Ribavirin potentiates interferon action by augmenting interferon-stimulated gene induction in hepatitis C virus cell culture models

The combination of pegylated interferon (PEG-IFN) and ribavirin is the standard treatment for chronic hepatitis C. Our recent clinical study suggests that ribavirin augments the induction of interferon-stimulated genes (ISGs) in patients treated for hepatitis C virus (HCV) infection. In order to further characterize the mechanisms of action of ribavirin, we examined the effect of ribavirin treatment on ISG induction in cell culture. In addition, the effect of ribavirin on infectious HCV cell culture systems was studied. Similar to interferon (IFN)-α, ribavirin potently inhibits JFH-1 infection of Huh7.5.1 cells in a dose-dependent manner, which spans the physiological concentration of ribavirin in vivo. Microarray analysis and subsequent quantitative polymerase chain reaction assays demonstrated that ribavirin treatment resulted in the induction of a distinct set of ISGs. These ISGs, including IFN regulatory factors 7 and 9, are known to play an important role in anti-HCV responses. When ribavirin is used in conjunction with IFN-α, induction of specific ISGs is synergistic when compared with either drug applied separately. Direct up-regulation of these antiviral genes by ribavirin is mediated by a novel mechanism different from those associated with IFN signaling and intracellular double-stranded RNA sensing pathways such as RIG-I and MDA5. RNA interference studies excluded the activation of the Toll-like receptor and nuclear factor κB pathways in the action of ribavirin. CONCLUSION: Our study suggests that ribavirin, acting by way of a novel innate mechanism, potentiates the anti-HCV effect of IFN. Understanding the mechanism of action of ribavirin would be valuable in identifying novel antivirals.     

Viral and host immune regulatory mechanisms in hepatitis C virus infection

Recent studies suggest that liver inflammation in chronic hepatitis C virus (HCV) infection is controlled by several mechanisms, including host regulatory immune responses and viral polypeptides interacting with cells involved in innate and adaptive immunity. This article provides an overview about current thinking on host-pathogen symbiotic relationship in HCV infection and its significance with respect to pathogenesis. Special emphasis is given to regulatory T-cell subsets which have recently received attention and which are thought to play a major role in persistent viral infections such as HCV.     

Interferon alfa regulated gene expression in patients initiating interferon treatment for chronic hepatitis C

Interferon alfa (IFN-alpha) is an approved therapeutic agent for chronic hepatitis C. To directly characterize the effects of IFN-alpha in humans, we used microarrays to profile gene expression in peripheral blood mononuclear cells (PBMCs) from hepatitis C patients treated with IFN-alpha. Seven patients were studied using two strategies: (1) in vivo: PBMCs were collected immediately before the first dose of IFN-alpha, and 3 and 6 hours after the dose; (2) ex vivo: PBMCs that were collected before the first IFN-alpha dose were incubated with IFN-alpha for 3 and 6 hours. The microarray datasets were analyzed with significance analysis of microarrays (SAM) to identify genes regulated by IFN-alpha. We identified 516 named genes up-regulated at least 2-fold, at a false discovery rate (FDR) of less than 1%. In vivo and ex vivo studies generated similar results. No genes were identified as regulated differently between these 2 experimental conditions. The up-regulated genes belonged to a broad range of functional pathways and included multiple genes thought to be involved in the direct antiviral effect of IFN-alpha. Of particular interest, 88 genes directly relating to functions of immune cells were up-regulated, including genes involved in antigen processing and presentation, T-cell activation, lymphocyte trafficking, and effector functions, suggesting that IFN-alpha up-regulates multiple genes involving different aspects of immune responses to enhance immunity against hepatitis C virus. In conclusion, IFN-alpha-inducible genes can be identified in human PBMCs in vivo as well as ex vivo. Signature changes associated with different treatment outcomes may be found among these genes.     

丙型肝炎病毒NS3基因酵母表达载体构建及表达

目的　为探讨丙型肝炎病毒 (HCV)非结构蛋白NS3的功能 ,在真核生物酵母细胞中表达HCVNS3基因。方法　用聚合酶链反应 (PCR)的方法以HCV全长质粒pBRTM/HCV 1为模板扩增HCVNS3基因 ,克隆到 pGEM T载体中 ,双酶切后回收连接到酵母表达质粒 pGBKT7中表达。提取酵母蛋白质 ,进行十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和Western免疫印迹分析。结果　成功构建HCVNS3基因酵母表达载体 ,Western免疫印迹显示了HCVNS3在酵母细胞中表达。表达产物在胞内存在 ,相对分子质量为 70 0 0 0。结论　HCVNS3蛋白在酵母中表达成功。     

Mycophenolic acid augments interferon-stimulated gene expression and inhibits hepatitis C Virus infection in vitro and in vivo

Mycophenolic acid (MPA) is a highly effective immunosuppressant that has broad antiviral activity against different viruses and can act in synergy with interferon-α (IFN-α) on hepatitis C virus (HCV) replication. MPA is a potent inosine monophosphate dehydrogenase (IMPDH) inhibitor but the antiviral mechanisms are less understood. The aim of this study was to investigate the inhibition of HCV infection by MPA and the molecular basis for its synergy with IFN-α. The role of IMPDH and interferon-stimulated genes (ISGs) was investigated in two HCV models using gain- or loss-of-function approaches. The in vivo effect of MPA treatment was studied in NOD/SCID mice engrafted with HCV replicon cells. Potent antiviral effects of MPA at clinically relevant concentrations were observed with both the subgenomic and JFH1-derived infectious HCV models. MPA treatment in mice resulted in a specific and robust inhibition of HCV replication. Ectopic expression of an MPA-resistant IMPDH2 mutant in HCV host cells completely reversed the antiproliferative effect of MPA but only partially affected the antiviral potency. However, similar to ribavirin, MPA induced expression of multiple antiviral ISGs, including interferon regulatory factor 1 (IRF1). Cotreatment of MPA with IFN-α resulted in additive effects on ISG expression and enhanced IFN-induced luciferase reporter activity. Knockdown of IRF1, but not IFITM3, significantly attenuated the inhibition of HCV replication by MPA. CONCLUSION: MPA exerts a potent anti-HCV effect in vitro and in mice and acts in synergy with IFN-α. MPA's antiviral activity partially depends on IMPDH but also involves stimulation of ISGs, providing a molecular basis for its synergy with IFN-α.     

丙型肝炎病毒NS3区基因的克隆、表达及产物的纯化和抗原性分析

目前，我国阻断丙型肝炎传播的主要方法是用抗丙型肝炎病毒(HCV)酶联免疫吸附法(ELISA)试剂筛选供血员，国产试剂盒所用NS3抗原质量不太理想，使得国产试剂对NS3抗体漏检较多，提高该试剂盒质量的当务之急是开发更好的14CV抗原，特别是非结构区抗原。为分析和比较不同长度和型别的NS3片段的抗原性，我们在大肠杆菌中表达了5个不同长度和型别的HCV NS3抗原，并作为包被抗原，按间接ELISA原理检测抗HCV国家参考品血清及部分临床样本，比较其抗原性。     

慢性HBV/HCV感染人群伴随的免疫相关表现

慢性HBV/HCV感染者常常伴有自身免疫系统紊乱,在丙型肝炎中尤为常见。介绍了慢性丙型肝炎患者免疫状态紊乱的机制,出现非器官特异性自身抗体的比例,以及伴随的免疫相关疾病,如混合型冷球蛋白血症、肾小球肾炎、干燥综合征、甲状腺疾病、2型糖尿病的临床表现、诊断和治疗等。简述了慢性乙型肝炎患者免疫状态紊乱的机制、相关的免疫表现以及抗病毒治疗对其的影响。慢性乙型或丙型肝炎的抗病毒治疗可以减轻伴随的免疫系统疾病,但是不宜采用干扰素治疗,因此,乙型肝炎患者应采用核苷和核苷酸类药物治疗,丙型肝炎患者应采用直接抗病毒药物治疗。     

Intrahepatic expression of the hepatic stellate cell marker fibroblast activation protein correlates with the degree of fibrosis in hepatitis C virus infection

BACKGROUND: Activated hepatic stellate cells (HSCs), recognised by their alpha smooth muscle actin immunoreactivity, are primarily responsible for liver fibrosis. However, the presence of alpha smooth muscle actin positive HSCs is not always associated with the development of liver fibrosis. Recently, other markers of human HSCs including the gelatinase fibroblast activation protein (FAP) and glial fibrillary acidic protein have been identified. AIMS: We examined the relationship between the expression of these HSC markers and the severity of liver injury in patients with chronic hepatitis C virus infection. METHODS: Liver tissue from 27 patients was examined using immunohistochemistry. Linear correlation analysis was used to compare staining scores with the stage and grade of liver injury. RESULTS-CONCLUSIONS: FAP expression, seen at the tissue-remodelling interface, was strongly and significantly correlated with the severity of liver fibrosis. A weaker correlation was seen between glial fibrillary acidic protein expression and fibrosis stage. This contrasted with the absence of a relationship between alpha smooth muscle actin and the fibrotic score. A correlation was also observed between FAP expression and necroinflammatory score. In summary, FAP expression identifies a HSC subpopulation at the tissue-remodelling interface that is related to the severity of liver fibrosis.     

Differential gene expression between chronic hepatitis B and C hepatic lesion

BACKGROUND & AIMS: Complementary DNA (cDNA) microarray technology allows simultaneous expression analysis of hundreds to thousands of genes. We applied the cDNA microarray technique to clarify gene expression profiles in chronic viral hepatitis tissue lesions. METHODS: We made cDNA microarrays consisting of 1080 human cDNAs and analyzed gene expression using labeled cDNAs prepared from 6 normal, 12 chronic hepatitis B, and 14 chronic hepatitis C liver tissues. Relative expression ratios of individual genes were obtained by comparing hybridization of Cy5-labeled cDNAs from chronic hepatitis lesions and Cy3-labeled cDNA from normal liver tissue. RESULTS: Hierarchical clustering analysis of the gene expression profiles in 26 patients showed that the patients were clustered into 2 groups with respect to similarities in differentially expressed genes. Hepatitis B and C virus infection, but not age, sex, or histology of hepatitis, were significant factors determining clustering (P < 0.05). In hepatitis B tissue lesions, genes involved in inflammation were predominant, whereas in hepatitis C, expression of anti-inflammatory response genes was relatively dominant. CONCLUSIONS: These findings shed new light on the possible differential molecular mechanisms in the pathogenesis of hepatitis caused by hepatitis B virus and hepatitis C virus infection, from which hepatocellular carcinoma frequently develops.     

HBV X基因-HCV C基因cDNA融合基因真核表达载体的构建及其表达

目的：构建HBV X-HCV C融合基因真核表达载体，并获得稳定表达该基因的HepG2细胞株。方法：双酶切质粒pXT1-X，得到完整的HBV X基因片段后，将其插入到质粒PBK-CMV和PBK-HCVC的相应酶切位点，得到重组质粒PBK-X和PBK-X-C；再将质粒RBK-CMV、PBK-X、PBK-HCV C和PBK-X-C分别导入肝癌细胞株HepG2中，G418筛选，RT-PCR、蛋白印迹鉴定HBV X和HCV C蛋白表达。结果：质粒PBK-CMV、PBK-X、PBK-HCV C和PBK-X-C在HepG2细胞中有稳定表达。结论：成功构建HBV X-HCVC融合基因真核表达载体，并获得稳定表达该基因的HepG2细胞株。     

丙型肝炎病毒核心蛋白、Bcl-2、P21Waf1在丙型肝炎中的表达及相互关系

目的　调查丙型肝炎、肝硬化病人组织中的丙型肝炎病毒 (HCV)核心蛋白、P2 1、Bcl 2的表达以及相互间关系 ,探讨HCV核心蛋白是否抑制P2 1和促进Bcl 2表达。方法　收集 2 3例HCV表达阳性的肝炎、肝硬化组织 ,采用免疫组化Envision法检测核心蛋白、P2 1、Bcl 2表达 ,并用统计学方法分析三者间的表达关系。结果　HCV核心蛋白和突变P2 1的阳性表达主要位于细胞核中 ,Bcl 2的阳性表达主要位于细胞质 ;HCV核心蛋白表达阳性组织中 ,P2 1阳性表达率仅 13 % ,Bcl 2阳性表达率则达 95 .7% ,三组间比较差异有显著性 (P =0 .0 12 )。HCV核心蛋白和Bcl 2二组间比较差异无显著性 (P =0 .5 61) ;HCV核心蛋白与Bcl 2、p2 1阳性强度两者间P值分别为 0 .0 12和 0 .2。结论　三种蛋白的表达有相关性 ,HCV核心蛋白可能促进Bcl 2蛋白的表达 ,抑制P2 1蛋白表达