对比分析5'-UTR和NS5b区序列确定广东地区HCV基因亚型

［摘要］ 目的 对比丙型肝炎病毒（HCV）不同区段系统进化分析法基因分型的差异,探讨广东地区丙型肝炎患者的HCV基因亚型分布。方法 RT-PCR分别扩增HCV5'-UTR（71nt-311nt）共241bp和NS5b区（8249nt-8650nt）共402bp的片段。序列分析后进行系统进化分析以确定病毒基因型及亚型。结果 HCV NS5b区段分型74例,其中1b亚型51例,2a亚型10例,6a亚型8例,3a亚型2例,3b亚型2例,1a亚型1例;5'UTR区分型74例,其中1型45例,2型11例,6a亚型10例,3a亚型3例,3b亚型5例;两区段共同分型49例,47例基因型一致,2例不同。结论 对比分析HCV不同区段基因序列,可深化对HCV基因型和亚型的了解。初步表明广东地区慢性丙型肝炎患者HCV基因亚型分布以1b为主;其次为2a、6a,两者比例相当;3b、3a也占有一定比例。 ［关键词］丙型肝炎病毒;基因型;5'-非编码区;NS5b区;系统进化分析     

丙型肝炎病毒NS5区部分基因的克隆和表达及其抗体动态研究

分析丙型肝炎病毒ＮＳ５蛋白的抗原性,研究抗－ＮＳ５抗体的性质,动态变化规律及临床诊断意义。方法 用Ｇｏｌｄｋｅｙ程序对ＮＳ５蛋白的抗原决定簇进行预测分析,以ＲＴ－Ｎｅｓｔｅｄ ＰＣＲ方法扩增ＮＳ５基因片段,并进行核酸序列分析。通过基因体外重组技术克隆表达目的的基因。     

中国人丙型肝炎病毒基因组λgtll cDNA库的免疫筛选与鉴定

丙型肝炎病毒(Hepatitis C virus,HCV)是散发性及输血后肝炎的主要病原体,易致慢性肝炎、肝硬变或肝细胞癌。为了研究中国HCV基因组的变异及结构特征,我们从中国丙型肝炎(丙肝,Hepatitis C,HC)患者血浆中提取HCV基因RNA,用随机引物合成HCV cDNA并构建其λgtll cDNA库。对此基因库进行了免疫筛选,并对2个阳性克隆(Q349和Q653)在pUC18质粒中进行了亚克隆。序列分析结果表明:Q349和Q653分别来自HCV基因组核心(C)区(第554～902位)和非结构3(NS3)区(第4175～4827位)。Q349和Q653与HCV原型相应序列在核苷酸和氨基酸水平上的同源性分别为86.8％,80.2％和97.3％,93.1％;与日本HCV相应序列间有更高的同源性,故属于HCVⅡ组。特异性灾验表明,Q349和Q653的编码多肽可与HC人群血清特异反应而不与正常人血清反应,且Q653与中国HC血清反应的阳性率(95.8％)比日本HC血清的阳性率(85.7％)高。表明根据中国HCV基因序列(特别是非结构区序列)设计引物或合成多肽将更适合中国人群HCV感染的检出。     

丙型肝炎病毒6a型感染的状态与分析

目的:初步探讨丙型肝炎病毒6a型感染的状态.方法:对经HCV 5'非编码区(5'NCR)复合酶切分型结果为6a的3例样本(95,126,150)进行5'NCR和NS5B区的扩增、测序,然后将5'NCR和NS5B区序列与24个HCV全基因参考序列(均来自GenBank)比对并构建遗传进化树.结果:对3例分型结果为6a型的样品进行5'NCR序列分析发现,这3例样品都具有6a型特征性的第-145位的CA碱基插入,且与6a型参考序列Y12083的同源性最高,分别为0.993,0.987,0.993;进化树分析表明,3例样本都属于6a型.然而NS5B区的序列分析结果表明,95,126,150在NS5B区与1b型参考序列HC-J4的同源性最高,分别为0.934,0.930,0.926;进化树分析结果也显示它们都属于1b型,两个区的分型结果完全不同.为排除引物扩增效率的问题,使用6a型特异的引物对3例样品进行NS5B区扩增,3例样本扩增结果均为阴性.结论:我们发现的HCV 6a型的感染中,存在3例样品5'NCR和NS5B区的分型结果不一致,尚无直接证据证明其是否发生了基因重组.但是,我们的结果提示,在两个或两个以上区域进行HCV分型是非常重要的,且HCV基因型之间的重组必须纳入到HCV分型的考虑中来.     

Single-strand conformation polymorphism for analysis of genomic variability of hepatitis C virus nonstructure 5A region

OBJECTIVE To establish a convenient method to detect the genomic population with hepatitis C virus (HCV) at nonstructure 5A (NS5A) region and to determine the correlation between the genomic population complexity at NS5A region and disease stage.

METHODS The sera from 52 patients with chronic hepatitis C virus infections were analysed using single strand conformation polymorphism (SSCP). In the SSCP, an asymmetric polymerase chain reaction (PCR) was carried out on the 455 bp products of the first round PCR at the NS5A region and the number of band of single strand deoxyribonucleic acid (DNA) which reacted with complemental DNA probe specific for the NS5A region after gel electrophoresis was analyzed.

RESULTS In 90% patients with chronic persistent hepatitis, the bands of single strand DNA was limited to one, and in those with chronic active hepatitis or liver cirrhosis, two or more bands of DNA were frequently detected. In about half of patients with hepatocellular-carcinoma, three or more bands were found. The number of bands increased with the progression of liver disease. The multivariate analysis showed that the progression of liver disease was the independent factor of viral diversity (P < 0.025) and was not related to the age, sex, the route of infection and the titer of hepatitis C virus ribonucleic acid (HCV RNA).

CONCLUSIONS These results suggest that the genomic variability of HCV at NS5A region increases with the progression of liver disease, and this may be closely related to the clinical features of type C liver disease.

     

HCV NS5b1b与1b混合感染指标在干扰素治疗中的应用

目的 :探讨丙型肝炎病毒 (HCV)感染的基因型及其与干扰素应答效果的关系。方法 :应用逆转录—聚合酶链反应 (RT PGR)技术对 2 69例肝炎病人血清进行HCVNS5b区基因片段扩增 ,并对 5 6例阳性PCR产物进行酶切分型 ,同时观察干扰素α 2b对其中 48例慢性丙型肝炎的疗效。结果 :HCV单纯 1b型感染、1b与 1b混合感染及 2a型感染对干扰素的应答率分别为 70 % (14 /2 0 )、9 5 % (2 /2 1)、71 4% (5 /7) ;其中完全应答分别为 45 % (9/2 0 )、4 8% (1/2 1)、5 7 1% (4 /7) ;部分应答分别为 2 5 % (5 /2 0 )、4 8% (1/2 1)、14 3 % (1/7)。 1b与 1b混合感染组同单纯 1b型及 2a型感染组比较有显著性差异 (P <0 0 1) ,而单纯 1b型感染组与 2a型感染组比较无明显差异 (P >0 0 5 )。结论 :HCV单纯 1b型感染或 2a型感染者对干扰素敏感 ,而 1b与 1b混合感染者干扰素治疗效果极差 ,可作为预测干扰素疗效的一项新的重要指标     

淮北市HCV感染者基因亚型分布特征及1b型天然耐药突变分析

目的：分析淮北市HCV感染病人基因亚型分布特征及1b型天然耐药突变情况，为HCV临床抗病毒治疗和感染防控提供依据。方法：选取2019-2022年就诊于淮北矿工总医院住院的HCV感染者(未经任何DAAs治疗)HCV-RNA阳性血清样本，抽提RNA后逆转录，采用巢式PCR扩增NS5B、NS5A及NS3/4A区域基因序列，产物送交测序公司测序。采用Mega11.0软件对测序结果进行序列比对分析。结果：结果 HCV 1b型(68.63%)为主要亚型，2a型次之(29.41%),此外检出3a型2例(1.96%)。HCV亚型的分布与性别、年龄无明显相关(P>0.05),1b型多见于≥55岁年龄组中。RASs主要类型为：NS5B区C316N(100.00%);NS5A区Q54H(40.47%),Y93H(19.05%);NS3/4A区Q86P(30.88%),I71V+Q86P(19.12%),V170I+Q86P(14.71%)及T72I/A+Q86P(13.24%)。其中突变频率较高的RASs依次为：C316N(100.00%)、Q54H(60.00%)、Q86P(53.78%)、Y93H...     

丙型肝炎病毒1b型NS3区与原发性肝癌的关系

目的：探讨丙型肝炎病毒（HCV）1b型的非结构蛋白3（NS3）区与原发性肝癌（HCC）的关系。方法：将患者分为丙型肝炎病毒携带者组、非HCC组及HCC组,从患者血清中提取HCV-RNA,采用巢式聚合酶链法（nested -PCR）进行HCV基因分型并扩增HCV-NS3片段,分析HCV-NS3区片段的核苷酸序列、氨基酸序列、蛋白质二级结构。结果：3组均以1b型为主,3组间1b型所占百分率比较差异无显著性(P>0.05）;3组核苷酸序列同源性95%,3组氨基酸序列的同源性为95%,且3组在NS3_1227-1206区间都无特殊的氨基酸突变;NS3_1227-1206区的蛋白质二级结构分为A、B 2型,携带组及非HCC组以A型为主,而HCC组以B型为主;携带组与非HCC组蛋白质二级结构分型差异无显著性（P>0.05）,而HCC组与携带组、非HCC组之间差异有显著性（P<0.01）。结论：1b型HCV携带者及非HCC患者与HCC患者在蛋白质二级结构上有差异,HCV-NS3与HCC的发生有关联。     

Study of hepatitis C virus genotype in Guizhou area of southwestern China

OBJECTIVE: To determine the concordance in various hepatitis C (HCV) genotyping methods and to investigate the distribution of HCV genotypes in Guizhou area of Southwest China. METHODS: Serum samples from 206 patients (100 with chronic hepatitis and 106 with hemopathy) were detected for antibody of HCV by second generation enzyme-labelled immunosorbent assay (ELISA). Thirty-five anti-HCV positive samples were detected for HCV RNA by RT-polymerase chain reaction (PCR) and 30 HCV RNA positive samples were determined for their genotypes by three various genotyping methods [PCR with type-specific primers at the core region (primer-set), slot-blot hybridization with type-specific probes at NS5B region (blotting) and the restric fragment length polymorphism analysis of PCR products of 5' NC region (RFLP)]. Ten samples with the known genotype were analysed by the direct sequencing. RESULTS: Of 30 samples with positive HCV RNA, the types of 22 could be classified by three methods, and the genotypes determined by various methods had complete concordance. The types of 6 samples could be classified by two methods and 5 had agreement subtypes. The types of two samples could be classified only by RFLP. Overall, 27 (90.0%) had subtype 1b infection and 3 (10.0%) had subtype 2a infection. The nucleotide sequence of 8 samples with subtype 1b and one with subtype 2a were analysed by the direct sequencing. The subtypes determined by sequence analysis were in complete concordance with those decided by various genotyping methods. CONCLUSIONS: Subtype 1b is the predominent HCV genotype in Guizhou area, while subtype 2a is less common. There was a good concordance with the genotyping results obtained by various HCV genotyping methods.     

HCV2a(JFH1)NS5A蛋白在原核和真核细胞中的表达、鉴定及分析

目的在原核与真核细胞中表达丙型肝炎病毒(hepatitis C virus,HCV)2a型JFH1(japanese fulminanthepatitis 1)株NS5A蛋白,分析JFH1NS5A蛋白与其它基因型NS5A蛋白间的差异,为进一步研究NS5A蛋白在HCV病毒复制中的作用奠定基础。方法PCR特异扩增JFH1NS5A基因,克隆入pET-32a和pEGFP-N1载体中,构建JFH1NS5A的原核和真核重组表达质粒pET-JFH1NS5A和pEGFP-JFH1NS5A。将pET-JFH1NS5A转化大肠杆菌BL21(DE3),pEGFP-JFH1NS5A转染HEK293T细胞,在原核与真核系统中进行表达,并以SDS-PAGE、荧光显微术和Westernblot方法检测。利用PubMedBLAST软件对JFH1与HCV1a、1b、2b和2c型NS5A蛋白间的同源性进行了分析。结果酶切鉴定及测序结果表明成功构建了重组质粒pET-JFH1NS5A和pEGFP-JFH1NS5A。SDS-PAGE电泳检测到融合蛋白在原核细胞中的表达,荧光显微镜观察和Westernblot检测到了GFP-JFH1NS5A融合蛋白在HEK293T细胞中表达。BLAST分析显示,JFH1和H77、HC-J4、HCV-J8和BEBE1病毒株NS5A蛋白的同源性分别为57%、60%、68%和74%。结论JFH1NS5A蛋白在原核与真核细胞中表达成功,为研究HCVNS5A在JFH1株病毒高效复制中的作用提供了材料。     

山东省HCV分离株C 区及NS5 区核苷酸序列分析

①目的 探讨山东省丙型肝炎病毒（ＨＣＶ）分离株Ｃ区、ＮＳ５区的核苷酸序列及其基因型别。②方法 以ＲＴ－ｎｅｓｔ－ＰＣＲ方法从１例临床ＨＣＶ感染者血清中分别扩增出ＨＣＶＣ区（４３２ｂｐ）及ＮＳ５区（３１９ｂｐ）的基因片段,将其克隆于Ｔ载体上,以双脱氧末端终止法自动测序并进行序列分析。③结果 该分离株与ＧｅｎＢａｎｋ中５０多个１ｂ型分离株同源性均在９０％以上,其中Ｃ区与中国北京的１ｂ型株ＨＣＵ６３３７     

青岛地区丙型肝炎基因分型的研究

目的 探讨青岛地区丙型肝炎基因型的分布状况,发现丙型肝炎病毒（HCV）流行优势株,分析丙型肝炎基因型与病人性别、感染途径、炎症活动度、病毒载量等的相关性。方法 应用RT-PCR方法,对46例青岛地区丙型肝炎病人的血清RNA提取物NS5B区和（或）CORE-E1区进行扩增,扩增片段进行基因测序,根据测序结果在PUBMED网站中获得基因分型结果,分析不同丙型肝炎基因型与病人性别、感染途径、肝病程度、HCV RNA含量及谷丙转氨酶（ALT）、谷草转氨酶（AST）水平的关系。结果 46例血清标本,HCV RNA均为阳性,44例可以分型,分型率为95.65%,其中基因1b型28例（63.64％）,2a型14例（31.81％）,3b型2例（4.55％）。丙型肝炎基因型的分布与病人性别、感染途径、肝病程度、HCV RNA含量及ALT、AST水平均无关（P〉0.05）。结论 青岛地区HCV流行株为基因 1b型和2a型,基因1b型为优势株,同时发现3b基因型。基因型与丙型肝炎的疾病进展程度及炎症活动度无关。     

SEQUENCE ANALYSIS OF THE NS5 REGION OF GBV-C／HGV AND DETECTION OF THE VIRUS BY REVERSE TRANSCRIPTASE PCR

GBV-C/HGV is a newly identified virus associated with human hepatitis.In this study,the nucleotide sequences of the partial NS5 gene of GBV-C/HGV derived from sera of 8 Chinese patients were determined.The nucleotide homology among the 8 isolates were 92% on average.On the basis of sequence analysis,two sets of oligonucleotide primers derived from highly conserved region of GBV-C/HGV NS5 gene were designed to establish both sensitive and specific nested PCR for detection of GBV-C/HGV RNA.253 Chinese patients were examined for the virus RNA.GBV-C/HGV RNA positive rates in patients infected with HBV,HCV and patients with chronic non-B,non-C hepatitis were 18.4%,19.8% and 8.9% respectively.This result suggested that HBV,HCV and GBV-C/HGV shared the same transmission risk factors.8 patients with GBV-C/HGV and HCV coinfection were retrospectively observed for the response to interferon.Coinfection with GBV/HGV did not negatively influence the responsiveness of HCV,and GBV-C/HGV was sensitive to interferon to a certain degree.     

New hepatitis C therapies in clinical development

With the current standard of care for the treatment of chronic hepatitis C, a combination of pegylated interferon alfa and ribavirin, sustained virologic response rates can be achieved in approximately 50% of patients only. - Improved understanding of the viral life cycle has led to the identification of numerous potential targets for novel, direct-acting antiviral compounds. Inhibitors of the NS3/4A protease are currently the most advanced in clinical development. Recently completed phase 3 studies of the two protease inhibitors telaprevir and boceprevir, each given in combination with standard of care, yielded sustained virologic response rates in the range of 66-75% in treatment-naive patients and 59-66% in treatment-experienced patients with HCV genotype 1 infection. Studies of second-generation protease inhibitors, with the potential advantage of improved potency, drug metabolism and pharmacokinetics profile, are already underway. - Inhibitors of the HCV NS5A protein and NS5B polymerase are potentially active across different HCV genotypes and have shown promising antiviral efficacy in early clinical studies. Other emerging mechanisms include silymarin components and inhibitors of cell proteins required for HCV replication. - While improved formulations of current HCV therapies are also being developed, future hopes lie on the combination of direct-acting antivirals with the eventual possibility of interferon-free treatment regimens.     

某地区吸毒人员丙型肝炎病毒的流行病学和基因分型研究

目的：了解吸毒人群中丙型肝炎病毒（HCV）的流行和基因分布现状,探索HCV的传播规律。方法：采集本市强制戒毒所576份吸毒人员和本站体检的从业人员210份血清标本,用ELISA法对所有标本进行抗HCV的检测;检出抗HCV阳性的血清进一步用特异性引物RT－PCR法进行基因分型。结果：576份标本中,经血途径（静脉吸毒、共用注射器）吸毒或有输血史和用血制品史的吸毒者,其抗HCV检出率（13．8％）明显高于经口吸毒者（6．3％）,说明静脉吸毒、共用注射器吸毒、用血制品、输血都是感染HCV的危险因素。经口吸毒者抗HCB检出率明显高于从业人员,提示经口吸毒在传播HCV上具有重要意义。此外,本地籍吸毒者的抗HCV检出率（5．7％）明显低于外省吸毒者（27．7％）、外省有血途径感染机会的吸毒者,83．3％都感染了HCV,本地有同样途径有吸毒者,仅有8．6％感染了HCV;这可能与外省有关城市的高感染率有关。结果还显示,山西省吸毒人群的HCV基因型的分布与从业人员较一致,均以1b型为主（78．9％）,其次为2a型（15．8％）和1b/2a混合型（5．3％）;未检出1b型、2b型和3a型。结论：该吸毒人群中HCV基因以1b型占优势。其分布与南方城市和本省的其他人群相近^[6]。血途径仍是HCV感染的主要途径,但经口吸毒在HCV传播上的重要意义应引起重视。     

庚型肝炎病毒RNA NS3区基因分型

目的：探讨HGVNS3区基因变异与基因型的关系。方法：对32株NS3区序列进行酶切位点分析，比较中国株与GBV－C株和已发表的HGVNS3区酶切位点，选择特异性内切酶切点。结果：32株NS3序列酶切位点分析表明存在5种基因型。1组：在4338位无HhaI切点，而在4360位有HhaI切点；2组：4338位及4360位均无HhaI切点；3组：仅在4338位有HhaI切点；4组：4338位及4351位有HhaI及Tsp509I切点；5组：4351位有Tsp509I切点。其核苷酸变异率分别为0．85％、19．0％、20．5％、18．2％、19．6％等。结论：中国株与西非、东非、加拿大、美国及日本株不是同一基因型。     

广州地区丙型肝炎患者HCV基因分型研究

目的研究广州地区丙型肝炎患者HCV基因型分布的变化情况。方法收集207例HCV RAN阳性丙型肝炎患者血清,对其HCV Core区(C区)和NS5B区基因序列进行RT-nPCR扩增,两区扩增成功的PCR产物进行基因测序,测序结果分别与相应的参照株序列比对,构建系统进化树,两区基因分型结果一致者确定HCV基因亚型。结果 185例HCV C区和NS5B区都扩增成功,其中179例两区基因分型结果一致。共检出1、2、3、6型4种基因型和8种基因亚型,其中1b型占40.78%(73/179),6a型占27.37%(49/179),3b型占11.73%(21/179),3a型占8.94%(16/179),2a型占6.15%(11/179),1a型占3.91%(7/179),2b型占0.56%(1/179),6n型占0.56%(1/179)。1b株可分为A、B、C三大簇,A簇与中国广泛流行1b株接近、B簇与中国中部及南部地区流行1b株接近,6a株分为Ⅰ、Ⅱ两大簇,与献血人群及静脉吸毒人群中6a型株接近。结论广州地区丙型肝炎患者HCV基因型以1b、6a、3型为主,存在多种亚型;1b型株包含中国大部分地区的1b型株,6a、3型已取代2a型分别成为第二、三主要流行基因型;未发现6型变异株。     

慢性丙型肝炎患者对NS3／4A蛋白酶抑制剂预存耐药的研究

目的对我国南方184例无抗病毒治疗史的慢性丙型肝炎患者进行NS3／4A蛋白酶抑制剂相关耐药位点检测。方法NS3／4A蛋白酶抑制剂相关的耐药位点分析采用自行设计型别特异性引物行巢式PCR．PCR产物经纯化后采用直接测序法鉴定。结果184例样本中有162例扩增成功,其中125例（77．16％）患者共检出266个变异位点。HCVlb型A156S变异率为18．33％,V170I为16．67％;HCV2a型V36L、Q80G、V170I变异率为100％,A156S为64．29％;HCV6a型Q80K变异率为95．45％,V170I为98．86％。结论未使用抗病毒药的慢性丙型肝炎患者对NS3／4A蛋白酶抑制剂也存在预存耐药,变异率因HCV基因型而不同。1b型发生变异较少,HCV2a型及6a型普遍发生变异。     

Prevalence of hepatitis C virus isolate genotypes from chronically infected Lebanese patients: a hospital-based study

To assess percentages of hepatitis C virus (HCV) genotypes in infected Lebanese patients referred to St. George Hospital, Beirut, Lebanon, 77 infected cases were studied. Of those, 27 were hemodialysis patients. Genotyping was performed by nested PCR of the HCV core-region with specific primers, followed by DNA enzyme-immunoassay using HCV type and subtype-specific probes. Single genotype infections were detected in 52 patients (67.5%). In these cases, types 1, 2, 3 and 4 were detected in 19.5%, 32.5%, 5.1% and 10.4% of the cases respectively. Twenty-five (32.5%) samples showed mixed genotype infections. Single genotype distribution was significantly different among dialysis and non-dialysis patients. In the dialysis group, genotype 2 was predominant (80%, p < 0.001). In single HCV genotype-infected patients, subtype 1b was frequently detected in nondialysis cases (34.4%) whereas this genotype was found in only 5% of dialysis cases. Genotypes 5 and 6 were not detected in any of the cases studied. This pilot hospital-based study provides evidence for the diversity of HCV genotypes in the Lebanese population and establishes differences in distribution depending on the risk group.