微小RNA-122对丙型肝炎病毒复制及感染治疗影响的研究进展

丙型肝炎病毒(HCV)感染后易演变为慢性肝炎,甚至进展为肝硬化、肝癌。目前尚无有效的预防疫苗,抗病毒药物的疗效也较局限。因此,直接靶向抗病毒且无毒副作用的治疗方法是目前研究的重点。微小RNA(miRNA)是一类小分子非编码RNA,主要通过下调宿主基因表达而发挥生物学功能。miRNA-122(miR-122)在HCV感染中的作用受到关注,探讨其影响HCV复制的具体分子机制对将其作为抗病毒治疗的一个靶目标、研发新型靶向抗HCV治疗药物有重要意义。本文主要就miR-122对HCV复制的影响及其成为潜在治疗靶点的研究现状作一综述。     

丙型肝炎病毒体外培养技术的新进展

丙型肝炎病毒(hepatitis C virus,HCV)是引起丙型肝炎的病原体,全球约1.7亿人感染HCV.高效体外细胞培养体系的缺乏,严重影响了HCV病毒生命周期的阐明以及抗病毒药物和预防性疫苗的研发.2005年Wakita T建立了JFH1/Huh7真正的HCV体外培养体系,为HCV研究提供了必要的研究条件.其后,研究者为提高病毒复制效率,简便病毒检测方法,在病毒株的选择替换、重组病毒构建、宿主细胞的改造筛选及病毒培养操作技术改进方面做了大量工作,取得了较好进展,为HCV的病毒学研究、药物筛选以及疫苗的研发提供了更有效的技术途径.     

抗HCV药物筛选系统研究进展

丙型肝炎病毒(Hepatitis C virus,HCV)感染的持久性引发慢性肝病疾病,并可能发展成为肝硬化和肝癌。目前对HCV的治疗不能达到理想的治疗效果,所以开发新型抗HCV药物迫在眉睫。抗HCV药物筛选的细胞模型,如复制子系统、假病毒系统、细胞培养系统,动物模型,如黑猩猩、uPA-SCID小鼠等,取得了快速的进展,并推动丙型肝炎的研究和抗HCV药物的发现。     

树鼩作为丙型肝炎动物模型的HCV受体研究进展

丙型肝炎病毒(hepatitis C virus,HCV)全球流行、危害严重,合适的小动物模型的缺乏严重阻碍了药物和疫苗的研发。该文介绍丙型肝炎危害与病毒复制特点,以HCV入胞受体为重点,通过比较现有丙型肝炎动物模型,从分子水平探讨树鼩作为丙型肝炎动物模型的可能性。     

丙型肝炎病毒临床检测方法的研究及临床意义

丙型肝炎病毒(HCV)是输血后肝炎的主要病原体,丙型肝炎易复发和慢性化.检测HCV标志物,可有效地筛选献血员,保证输血安全,降低丙型肝炎病毒感染率.本文就丙型肝炎病毒的检测方法以及其临床意义作一简要概述.     

HCV 5′NCR转基因细胞HepG2.9706的特性分析

丙型肝炎是由丙型肝炎病毒(hepatitis C virus,HCV)引起的威胁人类健康的重要传染病,迄今尚无有效的疫苗及特异性抗病毒治疗药物.抗HCV药物的研究和开发面临的关键问题是缺乏合适的HCV感染细胞及动物评价模型.转基因细胞模型是研究人类疾病常用的有效手段,因此,在HCV分子生物学进展的基础上,我们以对HCV翻译与复制有明显调控功能的HCV 5′NCR及部分翻译起始区序列为靶标,构建了HCV 5′NCR及部分多聚蛋白起始区序列与荧光素酶基因的融合基因,插入真核表达载体,转染HepG2细胞,获得了HCV 5′NCR转基因细胞HepG2.9706细胞株[1].本文对该细胞的某些特性进行了分析,以便更好地利用该细胞模型进行药物评价.     

JCI：预测丙型肝炎病毒复发的标志物

在世界各地,百万计人感染了丙型肝炎病毒（HCV）,HCV可导致肝硬化和肝癌症。直接作用的抗病毒剂能抑制病毒蛋白,并已成功地用于治疗HCV。不幸的是,抗病毒治疗在一些患者中失败,导致丙型肝炎病毒复发。     

HCV复制子与细胞培养体系研究进展

丙型肝炎是由丙型肝炎病毒(hepatitis C virus,HCV)感染所导致的传染性肝病,呈现世界性流行态势,严重危害人类健康。由于病毒自身高度突变,以及广泛高效的细胞培养体系和合适的小动物模型缺乏,目前尚无可有效预防的疫苗。自1989年丙型肝炎病毒基因组首次被确定以来,Con1(1b)亚基因组复制子和JFH1(2a)毒株细胞培养体系相继建立。以此为工具,HCV生活周期多个关键环节得以阐明。近年来,研究者在Con1亚基因组复制子、JFH1和J6/JFH1细胞培养体系的基础上,构建出多个基因型和亚型的复制子和细胞培养体系。不同的体系在HCV复制与致病机制研究、抗病毒药物筛选方面,具有不同的用途及优缺点。针对HCV复制子与细胞培养体系的研究进展进行综述,可为HCV的相关研究提供参考。     

丙型肝炎病毒的非结构蛋白3抑制剂

丙型肝炎严重威胁人类健康,非结构蛋白3（NS3）在丙型肝炎病毒（HCV）多聚蛋白水解过程中起重要作用,被公认为治疗丙型肝炎的有效药物靶标。该文介绍目前国内外有关NS3蛋白酶抑制剂（包括寡肽类抑制剂和非肽小分子抑制剂）的研究进展。     

天然来源的乙型和丙型肝炎病毒抑制剂的研究进展

慢性病毒性肝炎是威胁人类健康的主要流行病之一,目前全球约有 4 亿慢性乙型和丙型肝炎患者,现有临床药物仍不能很好地治愈 病毒性肝炎。作为药物发现的重要来源,天然产物提供了许多能强效抑制乙型肝炎病毒 (hepatitis B virus, HBV) 和丙型肝炎病毒（hepatitis C virus, HCV）的抑制剂。综述近 10 年来国内外报道的抗 HBV 和 HCV 的活性中药成分和天然产物的研究进展。     

Replicative Homeostasis III: implications for antiviral therapy and mechanisms of response and non-response

While improved drug regimens have greatly enhanced outcomes for patients with chronic viral infection, antiviral therapy is still not ideal due to drug toxicities, treatment costs, primary drug failure and emergent resistance. New antiviral agents, alternative treatment strategies and a better understanding of viral pathobiology, host responses and drug action are desperately needed. Interferon (IFN) and ribavirin, are effective drugs used to treat hepatitis C (HCV), but the mechanism(s) of their action are uncertain. Error catastrophe (EC), or precipitous loss of replicative fitness caused by genomic mutation, is postulated to mediate ribavirin action, but is a deeply flawed hypothesis lacking empirical confirmation. Paradoxically ribavirin, a proven RNA mutagen, has no impact on HCV viraemia long term, suggesting real viruses, replicating in-vitro, as opposed to mathematical models, replicating in-silico, are likely to resist EC by highly selective replication of fit (~consensus sequence) genomes mediated, in part, by replicative homeostasis (RH), an epicyclic mechanism that dynamically links RNApol fidelity and processivity and other viral protein functions. Replicative homeostasis provides a rational explanation for the various responses seen during treatment of HCV, including genotype-specific and viral load-dependent differential response rates, as well as otherwise unexplained phenomena like the transient inhibition and rebound of HCV viraemia seen during ribavirin monotherapy. Replicative homeostasis also suggests a primarily non-immunological mechanism that mediates increased immune responsiveness during treatment with ribavirin (and other nucleos(t)ide analogues), explicating the enhanced second-phase clearance of HCV ribavirin promotes and, thus, the apparent immunomodulatory action of ribavirin. More importantly, RH suggests specific new antiviral therapeutic strategies.     

Effect of Retreatment with Interferon Alone or Interferon plus Ribavirin on Hepatitis C Virus Quasispecies Diversification in Nonresponder Patients with Chronic Hepatitis C

Alpha interferon (IFN-alpha) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-alpha is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-alpha (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P = 0.017) and in patients who received IFN alone (P = 0. 05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 months of IFN therapy.     

Cell adhesion molecules and hyaluronic acid as markers of inflammation, fibrosis and response to antiviral therapy in chronic hepatitis C patients.

OBJECTIVE: Cell adhesion molecules (intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1)) and hyaluronic acid, markers of inflammation and fibrosis were monitored in hepatitis C patients to determine whether changes in plasma levels, during antiviral treatment, can predict long-term response to therapy. METHODS: In 55 patients with chronic hepatitis C virus (HCV), 33 treated with interferon (IFN) and 22 treated with IFN + ribavirin, sera was collected prior to treatment, at 3 + 6 months of therapy and 6 months post-treatment. Levels of ICAM-1, VCAM-1 and hyaluronic acid were correlated with alanine aminotransferase levels, HCV-RNA-polymerase chain reaction status and histological fibrosis scoring. RESULTS: A decrease in ICAM-1 levels at 3 and 6 months of therapy, compared with pretreatment levels, was observed in responders to IFN + ribavirin therapy but this decrease in ICAM-1 levels was not evident following cessation of treatment. Hyaluronic acid levels, in both treatment groups, did not differ significantly between responders and non-responders. Hyaluronic acid levels did correlate, significantly, with degree of fibrosis whereas VCAM-1 levels were marginally increased only in patients with moderate (grade III) fibrosis. CONCLUSIONS: Monitoring of VCAM-1 and hyaluronic acid, during antiviral therapy, does not differentiate between responders and non-responders. A decrease in ICAM-1 levels during IFN + ribavirin treatment is associated with response to therapy, and its efficacy in predicting long-term response should be further substantiated.     

Ribavirin Contributes to Hepatitis C Virus Suppression by Augmenting pDC Activation and Type 1 IFN Production

Ribavirin is used as a component of combination therapies for the treatment of chronic hepatitis C virus (HCV) infection together with pegylated interferon and/or direct-acting antiviral drugs. Its mechanism of action, however, is not clear. Direct antiviral activity and immunomodulatory functions have been implicated. Plasmacytoid dendritic cells (pDCs) are the principal source of type 1 interferon during viral infection. The interaction of pDCs with HCV-infected hepatocytes is the subject of intense recent investigation, but the effect of ribavirin on pDC activation has not been evaluated. In this study we showed that ribavirin augments toll-like receptors 7 and 9-mediated IFNα/β expression from pDCs and up-regulated numerous interferon-stimulated genes. Using the H77S.3 HCV infection and replication system, we showed that ribavirin enhanced the ability of activated pDCs to inhibit HCV replication, correlated with elevated induction of IFNα. Our findings provide novel evidence that ribavirin contributes to HCV inhibition by augmenting pDCs-derived type 1 IFN production.     

Mizoribine inhibits hepatitis C virus RNA replication: effect of combination with interferon-alpha

Interferon (IFN)-alpha monotherapy, as well as the more effective combination therapy of IFN-alpha and ribavirin, are currently used for patients with chronic hepatitis C caused by hepatitis C virus (HCV) infection, although the mechanisms of the antiviral effects of these reagents on HCV remain ambiguous, and side effects such as anemia due to the administration of ribavirin present a problem for patients who are advanced in years. Using a recently developed reporter assay system in which genome-length dicistronic HCV RNA encoding Renilla luciferase gene was found to replicate efficiently, we found that mizoribine, an imidazole nucleoside, inhibited HCV RNA replication. The anti-HCV activity of mizoribine (IC50: approximately 100 microM) was similar to that of ribavirin. Using this genome-length HCV RNA replication monitor system, we were the first to demonstrate that the combination of IFN-alpha and ribavirin exhibited more effective anti-HCV activity than the use of IFN-alpha alone. Moreover, we found that the anti-HCV activity of mizoribine in co-treatment with IFN-alpha was at least equivalent to that of ribavirin. This effect was apparent in the presence of at least 5 microM mizoribine. Since mizoribine is currently used in several clinical applications and has not been associated with severe side effects, mizoribine is considered to be of potential use as a new anti-HCV reagent in combination with IFN-alpha.     

Discovery and development of telaprevir: an NS3-4A protease inhibitor for treating genotype 1 chronic hepatitis C virus

Infection with hepatitis C virus (HCV) is a major medical problem with over 170 million people infected worldwide. Substantial morbidity and mortality are associated with hepatic manifestations (cirrhosis and hepatocellular carcinoma), which develop with increasing frequency in people infected with HCV for more than 20 years. Less well known is the burden of HCV disease associated with extrahepatic manifestations (diabetes, B-cell proliferative disorders, depression, cognitive disorders, arthritis and Sj?gren's syndrome). For patients infected with genotype 1 HCV, treatment with polyethylene glycol decorated interferon (peginterferon) α and ribavirin (PR) is associated with a low (40-50%) success rate, substantial treatment-limiting side effects and a long (48-week) duration of treatment. In the past 15 years, major scientific advances have enabled the development of new classes of HCV therapy, the direct-acting antiviral agents, also known as specifically targeted antiviral therapy for hepatitis C (STAT-C). In combination with PR, the HCV NS3-4A protease inhibitor telaprevir has recently been approved for treatment of genotype 1 chronic HCV in the United States, Canada, European Union and Japan. Compared with PR, telaprevir combination therapy offers significantly improved viral cure rates and the possibility of shortened treatment duration for diverse patient populations. Developers of innovative drugs have to blaze a new path with few validated sign posts to guide the way. Indeed, telaprevir's development was once put on hold because of its performance in a standard IC(50) assay. Data from new hypotheses and novel experiments were required to justify further investment and reduce risk that the drug might fail in the clinic. In addition, the poor drug-like properties of telaprevir were a formidable hurdle, which the manufacturing and formulation teams had to overcome to make the drug. Finally, novel clinical trial designs were developed to improve efficacy and shorten treatment in parallel instead of sequentially. Lessons learned from the development of telaprevir suggest that makers of innovative medicines cannot rely solely on traditional drug discovery metrics, but must develop innovative, scientifically guided pathways for success.     

Human uridine-cytidine kinase phosphorylation of ribavirin: a convenient method for activation of ribavirin for conjugation to proteins

Ribavirin is a synthetic nucleoside analog that is used for the treatment of hepatitis C virus (HCV) infection. Its primary toxicity is hemolytic anemia, which sometimes necessitates dose reduction or discontinuation of therapy. Selective delivery of ribavirin into liver cells would be desirable to enhance its antiviral activity and avoid systemic side effects. One approach to liver-specific targeting is conjugation of the ribavirin with asialoglycoprotein that is taken up specifically by liver cells. Human uridine-cytidine kinase-1 (UCK-1) was used for ribavirin phosphorylation to its monophosphate form. 1-Ethyl-3-diisopropylaminocarbodiimide (EDC) was used as a coupling agent. The best results were obtained using direct conjugation protocol with a molar ratio of 6.5 ribavirin monophosphate (RMP) molecules per one asialoorosomucoid (AsOR) molecule. Our findings show that ribavirin is a potential substrate of UCK-1, and RMP formed could be chemically coupled to AsOR to form a conjugate for liver specific targeting.     

Hepatitis C Virus Experimental Model Systems and Antiviral drug Research

An estimated 130 million people worldwide are chronically infected with hepatitis C virus (HCV) making it a leading cause of liver disease worldwide. Because the currently available therapy of pegylated interferon-alpha and ribavirin is only effective in a subset of patients, the development of new HCV antivirals is a healthcare imperative. This review discusses the experimental models available for HCV antiviral drug research, recent advances in HCV antiviral drug development, as well as active research be...     

Hepatitis C virus experimental model systems and antiviral drug research

An estimated 130 million people worldwide are chronically infected with hepatitis C virus (HCV) making it a leading cause of liver disease worldwide. Because the currently available therapy of pegylated interferon-alpha and ribavirin is only effective in a subset of patients, the development of new HCV antivirals is a healtheare imperative. This review discusses the experimental models available for HCV antiviral drug research, reeent advanees in HCV antiviral drug development, as well as active research being pursued to facilitate development of new HCV-speeifie therapeutics.     

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon

We developed a reverse genetics system of hepatitis C virus (HCV) genotypes 1a and 2a using infectious clones and human hepatocyte chimeric mice. We inoculated cell culture-produced genotype 2a (JFH-1) HCV intravenously. We also injected genotype 1a CV-H77C clone RNA intrahepatically. Mice inoculated with HCV by both procedures developed measurable and transmissible viremia. Interferon (IFN) alpha treatment resulted in greater reduction of genotype 2a HCV levels than genotype 1a, as seen in clinical practice. Genetically engineered HCV infection system should be useful for analysis of the mechanisms of resistance of HCV to IFN and other drugs.