaz, et al., Indigenous hepatitis E virus infection in England and Wales. BMJ (Clinical Research Ed.)[J],2006,332 (7556):1509-1510.
[63]De Silva, A., A. Muddu, J. Iredale, et al., Unexpectedly high incidence of indigenous acute hepatitis E within South Hampshire:Time for routine testing? Journal of Medical Virology[J],2008,80 (2):283-288.
[64]Peron, J.M., J.M. Mansuy, H. Poirson, et al., Hepatitis E is an autochthonous disease in industrialized countries. Analysis of 23 patients in South-West France over a 13-month period and comparison with hepatitis A. Gastroenterologie Clinique et Biologique[J], 2006,30 (5):757-762.
[65]Takahashi, K., J.H. Kang, S. Ohnishi, et al., Genetic heterogeneity of hepatitis E virus recovered from Japanese patients with acute sporadic hepatitis. The Journal of infectious diseases[J],2002,185 (9):1342-1345.
[66]Takahashi, K., K. Iwata, N. Watanabe, et al., Full-genome nucleotide sequence of a hepatitis E virus strain that may be indigenous to Japan. Virology[J],2001,287 (1):9-12.
[67]Matsubayashi, K., Y. Nagaoka, H. Sakata, et al., Transfusion-transmitted hepatitis E caused by apparently indigenous hepatitis E virus strain in Hokkaido, Japan. Transfusion[J],2004,44 (6):934-940.
[68]Takahashi, M., T. Nishizawa, and H. Okamoto, Identification of a genotype Ⅲ swine hepatitis E virus that was isolated from a Japanese pig born in 1990 and that is most closely related to Japanese isolates of human hepatitis E virus. Journal of Clinical Microbiology[J],2003,41 (3):1342-1343.
[69]Okamoto, H., M. Takahashi, T. Nishizawa, et al., Analysis of the complete genome of indigenous swine hepatitis E virus isolated in Japan. Biochemical and Biophysical Research Communications[J],2001,289 (5):929-936.
[70]Tokita, H., H. Harada, Y. Gotanda, et al., Molecular and serological characterization of sporadic acute hepatitis E in a Japanese patient infected with a genotype III hepatitis E virus in 1993. Journal of General Virology[J],2003,84 (Pt 2):421-427.
[71]Takahashi, M., T. Nishizawa, H. Miyajima, et al., Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. Journal of General Virology[J],2003,84 (Pt 4):851-862.
[72]Tamada, Y, K. Yano, H. Yatsuhashi, et al., Consumption of wild boar linked to cases of hepatitis E. Journal of Hepatology[J],2004,40 (5):869-870.
[73]Ning, H., S. Yu, Y. Zhu, et al., Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs. Veterinary Microbiology[J],2008,126 (1-3):257-263.
[74]Yazaki, Y, H. Mizuo, M. Takahashi, et al., Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. Journal of General Virology[J],2003,84 (Pt 9):2351-2357.
[75]Arankalle, V.A., L.P. Chobe, M.V. Joshi, et al., Human and swine hepatitis E viruses from Western India belong to different genotypes. Journal of Hepatology[J],2002,36 (3): 417-425.
[76]Arankalle, V.A., L.P. Chobe, A.M. Walimbe, et al., Swine HEV infection in south India and phylogenetic analysis (1985-1999). Journal of Medical Virology[J],2003,69 (3): 391-396.
[77]Li, K., H. Zhuang, and W. Zhu, Partial nucleotide sequencing of hepatitis E viruses detected in sera of patients with hepatitis E from 14 cities in China. Chinese Medical Journal[J],2002,115 (7):1058-1063.
[78]Wang, Y.C., H.Y. Zhang, N.S. Xia, et al., Prevalence, isolation, and partial sequence analysis of hepatitis E virus from domestic animals in China. Journal of Medical Virology[J],2002,67 (4):516-521.
[79]Nishizawa, T., M. Takahashi, H. Mizuo, et al., Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99% identity over the entire genome. Journal of General Virology[J],2003,84 (Pt 5):1245-1251.
[80]Wibawa, I.D., D.H. Muljono, Mulyanto, et al., Prevalence of antibodies to hepatitis E virus among apparently healthy humans and pigs in Bali, Indonesia:Identification of a pig infected with a genotype 4 hepatitis E virus. Journal of Medical Virology[J],2004,73 (1):38-44.
[81]Hijikata, M., S. Hayashi, N.T. Trinh, et al., Genotyping of hepatitis E virus from Vietnam. Intervirology[J],2002,45 (2):101-104.
[82]Amon, J.J., J. Drobeniuc, W.A. Bower, et al., Locally acquired hepatitis E virus infection, El Paso, Texas. Journal of Medical Virology[J],2006,78 (6):741-746.
[83]Mizuo, H., K. Suzuki, Y. Takikawa, et al., Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan. Journal of Clinical Microbiology[J],2002,40 (9):3209-3218.
[84]Kabrane-Lazizi, Y, M. Zhang, R.H. Purcell, et al., Acute hepatitis caused by a novel strain of hepatitis E virus most closely related to United States strains. Journal of General Virology[J],2001,82 (Pt 7):1687-1693.
[85]Inoue, J., T. Nishizawa, M. Takahashi, et al., Analysis of the full-length genome of genotype 4 hepatitis E virus isolates from patients with fulminant or acute self-limited hepatitis E. Journal of Medical Virology[J],2006,78 (4):476-484.
[86]Ahn, J.M., S.G Kang, D.Y. Lee, et al., Identification of novel human hepatitis E virus (HEV) isolates and determination of the seroprevalence of HEV in Korea. Journal of Clinical Microbiology[J],2005,43 (7):3042-3048.
[87]Suzuki, K., T. Aikawa, and H. Okamoto, Fulminant hepatitis E in Japan. New England Journal of Medicine[J],2002,347 (18):1456.
[88]Takahashi, M., T. Nishizawa, A. Yoshikawa, et al., Identification of two distinct genotypes of hepatitis E virus in a Japanese patient with acute hepatitis who had not travelled abroad. Journal of General Virology[J],2002,83 (Pt 8):1931-1940.
[89]Michitaka, K., K. Takahashi, S. Furukawa, et al., Prevalence of hepatitis E virus among wild boar in the Ehime area of western Japan. Hepatol Res[J],2007,37 (3):214-220.
[90]Martelli, F., A. Caprioli, M. Zengarini, et al., Detection of hepatitis E virus (HEV) in a demographic managed wild boar (Sus scrofa scrofa) population in Italy. Veterinary Microbiology[J],2008,126 (1-3):74-81.
[91]Agrawal, S., D. Gupta, and S.K. Panda, The 3'end of hepatitis E virus (HEV) genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp). Virology[J], 2001,282(1):87-101.
[92]Koonin, E.V., A.E. Gorbalenya, M.A. Purdy, et al., Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus:delineation of an additional group of positive-strand RNA plant and animal viruses. Proceedings of the National Academy of Sciences of the United States of America[J],1992,89 (17): 8259-8263.
[93]Magden, J., N. Takeda, T. Li, et al., Virus-specific mRNA capping enzyme encoded by hepatitis E virus. Journal of Virology[J],2001,75 (14):6249-6255.
[94]Emerson, S.U., M. Zhang, X.J. Meng, et al., Recombinant hepatitis E virus genomes infectious for primates:importance of capping and discovery of a cis-reactive element. Proceedings of the National Academy of Sciences of the United States of America[J], 2001,98 (26):15270-15275.
[95]Zhang, M., R.H. Purcell, and S.U. Emerson, Identification of the 5'terminal sequence of the SAR-55 and MEX-14 strains of hepatitis E virus and confirmation that the genome is capped. Journal of Medical Virology[J],2001,65 (2):293-295.
[96]Graff, J., Y.H. Zhou, U. Torian, et al., Mutations within potential glycosylation sites in the capsid protein of hepatitis E virus prevent the formation of infectious virus particles. Journal of Virology[J],2008,82 (3):1185-1194.
[97]Robinson, R.A., W.H. Burgess, S.U. Emerson, et al., Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells. Protein Expression and Purification[J],1998,12 (1):75-84.
[98]Zafrullah, M., M.H. Ozdener, S.K. Panda, et al., The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton. Journal of Virology [J],1997,71 (12):9045-9053.
[99]Tyagi, S., H. Korkaya, M. Zafrullah, et al., The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. Journal of Biological Chemistry[J],2002,277 (25):22759-22767.
[100]Korkaya, H., S. Jameel, D. Gupta, et al., The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK. Journal of Biological Chemistry [J],2001, 276 (45):42389-42400.
[101]Huang, F.F., Z.F. Sun, S.U. Emerson, et al., Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV. Journal of General Virology[J],2004,85 (Pt 6):1609-1618.
[102]Stoszek, S., R. Engle, M. Abdel-Hamid, et al., Hepatitis E antibody seroconversion without disease in highly endemic rural Egyptian communities. Transactions of the Royal Society of Tropical Medicine and Hygiene[J],2006,100 (2):89-94.
[103]Drobeniuc, J., M.O. Favorov, C.N. Shapiro, et al., Hepatitis E virus antibody prevalence among persons who work with swine. The Journal of infectious diseases[J],2001,184 (12):1594-1597.
[104]Meng, X.J., B. Wiseman, F. Elvinger, et al., Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. Journal of Clinical Microbiology[J],2002,40 (1):117-122.
[105]Arankalle, V.A., S.A. Tsarev, M.S. Chadha, et al., Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India,1982 and 1992. The Journal of infectious diseases[J],1995,171 (2):447-450.
[106]Bouwknegt, M., K. Frankena, S.A. Rutjes, et al., Estimation of hepatitis E virus transmission among pigs due to contact-exposure. Veterinary Research[J],2008,39 (5): 40.*
[107]Kasorndorkbua, C., D. Guenette, F. Huang, et al., Routes of transmission of swine hepatitis E virus in pigs. Journal of Clinical Microbiology[J],2004,42 (11):5047.
[108]Peralta, B., M. Biarnes, G Ordonez, et al., Evidence of widespread infection of avian hepatitis E virus (avian HEV) in chickens from Spain. Veterinary Microbiology[J],2009, 137 (1-2):31-36.
[109]Huang, F.F., G Haqshenas, H.L. Shivaprasad, et al., Heterogeneity and seroprevalence of a newly identified avian hepatitis e virus from chickens in the United States. Journal of Clinical Microbiology[J],2002,40 (11):4197-4202.
[110]Sun, Z.F., C.T. Larsen, A. Dunlop, et al., Genetic identification of avian hepatitis E virus (HEV) from healthy chicken flocks and characterization of the capsid gene of 14 avian HEV isolates from chickens with hepatitis-splenomegaly syndrome in different geographical regions of the United States. Journal of General Virology[J],2004,85 (Pt 3): 693-700.
[111]Billam, P., F.F. Huang, Z.F. Sun, et al., Systematic pathogenesis and replication of avian hepatitis E virus in specific-pathogen-free adult chickens. Journal of Virology[J],2005, 79 (6):3429-3437.
[112]Harrison, T.J., Hepatitis E virus-an update. Liver[J],1999,19 (3):171-176.
[113]Smith, J.L., A review of hepatitis E virus. Journal of Food Protection[J],2001,64 (4): 572-586.
[114]Mizuo, H., Y. Yazaki, K. Sugawara, et al., Possible risk factors for the transmission of hepatitis E virus and for the severe form of hepatitis E acquired locally in Hokkaido, Japan. Journal of Medical Virology[J],2005,76 (3):341-349.
[115]Tsarev, S.A., T.S. Tsareva, S.U. Emerson, et al., Experimental hepatitis E in pregnant rhesus monkeys:failure to transmit hepatitis E virus (HEV) to offspring and evidence of naturally acquired antibodies to HEV. The Journal of infectious diseases[J],1995,172 (1): 31-37.
[116]Halbur, P.G., C. Kasorndorkbua, C. Gilbert, et al., Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human. Journal of Clinical Microbiology[J],2001,39 (3):918-923.
[117]Kawai, H.F., T. Koji, F. Iida, et al., Shift of hepatitis E virus RNA from hepatocytes to biliary epithelial cells during acute infection of rhesus monkey. Journal of Viral Hepatitis[J],1999,6 (4):287-297.
[118]Williams, T.P., C. Kasorndorkbua, P.G Halbur, et al., Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model. Journal of Clinical Microbiology[J], 2001,39 (9):3040-3046.
[119]Reyes, G.R., C.C. Huang, A.W. Tam, et al., Molecular organization and replication of hepatitis E virus (HEV). Archives of Virology. Supplementum[J],1993,7:15-25.
[120]Kalia, M., V. Chandra, S.A. Rahman, et al., Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection. Journal of Virology[J],2009,83 (24):12714-12724.
[121]Zheng, Z.Z., J. Miao, M. Zhao, et al., Role of heat-shock protein 90 in hepatitis E virus capsid trafficking. Journal of General Virology[J],2010,91 (Pt 7):1728-1736.
[122]Panda, S., I. Ansari, H. Durgapal, et al., The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious. Journal of Virology[J],2000,74 (5):2430.
[123]Nanda, S.K., S.K. Panda, H. Durgapal, et al., Detection of the negative strand of hepatitis E virus RNA in the livers of experimentally infected rhesus monkeys:evidence for viral replication. Journal of Medical Virology[J],1994,42 (3):237-240.
[124]Meng, X.J., P.G. Halbur, J.S. Haynes, et al., Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV. Archives of Virology[J],1998,143 (7):1405-1415.
[125]Graff, J., U. Torian, H. Nguyen, et al., A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. Journal of Virology[J],2006,80 (12): 5919-5926.
[126]Huang, Y.W., T. Opriessnig, P.G Halbur, et al., Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. Journal of Virology[J],2007,81 (6):3018-3026.
[127]Ichiyama, K., K. Yamada, T. Tanaka, et al., Determination of the 5'-terminal sequence of subgenomic RNA of hepatitis E virus strains in cultured cells. Archives of Virology[J], 2009,154(12):1945-1951.
[128]Huang, R.T., D.R. Li, J. Wei, et al., Isolation and identification of hepatitis E virus in Xinjiang, China. Journal of General Virology[J],1992,73 (Pt 5):1143-1148.
[129]Huang, R., N. Nakazono, K. Ishii, et al., Hepatitis E virus (87A strain) propagated in A549 cells. Journal of Medical Virology[J],1995,47 (4):299-302.
[130]Tanaka, T., M. Takahashi, E. Kusano, et al., Development and evaluation of an efficient cell-culture system for Hepatitis E virus. Journal of General Virology[J],2007,88 (Pt 3): 903-911.
[131]Kazachkov Yu, A., M.S. Balayan, T.A. Ivannikova, et al., Hepatitis E virus in cultivated cells. Archives of Virology[J],1992,127 (1-4):399-402.
[132]Tam, A.W., R. White, E. Reed, et al., In vitro propagation and production of hepatitis E virus from in vivo-infected primary macaque hepatocytes. Virology[J],1996,215 (1):1-9.
[133]Divizia, M., R. Gabrieli, A.M. Degener, et al., Evidence of hepatitis E virus replication on cell cultures. New Microbiologica[J],1999,22 (2):77-83.
[134]乐耕耘,吴娟,马雁冰,等.戊型肝炎病毒在人胚肺二倍体细胞KMB17等传代细胞中的繁殖.中国医学科学院学报[J],2001,(06)：590-593.
[135]Wei, S., P. Walsh, R. Huang, et al.,93G, a novel sporadic strain of hepatitis E virus in South China isolated by cell culture. Journal of Medical Virology[J],2000,61 (3): 311-318.
[136]Meng, J., J. Pillot, X. Dai, et al., Neutralization of Different Geographic Strains of the Hepatitis E Virus with Anti-Hepatitis E Virus-Positive Serum Samples Obtained from Different Sources. Virology[J],1998,249 (2):316-324.
[137]Emerson, S.U., H. Nguyen, J. Graff, et al., In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. Journal of Virology[J],2004,78 (9):4838-4846.
[138]Emerson, S.U., V.A. Arankalle, and R.H. Purcell, Thermal stability of hepatitis E virus. The Journal of infectious diseases[J],2005,192 (5):930-933.
[139]Purdy, M.A., K.A. McCaustland, K. Krawczynski, et al., Preliminary evidence that a trpE-HEV fusion protein protects cynomolgus macaques against challenge with wild-type hepatitis E virus (HEV). Journal of Medical Virology[J],1993,41 (1):90-94.
[140]毕胜利,鲁健,蒋琳,等.原核表达戊型肝炎病毒基因重组结构蛋白免疫保护恒河猴的初步实验研究.中华实验和临床病毒学杂志[J],2002,(01)：31-32.
[141]徐国民,李奎,庄辉,等.戊型肝炎病毒重组疫苗的初步实验研究.中国公共卫生[J],2000,(09)：57-58.
[142]Li, T.C., Y. Yamakawa, K. Suzuki, et al., Expression and self-assembly of empty virus-like particles of hepatitis E virus. Journal of Virology [J],1997,71 (10):7207-7213.
[143]Zhang, M., S. Emerson, H. Nguyen, et al., Immunogenicity and protective efficacy of a vaccine prepared from 53 kDa truncated hepatitis E virus capsid protein expressed in insect cells. Vaccine[J],2001,20 (5-6):853-857.
[144]Tsarev, S.A., T.S. Tsareva, S.U. Emerson, et al., Recombinant vaccine against hepatitis E: dose response and protection against heterologous challenge. Vaccine[J],1997,15 (17-18): 1834-1838.
[145]He, J., S.L. Hoffman, and C.G. Hayes, DNA inoculation with a plasmid vector carrying the hepatitis E virus structural protein gene induces immune response in mice. Vaccine[J], 1997,15 (4):357-362.
[146]洪艳,阮冰,杨连华,等.戊型肝炎病毒嵌合基因疫苗的初步研究.中国公共卫生[J],2004,(07)：123-124.
[147]Lu, F., H. Zhuang, Y. Zhu, et al., A preliminary study on immune response to hepatitis E virus DNA vaccine in mice. Chinese Medical Journal[J],1996,109 (12):919-921.
[148]Zhu, F.C., J. Zhang, X.F. Zhang, et al., Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults:a large-scale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet[J],2010,376 (9744):895-902.
[149]Shrestha, M., R. Scott, D. Joshi, et al., Safety and efficacy of a recombinant hepatitis E vaccine. The New England journal of medicine[J],2007,356 (9):895.
[150]Favorov, M.O., M.Y. Kosoy, S.A. Tsarev, et al., Prevalence of antibody to hepatitis E virus among rodents in the United States. The Journal of infectious diseases[J],2000,181 (2):449-455.
[151]Meng, X.J., S. Dea, R.E. Engle, et al., Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. Journal of Medical Virology[J],1999,59 (3):297-302.
[1]黄耀伟,人类及动物RNA病毒的反向遗传系统.生物工程学报[J],2004,20(3)：311-318.
[2]韩剑峰,SARS-CoV中国株基因组全长cDNA的构建及其恢复病毒的生物学性质.微生物学报[J],2006,46(6)：922-927.
[3]Zibert, A., G. Maass, K. Strebel, et al., Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA. Journal of Virology [J],1990,64 (6):2467-2473.
[4]Cohen, J., J. Ticehurst, S. Feinstone, et al., Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture. Journal of Virology[J],1987,61 (10):3035.
[5]胡建和,猪瘟病毒兔化弱毒株全长cDNA克隆的感染性鉴定.病毒学报[J],2004,20(2)：143-147.
[6]Yamshchikov, V.F., G. Wengler, A.A. Perelygin, et al., An infectious clone of the West Nile flavivirus. Virology[J],2001,281 (2):294-304.
[7]Liu, G., Z. Liu, Q. Xie, et al., Generation of an infectious cDNA clone of an FMDV strain isolated from swine. Virus Research[J],2004,104 (2):157-164.
[8]Kassimi, L.B., A. Boutrouille, M. Gonzague, et al., Nucleotide sequence and construction of an infectious cDNA clone of an EMCV strain isolated from aborted swine fetus. Virus Research[J],2002,83 (1-2):71-87.
[9]Liu, G., Y. Zhang, Z. Ni, et al., Recovery of infectious rabbit hemorrhagic disease virus from rabbits after direct inoculation with in vitro-transcribed RNA. Journal of Virology[J], 2006,80 (13):6597-6602.
[10]Huang, Y.W., G. Haqshenas, C. Kasomdorkbua, et al., Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs. Journal of Virology[J],2005,79 (3):1552-1558.
[11]Ruggli, N., J.D. Tratschin, C. Mittelholzer, et al., Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. Journal of Virology[J],1996,70 (6):3478-3487.
[12]Meulenberg, J.J., J.N. Bos-de Ruijter, R. van de Graaf, et al., Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. Journal of Virology[J],1998,72 (1):380-387.
[13]Almazan, F., J.M. Gonzalez, Z. Penzes, et al., Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proceedings of the National Academy of Sciences of the United States of America[J],2000,97 (10):5516-5521.
[14]Van Dinten, L., J. Den Boon, A. Wassenaar, et al., An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (3):991.
[15]Yount, B., K. Curtis, E. Fritz, et al., Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proceedings of the National Academy of Sciences of the United States of America[J],2003,100 (22):12995.
[16]Racaniello, V. and D. Baltimore, Cloned poliovirus complementary DNA is infectious in mammalian cells. Science[J],1981,214:916-919.
[17]Racaniello, V.R. and D. Baltimore, Cloned poliovirus complementary DNA is infectious in mammalian cells. Science[J],1981,214 (4523):916-919.
[18]Hamilton, W. and D. Baulcombe, Infectious RNA produced by in vitro transcription of a full-length tobacco rattle virus RNA-1 cDNA. Journal of General Virology[J],1989,70 (4):963.
[19]van der Werf, S., J. Bradley, E. Wimmer, et al., Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America[J],1986,83 (8):2330-2334.
[20]Hayes, R. and K. Buck, Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction. Journal of General Virology[J],1990,71 (11):2503.
[21]Mizutani, S. and R. Colonno, In vitro synthesis of an infectious RNA from cDNA clones of human rhinovirus type 14. Journal of Virology[J],1985,56 (2):628.
[22]Klump, W.M., I. Bergmann, B.C. Muller, et al., Complete nucleotide sequence of infectious Coxsackievirus B3 cDNA:two initial 5'uridine residues are regained during plus-strand RNA synthesis. Journal of Virology[J],1990,64 (4):1573-1583.
[23]Duechler, M., T. Skern, D. Blaas, et al., Human rhinovirus serotype 2:in vitro synthesis of an infectious RNA. Virology[J],1989,168 (1):159-161.
[24]Sarnow, P., Role of 3'-end sequences in infectivity of poliovirus transcripts made in vitro. Journal of Virology[J],1989,63 (1):467.
[25]Dzianott, A.M. and J.J. Bujarski, Derivation of an infectious viral RNA by autolytic cleavage of in vitro transcribed viral cDNAs. Proceedings of the National Academy of Sciences of the United States of America[J],1989,86 (13):4823-4827.
[26]袁海峰,姚火春,高飞,等.poly(A)尾对猪繁殖与呼吸综合征病毒基因组感染性的影响.中国预防兽医学报[J],2008,(02)：86-90.
[27]Emerson, S.U., M. Zhang, X.J. Meng, et al., Recombinant hepatitis E virus genomes infectious for primates:importance of capping and discovery of a cis-reactive element. Proceedings of the National Academy of Sciences of the United States of America[J], 2001,98 (26):15270-15275.
[28]Angenent, G.C., E. Posthumus, and J.F. Bol, Biological activity of transcripts synthesized in vitro from full-length and mutated DNA copies of tobacco rattle virus RNA 2. Virology[J],1989,173 (1):68-76.
[29]Chambers, T.J., R.C. Weir, A. Grakoui, et al., Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein. Proceedings of the National Academy of Sciences of the United States of America[J],1990,87 (22):8898-8902.
[30]Chambers, T.J., A. Nestorowicz, and C.M. Rice, Mutagenesis of the yellow fever virus NS2B/3 cleavage site:determinants of cleavage site specificity and effects on polyprotein processing and viral replication. Journal of Virology [J],1995,69 (3):1600-1605.
[31]Muylaert, I.R., T.J. Chambers, R. Galler, et al., Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein:effects on virus replication and mouse neurovirulence. Virology[J],1996,222 (1):159-168.
[32]Leippert, M., E. Beck, F. Weiland, et al., Point mutations within the betaG-betaH loop of foot-and-mouth disease virus O1K affect virus attachment to target cells. Journal of Virology[J],1997,71 (2):1046-1051.
[33]Dobbe, J.C., Y. van der Meer, W.J. Spaan, et al., Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture. Virology[J],2001,288 (2):283-294.
[34]Zeng, L., B. Falgout, and L. Markoff, Identification of specific nucleotide sequences within the conserved 3'-SL in the dengue type 2 virus genome required for replication. Journal of Virology[J],1998,72 (9):7510-7522.
[35]Kanno, T., D. Mackay, G. Wilsden, et al., Virulence of swine vesicular disease virus is determined at two amino acids in capsid protein VP1 and 2A protease. Virus Research[J], 2001,80(1-2):101-107.
[36]Rieder, E., T. Bunch, F. Brown, et al., Genetically engineered foot-and-mouth disease viruses with poly(C) tracts of two nucleotides are virulent in mice. Journal of Virology [J], 1993,67 (9):5139-5145.
[37]McKenna, T., J. Lubroth, E. Rieder, et al., Receptor binding site-deleted foot-and-mouth disease (FMD) virus protects cattle from FMD. Journal of Virology[J],1995,69 (9): 5787.
[38]Ward, G., E. Rieder, and P.W. Mason, Plasmid DNA encoding replicating foot-and-mouth disease virus genomes induces antiviral immune responses in swine. Journal of Virology[J],1997,71 (10):7442-7447.
[39]Mandl, C.W., J.H. Aberle, S.W. Aberle, et al., In vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirus model. Nature Medicine[J],1998,4 (12): 1438-1440.
[40]Bray, M., R. Men, and C.J. Lai, Monkeys immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge. Journal of Virology[J],1996,70 (6):4162-4166.
[41]Marchevsky, R.S., J. Mariano, V.S. Ferreira, et al., Phenotypic analysis of yellow fever virus derived from complementary DNA. American Journal of Tropical Medicine and Hygiene[J],1995,52 (1):75-80.
[42]卢建红,以BAC为基础的疱疹病毒感染性克隆技术.中国生物工程杂志[J],2006,26(6)：78-82.
[43]Porter, D.C., J. Wang, Z. Moldoveanu, et al., Immunization of mice with poliovirus replicons expressing the C-fragment of tetanus toxin protects against lethal challenge with tetanus toxin. Vaccine[J],1997,15 (3):257-264.
[44]Yount, B., K.M. Curtis, and R.S. Baric, Strategy for systematic assembly of large RNA and DNA genomes:transmissible gastroenteritis virus model. Journal of Virology[J], 2000,74(22):10600-10611.
[45]Panda, S., I. Ansari, H. Durgapal, et al., The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious. Journal of Virology[J],2000,74 (5):2430.
[46]Yount, B., M.R. Denison, S.R. Weiss, et al., Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. Journal of Virology[J],2002,76 (21):11065-11078.
[47]Huang, F.F., F.W. Pierson, T.E. Toth, et al., Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV. Journal of General Virology[J],2005,86 (Pt 9):2585-2593.
[48]Yamada, K., M. Takahashi, Y. Hoshino, et al., Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells. Journal of General Virology[J],2009,90 (Pt2):457-462.
[49]Kwon, H.M., T. Leroith, R.S. Pudupakam, et al., Construction of an infectious cDNA clone of avian hepatitis E virus (avian HEV) recovered from a clinically healthy chicken in the United States and characterization of its pathogenicity in specific-pathogen-free chickens. Veterinary Microbiology[J],2010.
[50]Balayan, M.S., R.K. Usmanov, N.A. Zamyatina, et al., Brief report:experimental hepatitis E infection in domestic pigs. Journal of Medical Virology[J],1990,32 (1):58-59.
[51]Meng, X.J., R.H. Purcell, P.G. Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[52]Meng, X.J., P.G Halbur, M.S. Shapiro, et al., Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. Journal of Virology[J],1998,72 (12): 9714-9721.
[53]Yazaki, Y., H. Mizuo, M. Takahashi, et al., Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. Journal of General Virology[J],2003,84 (Pt 9):2351-2357.
[54]Feagins, A.R., T. Opriessnig, D.K. Guenette, et al., Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA. Journal of General Virology[J],2007,88 (Pt 3):912-917.
[55]Kato, M., K. Taneichi, and K. Matsubayashi, A mini-outbreak of hepatitis E infection in those who enjoyed Yokiniko party:One died of fulminant hepatitis. Kanzo[J],2004,45: 1326-1332.
[56]Mizuo, H., Y. Yazaki, K. Sugawara, et al., Possible risk factors for the transmission of hepatitis E virus and for the severe form of hepatitis E acquired locally in Hokkaido, Japan. Journal of Medical Virology [J],2005,76 (3):341-349.
[57]Favorov, M.O., M.Y. Kosoy, S.A. Tsarev, et al., Prevalence of antibody to hepatitis E virus among rodents in the United States. The Journal of infectious diseases[J],2000,181 (2):449-455.
[58]Saad, M.D., H.A. Hussein, M.M. Bashandy, et al., Hepatitis E virus infection in work horses in Egypt. Infection, Genetics and Evolution[J],2007,7 (3):368-373.
[59]Khuroo, M.S., S. Kamili, and S. Jameel, Vertical transmission of hepatitis E virus. Lancet[J],1995,345 (8956):1025-1026.
[60]Khuroo, M.S., M.R. Teli, S. Skidmore, et al., Incidence and severity of viral hepatitis in pregnancy. American Journal of Medicine[J],1981,70 (2):252-255.
[61]Khuroo, M.S. and S. Kamili, Aetiology, clinical course and outcome of sporadic acute viral hepatitis in pregnancy. Journal of Viral Hepatitis[J],2003,10 (1):61-69.
[62]Ning, H., Z. Niu, R. Yu, et al., Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb. Veterinary Microbiology[J],2007, 121 (1-2):125-130.
[63]Ning, H., S. Yu, Y. Zhu, et al., Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs. Veterinary Microbiology[J],2008,126 (1-3):257-263.
[64]Zhang, W., S. Yang, Q. Shen, et al., Genotype 3 hepatitis E virus existed among swine groups in 4 geographically far regions in China. Veterinary Microbiology[J],2010,140 (1-2):193-195.
[65]Zhang, W., Y. He, H. Wang, et al., Hepatitis E virus genotype diversity in eastern China. Emerging Infectious Diseases[J],2010,16(10):1630-1632.
[66]于水生,司伏生,朱于敏,等.上海某猪场发现基因3型和基因4型戊型肝炎病毒共存.上海农业学报[J],2008,24(04)：29-32.
[1]Das, K., A. Agarwal, R. Andrew, et al., Role of hepatitis E and other hepatotropic virus in aetiology of sporadic acute viral hepatitis:a hospital based study from urban Delhi. European Journal of Epidemiology[J],2000,16 (10):937-940.
[2]Balayan, M.S., A.G. Andjaparidze, S.S. Savinskaya, et al., Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology[J],1983,20 (1):23-31.
[3]Meng, X. J., R.H. Purcell, P.G. Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[4]Favorov, M.O., M.Y. Kosoy, S.A. Tsarev, et al., Prevalence of antibody to hepatitis E virus among rodents in the United States. The Journal of infectious diseases[J],2000,181 (2):449-455.
[5]Arankalle, V.A., M.V. Joshi, A.M. Kulkarni, et al., Prevalence of anti-hepatitis E virus antibodies in different Indian animal species. Journal of Viral Hepatitis[J],2001,8 (3): 223-227.
[6]Meng, X.J., S. Dea, R.E. Engle, et al., Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. Journal of Medical Virology[J],1999,59 (3):297-302.
[7]Meng, X.J., Novel strains of hepatitis E virus identified from humans and other animal species:is hepatitis E a zoonosis? Journal of Hepatology[J],2000,33 (5):842-845.
[8]庄辉,毕胜利,王佑春,等.我国戊型肝炎研究.北京大学学报(医学版)[J],2002,34(05)：434-439.
[9]李金,胡志东,钟述猷,戊型肝炎研究新进展.国外医学.流行病学.传染病学分册[J],2004,(04).
[10]曹学义,马学众,刘玉璋,新疆南部地区肠道传播的非甲非乙型肝炎的流行病学研究.中国公共卫生学报[J],1989,8(04)：193-199.
[11]Cao, X., X. Ma, Y. Liu, et al., Epidemiological and etiological studies on enterically transmitted non-A, non-B hepatitis in the south part of Xinjiang. Viral Hepatitis C, D, and E. New York:Elsevier Science[J],1991:297-312.
[12]Ning, H., Z. Niu, R Yu, et al., Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb. Veterinary Microbiology [J],2007, 121 (1-2):125-130.
[13]Inoue, J., M. Takahashi, Y. Yazaki, et al., Development and validation of an improved RT-PCR assay with nested universal primers for detection of hepatitis E virus strains with significant sequence divergence. Journal of Virological Methods[J],2006,137 (2): 325-333.
[14]Lu, L., C. Li, and C.H. Hagedorn, Phylogenetic analysis of global hepatitis E virus sequences:genetic diversity, subtypes and zoonosis. Reviews in Medical Virology[J], 2006,16 (1):5-36.
[15]张军,戊型肝炎研究动态第12届国际病毒性肝炎和肝病研讨会(巴黎)信息.国际病毒学杂志[J],2006,13(04)：97-100.
[16]Meng, X.J., B. Wiseman, F. Elvinger, et al., Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. Journal of Clinical Microbiology[J],2002,40 (1):117-122.
[17]Zhuang, H., X.Y. Cao, C.B. Liu, et al., Epidemiology of hepatitis E in China. Gastroenterologia Japonica[J],1991,26 Suppl 3:135-138.
[18]Tei, S., N. Kitajima, S. Ohara, et al., Consumption of uncooked deer meat as a risk factor for hepatitis E virus infection:an age- and sex-matched case-control study. Journal of Medical Virology[J],2004,74 (1):67-70.
[19]Li, T.C., K. Chijiwa, N. Sera, et al., Hepatitis E virus transmission from wild boar meat. Emerging Infectious Diseases[J],2005,11 (12):1958-1960.
[20]Drobeniuc, J., M.O. Favorov, C.N. Shapiro, et al., Hepatitis E virus antibody prevalence among persons who work with swine. The Journal of infectious diseases[J],2001,184 (12):1594-1597.
[21]Zheng, Y., S. Ge, J. Zhang, et al., Swine as a principal reservoir of hepatitis E virus that infects humans in eastern China. The Journal of infectious diseases[J],2006,193: 1643-1649.
[22]高橘雅春,谷仁烨,朱丽影,戊型肝炎病毒研究的新进展.日本医学介绍[J],2005,26(06)：253-255.
[23]Li, Z., S. Yu, S. Dong, et al., Reduced prevalence of genotype 3 HEV in Shanghai pig farms and hypothetical homeostasis of porcine HEV reservoir. Veterinary Microbiology [J], 2009,137 (1-2):184-189.
[24]Zhang, W., S. Yang, Q. Shen, et al., Genotype 3 hepatitis E virus existed among swine groups in 4 geographically far regions in China. Veterinary Microbiology[J],2010,140 (1-2):193-195.
[25]Zhang, W., Y. He, H. Wang, et al., Hepatitis E virus genotype diversity in eastern China. Emerging Infectious Diseases[J],2010,16(10):1630-1632.
[26]Tei, S., N. Kitajima, K. Takahashi, et al., Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet[J],2003,362 (9381):371-373.
[1]Tam, A.W., M.M. Smith, M.E. Guerra, et al., Hepatitis E virus (HEV):molecular cloning and sequencing of the full-length viral genome. Virology[J],1991,185 (1):120-131.
[2]Torresi, J., F. Li, S.A. Locarnini, et al., Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells. Journal of General Virology[J],1999,80 (Pt 5):1185-1188.
[3]Tyagi, S., H. Korkaya, M. Zafrullah, et al., The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. Journal of Biological Chemistry[J],2002,277 (25):22759.
[4]Meng, X.J., P.G. Halbur, M.S. Shapiro, et al., Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. Journal of Virology[J],1998,72 (12): 9714-9721.
[5]Tei, S., N. Kitajima, K. Takahashi, et al., Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet[J],2003,362 (9381):371-373.
[6]Meng, J., X. Dai, J.C. Chang, et al., Identification and characterization of the neutralization epitope(s) of the hepatitis E virus. Virology[J],2001,288 (2):203-211.
[7]Ning, H., Z. Niu, R. Yu, et al., Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb. Veterinary Microbiology[J],2007, 121 (1-2):125-130.
[8]Graff, J., U. Torian, H. Nguyen, et al., A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. Journal of Virology[J],2006,80 (12): 5919-5926.
[9]Huang, Y.W., T. Opriessnig, P.G. Halbur, et al., Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. Journal of Virology[J],2007,81 (6):3018-3026.
[10]Aye, T., T. Uchida, X. Ma, et al., Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986-1988) of China. Nucleic Acids Research[J], 1992,20 (13):3512.
[11]Huang, C.C., D. Nguyen, J. Fernandez, et al., Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology[J],1992,191 (2):550-558.
[12]Schlauder, GG, GJ. Dawson, J.C. Erker, et al., The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. The Journal of general virology[J],1998,79:447.
[13]Wang, Y, H. Zhang, R. Ling, et al., The complete sequence of hepatitis E virus genotype 4 reveals an alternative strategy for translation of open reading frames 2 and 3. Journal of General Virology[J],2000,81 (Pt 7):1675-1686.
[14]Bi, S.L., M.A. Purdy, K.A. McCaustland, et al., The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China. Virus Research[J],1993,28 (3): 233-247.
[15]Wang, Y., R. Ling, J.C. Erker, et al., A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. Journal of General Virology[J],1999,80 (Pt 1):169-177.
[16]Wang, Y, H. Zhang, Z. Li, et al., Detection of sporadic cases of hepatitis E virus (HEV) infection in China using immunoassays based on recombinant open reading frame 2 and 3 polypeptides from HEV genotype 4. Journal of Clinical Microbiology[J],2001,39 (12): 4370-4379.
[17]Huang, R., N. Nakazono, K. Ishii, et al., Existing variations on the gene structure of hepatitis E virus strains from some regions of China. Journal of Medical Virology[J], 1995,47 (4):303-308.
[18]Meng, X.J., R.H. Purcell, P.G Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[19]Nishizawa, T., M. Takahashi, H. Mizuo, et al., Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99% identity over the entire genome. Journal of General Virology[J],2003,84 (Pt 5):1245-1251.
[20]Takahashi, M., T. Nishizawa, H. Miyajima, et al., Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. Journal of General Virology[J],2003,84 (4):851.
[21]Okamoto, H., M. Takahashi, T. Nishizawa, et al., Analysis of the complete genome of indigenous swine hepatitis E virus isolated in Japan. Biochemical and Biophysical Research Communications[J],2001,289 (5):929-936.
[22]Hsieh, S.Y., X.J. Meng, YH. Wu, et al., Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus. Journal of Clinical Microbiology[J],1999,37 (12):3828-3834.
[23]Takahashi, M., T. Nishizawa, and H. Okamoto, Identification of a genotype III swine hepatitis E virus that was isolated from a Japanese pig born in 1990 and that is most closely related to Japanese isolates of human hepatitis E virus. Journal of Clinical Microbiology[J],2003,41 (3):1342-1343.
[24]van der Poel, W.H., F. Verschoor, R. van der Heide, et al., Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerging Infectious Diseases[J], 2001,7 (6):970-976.
[25]Huang, F.F., G. Haqshenas, D.K. Guenette, et al., Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. Journal of Clinical Microbiology[J], 2002,40 (4):1326-1332.
[1]Purcell, R.H. and S.U. Emerson, Hepatitis E:an emerging awareness of an old disease. Journal of Hepatology[J],2008,48 (3):494-503.
[2]Tam, A.W., M.M. Smith, M.E. Guerra, et al., Hepatitis E virus (HEV):molecular cloning and sequencing of the full-length viral genome. Virology[J],1991,185 (1):120-131.
[3]Huang, F.F., G Haqshenas, D.K. Guenette, et al., Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. Journal of Clinical Microbiology[J], 2002,40(4):1326-1332.
[4]Aggarwal, R., D. Kini, S. Sofat, et al., Duration of viraemia and faecal viral excretion in acute hepatitis E. Lancet[J],2000,356 (9235):1081-1082.
[5]Emerson, S.U. and R.H. Purcell, Hepatitis E virus. Reviews in Medical Virology[J],2003, 13 (3):145-154.
[6]Meng, X.J., R.H. Purcell, P.G. Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[7]Hsieh, S.Y., X.J. Meng, Y.H. Wu, et al., Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus. Journal of Clinical Microbiology[J],1999,37 (12):3828-3834.
[8]Wu, J.C., C.M. Chen, T.Y. Chiang, et al., Clinical and epidemiological implications of swine hepatitis E virus infection. Journal of Medical Virology [J],2000,60 (2):166-171.
[9]van der Poel, W.H., F. Verschoor, R. van der Heide, et al., Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerging Infectious Diseases[J], 2001,7 (6):970-976.
[10]Choi, C. and C. Chae, Localization of swine hepatitis E virus in liver and extrahepatic tissues from naturally infected pigs by in situ hybridization. Journal of Hepatology[J], 2003,38 (6):827-832.
[11]董晨,孟继鸿,一种新型戊型肝炎病毒样颗粒的表达、纯化及其免疫原性.细胞与分子免疫学杂志[J],2006,(03)：339-342.
[12]Robinson, R.A., W.H. Burgess, S.U. Emerson, et al., Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells. Protein Expression and Purification[J],1998,12 (1):75-84.
[13]Xing, L., K. Kato, T. Li, et al., Recombinant hepatitis E capsid protein self-assembles into a dual-domain T=1 particle presenting native virus epitopes. Virology[J],1999,265 (1): 35-45.
[14]Tsarev, S.A., T.S. Tsareva, S.U. Emerson, et al., ELISA for antibody to hepatitis E virus (HEV) based on complete open-reading frame-2 protein expressed in insect cells: identification of HEV infection in primates. The Journal of infectious diseases[J],1993, 168 (2):369-378.
[15]McAtee, C.P., Y. Zhang, P.O. Yarbough, et al., Purification of a soluble hepatitis E open reading frame 2-derived protein with unique antigenic properties. Protein Expression and Purification[J],1996,8 (2):262-270.
[16]Li, T.C., Y. Yamakawa, K. Suzuki, et al., Expression and self-assembly of empty virus-like particles of hepatitis E virus. Journal of Virology[J],1997,71 (10):7207-7213.
[17]Monga, R., S. Garg, P. Tyagi, et al., Superimposed acute hepatitis E infection in patients with chronic liver disease. Indian Journal of Gastroenterology[J],2004,23 (2):50-52.
[18]Kaur, M., K.C. Hyams, M.A. Purdy, et al., Human linear B-cell epitopes encoded by the hepatitis E virus include determinants in the RNA-dependent RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America[J], 1992,89 (9):3855-3858.
[19]Khudyakov, Y.E., N.S. Khudyakova, H.A. Fields, et al., Epitope mapping in proteins of hepatitis E virus. Virology[J],1993,194 (1):89-96.
[20]Khudyakov Yu, E., M.O. Favorov, D.L. Jue, et al., Immunodominant antigenic regions in a structural protein of the hepatitis E virus. Virology[J],1994,198 (1):390-393.
[21]Purdy, M.A., K.A. McCaustland, K. Krawczynski, et al., Expression of a hepatitis E virus (HEV)-trpE fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates. Archives of Virology[J],1992,123 (3-4):335-349.
[22]Li, F., H. Zhuang, S. Kolivas, et al., Persistent and transient antibody responses to hepatitis E virus detected by western immunoblot using open reading frame 2 and 3 and glutathione S-transferase fusion proteins. Journal of Clinical Microbiology[J],1994,32 (9):2060-2066.
[23]葛胜祥,张军,黄果勇,等.大肠杆菌表达的戊型肝炎病毒ORF2多肽对恒河猴的免疫保护研究.微生物学报[J],2003,(01)：35-42.
[1]Tam, A.W., R. White, E. Reed, et al., In vitro propagation and production of hepatitis E virus from in vivo-infected primary macaque hepatocytes. Virology[J],1996,215 (1):1-9.
[2]Meng, J., J. Pillot, X. Dai, et al., Neutralization of Different Geographic Strains of the Hepatitis E Virus with Anti-Hepatitis E Virus-Positive Serum Samples Obtained from Different Sources. Virology[J],1998,249 (2):316-324.
[3]Huang, R.T., D.R. Li, J. Wei, et al., Isolation and identification of hepatitis E virus in Xinjiang, China. Journal of General Virology[J],1992,73 (Pt 5):1143-1148.
[4]Wei, S., P. Walsh, R. Huang, et al.,93G, a novel sporadic strain of hepatitis E virus in South China isolated by cell culture. Journal of Medical Virology[J],2000,61 (3): 311-318.
[5]乐耕耘,吴娟,马雁冰,等.戊型肝炎病毒在人胚肺二倍体细胞KMB17等传代细胞中的繁殖.中国医学科学院学报[J],2001,(06)：590-593.
[6]Keohavong, P. and W.G. Thilly, Fidelity of DNA polymerases in DNA amplification. Proceedings of the National Academy of Sciences of the United States of America[J], 1989,86 (23):9253-9257.
[1]Balayan, M.S., A.G. Andjaparidze, S.S. Savinskaya, et al., Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology[J],1983,20 (1):23-31.
[2]林晓田,候金林,骆抗先,戊型肝炎动物模型研究概况.上海实验动物科学[J],1997,17(03)：170-173.
[3]毕胜利,McCaustland Karen A,江永珍,等.戊型肝炎病毒黑猩猩动物模型的建立.中华实验和临床病毒学杂志[J],1998,12(01)：5-6.
[4]Bi, S., K.A. McCaustland, and Y. Jiang, Chimpanzee model of hepatitis E virus infection. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi[J],1998,12 (1):5-6.
[5]Tsarev, S.A., S.U. Emerson, T.S. Tsareva, et al., Variation in course of hepatitis E in experimentally infected cynomolgus monkeys. The Journal of infectious diseases[J],1993, 167(6):1302-1306.
[6]Kabrane-Lazizi, Y., J.B. Fine, J. Elm, et al., Evidence for widespread infection of wild rats with hepatitis E virus in the United States. American Journal of Tropical Medicine and Hygiene[J],1999,61 (2):331-335.
[7]Hirano, M., X. Ding, T.C. Li, et al., Evidence for widespread infection of hepatitis E virus among wild rats in Japan. Hepatol Res[J],2003,27 (1):1-5.
[8]Ning, H., Z. Niu, R. Yu, et al., Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb. Veterinary Microbiology[J],2007, 121 (1-2):125-130.
[9]Meng, X.J., Swine hepatitis E virus:cross-species infection and risk in xenotransplantation. Current Topics in Microbiology and Immunology[J],2003,278: 185-216.
[10]Balayan, M.S., R.K. Usmanov, N.A. Zamyatina, et al., Brief report:experimental hepatitis E infection in domestic pigs. Journal of Medical Virology[J],1990,32 (1):58-59.
[11]Favorov, M.O., M.Y. Kosoy, S.A. Tsarev, et al., Prevalence of antibody to hepatitis E virus among rodents in the United States. The Journal of infectious diseases[J],2000,181 (2):449-455.
[12]Saad, M.D., H.A. Hussein, M.M. Bashandy, et al., Hepatitis E virus infection in work horses in Egypt. Infection, Genetics and Evolution[J],2007,7 (3):368-373.
[13]Maneerat, Y., E.T. Clayson, K.S. Myint, et al., Experimental infection of the laboratory rat with the hepatitis E virus. Journal of Medical Virology[J],1996,48 (2):121-128.
[14]Huang, F., W. Zhang, G. Gong, et al., Experimental infection of Balb/c nude mice with Hepatitis E virus. BMC Infectious Diseases[J],2009,9:93.
[15]Bouwknegt, M., K. Frankena, S.A. Rutjes, et al., Estimation of hepatitis E virus transmission among pigs due to contact-exposure. Veterinary Research[J],2008,39 (5): 40.
[16]Kasorndorkbua, C., D. Guenette, F. Huang, et al., Routes of transmission of swine hepatitis E virus in pigs. Journal of Clinical Microbiology[J],2004,42 (11):5047.
[2]Irshad, M., Hepatitis E virus:an update on its molecular, clinical and epidemiological characteristics. Intervirology[J],1999,42 (4):252-262.
[3]Balayan, M.S., A.G. Andjaparidze, S.S. Savinskaya, et al., Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology[J],1983,20 (1):23-31.
[4]Reyes, G.R., M.A. Purdy, J.P. Kim, et al., Isolation of a cDNA from the virus responsible for enterically transmitted non-A, non-B hepatitis. Science[J],1990,247 (4948): 1335-1339.
[5]Purcell, R.H. and S.U. Emerson, Hepatitis E:an emerging awareness of an old disease. Journal of Hepatology[J],2008,48 (3):494-503.
[6]Navaneethan, U., M. Al Mohajer, and M. Shata, Hepatitis E and pregnancy: understanding the pathogenesis. Liver International[J],2008,28 (9):1190-1199.
[7]Vitral, C.L., M.A. Pinto, L.L. Lewis-Ximenez, et al., Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil. Memorias do Institute Oswaldo Cruz[J],2005,100 (2):117-122.
[8]Ellis, J., E. Clark, D. Haines, et al., Porcine circovirus-2 and concurrent infections in the field. Veterinary Microbiology[J],2004,98 (2):159-163.
[9]Tan, D., S.W. Im, J.L. Yao, et al., Acute sporadic hepatitis E virus infection in southern China. Journal of Hepatology[J],1995,23 (3):239-245.
[10]Tam, A.W., M.M. Smith, M.E. Guerra, et al., Hepatitis E virus (HEV):molecular cloning and sequencing of the full-length viral genome. Virology[J],1991,185 (1):120-131.
[11]Balayan, M.S., R.K. Usmanov, N.A. Zamyatina, et al., Brief report:experimental hepatitis E infection in domestic pigs. Journal of Medical Virology[J],1990,32 (1):58-59.
[12]Lu, L., J. Drobeniuc, N. Kobylnikov, et al., Complete sequence of a Kyrgyzstan swine hepatitis E virus (HEV) isolated from a piglet thought to be experimentally infected with human HEV. Journal of Medical Virology[J],2004,74 (4):556-562.
[13]Meng, X.J., Swine hepatitis E virus:cross-species infection and risk in xenotransplantation. Current Topics in Microbiology and Immunology[J],2003,278: 185-216.
[14]Meng, X.J., R.H. Purcell, P.G Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[15]Payne, C.J., T.M. Ellis, S.L. Plant, et al., Sequence data suggests big liver and spleen disease virus (BLSV) is genetically related to hepatitis E virus. Veterinary Microbiology[J],1999,68 (1-2):119-125.
[16]Haqshenas, G., H.L. Shivaprasad, P.R. Woolcock, et al., Genetic identification and characterization of a novel virus related to human hepatitis E virus from chickens with hepatitis-splenomegaly syndrome in the United States. Journal of General Virology[J], 2001,82 (Pt 10):2449-2462.
[17]Erker, J.C., S.M. Desai, G.G Schlauder, et al., A hepatitis E virus variant from the United States:molecular characterization and transmission in cynomolgus macaques. Journal of General Virology[J],1999,80 (Pt 3):681-690.
[18]Gouvea, V., C.H. Hoke, Jr., and B.L. Innis, Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis. Journal of Virological Methods[J],1998, 70 (1):71-78.
[19]Schlauder, G.G. and I.K. Mushahwar, Genetic heterogeneity of hepatitis E virus. Journal of Medical Virology[J],2001,65 (2):282-292.
[20]Schlauder, G.G, S.M. Desai, A.R. Zanetti, et al., Novel hepatitis E virus (HEV) isolates from Europe:evidence for additional genotypes of HEV. Journal of Medical Virology[J], 1999,57 (3):243-251.
[21]Schlauder, G.G., B. Frider, S. Sookoian, et al., Identification of 2 novel isolates of hepatitis E virus in Argentina. The Journal of infectious diseases[J],2000,182 (1): 294-297.
[22]Tsarev, S.A., L.N. Binn, P.J. Gomatos, et al., Phylogenetic analysis of hepatitis E virus isolates from Egypt. Journal of Medical Virology[J],1999,57 (1):68-74.
[23]van Cuyck-Gandre, H., H.Y. Zhang, S.A. Tsarev, et al., Short report:phylogenetically distinct hepatitis E viruses in Pakistan. American Journal of Tropical Medicine and Hygiene[J],2000,62 (2):187-189.
[24]Wang, Y, R. Ling, J.C. Erker, et al., A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. Journal of General Virology[J],1999,80 (Pt 1):169-177.
[25]Zhai, L., X. Dai, and J. Meng, Hepatitis E virus genotyping based on full-length genome and partial genomic regions. Virus Research[J],2006,120 (1-2):57-69.
[26]Lu, L., C. Li, and C.H. Hagedorn, Phylogenetic analysis of global hepatitis E virus sequences:genetic diversity, subtypes and zoonosis. Reviews in Medical Virology[J], 2006,16 (1):5-36.
[27]Jameel, S., M. Zafrullah, Y.K. Chawla, et al., Reevaluation of a North India isolate of hepatitis E virus based on the full-length genomic sequence obtained following long RT-PCR. Virus Research[J],2002,86 (1-2):53-58.
[28]Aggarwal, R., K. McCaustland, J. Dilawari, et al., Genetic variability of hepatitis E virus within and between three epidemics in India. Virus Research[J],1999,59 (1):35-48.
[29]Arankalle, V.A., S. Paranjape, S.U. Emerson, et al., Phylogenetic analysis of hepatitis E virus isolates from India (1976-1993). Journal of General Virology[J],1999,80 (Pt 7): 1691-1700.
[30]Vaidya, S.R., S.D. Chitambar, and V.A. Arankalle, Polymerase chain reaction-based prevalence of hepatitis A, hepatitis E and TT viruses in sewage from an endemic area. Journal of Hepatology[J],2002,37 (1):131-136.
[31]Tsarev, S.A., S.U. Emerson, G.R. Reyes, et al., Characterization of a prototype strain of hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1992,89 (2):559-563.
[32]Shrestha, S.M., S. Shrestha, F. Tsuda, et al., Molecular investigation of hepatitis E virus infection in patients with acute hepatitis in Kathmandu, Nepal. Journal of Medical Virology[J],2003,69 (2):207-214.
[33]Shrestha, S.M., S. Shrestha, F. Tsuda, et al., Genetic changes in hepatitis E virus of subtype 1a in patients with sporadic acute hepatitis E in Kathmandu, Nepal, from 1997 to 2002. Journal of General Virology[J],2004,85 (Pt 1):97-104.
[34]Drabick, J., J. Gambel, V. Gouvea, et al., A cluster of acute hepatitis E infection in United Nations Bangladeshi peacekeepers in Haiti. The American journal of tropical medicine and hygiene[J],1997,57 (4):449.
[35]Bi, S.L., M.A. Purdy, K.A. McCaustland, et al., The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China. Virus Research[J],1993,28 (3): 233-247.
[36]Wang, Y., H. Zhang, Z. Li, et al., Detection of sporadic cases of hepatitis E virus (HEV) infection in China using immunoassays based on recombinant open reading frame 2 and 3 polypeptides from HEV genotype 4. Journal of Clinical Microbiology[J],2001,39 (12): 4370-4379.
[37]Yin, S., R.H. Purcell, and S.U. Emerson, A new Chinese isolate of hepatitis E virus: comparison with strains recovered from different geographical regions. Virus Genes[J], 1994,9(1):23-32.
[38]Chatterjee, R., S. Tsarev, J. Pillot, et al., African strains of hepatitis E virus that are distinct from Asian strains. Journal of Medical Virology[J],1997,53 (2):139-144.
[39]Grandadam, M., S. Tebbal, M. Caron, et al., Evidence for hepatitis E virus quasispecies. Journal of General Virology[J],2004,85 (Pt 11):3189-3194.
[40]He, J., L.N. Binn, S.A. Tsarev, et al., Molecular characterization of a hepatitis E virus isolate from Namibia. Journal of Biomedical Science[J],2000,7 (4):334-338.
[41]Nicand, E., GL. Armstrong, V. Enouf, et al., Genetic heterogeneity of hepatitis E virus in Darfur, Sudan, and neighboring Chad. Journal of Medical Virology[J],2005,77 (4): 519-521.
[42]van Cuyck, H., F. Juge, and P. Roques, Phylogenetic analysis of the first complete hepatitis E virus (HEV) genome from Africa. FEMS Immunology and Medical Microbiology[J],2003,39 (2):133-139.
[43]Huang, C.C., D. Nguyen, J. Fernandez, et al., Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology[J],1992,191 (2):550-558.
[44]Buisson, Y, M. Grandadam, E. Nicand, et al., Identification of a novel hepatitis E virus in Nigeria. Journal of General Virology[J],2000,81 (Pt 4):903-909.
[45]Maila, H.T., S.M. Bowyer, and R. Swanepoel, Identification of a new strain of hepatitis E virus from an outbreak in Namibia in 1995. Journal of General Virology [J],2004,85 (Pt 1):89-95.
[46]Worm, H.C., H. Wurzer, and G Frosner, Sporadic hepatitis E in Austria. New England Journal of Medicine[J],1998,339 (21):1554-1555.
[47]Mansuy, J.M., J.M. Peron, F. Abravanel, et al., Hepatitis E in the south west of France in individuals who have never visited an endemic area. Journal of Medical Virology[J],2004, 74 (3):419-424.
[48]Dalton, H.R., H.J. Fellows, E.J. Gane, et al., Hepatitis e in new zealand. Journal of Gastroenterology and Hepatology[J],2007,22 (8):1236-1240.
[49]Schlauder, GG, GJ. Dawson, J.C. Erker, et al., The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. Journal of General Virology[J],1998,79 (Pt 3):447-456.
[50]Zanetti, A.R., GG Schlauder, L. Romano, et al., Identification of a novel variant of hepatitis E virus in Italy. Journal of Medical Virology[J],1999,57 (4):356-360.
[51]Ijaz, S., E. Arnold, M. Banks, et al., Non-travel-associated hepatitis E in England and Wales:demographic, clinical, and molecular epidemiological characteristics. The Journal of infectious diseases[J],2005,192 (7):1166-1172.
[52]Dalton, H.R., P.H. Thurairajah, H.J. Fellows, et al., Autochthonous hepatitis E in southwest England. Journal of Viral Hepatitis[J],2007,14 (5):304-309.
[53]Buti, M., P. Clemente-Casares, R. Jardi, et al., Sporadic cases of acute autochthonous hepatitis E in Spain. Journal of Hepatology[J],2004,41 (1):126-131.
[54]McCrudden, R., S. O'Connell, T. Farrant, et al., Sporadic acute hepatitis E in the united kingdom:an underdiagnosed phenomenon? Gut[J],2000,46 (5):732-733.
[55]Kwo, P.Y., G.G. Schlauder, H.A. Carpenter, et al., Acute hepatitis E by a new isolate acquired in the United States. Mayo Clinic Proceedings[J],1997,72 (12):1133-1136.
[56]Herremans, M., H. Vennema, J. Bakker, et al., Swine-like hepatitis E viruses are a cause of unexplained hepatitis in the Netherlands. Journal of Viral Hepatitis[J],2007,14 (2): 140-146.
[57]Munne, M.S., S. Vladimirsky, L. Otegui, et al., Identification of the first strain of swine hepatitis E virus in South America and prevalence of anti-HEV antibodies in swine in Argentina. Journal of Medical Virology[J],2006,78 (12):1579-1583.
[58]Cooper, K., F.F. Huang, L. Batista, et al., Identification of genotype 3 hepatitis E virus (HEV) in serum and fecal samples from pigs in Thailand and Mexico, where genotype 1 and 2 HEV strains are prevalent in the respective human populations. Journal of Clinical Microbiology[J],2005,43 (4):1684-1688.
[59]Banks, M., GS. Heath, S.S. Grierson, et al., Evidence for the presence of hepatitis E virus in pigs in the United Kingdom. Veterinary Record[J],2004,154 (8):223-227.
[60]van der Poel, W.H., F. Verschoor, R. van der Heide, et al., Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerging Infectious Diseases[J], 2001,7 (6):970-976.
[61]Tei, S., N. Kitajima, K. Takahashi, et al., Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet[J],2003,362 (9381):371-373.
[62]Lewis, H., D. Morgan, S. Ijaz, et al., Indigenous hepatitis E virus infection in England and Wales. BMJ (Clinical Research Ed.)[J],2006,332 (7556):1509-1510.
[63]De Silva, A., A. Muddu, J. Iredale, et al., Unexpectedly high incidence of indigenous acute hepatitis E within South Hampshire:Time for routine testing? Journal of Medical Virology[J],2008,80 (2):283-288.
[64]Peron, J.M., J.M. Mansuy, H. Poirson, et al., Hepatitis E is an autochthonous disease in industrialized countries. Analysis of 23 patients in South-West France over a 13-month period and comparison with hepatitis A. Gastroenterologie Clinique et Biologique[J], 2006,30 (5):757-762.
[65]Takahashi, K., J.H. Kang, S. Ohnishi, et al., Genetic heterogeneity of hepatitis E virus recovered from Japanese patients with acute sporadic hepatitis. The Journal of infectious diseases[J],2002,185 (9):1342-1345.
[66]Takahashi, K., K. Iwata, N. Watanabe, et al., Full-genome nucleotide sequence of a hepatitis E virus strain that may be indigenous to Japan. Virology[J],2001,287 (1):9-12.
[67]Matsubayashi, K., Y. Nagaoka, H. Sakata, et al., Transfusion-transmitted hepatitis E caused by apparently indigenous hepatitis E virus strain in Hokkaido, Japan. Transfusion[J],2004,44 (6):934-940.
[68]Takahashi, M., T. Nishizawa, and H. Okamoto, Identification of a genotype Ⅲ swine hepatitis E virus that was isolated from a Japanese pig born in 1990 and that is most closely related to Japanese isolates of human hepatitis E virus. Journal of Clinical Microbiology[J],2003,41 (3):1342-1343.
[69]Okamoto, H., M. Takahashi, T. Nishizawa, et al., Analysis of the complete genome of indigenous swine hepatitis E virus isolated in Japan. Biochemical and Biophysical Research Communications[J],2001,289 (5):929-936.
[70]Tokita, H., H. Harada, Y. Gotanda, et al., Molecular and serological characterization of sporadic acute hepatitis E in a Japanese patient infected with a genotype III hepatitis E virus in 1993. Journal of General Virology[J],2003,84 (Pt 2):421-427.
[71]Takahashi, M., T. Nishizawa, H. Miyajima, et al., Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. Journal of General Virology[J],2003,84 (Pt 4):851-862.
[72]Tamada, Y, K. Yano, H. Yatsuhashi, et al., Consumption of wild boar linked to cases of hepatitis E. Journal of Hepatology[J],2004,40 (5):869-870.
[73]Ning, H., S. Yu, Y. Zhu, et al., Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs. Veterinary Microbiology[J],2008,126 (1-3):257-263.
[74]Yazaki, Y, H. Mizuo, M. Takahashi, et al., Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. Journal of General Virology[J],2003,84 (Pt 9):2351-2357.
[75]Arankalle, V.A., L.P. Chobe, M.V. Joshi, et al., Human and swine hepatitis E viruses from Western India belong to different genotypes. Journal of Hepatology[J],2002,36 (3): 417-425.
[76]Arankalle, V.A., L.P. Chobe, A.M. Walimbe, et al., Swine HEV infection in south India and phylogenetic analysis (1985-1999). Journal of Medical Virology[J],2003,69 (3): 391-396.
[77]Li, K., H. Zhuang, and W. Zhu, Partial nucleotide sequencing of hepatitis E viruses detected in sera of patients with hepatitis E from 14 cities in China. Chinese Medical Journal[J],2002,115 (7):1058-1063.
[78]Wang, Y.C., H.Y. Zhang, N.S. Xia, et al., Prevalence, isolation, and partial sequence analysis of hepatitis E virus from domestic animals in China. Journal of Medical Virology[J],2002,67 (4):516-521.
[79]Nishizawa, T., M. Takahashi, H. Mizuo, et al., Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99% identity over the entire genome. Journal of General Virology[J],2003,84 (Pt 5):1245-1251.
[80]Wibawa, I.D., D.H. Muljono, Mulyanto, et al., Prevalence of antibodies to hepatitis E virus among apparently healthy humans and pigs in Bali, Indonesia:Identification of a pig infected with a genotype 4 hepatitis E virus. Journal of Medical Virology[J],2004,73 (1):38-44.
[81]Hijikata, M., S. Hayashi, N.T. Trinh, et al., Genotyping of hepatitis E virus from Vietnam. Intervirology[J],2002,45 (2):101-104.
[82]Amon, J.J., J. Drobeniuc, W.A. Bower, et al., Locally acquired hepatitis E virus infection, El Paso, Texas. Journal of Medical Virology[J],2006,78 (6):741-746.
[83]Mizuo, H., K. Suzuki, Y. Takikawa, et al., Polyphyletic strains of hepatitis E virus are responsible for sporadic cases of acute hepatitis in Japan. Journal of Clinical Microbiology[J],2002,40 (9):3209-3218.
[84]Kabrane-Lazizi, Y, M. Zhang, R.H. Purcell, et al., Acute hepatitis caused by a novel strain of hepatitis E virus most closely related to United States strains. Journal of General Virology[J],2001,82 (Pt 7):1687-1693.
[85]Inoue, J., T. Nishizawa, M. Takahashi, et al., Analysis of the full-length genome of genotype 4 hepatitis E virus isolates from patients with fulminant or acute self-limited hepatitis E. Journal of Medical Virology[J],2006,78 (4):476-484.
[86]Ahn, J.M., S.G Kang, D.Y. Lee, et al., Identification of novel human hepatitis E virus (HEV) isolates and determination of the seroprevalence of HEV in Korea. Journal of Clinical Microbiology[J],2005,43 (7):3042-3048.
[87]Suzuki, K., T. Aikawa, and H. Okamoto, Fulminant hepatitis E in Japan. New England Journal of Medicine[J],2002,347 (18):1456.
[88]Takahashi, M., T. Nishizawa, A. Yoshikawa, et al., Identification of two distinct genotypes of hepatitis E virus in a Japanese patient with acute hepatitis who had not travelled abroad. Journal of General Virology[J],2002,83 (Pt 8):1931-1940.
[89]Michitaka, K., K. Takahashi, S. Furukawa, et al., Prevalence of hepatitis E virus among wild boar in the Ehime area of western Japan. Hepatol Res[J],2007,37 (3):214-220.
[90]Martelli, F., A. Caprioli, M. Zengarini, et al., Detection of hepatitis E virus (HEV) in a demographic managed wild boar (Sus scrofa scrofa) population in Italy. Veterinary Microbiology[J],2008,126 (1-3):74-81.
[91]Agrawal, S., D. Gupta, and S.K. Panda, The 3'end of hepatitis E virus (HEV) genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp). Virology[J], 2001,282(1):87-101.
[92]Koonin, E.V., A.E. Gorbalenya, M.A. Purdy, et al., Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus:delineation of an additional group of positive-strand RNA plant and animal viruses. Proceedings of the National Academy of Sciences of the United States of America[J],1992,89 (17): 8259-8263.
[93]Magden, J., N. Takeda, T. Li, et al., Virus-specific mRNA capping enzyme encoded by hepatitis E virus. Journal of Virology[J],2001,75 (14):6249-6255.
[94]Emerson, S.U., M. Zhang, X.J. Meng, et al., Recombinant hepatitis E virus genomes infectious for primates:importance of capping and discovery of a cis-reactive element. Proceedings of the National Academy of Sciences of the United States of America[J], 2001,98 (26):15270-15275.
[95]Zhang, M., R.H. Purcell, and S.U. Emerson, Identification of the 5'terminal sequence of the SAR-55 and MEX-14 strains of hepatitis E virus and confirmation that the genome is capped. Journal of Medical Virology[J],2001,65 (2):293-295.
[96]Graff, J., Y.H. Zhou, U. Torian, et al., Mutations within potential glycosylation sites in the capsid protein of hepatitis E virus prevent the formation of infectious virus particles. Journal of Virology[J],2008,82 (3):1185-1194.
[97]Robinson, R.A., W.H. Burgess, S.U. Emerson, et al., Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells. Protein Expression and Purification[J],1998,12 (1):75-84.
[98]Zafrullah, M., M.H. Ozdener, S.K. Panda, et al., The ORF3 protein of hepatitis E virus is a phosphoprotein that associates with the cytoskeleton. Journal of Virology [J],1997,71 (12):9045-9053.
[99]Tyagi, S., H. Korkaya, M. Zafrullah, et al., The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. Journal of Biological Chemistry[J],2002,277 (25):22759-22767.
[100]Korkaya, H., S. Jameel, D. Gupta, et al., The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK. Journal of Biological Chemistry [J],2001, 276 (45):42389-42400.
[101]Huang, F.F., Z.F. Sun, S.U. Emerson, et al., Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV. Journal of General Virology[J],2004,85 (Pt 6):1609-1618.
[102]Stoszek, S., R. Engle, M. Abdel-Hamid, et al., Hepatitis E antibody seroconversion without disease in highly endemic rural Egyptian communities. Transactions of the Royal Society of Tropical Medicine and Hygiene[J],2006,100 (2):89-94.
[103]Drobeniuc, J., M.O. Favorov, C.N. Shapiro, et al., Hepatitis E virus antibody prevalence among persons who work with swine. The Journal of infectious diseases[J],2001,184 (12):1594-1597.
[104]Meng, X.J., B. Wiseman, F. Elvinger, et al., Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. Journal of Clinical Microbiology[J],2002,40 (1):117-122.
[105]Arankalle, V.A., S.A. Tsarev, M.S. Chadha, et al., Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India,1982 and 1992. The Journal of infectious diseases[J],1995,171 (2):447-450.
[106]Bouwknegt, M., K. Frankena, S.A. Rutjes, et al., Estimation of hepatitis E virus transmission among pigs due to contact-exposure. Veterinary Research[J],2008,39 (5): 40.*
[107]Kasorndorkbua, C., D. Guenette, F. Huang, et al., Routes of transmission of swine hepatitis E virus in pigs. Journal of Clinical Microbiology[J],2004,42 (11):5047.
[108]Peralta, B., M. Biarnes, G Ordonez, et al., Evidence of widespread infection of avian hepatitis E virus (avian HEV) in chickens from Spain. Veterinary Microbiology[J],2009, 137 (1-2):31-36.
[109]Huang, F.F., G Haqshenas, H.L. Shivaprasad, et al., Heterogeneity and seroprevalence of a newly identified avian hepatitis e virus from chickens in the United States. Journal of Clinical Microbiology[J],2002,40 (11):4197-4202.
[110]Sun, Z.F., C.T. Larsen, A. Dunlop, et al., Genetic identification of avian hepatitis E virus (HEV) from healthy chicken flocks and characterization of the capsid gene of 14 avian HEV isolates from chickens with hepatitis-splenomegaly syndrome in different geographical regions of the United States. Journal of General Virology[J],2004,85 (Pt 3): 693-700.
[111]Billam, P., F.F. Huang, Z.F. Sun, et al., Systematic pathogenesis and replication of avian hepatitis E virus in specific-pathogen-free adult chickens. Journal of Virology[J],2005, 79 (6):3429-3437.
[112]Harrison, T.J., Hepatitis E virus-an update. Liver[J],1999,19 (3):171-176.
[113]Smith, J.L., A review of hepatitis E virus. Journal of Food Protection[J],2001,64 (4): 572-586.
[114]Mizuo, H., Y. Yazaki, K. Sugawara, et al., Possible risk factors for the transmission of hepatitis E virus and for the severe form of hepatitis E acquired locally in Hokkaido, Japan. Journal of Medical Virology[J],2005,76 (3):341-349.
[115]Tsarev, S.A., T.S. Tsareva, S.U. Emerson, et al., Experimental hepatitis E in pregnant rhesus monkeys:failure to transmit hepatitis E virus (HEV) to offspring and evidence of naturally acquired antibodies to HEV. The Journal of infectious diseases[J],1995,172 (1): 31-37.
[116]Halbur, P.G., C. Kasorndorkbua, C. Gilbert, et al., Comparative pathogenesis of infection of pigs with hepatitis E viruses recovered from a pig and a human. Journal of Clinical Microbiology[J],2001,39 (3):918-923.
[117]Kawai, H.F., T. Koji, F. Iida, et al., Shift of hepatitis E virus RNA from hepatocytes to biliary epithelial cells during acute infection of rhesus monkey. Journal of Viral Hepatitis[J],1999,6 (4):287-297.
[118]Williams, T.P., C. Kasorndorkbua, P.G Halbur, et al., Evidence of extrahepatic sites of replication of the hepatitis E virus in a swine model. Journal of Clinical Microbiology[J], 2001,39 (9):3040-3046.
[119]Reyes, G.R., C.C. Huang, A.W. Tam, et al., Molecular organization and replication of hepatitis E virus (HEV). Archives of Virology. Supplementum[J],1993,7:15-25.
[120]Kalia, M., V. Chandra, S.A. Rahman, et al., Heparan sulfate proteoglycans are required for cellular binding of the hepatitis E virus ORF2 capsid protein and for viral infection. Journal of Virology[J],2009,83 (24):12714-12724.
[121]Zheng, Z.Z., J. Miao, M. Zhao, et al., Role of heat-shock protein 90 in hepatitis E virus capsid trafficking. Journal of General Virology[J],2010,91 (Pt 7):1728-1736.
[122]Panda, S., I. Ansari, H. Durgapal, et al., The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious. Journal of Virology[J],2000,74 (5):2430.
[123]Nanda, S.K., S.K. Panda, H. Durgapal, et al., Detection of the negative strand of hepatitis E virus RNA in the livers of experimentally infected rhesus monkeys:evidence for viral replication. Journal of Medical Virology[J],1994,42 (3):237-240.
[124]Meng, X.J., P.G. Halbur, J.S. Haynes, et al., Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV. Archives of Virology[J],1998,143 (7):1405-1415.
[125]Graff, J., U. Torian, H. Nguyen, et al., A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. Journal of Virology[J],2006,80 (12): 5919-5926.
[126]Huang, Y.W., T. Opriessnig, P.G Halbur, et al., Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. Journal of Virology[J],2007,81 (6):3018-3026.
[127]Ichiyama, K., K. Yamada, T. Tanaka, et al., Determination of the 5'-terminal sequence of subgenomic RNA of hepatitis E virus strains in cultured cells. Archives of Virology[J], 2009,154(12):1945-1951.
[128]Huang, R.T., D.R. Li, J. Wei, et al., Isolation and identification of hepatitis E virus in Xinjiang, China. Journal of General Virology[J],1992,73 (Pt 5):1143-1148.
[129]Huang, R., N. Nakazono, K. Ishii, et al., Hepatitis E virus (87A strain) propagated in A549 cells. Journal of Medical Virology[J],1995,47 (4):299-302.
[130]Tanaka, T., M. Takahashi, E. Kusano, et al., Development and evaluation of an efficient cell-culture system for Hepatitis E virus. Journal of General Virology[J],2007,88 (Pt 3): 903-911.
[131]Kazachkov Yu, A., M.S. Balayan, T.A. Ivannikova, et al., Hepatitis E virus in cultivated cells. Archives of Virology[J],1992,127 (1-4):399-402.
[132]Tam, A.W., R. White, E. Reed, et al., In vitro propagation and production of hepatitis E virus from in vivo-infected primary macaque hepatocytes. Virology[J],1996,215 (1):1-9.
[133]Divizia, M., R. Gabrieli, A.M. Degener, et al., Evidence of hepatitis E virus replication on cell cultures. New Microbiologica[J],1999,22 (2):77-83.
[134]乐耕耘,吴娟,马雁冰,等.戊型肝炎病毒在人胚肺二倍体细胞KMB17等传代细胞中的繁殖.中国医学科学院学报[J],2001,(06)：590-593.
[135]Wei, S., P. Walsh, R. Huang, et al.,93G, a novel sporadic strain of hepatitis E virus in South China isolated by cell culture. Journal of Medical Virology[J],2000,61 (3): 311-318.
[136]Meng, J., J. Pillot, X. Dai, et al., Neutralization of Different Geographic Strains of the Hepatitis E Virus with Anti-Hepatitis E Virus-Positive Serum Samples Obtained from Different Sources. Virology[J],1998,249 (2):316-324.
[137]Emerson, S.U., H. Nguyen, J. Graff, et al., In vitro replication of hepatitis E virus (HEV) genomes and of an HEV replicon expressing green fluorescent protein. Journal of Virology[J],2004,78 (9):4838-4846.
[138]Emerson, S.U., V.A. Arankalle, and R.H. Purcell, Thermal stability of hepatitis E virus. The Journal of infectious diseases[J],2005,192 (5):930-933.
[139]Purdy, M.A., K.A. McCaustland, K. Krawczynski, et al., Preliminary evidence that a trpE-HEV fusion protein protects cynomolgus macaques against challenge with wild-type hepatitis E virus (HEV). Journal of Medical Virology[J],1993,41 (1):90-94.
[140]毕胜利,鲁健,蒋琳,等.原核表达戊型肝炎病毒基因重组结构蛋白免疫保护恒河猴的初步实验研究.中华实验和临床病毒学杂志[J],2002,(01)：31-32.
[141]徐国民,李奎,庄辉,等.戊型肝炎病毒重组疫苗的初步实验研究.中国公共卫生[J],2000,(09)：57-58.
[142]Li, T.C., Y. Yamakawa, K. Suzuki, et al., Expression and self-assembly of empty virus-like particles of hepatitis E virus. Journal of Virology [J],1997,71 (10):7207-7213.
[143]Zhang, M., S. Emerson, H. Nguyen, et al., Immunogenicity and protective efficacy of a vaccine prepared from 53 kDa truncated hepatitis E virus capsid protein expressed in insect cells. Vaccine[J],2001,20 (5-6):853-857.
[144]Tsarev, S.A., T.S. Tsareva, S.U. Emerson, et al., Recombinant vaccine against hepatitis E: dose response and protection against heterologous challenge. Vaccine[J],1997,15 (17-18): 1834-1838.
[145]He, J., S.L. Hoffman, and C.G. Hayes, DNA inoculation with a plasmid vector carrying the hepatitis E virus structural protein gene induces immune response in mice. Vaccine[J], 1997,15 (4):357-362.
[146]洪艳,阮冰,杨连华,等.戊型肝炎病毒嵌合基因疫苗的初步研究.中国公共卫生[J],2004,(07)：123-124.
[147]Lu, F., H. Zhuang, Y. Zhu, et al., A preliminary study on immune response to hepatitis E virus DNA vaccine in mice. Chinese Medical Journal[J],1996,109 (12):919-921.
[148]Zhu, F.C., J. Zhang, X.F. Zhang, et al., Efficacy and safety of a recombinant hepatitis E vaccine in healthy adults:a large-scale, randomised, double-blind placebo-controlled, phase 3 trial. Lancet[J],2010,376 (9744):895-902.
[149]Shrestha, M., R. Scott, D. Joshi, et al., Safety and efficacy of a recombinant hepatitis E vaccine. The New England journal of medicine[J],2007,356 (9):895.
[150]Favorov, M.O., M.Y. Kosoy, S.A. Tsarev, et al., Prevalence of antibody to hepatitis E virus among rodents in the United States. The Journal of infectious diseases[J],2000,181 (2):449-455.
[151]Meng, X.J., S. Dea, R.E. Engle, et al., Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. Journal of Medical Virology[J],1999,59 (3):297-302.
[1]黄耀伟,人类及动物RNA病毒的反向遗传系统.生物工程学报[J],2004,20(3)：311-318.
[2]韩剑峰,SARS-CoV中国株基因组全长cDNA的构建及其恢复病毒的生物学性质.微生物学报[J],2006,46(6)：922-927.
[3]Zibert, A., G. Maass, K. Strebel, et al., Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA. Journal of Virology [J],1990,64 (6):2467-2473.
[4]Cohen, J., J. Ticehurst, S. Feinstone, et al., Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture. Journal of Virology[J],1987,61 (10):3035.
[5]胡建和,猪瘟病毒兔化弱毒株全长cDNA克隆的感染性鉴定.病毒学报[J],2004,20(2)：143-147.
[6]Yamshchikov, V.F., G. Wengler, A.A. Perelygin, et al., An infectious clone of the West Nile flavivirus. Virology[J],2001,281 (2):294-304.
[7]Liu, G., Z. Liu, Q. Xie, et al., Generation of an infectious cDNA clone of an FMDV strain isolated from swine. Virus Research[J],2004,104 (2):157-164.
[8]Kassimi, L.B., A. Boutrouille, M. Gonzague, et al., Nucleotide sequence and construction of an infectious cDNA clone of an EMCV strain isolated from aborted swine fetus. Virus Research[J],2002,83 (1-2):71-87.
[9]Liu, G., Y. Zhang, Z. Ni, et al., Recovery of infectious rabbit hemorrhagic disease virus from rabbits after direct inoculation with in vitro-transcribed RNA. Journal of Virology[J], 2006,80 (13):6597-6602.
[10]Huang, Y.W., G. Haqshenas, C. Kasomdorkbua, et al., Capped RNA transcripts of full-length cDNA clones of swine hepatitis E virus are replication competent when transfected into Huh7 cells and infectious when intrahepatically inoculated into pigs. Journal of Virology[J],2005,79 (3):1552-1558.
[11]Ruggli, N., J.D. Tratschin, C. Mittelholzer, et al., Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. Journal of Virology[J],1996,70 (6):3478-3487.
[12]Meulenberg, J.J., J.N. Bos-de Ruijter, R. van de Graaf, et al., Infectious transcripts from cloned genome-length cDNA of porcine reproductive and respiratory syndrome virus. Journal of Virology[J],1998,72 (1):380-387.
[13]Almazan, F., J.M. Gonzalez, Z. Penzes, et al., Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proceedings of the National Academy of Sciences of the United States of America[J],2000,97 (10):5516-5521.
[14]Van Dinten, L., J. Den Boon, A. Wassenaar, et al., An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (3):991.
[15]Yount, B., K. Curtis, E. Fritz, et al., Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proceedings of the National Academy of Sciences of the United States of America[J],2003,100 (22):12995.
[16]Racaniello, V. and D. Baltimore, Cloned poliovirus complementary DNA is infectious in mammalian cells. Science[J],1981,214:916-919.
[17]Racaniello, V.R. and D. Baltimore, Cloned poliovirus complementary DNA is infectious in mammalian cells. Science[J],1981,214 (4523):916-919.
[18]Hamilton, W. and D. Baulcombe, Infectious RNA produced by in vitro transcription of a full-length tobacco rattle virus RNA-1 cDNA. Journal of General Virology[J],1989,70 (4):963.
[19]van der Werf, S., J. Bradley, E. Wimmer, et al., Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America[J],1986,83 (8):2330-2334.
[20]Hayes, R. and K. Buck, Infectious cucumber mosaic virus RNA transcribed in vitro from clones obtained from cDNA amplified using the polymerase chain reaction. Journal of General Virology[J],1990,71 (11):2503.
[21]Mizutani, S. and R. Colonno, In vitro synthesis of an infectious RNA from cDNA clones of human rhinovirus type 14. Journal of Virology[J],1985,56 (2):628.
[22]Klump, W.M., I. Bergmann, B.C. Muller, et al., Complete nucleotide sequence of infectious Coxsackievirus B3 cDNA:two initial 5'uridine residues are regained during plus-strand RNA synthesis. Journal of Virology[J],1990,64 (4):1573-1583.
[23]Duechler, M., T. Skern, D. Blaas, et al., Human rhinovirus serotype 2:in vitro synthesis of an infectious RNA. Virology[J],1989,168 (1):159-161.
[24]Sarnow, P., Role of 3'-end sequences in infectivity of poliovirus transcripts made in vitro. Journal of Virology[J],1989,63 (1):467.
[25]Dzianott, A.M. and J.J. Bujarski, Derivation of an infectious viral RNA by autolytic cleavage of in vitro transcribed viral cDNAs. Proceedings of the National Academy of Sciences of the United States of America[J],1989,86 (13):4823-4827.
[26]袁海峰,姚火春,高飞,等.poly(A)尾对猪繁殖与呼吸综合征病毒基因组感染性的影响.中国预防兽医学报[J],2008,(02)：86-90.
[27]Emerson, S.U., M. Zhang, X.J. Meng, et al., Recombinant hepatitis E virus genomes infectious for primates:importance of capping and discovery of a cis-reactive element. Proceedings of the National Academy of Sciences of the United States of America[J], 2001,98 (26):15270-15275.
[28]Angenent, G.C., E. Posthumus, and J.F. Bol, Biological activity of transcripts synthesized in vitro from full-length and mutated DNA copies of tobacco rattle virus RNA 2. Virology[J],1989,173 (1):68-76.
[29]Chambers, T.J., R.C. Weir, A. Grakoui, et al., Evidence that the N-terminal domain of nonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein. Proceedings of the National Academy of Sciences of the United States of America[J],1990,87 (22):8898-8902.
[30]Chambers, T.J., A. Nestorowicz, and C.M. Rice, Mutagenesis of the yellow fever virus NS2B/3 cleavage site:determinants of cleavage site specificity and effects on polyprotein processing and viral replication. Journal of Virology [J],1995,69 (3):1600-1605.
[31]Muylaert, I.R., T.J. Chambers, R. Galler, et al., Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein:effects on virus replication and mouse neurovirulence. Virology[J],1996,222 (1):159-168.
[32]Leippert, M., E. Beck, F. Weiland, et al., Point mutations within the betaG-betaH loop of foot-and-mouth disease virus O1K affect virus attachment to target cells. Journal of Virology[J],1997,71 (2):1046-1051.
[33]Dobbe, J.C., Y. van der Meer, W.J. Spaan, et al., Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture. Virology[J],2001,288 (2):283-294.
[34]Zeng, L., B. Falgout, and L. Markoff, Identification of specific nucleotide sequences within the conserved 3'-SL in the dengue type 2 virus genome required for replication. Journal of Virology[J],1998,72 (9):7510-7522.
[35]Kanno, T., D. Mackay, G. Wilsden, et al., Virulence of swine vesicular disease virus is determined at two amino acids in capsid protein VP1 and 2A protease. Virus Research[J], 2001,80(1-2):101-107.
[36]Rieder, E., T. Bunch, F. Brown, et al., Genetically engineered foot-and-mouth disease viruses with poly(C) tracts of two nucleotides are virulent in mice. Journal of Virology [J], 1993,67 (9):5139-5145.
[37]McKenna, T., J. Lubroth, E. Rieder, et al., Receptor binding site-deleted foot-and-mouth disease (FMD) virus protects cattle from FMD. Journal of Virology[J],1995,69 (9): 5787.
[38]Ward, G., E. Rieder, and P.W. Mason, Plasmid DNA encoding replicating foot-and-mouth disease virus genomes induces antiviral immune responses in swine. Journal of Virology[J],1997,71 (10):7442-7447.
[39]Mandl, C.W., J.H. Aberle, S.W. Aberle, et al., In vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirus model. Nature Medicine[J],1998,4 (12): 1438-1440.
[40]Bray, M., R. Men, and C.J. Lai, Monkeys immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge. Journal of Virology[J],1996,70 (6):4162-4166.
[41]Marchevsky, R.S., J. Mariano, V.S. Ferreira, et al., Phenotypic analysis of yellow fever virus derived from complementary DNA. American Journal of Tropical Medicine and Hygiene[J],1995,52 (1):75-80.
[42]卢建红,以BAC为基础的疱疹病毒感染性克隆技术.中国生物工程杂志[J],2006,26(6)：78-82.
[43]Porter, D.C., J. Wang, Z. Moldoveanu, et al., Immunization of mice with poliovirus replicons expressing the C-fragment of tetanus toxin protects against lethal challenge with tetanus toxin. Vaccine[J],1997,15 (3):257-264.
[44]Yount, B., K.M. Curtis, and R.S. Baric, Strategy for systematic assembly of large RNA and DNA genomes:transmissible gastroenteritis virus model. Journal of Virology[J], 2000,74(22):10600-10611.
[45]Panda, S., I. Ansari, H. Durgapal, et al., The in vitro-synthesized RNA from a cDNA clone of hepatitis E virus is infectious. Journal of Virology[J],2000,74 (5):2430.
[46]Yount, B., M.R. Denison, S.R. Weiss, et al., Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. Journal of Virology[J],2002,76 (21):11065-11078.
[47]Huang, F.F., F.W. Pierson, T.E. Toth, et al., Construction and characterization of infectious cDNA clones of a chicken strain of hepatitis E virus (HEV), avian HEV. Journal of General Virology[J],2005,86 (Pt 9):2585-2593.
[48]Yamada, K., M. Takahashi, Y. Hoshino, et al., Construction of an infectious cDNA clone of hepatitis E virus strain JE03-1760F that can propagate efficiently in cultured cells. Journal of General Virology[J],2009,90 (Pt2):457-462.
[49]Kwon, H.M., T. Leroith, R.S. Pudupakam, et al., Construction of an infectious cDNA clone of avian hepatitis E virus (avian HEV) recovered from a clinically healthy chicken in the United States and characterization of its pathogenicity in specific-pathogen-free chickens. Veterinary Microbiology[J],2010.
[50]Balayan, M.S., R.K. Usmanov, N.A. Zamyatina, et al., Brief report:experimental hepatitis E infection in domestic pigs. Journal of Medical Virology[J],1990,32 (1):58-59.
[51]Meng, X.J., R.H. Purcell, P.G. Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[52]Meng, X.J., P.G Halbur, M.S. Shapiro, et al., Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. Journal of Virology[J],1998,72 (12): 9714-9721.
[53]Yazaki, Y., H. Mizuo, M. Takahashi, et al., Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. Journal of General Virology[J],2003,84 (Pt 9):2351-2357.
[54]Feagins, A.R., T. Opriessnig, D.K. Guenette, et al., Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA. Journal of General Virology[J],2007,88 (Pt 3):912-917.
[55]Kato, M., K. Taneichi, and K. Matsubayashi, A mini-outbreak of hepatitis E infection in those who enjoyed Yokiniko party:One died of fulminant hepatitis. Kanzo[J],2004,45: 1326-1332.
[56]Mizuo, H., Y. Yazaki, K. Sugawara, et al., Possible risk factors for the transmission of hepatitis E virus and for the severe form of hepatitis E acquired locally in Hokkaido, Japan. Journal of Medical Virology [J],2005,76 (3):341-349.
[57]Favorov, M.O., M.Y. Kosoy, S.A. Tsarev, et al., Prevalence of antibody to hepatitis E virus among rodents in the United States. The Journal of infectious diseases[J],2000,181 (2):449-455.
[58]Saad, M.D., H.A. Hussein, M.M. Bashandy, et al., Hepatitis E virus infection in work horses in Egypt. Infection, Genetics and Evolution[J],2007,7 (3):368-373.
[59]Khuroo, M.S., S. Kamili, and S. Jameel, Vertical transmission of hepatitis E virus. Lancet[J],1995,345 (8956):1025-1026.
[60]Khuroo, M.S., M.R. Teli, S. Skidmore, et al., Incidence and severity of viral hepatitis in pregnancy. American Journal of Medicine[J],1981,70 (2):252-255.
[61]Khuroo, M.S. and S. Kamili, Aetiology, clinical course and outcome of sporadic acute viral hepatitis in pregnancy. Journal of Viral Hepatitis[J],2003,10 (1):61-69.
[62]Ning, H., Z. Niu, R. Yu, et al., Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb. Veterinary Microbiology[J],2007, 121 (1-2):125-130.
[63]Ning, H., S. Yu, Y. Zhu, et al., Genotype 3 hepatitis E has been widespread in pig farms of Shanghai suburbs. Veterinary Microbiology[J],2008,126 (1-3):257-263.
[64]Zhang, W., S. Yang, Q. Shen, et al., Genotype 3 hepatitis E virus existed among swine groups in 4 geographically far regions in China. Veterinary Microbiology[J],2010,140 (1-2):193-195.
[65]Zhang, W., Y. He, H. Wang, et al., Hepatitis E virus genotype diversity in eastern China. Emerging Infectious Diseases[J],2010,16(10):1630-1632.
[66]于水生,司伏生,朱于敏,等.上海某猪场发现基因3型和基因4型戊型肝炎病毒共存.上海农业学报[J],2008,24(04)：29-32.
[1]Das, K., A. Agarwal, R. Andrew, et al., Role of hepatitis E and other hepatotropic virus in aetiology of sporadic acute viral hepatitis:a hospital based study from urban Delhi. European Journal of Epidemiology[J],2000,16 (10):937-940.
[2]Balayan, M.S., A.G. Andjaparidze, S.S. Savinskaya, et al., Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology[J],1983,20 (1):23-31.
[3]Meng, X. J., R.H. Purcell, P.G. Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[4]Favorov, M.O., M.Y. Kosoy, S.A. Tsarev, et al., Prevalence of antibody to hepatitis E virus among rodents in the United States. The Journal of infectious diseases[J],2000,181 (2):449-455.
[5]Arankalle, V.A., M.V. Joshi, A.M. Kulkarni, et al., Prevalence of anti-hepatitis E virus antibodies in different Indian animal species. Journal of Viral Hepatitis[J],2001,8 (3): 223-227.
[6]Meng, X.J., S. Dea, R.E. Engle, et al., Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population. Journal of Medical Virology[J],1999,59 (3):297-302.
[7]Meng, X.J., Novel strains of hepatitis E virus identified from humans and other animal species:is hepatitis E a zoonosis? Journal of Hepatology[J],2000,33 (5):842-845.
[8]庄辉,毕胜利,王佑春,等.我国戊型肝炎研究.北京大学学报(医学版)[J],2002,34(05)：434-439.
[9]李金,胡志东,钟述猷,戊型肝炎研究新进展.国外医学.流行病学.传染病学分册[J],2004,(04).
[10]曹学义,马学众,刘玉璋,新疆南部地区肠道传播的非甲非乙型肝炎的流行病学研究.中国公共卫生学报[J],1989,8(04)：193-199.
[11]Cao, X., X. Ma, Y. Liu, et al., Epidemiological and etiological studies on enterically transmitted non-A, non-B hepatitis in the south part of Xinjiang. Viral Hepatitis C, D, and E. New York:Elsevier Science[J],1991:297-312.
[12]Ning, H., Z. Niu, R Yu, et al., Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb. Veterinary Microbiology [J],2007, 121 (1-2):125-130.
[13]Inoue, J., M. Takahashi, Y. Yazaki, et al., Development and validation of an improved RT-PCR assay with nested universal primers for detection of hepatitis E virus strains with significant sequence divergence. Journal of Virological Methods[J],2006,137 (2): 325-333.
[14]Lu, L., C. Li, and C.H. Hagedorn, Phylogenetic analysis of global hepatitis E virus sequences:genetic diversity, subtypes and zoonosis. Reviews in Medical Virology[J], 2006,16 (1):5-36.
[15]张军,戊型肝炎研究动态第12届国际病毒性肝炎和肝病研讨会(巴黎)信息.国际病毒学杂志[J],2006,13(04)：97-100.
[16]Meng, X.J., B. Wiseman, F. Elvinger, et al., Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries. Journal of Clinical Microbiology[J],2002,40 (1):117-122.
[17]Zhuang, H., X.Y. Cao, C.B. Liu, et al., Epidemiology of hepatitis E in China. Gastroenterologia Japonica[J],1991,26 Suppl 3:135-138.
[18]Tei, S., N. Kitajima, S. Ohara, et al., Consumption of uncooked deer meat as a risk factor for hepatitis E virus infection:an age- and sex-matched case-control study. Journal of Medical Virology[J],2004,74 (1):67-70.
[19]Li, T.C., K. Chijiwa, N. Sera, et al., Hepatitis E virus transmission from wild boar meat. Emerging Infectious Diseases[J],2005,11 (12):1958-1960.
[20]Drobeniuc, J., M.O. Favorov, C.N. Shapiro, et al., Hepatitis E virus antibody prevalence among persons who work with swine. The Journal of infectious diseases[J],2001,184 (12):1594-1597.
[21]Zheng, Y., S. Ge, J. Zhang, et al., Swine as a principal reservoir of hepatitis E virus that infects humans in eastern China. The Journal of infectious diseases[J],2006,193: 1643-1649.
[22]高橘雅春,谷仁烨,朱丽影,戊型肝炎病毒研究的新进展.日本医学介绍[J],2005,26(06)：253-255.
[23]Li, Z., S. Yu, S. Dong, et al., Reduced prevalence of genotype 3 HEV in Shanghai pig farms and hypothetical homeostasis of porcine HEV reservoir. Veterinary Microbiology [J], 2009,137 (1-2):184-189.
[24]Zhang, W., S. Yang, Q. Shen, et al., Genotype 3 hepatitis E virus existed among swine groups in 4 geographically far regions in China. Veterinary Microbiology[J],2010,140 (1-2):193-195.
[25]Zhang, W., Y. He, H. Wang, et al., Hepatitis E virus genotype diversity in eastern China. Emerging Infectious Diseases[J],2010,16(10):1630-1632.
[26]Tei, S., N. Kitajima, K. Takahashi, et al., Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet[J],2003,362 (9381):371-373.
[1]Tam, A.W., M.M. Smith, M.E. Guerra, et al., Hepatitis E virus (HEV):molecular cloning and sequencing of the full-length viral genome. Virology[J],1991,185 (1):120-131.
[2]Torresi, J., F. Li, S.A. Locarnini, et al., Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells. Journal of General Virology[J],1999,80 (Pt 5):1185-1188.
[3]Tyagi, S., H. Korkaya, M. Zafrullah, et al., The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2. Journal of Biological Chemistry[J],2002,277 (25):22759.
[4]Meng, X.J., P.G. Halbur, M.S. Shapiro, et al., Genetic and experimental evidence for cross-species infection by swine hepatitis E virus. Journal of Virology[J],1998,72 (12): 9714-9721.
[5]Tei, S., N. Kitajima, K. Takahashi, et al., Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet[J],2003,362 (9381):371-373.
[6]Meng, J., X. Dai, J.C. Chang, et al., Identification and characterization of the neutralization epitope(s) of the hepatitis E virus. Virology[J],2001,288 (2):203-211.
[7]Ning, H., Z. Niu, R. Yu, et al., Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb. Veterinary Microbiology[J],2007, 121 (1-2):125-130.
[8]Graff, J., U. Torian, H. Nguyen, et al., A bicistronic subgenomic mRNA encodes both the ORF2 and ORF3 proteins of hepatitis E virus. Journal of Virology[J],2006,80 (12): 5919-5926.
[9]Huang, Y.W., T. Opriessnig, P.G. Halbur, et al., Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. Journal of Virology[J],2007,81 (6):3018-3026.
[10]Aye, T., T. Uchida, X. Ma, et al., Complete nucleotide sequence of a hepatitis E virus isolated from the Xinjiang epidemic (1986-1988) of China. Nucleic Acids Research[J], 1992,20 (13):3512.
[11]Huang, C.C., D. Nguyen, J. Fernandez, et al., Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology[J],1992,191 (2):550-558.
[12]Schlauder, GG, GJ. Dawson, J.C. Erker, et al., The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. The Journal of general virology[J],1998,79:447.
[13]Wang, Y, H. Zhang, R. Ling, et al., The complete sequence of hepatitis E virus genotype 4 reveals an alternative strategy for translation of open reading frames 2 and 3. Journal of General Virology[J],2000,81 (Pt 7):1675-1686.
[14]Bi, S.L., M.A. Purdy, K.A. McCaustland, et al., The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China. Virus Research[J],1993,28 (3): 233-247.
[15]Wang, Y., R. Ling, J.C. Erker, et al., A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. Journal of General Virology[J],1999,80 (Pt 1):169-177.
[16]Wang, Y, H. Zhang, Z. Li, et al., Detection of sporadic cases of hepatitis E virus (HEV) infection in China using immunoassays based on recombinant open reading frame 2 and 3 polypeptides from HEV genotype 4. Journal of Clinical Microbiology[J],2001,39 (12): 4370-4379.
[17]Huang, R., N. Nakazono, K. Ishii, et al., Existing variations on the gene structure of hepatitis E virus strains from some regions of China. Journal of Medical Virology[J], 1995,47 (4):303-308.
[18]Meng, X.J., R.H. Purcell, P.G Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[19]Nishizawa, T., M. Takahashi, H. Mizuo, et al., Characterization of Japanese swine and human hepatitis E virus isolates of genotype IV with 99% identity over the entire genome. Journal of General Virology[J],2003,84 (Pt 5):1245-1251.
[20]Takahashi, M., T. Nishizawa, H. Miyajima, et al., Swine hepatitis E virus strains in Japan form four phylogenetic clusters comparable with those of Japanese isolates of human hepatitis E virus. Journal of General Virology[J],2003,84 (4):851.
[21]Okamoto, H., M. Takahashi, T. Nishizawa, et al., Analysis of the complete genome of indigenous swine hepatitis E virus isolated in Japan. Biochemical and Biophysical Research Communications[J],2001,289 (5):929-936.
[22]Hsieh, S.Y., X.J. Meng, YH. Wu, et al., Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus. Journal of Clinical Microbiology[J],1999,37 (12):3828-3834.
[23]Takahashi, M., T. Nishizawa, and H. Okamoto, Identification of a genotype III swine hepatitis E virus that was isolated from a Japanese pig born in 1990 and that is most closely related to Japanese isolates of human hepatitis E virus. Journal of Clinical Microbiology[J],2003,41 (3):1342-1343.
[24]van der Poel, W.H., F. Verschoor, R. van der Heide, et al., Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerging Infectious Diseases[J], 2001,7 (6):970-976.
[25]Huang, F.F., G. Haqshenas, D.K. Guenette, et al., Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. Journal of Clinical Microbiology[J], 2002,40 (4):1326-1332.
[1]Purcell, R.H. and S.U. Emerson, Hepatitis E:an emerging awareness of an old disease. Journal of Hepatology[J],2008,48 (3):494-503.
[2]Tam, A.W., M.M. Smith, M.E. Guerra, et al., Hepatitis E virus (HEV):molecular cloning and sequencing of the full-length viral genome. Virology[J],1991,185 (1):120-131.
[3]Huang, F.F., G Haqshenas, D.K. Guenette, et al., Detection by reverse transcription-PCR and genetic characterization of field isolates of swine hepatitis E virus from pigs in different geographic regions of the United States. Journal of Clinical Microbiology[J], 2002,40(4):1326-1332.
[4]Aggarwal, R., D. Kini, S. Sofat, et al., Duration of viraemia and faecal viral excretion in acute hepatitis E. Lancet[J],2000,356 (9235):1081-1082.
[5]Emerson, S.U. and R.H. Purcell, Hepatitis E virus. Reviews in Medical Virology[J],2003, 13 (3):145-154.
[6]Meng, X.J., R.H. Purcell, P.G. Halbur, et al., A novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences of the United States of America[J],1997,94 (18):9860-9865.
[7]Hsieh, S.Y., X.J. Meng, Y.H. Wu, et al., Identity of a novel swine hepatitis E virus in Taiwan forming a monophyletic group with Taiwan isolates of human hepatitis E virus. Journal of Clinical Microbiology[J],1999,37 (12):3828-3834.
[8]Wu, J.C., C.M. Chen, T.Y. Chiang, et al., Clinical and epidemiological implications of swine hepatitis E virus infection. Journal of Medical Virology [J],2000,60 (2):166-171.
[9]van der Poel, W.H., F. Verschoor, R. van der Heide, et al., Hepatitis E virus sequences in swine related to sequences in humans, The Netherlands. Emerging Infectious Diseases[J], 2001,7 (6):970-976.
[10]Choi, C. and C. Chae, Localization of swine hepatitis E virus in liver and extrahepatic tissues from naturally infected pigs by in situ hybridization. Journal of Hepatology[J], 2003,38 (6):827-832.
[11]董晨,孟继鸿,一种新型戊型肝炎病毒样颗粒的表达、纯化及其免疫原性.细胞与分子免疫学杂志[J],2006,(03)：339-342.
[12]Robinson, R.A., W.H. Burgess, S.U. Emerson, et al., Structural characterization of recombinant hepatitis E virus ORF2 proteins in baculovirus-infected insect cells. Protein Expression and Purification[J],1998,12 (1):75-84.
[13]Xing, L., K. Kato, T. Li, et al., Recombinant hepatitis E capsid protein self-assembles into a dual-domain T=1 particle presenting native virus epitopes. Virology[J],1999,265 (1): 35-45.
[14]Tsarev, S.A., T.S. Tsareva, S.U. Emerson, et al., ELISA for antibody to hepatitis E virus (HEV) based on complete open-reading frame-2 protein expressed in insect cells: identification of HEV infection in primates. The Journal of infectious diseases[J],1993, 168 (2):369-378.
[15]McAtee, C.P., Y. Zhang, P.O. Yarbough, et al., Purification of a soluble hepatitis E open reading frame 2-derived protein with unique antigenic properties. Protein Expression and Purification[J],1996,8 (2):262-270.
[16]Li, T.C., Y. Yamakawa, K. Suzuki, et al., Expression and self-assembly of empty virus-like particles of hepatitis E virus. Journal of Virology[J],1997,71 (10):7207-7213.
[17]Monga, R., S. Garg, P. Tyagi, et al., Superimposed acute hepatitis E infection in patients with chronic liver disease. Indian Journal of Gastroenterology[J],2004,23 (2):50-52.
[18]Kaur, M., K.C. Hyams, M.A. Purdy, et al., Human linear B-cell epitopes encoded by the hepatitis E virus include determinants in the RNA-dependent RNA polymerase. Proceedings of the National Academy of Sciences of the United States of America[J], 1992,89 (9):3855-3858.
[19]Khudyakov, Y.E., N.S. Khudyakova, H.A. Fields, et al., Epitope mapping in proteins of hepatitis E virus. Virology[J],1993,194 (1):89-96.
[20]Khudyakov Yu, E., M.O. Favorov, D.L. Jue, et al., Immunodominant antigenic regions in a structural protein of the hepatitis E virus. Virology[J],1994,198 (1):390-393.
[21]Purdy, M.A., K.A. McCaustland, K. Krawczynski, et al., Expression of a hepatitis E virus (HEV)-trpE fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates. Archives of Virology[J],1992,123 (3-4):335-349.
[22]Li, F., H. Zhuang, S. Kolivas, et al., Persistent and transient antibody responses to hepatitis E virus detected by western immunoblot using open reading frame 2 and 3 and glutathione S-transferase fusion proteins. Journal of Clinical Microbiology[J],1994,32 (9):2060-2066.
[23]葛胜祥,张军,黄果勇,等.大肠杆菌表达的戊型肝炎病毒ORF2多肽对恒河猴的免疫保护研究.微生物学报[J],2003,(01)：35-42.
[1]Tam, A.W., R. White, E. Reed, et al., In vitro propagation and production of hepatitis E virus from in vivo-infected primary macaque hepatocytes. Virology[J],1996,215 (1):1-9.
[2]Meng, J., J. Pillot, X. Dai, et al., Neutralization of Different Geographic Strains of the Hepatitis E Virus with Anti-Hepatitis E Virus-Positive Serum Samples Obtained from Different Sources. Virology[J],1998,249 (2):316-324.
[3]Huang, R.T., D.R. Li, J. Wei, et al., Isolation and identification of hepatitis E virus in Xinjiang, China. Journal of General Virology[J],1992,73 (Pt 5):1143-1148.
[4]Wei, S., P. Walsh, R. Huang, et al.,93G, a novel sporadic strain of hepatitis E virus in South China isolated by cell culture. Journal of Medical Virology[J],2000,61 (3): 311-318.
[5]乐耕耘,吴娟,马雁冰,等.戊型肝炎病毒在人胚肺二倍体细胞KMB17等传代细胞中的繁殖.中国医学科学院学报[J],2001,(06)：590-593.
[6]Keohavong, P. and W.G. Thilly, Fidelity of DNA polymerases in DNA amplification. Proceedings of the National Academy of Sciences of the United States of America[J], 1989,86 (23):9253-9257.
[1]Balayan, M.S., A.G. Andjaparidze, S.S. Savinskaya, et al., Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology[J],1983,20 (1):23-31.
[2]林晓田,候金林,骆抗先,戊型肝炎动物模型研究概况.上海实验动物科学[J],1997,17(03)：170-173.
[3]毕胜利,McCaustland Karen A,江永珍,等.戊型肝炎病毒黑猩猩动物模型的建立.中华实验和临床病毒学杂志[J],1998,12(01)：5-6.
[4]Bi, S., K.A. McCaustland, and Y. Jiang, Chimpanzee model of hepatitis E virus infection. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi[J],1998,12 (1):5-6.
[5]Tsarev, S.A., S.U. Emerson, T.S. Tsareva, et al., Variation in course of hepatitis E in experimentally infected cynomolgus monkeys. The Journal of infectious diseases[J],1993, 167(6):1302-1306.
[6]Kabrane-Lazizi, Y., J.B. Fine, J. Elm, et al., Evidence for widespread infection of wild rats with hepatitis E virus in the United States. American Journal of Tropical Medicine and Hygiene[J],1999,61 (2):331-335.
[7]Hirano, M., X. Ding, T.C. Li, et al., Evidence for widespread infection of hepatitis E virus among wild rats in Japan. Hepatol Res[J],2003,27 (1):1-5.
[8]Ning, H., Z. Niu, R. Yu, et al., Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb. Veterinary Microbiology[J],2007, 121 (1-2):125-130.
[9]Meng, X.J., Swine hepatitis E virus:cross-species infection and risk in xenotransplantation. Current Topics in Microbiology and Immunology[J],2003,278: 185-216.
[10]Balayan, M.S., R.K. Usmanov, N.A. Zamyatina, et al., Brief report:experimental hepatitis E infection in domestic pigs. Journal of Medical Virology[J],1990,32 (1):58-59.
[11]Favorov, M.O., M.Y. Kosoy, S.A. Tsarev, et al., Prevalence of antibody to hepatitis E virus among rodents in the United States. The Journal of infectious diseases[J],2000,181 (2):449-455.
[12]Saad, M.D., H.A. Hussein, M.M. Bashandy, et al., Hepatitis E virus infection in work horses in Egypt. Infection, Genetics and Evolution[J],2007,7 (3):368-373.
[13]Maneerat, Y., E.T. Clayson, K.S. Myint, et al., Experimental infection of the laboratory rat with the hepatitis E virus. Journal of Medical Virology[J],1996,48 (2):121-128.
[14]Huang, F., W. Zhang, G. Gong, et al., Experimental infection of Balb/c nude mice with Hepatitis E virus. BMC Infectious Diseases[J],2009,9:93.
[15]Bouwknegt, M., K. Frankena, S.A. Rutjes, et al., Estimation of hepatitis E virus transmission among pigs due to contact-exposure. Veterinary Research[J],2008,39 (5): 40.
[16]Kasorndorkbua, C., D. Guenette, F. Huang, et al., Routes of transmission of swine hepatitis E virus in pigs. Journal of Clinical Microbiology[J],2004,42 (11):5047.