HCV dsRNA-Activated Macrophages Inhibit HCV Replication in Hepatocytes

Background:

Macrophages play critical roles in innate immune response in the liver. Whether macrophages participate in liver innate immunity against HCV replication is poorly understood

Objectives:

The aim of this study was to investigate the role of macrophages in liver innate immunity against HCV replication.

Materials and Methods:

Freshly isolated monocytes were purified from peripheral blood of healthy adult donors. Macrophages refer to 7-day-cultured monocytes in vitro. A hepatoma cell line (Huh7) was infected with HCV JFH-1 to generate in vitro HCV infectious system. RT-PCR was used to determine HCV RNA and mRNA levels of genes expression. ELISA was used to measure the protein level of interferon-α (IFN-α) and western blot was used to determine protein expression level of Toll-like receptor 3 (TLR3).

Results:

HCV dsRNA induced the expression of type I IFN (IFN-α/β) in monocyte-derived macrophages. HCV dsRNA also induced the expression of TLR3 and IFN regulatory factor-7 (IRF-7), the key regulators of the IFN signaling pathway. When HCV JFH-1-infected Huh7 cells were co-cultured with macrophages activated with HCV dsRNA or incubated in media conditioned with supernatant (SN) from HCV dsRNA-activated macrophages, HCV replication was significantly suppressed. This macrophage SN action on HCV inhibition was mediated through type I IFN, which was evidenced by the observation that antibody to type I IFN receptor could neutralize the macrophages-mediated anti-HCV effect. The role of type I IFN in macrophages-mediated anti-HCV activity is further supported by the observation that HCV dsRNA-activated macrophages SN treatment induced the expression of several IFN-stimulated genes (ISGs), ISG15, ISG56, OAS-1, OAS-2, MxA and Viperin in HCV-infected Huh7 cells.

Conclusions:

Macrophages may play an important role in liver innate immunity against HCV replication through a type I IFN-dependent mechanism.     

丙型肝炎病毒与其合并人类免疫缺陷病毒感染者的病毒基因型差异

HCV与HIV具有相似的传播途径,都可以通过使用污染的血制品、注射用具、性接触及母婴传播,因此,HIV与HCV混合感染较常见.HIV感染改变HCV的自然病程,加速肝纤维化、肝硬化及肝细胞癌的进程已达成共识;HCV基因型对肝病的进程和预后亦起到重要作用.那么HCV基因型的分布在HⅣ/HCV混合感染与HCV单纯感染之间是否存在差异?各自的感染途径有何不同?值得我们作进一步的探讨.我们对昆明市第三人民医院收治的昆明地区85例HCV感染住院患者采用PCR等分子生物学方法研究了HIV/HCV感染与HCV单纯感染患者HCV基因型分布的差异.     

特异性脱氧核酶对丙型肝炎病毒的体外消化作用

目的研究特异性脱氧核酶对丙型肝炎病毒（HCV）的体外消化作用。方法设计3个分别作用于HCV RNA 5＇非编码区（5＇ NCR）上157、168、173位点的脱氧核酶（DRz），分别命名为DRz1，DRz2,DRz3，均以5＇GGCTAGCTACAACGA-3’为脱氧核酶的催化活性中心，用内切酶XbaⅠ将含有HCV病毒序列的质粒pCMV/T7 NCRC,△Luc消化成线性，以此为模板体外转录出用α-^32PIUTP进行标记的靶HCV RNA。在37℃,PH7.5，Mg^2 10mmol/L等条件下，将HCV RNA（10nmol/L）与3个脱氧核酶（1μmol/L）分别混合进行切割反应，并进一步比较不同Mg^2浓度下DRz3的切割反应效率，反应产物在8%变性聚丙烯酰胺凝胶上电泳分离并行放射自显影，应用凝胶成像分析仪评价脱氧核酶的切割效率。结果在设定反应条件下，3个脱氧核酶对HCV病毒均有切割活性，随着Mg^2浓度的增加，DRz3的切割活性增强，结论特异性脱氧核酶可有效切割HCV RNA 5＇NCR的RNA，且切割效率与Mg^2浓度呈正相关。     

Effects of HCV proteins in current HCV transgenic models

Hepatits C virus (HCV) is an enveloped virus with positive‐sense single‐stranded RNA genome that causes both acute and persistent infections associated with chronic hepatitis, cirrhosis and hepatocellular carcinoma, which needs fully functional human hepatocytes for its development. Due to the strict human tropism of HCV, only human and higher primates such as chimpanzees have been receptive to HCV infection and development, cognition about pathophysiololgy and host immune responses of HCV infection is limited by lacking of simple laboratory models of infection for a long time. During the past decade, gene transfer approaches have been helpful to the understanding of the molecular basis of human disease. Transgenic cell lines, chimeric and transgenic animal models were developed and had been demonstrated their invaluable benefits. This review focuses on the existing HCV transgenic models and summarize the relative results about probable pathophysical changes induced by HCV proteins.     

HIV/HCV混合感染者与HCV感染者感染HCV基因型分析*

目的 使用二代测序技术检测广州地区人类免疫缺陷病毒（HIV）/丙型肝炎病毒（HCV）混合感染者和HCV感染者HCV基因型,了解本地区HCV基因型的流行特点。方法 在233例HCV感染者中,包括95例HIV/HCV混合感染者和138例HCV感染者,使用Illumina Miseq平台PE300模式对HCV Core和NS5B片段进行测序,采用生物信息学分析确定HCV基因型,对测序结果提示混合基因型感染的样本,使用NS5A基因型特异引物进行验证。结果 HIV/HCV混合感染者静脉吸毒感染构成比显著高于HCV感染者（77.7%对19.6% P<0.001）,HCV感染者输血感染的构成比显著高于HIV/HCV混合感染者（48.6%对3.2% P<0.001）;HIV/HCV混合感染者HCV Core基因片段平均数据量为（15456±6689） reads,HCV感染者为（14323±5321） reads,两组无显著差异（P>0.05）;HIV/HCV混合感染者HCV NS5B基因片段平均数据量为（16432±3467） reads,HCV感染者为（17611±5632） reads,也无显著差异（P>0.05）;本组228例（97.9%）HCV感染者为单一HCV基因型感染,HIV/HCV混合感染者HCV 6a型和3b型感染率显著高于HCV感染者（分别为47.4%对26.8%,P=0.001和13.7%对3.6%,P=0.005）,HCV 1b型感染率显著低于HCV感染者（20.0%对59.4%,P<0.001）;在233例入组的HCV感染者中,发现5例（2.1%）为混合基因型感染,其中4例为HIV/HCV混合感染者,1例为HCV感染者。结论 广州地区HIV/HCV混合感染者与HCV感染者感染HCV基因型构成比存在差异,其临床意义还需要探讨。     

HCV基因型和病毒量与干扰素疗效的关系

用α１型基因工程干扰素（ＩＦＮ－α１）治疗２６例慢性丙型肝炎（丙肝），观察丙型肝炎病毒（ＨＣＶ）基因型和ＨＣＶＲＮＡ量和疗效的关系。其中２０例作ＨＣＶ的基因型，结果表明ＨＣＶⅠ型中病毒清除反应属Ａ级（完全清除）为４／６，Ⅱ型为０／６，Ⅲ型为５／７，提示Ⅰ、Ⅲ型较Ⅱ型易于清除病毒（Ｐ＜０．０５）；基因型和ＡＬＴ反应关系不明显。２６例作ＨＣＶＲＮＡ，半定量结果表明病毒含量低者，病毒清除反应佳（Ｐ＜０．０５），而和ＡＬＴ关系不明显。作者认为ＨＣＶ基因型和血清病毒量可作为预测干扰素疗效的参考指标，定量检测血清中ＨＣＶＲＮＡ在评价ＩＦＮ疗效中十分重要。     

HCV viral kinetics

Viral kinetics has been and continues to be an important tool for studying hepatitis C virus (HCV) infection and treatment. In this review article the basic principles of viral kinetics as applied to HCV are reviewed. In addition, key insights gained from studies concerning both the mechanism of action of interferon and ribavirin and the life cycle of the virus are discussed. Finally, clinical questions, such as differences in response based on race, genotype, and coinfection that have been examined using kinetics, are discussed.     

HCV C-Fc融合基因修饰的树突状细胞疫苗抗HCV功能研究

目的　探讨丙型肝炎病毒 (HCV)C Fc基因修饰的树突状细胞 (DCs)能否诱导抗HCV细胞和体液免疫反应。方法　将pcDNA3 HCV Fc质粒用电穿孔法转染经过白介素 4 (IL 4 ) ,粒 巨噬细胞集落刺激因子 (GM CSF)刺激增殖的小鼠DCs前体细胞 ,观察HCV、Fc抗原表达 ;将制备的 5×10 5/ 10 0 μlDC疫苗皮下免疫Balb/c小鼠 ,两周后检测特异性抗体、脾脏CD4 、CD8 细胞增殖作用以及诱导的细胞毒性T淋巴细胞 (CTL)反应。结果　HCVC Fc基因电穿孔法转染细胞在上述细胞因子作用下发育成能有效表达HCVC Fc并具有典型形态学与表型特征的DCs。免疫小鼠后 ,HCVC Fc基因转染DCs能诱导产生抗HCV特异性抗体和较强的CTL反应。结论　HCVC Fc基因修饰的DCs能增强对HCV特异性CTL效应的诱导能力 ,提示它在抗病毒疫苗发展中的潜力     

HCV不同功能区抗体与HCVRNA的关系探讨

探讨HCV不同功能区抗体(抗-C、抗-NS3、抗-NS4及抗-NS5)与HCVRNA的关系。HCV不同功能区抗体测定采用ELISA法,用RT-PCR进行HCV RNA检测。结果显示55例抗HCV阳性血清中有7种抗体阳性组合。抗-NS3、抗-C、抗-NS5及抗-NS4在7种阳性组合中的检出率分别为96．4%、89．1%、58．2%、56．4%。HCVRNA阳性血清中抗-NS3、抗-C、抗-NS5及抗NS4的检出率分别为96．2%,88．5%,57．7%,57．7%。HCV不同功能区抗体ELISA法有很高的敏感性和特异性,与RT-PCR法基本一致。抗-NS3、抗-C在HCV的诊断中有重要的意义,抗-NS5和抗-NS4有诊断的互补作用。     

HBV plus HCV, HCV plus HIV, HBV plus HIV

Coinfection of hepatitis C virus (HCV), hepatitis B virus (HBV), and HIV is common due to shared modes of transmission. These coinfections accelerate the course of chronic liver disease and facilitate progression to cirrhosis and hepatocellular carcinoma. The viral interactions between these viruses are complex, and their treatment may be challenging for clinicians.     

不同HCV感染者各区段抗HCV抗体的反应性及HCV-RNA检测结果分析

利用丙型肝炎病毒（ＨＣＶ）不同区段抗原的单片段酶免疫（ＥＩＡ）试剂及反转录聚合酶链反应（ＲＴ－ＰＣＲ）技术,对不同ＨＣＶ感染者血清不同区段抗ＨＣＶ抗体及丙型肝炎病毒核糖核酸（ＨＣＶ－ＲＮＡ）进行检测。结果显示,慢性肝炎患者各抗体检出率及Ｓ／Ｃｏ（Ｓ为样品光密度值,Ｃｏ为临界值）值均高,肝癌患者各抗体检出率及ＨＣＶ－ＲＮＡ阳性率均较低;ＡＬＴ正常而抗ＨＣＶ阳性者各区段抗体阳性率低于急、慢性肝炎患者,而其ＨＣＶ－ＲＮＡ的阳性率高于其他各组;抗－ＮＳ３和抗－Ｃ抗体在各组的检出率和反应性均强。表明抗－Ｃ和抗－ＮＳ３抗体的检测对ＨＣＶ感染最有诊断价值,同时用ＲＴ－ＰＣＲ技术检测血清ＨＣＶ－ＲＮＡ有助于ＨＣＶ感染的早期诊断     

HCV感染引起的HCV感染相关性肾炎1例报道

患者因输血而感染HCV,在应用泼尼松治疗肾病同时,HCV复制激活,引起肾功能损害,肾病理检查提示膜型肾小球肾炎、系膜增生性病变伴肾小球硬化、肾小管-间质慢性病变及血管壁内膜增厚、肾组织有HCV抗体;给予IFNα300万U隔日1次,皮下注射,联合利巴韦林0．3mg,3次／d,疗程12个月,尿蛋白降低,肾功能改善,浮肿消退。