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

目的 观察基因重组戊型肝炎病毒（HEV）结构蛋白对恒河猴戊型肝炎野病毒攻击的保护作用。方法 用HEV重组结构蛋白免疫恒河猴，猴血清中抗-HEV升高后，与空白对照组一同用HEV野病毒攻击，采血观察野病毒攻击前后ALT和HEV抗体等的动态变化。结果 野病毒攻击后第3周，空白对照组5只猴ALT均出现明显异常，而免疫接种组5只猴ALT均正常，未观察到明显的肝脏炎症表现，结论 HEV重组结构蛋白免疫恒河猴之后，可以有效地保护HEV野病毒的攻击，该HEV重组结构蛋白可作为戊型肝炎基因工程疫苗的候选蛋白。     

酵母表达的重组戊型肝炎病毒结构区ORF2蛋白的抗原性鉴定

目的 对应用巴氏毕赤酵母表达系统制备的重组戊型肝炎病毒(HEV)结构区ORF2蛋白的抗原性进行鉴定。方法 以原核细胞表达的重组HEV ORF2抗原作为对照，应用间接ELISA方法鉴定重组HEV ORF2蛋白在HEV IgM和IgG抗体检测中的特异性和敏感性；并测定不同保存时间对抗原稳定性的影响。同时，比较了两种重组抗原与国外5株HEV ORF2单克隆抗体的反应特性。结果 应用酵母抗原可检测到HEV抗体的最低包被量为12．5ng／ml，可检测到HEV IgM、IgG抗体的血清最大稀释度皆为l：51200重组蛋白与其他类型肝炎患者血清无交叉反应，37℃加速试验证实重组蛋白4℃保存12个月可保持良好的抗原性。各株单克隆抗体与酵母表达的HEV ORF2蛋白有更好的反应性，其中4B2、2E2与酵母表达的HEV ORF2蛋白的反应性比与原核表达抗原的反应性分别高出125倍和25倍。结论 应用巴氏毕赤酵母表达系统制备的重组HEV ORF2蛋白可能包含了更为广泛的构象依赖性抗原表位，具有良好抗原性、特异性和稳定性。提示其可作为开发戊型肝炎诊断试剂的一个独具优势的候选抗原。     

热休克蛋白90与戊型肝炎病毒重组衣壳蛋白P239相互作用的初步研究

目的 对前期研究中所筛选出的能够与戊型肝炎病毒(hepatitis E virus,HEV)重组衣壳蛋白颗粒P239存在特异性的相互作用的蛋白进行分析验证.方法 使用pull-down、MALDI-TOF-MS、免疫共沉淀技术及免疫荧光技术对与P239有潜在相互作用的蛋白进行鉴定和进一步分析.结果 MALDI-TOF-MS及免疫共沉淀技术,均表明该蛋白为热休克蛋白90(HSP90)并与P239相互作用.随后,对P239进入细胞的过程中胞内HSP90蛋白量及其分布的变化做了初步研究.发现虽然HSP90在蛋白总量上并没有发生明显的变化,但其定位却伴随着P239在胞内的迁移发生了由细胞膜向细胞核核周的转移.结论 HSP90可能参与了P239入胞后的转运并介导了HEV入胞后向效应细胞器的转运,为研究HEV的感染机制及对HEV的预防和控制提供了有益的线索.     

用杆状病毒系统表达戊型肝炎病毒全长结构基因的研究

目的：获取含有戊型肝炎病毒（HEV）全长结构基因的重组杆状病毒，表达戊型肝炎病毒结构蛋白。方法：将HEV全长结构基因（5147－7126nt）插入杆状病毒表达载体pAcUW51，与线性化杆状病毒DNA（Baculo Gold DNA）共转染Sf9昆虫细胞，挑病毒斑进一步感染Sf9细胞，用SDS－PAGE，Western Blot及免疫荧光检测戊型肝炎病毒结构蛋白的表达及活性。结果：SDS－PAGE分析表明HEV结构基因在杆状病毒系统中高效表达，有相对分子质量约为73000和63000两种形式；Western Blot及免疫荧光检测证明表达产物可与HEV阳性血清特异反应，说明具有HEV特异抗原性。结论：应用杆状病毒表达系统成功表达了HEV结构蛋白。     

戊型肝炎病毒与戊型肝炎

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

戊型肝炎病毒衣壳蛋白特异性人源单克隆抗体的筛选与鉴定

目的:建立从外周血快速筛选戊型肝炎病毒(Hepatitis E virus,HEV)衣壳蛋白特异性人源抗体的方法,从疫苗免疫者外周血中筛选出相应抗体并进行鉴定。方法:采用分选型流式细胞仪获得外周血中HEV 衣壳蛋白特异性的记忆B细胞,通过单细胞RT-PCR 的方法获得抗体序列,并进行重组表达,最后对获得的人源单克隆抗体进行初步性质鉴定。结果:成功筛选到识别HEV 衣壳蛋白的6 株人源单克隆抗体,6 株抗体均具有抗原结合活性,4 株抗体具有中和活性。结论:成功获得HEV 衣壳蛋白特异性人源单克隆抗体序列,并进行真核表达,对抗体的性质进行初步鉴定,为后期研究疫苗免疫的人体内的抗体演化打下基础。     

戊型肝炎病毒研究进展

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

戊型肝炎病毒嵌合重组蛋白的表达及免疫原性研究

我国是戊型肝炎主要流行区之一，人群感染率达17．2％，且呈上升趋势。由于缺乏有效的病毒体外长期培养系统，阻碍了戊型肝炎病毒(HEV)疫苗的研制和诊断试剂的开发。用基因工程手段表达HEV主要结构蛋白已成为研究的热点。本文报道用原核表达系统表达含HEV主要抗原表位的ORF2和ORF3的嵌合蛋白，并对其免疫原性进行研究。     

戊型肝炎病人和实验感染戊型肝炎病毒的猕猴血清抗——H…

应用人工合成的戊型肝炎病毒（ＨＥＶ）基因组开放读码框架２（ＯＲＦ２）和３（ＯＲＦ３）两段多肽，分别建立了酶联免疫试验（ＥＩＡ），检测８５份实验感染ＨＥＶ的猕猴和１４３份戊型肝炎（简称戊肝）病人血清，结果两组抗－ＨＥＶ ＯＲＦ２阳性率分别为７０．８９％和６８．５３％，均显著高于抗－ＨＥＶ ＯＲＦ３，且前者阳转时间较早，持续阳性时间也较长。虽然多数（９７．３％）抗－ＨＥＶ ＯＲＦ３阳性血清，抗－ＨＥＶ     

人、猪、禽戊型肝炎病毒血清学关系的研究

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

不同基因型戊型肝炎病毒重组抗原p166用于抗体检测的价值

目的探讨不同基因型和亚型戊型肝炎病毒（HEV）ORF2重组蛋白p166用于抗体检测的价值,为研发准确可靠的戊型肝炎诊断试剂提供新的途径.方法用等浓度的不同基因型和亚型HEV p166作为包被抗原,对血清标本进行酶联免疫吸附试验测定.用HEV多基因型通用性引物逆转录套式PCR（RT-nPCR）扩增标本中HEV RNA,并测序、分型.结果8种不同p166抗原对30份健康献血者血清无抗原性,对182份来自世界不同国家和地区的已知HEV抗体阳性血清和7份HEV实验感染动物血清标本均呈阳性反应,但所得血清抗体滴度的高低与所用抗原的基因型有明显关系.RT-nPCR检测的50份中国血清标本中,19份阳性,基因分型均为Ⅳ型,与Ⅳ型中国株p166抗原反应最好.而以同属于第Ⅲ基因型的猪HEV新西兰株和人HEV美国株重组p166检测血清标本,两者结果差异无统计学意义.以多基因型p166混合抗原建立的ELISA抗体检测法与两种市售试剂盒比较,前者敏感性高,特异性好.结论不同基因型和亚型的HEV重组蛋白p166对不同血清标本HEV抗体检测的敏感性高低不同,因此多基因型和亚型p166的混合抗原是HEV抗体检测的最佳抗原.     

戊型肝炎病毒Ⅰ、Ⅱ与Ⅲ、Ⅳ基因型B细胞抗原表位的异同

目的：制备戊型肝炎病毒(HEV)缅甸株和墨西哥株ORF2重组蛋白(p166Bur和p166Mex)的单克隆抗体(McAbs)，用于分析HEV不同基因型B细胞抗原表位的特点。方法：将免疫BALB/C小鼠脾细胞与SP2/0骨髓瘤细胞融合，获得分泌抗-p166Bur和抗-p166Mex McAbs的杂交瘤细胞株，然后采用ELISA和免疫印迹法测定McAbs与不同基因型HEV ORF2编码蛋白p166的免疫反应性。结果：获得4株杂交瘤细胞株，即分泌抗-p166Bur McAbs的2G2、2B1以及分泌抗-p166Mex McAbs的D8G10和E5E12，其中2B1分泌的McAb仅能与第Ⅰ、Ⅱ基因型编码的重组蛋白结合，而其余3株分泌的McAbs既能与Ⅰ、Ⅱ基因型HEV的p166重组蛋白发生反应，也能与Ⅲ、Ⅳ基因型的p166蛋白反应。结论：HEV第Ⅰ、Ⅱ基因型与Ⅲ、Ⅳ基因型ORF2编码蛋白既有共同又有不同的B细胞抗原表位。     

戊型肝炎病毒Ⅳ型的ORF3及ORF2蛋白的分片段表达、纯化及抗原性分析

目的：对戊型肝炎病毒（HEV）Ⅳ型ORF3的全长基因片段和4个覆盖全长ORF2的互相重叠的基因片段进行了表达，对表达产物进行了纯化及抗原性分析。方法：将O3、FB5、E4、F2－2和E5基因片段分别连接到融合性表达质粒pThioHis，用IPTG进行诱导表达，并用反向、分子筛、离子交换和亲和层析方法进行纯化，用纯化的蛋白分别制备检测抗HEV IgG的诊断试剂，用该试剂检测急性散发性的戊肝病人和非戊肝病人血清。结果：位于ORF2－N端的FB5蛋白在急性期的戊肝病人血清中具有最强的反应性；而且仍有部分被HEV I型或／和Ⅱ型的诊断试剂排除的急性非戊肝病人血清对HEV Ⅳ型的多肽吴阳性反应。结论：为早期诊断戊肝病毒的感染，其诊断试剂应含有HEVⅣ型的ORF2N－端的蛋白片段。     

Genotypic characterization of symptomatic hepatitis E virus (HEV) infections in Egypt

BackgroundHepatitis E virus (HEV) is a common cause of acute viral hepatitis (AVH) in many developing countries. In Egypt, HEV seroprevalence is among the highest in the world; however, only a very limited number of Egyptian HEV sequences are currently available.ObjectivesThe objectives were to determine the HEV genotype(s) currently circulating in Egypt.Study designAVH patients without serologic evidence of hepatitis A, B, and C viruses were evaluated for possible HEV infection using serologic assays for anti-HEV IgM and anti-HEV IgG and real-time PCR for HEV RNA. Stool suspensions from suspected cases were inoculated into rhesus macaques to confirm the presence of HEV. Sequence analysis was utilized to determine HEV genotype.ResultsOf 287 subjects with AVH enrolled, 58 had serologic evidence of acute HEV infection. Stool samples for two of these patients were repeatedly positive for HEV RNA by real-time PCR. Macaques experimentally inoculated with these human stools also developed viremia. Sequence analysis of open reading frame (ORF) 1 demonstrated that these isolates belonged to HEV genotype 1 and were 3.9–9.5% divergent from other genotype 1 isolates. ORF2 was 5.3–8.7% divergent from previously reported Egyptian isolates.ConclusionsThis study strongly suggests that genotype 1 HEV related to other North African isolates is circulating in acute symptomatic patients in Egypt. Further evaluation of genotypic variability is underway in this highly endemic cohort and is considered an important component of our increased understanding of HEV pathogenesis.     

An overview: Rabbit hepatitis E virus (HEV) and rabbit providing an animal model for HEV study

Hepatitis E virus (HEV) is a single‐stranded, positive‐sense RNA virus and the causative agent of hepatitis E. The virus belongs to genus Orthohepevirus in the family Hepeviridae, which contains 4 major genotypes closely relating to humans. Genotypes 1 and 2 only infect humans whereas genotypes 3 and 4 HEV are harbored in a wide range of animal species worldwide and are zoonotic to humans. Recently, a novel animal strain of HEV has been isolated in farmed rabbits in China, and subsequently more strains were discovered in the rabbit populations in at least 7 other countries. Due to high sequence similarity to genotype 3 HEV, rabbit HEV (rHEV) has been assigned to genotype 3. Experimental study showed that rHEV could infect non‐human primate and human, which pose a direct threat to human. Further pathogenesis studies showed laboratory rabbits infected with rHEV and genotype 4 HEV could present similar signs of acute and chronic hepatitis E along with extra‐hepatic replication as observed in humans. High mortality and vertical transmission were reproduced in rHEV infected pregnant rabbits. Furthermore, rabbit model was also found suitable for evaluating HEV vaccine efficacy in order to manage zoonotic transmission. These data showed laboratory rabbits could serve as an alternative animal model for HEV study under the current circumstances that HEV propagation is limited in vitro. In general, this review aims at presenting comprehensive up‐to‐date information about rHEV strains and rabbit model for HEV studies.     

IgM and IgG antibodies to hepatitis E virus (HEV) detected by an enzyme immunoassay based on an HEV-specific artificial recombinant mosaic protein

To develop an enzyme immunoassay (EIA) for IgM antibody to hepatitis E virus (HEV) (IgM anti-HEV) and IgG antibody to HEV (IgG anti-HEV), a synthetic gene encoding several liner immuno-dominant antigenic epitopes from HEV structural proteins was assembled as a chimeric recombinant mosaic protein (Mpr) with glutathione S-transferase and used as an immunodiagnostic target. In addition, a neutralization confirmation test was developed using individual synthetic peptides. Among 614 patients with acute hepatitis from 10 geographically distinct outbreaks, IgG anti-HEV was found in 546 (88.9%), with a range of 77–100% depending on the outbreak. Of 130 patients tested for IgM anti-HEV, 126 (96.9%) were positive. Among patients tested within 4 months of onset of jaundice, 37/37 (100%) were IgG anti-HEV positive. For patients from whom sera were collected 1–16 days after onset of jaundice, the geometric mean IgG titer (GMT) was 1:47,000; the GMT increased to 1:70,710 30–40 days after onset of jaundice and decreased to 1:1,778 3–4 months after the onset of jaundice. For patients tested 6–8 months after onset of jaundice, 11/12 (92%) were IgG anti-HEV positive, and the GMT was 1:2,908. IgM anti-HEV was detected in 43/43 (100%) sera collected 1–40 days after onset of jaundice, and the GMT for IgM anti-HEV was 1:10,000 at that time. For sera collected 3–4 and 6–12 months after onset of jaundice, 7/14 (50%) and 5/12 (40%) respectively, were IgM anti-HEV positive. In conclusion, an artificial mosaic protein composed of linear antigenic epitopes from open reading frame 2 (ORF2) and ORF3 of HEV has been successfully applied to the development of a sensitive and specific EIA for the detection of IgG and IgM anti-HEV activity. These assays were used for the verification of HEV infection in outbreak settings and for the diagnosis of HEV infection in sporadic cases. © 1996 Wiley-Liss, Inc.     

The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus for replication and movement in host plants. The positive-sense single-stranded genomic RNA of SPMV encodes a 17-kDa capsid protein (CP) to form 16-nm virions. We determined that SPMV CP accumulates in both cytosolic and non-cytosolic fractions, but cytosolic accumulation of SPMV CP is exclusively associated with virions. An N-terminal arginine-rich motif (N-ARM) on SPMV CP is used to bind its cognate RNA and to form virus particles. Intriguingly, virion formation is dispensable for successful systemic SPMV RNA accumulation, yet this process still depends on an intact N-ARM. In addition, a C-terminal domain on the SPMV CP is necessary for self-interaction. Biochemical fractionation and fluorescent microscopy of green fluorescent protein-tagged SPMV CP demonstrated that the non-cytosolic SPMV CP is associated with the cell wall, the nucleus and other membranous organelles. To our knowledge, this is the first report that a satellite virus CP not only accumulates exclusively as virions in the cytosol but also is directed to the nucleolus and membranes. That SPMV CP is found both in the nucleus and the cell wall suggests its involvement in viral nuclear import and cell-to-cell transport.     

Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies

Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs.