Determining hepatitis C virus genotype distribution among high-risk groups in Iran using real-time PCR

AIM: To assess hepatitis C virus (HCV) genotype patterns among high-risk Iranian groups, using real-time RT-PCR.

Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C

AIM To explore the status of extrahepatichepatitis C virus(HCV)infection and replicationin hepatitis C patients,and its potentialimplication in HCV infection and pathogenicity.METHODS By reverse-transcriptase poly-merase chain reaction(RT-PCR),in situhybridization(ISH)and immunohistochemistry,HCV RNA,HCV replicative intermediate(minus-strand of HCV RNA),and HCV antigens weredetected in 38 autopsy extrahepatic tissuespecimens(including 9 kidneys,9 hearts,9pancreas,5 intestines,2 adrenal glands,2spleens,1 lymph node,and 1 gallbladder)from 9hepatitis C patients,respectively;and thestatus of HCV replication in extrahepatic tissueswas studied.RESULTS By RT-PCR,all 9 patients werepositive for HCV RNA in kidney,heart,pancreas,and intestine,but only 6(66.7%)patients were positive for HCV replicativeintermediate.HCV RNA and HCV antigens weredetected in kidney,heart,pancreas,intestine,adrenal gland,lymph node,and gallbladder in 5(55.6%)and 6(66.7%)patients by ISH andimmunohistochemistry,respectively.HCV RNA and HCV antigens were not detected in theseextrahepatic organs in 3(33.3%)patients,although their livers were positive for HCV.HCVreplicative intermediate detected by RT-PCR wasconsistent with HCV RNA and HCV antigensdetected by ISH and immunohistochemistry(Kappa=0.42-0.75).HCV RNA and HCVantigens were detected in myocardial cells,epithelial cells of intestinal gladular,interstitialcells of kidney,epithelial cells of tubules andglomerulus,pancreas acinar cells and epithelialcells of pancreatic duct,epithelial cells ofmucous membrane sinus of gallbladder,cortexand medulla cells in adrenal gland,andmononuclear cells in lymph node.HCV RNA wasalso detected in bile duct epithelial cells,sinusoidal cells,and mononuclear cells in livertissues by ISH.CONCLUSION HCV can infect extrahepatictissues,and many various tissue cells maysupport HCV replication;extrahepatic HCVinfection and replication may be of'concomitantstate'in most of patients with hepatitis C.Theinfected extrahepatic tissues might act as areservoir for HCV,and play a role in both HCVpersistence and reactivation of infection.HCVas an etiologic agent replicating and expressingviral proteins in extrahepatic tissues itselfcontributes to extrahepatic syndromeassociated.HCV infection in a few patients withchronic HCV infection.

Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

Inhibition of hepatitis C virus replication by single-stranded RNA structural mimics

AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were transfected with expression vectors generating HCV mimics and controls.Cellular HCV RNA and protein levels were quantified by real-time polymerase chain reaction and Western blotting,respectively.To evaluate possible antisense effects,complementary RNAs spanning a mimic were prepared.RESULTS: In the BB7 genotype 1b replicon system,mimics of the polymerase (NS-5B),X and BA regions inhibited replication by more than 90%,50%,and 60%,respectively.In the JFH-1 genotype 2 infection system,mimics that were only 74% and 46% identical in sequence relative to the corresponding region in JFH-1 inhibited HCV replication by 91.5% and 91.2%,respectively,as effectively as a mimic with complete identity to HCV genotype 2a.The inhibitory effects were confirmed by NS3 protein levels.Antisense RNA molecules spanning the 74% identical mimic had no significant effects.CONCLUSION: HCV RNA structural mimics can inhibit HCV RNA replication in replicon and infectious HCV systems and do so independent of close sequence identity with the target.

Antiviral effect of Chinese medicine jiaweisinisan in hepatitis B virus transgenic mice

AIM： To study the antiviral effect of Chinese medicine jiaweisinisan （JWSNS） on hepatitis B virus （HBV） infection in transgenic mice （TGM）. METHODS： Twenty two 6-8 wk old HBV TGM in the third generation were divided into TGM control group and TGM treated group randomly. The normal control group included ten normal BC 57L/6 mice at the same age. The mice in treated group were administrated with JWSNS at the concentration of 4 g/mL and the dosage of 50 g/kg per d for 30 d, while the mice in TGM control group and normal control group were administrated with normal saline at the same dosage and the same time. Polymerase chain reaction （PCR） was used to assess the contents of HBV DNA in serum of HBV TGM before and after treatments, whereas blot hybridization was utilized to measure the contents of HBV DNA in the liver of both HBV TGM and normal BC 57L/6 mice. RESULTS： The levels of serum HBV DNA in TGM treated group were remarkably decreased after the treatment of JWSNS （7.662±0.78 vs 5.22±3.14, P〈0.05）, while there was no obvious change after administration of normal saline in TGM control group （7.125±4.26 vs 8.932 ± 5.12, P〉 0.05）. The OD values of HBV DNA in the livers of the mice in TGM treated group were significantly lower than those of TGM control group （0.274±0.096 vs 0.432 ± 0.119, P 〈 0.01）. CONCLUSION： JWSNS exerts suppressive effects on HBV DNA in the serum and liver of TGM.

Extrahepatic and Intrahepatic Replication and Expression of Hepatitis C Virus

In order to determine the replication sites of hepatitis C virus, the in situ hybridization and immunohistochemical technique using digoxin-labeled 531bp plus-strand and minus-strand HCVRNA probes were employed to detect HCVRNA in the liver tissues, bone marrow mononuclear cells and peripheral blood mononuclear cells (PBMCs) from the patients with chronic hepatitis C, and in HCV transfected COS cells. The results showed that both plus-strand and minusstrand HCVRNA were detected in 80% of liver tissues (4/5). Plus-strand HCVRNA could be detected in 90% of PBMCs and bone marrow mononuclear cells (18/20), minus-strand HCVRNA in 25% of PBMCs. In HCV transfected COS cells, plus-strand HCVRNA distributed evenly in 20% cellular nuclei and cytoplasms. No minus-strand HCVRNA was detected in the bone marrow mononuclear cells and HCV transfected COS cells. The positive signal appeared in more cells when the liver tissues, PBMCs and marrow mononuclear cells were hybridized by plus-strand probes than when hybridized by minus-strand probes. Our results suggested that the hepatocytic cytoplasms and PBMC cytoplasms were the replication sites of HCV, but the marrow mononuclear cells were not the replication sites of HCV although they were infected by HCV. HCV infection might be accounted for the pathogenesis of chronic hepatitis and relapse of hepatitis C after liver transplantation.

丙型肝炎病毒(HCV)荧光PCR(F-PCR)试剂盒的研制及与免疫学方法的比较

目的:用荧光PCR（Fluorescence PCR,F-PCR）方法研制丙型肝炎病毒（Hepatitis C Virus,HCV）RNA检测试剂盒,通过临床验证,考核其性能,并与免疫学方法比较。方法:F-PCR是PCR和荧光探针杂交技术结合所产生的PCR方法。由于采用完全闭管荧光检测,避免了PCR后处理导致的假阳性污染;又采用了荧光检测技术,可以提高检测的灵敏度。结果:设计合成了HLV F-PCR诊断试剂盒。检测了 512份临床血清标本,以美国 Abbott公司的HCV ELISA诊断试剂盒和美国Biotronics公司的HCV荧光RT-PCR诊断试剂盒（B-PCR）为对照。阳性率30.5％,灵敏度97.3％,特异性98.1％。结论:F-PC的灵敏度显著优于ELISA,也高于B-PCR;三者的特异性无统计学差异。F-PCR试剂盒可以检测HCV的真实感染情况,对于临床诊断和疗效考察有一定的指导意义。

RT套式PCR检测血浆HCV RNA及与抗HCV检测的比较

应用微量血清热变性法提取核酸 ,逆转录套式聚合酶链反应 (RT nestPCR)检测血浆HCVRNA ,并与抗HCVELISA检测结果比较 ,对HCVRNA阳性标本进行HGVRNA的筛查。结果在 32例抗HCV阳性和 2 0例抗HCV阴性血浆中 ,HCVRNA分别检出 18例和 2例 ,总符合率为 70 % ,2 0例HCVRNA阳性者中有 2例合并感染HBV ,1例合并感染HGV。

用套式PCR分析我国不同地区的HCV基因型

目的:了解我国不同地区HCV基因型分布的特点.方法:根据不同基因型丙型肝炎病毒(HCV)核心区基因序列,合成一条通用引物和四条型特异性引物用于套式PCR扩增反应,所得不同长度的扩增产物分别对应于HCVⅠ、Ⅱ、Ⅲ和Ⅳ基因型,用此方法对采自北京、上海、天津、山西、西藏和新疆共455份丙型肝炎病毒阳性血清进行了基因型分析.结果:在我国HCV基因型分布以Ⅱ型(66.2%)为主,其次为Ⅲ型(20.2%),并有一些混合型(11.4%)及少量Ⅰ型(2.2%).结论:除新疆地区HCV混合型(43.1%)较多外,其它地区HCV基因型分布差别不大.

鸭甲肝病毒基因A型和C型双重RT-PCR检测方法的建立

为建立同时检测基因A型和C型鸭甲肝病毒(DHAV-A,DHAV-C)的双重RT-PCR方法,本实验根据GenBank中DHAV-A和DHAV-C全基因组序列,针对DHAV-A的3C、3D和DHAV-C的2C区域分别设计了2对引物,通过对反应体系和反应条件的优化及特异性、灵敏度评价,建立了检测DHAV-A和DHAV-C的双重RT-PCR方法。该方法能够分别从DHAV-A、DHAV-C阳性样本中扩增出DHAV-A(259 bp)和DHAV-C(194 bp)的特异片段,而不与其它被检的无关病原发生非特异反应;对DHAV-A和DHAV-C的最低检出限分别为4.98×104拷贝/μL、1.68×104拷贝/μL;对2009年8月至2011年7月送检病料检出DHAV-A和DHAV-C的阳性率分别为20%(14/70)、70%(49/70);随机抽取的DHAV-C PCR阳性样本的病毒分离结果与双重RT-PCR检测结果的符合率为100%(16/16)。本研究结果显示,建立的双重RT-PCR方法具有良好的特异性和敏感性,可用于临床DHAV的快速鉴别诊断。

实时荧光PCR技术在丙型肝炎病毒基因型检测中的应用

目的探讨实时荧光PCR技术在丙型肝炎病毒(HCV)基因型检测中的应用价值。方法采用一步法逆转录聚合酶链反应(RT-PCR)结合TaqMan技术,对HCV基因型进行实时荧光PCR检测;巢式RT-PCR扩增HCV的NS5B基因区域,测序后进行HCV基因亚型分析并与实时荧光PCR分型结果进行比较。结果实时荧光PCR共分型119例,1型71例(59.7%),非1型48例(40.3%)其中110例成功进行测序分型;测序分型的110例中,1型73例(均为1b型),占66.4%,非1型37例(其中2a型7例、3a型6例、3b型3例、4a型1例、4k型1例、5a型1例、6a型17例、6n型1例),占33.6%。与110例测序分型的结果比较,实时荧光PCR分型总的符合率为71.8%(79/110),其中1型的符合率为80.9%(55/68),非1型的符合率为57.1%(24/42)。结论实时荧光PCR进行HCV基因型检测操作简便、快速,但分型结果特异性不高。

献血者HCV RNA实时荧光定量PCR方法的建立

目的建立快速检测献血者中丙型肝炎病毒(hepatitis C virus,HCV)感染的方法。方法利用体外转录制备的HCV RNA转录体进行病毒RNA抽提方法及抽提效率的比较;将RNA转录体与正常血清进行不同数目的混合,对最佳混合标本数进行了摸索;建立HCV荧光定量PCR方法(fluorescence quantitative PCR,FQ-PCR),并对献血者进行混合标本HCV核酸检测。结果确定了比较理想的病毒RNA抽提方法;用于HCV核酸检测的混合标本数为24例;建立的荧光定量PCR方法能最低检测出10个copies/ml;采取24例混合血标本方法,FQ-PCR检测HCV RNA,576例ELISA检测HCV阴性的献血者未检测出HCV RNA。结论初步建立了献血者HCV感染的混合标本荧光定量PCR检测方法。

PCR扩增杂交试验检测HCV-RNA

目的：评价ＰＣＲ扩增杂交试验（ＰＣＲ－Ｈｙｂ）在ＨＣＶ－ＲＮＡ检测中的应用。方法：对２８例丙型肝炎患者和５例非丙型肝炎患者采用ＰＣＲ扩增杂交（ＰＣＲ－Ｈｙｂ）试验、套式ＲＴ－ＰＣＲ及ｂＤＮＡ信号扩增试验检测ＨＣＶ－ＲＮＡ。结果：三种方法的阳性率分别为７１．３４％（２０／２８）、７８．５７％（２２／２８）、１００％（２７／２７）。ＰＣＲ－Ｈｙｂ与套式ＲＴ－ＰＣＴ的阳性符合率为９３．９３％（３１／３３），与ｂＤＮＡ结果呈现较好的相关性（ｒ＝０．８０８３）。结论：ＰＣＲ－Ｈｙｂ具有较好的特异性和敏感性，并在抗病毒治疗时的动态观察有一定价值。

弓形虫乙肝病毒多重PCR及芯片杂交技术的研究

目的建立同时检测弓形虫和乙肝DNA的多重PCR和芯片杂交检测方法,为孕产妇TORCH检测的进一步研究提供实验依据。方法以病人血液及弓形虫RH腹水为样本,多重PCR方法为基础,通过基因片段筛选及引物比例调整,进行弓形虫和乙肝病毒基因片段的扩增。然后进一步建立生物芯片检测法以排除PCR假阳性。结果在确定合适的多重PCR引物比例后,扩增结果清晰可辨。在PCR体系中加入Norwalk扩增监控进一步证实了扩增结果的可靠性。芯片与PCR产物杂交的结果具有较好的灵敏性和特异性。结论建立的针对弓形虫和乙型肝炎病毒检测的多重PCR和芯片杂交方法是有效的,芯片杂交可用于对PCR检测结果的进一步确认。

二重RT-PCR同时检测HGV与HCVRNA

庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）具有相近的传播途径与类似的检测方法，本文建立了二重ＲＴ－ＰＣＲ法，用于同时检测ＨＧＶ与ＨＣＶ病毒核酸，其扩增产物分别为６０９ｂｐ与２５７ｂｐ，在琼脂糖或聚丙烯酰胺凝胶上均能完全分离。ＨＧＶ产物经测序显示与报道的核酸序列同源性为８９．２％～９２．６％，该法具较好的批间重复性。二重ＰＣＲ法减轻了实验人员的操作强度，可降低测定费用近５０％。

肝细胞癌组织中HCV基因型的逆转录原位PCR检测

目的从分子病理学水平探讨 HCV 基因型与 HCC 发生的关系。方法采用逆转录原位 PCR 法对95例 HCC 及其非癌组织中1b 型和2a 型 HCV 进行检测,同时采用 Okamoto 法对 HCC 组织 RNA 抽提物中 HCV 基因型加以验证。结果 HCC 中 HCV 阳性率为44%(42/95),检出3种 HCV 基因型,包括1b(13例,31%)、2a(23例,55%)、混合型(1b+2a,2例,5%)和3a(4例,10%),1b 型检出率(14%)与2a 型者(24%)无显著性差异(X^2=3.427,P>0.05),两型与 HCC 主要病理学参数的相关性相似,2a 型阳性细胞在癌和非癌区的分布相似,而1b 型在癌组织多呈局灶分布,在非癌区多呈局灶/弥漫分布,HCC 中1b 型 HCV 的核-胞质型/核型阳性信号较2a 型者明显多见(X^2=4.073,P<0.05),去除逆转录并用RNase A 处理后,1b 型和2a 型 HCV 仍分别有5例和9例可见阳性信号。结论我国两种常见的 HCV 基因型与 HCC 发生的相关性相似,HCC 中 HCV 相关 DNA 及其与宿主基因组整合的可能性不能除外。

应用巢式PCR检测原发性肝癌患者血清中HCV—RNA

为了探讨原发性肝癌的发生是否与丙型肝炎病毒有关,应用巢式PCR技术和ELISA检测40例原发性肝癌患者及50例除外肝病患者血清中HCV—RNA及抗HCV抗体和乙肝二对半。结果:原发性肝癌组中,乙肝二对半阳性37例(92.5％),抗HCV阳性8例(20％),HCV—RNA阳性13例(32.5％),HCV总感染率为35.0％(14例);对照组分别为16例(40.0％),2例(4.0％)及3例(6.0％)。在3例HBV—M阴性PHC患者中,HCV—RNA阳性2例。结论:在我国PHC的发生不仅与乙肝病毒感染有关,还与丙型肝炎病毒有关。

荧光定量PCR检测HCVRNA的测量不确定度评定及应用

目的对荧光定量PCR测定HCVRNA的测量不确定度进行评定及临床应用。方法利用罗氏LightCycler1．0荧光定量PCR分析仅选取2个HCVRNA结果阳性的临床样本，HCVRNA拷贝数分别为1×10^3 copies／ml～1×10^5 copies／ml和1×10^4 copies／ml～1×10^7 copies／ml，各重复测定10次，对测定HCVRNA潮量过程的分析，确定并简化测量不确定度的来源，采用不确定度A类评定方法以及不确定度B美评定方法，量化各不确定度分量，确定合成不确定度与扩展不确定度。结果测量不确定度来源主要有：批内重复性、批间重复性，温度时间，消毒前消毒后和方法偏倚等因素。在各分量中，由温度时间影响导致的不确定度所占比例最大，批间重复性试验所占比例最小。荧光定量PCR测定HCVRNA的扩展不确定度U=0．268。结论通过荧光定量PCR测定HCVRNA测量不确定的研究有助于各实验室结果的表达趋于一致，可使来自不同实验室的结果或同一实验室不同时期的结果具有可比性。