戊型肝炎病毒ORF3及其蛋白的研究进展

戊型肝炎病毒(HEV)是发展中国家急性肝炎流行的重要病原体之一,由于缺少有效的细胞培养系统和廉价的小型动物模型,对病毒的生物学特性及发病机理了解较少。近几年在HEV ORF3及其蛋白的分子生物学特性和功能研究方面取得了一些进展,为深入了解HEV的感染和致病机理提供了新的理论依据,对这些研究成果进行了综述。     

西北农林科技大学动物医学院，兽医免疫学研究所，杨凌712100

禽戊型肝炎病毒(Hepatitis E virus,HEV)与人、猪HEV同属于肝炎病毒属,它们在遗传性和抗原性上有一定的相关性。自禽HEV被分离鉴定以来,许多国家从血清学或分子流行病学方面证实了该病毒的存在和流行。目前,GenBank上共有5个禽HEV的全基因组或接近全基因组的序列,分为3个基因型,并且其全基因组包含3个ORFs,其中ORF2基因编码病毒的衣壳蛋白,包含病毒主要的抗原表位,是血清学检测和疫苗设计的主要靶蛋白。禽HEV由于其对家禽养殖业的危害以及人畜共患的可能性,正被引起越来越多的关注。本文结合国内禽HEV的分离鉴定从禽HEV病原学、致病性以及衣壳蛋白抗原性等方面进行了总结概述。     

戊型肝炎病毒细胞培养模型的研究进展

戊型肝炎病毒（ HEV）体外细胞培养模型的建立有助于阐明HEV的感染、复制及其致病机理,有利于新型抗病毒药物和疫苗的研发与评估。近年来国内外对HEV细胞培养模型的报道很多,但所用细胞系、感染方法及病毒的复制效果等不尽相同。因此,对近年来HEV细胞培养模型的研究进展进行了综述。     

Management of Hepatitis E Virus (HEV) Zoonotic Transmission: Protection of Rabbits against HEV Challenge following Immunization with HEV 239 Vaccine

Hepatitis E virus (HEV) constitutes a significant health burden worldwide, with an estimated approximately 33% of the world’s population exposed to the pathogen. The recent licensed HEV 239 vaccine in China showed excellent protective efficacy against HEV of genotypes 1 and 4 in the general population and pregnant women. Because hepatitis E is a zoonosis, it is also necessary to ascertain whether this vaccine can serve to manage animal sources of human HEV infection. To test the efficacy of the HEV 239 vaccine in protecting animal reservoirs of HEV against HEV infection, twelve specific-pathogen-free (SPF) rabbits were divided randomly into two groups of 6 animals and inoculated intramuscularly with HEV 239 and placebo (PBS). All animals were challenged intravenously with swine HEV of genotype 4 or rabbit HEV seven weeks after the initial immunization. The course of infection was monitored for 10 weeks by serum ALT levels, duration of viremia and fecal virus excretion and HEV antibody responses. All rabbits immunized with HEV 239 developed high titers of anti-HEV and no signs of HEV infection were observed throughout the experiment, while rabbits inoculated with PBS developed viral hepatitis following challenge, with liver enzyme elevations, viremia, and fecal virus shedding. Our data indicated that the HEV 239 vaccine is highly immunogenic for rabbits and that it can completely protect rabbits against homologous and heterologous HEV infections. These findings could facilitate the prevention of food-borne sporadic HEV infection in both developing and industrialized countries.     

Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody

Hepatitis E virus (HEV), a non-enveloped, positive-sense, single-stranded RNA virus, is a major cause of enteric hepatitis. Classified into the family Hepeviridae, HEV comprises four genotypes (genotypes 1-4), which belong to a single serotype. We describe a monoclonal antibody (mAb), 8G12, which equally recognizes all four genotypes of HEV, with ∼2.53–3.45 nM binding affinity. The mAb 8G12 has a protective, neutralizing capacity, which can significantly block virus infection in host cells. Animal studies with genotypes 1, 3 and 4 confirmed the cross-genotype neutralizing capacity of 8G12 and its effective prevention of hepatitis E disease. The complex crystal structures of 8G12 with the HEV E2s domain (the most protruded region of the virus capsid) of the abundant genotypes 1 and 4 were determined at 4.0 and 2.3 Å resolution, respectively. These structures revealed that 8G12 recognizes both genotypes through the epitopes in the E2s dimerization region. Structure-based mutagenesis and cell-model assays with virus-like particles identified several conserved residues (Glu549, Lys554 and Gly591) that are essential for 8G12 neutralization. Moreover, the epitope of 8G12 is identified as a key epitope involved in virus-host interactions. These findings will help develop a common strategy for the prevention of the most abundant form of HEV infection.     

Experimental Infection of Rabbits with Rabbit and Genotypes 1 and 4 Hepatitis E Viruses

Background

A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77–79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.

Methods

Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.

Findings

Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.

Conclusions

These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.     

Complete Genome Sequence of Hepatitis E Virus from Rabbits in the United States

Hepatitis E virus (HEV) is a single-strand positive-sense RNA virus in the family Hepeviridae. The disease caused by HEV, hepatitis E, is an important public health problem in developing countries of Asia and Africa and is also endemic in many industrialized countries, including the United States. HEV has been identified from several other animal species in addition to humans, including the pig, chicken, mongoose, deer, rabbit, ferret, bat, and fish. Here we report the complete genome sequence of the first strain of HEV from rabbits in the United States. Sequence and phylogenetic analyses revealed that the U.S. rabbit HEV is a distant member of the zoonotic genotype 3 HEV, thus raising a concern for potential zoonotic human infection. A unique 90-nucleotide insertion within the X domain of the ORF1 was identified in the rabbit HEV, and this insertion may play a role in the species tropism of HEV.     

Detection of Hepatitis E Virus Antibodies in Dogs in the United Kingdom

Hepatitis E virus (HEV) genotypes 3 and 4 are zoonotic pathogens, with pigs predominantly implicated in disease transmission. The rapid rise in human cases in developed countries over the past decade indicates a change in epidemiology of HEV, and it has been suggested that additional animal species may be involved in transmission of infection. Multiple studies have identified contact with dogs as a risk factor for HEV infection in industrialised nations, and a low seroprevalence to HEV has previously been reported in dogs in low-income countries. In this study we aimed to evaluate the possibility that dogs are susceptible to HEV, and determine the frequency with which this occurs. Serum samples from UK dogs with and without hepatitis were screened for HEV-specific antibodies, and canine liver and stool samples were analysed by qPCR for the presence of HEV RNA. We describe evidence to show HEV infection occurs at low levels in dogs in the UK, but the strain of origin is undetermined. The low seroprevalence level of HEV in dogs implies the risk of zoonotic disease transmission is likely to be limited, but further investigations will be required to determine if HEV-infected dogs can transmit HEV to man.     

The hepatitis E virus ORF3 protein regulates the expression of liver-specific genes by modulating localization of hepatocyte nuclear factor 4

The hepatitis E virus (HEV) is a small RNA virus and the cause of acute viral hepatitis E. The open reading frame 3 protein (pORF3) of HEV appears to be a pleiotropic regulatory protein that helps in the establishment, propagation and progression of viral infection. However, the global cellular effects of this protein remain to be explored. In the absence of traditional in vitro viral infection systems or efficient replicon systems, we made an adenovirus based ORF3 protein expression system to study its effects on host cell gene expression. We infected Huh7 hepatoma cells with recombinant adenoviruses expressing pORF3 and performed microarray-based gene expression analyses. Several genes down regulated in pORF3-expressing cells were found to be under regulation of the liver-enriched hepatocyte nuclear factor 4 (HNF4), which regulates hepatocyte-specific gene expression. While HNF4 localizes to the nucleus, its phosphorylation results in impaired nuclear localization of HNF4. Here we report that pORF3 increases HNF4 phosphorylation through the ERK and Akt kinases, which results in impaired nuclear translocation of HNF4 and subsequently the down modulation of HNF4-responsive genes in pORF3-expressing cells. We propose that modulation of several hepatocyte specific genes by pORF3 will create an environment favorable for viral replication and pathogenesis.     

Characterization of hepatitis E virus recombinant ORF2 proteins expressed by vaccinia viruses

Hepatitis E virus (HEV), an enterically transmitted pathogen, is one of the major causes of acute hepatitis in humans worldwide, being responsible for outbreaks and epidemics in regions with suboptimal sanitary conditions, in many of which it is endemic. In industrialized countries, hepatitis E is rarely reported, but recent studies have revealed quite high human seroprevalence rates and the possibility of porcine zoonotic transmission. There is currently no specific therapy or licensed vaccine against HEV infection, and little is known about its intracellular growth cycle, as until very recently no efficient cell culture system has been available. In the present study, vaccinia viruses have been used to express recombinant HEV ORF2 proteins, allowing the study of their glycosylation patterns and subcellular localization. Furthermore, the expressed proteins have been shown to be good antigens for diagnostic purposes and to elicit high and long-lasting specific anti-HEV titers of antibodies in mice that are passively transferred to the offspring by both transplacental and lactation routes.     

Establishment of hepatitis E virus infection‐permissive and ‐non‐permissive human hepatoma PLC/PRF/5 subclones

PLC/PRF/5 cells show limited permissiveness, meaning that almost all subclones are permissive; however, some subclones do not exhibit permissiveness for hepatitis E virus (HEV) infection. In this study, the single‐cell cloning of PLC/PRF/5 was performed and heterogeneous subclones characterized. Notably, the efficiency of intracellular virus replication did not correlate with the permissiveness for HEV infection. However, as well as binding permissive subclones, virus‐like particles bound non‐permissive subclones on various levels, suggesting that these subclones have some deficiencies in the attachment and entry steps of infection. Our data would be useful for investigating the HEV life cycle.     

Hepatitis E Virus: An emerging enigmatic and underestimated pathogen

Hepatitis E virus (HEV) is an RNA virus causing hepatitis E disease. The virus is of one serotype but has diverse genotypes infecting both humans and animals. Based on evidence from seroprevalence studies, about 2 billion people are estimated to have been infected with HEV globally. HEV, therefore, poses a significant public health and economic challenge worldwide. HEV was discovered in the 1980s and was traced back to the 1955 – 1956 outbreak of hepatitis that occurred in India. Subsequently, several HEV epidemics involving thousands of individuals have occurred nearly annually in different countries in Asia and Africa. Initially, the virus was thought to be only enterically transmitted, and endemic in developing countries. Due to the environmental hygiene and sanitation challenges in those parts of the world. However, recent studies have suggested otherwise with the report of autochthonous cases in industrialised countries with no history of travel to the so-called endemic countries. Thus, suggesting that HEV has a global distribution with endemicity in both developing and industrialised nations. Studies have also revealed that HEV has multiple risk factors, and modes of transmission as well as zoonotic potentials. Additionally, recent findings have shown that HEV leads to severe disease, particularly among pregnant women. In contrast to the previous narration of a strictly mild and self-limiting infection. Studies have likewise demonstrated chronic HEV infection among immunocompromised persons. Consequent to these recent discoveries, this pathogen is considered a re – emerging virus, particularly in the developed nations. However, despite the growing public health challenges of this pathogen, the burden is still underestimated. The underestimation is often attributed to poor awareness among clinicians and a lack of routine checks for the disease in the hospitals. Thus, leading to misdiagnosis and underdiagnosis. Hence, this review provides a concise overview of epidemiology, diagnosis, and prevention of hepatitis E.     

Hepatitis E virus infection in Europe: regional situation regarding laboratory diagnosis and epidemiology

Hepatitis E virus (HEV) was first identified in the excreta of an experimentally infected human volunteer and further confirmed by similar findings in clinical specimens from patients with acute jaundice disease different from hepatitis A and B. The HEV is a 27- to 34-nm spherical non-enveloped virus obviously represented by a single serotype; however, its final taxonomic definition remains to be established. Studies on molecular biology of this virus revealed some peculiar characteristics showing no homologies in its nucleotide sequence to any entries in the Genbank database. The HEV infection was experimentally transmitted to non-human primates producing a disease in many features similar to that occurring in humans. Recently cell lines persistently infected with the HEV have also been obtained. These studies provided valuable virus-specific reagents which were used in diagnostic tests. Currently immune electron microscopy, fluorescent antibody technique, latex agglutination, cDNA hybridization, and Western blotting are employed to prove the etiological involvement of HEV in suspected hepatitis cases; serological tests with synthetic substances analogous to HEV antigens are expected to be available soon. Reliable diagnostic procedures can be carried out in a number of laboratories with the locally produced reagents. The HEV infection is common in many hot climate countries being responsible for more than 50% of jaundice cases among young adults. The European region is considered to be free of natural foci of this infection, however, several sporadic cases of HEV disease were reported to occur in Europeans who developed jaundice shortly after returning from endemic areas. It is suspected that in the Mediterranean countries (Italy and Spain) the cases of HEV infection could be causatively related to the consumption of shell-fish cultivated in sewage-polluted waters.     

CD63在戊型肝炎病毒感染中的作用机制

本研究旨在寻找戊型肝炎病毒(hepatitis E virus,HEV)衣壳蛋白ORF2的相互作用蛋白,探讨其在HEV感染中的作用。采用酵母双杂交方法从人肝细胞文库中筛选与HEV ORF2相互作用的蛋白,结果显示CD63与HEV ORF2相互作用。Pull-down实验提示原核表达的ORF2与CD63结合较弱,而免疫共沉淀实验提示真核表达的ORF2能与CD63结合。流式细胞术检测结果显示,HEV易感细胞PLC/PRF/5细胞膜表面的CD63表达水平普遍低于HEV非易感细胞。过表达CD63抑制PLC/PRF/5细胞的HEV感染,而小干扰RNA(small interfering RNA,siRNA)干扰CD63表达则促进HEV感染。结果提示,CD63能与HEV ORF2相互作用,可能抑制HEV感染肝细胞。     

中缅树鼩自然感染六种病毒的血清流行病学

中缅树鼩作为一种新型实验动物,在医学生物学上,尤其是病毒学方面的应用受到越来越多的重视.实验动物自身病毒感染会影响动物健康和干扰实验结果,甚至危害实验人员生命安全.所以,实验动物病毒检测一直是动物质量控制的重要部分.中缅树鼩研究迄今缺乏清晰的病毒自然感染资料.为调查中缅树鼩的病毒感染状况,采集野生俘获和人工繁殖的中缅树鼩血清样本272份,全部血清样本通过ELISA方法对乙型肝炎病毒(HBV)表面抗原,丙型肝炎病毒(HCV)总抗体,以及戊型肝炎病毒(HEV)、腺病毒(ADV)、单纯疱疹病毒1型(HSV-1)和2型(HSV-2)的IgG抗体进行了检测.结果表明,ELISA初筛HBV表面抗原有3份阳性样本,但通过乙型肝炎两对半定量检测进一步确认为阴性:抗HCV抗体和抗HEV、ADV、HSV-1 IgO抗体检测均为阴性;抗HSV-2 IgG检测有1份阳性样本.提示仪抗原或抗体血清学指标检测树鼩肝炎结果并不能反应个体携带病毒的状态,应该再进行病毒学指标确认.同时建议中缅树嗣繁殖群应进行HSV-2的筛选,以便杜绝和控制该病毒的感染.     

The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK

The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85alpha regulatory subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3.SH3 complex. In pORF3-expressing cells, pp60(src) was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.     

HepG2细胞中与戊型肝炎病毒衣壳蛋白相互作用蛋白的初步研究

利用重组戊型肝炎病毒(HEV)衣壳蛋白片段p239(368~606 aa)形成的类病毒颗粒作为亲和层析诱饵蛋白,HepG2为细胞模型,筛选与p239类病毒颗粒特异性相互作用蛋白。经过二维电泳分离,MALDI-TOF-MS分析鉴定得到GRP78/Bip,HSP90,alpha-tubulin及P43四个与p239有相互作用的候选蛋白。GRP78/Bip为热休克蛋白家族成员,体外免疫共沉淀实验结果证实,其与p239有特异性的结合。此研究结果为深入研究HEV的感染过程如吸附、入胞,以及HEV的致病机理提供了有益线索。     

Genetic and Experimental Evidence for Cross-Species Infection by Swine Hepatitis E Virus

Prior to the recent discovery of the swine hepatitis E virus (swine HEV) in pigs from the midwestern United States, HEV was not considered endemic to this country. Since swine HEV is antigenically and genetically related to human strains of HEV, it was important to characterize this new virus further. The infectivity titer of a pool of swine HEV in pigs was determined in order to prepare a standardized reagent and to evaluate the dose response in pigs. Although the sequence of swine HEV varied extensively from those of most human strains of HEV, it was very closely related to the two strains of human HEV (US-1 and US-2) isolated in the United States. The U.S. strains which were recently recovered from two patients with clinical hepatitis E in the United States shared ≥97% amino acid identity with swine HEV in open reading frames 1 and 2. Phylogenetic analyses of different regions of the genome revealed that swine HEV and the U.S. strains grouped together and formed a distinct branch. These results suggested that swine HEV may infect humans. When we inoculated rhesus monkeys and a chimpanzee, experimental surrogates of humans, with swine HEV, the primates became infected. Furthermore, in a reciprocal experiment, specific-pathogen-free pigs were experimentally infected with the US-2 strain of human HEV that is genetically similar to swine HEV. These results provided experimental evidence for cross-species infection by the swine virus. Thus, humans appear to be at risk of infection with swine HEV or closely related viruses.     

Serological evidence of hepatitis E virus infection in different animal species from the Southeast of Brazil

Serological evidence of hepatitis E virus infection (HEV) has been observed in both humans and different animal species living in non-endemic areas, suggesting that animals could be important reservoir for virus transmission to man. Antibodies to HEV have been detected in some Brazilian population groups. Nevertheless, sporadic cases of acute HEV infection have never been reported. We collected 271 serum samples from several domestic animals and also from pig handlers from Southeast of Brazil in order to investigate the seroprevalence of HEV infection. Anti-HEV IgG was detected in cows (1.42%), dogs (6.97%), chickens (20%), swines (24.3%), and rodents (50%), as well as in pig handlers (6.3%). The recognition of swine HEV infections in pigs in many countries of the world led us to investigate a larger sample of pigs (n = 357) from the same Brazilian region with ages ranging from 1 to > 25 weeks. IgG anti-HEV was detected in 100% of 7-day old pigs. Following a gradual decline between weeks 2 and 8 (probably due to loss of maternal IgG), the prevalence then steady increased until it reached 97.3% of animals older than 25 weeks. Besides the detection of anti-HEV antibodies in different animal species, the results showed that swine HEV infection seems to be almost universal within this Brazilian pig population. This is the first report that shows evidences of HEV circulation in Brazilian animal species and pig handlers.     

Epidemiology of Hepatitis E Virus in China: Results from the Third National Viral Hepatitis Prevalence Survey, 2005–2006

In China, hepatitis E virus (HEV) is prevalent and causes disease, but its epidemiological profile is not well understood. We used a commercial enzyme-linked immunosorbent assay to detect total antibodies to hepatitis E virus in 15,862 serum samples collected during the Third National Viral Hepatitis Prevalence Survey. The results were analyzed to calculate estimates of HEV seroprevalence and to examine the effects of some putative risk factors. The seroprevalence of HEV in the general Chinese population during the period from 2005 through 2006 was 23.46% (95% confidence interval [CI], 18.41%–28.50%). The farming population, the age group of 15–60 year olds, and those living in the Midwest or Mideast region and in Xinjiang province had the highest seroprevalence estimates. The prevalence of HEV is high in China. The seroprevalence rate of HEV shows an unbalanced distribution among areas with different geographic location and economic development levels. The characteristics of the distribution associated may be due to the route of HEV transmission (via contaminated water or animal reservoirs). Within the same region, the seroprevalence of HEV is generally increased with age.