猪源戊型肝炎病毒上海株全基因组序列分析

目的分析猪源戊型肝炎病毒(HEV)上海株swChinash与已知其它HEV基因型的差异和人源HEV的关系。方法设计22对PCR引物,分片段进行扩增,两末端采用快速扩增方法(RACE)扩增,扩增产物克隆到pMD18-T载体并测序,用DNASTAR软件与53株人源和猪源HEV的基因组进行同源性分析,用Clustalx和Mega3.0软件绘制基因进化树。结果swChinash全序列除去poly(A)尾,由7235个核苷酸组成,ORF1～3分别编码1707,660,122个氨基酸。全基因序列与Ⅳ型的核苷酸同源性为83.4%～84.7%,与其他三型为73.3%～76.3%,其中与中国人源长春株最高,为84.7%。结论基于ORF1～3和全长基因组的进化树,猪源HEV上海株swChinash属于基因Ⅳ型,与其他Ⅳ型HEV亲缘关系较远,单独在一个进化分支上。     

云南某农村猪戊型肝炎病毒分子流行病学调查

目的 了解云南农村散养猪群HEV感染情况,为HEV防控提供理论依据。方法 利用反转录套式聚合酶链反应（RT-nested PCR）技术,对所采集云南猪群78份粪便样品进行HEV ORF2基因部分片段扩增,经琼脂糖凝胶电泳后对目的条带进行回收纯化及克隆测序。序列利用DNAStar和MEGA4.0软件进行同源性比较和系统进化树构建。结果 RT-nested PCR方法扩增出了350bp目的基因序列,该序列与GenBank中HEV各型的同源性在77.4~97.4%之间,与基因4型同源性最高（97.4%）,与3型同源性最低（77.4%）。系统进化树显示测定的序列与基因4型聚为一支,表明该序列属于基因4型。本次实验共检测了78份猪粪便样品,其中8份为阳性,阳性率为10.26%。结论 云南农村人群存在感染HEV的风险,应该加以防控以免HEV暴发流行。     

与人类关系密切的7种动物对戊型肝炎病毒易感性的初步研究

目的　用双抗原夹心法ELISA (DS -ELISA)和逆转录巢式PCR法 (RT -nPCR)研究与人类生活关系密切的猪、犬、山羊、鸡、鸭、鸽子和兔等 7种动物对戊型肝炎病毒 (HEV)的易感性。方法　用多基因型HEV重组蛋白建立检测HEV抗体的DS -ELISA ,并用于 5 91份猪、警犬、家犬、宠物犬、山羊、鸡、鸭、鸽子和兔等多种动物血清标本的HEV抗体检测 ;用HEV多基因型通用性引物RT -nPCR扩增 14 3份犬血清标本HEVRNA。结果　在猪、家犬和宠物犬血清标本中检测到HEV抗体 ,阳性率分别为 4 3 93%、15 19%和 4 6 5 % ,但在警犬、山羊、鸡、鸭、鸽子和兔血清标本中未检测到HEV抗体 ;14 3份犬血清标本用RT -nPCR未扩增出HEVRNA。结论　用多基因型HEV重组蛋白建立的双抗原夹心法ELISA适用于多种动物血清标本HEV抗体的检测 ;猪和犬对HEV易感 ,在HEV传播中可能起重要作用。     

深圳地区散发性戊型肝炎临床特点及戊型肝炎病毒部分核苷酸序列分析

深圳地区散发性戊型肝炎临床特点及戊型肝炎病毒部分核苷酸序列分析许诚马为民朱晓洁庄辉为探讨散发性戊型肝炎的临床、血清病原学及戊型肝炎病毒（ＨＥＶ）核苷酸序列的特点，对１９９４年１月至１９９５年５月我院收治的散发性戊型肝炎１８２例进行了分析，报告如下。一...     

全长乙型肝炎病毒基因组的扩增及序列分析

目的 建立一种全长乙型肝炎病毒(HBV)基因组扩增及序列分析的新方法。方法 在HBV负链开环缺口处设计引物，使用TaKaRa LA Taq^TM DNA聚合酶进行扩增，聚合酶链反应产物克隆后进行全长序列测定。结果 用此方法成功获得HBV全基因组DNA序列。将已知序列的HBV全基因组重组质粒作为模板进行敏感性和保真性测定，其敏感性为10^2个初始模板，核苷酸的人为突变率为1．2bp／kb。结论 此方法可用于大规模HBV全基因组扩增和序列分析，为HBV的基础和临床研究提供了一种新方法。     

戊型肝炎合并甲乙丙丁肝炎病毒感染病例的分析

本文对３５４例各型肝炎病人检测了甲，乙，丙，丁，戊各型肝炎病毒的标志物，发现戊型肝炎（抗－ＨＥＢ阳性）６６例，占１８．６４％，戊型肝炎临床主要表现为急性黄疸型（６３．６４％）另还有急性无黄疸型，胆汁瘀积型及重症型，戊型肝为具有典型肝炎症状者多见（９３．９４％），黄疸多见（８３．３３％），ＡＬＴ均有升高，ＨＥＶ可与ＨＡＶ，ＨＢＶ，ＨＣＶ混合感染，总的混合感染率为３４．８５％，以乙＋戊为最多（１６．６     

实时荧光PCR定量检测戊型肝炎病毒方法的建立

目的建立灵敏、稳定、特异的实时荧光PCR方法,用于戊型肝炎病毒的定量检测。方法采用生物信息学方法,分析比对戊型肝炎病毒全序列,并针对国内流行的戊型肝炎病毒株序列,选择位于ORF2的保守区域设计引物和Taqman探针,建立实时荧光定量PCR方法。并从灵敏度、稳定性及特异性等方面对建立的方法进行综合评价。结果戊型肝炎定量检测的灵敏度为5个目的拷贝,定量的线性范围为5×(100~108)9个数量级。批内重复性检测显示其变异系数为1.49%;批间重复性检测显示其变异系数为1.98%和2.23%。对22份临床标本的检测结果显示具有良好的特异性。结论建立的实时荧光PCR方法具有灵敏度高、稳定性好、特异性强等特点,适用于戊型肝炎病毒的定量检测。     

云南省大理市啮齿动物携带冠状病毒和戊型肝炎病毒的调查

目的 调查云南省大理市啮齿动物冠状病毒(Coronavirus, CoV)和戊型肝炎病毒(Hepatitis E Virus, HEV)感染率。方法 2020年8月～2021年8月在大理市4个乡镇采集啮齿动物样本,采用巢式PCR扩增CoV和HEV的保守序列基因,用生物信息学软件进行同源性与遗传进化分析。结果 共捕获76只啮齿动物属于5属6种,CoV感染动物为褐家鼠(Rattus norvegicus)和黄胸鼠(Rattus tanezumi),感染率分别为40.74%(11/27)、2.38%(1/42);HEV感染动物为褐家鼠和黄胸鼠,感染率分别为14.81%(4/27)、2.38%(1/42)。在银桥镇捕获的2只褐家鼠中同时检测到CoV和HEV,感染率为7.41%(2/27)。基于CoV的部分RNA依赖的RNA酶(RNA-dependent RNA polymerase, RdRp)的434 bp核苷酸片段分析,11株CoV阳性样本均来自褐家鼠,为α-CoV,与来自中国福建黄毛鼠(Rattus losea)RtRl/FJ2015同源性最高,为99.73%～99.74%;1株CoV阳...     

家畜戊型肝炎病毒诊断方法的建立及应用

目的建立检测家畜戊型肝炎病毒（HEV）反转录套式聚合酶链式反应（RT-nPCR）方法,并应用于广西地区家畜戊型肝炎感染调查。方法参考GenBank上4种基因型HEV核苷酸序列,应用生物软件Oligo6.0在ORF2保守区设计两对简并引物,扩增目的片段为436bp,进行了特异性、敏感性和重复性试验,并对采自广西地区猪、牛和羊的肝粪样本进行HEV检测。结果检测家畜粪便样本阳性率31.49%（285/905）;家畜肝脏样本阳性率18.01%（49/272）。结论所建立的RT-nPCR方法适用于家畜肝粪样本HEV检测,临床检测应用表明广西家畜存在HEV感染。     

Molecular detection and sequence analysis of a new hepatitis E virus isolate from Pakistan

Sporadic and epidemic acute viral hepatitis E in many developing countries is caused by hepatitis E virus (HEV). The HEV genome has been classified into three major genotypes. However, extensive diversity has been noted among HEV isolates from patients with acute hepatitis in China and Taiwan. Some reports indicated that multiple genotypes of HEV could cocirculate in the same area; even distinct genotypes of HEV could exist in the same patient. Pakistan is a highly endemic area for hepatitis E. So far only two Pakistan HEV isolates Sar-55 (87-Pakistan-A) and Abb-2B (88-Pakistan-2B) have been characterized, and the nucleotide sequences of these two HEV isolates show only 90% homology. In this study, a third HEV isolate from Pakistan (87-Pakistan-B) is reported. The sequences of a 438-bp fragment from ORF-2 and a 259-bp fragment from the ORF-1-3 region of this new HEV isolate were obtained and sequenced. The sequence analysis showed that this new HEV isolate was very closely related to the Sar-55 but different from the Abb-2B HEV isolate. These results indicated that the Sar-55 (87-Pakistan-A) genotype is the main endemic HEV strain in the Sargodha area. These data will be useful for HEV epidemiological studies, diagnosis and vaccine development.     

Detection of hepatitis E virus in genome and gene products in two patients with fulminant hepatitis E

Non-isotopic in situ hybridization (digoxigenin-labeled probe directed towards hepatitis E virus ORF1) and immunohistochemistry (against hepatitis E virus ORF2 and ORF3) were applied to detect hepatitis E virus genome and gene product in the liver tissue of two patients with fulminant hepatitis E seropositive for hepatitis E virus RNA. Both hepatitis E virus RNA and hepatitis E virus antigens were detected exclusively in the cytoplasm of hepatocytes and not detected in other cell types. In both patients, more than 50% of the hepatocytes were positive for both hepatitis E virus RNA and hepatitis E virus antigens, most of which showed degenerative changes. This is consistent with the histological appearance of marked loss of hepatocytes with acinar collapse. Interestingly, denaturation of the RNA before in situ hybridization was found to enhance hepatitis E virus RNA detection. We conclude that: (1) hepatitis E virus RNA and hepatitis E virus antigens can be demonstrated in the liver in hepatitis E virus-related fulminant hepatitic failure, (2) hepatitis E virus is hepatocyte-tropic within the liver, (3) cytoplasmic localization of hepatitis E virus RNA and hepatitis E virus antigens is consistent with cytoplasmic replication, and (4) the presence of degenerative changes in hepatitis E virus positive cells, together with the histological appearance of hepatocyte loss in the absence of significant inflammatory infiltrate, suggests that hepatitis E virus-related fulminant failure is mediated by a cytopathic mechanism.     

Structural and functional analysis of full-length hepatitis B virus genomes in patients: implications in pathogenesis

The structural analysis, replicative efficiency and immunogenicity of hepatitis B virus (HBV) full-length genomes isolated from different patients or asymptomatic carriers are presented in the present review. Data indicate the importance of viral genome-based studies in elucidating the pathogenesis of HBV infections. Comparison of structural and functional characteristics of viral genomes isolated from various geographical regions might contribute to explaining the differences in HBV clinical manifestation and prognosis in different geographical regions.