中国人HCV标本库的建立

目的 建立中国人HCV感染患者系列标本库.方法 系列收集门诊及住院的HCV感染患者血液标本.所有血清标本进行基本的生化和病毒学检测.记录每一位患者的流行病学资料和临床与诊断检测结果.定期随访,不断扩充标本库.同时.对大部分HCV RNA阳性血清标本进行HCV基因型和亚型的确定.结果 建立了中国人HCV标本库,收集605例HCV慢性感染患者的934份血清标本.完整记录了流行病学、临床及实验室资料,可提供研究所需的血清标本.对其中206份HCV RNA阳性感染者血清标本进行了基因型别的检测.序列与系谱分析结果显示Ib 87例(42.2%),2a 26例(12.6%),3a 32例(15.5%),3b 30例(14.6%),6a 31例(15.1%),未见la和2b型.结论 建立了中国人HCV标本库.为HCV的基础与临床研究奠定了良好的基础,也为其他疾病相关标本库的构建提供了参考.     

新疆维吾尔族人群丙型肝炎病毒基因分型分布分析

目的 了解新疆维吾尔族人群感染的丙型肝炎病毒（hepatitis C virus,HCV）的基因型别,为采取相应的临床治疗措施提供科学依据.方法 从医院收集维吾尔族丙型肝炎（丙肝）患者的血清样本基本信息,通过反转录巢式PCR法扩增HCV NS5b区并进行序列测定,与HCV标准株比较分析、绘制系统发生树、确定其基因型并进行比较分析.结果 39份丙型肝炎患者的样本中,HCVRNA阳性者为20份,基因型的分布状况为：1型17例,占85.0％,2型2例,只占10.0％,3型1例,占5.0％;亚型分析发现lb亚型14例（70.0％）,la亚型3例（15.0％）,2a亚型2例（10.0％）,还发现1例3a亚型（5.0％）.结论 新疆地区维吾尔族人群中流行的HCV基因型构成较为复杂,1b亚型为主要型别.     

广州无偿献血人群中丙型肝炎抗体阳性者HCV基因型与病毒载量的关系

目的:了解最新广州地区无偿献血人群丙型肝炎病毒(HCV)基因型与病毒载量的关联性。方法:收集2008～2011年广州地区无偿献血人群中抗-HCV阳性标本605份,采用荧光定量PCR(Q-PCR)的方法对其进行核酸及病毒载量检测,阳性标本作NS5B基因扩增;核苷酸序列测定后运用DNASTAR、BioEdit和Mega4.0等软件作序列分析和基因分型,采用SPSS16.0软件对病毒载量与基因型(亚型)的关联性进行分析。结果:337份HCV RNA阳性的标本扩增出NS5B基因320份,HCV 1b、6a、3a、2a、3b、1a、6n比例依次为45.00%、33.44%、8.75%、7.81%、4.38%、0.31%和0.31%。HCV1b与2a、3a、6a、6a与2a、3a之间病毒载量存在显著差异:HCVba病毒载量高于2a、3a和6a,HCV6a病毒载量高于2a和3a。结论:广州地区无偿献血人群中HCV1b和6a为主要亚型且其病毒载量高于其他亚型。     

湖北地区丙型肝炎患者HCV基因分型及HCV NS5B耐药突变位点分析

目的:检测湖北地区丙型肝炎(丙肝)患者丙肝病毒(HCV)基因分型及HCV NS5B基因耐药突变位点的分布特征。方法:收集自2011-03-2014-05在本院确诊的来自湖北地区的丙肝患者外周静脉血标本273例,采用一代测序法检测每例样本的HCV和NS5B基因序列,将测得的序列与Blast进行在线比对后,统计分析HCV基因型和各亚型检出率,以及HCV NS5B耐药突变位点在HCV各基因亚型中的分布差异。结果:273例丙肝患者共检出1、2、3、6四种基因型和1a、1b、2a、3a、3b、6a六种基因亚型,其中1b亚型检出率较高,占76.19%(208/273),其次是2a亚型,占15.02%(41/273),其它各亚型检出率均不超过4.40%,各亚型检出率差异有统计学意义(P0.05)。HCV NS5B基因以L159F突变率较高,占17.95%(42/234),与其它位点的基因突变率差异有统计学意义(P0.05)。同时各个位点的耐药突变发生在2a亚型中的比例较高,达57.26%(134/234),显著高于其它亚型的耐药突变(P0.05)。结论:分析湖北地区丙肝患者HCV基因分型及HCV NS5B耐药突变位点的分布特点,可为该地区丙肝患者的个体化治疗提供指导依据。     

PCR-荧光探针法检测庆阳地区丙型肝炎病毒基因型

目的:应用PCR-荧光探针法分析庆阳地区丙型肝炎患者基因型,并对PCR-荧光探针法的各种性能进行评价。方法:收集庆阳地区289 例各种丙型肝炎患者的临床资料和外周静脉血,采用PCR-荧光探针法检测其基因型,并和PCR-反向点杂交法、测序法进行比较。结果:289 份HCV RNA 阳性血清标本中,PCR-荧光探针法基因型及亚型检出率为99.3%(287/289),其中1b 型139 例(48.1%),2a 型136 例(47.1%),3a 型7 例(2.4%),3b 型5 例(1.7%),未分出型2 例(0.7%)。PCR-荧光探针法的特异度和准确度为100%,重复性良好,并与PCR-反向点杂交法、巢式PCR 测序法分型结果均一致,三种方法的一致率为98.2%(56/57),差异无统计学意义(P>0.05)。1b 基因型患者ALT、AST、PLT 和HCVRNA(lg)水平均高于2a基因型患者,差异具有统计学意义(P<0.05)。结论:庆阳地区HCV 基因型呈现多基因型分布特点,主要为1b 型和2a 型,且2a 型和1b 型的比例相当,呈现出2a 型比例升高,1b 型下降的趋势;PCR-荧光探针法HCV 基因分型敏感度和特异度高,方法简单,适合临床实验中应用。     

山西省不同人群丙型肝炎病毒的基因分型研究

目的探索丙型肝炎病毒(HCV)基因型在山西省不同人群中的分布规律及流行的优势型。方法用RT-PCR和型特异性引物逆转录巢式PCR法,对山西省271例抗HCV阳性的丙型肝炎病人、原发性肝细胞癌患者、非肝癌癌症患者、性关系混乱者和性病患者、职业献血员、吸毒者及公共场所从业人员进行了HCVRNA的检测和基因分型。结果271份抗HCV阳性标本中,HCVRNA检出率为45.45%～89.66%,平均67.52%。以丙型肝炎病人、献血员和吸毒者的HCVRNA检出率较高(76.9%～89.7%),χ2=30.44,P<0.01。在133份HCVRNA阳性血清中,仅检出了108例1b型、2a型和此两种基因型的混合感染者。未检出1a型、2b型和3a型。其中1b型占80.00%(88例),2a型占11.81%(13例),混合型占6.36%(7例)。在肝癌患者和献血员中,仅检出1b型的感染;非肝癌的其他癌症患者中,未发现混合感染。各基因型在各人群中的分布比例也有差别,丙型肝炎患者、非肝癌的其他癌症患者、吸毒者和从业人员的各基因型构成比较接近,均以1b型为主。而性病患者和性关系混乱者中1b型和2a型感染者比例相等。结论山西省HCV的基因以1b型占优势。     

山东烟台地区HCV感染的基因分型研究

目的 了解山东烟台地区丙型肝炎病毒的基因分型,结合受试者的肝功能指标观察基因型别与肝损情况是否相关.方法 采用特异性PCR引物对HCV RNA5'UTR区和(或)NS5B区进行扩增,PCR产物进行序列分析,通过与GenBank中参考序列的比对,联合遗传进化树对标本予以分型.结果 9例无偿献血员中检出1b和3a两种基因亚型,分别为8例和1例.33例丙肝患者中,检出1b、2a和6a三种基因亚型,分别为22(66.7％)、10(30.3％)和1(3.03％)例.1b亚型是烟台地区HCV携带者的优势流行基因亚型,在不同人群中分布差异无统计学意义(x2=0.796,P=0.373);不同基因分型的受试者其肝损指标的差异有统计学意义(P＜0.05),2a型携带者的ALT、AST均值明显高于1b型.结论 山东烟台地区HCV基因型呈现多样性,以1b为主,并首次检出3a和6a亚型.HCV基因型与肝损指标具有相关性,2a型HCV感染可能在肝细胞的病变过程中起着重要作用.     

北京市吸毒人群HCV感染状况及基因特征分析

目的 分析北京市吸毒人群HCV感染情况及其HCV的病毒基因特征.方法 利用ELISA方法和Real-time PCR方法同时检测684名吸毒人群血清中HCV抗体和HCV RNA,确定该人群的HCV感染情况.对HCV抗体或RNA检测阳性的样本进行C/E1和NS5B基因区扩增并对扩增产物进行序列测定,分析吸毒人群HCV基因亚型构成.结果 吸毒人群HCV感染率为26.2％(179/684).142例样本的C/E1或NS5B基因区扩增成功,基因进化分析发现8种HCV基因亚型:1a、1b、2a、3a、3b、6a、6n和6u.结论 伴随着人口流动性的增加,目前北京市吸毒人群HCV流行情况复杂,具有多种基因亚型.     

山东省HCV分离株C区及NS5区核苷酸序列分析及其基因分型

目的 研究山东省丙型肝炎病毒（HCV）流行株的基因型别,方法 用反转录套式聚合酶链反应（PCR）方法分别扩增山东省HCV流行株C区（432bp)NS5区（319bp)的两个基因片段,将其克隆人T载体上并自动测序,进行同源性分析及基因型别鉴定。结果 4株HCVC区基因片段有3株为1b基因亚型,1株为2a基因亚型,10株NS5区的基因片段分析均为1b基因亚型,并且与GenBank中多个1b亚型代表株核苷酸序列同源性达90%以上,结论 山东省HCV流行株以1b亚型为主,兼有2a亚型,同一亚型中也有较大的变异,NS5区1b亚型中基因散率可达5%以上。     

RT-PCR-RFLP用于麻疹病毒疫苗株和H1基因型的分析

目的 建立RT-PCR-RFLP方法 用于天津地区2002-2008年流行的麻疹野病毒基因型研究.方法 采集疑似麻疹患者的尿标本和咽拭子,传代细胞分离病毒.提取病毒液中的RNA,用一步RT-PCR法扩增麻疹病毒核蛋白(nucleoprotein,N)基因C端594个核苷酸片段,扩增产物经Bcn I酶切后琼脂糖凝胶电泳进行限制性片段多态性分析(RFLP),同时与序列分析进行对比验证.根据结果 构建基因系统发生树进行亲缘关系和遗传距离分析.结果 2002-2008年189份标本,分离获得69株麻疹病毒,N基因C端RT-PCR检测全部阳性,RFLP分析H1a是优势流行亚型,占98.55%(68/69),仅1株(1.45%)为H1b基因亚型,分型结果 与序列测定完全一致.系统发生树分析显示H1a亚型毒株分为2个亚枝,变异范围为0.2%～3.8%,存在不同病毒株引起的传播链共循环.结论 基于Bcn I限制性内切酶的RT-PCR-RFLP方法 能够特异性区分A基因型、H1a、H1b基因亚型,具有快速、简便、准确和经济的优点,更适用于国内大规模麻疹病毒的监测.     

Genotype distribution and comparison of the putative envelope region of hepatitis C virus from Korean patients

Comparative nucleotide sequence studies of the genomes of hepatitis C virus (HCV) revealed that there are at least 6 different genotypes of HCV. The prevalence of HCV genotypes among the patients with liver diseases in Korea was investigated using the polymerase chain reaction (PCR) for the NS5 region. In the 75 HCV RNA positive samples, two genotypes, type 1b and type 2a, were the major causative agents which accounted for 60% and 33% of infections respectively, while 7% could not be assigned a genotype by the methods used. The nucleotide sequences of cDNAs encoding the putative envelope proteins from 10 type 1b and 5 type 2a genotype samples were analyzed. Approximately 31–42% of the nucleotide sequences of type 1b samples examined differed from those of different genotypes, In the case of type 2a samples, 36–42% of the nucleotide sequences differed from those of different genotypes. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. Two hypervariable regions (HVR1 and HVR2) were recognized in both HCV genomes of genotypes 1b and 2a. However, the sequence divergence within the HVR2 region of genotype 2a was less than that of genotype 1b. © 1995 Wiley-Liss, Inc.     

High prevalence of HBV and HCV infection among intravenous drug users in Korea

Although intravenous drug users are a well-known route of hepatitis C virus (HCV) and hepatitis B virus (HBV) transmission, there is no data on the prevalence of HBV and HCV infection among intravenous drug users in Korea. In order to describe the prevalence of HBV and HCV infection, and to determine HCV genotypes in the population, serum samples were collected from 107 intravenous drug users during 2005-2006. Fifty-seven percent (n = 61) were HCV RNA positive and 51% (n = 55) were HBV DNA positive. Co-infection of HBV and HCV were found in 23% (n = 25). HCV genotypes 1b, 2a/2c, 2, 2b, and 3a were found in 38% (n = 23), 44% (n = 27), 8% (n = 5), 2% (n = 1), and 3% (n = 2), respectively. Moreover, mixed infections of genotypes 1b and 2a/2c were found in 5% (n = 3). When the number of patients with HCV genotype 1b compared with that of patients with genotype 2a/2c, HBV DNA titer was not significantly different by independent t-test (t = -0.881, P = 0.392 > 0.05) between the two patient groups. These results suggest that the prevalence of HBV and HCV infection among intravenous drug users is high showing over 50% in Korea and a strategic prevention program should be performed in this group to prevent further infection into local community.     

丙型肝炎病毒基因分型及其与干扰素治疗应答的关系

目的为了解山西省丙型肝炎病毒的基因型和基因型对干扰素疗效的预示价值。方法用ＨＣＶ５’ＮＣ区酶切分型方法对９４例丙型肝炎病人进行基因分型，并观察其中４５例患者对干扰素α１ｂ治疗的应答。结果显示ＨＣＶⅠ组（Ⅰ、Ⅱ型）感染８０例（８５１％），ＨＣＶⅡ组（Ⅲ、Ⅳ型）感染１２例（１２８％），ＨＣＶⅠ／Ⅱ组混合感染２例（２１％）。在接受干扰素治疗的病例中，ＨＣＶⅠ组感染（３５例）的应答率为３７１％，持续应答率为１７１％，而Ⅱ组感染（１０例）的应答率为８０％，持续应答率为６０％，两组相比，有显著性差异（Ｐ＜００５，Ｐ＜００２５）。结论表明山西省以ＨＣＶⅠ组感染为主，干扰素对ＨＣＶⅡ组感染的疗效优于ＨＣＶⅠ组感染，ＨＣＶ基因型有预测干扰素疗效的意义。     

Development of an improved genotyping assay for the detection of hepatitis C virus genotypes and subtypes in Pakistan

A new genotyping system was established for the specific detection of HCV genotypes 1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4a–h, 5a and 6a during the course of this study. The system is based on entire core region and a part of 5′ noncoding region (5′NCR) with genotype-specific primers. Genotype-specific primers were designed on the basis of 114 HCV isolates. Serum samples with known genotypes were used as positive controls to validate the assay developed and to generate PCR band patterns. Band patterns generated from the clinical serum samples from HCV patients were compared to the patterns produced from these control samples. In addition, the type-specific bands were sequenced from the test patients and control clinical samples to validate further the test results. To determine sensitivity and specificity of the assay, a total 260 samples were analyzed simultaneously by this HCV genotyping method and that developed by Ohno and Murex HCV Serotyping 1–6 Assay. The system showed 79.2% concordance with Ohno's system and 65.38% with serotyping system. Samples with discordant results were sequenced and their genotypes were determined by molecular evolutionary analysis. The data indicate that the method described in this study may offer better sensitivity and specificity for the detection directly of HCV genotypes present at low levels in HCV patient samples.     

Distribution of hepatitis C virus genotypes among hemodialysis patients in Tehran--a multicenter study

Hepatitis C virus has substantial heterogeneity of genotypes throughout the world. The aim of this study was to determine the frequency of HCV genotypes, risk factors and clinical implications in cases of hemodialysis living in Tehran. A total of 155 patients treated by hemodialysis, who had been identified to be anti-HCV positive at 45 medical centers in Tehran, were enrolled. Genotyping was using restriction fragment length polymorphism (RFLP) on HCV-RNA positive samples. HCV-RNA was detected in 66 (42.6%) patients. Genotyping of HCV-RNA positive serum samples demonstrated that subtypes 3a and 1a were predominant accounting for 30.3 and 28.8%, respectively. The distribution of other HCV genotypes showed genotype 1b, 18.2%; genotype 4, 16.7%; mixed genotypes 1a and 1b, 3%; and genotype 3b, 3%. Genotype 2 was not detected in this study. Statistically significant differences were identified between HCV infected and non-HCV infected patients regarding history of hemodialysis unit changes more than two times (P = 0.01), and history of hemodialysis for more than 20 years (P = 0.02). However, blood transfusion, mean duration of hemodialysis therapy and the history of solid organ transplantation did not differ between these two groups. This study indicates that the dominant HCV genotypes among patients treated by hemodialysis living in Tehran were 3a and 1a, and considering previous reports from the general population, genotype 4 was strongly associated with hemodialysis. The duration of treatment by hemodialysis and, in turn, more hemodialysis unit changes will lead to more frequent HCV infections.     

Molecular and epidemiological aspects of hepatitis C virus infection among crack cocaine users

The aim is to investigate the prevalence, risk factors, and hepatitis C virus (HCV) genotypes/subtypes among crack users in-treatment in Central Brazil. A cross-sectional survey in which 600 in-treatment crack users were interviewed and tested for anti-HCV Ab by enzyme-linked immunosorbent assay was conducted between August 2012 and April 2013. Anti-HCV-positive samples were also submitted for HCV RNA detection by polymerase chain reaction. Positive HCV RNA samples were genotyped by direct sequencing analysis of the NS5B region of the viral genome, followed by phylogenetic analysis. Of the total, 3.7% (95.0% CI, 2.4%-5.6%) were anti-HCV positive. Age over 40 years and history of injecting drugs were risk factors for HCV, while snorting cocaine was a protector variable. HCV RNA was detected in 14 of 22 anti-HCV-positive samples, and the genotypes 1 (n = 10) and 3 (n = 2), subtypes 1a (n = 7), 1b (n = 3), and 3a (n = 2) were identified. The HCV prevalence found among crack users is almost threefold that observed in the general population in Brazil supporting that this population is at higher risk for HCV. The findings of cocaine insufflation as a protective behavior for HCV infection in this population should be explored.