庚型肝炎病毒5‘端非编码区cDNA的序列分析

为了解国CBV-C/HCV株的基因序列与国外分离株的同源性状况,对我国GBV-C/HCV的5’端非编码区（5’NCR）。cDNA进行了序列测定。参考国外发表的序列资料,在相对保守的5’NCR设计两对HGV特异性引物。采用热变性法提取云南省一个静脉吸毒者血浆中的GBV－C／HGVRNA逆转录为cDNA后进行巢式聚合酶链反应（PCR）扩增,获得238bp的片段。PCR产物纯化后直接经双脱氧链末端终止法测定核苷酸序列。与国外分离株比较,同源性为86.36％～90.9t％,微分区域变异较大。结果表明,所扩增片段属于GBV－C／HGV基因,所测序列可为引物设计据供依据。对核酸变异性分析有一定意义。     

抗—HCV阴性献血员中丙型肝炎病毒RNA检测及序列分析

对95份抗—HCVIgG阴性献血员采用逆转录聚合酶链反应法（PCR）检测丙型肝炎病毒RNA,结果8次中有6次其检测出17份阳性标本（17／95,17．9％）,复查抗—HCVIgG仍为阴性。对其中8份阳性产物中高变区1的序列分析结果表明均为不同株HCV序列．排除了PCR污染的可能性。对其中2份阳性产物测定了全序列并与HCV各基因型代表株的相应序列比较,与HCVⅡ型相应序列的核苷酸同源性为77％～79％。而与HCVⅠ、Ⅲ、Ⅳ相应序列间的同源性为62％～69％,表明为HCVⅡ型序列。结果提示献血员抗—HCVIgG筛选不能完全排除HCV感染者,漏检不是由于HCV基因序列变异．而是检测方法本身缺陷所致。     

两个猪瘟病毒野毒株gp55基因抗原编码区序列的分析及比较

利用反转录－PCR方法扩增了吉林省猪瘟病毒（HCV）两个野毒株gp55基因的主要保护性抗原编码区,并将其克隆到pGEM－T载体中,然后用Sanger双脱氧法测定了其核苷酸序列,并推导了其氨基酸序列。将测定的这两个HCV野毒株的部分序列（350bp）与国内外已知的HCV序列进行比较,结果表明：这两个野毒株的核苷酸序列的同源性为94．9％,氨基酸序列同源性为97．4％,与1985～1992年意大利中部分离4个野毒株的同源性明显高于其它HCV毒株,核苷酸同源性分别为97．2％～98．3％和94.0％～949％,氨基酸同源性分别为98．3％～991％和97.4％～98.3％,而与我国的HCV标准强毒株即石门株的核苷酸同源性仅分别为83.1％和83.1％,氨基酸同源性仅分别为90.6％和91.4％。因此认为吉林省这两个野毒株与意大利中部的4个野毒株具有密切的关系,而与石门株很可能来源不同。     

猪Fas基因的克隆及序列分析

广西大学动物科技学院李军、李晓宁、杨坚和罗建荣四位科研工作者从pk-15细胞中提取总RNA,用RT—PCR方法扩增出猪Fas基因,将其克隆到PMD18-T载体上,再进行序列分析,结果表明：克隆的猪Fas基因序列与genBank上登录的猪Fas基因同源性为100％,与人、牛、羊的Fas基因核苷酸及推导的氨基酸序列同源性分别为73．4％、79．2％、76．4％和56．2％、67．0％、64．6％。Fas蛋白胞内区的死亡域,其氨基酸序列在猪、牛、羊与人的基因中呈现较高的同源性。[第一段]     

我国西部1a型丙肝病毒结构区的cDNA克隆和序列分析

克隆我国西部丙型肝炎病毒全部结构区基因,为探讨HCV的变异和致癌作用,研制丙肝疫苗作准各。从1名西安市丙肝感染血液中,用Trizol试剂裂解HCV病毒颗粒,用糖原与RNA共沉淀提取HCVRNA,用AMV逆转录酶和随机引物逆转录为eDNA,用RT-PCR方法扩增目的片段并转化到pGEM-T质粒,得到pGEM-T-HCVc、pGEM-T-HCVel和pGEM-T-HCVe2克隆。将以上3个克隆用特定的酶降解,用连结酶进行连接,构建了HCV结构区的完整克隆。将克隆好的质粒pGEM-T-HCVjg从两端双向测广手,得到完整的HCV结构基因eDNA核苷酸序列,总长度为2238bp,与已发表的HCV序列长度一致,未发现缺失或移码突变,序列中间亦未发现转录终止子。本序列与HCVla、lb序列核苷酸的同源性分别为91．8％、84．5％;氨基酸的同源性分别为94.2％、86．3％;应为HCVla亚型。以上结果说明我国西部地区存在HCVla亚型,所克隆HCV结构区eDNA克隆可以用于基因工程表达。     

丙型肝炎病毒C区基因的克隆与分析

通过RT－PCR从1份来自甘肃武威地区献血员HCV阳性血清顺克隆到573bp的HCV核心基因全片段,用T－A克隆载体法将该片段接入克隆载体pUC19中并测序,结果表明武威地区分离株与Ⅰ型株HCV－Ⅰ和Ⅱ型株HCV－HeBei在该基因区段的核苷酸／氨基酸序列同源性分别为89.6%／95.4%、97.3%／97.4%,属于Ⅱ型株。     

兔出血症病毒中国株衣壳蛋白基因的克隆和序列分析

应用RT—PCR技术,从兔出血症病毒中国分离株WX84中成功扩增出预期大小为1．7kb的特异性条带,将扩增产物提纯后克隆入pGEM^R—T载体,经转化、筛选及酶切鉴定后,获得了该株病毒衣壳蛋白基因的克隆,序列分析表明扩增的中国株BHD衣壳蛋白基因片段长度为1740bp,共编码580个氨基酸。该核酸序列与其它国家报道的多株BHDV序列相互间同源性高达98．2％一99．0％,其推导的氨基酸序列同源性也达98．3％--99．1％,为极度保守片段。     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT—PCR和PCR—RACE技术,从菊科植物甘菊（Dendranthema lavandulifolium）中克隆到2个甜菜碱醛脱氢酶（betaine aldehyde dehydrogenase,BADH）基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152。DlBADH1的cDNA全长1821bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918bp,编码506个氨基酸的蛋白质。两个基因核苷酸序列的同源性为97％,推导的氨基酸序列的同源性为98％。与已发表的其它植物BADH基因氨基酸序列的同源性在64％以上。在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽（VTLELGGKSP）以及与酶功能有关的半胱氨酸残基（C）。在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合。RT—PCR—Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员。     

丙型肝炎病毒NS4区基因的克隆,序列分析及其表达

用RT－PCR法从一名抗-HCV阳性献血员血清中扩增到约900bp的DNA片段,经EcoRI酶切后插入pET17Xb表达质粒。SDS－PAGE表明重组质粒在约65kD处有融合蛋白表达,表达蛋白含量约为24％。对其插入片段进行序列分析：核着酸长度为945bp,A：T：G：C比例为18．6：21.9:30.5：29.0,G/C含量占59％,与其它一些HCV殊比较,核着酸同源性Ⅰ型为77．1％．Ⅲ、Ⅳ、Ⅴ、Ⅵ型均＜70％．Ⅱ型各株均＞90％;氨基酸序列比较的同源性Ⅰ型为857％,Ⅲ、Ⅳ、ⅤⅥ型各株均＞70％,Ⅱ型各株均＞93％。NS4基因的克隆和表达为其基因和产物作用的进一步研究奠定了基础。     

The occurrence rate of HGV/GBV-C RNA and risk factors in patients of narcological dispensary in Novosibirsk

The occurrence rate of HGV/GBV-C RNA, genotypic variety of isolates and various risk factors of infection with HGV/GBV-C were evaluated in 500 patients of the narcological dispensary of Novosibirsk. The occurrence rate of HGV/GBV-C RNA among all examined blood sera was 33.6%. At the same time in blood sera with HCV markers the occurrence rate of HGV/ GBV-C was 42.9% and in sera with negative results for markers HCV--25%. For gene typing of obtained isolates the direct sequencing of the amplification products of fragment NS3B and the phylogenetic analysis of the sequences thus obtained were used. Almost all isolates subjected to gene typing belonged to genotype 2, widespread in Europe, and only 1 isolate was classified with genotype 4. Statistically significant (p<0.05) risk of HGV/GBV-C infection among the examined subjects was linked with the intravenous use of drugs (OR 2.15), risky sexual behavior (OR 1.8) and the presence of virus hepatitis C (OR 2.26).     

Sequence variation within a nonstructural region of the hepatitis G virus genome.

Nine sets of nested PCR primers from a 2.6-kb region of the hepatitis G virus (HGV) genome at nucleotide positions 5829 to 8421 were designed and used to analyze serum specimens obtained from patients with community-acquired non-A, non-B hepatitis who were HGV RNA positive. One set of primers was found to be most efficient in detecting HGV and was subsequently used to test 162 HCV-positive and 11 HCV-negative plasma units obtained from individual paid donors. HGV RNA was detected in 30 (17.3%) plasma units, 2 of which were found among the 11 HCV-negative specimens. A complete set of nine PCR fragments was obtained from two patients with community-acquired acute non-A, non-B hepatitis and from four paid donors. All PCR fragments were sequenced and were shown to have a nucleotide similarity of 85.9 to 92.3% and a derived amino acid similarity of 96.0 to 99.0%. The majority of nucleotide changes occurred in the third position of codons. The HGV nucleotide and protein sequences obtained in this study were compared with HCV sequences. Based on this analysis the 2.6-kb fragment was predicted to encode the C-terminal part of the putative NS4b, the entire NS5a, and almost the complete NS5b proteins. Putative protease cleavage sites separating these proteins were also predicted. In serial specimens obtained from the two HGV-infected patients, no significant variations were found in the HGV nucleotide and derived amino acid sequences over time. The HGV sequences obtained from one patient showed no changes over 6 months, whereas more than 99.0% homology was observed for sequences from the second patient over 2.5 years. Heterogeneity analysis performed on 10 sequences obtained in this study and corresponding regions from 6 known full-size sequences of the HGV genomes demonstrated notable discrete heterogeneity consistent with the existence of HGV genetic groups or types.     

丙型肝炎患者血浆和外周血单个核细胞同步检测HCV—RNA和HGV—RNA

庚型肝炎和丙型肝炎传播途径是一致的 ,主要通过血制品和注射途径 ,有可能同时或重叠感染 ,尽管ＨＧＶ感染能否导致肝损害仍有争论 ,由于国外近年做了许多研究 ,而国内仅有血清检测丙肝和庚肝重叠感染的个别报道 ,但对外周血单个核细胞和血浆未作同步检测。我们对此进行了探讨 ,现将结果报道如下。1　方法观察组 72例患者均为我院住院治疗或门诊追踪观察病例 ,所有病例通过血清学检查排除甲、乙、丁、戊型肝炎。 70 %有输血史 ,肝功能反复异常 ,抗ＨＣＶ阳性 ,临床诊断为慢性或急性丙型肝炎 ,以 2 0例健康献血员作阴性对照 ,用比重 1.0 77的…     

Hepatitis G virus (GBV-C) infection among Brazilian patients with chronic liver disease and blood donors

Background: The recently discovered hepatitis G virus (HGV) belongs, as hepatitis C virus (HCV), to the Flaviviridae family. HGV has been isolated from the serum of patients with non A-E hepatitis. However, the association of HGV with hepatitis is uncertain.Objective: To determine the HGV prevalence in blood donors and in patients with liver disease and to evaluate a possible correlation between HGV infection and liver disease.Study design: Sera from a total of 113 consecutive patients with chronic liver disease were submitted to a series of liver enzymes and function tests and analyzed for the presence of HBsAg, anti-HBs, anti-HBc, anti-HCV, HCV RNA and HGV RNA. Prevalence of HGV RNA was determined in a group of 87 blood donors.Results: Nine (10%) sera from blood donors and 15 (13%) sera from patients with chronic liver disease were HGV RNA positive. Some 28 (25%) patients were HCV RNA positive, with genotypes 1a, 1b and 3 present in 10, 12 and 5 patients, respectively. A total of 20 (18%) patients were HBsAg carriers. Five (4%) patients were double infected (one with HBV+HCV, one with HBV+HGV and three with HCV+HGV).Conclusion: The proportion (10%) of HGV-infected blood donors was very high when compared with other countries. The results did not allow to establish HGV as an etiologic agent for chronic liver disease. The parenteral route was the presumed means of HGV transmission for only one-third of the patients.     

庚型肝炎病毒—中国河北分离株部分NS3区基因序列分析

在急慢性输血后肝炎、散发性肝炎及爆发型肝炎中,大约１０～２０％的病人不属于已发现的Ａ～Ｅ型肝炎,提示存在新型肝炎病毒。在美国１９９５年第４６届医学年会上,已有了一些有关庚型肝炎病毒（ＨＧＶ）分子生物学及庚型肝炎血清学方面的摘要报道。１９９６年初Ｌｉｎ...     

Co-infections with hepatitis G and TT virus in patients with chronic hepatitis C in Hungary

The significance of co-infections with novel hepatitis viruses Hepatitis G (GBV-C, HGV) and TT virus (TTV) in chronic hepatitis C is not clear. We determined the prevalence of HGV RNA and TTV DNA in chronic hepatitis C patients and in asymptomatic hepatitis C virus (HCV) carriers, and assessed the influence of these agents on the course of HCV infection. Seventy-seven patients with chronic hepatitis C--50 of them treated with interferon (IFN)--and 33 HCV carriers with normal alanine aminotransferase have been investigated. Previous HBV infection was detected by testing serum HBsAg and aHBc. HGV RNA and TTV DNA were detected by PCR. In the healthy population, the prevalence of anti-HCV was 0.3%, HGV RNA 8.0% and TTV DNA 18.5%. In chronic hepatitis C HGV RNA occurred in 9.09% and TTV DNA in 40.25% of cases. In IFN-treated patients with sustained remission, the frequency of TTV was 20% vs. 45.7% found in non-responders. Among asymptomatic HCV-carriers, the prevalence of HGV RNA was 9.09% and TTV DNA 75.7%. Neither HGV RNA nor TTV DNA had apparent effect on the HCV infection. TTV was detected with the lowest frequency in persons with sustained remission due to IFN, suggesting antiviral effect of IFN on TTV.     

GB virus C/hepatitis G virus infection in dialysis patients and kidney transplant recipients in Central Brazil

In order to investigate the prevalence of GB virus C (GBV-C)/hepatitis G virus (HGV) infection in dialysis patients and kidney transplant recipients in Central Brazil and also to analyze the virus genotypes distribution, a total of 123 patients including 98 on hemodialysis, 13 on continuous ambulatory peritoneal dialysis treatment, and 12 who received kidney transplantation were interviewed in one unit of dialysis treatment in Goiania city. Blood samples were collected and serum samples tested for GBV-C/HGV RNA by polymerase chain reaction. Genotypes were determined by restriction fragment length polymorphism (RFLP) analysis. Eighteen samples were GBV-C/HGV RNA-positive, resulting in an overall prevalence of 14.6% (95% CI: 9.2-21.7). A high positivity for GBV-C/HGV RNA was observed in patients who had received kidney transplant (16.7%), followed by those on hemodialysis (15.3%), and peritoneal dialysis (7.7%). RFLP analysis revealed the presence of genotypes 1, 2, and 3 of GBV-C/HGV; more precisely, 9 (50%) samples were found belonging to the 2b subtype, 4 (22%) to the 2a subtype, 3 (17%) to genotype 1, and 2 (11%) to genotype 3. The present data indicate an intermediate prevalence of GBV-C/HGV infection among dialysis patients and kidney transplant recipients in Central Brazil. Genotype 2 (subtype 2b) seems to be the most prevalent GBV-C/HGV genotype in our region.