In-vitro model systems to study Hepatitis C Virus

Hepatitis C virus (HCV) is a major cause of chronic liver diseases including steatosis, cirrhosis and hepatocellular carcinoma. Currently, there is no vaccine available for prevention of HCV infection due to high degree of strain variation. The current treatment of care, Pegylated interferon α in combination with ribavirin is costly, has significant side effects and fails to cure about half of all infections. The development of in-vitro models such as HCV infection system, HCV sub-genomic replicon, HCV producing pseudoparticles (HCVpp) and infectious HCV virion provide an important tool to develop new antiviral drugs of different targets against HCV. These models also play an important role to study virus lifecycle such as virus entry, endocytosis, replication, release and HCV induced pathogenesis. This review summarizes the most important in-vitro models currently used to study future HCV research as well as drug design.     

A weak neutralizing antibody response to hepatitis C virus envelope glycoprotein enhances virus infection

We have completed a phase 1 safety and immunogenicity trial with hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, with MF59 adjuvant as a candidate vaccine. Neutralizing activity to HCV genotype 1a was detected in approximately 25% of the vaccinee sera. In this study, we evaluated vaccinee sera from poor responders as a potential source of antibody dependent enhancement (ADE) of HCV infection. Sera with poor neutralizing activity enhanced cell culture grown HCV genotype 1a or 2a, and surrogate VSV/HCV pseudotype infection titer, in a dilution dependent manner. Surrogate pseudotypes generated from individual HCV glycoproteins suggested that antibody to the E2 glycoprotein; but not the E1 glycoprotein, was the principle target for enhancing infection. Antibody specific to FcRII expressed on the hepatic cell surface or to the Fc portion of Ig blocked enhancement of HCV infection by vaccinee sera. Together, the results from in vitro studies suggested that enhancement of viral infectivity may occur in the absence of a strong antibody response to HCV envelope glycoproteins.     

Use of human hepatocyte-like cells derived from induced pluripotent stem cells as a model for hepatocytes in hepatitis C virus infection

Host tropism of hepatitis C virus (HCV) is limited to human and chimpanzee. HCV infection has never been fully understood because there are few conventional models for HCV infection. Human induced pluripotent stem cell-derived hepatocyte-like (iPS-Hep) cells have been expected to use for drug discovery to predict therapeutic activities and side effects of compounds during the drug discovery process. However, the suitability of iPS-Hep cells as an experimental model for HCV research is not known. Here, we investigated the entry and genomic replication of HCV in iPS-Hep cells by using HCV pseudotype virus (HCVpv) and HCV subgenomic replicons, respectively. We showed that iPS-Hep cells, but not iPS cells, were susceptible to infection with HCVpv. The iPS-Hep cells expressed HCV receptors, including CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin; in contrast, the iPS cells showed no expression of SR-BI or claudin-1. HCV RNA genome replication occurred in the iPS-Hep cells. Anti-CD81 antibody, an inhibitor of HCV entry, and interferon, an inhibitor of HCV genomic replication, dose-dependently attenuated HCVpv entry and HCV subgenomic replication in iPS-Hep cells, respectively. These findings suggest that iPS-Hep cells are an appropriate model for HCV infection.     

丙型肝炎病毒感染实验动物模型的研究进展

丙型肝炎病毒（HCV）感染是导致人类慢性病毒性肝炎、肝硬化和肝癌的最主要病因之一。由于缺乏合适的HCV感染实验动物模型,使得针对HCV感染更为有效的疗法及疫苗的研发滞后。黑猩猩是HCV感染研究的最佳实验动物,但由于其来源有限、价格昂贵及临床症状等诸多问题,其应用受限,因此发展新的实验动物模型用于HCV感染相关的基础和应用研究迫在眉睫。近年来,以啮齿类等动物为替代模型取得了不少进展,应用转基因等实验技术使替代动物感染了HCV,并成功应用于多个学科领域的研究。本文分析了HCV自然感染的实验动物、自然感染和非自然感染的替代实验动物在致病机制研究、药物评价和疫苗研发应用中的优缺点及未来研究趋势。     

Initiation of hepatitis C virus infection is dependent on cholesterol and cooperativity between CD81 and scavenger receptor B type I

In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol.     

Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes.

The buoyant density of hepatitis C virus (HCV), with high in vivo infectivity (strain H) or low in vivo infectivity (strain F), was determined by sucrose gradient equilibrium centrifugation. Viral RNA of strain H was detected in fractions with densities of < or = 1.09 g/ml (principally approximately 1.06 g/ml), while that of strain F was found in fractions with densities of approximately 1.06 and approximately 1.17 g/ml. The observed difference was confirmed by differential flotation centrifugation; in NaCl solution with a density of 1.063 g/ml, most of the HCV RNA of strain H was detected in the top fraction, while that of strain F appeared in the bottom. The same relationship between buoyant density and infectivity was observed in flotation centrifugation experiments with other HCV strains. In immunoprecipitation experiments with anti-human immunoglobulin, HCV (as measured by HCV RNA) was precipitated from the samples with low infectivity and high density but not from those with high infectivity and low density. Examination of serial sera from a chimpanzee infected with HCV revealed parallel changes in the buoyant density and immunoprecipitability of HCV-associated RNA during the course of infection. These data suggest that HCV is bound to anti-HCV antibodies as antigen-antibody complexes in chronic hepatitis C.     

Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection

Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.     

Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities

Hepatitis C virus (HCV) infection causes chronic hepatitis and is currently treated with alpha interferon (IFN-alpha)-based therapies. The underlying mechanisms of chronic HCV infection and IFN-based therapies, however, have not been defined. Protein kinase R (PKR) was implicated in the control of HCV replication and mediation of IFN-induced antiviral response. In this report, we demonstrate that a subgenomic RNA replicon of genotype 2a HCV replicated efficiently in mouse embryonic fibroblasts (MEFs), as determined by cell colony formation efficiency and the detection of HCV proteins and both positive- and negative-strand RNAs. Additionally, the subgenomic HCV RNA was found to replicate more efficiently in the PKR knockout (PKR(-/-)) MEF than in the wild-type (PKR(+/+)) MEF. The knockdown expression of PKR by specific small interfering RNAs significantly enhanced the level of HCV RNA replication, suggesting that PKR is involved in the control of HCV RNA replication. The level of ISG56 (p56) was induced by HCV RNA replication, indicating the activation of PKR-independent antiviral pathways. Furthermore, IFN-alpha/beta inhibited HCV RNA replication in PKR(-/-) MEFs as efficiently as in PKR(+/+) MEFs. These findings demonstrate that PKR-independent antiviral pathways play important roles in controlling HCV replication and mediating IFN-induced antiviral effect. Our findings also provide a foundation for the development of transgenic mouse models of HCV replication and set a stage to further define the roles of cellular genes in the establishment of chronic HCV infection and the mediation of intracellular innate antiviral response by using MEFs derived from diverse gene knockout animals.     

Hepatitis C Virus Infection Causes Iron Deficiency in Huh7.5.1 Cells

Patients with chronic hepatitis C virus (HCV) infection frequently develop systemic iron overload, which exacerbates morbidity. Nevertheless, iron inhibits HCV replication in cell culture models and thereby exerts antiviral activity. We hypothesized that the cellular iron status is crucial for the establishment of HCV infection. We show that HCV infection of permissive Huh7.5.1 hepatoma cells promotes an iron deficient phenotype. Thus, HCV leads to increased iron regulatory protein (IRP) activity, accumulation of IRP2 and suppression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) in the host. These data suggest that HCV regulates cellular iron levels to bypass iron-mediated inhibition in viral replication.     

The potential use of Toll-like receptor agonists to restore the dysfunctional immunity induced by hepatitis C virus

Hepatitis C virus (HCV) infection is a major public health concern with approximately 3% of the world’s population is infected, posing social, economical and health burden. Less than 20% of the infected individuals clear the virus during the acute infection, while the rest develop chronic infection. The treatment of choice for HCV infection is pegylated interferon-α (IFN-α) in combination with ribavarin. Despite the cost and side effects of this treatment regimen, many patients fail this therapy and develop persistent HCV infection, leading to cirrhosis and hepatocellular carcinoma. Although the mechanisms underlying the failure to resolve HCV infection are poorly understood, the incapability of patients to develop effective anti-HCV immunity is a potential cause. We hypothesize that the dysfunctional anti-HCV immunity is due to the emergence of immunosuppressive cells coinciding with a decrease in the stimulatory dendritic cells (DCs) and natural killer (NK) cells. We further hypothesize that applying agents that can correct the imbalance between the immunosuppressive cells and stimulatory cells can results in resolution of chronic HCV. In this review article, we will discuss potential approaches, focusing on the use of Toll-like receptor agonists, to block the suppressive effects of the regulatory cells and restore the stimulatory effects of DCs and NK cells.     

Systems virology identifies a mitochondrial fatty acid oxidation enzyme, dodecenoyl coenzyme A delta isomerase, required for hepatitis C virus replication and likely pathogenesis

We previously employed systems biology approaches to identify the mitochondrial fatty acid oxidation enzyme dodecenoyl coenzyme A delta isomerase (DCI) as a bottleneck protein controlling host metabolic reprogramming during hepatitis C virus (HCV) infection. Here we present the results of studies confirming the importance of DCI to HCV pathogenesis. Computational models incorporating proteomic data from HCV patient liver biopsy specimens recapitulated our original predictions regarding DCI and link HCV-associated alterations in cellular metabolism and liver disease progression. HCV growth and RNA replication in hepatoma cell lines stably expressing DCI-targeting short hairpin RNA (shRNA) were abrogated, indicating that DCI is required for productive infection. Pharmacologic inhibition of fatty acid oxidation also blocked HCV replication. Production of infectious HCV was restored by overexpression of an shRNA-resistant DCI allele. These findings demonstrate the utility of systems biology approaches to gain novel insight into the biology of HCV infection and identify novel, translationally relevant therapeutic targets.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

Hepatitis C virus (HCV) employs multiple strategies to subvert the host innate antiviral response

Hepatitis C virus (HCV) is a serious global health problem which accounts for approximately 40% of chronic liver diseases worldwide. HCV frequently establishes a persistent infection, although it is recognized and targeted by innate immunity as well as cellular and humoral immune mechanisms. This suggests that HCV has developed powerful strategies to escape elimination by innate and adaptive immunity. HCV-induced liver injury is thought to be mainly immune-mediated rather than due to direct cytopathic effects of the virus. Hence, therapeutic strategies should target those mechanisms favoring viral persistence since unspecific enhancement of host antiviral immunity may theoretically also promote liver injury. The present review summarizes our current understanding of how the hepatitis C virus interferes with the innate antiviral host-response to establish persistent infection.     

Seroepidemiology of hepatitis C virus infection in Japan and HCV infection in haemodialysis patients

Abstract: Since January 1990, Japanese Red Cross Blood Centres have introduced hepatitis C virus screening with a first-generation ELISA. From April to December 1992, approximately 0.98% among 10905 489 blood donations screened by a second-generation assay were anti-HCV-positive in all Japan. Seropositivity of anti-HCV increased with the age and serum transminase value in both sexes. In blood donors having a history of transfusion, the anti-HCV reactive rate was 7.4%. The results of the study made by the Japanese Red Cross Non-A, Non-B Hepatitis Research Group show the effectiveness of implementation of HCV screening to prevent posttransfusion hepatitis. Consecutive haemodialysis patients with chronic renal failure are at risk for inflection by a variety of blood-borne agents transmitted within dialysis units. Because of their immunocompromised state, they frequently also have an unusual susceptibility to a variety of nosocomial infections, such as HBV, and HTLV-I. We tested the prevalence of anti-HCV in 1423 (848 males and 575 females) haemodialiysis patients from 18 hospitals in Kumamoto Prefecture, Japan using the Orhto first generation anti- HCV screening assay. There were 316 patients (22.2%) positive for HCV antibodies. The second-generation test was positive in most haemodialysis patients who were eractive to the firs-generation assay. The prevalence of HCV infection increased with the duration of haemodialysis, yet there was a high frequency of HCV seropositivity even wihtout blood transfusion. Acquisition of HCV in dialysis patients could be explained by HCV seropositivity even without blood (all haemodialysis are done with disposable kits, and needles), by secondary HCV infection after the immunodeficiency of haemodialysis, or by HCV infection of the kidney or glomerular deposition of immune HCV/anti-HCV complexes leading to chronic renal failure (as with HBV infection of the liver and kidney).     

Virus-host Interactions during Hepatitis C Virus Entry - Implications for Pathogenesis and Novel Treatment Approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.     

Scavenger receptor class B type I and hepatitis C virus infection of primary tupaia hepatocytes

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.     

Virus-host Interactions during Hepatitis C Virus Entry - Implications for Pathogenesis and Novel Treatment Approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.     

Virus-host interactions during hepatitis C virus entry — implications for pathogenesis and novel treatment approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.      

丙型肝炎病毒细胞和动物模型的研究进展

丙型肝炎病毒(HCV)是造成慢性肝炎、肝硬化及肝癌的重要病因之一,目前全世界约1.7亿人感染HCV.HCV的自然宿主范围很窄,由于缺少有效的HCV细胞培养系统和小动物模型,人们对其生活周期、作用机制等仍不是很清楚,从而严重阻碍了HCV疫苗及治疗药物的开发与研制.我们简要综述了近几年在HCV细胞和动物模型方面的进展,同时...