吉林省延边地区朝鲜族与汉族HCV基因型特征分析

目的分析吉林省延边地区朝鲜族与汉族丙型肝炎病毒（HCV）基因型特征。方法采用荧光定量PCR方法和探针反向杂交技术对延边地区收治的62例朝鲜族和57例汉族慢性丙型肝炎患者进行HCV病毒载量检测和HCV基因型分析,比较HCV基因型在朝鲜族与汉族之间的分布差异,并分析HCV基因型与病毒载量、2型糖尿病和肝病严重程度之间的相关性。结果朝鲜族HCV基因型显示,1b型占45．16％（28／62）、2a型占38．71％（24／62）、未分型占16．13％（（10／62）;汉族HCV基因型显示,1b型占45．61％（26／57）,2a型占40．35％（23／57）,未分型占14．14％（8／57）,朝、汉族之间各基因型分布差异无统计学意义（P〉0．05）。HCV各基因型之间HCV病毒载量差异和合并2型糖尿病分布差异之间无统计学意义（P〉0．05）,而HCV1b型在中、重度慢性丙型肝炎患者与轻度慢性丙型肝炎患者中所占的比例差异具有统计学意义（P〈0．05）。结论①吉林省延边地区HCV基因型中以1b型最多,2a型次之,还有少数其他基因型;②HCV基因型在朝、汉民族之间分布无差异;③HCV基因型与HCV—RNA载量无关;④HCV基因型在合并糖尿病者与未合并糖尿病者之间分布无差异;⑤1b型HCV感染者病情相对较重。     

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.     

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.     

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。     

我国六省市（区）部分人群丙型肝炎病毒感染调查

目的 了解我国健康人群丙型肝炎病毒感染现状.方法 采用北京万泰生物药业的HCV EIA诊断试剂检测人群血清丙型肝炎病毒（丙肝,HCV）IgG抗体.结果 六省市（区）人群血清共9 538份,HCV抗体阳性数37份,总阳性率为0.39％,其中北京人群阳性率0.23％,黑龙江人群为0.74％,山东人群为0.26％,宁夏人群为0.10％,甘肃和四川人群阳性率均为0.44％.对37例HCV阳性者性别及年龄分析,男性19例占51.35％,女性18例占48.65％,＜10岁年龄组1例,占2.70％,10～19岁组5例,占13.51％,20～29岁4例占10.81％,30～39岁组6例,占16.22％,40～49岁组9例,占24.32％,≥50岁有12例占32.43％.对HCV阳性者重叠其他型别肝炎病毒感染分析,单独HCV阳性2例,占5.41％,伴有HAV-IgG抗体阳性35例,占94.59％,伴有HEV-IgG抗体阳性10例,占27.03％,伴有HBsAg、HBcAb和HbeAb阳性者2例,占5.41％,该2例HAV和HEV抗体也阳性.结论 六省市（区）人群HCV感染率均在1％以下,感染者年龄以50岁以上最高,决大多数HCV阳性者重叠有其他型别的肝炎病毒感染.     

HCV反向点杂交基因分型方法的建立

目的利用反向点杂交技术建立HCV基因分型新方法。方法在HCV5’端非编码区（5’NCR）设计引物和分型探针（1．2．3．6型）,采用反向点杂交分型技术对53例HCVRNA阳性（浓度均在102～107IU／mL之间）血清进行分型,并以基因序列分析作为金标准,对新方法的敏感性、特异性和重复性进行评价。结果反向点杂交基因分型新方法检出的基因型有52例：1b型32例占60．38％,2a型4例占7．55％,6a型8例占15．09％．3b型8例占15．09％;未检出的基因型有1例,用基因序列分析也未能确定型别（失败的原因应是该HCVRNA扩增产物浓度偏低2．24×10^2IU／mL）。本研究的方法敏感性为98．1％,特异性为100％,随机抽取20份标本再次检测的结果与前次检测的结果完全一致,重复性好。结论相对现有的型特异性PCR分型、基因芯片、核酸序列分析等各种方法,反向点杂交技术用于HCV基因分型是一种准确有效和简便经济的方法。     

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。     

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.     

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。     

Nested restriction site-specific PCR to detect and type hepatitis C virus (HCV): a rapid method to distinguish HCV subtype 1b from other genotypes

Genotypic differentiation of hepatitis C virus (HCV) has become an integral part of clinical management and epidemiologic studies of hepatitis C infections. Thus, it is extremely important in areas such as the Czech Republic, where current instrumentation and kits for assessing HCV infection are too costly for widespread use. We describe a new and relatively inexpensive method called nested restriction site-specific PCR (RSS-PCR) that generates a "fingerprint" pattern to represent an HCV genotype without the use of restriction endonucleases and that specifically differentiates HCV genotype 1b from the other HCV genotypes. The RSS-PCR method was applied directly to serum samples from patients with hepatitis C from the Czech Republic and from patients with known HCV genotypes from the United States. The method was validated by comparison of the subtype determined by RSS-PCR to the subtype determined from analysis of the 5' noncoding region (NC) or the nonstructural protein gene (NS5b) nucleotide sequence of HCV in these clinical samples. From 75 Czech samples containing HCV RNA, three distinct RSS-PCR patterns were observed; 54 were predicted to contain subtype 1b, 19 were predicted to contain subtype 1a, and 2 were predicted to contain subtype 3a. Among 54 samples predicted to contain HCV genotype 1b, all were confirmed by their 5' NC or NS5b sequences to be subtype 1b. Thus, both the sensitivity and specificity of the RSS-PCR test for the differentiation of HCV subtype 1b from the others were 100%. While the assay described here was designed to specifically differentiate HCV subtype 1b from the other HCV genotypes, the RSS-PCR method can be modified to differentiate any HCV genotype or subtype of interest. Its simplicity and speed may provide new opportunities to study the epidemiology of HCV infections and the relationship between HCV genotypes and clinical outcome by more laboratories throughout the world.     

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.     

The Prevalence of Anti-Hepatitis C Antibody among Acute Febrile Illness Cases in Idar Taluk,Gujarat, West India

Purpose: The major cause of chronic hepatitis is infections with hepatitis B virus and hepatitis C virus (HCV) globally. However, there exists sparse epidemiological data regarding the prevalence of HCV infection from India. Methodology: We carried out a cross-sectional study to estimate the prevalence of anti-HCV antibody among acute febrile illness cases aged between 1 and 65 years in Idar Taluk, Sabarkantha district, Gujarat state located in West India. A total of 702 serum samples collected from the study area during the year 2017, were screened for anti-hepatitis C IgG by enzyme-linked immunosorbent assay. The serum samples screened positive were then subjected to molecular testing for confirmation. Results: Among the 702 study participants screened, 16 cases were reported to be anti-HCV IgG positive with an estimated seroprevalence rate of 2.3% (95% confidence interval: 1.4%–3.7%). Out of the 16 cases, two samples were confirmed positive by molecular testing indicating active infection. When analysed phylogenetically, one strain was genotyped as HCV1b genotype, and the other one was clustered along with HCV3a genotype. Both the patients with hepatitis C infection were observed to be having a probable 1-year survival rate of 100% and a 2-year survival rate of 85% when the Child-Turcotte-Pugh classification was applied. Conclusion: The estimated seroprevalence of hepatitis C in Idar Taluk, Sabarkantha district, west India was 2.3%. HCV genotypes 1b and 3a were observed to be circulating in the study area.     

Hepatitis C virus genotype distribution in China: predominance of closely related subtype 1b isolates and existence of new genotype 6 variants

To determine hepatitis C virus (HCV) genotype distribution in China, a total of 148 HCV RNA positive serum samples were collected from nine geographic areas and subjected to RT-PCR followed by direct DNA sequencing and phylogenetic analysis of the core, E1, and NS5B regions. HCV was genotyped in 139 (93.9%) samples. Among them subtype 1b was the most predominant [66% (92/139)] followed by 2a [14% (19/139)]. Of 92 subtype 1b isolates, 35 (38%) and 30 (33%) formed two clusters, designated groups A and B. Group A was prevalent throughout China, while group B was predominant in the central and southern regions. In three cities in the Pearl River Delta, subtype 6a replaced 2a as the second most predominant subtype, and in Kunming (southwest) multiple HCV genotypes/subtypes were present. New variants of HCV genotype 6 were discovered in three samples from Kunming and one in Guangzhou in the Pearl River Delta.     

Molecular epidemiology of hepatitis B, C and D viruses in Turkish patients

Summary. Different genotypes of the hepatitis viruses may influence the clinical outcome of the disease. The distribution of genotypes may vary according to geographical regions. The aim of this study was to evaluate hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis D virus (HDV) genotypes in Turkish patients with chronic hepatitis in a large cohort of patients. Genotyping was performed in 41, 59 and 365 patients with chronic hepatitis B, D and C, respectively, and 36 hemodialysis patients with chronic hepatitis C. Genotypes were determined by direct sequencing in hepatitis B and by polymerase chain reaction-restriction fragment length polymorphism in hepatitis C and D patients. In addition, HBV subtyping by multiplex PCR and subtype specific ELISA were performed in 83 and 71 HBsAg (+) blood donors, respectively. All hepatitis B (100%) and hepatitis D (100%) patients had genotype D and type I, respectively. HBsAg subtyping by two methods yielded that 99% of the patients were subtype ayw. S gene amino acid sequence in the 41 patients included for HBV genotyping revealed the ayw2 subtype. Genotype distribution of 365 patients with chronic C hepatitis were as follows: 306 (84%) patients genotype 1b, 43 (11%) patients genotype 1a, 10 (3%) patients genotype 2, 3 (1%) patients genotype 3, 3 (1%) patients genotype 4. Among 36 patients receiving hemodialysis, 28 (78%) patients had genotype 1b and 8 (22%) patients had genotype 1a. The study indicates that Turkish patients with chronic viral hepatitis show very little genotypic heterogeneity. Subtype ayw and the genotype D of HBV DNA, and the type I of HDV RNA represent almost 100% of related infections. The genotype 1b of HCV RNA was found to be significantly dominant in Turkish patients.     

Genotype distribution and molecular epidemiology of hepatitis C virus in blood donors from southeast France

The genotype distribution of hepatitis C virus (HCV) in blood donors from southeast France was tracked for a period of 13 years (1991 to 2003). Virus genomes from 321 samples were analyzed by amplification and sequencing of the NS5b and E1 regions. The most frequent genotypes were 1b (30.2%), 1a (27.7%), and 3a (22.4%). Although it was less common, genotype 2 was characterized by the presence of strains belonging to 11 different subtypes, including 5 that had never been characterized. Genotypes 1a, 1b, 3a, and 4a presented typical "epidemic" profiles, with a large number of isolates per subtype and short mean genetic distances between isolates. Type 2 isolates displayed a typical "endemic" profile, with a large number of subtypes and very few isolates in each subtype. The epidemiology of HCV infection in southeast France changed radically during the study period in relation to modifications in the etiology of infection. We observed the emergence of new epidemic subtypes (subtypes 1a and 3) linked to intravenous drug use and a decrease in the types linked to blood transfusion and nosocomial infection (epidemic subtype 1b and endemic type 2). Comparison of strains from blood donors with strains from a cohort of inpatients in the same region during 2001 and 2002 demonstrated for the first time that the monitoring of blood donors is a generally valid indicator of HCV epidemiology in terms of genotype distribution.     

Use of sequence analysis of the NS5B region for routine genotyping of hepatitis C virus with reference to C/E1 and 5' untranslated region sequences

Nucleotide sequence analysis of the NS5B region was performed to identify genotypes of 8,479 hepatitis C virus (HCV) RNA-positive patient samples collected in the Canadian province of Quebec. Genotypes could be determined for 97.3% of patients. Genotypes 1 to 6 were found in 59.4, 9.0, 25.7, 3.6, 0.6, and 1.8% of patients, respectively. Two isolates did not classify within the six genotypes. The subtype 1 distribution was 76.7% 1a, 22.6% 1b, and 0.7% others, while the subtype 2 distribution was 31.8% 2a, 47.6% 2b, 10.9% 2c, 4.1% 2i, and 5.6% others. Subtype 3a accounted for 99.1% of genotype 3 strains, while all genotype 5 samples were of subtype 5a. The subtype 4 distribution was 39.2% 4a, 15.4% 4k, 11.6% 4d, 10.2% 4r, and 23.6% others. The subtype 6 distribution was 40.4% 6e, 20.5% 6a, and 39.1% others. The 5' untranslated region (5'UTR) sequences of subtype 6e were indistinguishable from those of genotype 1. All samples that did not classify within the established subtypes were also sequenced in C/E1 and 5'UTR. C/E1 phylogenetic reconstructions were analogous to those of NS5B. The sequences identified in this study allowed the provisional assignments of subtypes 1j, 1k, 2m, 2r, 3i, 4q, 6q, 6r, and 6s. Sixty-four (0.8%) isolates classifying within genotypes 1 to 6 could not be assigned to one of the recognized subtypes. Our results show that genotyping of HCV by nucleotide sequence analysis of NS5B is efficient, allows the accurate discrimination of subtypes, and is an effective tool for studying the molecular epidemiology of HCV.