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。     

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.     

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。     

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.     

慢性丙型肝炎患者丙型肝炎病毒NS5A区序列与干扰素疗效的关系

目的　探讨上海地区丙型肝炎病毒 (HCV) 1b亚型慢性感染者的血清HCV非结构基因5A(NS5A)与干扰素 (IFN)疗效的关系。方法　收集上海地区 2 4例HCV1b慢性感染者在干扰素治疗前后及随访过程中的血清标本 ,定量检测治疗前血清HCVRNA ,用逆转录 聚合酶链反应方法扩增NS5A的干扰素敏感决定区 (ISDR)基因并进行测序和分析。另扩增干扰素应答类型不同的 3例患者治疗前后共 5株HCV病毒的NS5A全长序列 ,测序后作种系发生树分析及蛋白二级结构预测。结果　治疗前血清HCVRNA的定量结果显示 ,持续应答组的病毒滴度 (平均滴度 4 50× 1 0 4copies ml)明显低于复发组和无应答组 (平均滴度 1 82× 1 0 7copies ml)。 2 4例慢性丙型肝炎患者干扰素治疗前血清HCV的ISDR氨基酸序列与抗干扰素的HCV J株比较 ,1 3例为野生型 ,1 1例为中间型 ,无突变型。 6例完全应答者 3例感染的是野生型株 ,另 3例感染的是中间型病毒株。 5株HCV病毒的NS5A全长序列种系发生树显示 ,3种不同应答类型株在种系发生上分属 3个组别 ,无应答株与抗干扰素的HCV J株关系相近被归为 1组。蛋白质二级结构预测显示 ,上述病毒株NS5A蛋白在二级结构方面基本相似 ,仅在 2 2 55～ 2 2 89范围内有明显不同 ,这一区域与PKR结合域部分重叠。结论　HCVNS5A基因     

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。     

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.     

慢性丙型肝炎患者基因分型与病毒载量、肝功能、抗体检测结果的相关性分析

目的 分析慢性丙型肝炎患者丙型肝炎病毒(HCV)基因型分布概况,并探讨其与性别、年龄、病毒载量、肝功能、抗体检测结果的关系,为慢性丙型肝炎的诊疗提供实验室依据。方法 通过回顾性研究,收集2020年1月至2021年12月就诊于首都医科大学附属北京地坛医院未经过治疗的247例慢性丙型肝炎患者一般临床资料、基因分型、丙型肝炎病毒核酸定量结果、肝酶[丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、总胆红素(TBIL)、直接胆红素(DBIL)、碱性磷酸酶(ALP)、γ-谷氨酰转移酶(GGT)、白蛋白(ALB)]及丙型肝炎病毒抗体S/CO值,比较分析HCV不同基因型病毒载量、肝功能及丙型肝炎病毒抗体检测结果的差异。结果 在247例慢性丙型肝炎患者中,1b型143例(57.9%)、2a型71例(28.7%)、3a型19例(7.7%)、3b型14例(5.7%),不同基因型性别、年龄、病毒载量、TBIL、DBIL、ALP、ALB差异无统计学意义(P>0.05),3a型、3b型ALT、AST水平显著高于1b型、2a型(P<0.05),3a型GGT水平显著高于1b型、2a型(P<...     

全血与血液制品中HCV RNA检测及其基因分型的初步研究

对一组临床应用的全血（配血用标本）、血液制剂与丙种球蛋白制剂进行检测。血浆制剂抗ＨＣＶ阳性率显著高于全血（３２．６４％与１１．６８％、Ｐ＜０．０１）。三组标本中抗ＨＣＶ阳性者ＨＣＶ ＲＮＡ检出率基本相似。但丙球制剂组ＨＣＶ ＲＮＡ阳性标本在ＰＣＲ中的反应强度显著低于前两者。对部份标本作ＨＣＶ基因亚型检测，三组总体ＨＣＶ亚型表达频率为，原型：２．９４％；Ｋ１：４７．０６％；Ｋ２ａ：３２．３５％；Ｋ２     

A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma

Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined.     

Hepatitis C virus infection among pregnant women in Yaounde,Cameroon: prevalence,viremia, and genotypes

Central Africa is considered to be an area of high endemic hepatitis C infection. To determine the prevalence of anti-HCV antibodies, HCV RNA, and the genotype distribution in Cameroon, 1,494 pregnant women attending antenatal care units in Yaounde, Cameroon were screened for HCV infection. Anti-HCV antibodies were detected with a 3rd generation ELISA (Monolisa anti-HCV plus version 2, BioRad, Richmond, CA). All anti-HCV antibody-positive sera were then tested with another 3rd generation ELISA (AxSYM) HCV version 3, Abbott Laboratories, Abbott Park, IL) and subsequently for HCV RNA (Amplicor HCV, Roche Diagnostics, Basel, Switzerland). Genotype was determined by phylogenetic analysis of the NS5b gene. Seventy-three pregnant women were found to be anti-HCV antibody positive by the first ELISA, but only 28 were anti-HCV positive by both ELISA. The prevalence of anti-HCV antibodies was thus 1.9% (28/1,494) (95% CI: 1.3-2.7%). 21/28 (75%) of the positive samples by both ELISA were HCV RNA positive. The 45 samples that were HCV antibody negative by the second ELISA were also HCV RNA negative. The HCV subtypes identified were 1a (24%), 2f (38%) and 4f (38%). In contrast to previous studies, anti-HCV antibodies were rare among pregnant women in Cameroon. The percentage of HCV seropositive pregnant women who had circulating HCV RNA was similar to that observed in Europe. Several HCV genotypes were found in Cameroon.     

Prevalence of amino acid mutation in hepatitis C virus core region among Japanese volunteer blood donors

It is not known whether there is a trend of increasing or decreasing incidence of new hepatitis C virus (HCV) infections in Japan. From the treatment point of view, it is important to verify HCV genotypes and the prevalence of treatment-resistant clones of HCV. At the Japanese Red Cross blood centers, all blood samples obtained from blood donation have been screened using serological methods and the minipool nucleic acid amplification testing. One hundred and fourteen donors have been identified over the past 10 years to be HCV RNA-only positive without detectable anti-HCV and were considered to be in the acute phase of HCV infection. There was a trend of decreasing incidence of such new infections among the blood donors. HCV RNA-only-positive samples were examined further for genotyping and HCV RNA quantitation. Genotype 2 (2a plus 2b) was predominant (78.2%) among them followed by genotype 1b (21.2%). Direct sequencing was carried out to detect the possible treatment-resistant mutant clones 70Q and 91M, clones with amino acid substitutions at positions 70 and 91 of the HCV core protein, respectively. 70Q and 91M were found regularly in donors with genotype 1b, but not in those with other genotypes. No particular endemic areas for the mutant clones were identified. There was no significant difference in the mean viral titer between the 70Q mutant type and the non-70Q wild-type. Even in newly infected people, the mutant clone 70Q was detected frequently.     

血清中丙型肝炎NS3抗原ELISA检测方法的建立和初步应用

目的 评价血清中丙型肝炎病毒(HCV)游离NS3抗原的酶联免疫吸附(ELISA)检测方法的特异性和灵敏度,初步探讨该方法在临床应用中的意义.方法 对77例正常人血清标本,173例抗-HCV阳性标本和3708例抗-HCV阴性的其他类型肝炎血清标本检测HCV游离NS3抗原;对部分HCV NS3抗原阳性标本进行验证,包括HCV RNA测定、中和试验和免疫斑点试验;对11例患者的25份系列血清标本进行了HCV游离NS3抗原、HCV RNA和HCV抗体的联合检测,并结合临床资料综合分析.结果 3708例抗-HCV阴性的其他类型肝炎血清标本中有48例为HCV NS3抗原阳性,其中3030例单纯乙型肝炎和445例其他类型肝炎血清标本中分别有44例和4例为HCV NS3抗原阳性;173例HCV抗体阳性标本中有42例为HCV NS3抗原阳性;77例正常人血清标本的HCV NS3抗原检测结果均为阴性;15例HCV NS3抗原阳性标本中有9例为HCV RNA阳性;23例HCV NS3抗原阳性标本的中和率和免疫斑点试验的阳性率分别为87.0%和69.6%;25份系列血清标本的检测结果显示其HCV NS3抗原的吸光度值与时间呈负相关,并有2例HCV NS3抗原阳性标本随着血清中HCV NS3抗原的吸光度值下降,其HCV抗体转阳.结论 血清中HCV游离NS3抗原的ELISA检测方法有较好的特异性和敏感度,在发展中国家应用此方法进行HCV感染的早期诊断有一定的临床意义和推广价值.     

Analysis of hepatitis C virus isolates among healthy blood donors and drug addicts in Chiang Mai, Thailand.

Hepatitis C virus (HCV) isolates obtained from 25 anti-HCV antibody-positive healthy blood donors and 29 drug addicts in Chiang Mai, Thailand, were analyzed. HCV RNA was detected in 23 blood donor samples (92%) and 24 drug addict blood samples (83%) by PCR for a portion of the NS5 region. Subtype analysis revealed that HCV type 3a (HCV-3a) was the prevailing subtype (30%), which was followed in prevalence by HCV-1a (21%), -1b (13%), -3b (13%), and -6a (2%). Six (13%) of the 47 isolates showed low sequence similarities with known types and subtypes. The sequence variants could be grouped into four branches in a molecular evolutionary phylogenetic tree.     

Molecular characterization of Hepatitis C virus (HCV) core region in HCV-infected Thai blood donors

In order to investigate the distribution of Hepatitis C virus (HCV) genotypes in Thailand, we performed phylogenetic analysis based on the virus core region and in this way we identified and reliably distinguished HCV genotypes 1-6 as well as subtypes. Among 100 plasma samples randomly selected from blood donors positive for antibodies to HCV (anti-HCV) 90 (90%) were found positive for HCV RNA and 77 of them were subjected to nucleotide sequencing. The following types and subtypes were identified in this group: 1a in 16 samples (20.8%), 1b in 14 samples (18.2%), 3a in 29 samples (37.7%), 3b in 5 samples (6.5%), and 6a in 13 samples (16.9%). Although this study allowed identification and characterization of HCV among blood donors, more extensive studies are needed to explore the HCV distribution in other population groups and in other geographical regions and to exploit the virus core-based characterization of HCV for evaluation of treatment and clinical outcome and epidemiological purposes.     

Protective effect of HLA-B57 on HCV genotype 2 infection in a West African population

Recovery from Hepatitis C virus (HCV) infection is considered infrequent (<20%) in western populations but reaches 50% in West Africa where genotype 2 infection is predominant. To investigate the role of cellular immune responses and host genetics in this phenomenon, samples from 104 Ghanaian blood donors reactive with anti-HCV assays were collected between 2000 and 2005. HCV antibody was confirmed by Western blot using genotype 2 recombinant core, E2 and NS3 proteins. Viral load and genotype were determined. Samples were stratified into 37 chronic, 35 recovered infections and 32 false positive. Eighty-one percentage of subjects with chronic infection (RNA positive) carried genotype 2 HCV. Cellular immune response was investigated in 35 frozen peripheral blood mononuclear cell (PBMC) samples suitable for interferon-gamma ELISPOT assay. Twelve out of 24 confirmed recovered, 1 out of 5 chronically infected and none of the 6 false-positive controls reacted to recombinant proteins. HLA-A, -B and -DR types were determined by DNA methodology. HLA-B*57 was significantly more frequent in the group which had recovered from HCV infection compared with chronically infected subjects (P = 0.0053, OR = 8.02). In conclusion, it is hypothesized that the dominance of genotype 2 HCV strains may be an important factor explaining the high rate of recovery from HCV infections in Ghana via an efficient contribution of HLA-B*57 which is relatively frequent in the population.     

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.