河北某非法采血村人群感染HCV基因亚型调查

目的 了解河北某非法采血村村民感染丙型肝炎病毒(HCV)基因亚型分布情况.方法 对该村全体村民采集静脉血标本计520份,无菌分离血清;以RT-PCR法扩增其中可利用的483份血清HCV之C/E1基因片段,双脱氧链终止法测序;用Mega4.0软件进行HCV系统进化分析,构建系统进化树,判定基因亚型.结果 483份标本中,HCV RNA阳性者70份,阳性率14.5%;基因分型1b亚型36例,占51.4%;2a亚型34例,占48.6%.结论 该村村民血清HCV检出率约为14.5%,远高于一般人群;HCV亚型构成为1b、2a各半.     

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.     

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。     

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。     

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。     

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.     

广州地区人类白细胞抗原-Ⅰ类与丙型肝炎病毒-6a型感染相关性的研究

目的:广州地区HLA-Ⅰ类抗原和HCV-6a感染相关性的研究。方法:选取2002~2008年HCV抗体检测双试剂阳性无偿献血者250名,随机抽取621名广州地区无血缘关系的健康无偿献血者作为对照,用逆转录-套式聚合酶链反应扩增HCVE1和NS5B基因,对E1和NS5B区扩增阳性的PCR产物核苷酸序列测定,将所测定的HCVE1、NS5B进行基因分型。LU-MINEX-SSO方法检测HLA-A,B检测样本HLA-A,B位点。根据公式对HLA型别相关性的相对危险性进行评估。结果:250名无偿献血者中有69个样本的HCVE1和NS5B区分型结果为HCV-6a,占到分析总数的27.6%。A*33、A*30与HCV-6a感染相关评估的RR值均大于4,B*56、B*54、B*55、B*58、B*76与HCV-6a感染相关评估的RR值均大于4,与HCV-6a感染具有强关联性。结论:广州地区HCV-6a的感染与A*19和B*22存在强关联性,对HCV-6a的地域性流行、治疗和预后评价有一定预见作用。     

HCV在中国流行多样性和独特生长史的分析

目的 探讨中国丙型肝炎患者中HCV的基因型分布类型,并进行HCV在中国流行病学历史分析,进而研究HCV在中国的分子流行病学和进化动力学特征.方法 从423例丙型肝炎患者的血清中提取HCV RNA并进行cDNA反转录,扩增E1和NS5B两个基因区的核苷酸序列,排除不合格及不适合分析的病毒株序列,再对合格序列进行系统发育分析（phylogenetic analysis）.运用BEAST软件中的Bayesian MCMC （Markov Chain Monte Carlo）算法来进行Coalescence分析,通过重构BSPs（Bayesian skyline plots）图回溯HCV的流行病学历史.结果 HCV分离株的基因分型如下：共包括6个基因型,12个基因亚型（1b：65.9％,6a：17.1％,2a：7.4％,3a：3.6％,3b：3.3％,6e：0.76％,1a,1c,2b,2f,4d以及5a共占0.25％）,以及2个新基因型6变异体.所产生的5个BSP曲线图均凸显1993年至2000年这一时间段,中国的HCV感染数量呈现指数增长,随后则陡然下降.结论 证实了HUV在中国多样性的流行现状,反映了HCV不断变化的基因型流行模式;1993年至2000年期间由于“单采血浆”事件的影响,HCV在中国的感染数量经历了一段“指数”增长期.     

支持HCV 1b亚基因组复制的细胞模型的建立

目的 建立一个稳定支持中国人群HCV 1b亚基因组高效复制的体外细胞模型。方法 构建中国人群HCV 1b亚型亚基因组复制子质粒，通过体外转录的方法获得亚基因组复制子RNA，采用电穿孔技术把RNA转染到Huh-7.5.1细胞中，经过G418筛选含有HCV 1b亚基因组的细胞集落。RT-PCR检测细胞集落中的HCV亚基因组以及Western blot检测细胞集落中的HCV蛋白的表达。用干扰素（IFN）处理含有HCV亚基因组的细胞，观察细胞中HCV RNA的变化。结果 G418药物筛选得到了支持HCV1b亚基因组复制的细胞集落。RT-PCR 检测有HCV RNA NS4B的表达，Western blot检测有HCV的非结构蛋白NS5B的表达。IFN处理含有HCV复制子的细胞，HCV RNA水平明显降低。结论 成功建立了支持中国人群HCV 1b亚基因组高效复制的体外细胞模型，为进一步研究HCV致病机制、治疗药物筛选及疫苗方面的研究打下了基础。     

北京市男男性接触HIV-1感染者膜蛋白基因序列测定及亚型分析

目的 了解北京市男男性接触人群(MSM)中HIV-1的最新流行趋势及膜蛋白V3环序列特征.方法 巢式聚合酶链式反应(n-PER)扩增2007年提取的北京市男男性接触HIV感染者基因组DNA样品,对膜蛋白基因C2-V3区测序,进行病毒亚型及V3环序列特点分析.结果 11例样本中,4例是欧美B亚型,5例是AE重组亚型,1例是BC重组亚型,1例是01B重组亚型.V3环顶端四肽以GPGQ和GPGR为主.结论 北京市男男性接触HIV-1感染者中重组亚型呈蔓延流行趋势.     

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.     

New hepatitis C virus (HCV) genotyping system that allows for identification of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a.

Recent studies have focused on whether different hepatitis C virus (HCV) genotypes are associated with different profiles of pathogenicity, infectivity, and response to antiviral therapy. The establishment of a simple and precise genotyping system for HCV is essential to address these issues. A new genotyping system based on PCR of the core region with genotype-specific PCR primers for the determination of HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5a, and 6a was developed. A total of 607 samples (379 from Japan, 63 from the United States, 53 from Korea, 35 from Taiwan, 32 from China, 20 from Hong Kong, 15 from Australia, 6 from Egypt, 3 from Bangladesh, and 1 from South Africa) were tested by both the assay of Okamoto et al. (H. Okamoto, Y. Sugiyama, S. Okada, K. Kurai, Y. Akahane, Y. Sugai, T. Tanaka, K. Sato, F. Tsuda, Y. Miyamura, and M. Mayumi, J. Gen. Virol. 73:673-679, 1992) and this new genotyping system. Comparison of the results showed concordant results for 539 samples (88.8%). Of the 68 samples with discordant results, the nucleotide sequences of the HCV isolates were determined in 23, and their genotypes were determined by molecular evolutionary analysis. In all 23 samples, the assignment of genotype by our new genotyping system was correct. This genotyping system may be useful for large-scale determination of HCV genotypes in clinical studies.     

Assessment of HCV genotypes in Yunnan Province of Southwest China

Recently, we reported that the frequency of hepatitis C virus (HCV) genotypes and subtypes has rapidly changed among intravenous drug users (IDUs) in Yunnan Province over the last 5 years; this is especially true for subtype 6a which has increased in frequency from 5 to 15%. Here, we assessed 120 HCV-positive plasma samples from the general population (GP). HCV NS5B fragments were amplified and sequenced by PCR. We identified four HCV genotypes (1, 2, 3 and 6) and seven HCV subtypes (1b, 2a, 3a, 3b, 6a, 6n, and 6k) in this population. Genotype 3 was predominant, with a distribution frequency of 0.484, followed by genotype 1 (0.283), genotype 6 (0.133) and genotype 2 (0.100). HCV subtypes 3b (frequency 0.292) and 1b (frequency 0.283) were the most common subtypes. A comparison of the current data with previous results reported for IDUs showed that the distribution frequencies of genotypes 1, 2 and 6 were significantly different between patients in the GP and IDUs (P < 0.05). Among the HCV subtypes, the distribution frequencies of 1b, 2a, 6a, and 6n were significantly different between patients in the GP and IDU groups (P < 0.05). Moreover, Phylogenetic analyses showed that HCV subtype 6a strains isolated from IDUs and the GP were intermixed and not separately clustered. HCV subtype 6a was predominant not only among IDUs but also among those in the GP in the Guangdong Province and Vietnam. However, HCV subtype 6a was predominant only among IDUs and not among those in the GP in the Yunnan and Guangxi Provinces. Our results indicate that the HCV subtype 6a could rapidly spread across China.     

Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay

Hepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5'-untranslated region (5'UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5'UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5'UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5'UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.     

The nationwide distribution and trends of hepatitis C virus genotypes in mainland China

Comprehensive data on hepatitis C virus (HCV) genotypes distribution is critical for treatment regimen selection, vaccine design, and drug development. This study aimed to understand the dynamic distribution of HCV genotypes in Mainland China. Three hundred sixty-two studies published from January 1993 to December 2017 involving 64 891 samples (5133 injecting drug users, 2748 volunteer blood donors, 1509 former paid plasma donors, 160 sexually encounters, and 1992 human immunodeficiency virus (HIV)/HCV coinfection patients) were eligible for the quantitative synthesis estimation. Pooled proportion of HCV genotypes (and 95% confidence intervals [CIs]) was estimated through the Freeman-Tukey double arcsine transformation by period, region, and risk group. A sharp decline of the subtype 1b was observed in all regions except in northwestern and central regions. The genotypes 3 and 6 showed an obvious increase in southern and southwestern regions and have already spread nationwide. After 2010, subtype 1b was the most dominant variant in all regions and risk groups, accounting for 54.0% (95% CI, 51.9-56.1) of all national infections. Subtype 2a was the second most prevalent strain in all regions except in the south and southwest, with 15.4% (95% CI, 13.1-17.8) national infections. The subtype 6a in southern region and 3b and 3a in southwestern region had a higher proportion of infections than that in other regions. In addition, the genotypes 3 and 6 are already prevalent in almost all risk groups. The distribution of HCV genotypes were sharply shifting in China in the past three decades. The HCV subtype 1b posed a sharp decline, whereas genotypes 3 and 6 played an increasing role in the regional and populational HCV pandemic.     

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.     

Molecular epidemiology of HCV genotypes among injection drug users in Taiwan: Full‐length sequences of two new subtype 6w strains and a recombinant form_2b6w

Human immunodeficiency virus type 1 (HIV‐1) circulating recombinant form (CRF) 07_BC strain has caused serious outbreaks among injection drug users in Taiwan since 2004. The objective of this study was to conduct a molecular epidemiological study of HCV genotypes in intravenous drug users in Taiwan. Blood samples and questionnaires from 591 intravenous drug users infected with HIV‐1 were collected nationwide. In total, 180 samples were selected for HCV genotyping using multiplex PCR and phylogenetic analysis of the core, E1 and NS5B regions. The Inno‐Lipa assay was used to confirm multiple infections with different genotypes. Eighty percent had a single infection with subtype 1b being the most common subtype (24%), 12% had double infections and two had triple infections. In addition, three recombinant forms (RFs)‐2a1a, 3a1b, and 2b6w were identified. Phylogenetic analyses showed that the 3a, 6a, and 6n strains were clustered with strains present in Thailand and mainland China. Full‐length sequence analysis showed that two 6w strains shared 89.4–90.2% sequence homology with the 6(r) strain from the Guangdong Province, China. Bootscan analysis revealed that the recombination breakpoint of RF_2b6w was located at the NS2‐NS3 junction. In summary, the distribution of HCV genotypes among Taiwanese intravenous drug users was complex and more than 12% of the drug users were infected with more than one genotype of HCV. J. Med. Virol. 82:57–68, 2010. © 2009 Wiley‐Liss, Inc.     

Prevalence of hepatitis C virus and distribution of its genotypes in Northern Eurasia

Summary We tested hepatitis C virus (HCV) antibody in 4 216 sera collected from healthy people living in European part of Russia (including Northern, North-Western, Central, Central-Blacksoil, Volga-Vyatka, Volga, and North-Caucasian regions), non-European part of Russia (the Urals, East-Siberia, and the Far-East regions) and Mongolia. Prevalence of HCV antibody varied significantly by regions, ranging from 0.7% in Central region of European part of Russia to 10.7% in Mongolia. Genotyping of HCV (into 1a, 1b, 2a, 2b, and 3a) was performed on 469 sera from blood donors and patients (in Russia, Moldova, Turkmenistan, and Mongolia) who were positive for both HCV antibody and RNA. Genotype 1b was the most dominant genotype irrespective of regions (68.9%), with the highest rate in Moldova (96%). HCV unclassificable into genotypes 1a-to-3a was found in 28 (6.0%) samples: particularly 4 of 10 samples from Lipetzk were untypable. Overall, HCV genotypes in European part of Russia were more similar to those in European countries, while those in Eastern part of Russia more similar to China or Japan. Genotype distribution was not associated with the clinical expression of HCV disease: acute hepatitis, chronic hepatitis or liver cirrhosis.     

Identification of hepatitis C virus genotype 6 in Korean patients by analysis of 5' untranslated region using a matrix assisted laser desorption/ionization time of flight-based assay, restriction fragment mass polymorphism

Previous surveys of the prevalence of hepatitis C virus (HCV) in Korea have identified types 1 and 2, but little has been said of other genotypes and viral subtypes. In this study, HCV genotypes in Korea were investigated using Restriction Fragment Mass Polymorphism (RFMP) assay, a sensitive and specific method for genotyping based on MALDI-TOF mass spectrometry. A total of 1,043 independent serum samples from HCV-infected patients were analyzed. Of interest, 15 subjects (1.4%) were determined to contain HCV genotype 6 and 46 subjects (4.4%) contained mixed genotypes with the most prevalent genotypes being HCV 1b and 2a/c (45.0% and 35.4%, respectively). The 15 subjects with HCV genotype 6 comprised eight cases of subtype 6c, including one case of mixed infection with 1b, three cases of HCV 6a, and six cases of unassigned subtypes. Sequencing corroborated the identity of genotype 6 from 13 subjects, while the line probe assay (LiPA) mis-identified them as genotype 1b. The majority (7/9) of the genotype 6 patients enrolled for interferon/ribavirin therapy, achieved a sustained virologic response. The ability of the RFMP assay to differentiate various HCV genotypes should enable better analysis of the relationship between HCV genotype and disease prognosis.