Human DNA contains sequences homologous to the 5’-non-coding region of hepatitis C virus: characterization with restriction endonucleases reveals individual varieties

Objective To investigate a 272 base pair section of the 5‘-non-coding region of genomic DNA from the peripheral blood monounuclear cells of healthy hepatitis virus C (HCV)-negative human subjects( not patients).Mothods This sequence section bears interest because ① it harbors several potential methylation(Cp-rich) sites, and ② it represents the largest part of its internal ribosomal entry site. A pre-PCR digestion protocol was established making consistent use of four restriction endonucleases selected for certain features: Smal, XmaCI, Mspl, and Hpall are inhibited if methylation(s) are present at certain cytosines within their cutting sequences.Results The suspected HCV-specific sequence was found in the DNA of each subject tested. The pre-PCR digestion assay reveals individual differences in their pattern of methylation, which may be due to possible epigenetic phenomena.Conclusions The results provide formal proof that these HCV-specific sequences are contained in the genomic or extra chromosomal target DNA, and probably belong to a new class of endogenous sequences.     

Lyophilized standards for the calibration of real time PCR assay for hepatitis C virus RNA

Background Since October 1997, an international standard for hepatitis C virus （HCV） nucleic acid amplification technology assay, 96/790, has been available. We compared a series of lyophilized standards with known HCV RNA concentrations against the international standard in fluorescence quantitative PCR detection. Methods A series of lyophilized sera were calibrated by ROCHE COBAS AMPLICOR HCV Monitor test against the international standard and sent to various manufacturers to analyse the samples using their own kits. Then calibration curves from the series were compared with that obtained from the external standard calibration curve with the manufacture＇s series. Results The standard calibration curve with the series of lyophilized serum showed an excellent correlation （R^2〉0.98）, slope and intercept that were similar to those from the manufacture＇s series. When the standard calibration curve from the series of lyophilized standards were used to define the values of the given sample, lower coefficients of variation between kits from different manufactures were obtained. Conclusion The results showed that the lyophilized standards could be used to setup the standard calibration curve for clinical HCV RNA quantitative PCR detection.     

Detection of hepatitis G virus RNA in hepatitis C patients and hepatitis C virus infected hemodialysis patients

AlthoughsensitiveandspecificimmunoassayandmolecularbiologicaltechniquesforthedetectionofthehepatitisA--Evirusesareavailable,theetiologyofasubstantialfractionofpost--transfusionandcommunity--acquiredhepatitiscasesremainsundefined[1],suggestingtheexistenceofadditionalcausativeagents.Anewhumanhepatitisviruswasisolatedbytwoindependentgroups.ThenewvirusisprovisionallydesignatedashepatitisGvirus(HGV)[ZJorGBvirusC(GBV~C)[3'4),whichwasthoughttobetheagentofpartofthenon--A--Ehepatitispatients.Fro…     

Design and activity evaluation of deoxyribozymes specifically targeting hepatitis C virus RNA

Objective: To explore the cleaving and inhibitory activity of hepatitis C virus ( HCV)-specific deoxyribozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure of HCV 5′-noncoding region (5′-NCR) and the sites characterized with 5′…Y ↓ R…3′ ( Y = A/G, R = U/C), HCV-specific naive deoxyribozymes were designed and named DRz-232, DRz-127, DRz-84, DRzl, and the phosphorothioate deoxyribozymes (PSDRz) and mutated phosphorothioate deoxyribozymes (MPSDRz) were also designed. HCV RNA 5′-NCR was transcribed in vitro from linearized plasmid pHCV-neo and radiolabelled at its 5′-end. DRz, PSDRz or MPSDRz was respectively mixed with the substrate RNA and incubated under appropriate conditions, the cleaved products were displayed by 8% denaturated polyacrylamide gel electrophoresis (PAGE) and autoradiography, and the optical density of each band was measured to calculate cleavage rates. After that, every kind of DRz was added respectively to the cultured transgeneic HepG2 cells containing luciferase gene controlled by HCV 5′-NCR. The ceils were lysed at intended time points and the activity of luciferase was measured with chemiluminescence method for calculating inhibition rates. Results: After incubated for 90 rain in vitro, the cleavage rates of DRz-127, PSDRz-127, DRzl and PSDRzl reached 32.6%, 30.8%, 24.3% and 21.5%, respectively. No cleavage product was observed in any MPSDRz. DRz-127, PSDRz-127, DRzl and PSDRzl had an inhibitory rate of 53.2%, 50.6%, 44.7% and 43.3% respectively in transgeneic HepG2 cells in the first 24 h when the final dose of the DRz was 0.5 la.moL/L, higher than that of the corresponding MPSDRz. There was no significant difference between the inhibitory effect of each DRz and its PSDRz in HepG2 cells, but the inhibitory rate of DRz decreased more rapidly than that of the latter with the elapse of time. The results from transfection groups were significantly better than those of non-transfection groups. Conclusion:Rationally-designed HCV-specific deoxyribozymes are able to cleave target RNA at molecular level in vitro, and efficiently inhibit the expression of luciferase gene controlled by HCV 5′-NCR in transgeneic cells. Appropriate PSDRz may be more stable, and thus more suitable than the naive DRz in the application to cells. Introduction of the deoxyribozymes with transfection is more efficient than with direct delivering ways.     

形成丙型肝炎病毒负链复制体的相关蛋白的分析

【目的】确定丙型肝炎病毒 (HCV)非结构蛋白NS5B以及肝母细胞瘤株HepG2胞浆提取物中的宿主蛋白与HCV负链RNA 3′末端的特异性结合作用。【方法】用蛋白 核酸紫外交联试验分别检测NS5B以及HepG2胞浆提取物中的宿主蛋白与HCV负链RNA 3′末端的结合作用 ,用非同源RNA和非同源蛋白作为竞争物分析这种结合的特异性。【结果】NS5B以及宿主细胞内一约 4 5ku的蛋白质 (简称P4 5 )均可与HCV负链RNA 3′末端特异性结合。【结论】NS5B和P4 5是HCV负链RNA 3′末端复制体的两个成分     

庚型和丙型肝炎病毒的全长cDNA克隆在体内外的表达与复制

目的：研究HGV和HCV的复制和表达。方法：利用HGV全长cDNA克隆(HGVqz)及HCV1a／1b嵌合体cDNA克隆分别构建表达质粒p3．1HGV和p3.1HCV并转染张氏肝细胞,以HGVqz克隆建立HGV转基因小鼠。分别应用RT—PCR、免疫组化及Western印迹分析病毒正、负链RNA,蛋白表达和剪切。结果：在转染p3.1HCV的张氏肝细胞及HGV转基因小鼠某些组织中可检出相应病毒的负链RNA,但在转染p3．1HGV的张氏肝细胞中未能检出HGV负链RNA。Western印迹在转染p3.1HCV的张氏肝细胞中检测到针对HCV NS3蛋白、相对分子质量约70000的特异性条带,在HGV转基因小鼠某些组织中检测到2条特异性条带,相对分子质量约42000和100000,分别相当于HGV E2蛋白及其剪切中间体。然而,在转染p3．1HGV的张氏肝细胞中检测到针对HGV E2蛋白的特异性条带,其相对分子质量约为310000,相当于HGV整个前体蛋白。结论：HGV的表达与复制在体内、外存在差异。细胞内某些特异性因子在病毒前体蛋白剪切中起重要作用,HGV和HCV前体蛋白剪切所需宿主因子可能存在差异,故两种病毒体外培养时的嗜性细胞株并不完全一致。     

丙型肝炎患者血清中丙型肝炎病毒交叉中和抗体的检测

[摘要]目的分析HCV感染者血清中是否存在交叉反应性中和抗体。 方法以分泌表达HCV包膜E2蛋白真核表达质粒转染的293T 细胞培养上清液中的HCV E2蛋白作为检测抗原,建立检测HCV E2抗体的ELISA方法,检测32份HCV抗体阳性的慢性丙肝患者血清,然后用免疫荧光分析血清与HCV全长包膜蛋白表达质粒转染的293T细胞的结合反应,再用5株HCV假病毒(HCVpp)及两株细胞培养产生的HCV(HCVcc)为模型分析血清的病毒中和活性。 结果32份HCV抗体阳性血清中,26份血清可检测出E2抗体,阳性率81.3%。其中HCV RNA阳性的12份血清均为E2抗体阳性,E2抗体水平与HCV RNA水平负相关。HCV E2抗体阳性血清对5株HCVpp以及两株HCVcc的感染性均有不同程度的中和作用,中和活性与E2抗体水平相平行。结论HCV感染可诱导保护性体液免疫应答,丙肝患者血清中存在交叉中和抗体,提示开发能诱导广泛交叉中和抗体的丙肝疫苗具有可行性。     

Detection of hepatitis C virus RNA sequences in cholangiocarcinomas in Chinese and American patients

ＨｅｐａｔｉｔｉｓＣｖｉｒｕｓ(ＨＣＶ)ｉｎｆｅｃｔｉｏｎｉｓｒｅｃｏｇｎｉｚｅｄａｓａｍａｊｏｒｐａｔｈｏｇｅｎｗｏｒｌｄｗｉｄｅａｎｄａｆｒｅｑｕｅｎｔｅｔｉｏｌｏｇｉｃｆａｃｔｏｒｉｎｔｈｅｄｅｖｅｌｏｐｍｅｎｔａｎｄｐｒｏｇｒｅｓｓｉｏｎｏｆｃｈｒｏｎｉｃｈｅｐａｔｉｔｉｓ,ｃｉｒｒｈｏｓｉｓａｎｄｈｅｐａｔｏｃｅｌｌｕｌａｒｃａｒｃｉｎｏｍａ(ＨＣＣ)1　ＴｈｅｐｒｅｓｅｎｃｅｏｆＨＣＶＲＮＡｉｎｂｉｌｅｄｕｃｔｅｐｉｔｈｅｌｉａｌｃｅｌｌｓｉｎｃｈｒｏｎｉｃｈｅｐａｔｉｔｉｓＣｐａｔｉｅｎ…     

慢性丙型肝炎患者混合感染庚型肝炎病毒的临床研究

目的 研究庚型肝炎病毒（HGV）混合感染对慢性丙型肝炎（ＣＨＣ）临床、病毒复制及干扰素抗丙型肝炎病毒（ＨＣＶ）疗效的影响及ＨＧＶ对干扰素治疗的反应。方法 以逆转录套式降合酶链反应（ＲＴ－nested PCR）检测９０例ＣＨＣ患者血清ＨＧＶ ＲＮＡ，比较ＨＧＶ ＲＮＡ阳性及阴性两组９０例ＣＨＣ患者的临床资料和对干扰素治疗的反应。结果 ＨＧＶ ＲＮＡ阳性１８例，阳性率２０％，ＨＧＶ ＲＮＡ阳性及阴性两组ＣＨＣ患者在性别、年龄、病程、丙氨酸氨基转移酶（ＡＬＴ）水平、ＨＣＶ ＲＮＡ水平和对干扰素治疗的反应等方面无明显差异。５例ＨＧＶ ＲＮＡ阳性ＣＨＣ患者接受干扰素治疗过程中，ＨＧＶ ＲＮＡ均转为阴性，停止治疗后１年，３例ＨＧＶ ＲＮＡ恢复治疗前水平。结论 混合感染ＨＧＶ对ＣＨＣ的临床、病毒复制及干扰素抗ＨＣＶ疗效无明显影响。ＨＧＶ对干扰素敏感，但停药后易复发。     

乙型肝炎病毒X基因-丙型肝炎病毒C基因融合表达蛋白对肝癌细胞端粒酶活性的影响

目的建立HBV X-HCV C融合蛋白细胞表达模型,并探讨其对细胞端粒酶活性的影响.方法双酶切质粒pXT1-X,得到完整的HBV X基因片段后,将其插入到质粒PBK-CMV和PBK-HCV C的相应酶切位点,得到重组质粒PBK-X和PBK-X-C;再将质粒PBK-CMV、PBK-X、PBK-HCV C和PBK-X-C分别导入肝癌细胞株HepG2中,G418筛选,RT-PCR、蛋白印迹鉴定HBV X和HCV C蛋白表达.PCR-ELISA法检测端粒酶活性.结果质粒PBK-CMV、PBK-X、PBK-HCV C和PBK-X-C在HepG2细胞中有稳定表达.表达融合蛋白的细胞的端粒酶活性较转染空载体的细胞及单独表达HBV X、HCV C蛋白的细胞明显升高.结论HBV X-HCV C融合蛋白能显著上调端粒酶活性,提示HBV、HCV可能具有协同致癌作用.     

乙型肝炎病毒X基因-丙型肝炎病毒C基因融合表达蛋白对肝癌细胞胰岛素样生长因子1受体表达的影响

目的 建立HBV X-HCV C融合蛋白细胞表达模型,并探讨其对细胞胰岛素样生长因子-1的影响.方法 双酶切质粒pXT1 -X,得到完整的HBV X基因片段后,将其插入到质粒PBK-CMV和PBK-HCV C的相应酶切位点,得到重组质粒PBK-X和PBK-X-C;再将质粒PBK-CMV、PBK-X、PBK-HCV C和PBK-X-C分别导入肝癌细胞株HepG2中,G418筛选,RT-PCR、蛋白印迹鉴定HBV X和HCV C蛋白表达.流式细胞仪、蛋白印迹检测IGF-1受体蛋白表达.结果 质粒PBK-CMV、PBK-X、PBK-HCV C和PBK-X-C在HepG2细胞中有稳定表达.表达融合蛋白的细胞的胰岛素样生长因子-1受体活性较转染空载体的细胞及单独表达HBV X、HCV C蛋白的细胞明显升高.结论 HBV X-HCV C融合蛋白能显著上调胰岛素样生长因子-1受体活性,提示HBV、HCV可能具有协同致癌作用.     

微孔板固相杂交法检测丙型肝炎病毒

目的 寻找一种快速、准确、简便的丙型肝炎病毒（hepatitis C virus，HCV）RNA逆转录-聚合酶链反应产物的检测方法。方法 所有标本均作逆转录-套式聚合酶链反应，其产物分别以微孔板固相杂交法及聚丙烯酰胺凝胶电泳法检测，对结果可凝及阴性者均以HCV Amplification KIT对其血清再作扩增并检测，并以此为金标准进行比较。结果 144份血清（其中抗-HCV阳性64例），微孔板固相杂交法阳性70例，灵敏度97.18%，特异度98.63%，聚丙烯酰胺凝胶电泳法阳性61例，灵敏度81.69%，特异度95.89%，取同一份产物倍比长稀释后再作检测，前者可检测出2^-9稀释度，后者检测出2^-7稀释度。结论 微孔板固相杂交法检测丙型肝炎病毒快速、准确、简便，并且可定量，尤其适用于大批量标本的检测，但于基层推广。