戊型肝炎病毒Ⅳ型ORF2蛋白在汉逊酵母中的表达

为了寻求新型表达系统来研制戊型肝炎基因工程疫苗,利用甲基营养型汉逊酵母(Hansenulapolymorpha)系统表达戊型肝炎病毒(HepatitisEvirus,HEV)Ⅳ型结构区ORF2编码蛋白第112-607氨基酸片段。为实现目的基因在汉逊酵母中的高效表达,根据汉逊酵母偏爱密码子优化设计目的基因,用搭桥PCR法合成优化后的基因序列,并克隆到多拷贝表达载体上,转化汉逊酵母营养缺陷宿主菌ATCC26012(Ura3-),在选择培养基上培养,运用PCR法筛选得到携带外源基因的重组菌株,然后用含甲醇的培养基诱导表达,对表达产物进行SDS-PAGE、ELISA和Westernblot检测和鉴定。SDS-PAGE实验结果表明目的蛋白分子量约为56kD,表达量占菌体总蛋白的12%;ELISA检测结果表明表达产物为具有免疫反应性的HEVORF2蛋白,ELISA效价最高可达1∶2048,目的蛋白表达量随着基因拷贝数的增加呈升高的趋势;Westernblot鉴别实验结果证实表达产物与HEV多抗有特异性抗原抗体结合反应。HEV结构区ORF2蛋白在汉逊酵母中的成功表达,为研制基因工程戊型肝炎疫苗奠定了基础。     

合成肽抗原在戊型肝炎病毒感染诊断中的应用

An ELISA for the detection of anti HEV using synthetic peptide antigens was developed. The synthetic antigens were encoded by OFR2 and OFR3 genes of HEV. The purpose of this study was to determine the applicability of the synthetic antigens in the serodiagnosis of hepatitis E. The anti HEV detection using synthetic antigens was carried out in 47 healthy subjects and 89 patients with acute or chronic viral hepatitis. The results showed that the positive rate of anti HEV IgG in healthy subjects was 4.2%(2/47), and no IgM antibody to HEV was found. The positive rates of IgG and IgM antibodies to HEV in the hepatitis patients were 8.9% and 10% respectively. In addition, we compared the detecting efficacy of the synthetic antigens with that of the market reagent in 57 serum samples, the total coincident rate was 87.7% (50/57). All of the results accorded with the literatures reported. This study suggests that the ELISA based on the synthetic peptide antigens was specific, sensitive and convenient in diagnosis of HEV infection, it can be widely used in both clinical and epidemiological reseaches.     

Dimerization of Hepatitis E Virus Capsid Protein E2s Domain Is Essential for Virus–Host Interaction

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV–host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV.     

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.     

Hepatitis E virus DNA vaccine elicits immunologic memory in mice

Injection of an expression vector pJHEV containing hepatitis E virus (HEV) structural protein open reading frame 2 gene generates a strong antibody response in BALB/c mice that can bind to and agglutinate HEV. In this study, we tested for immunologic memory in immunized mice whose current levels of IgG to HEV were low or undetectable despite 3 doses of HEV DNA vaccine 18 months earlier. Mice previously vaccinated with vector alone were controls. All mice were administered a dose of HEV DNA vaccine to simulate an infectious challenge with HEV. The endpoint was IgG to HEV determined by ELISA. Ten days after the vaccine dose, 5 of 9 mice previously immunized with HEV DNA vaccine had a slight increase in IgG to HEV. By 40 days after the vaccine dose, the level of IgG to HEV had increased dramatically in all 9 mice (108-fold increase in geometric mean titer). In contrast, no control mice became seropositive. These results indicate that mice vaccinated with 3 doses of HEV DNA vaccine retain immunologic memory. In response to a small antigenic challenge delivered as DNA, possibly less than delivered by a human infective dose of virus, mice with memory were able to generate high levels of antibody in less time than the usual incubation period of hepatitis E. We speculate that this type of response could protect a human from overt disease.     

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.     

戊型肝炎病毒遗传多样性和进化机制的研究进展

戊型肝炎病毒(hepatitis E virus, HEV)是一种引发全球急性病毒性肝炎的人兽共患病病原。HEV具有丰富的遗传多样性,不同基因型或基因亚型的流行与地理位置、宿主物种以及防控策略等密切相关。欧洲和美洲HEV流行株为HEV-3,包括3a-i亚型,而亚洲流行株含HEV-3和HEV-4;我国的流行毒株已从HEV-1进化到HEV-4。近年来,研究发现HEV进化的影响机制,包括同义密码子使用模式、氨基酸突变和基因重组等,其中氨基酸突变是病毒持续流行的主要驱动力。因此,本文就HEV的分类、全球流行特征、进化机制等进行综述,以期为戊型肝炎的有效防控以及疫苗开发提供参考。     

Profile of acute infectious markers in sporadic hepatitis E

Laboratory diagnosis of acute infection of hepatitis E virus (HEV) is commonly based on the detection of HEV RNA, IgM and/or rising IgG levels. However, the profile of these markers when the patients present have not been well determined. To clarify the extent of misdiagnosed sporadic hepatitis E in the initial laboratory detection, serial sera of 271 sporadic acute hepatitis cases were collected, detected and the dynamics of each acute marker during the illness course were analyzed. 91 confirmed cases of hepatitis E were identified based on the presentation of HEV RNA, IgM or at least 4 fold rising of IgG levels. 21 (23.1%) hepatitis E cases were false negative for the viral RNA and 40 (44.0%) for rising IgG, because occurrence of these markers were confined to acute phase of infection and viremia had already subsided and antibody level peaked when these patients presented. IgM was detected in 82 (90.1%) cases. It is the most prevalent of the three markers, because the antibody persisted until early convalescence. Nine cases negative for IgM were positive for rising IgG and one was also positive for the viral RNA; all of these nine cases showed high avid IgG in their acute phase sera, which indicated re-infection. In summary, it is not practicable to determine the true occurrence of sporadic hepatitis E. Nevertheless, it could be closely approximated by approach using a combination of all three acute markers.     

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。     

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.     

上海部分地区戊型肝炎病毒（HEV）基因型的分析

为了解戊型肝炎病毒 (HEV)在上海部分地区流行的基因型 ,采用RT nPCR的方法检验 35例急性散发性戊型肝炎患者中HEVRNA ,并对阳性产物进行克隆测序 ,然后对其基因型进行分析。结果显示在 35例急性散发性戊型肝炎患者中PCR阳性为 9例 ,测序证实 8例为HEV的基因序列 ;其中 1例为HEV 1型 ,7例为HEV 4型。提示在上海部分地区的急性散发性戊型肝炎中以HEV 4型感染为主     

Prevalence and risk of hepatitis E virus infection in the HIV population of Nepal

Background

Infection with the hepatitis E virus (HEV) can cause acute hepatitis in endemic areas in immune-competent hosts, as well as chronic infection in immune-compromised subjects in non-endemic areas. Most studies assessing HEV infection in HIV-infected populations have been performed in developed countries that are usually affected by HEV genotype 3. The objective of this study is to measure the prevalence and risk of acquiring HEV among HIV-infected individuals in Nepal.

Methods

We prospectively evaluated 459 Human Immunodeficiency Virus (HIV)-positive individuals from Nepal, an endemic country for HEV, for seroprevalence of HEV and assessed risk factors associated with HEV infection. All individuals were on antiretroviral therapy and healthy blood donors were used as controls.

Results

We found a high prevalence of HEV IgG (39.4%) and HEV IgM (15.3%) in HIV-positive subjects when compared to healthy HIV-negative controls: 9.5% and 4.4%, respectively (OR: 6.17, 95% CI 4.42–8.61, p?<?0.001 and OR: 3.7, 95% CI 2.35–5.92, p?<?0.001, respectively). Individuals residing in the Kathmandu area showed a significantly higher HEV IgG seroprevalance compared to individuals residing outside of Kathmandu (76.8% vs 11.1%, OR: 30.33, 95% CI 18.02–51.04, p?=?0.001). Mean CD4 counts, HIV viral load and presence of hepatitis B surface antigen correlated with higher HEV IgM rate, while presence of hepatitis C antibody correlated with higher rate of HEV IgG in serum. Overall, individuals with HEV IgM positivity had higher levels of alanine aminotransferase (ALT) than IgM negative subjects, suggesting active acute infection. However, no specific symptoms for hepatitis were identified.

Conclusions

HIV-positive subjects living in Kathmandu are at higher risk of acquiring HEV infection as compared to the general population and to HIV-positive subjects living outside Kathmandu.

     

戊型肝炎病毒样颗粒在重组汉逊酵母中的发酵表达、纯化及其免疫原性分析

为了研制戊型肝炎新型基因工程疫苗,利用汉逊酵母表达系统表达重组戊型肝炎病毒样颗粒,成功构建了重组戊型肝炎疫苗工程菌株HP/HEV2.3,对该菌株的发酵条件和纯化工艺进行了研究。先将工作种子批进行发酵培养,收集发酵后的细胞培养物;对其先后进行细胞破碎、澄清和超滤、硅胶吸附和解吸附、超滤浓缩换液、色谱纯化及除菌过滤,制得重组汉逊酵母戊型肝炎病毒样颗粒,纯化收率为33%,纯度达99%;电镜观察显示该重组汉逊酵母戊型肝炎病毒样颗粒与天然戊型肝炎病毒颗粒理论大小一致,为32 nm;基因序列与理论一致;SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质分子量大小一致,均为56 k Da,表达量占细胞总蛋白的26%,表达水平为1.0 g/L发酵液;Western blotting、ELISA活性检测及小鼠免疫接种效力试验ED_(50)结果表明,此重组汉逊酵母戊型肝炎病毒样颗粒具有良好的抗原性和免疫原性,可用于制造戊型肝炎新型基因工程疫苗。     

基因重组戊肝病毒多价复合抗原的表达及初步应用

目的：利用基因工程技术体外表达高效HEV多价复合抗原,建立一套敏感和特异的抗HEV检测体系,方法：将前期构建的基因重组戊肝病毒多价复合抗原表达质粒转化大肠埃希菌JM109。筛选鉴定阳性克隆并进行诱导表达,表达产物经分子筛层析纯化后,利用western blot作抗原特异性鉴定,以此抗原建立ELISA检测卫生部生物药品检定所HEV血清参比品及临床血清标本,同时以Genelab公司抗HEVELISA试剂盒作为对照进行对比研究。结果：重组质粒转化大肠埃希菌JM109株后的阳性克隆,经诱导在SDS-PAGE中出现一条分子量大小约为31.5kD的蛋白条带,该蛋白纯化后经Western Blotting检测证实为HEV特异性抗原,以此抗原包被酶联板建立ELISA检测53份国家卫生部标准HEV参比血清,灵敏度达100%（38/38）,特异度达93.3%(14/15)。用此方法检测68份临床标本,并与Gene4labELISA试剂盒结果比较,两者阴阳性相符的有64份,总符合率为94.1%。结论：本实验克隆表达的基因重组戊肝病毒多价复合抗原具有良好抗原性,以此抗原建立的抗HEVELISA具有灵敏度高,特异性强的特点,可望为戊型肝炎的血清学诊断和流行病学调查提供一种更为有效的检测手段。     

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.