内蒙古呼和浩特地区丙型肝炎病毒基因型分布

为了解内蒙古呼和浩特地区丙型肝炎病毒(HCV)基因型的分布特征,为本地区丙型肝炎的治疗、预防提供基础数据资料。收集呼和浩特地区2014年1月至2015年1月就诊的门诊和住院丙型肝炎患者血清采样标本149份,均为HCV抗体检测阳性且HCVRNA定量检测阳性。QIAGEN柱式病毒RNA提取法提取HCV RNA,反转录成cDNA,巢式PCR法扩增HCVNS5B区,对扩增片段进行测序,在NCBI BLAST上进行序列比对得到相似度最大的参考序列并确定基因型,用Megalign,Clustal W进行序列分析并建立同源关系树,分析呼和浩特地区HCV感染基因型的分布特征,以及基因型与宿主性别、年龄的关系。在测序成功的94份样本中,共检出6个基因型,1b型占73.40%(69/94),2a型占19.14%(18/94),3a、3b、6a型各占2.12%(2/94),6u型占1.06%(1/94)。性别资料明确的93例样本,男女基因型分布无显著差异(P0.05)。年龄资料明确的90例HCV样本,1+2型的患病年龄高于3+6型的患病年龄有统计学意义(P0.05)。得出内蒙古呼和浩特地区HCV感染的基因型1b为主,其次为2a型,但也有3型、6型等基因型的传入。HCV的基因型4型和5型未检测到。     

HCV基因型的差异性流行与进化

丙型肝炎病毒(Hepatitis C virus,HCV)是导致慢性肝炎的主要病原体之一,全球感染人数大约为1.7亿。HCV基因组具有高度变异特性,利用现代遗传分类方法,可将HCV分为6个基因型和80多个基因亚型。不同HCV基因型、亚型的分布与流行具有明显地域特性:1型、2型呈全球流行态势,3型主要流行于亚洲、北美及欧洲部分地区,4型主要流行于中非、中东和欧洲地区,5型主要发现于非洲和欧洲部分国家,6型则主要在东南亚和北美地区流行。我国流行的HCV有1、2、3和6四种基因型,北方仍以1b和2a型为主要流行基因型,近年来3型和6型在华南、西南地区快速传播。据推断,云南将可能成为我国HCV流行与传播的重要源头,引起目前HCV基因型/亚型分布的较大变化,并呈现多样化的传播方式。通过溯祖理论和进化分子钟等分析方法,了解HCV不同基因型差异性流行与进化,对研究HCV的分子流行病学特征,对应性制定丙型肝炎的预防控制策略具有重要意义。     

应用基因重组抗原诊断丙型肝炎研究

丙型肝炎病毒(Hepatis C virus,HCV)感染者的血液中病毒含量极低,还没有适当的方法可以直接检测病毒。现在常用免疫学方法测定特异性抗体[1]或利用 PCR 技术检测病毒核酸。由于 HCV基因组变异高达33%,PCR检测易发生假阳性和假阴性。同时由于丙肝抗原高度的变异性[2],虽然目前ELISA方法应用广泛,但是单一的诊断抗原已不能满足临床要求,需要多价抗原联合应用作为诊断试剂。目前,在我国流行的丙型肝炎病毒主要是Ⅱ型和Ⅲ型,国外的丙型肝炎诊断试剂研制单位主要是以Ⅰ型为主[3],而且进口的丙型肝炎诊断试剂昂贵,不适合在我国大量使…     

苏南地区1b型丙型肝炎病毒包膜区变异进化研究

本研究旨在了解本地区丙型肝炎病毒(Hepatitis Cvirus,HCV)基因型构成的前提下,分析1b型丙型肝炎病毒包膜2(second envelope glycoprotein E2)区的变异和种系进化,并研究其准种变异与临床肝病活动度的关系.对宜兴市人民医院收集的抗HCV抗体阳性患者166名,RT-PCR方法检测HCVRNA,HCVRNA阳性患者采用型特异性引物分型法确定病毒基因型;选择其中未经干扰素治疗的43例1b型慢性丙型肝炎患者的血清标本,扩增E2区,从中选取肝硬化患者4例,慢性非肝硬化患者6例的E2区PCR产物纯化测序,序列采用CLUSTALW与GENBANK上多株不同型别的HCV序列进行比对分析,结果采用Phylip软件构建遗传进化树;并观察E2区高变区1(HVR-1)的氨基酸(amino acid,aa)残基序列的变异特征;采用单链构象多态性垂直电泳检测43例患者个体内HCV E2区准种的变异情况,比较不同肝病活动度患者准种变异情况.结果表明本地区HCV以1b型为主(84.3%),对E2区基因序列和氨基酸序列变异的分析显示其变异具有一定的规律性,种系进化树提示本地区HCV病毒序列与上海、湖南、日本等地的HCV株有较近的亲缘性.43例患者中ALT高于正常的丙型肝炎患者准种复杂程度明显高于ALT正常者(P＜0.05).故本地区HCV基因变异符合中国东南部的特点,基因变异与临床肝病活动度具有相关性.     

苏南地区1b型丙型肝炎病毒包膜区变异进化研究

本研究旨在了解本地区丙型肝炎病毒(Hepatitis C virus,HCV)基因型构成的前提下,分析1b型丙型肝炎病毒包膜2(second envelope glycoprotein E2)区的变异和种系进化,并研究其准种变异与临床肝病活动度的关系。对宜兴市人民医院收集的抗HCV抗体阳性患者166名,RT-PCR方法检测HCVRNA,HCVRNA阳性患者采用型特异性引物分型法确定病毒基因型;选择其中未经干扰素治疗的43例1b型慢性丙型肝炎患者的血清标本,扩增E2区,从中选取肝硬化患者4例,慢性非肝硬化患者6例的E2区PCR产物纯化测序,序列采用CLUSTALW与GENBANK上多株不同型别的HCV序列进行比对分析,结果采用Phylip软件构建遗传进化树;并观察E2区高变区1(HVR-1)的氨基酸(aminoacid,aa)残基序列的变异特征;采用单链构象多态性垂直电泳检测43例患者个体内HCVE2区准种的变异情况,比较不同肝病活动度患者准种变异情况。结果表明本地区HCV以1b型为主(84.3%),对E2区基因序列和氨基酸序列变异的分析显示其变异具有一定的规律性,种系进化树提示本地区HCV病毒序列与上海、湖南、日本等地的HCV株有较近的亲缘性。43例患者中ALT高于正常的丙型肝炎患者准种复杂程度明显高于ALT正常者(P<0.05)。故本地区HCV基因变异符合中国东南部的特点,基因变异与临床肝病活动度具有相关性。     

慢性丙型肝炎个体化治疗研究进展

丙型肝炎是由丙型肝炎病毒（HCV）引起的急、慢性传染病。丙型肝炎流行广泛,慢性化率高达50％～85％,并可转化为肝硬化和肝细胞肝癌。目前慢性丙型肝炎（CHC ）的标准治疗方案为聚乙二醇干扰素联合利巴韦林,但约50％的HCV 1型感染者不能获得持续病毒学应答。研究发现,HCV基因型、病毒载量,以及宿主的性别、年龄、乙醇摄入量、肝纤维化程度、合并感染、基因多态性等因素可影响治疗效果。其中,HCV病毒载量、HCV基因型及宿主基因多态性是预测持续病毒学应答的重要因素,可用于制订个体化治疗方案。     

三种丙型肝炎包膜感染性假病毒颗粒的制备及初步应用研究

本研究构建携带HCV代表株H77(1a)、中国HeBei株(1b)以及JFH-1株(2a)丙型肝炎病毒完整的E1-E2包膜糖蛋白基因的表达质粒,间接免疫荧光法及Western blot验证了其在细胞膜(293细胞)上的正确表达.三种包膜质粒分别与慢病毒包装质粒pHR'CMV△8.2及携带EGFP报告基因的自灭活(Self-Inactivating,SIN)转移质粒pCS-CG共转染293FT细胞,产生三种不同基因型的丙型肝炎包膜感染性假型(pseudotyped)病毒颗粒,免疫荧光与Western blot分析验证了E1/E2包膜糖蛋白在假病毒颗粒上的表达与掺人,利用p24 ELISA法及感染性实验对HCV假病毒进行滴定.从上清中获得的HCV假病毒可以在体外感染肝癌细胞系Huh7及Huh7-CD81(且后者上的感染效率约为前者上的2～3倍);利用针对HCV E2蛋白的具有广谱交叉中和活性的单克隆抗体AP33建立了基于上述假病毒颗粒的丙肝病毒体外中和抗体滴定方法,并应用于丙型肝炎患者体内的中和抗体水平的研究.本研究成功包装了包括中国流行株在内的3种不同基因型的HCV包膜感染性假病毒颗粒并建立了基于假病毒颗粒的HCV体外中和抗体检测方法,为研究HCV感染早期特性及特异抗病毒药物的体外筛选提供了有效模型,并可用于HCV感染者体内及HCV基因工程疫苗免疫后中和抗体水平的分析.     

锁核酸SPC3649在HCV抗感染治疗中的研究进展

丙型肝炎病毒(HCV)是由约9.6kb个核苷酸组成的单股正链RNA病毒,属于黄病毒科丙型肝炎病毒属,被分为7个基因型及若干亚型。丙型肝炎是由HCV感染引起的常见血液传播型疾病,全球约有1.8亿人感染,引起慢性肝炎、肝硬化、肝癌。至今没有针对HCV的保护性疫苗,目前批准的标准治疗方式主要是聚乙二醇-干扰素(PEG-IFN)和利巴韦林(RBV)的联合疗法。目前这种疗法对不同基因型的感染患者的作用效果差异很大,对HCV基因2型及3型的患者,80%可以获得持续的病毒学应答反应(SVR),然而     

中国HCV3b型包膜蛋白编码基因的克隆分析与假病毒制备北大核心CSCD

从中国丙型肝炎病毒(HCV)3b亚型感染者克隆分析包膜蛋白编码基因,并用于HCV 3b亚型假病毒制备。本研究从中国HCV感染血清中筛选克隆到2个3b亚型包膜蛋白编码基因,序列分析后构建表达质粒,以1b(Con1)作为对照,体外转染293T细胞后分析不同包膜蛋白表达水平,并制备了HCV 3b亚型假病毒(HCVpp),比较不同HCVpp感染效率。结果表明:2个3b亚型包膜蛋白编码基因(C27与C30)与国际3b参考株TrKj的进化关系较近。C27与C30核苷酸与氨基酸同源性较高(分别为98.5%与98.2%),包膜蛋白编码区存在10个氨基酸位点差异,且C27包膜蛋白体外表达水平明显高于C30,其中C27可制备出高感染效率的HCV 3b型假病毒,其感染效率明显高于C30与HCV 1型(Con1)制备的HCVpp。本研究分析了2个HCV 3b亚型感染者包膜蛋白编码基因序列及表达特性,并首次获得了高感染效率的HCV 3b亚型假病毒,为HCV研究及疫苗与药物研发提供了有效工具。     

HCV复制子与细胞培养体系研究进展

丙型肝炎是由丙型肝炎病毒(hepatitis C virus,HCV)感染所导致的传染性肝病,呈现世界性流行态势,严重危害人类健康。由于病毒自身高度突变,以及广泛高效的细胞培养体系和合适的小动物模型缺乏,目前尚无可有效预防的疫苗。自1989年丙型肝炎病毒基因组首次被确定以来,Con1(1b)亚基因组复制子和JFH1(2a)毒株细胞培养体系相继建立。以此为工具,HCV生活周期多个关键环节得以阐明。近年来,研究者在Con1亚基因组复制子、JFH1和J6/JFH1细胞培养体系的基础上,构建出多个基因型和亚型的复制子和细胞培养体系。不同的体系在HCV复制与致病机制研究、抗病毒药物筛选方面,具有不同的用途及优缺点。针对HCV复制子与细胞培养体系的研究进展进行综述,可为HCV的相关研究提供参考。     

Genotypic Distribution of Hepatitis C Virus in Thailand and Southeast Asia

The majority of hepatitis C virus (HCV) infection results in chronic infection, which can lead to liver cirrhosis and hepatocellular carcinoma. Global burden of hepatitis C virus (HCV) is estimated at 150 million individuals, or 3% of the world’s population. The distribution of the seven major genotypes of HCV varies with geographical regions. Since Asia has a high incidence of HCV, we assessed the distribution of HCV genotypes in Thailand and Southeast Asia. From 588 HCV-positive samples obtained throughout Thailand, we characterized the HCV 5’ untranslated region, Core, and NS5B regions by nested PCR. Nucleotide sequences obtained from both the Core and NS5B of these isolates were subjected to phylogenetic analysis, and genotypes were assigned using published reference genotypes. Results were compared to the epidemiological data of HCV genotypes identified within Southeast Asian. Among the HCV subtypes characterized in the Thai samples, subtype 3a was the most predominant (36.4%), followed by 1a (19.9%), 1b (12.6%), 3b (9.7%) and 2a (0.5%). While genotype 1 was prevalent throughout Thailand (27–36%), genotype 3 was more common in the south. Genotype 6 (20.9%) constituted subtype 6f (7.8%), 6n (7.7%), 6i (3.4%), 6j and 6m (0.7% each), 6c (0.3%), 6v and 6xa (0.2% each) and its prevalence was significantly lower in southern Thailand compared to the north and northeast (p = 0.027 and p = 0.030, respectively). Within Southeast Asia, high prevalence of genotype 6 occurred in northern countries such as Myanmar, Laos, and Vietnam, while genotype 3 was prevalent in Thailand and Malaysia. Island nations of Singapore, Indonesia and Philippines demonstrated prevalence of genotype 1. This study further provides regional HCV genotype information that may be useful in fostering sound public health policy and tracking future patterns of HCV spread.     

Hepatitis C virus protease gene diversity in patients coinfected with human immunodeficiency virus

The clonal variability of the hepatitis C virus (HCV) protease gene in 24 individuals with HCV genotypes 1a, 1b, 2b, and 3a who were coinfected with the human immunodeficiency virus was evaluated. Within-genotype variability at the nucleotide and amino acid levels ranged from 6.5 to 8.6% and 2.2 to 3.8%, respectively. After adjustments were made for correlation of intrapatient clonal variation, mixed-model analysis indicated that nucleotide and amino acid variability among patients with different genotypes did not differ significantly. However, within individual patients, clonal variability differed by up to 5.3% and 5.8% at the nucleotide and amino acid levels, respectively, and genotype 1a had significantly greater nucleotide variability than other genotypes (P = 0.01). Significant variability exists within HCV protease gene variants at the patient level and could affect the effectiveness of HCV protease inhibitors.     

Hepatitis C virus genotypes in patients with chronic hepatitis C infection in southern Iran from 2016 to 2019

Hepatitis C is a liver disease caused by the hepatitis C virus (HCV). The treatment of HCV infection has become more complicated due to various genotypes and subtypes of HCV. The treatment of HCV has made significant advances with direct-acting antivirals. However, for the choice of medicine or the combination of drugs for hepatitis C, it is imperative to detect and discriminate the crucial HCV genotypes. The main objective of this study was to determine the pattern of circulating HCV genotypes in southern Iran, from 2016 until 2019. The other aim of the study was to determine possible associations of patients’ risk factors with HCV genotypes. A total of 803 serum samples were collected in 4 years (2016–2019) from patients with HCV antibody positive results. A total of 728 serum samples were HCV-RNA positive. The prevalence of HCV genotypes was detected using the genotype-specific RT-PCR test for serum samples obtained from 615 patients. The HCV genotype 1 (G1) was the most prevalent (48.8%) genotype in the area, with G1a, G1b, and mixed G1a/b representing 38.4%, 10.1%, and 0.3%, respectively. Genotype 3a was the next most prevalent (47.2%). Mixed genotypes 1a/3a were detected in 22 (3.6%) and finally G4 was found in 3 (0.5%) patients. The other HCV genotypes were not detected in any patient. Genotype 1 (1a and 1b alone, 1a/1b and 1a/3a coinfections) is the most prevalent HCV genotype in southern Iran. HCV G1 shows a significantly higher rate in people under 40 years old.     

Detection of hepatitis C virus RNA by in situ hybridization in paraformaldehyde fixed biopsies

Fourteen hepatitis C virus (HCV) chronically infected patients were submitted to routine liver biopsy for histological evaluation. Liver samples were assayed to HCV-RNA by in situ hybridization, using digoxigenin labeled probe. HCV genotypes were found to be predominantly type 1 (71.4%), followed by genotype 3 (21.4%), and genotype 2 (7.2%). Alanine-aminotransferase levels were raised in 10 patients. The histopathological scores were minimal (21.4%), mild (57.2%), and moderate (21.4%). Viral RNA was detected in liver cells from nine patients (64.3%). ISH method provides localization and poor confirmation of HCV RNA in the liver tissue of HCV chronic patients.     

Activation and evasion of the antiviral 2'-5' oligoadenylate synthetase/ribonuclease L pathway by hepatitis C virus mRNA

Chronic hepatitis C virus (HCV) infections are a significant cause of morbidity and mortality worldwide. Interferon-alpha2b treatment, alone or in combination with ribavirin, eliminates HCV from some patients, but patients infected with HCV genotype 1 viruses are cured less frequently than patients infected with HCV genotype 2 or 3 viruses. We report that HCV mRNA was detected and destroyed by the interferon-regulated antiviral 2'-5' oligoadenylate synthetase/ ribonuclease L pathway present in cytoplasmic extracts of HeLa cells. Ribonuclease L cleaved HCV mRNA into fragments 200 to 500 bases in length. Ribonuclease L cleaved HCV mRNA predominately at UA and UU dinucleotides within loops of predicted stem-loop structures. HCV mRNAs from relatively interferon-resistant genotypes (HCV genotypes 1a and 1b) have fewer UA and UU dinucleotides than HCV mRNAs from more interferon-sensitive genotypes (HCV genotypes 2a, 2b, 3a, and 3b). HCV 2a mRNA, with 73 more UA and UU dinucleotides than HCV 1a mRNA, was cleaved by RNase L more readily than HCV 1a mRNA. In patients, HCV 1b mRNAs accumulated silent mutations preferentially at UA and UU dinucleotides during interferon therapy. These results suggest that the sensitivity of HCV infections to interferon therapy may correlate with the efficiency by which RNase L cleaves HCV mRNA.     

Identification of HCV genotypes in HCV infected blood donors

HCV infection is a leading cause of chronic liver disease, including cirrhosis of the liver. There are at least six major genotypes and more than 50 subtypes of HCV. The prevalence and distribution of HCV genotypes depend on geographical location. The aim of this study was to identify and compare the HCV genotypes in HCV infected blood donors and patients. In this cross-sectional study, 167 serum samples from 103 blood donors and 64 patients with hepatitis C were investigated for HCV genotypes. HCV genotyping was carried out using type-specific primers from the core region of the viral genome. The highest frequency was for genotype 1a, with 53 and 34 (51.5% versus 53.1%) of subjects in blood donors and patients respectively. Genotype 3a and 1b were the other frequent genotypes with 4 and 16 (3.9% versus 25%) and 39 and 10 (37.9% versus 15.6%) subjects, respectively. There was not any statistical significant association between the place of infection of the patients and genotype. The results of this study indicate that the distribution of genotypes in the two populations was similar. The dominant HCV genotypes between blood donors and patients were 1a, 3a and 1b respectively.     

Molecular characterization of human immunodeficiency virus type 1 and hepatitis C virus in paid blood donors and injection drug users in china

China is facing a rapid upsurge in cases of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection due to large numbers of paid blood donors (PBD), injection drug users (IDU), and sexual partners of infected individuals. In this report, a total of 236 HIV-1-positive blood samples were collected from PBD, IDU, and their sexual partners in the most severely affected provinces, such as Henan, Yunnan, Guangxi, and Xinjiang. PCR was used to amplify the p17 region of gag and the C2-V3 region of env of HIV-1 and the 5' noncoding region and a region of E1/E2 of HCV. Genetic characterization of viral sequences indicated that there are two major epidemics of HIV-1 and multiple HCV epidemics in China. The PBD and transfusion recipients in Henan harbored HIV-1 subtype B', which is similar to the virus found in Thailand, and HCV genotypes 1b and 2a, whereas the IDU in Yunnan, Guangxi, and Xinjiang carried HIV-1 circulating recombinant forms 07 and 08, which resemble those in India, and HCV genotypes 1b, 3a, and 3b. Our findings show that the epidemics of HIV-1 and HCV infection in China are the consequences of multiple introductions. The distinct distribution patterns of both the HIV-1 and HCV genotypes in the different high-risk groups are tightly linked to the mode of transmission rather than geographic proximity. These findings provide information relevant to antiviral therapy and vaccine development in China and should assist public health workers in implementing measures to reduce the further dissemination of these viruses in the world's most populous nation.     

Distribution of hepatitis C virus genotypes in Croatia--a 10 year retrospective study of four geographic regions

The aim of this 10-year retrospective study was to investigate the distribution of HCV genotypes in patients with chronic hepatitis C monitored in the largest center for molecular diagnostics of HCV infection in Croatia. The study enrolled 1163 anti-HCV positive adults with detectable HCV RNA in the plasma. The patients were classified in four regions: Zagreb and surrounding continental area, Split, Slavonija and Rijeka. HCV genotyping was performed by using VERSANT HCV Genotyping Assay (LIPA) (Bayer Diagnostics, Puteaux Cedex, France). Statistical analysis was performed by using Statistica for Windows V.5.1. The majority of HCV infections in the study population were caused by genotypes 1 (58.8% of infected patients) and 3 (35.6%). Percentages of patients infected with subtypes 1b and 1a were 37.4% and 13.1%, respectively. Genotypes 2 and 4 were present in a very low percentage of patients (2.2% and 3.4%, respectively) while genotypes 5 and 6 were not detected. Analysis of regional differences in the distribution of HCV genotypes revealed similar percentages of subtype 3a and 1b infections in the Split region while the majority of infections in other regions were caused by subtype 1b. Infections with genotypes 2 and 4 were present in less than 5% of patients in all geographic regions. Analysis of an association between risk factors for infection and distribution of genotypes and subtypes in a subset of patients from the Split region confirmed the association between IVDU and subtype 3a. We conclude that the prevalence of HCV genotypes and subtypes follows the pattern of other Southern and Eastern European Countries with the predominance of subtypes 1b, 3a and 1a.     

A shift in the Hepatitis C virus genotype dominance in blood donor samples from Thailand

Hepatitis C virus (HCV) can be classified into six major genotypes. The HCV genotypes variability accounts for its geographical distribution, its responses to treatments and the clinical outcomes. The aim of this study was to determine the distribution of HCV genotypes among volunteer blood donors in Thailand. Samples from 135 anti-HCV positive blood donors were analyzed. HCV RNA and genotyping was carried out using nested polymerase chain reaction (PCR) and genotype-specific primer PCR for a portion of the core region. HCV RNA was detected in 109 samples (80.7%). Genotype analysis demonstrated four different genotypes. The most common was genotype 3a (36.7%), followed by genotype 6 (29.4%), 1a (19.3%), 1b (6.4%) and mixed infection (1.8%). Seven samples were untyped (6.4%) in the present study. In several previous reports, the prevalence found in Thailand was HCV genotypes 3, 1 and 6. The present results show an increasing importance of the genotype 6 in HCV infections. This study has also described for the first time in Thailand mixed infections of HCV genotypes.