We constructed dicistronic, subgenomic hepatitis C virus (HCV) replicons in which the sequence encoding the human immunodeficiency virus (HIV) tat protein was placed in the upstream cistron, between the HCV 5'NTR and a picornaviral 2A proteinase sequence fused to the selectable marker Neo. Stably transformed Huh7 cells expressing secreted alkaline phosphatase (SEAP) under transcriptional control of the HIV LTR promoter actively secreted SEAP following transfection with these replicon RNAs. Extracellular SEAP activity correlated closely with intracellular HCV RNA levels, as determined by Northern blotting and real-time RT-PCR analysis. These RNAs replicated efficiently despite the absence of core-protein-coding sequence downstream of the HCV IRES. The replication efficiency of replicons derived from the HCV-N strain of HCV was significantly greater than those derived from Con1 in transiently transfected cells. Using this reporter system, we have demonstrated significant differences in the response to interferon alpha-2b in cell lines containing replicons derived from these two strains of HCV.

Progress in understanding the pathogenesis of hepatitis C virus (HCV) has been slowed by the absence of tractable small animal models. Whereas GB virus B (GBV-B, an unclassified flavivirus) shares a phylogenetic relationship and several biologic attributes with HCV, including hepatotropism, it is not known to cause persistent infection, a hallmark of HCV. Here, we document persistent GBV-B infection in one of two healthy tamarins (Saguinus oedipus) inoculated intrahepatically with infectious synthetic RNA. High-titer viremia (10 8 to 10 9 genome equivalents per ml) and transiently elevated serum alanine transaminase activities were present from weeks 4 to 12 postinoculation in both animals. However, whereas GBV-B was eliminated from one animal by 20 weeks, the second animal remained viremic (10 3 to 10 7 genome equivalents per ml) for >2 years, with alanine transaminase levels becoming elevated again before spontaneous resolution of the infection. A liver biopsy taken late in the course of infection demonstrated hepatitis with periportal mononuclear infiltrates, hepatocellular microvesicular changes, cytoplasmic lipid droplets, and disordered mitochondrial ultrastructure, findings remarkably similar to chronic hepatitis C. GBV-B-infected hepatocytes contained numerous small vesicular membranous structures resembling those associated with expression of HCV nonstructural proteins, and sequencing of GBV-B RNA demonstrated a rate of molecular evolution comparable to that of HCV. We conclude that GBV-B is capable of establishing persistent infections in healthy tamarins, a feature that substantially enhances its value as a model for HCV. Mitochondrial structural changes and altered lipid metabolism leading to steatosis are conserved features of the pathogenesis of chronic hepatitis caused by these genetically distinct flaviviruses.

Hepatitis C is the most common cause of clinically significant liver disease in the United States. Although acute infection with hepatitis C virus (HCV) is usually subclinical, persistent infection develops in up to 80% of initially infected patients (2). The majority of persistently infected individuals develop evidence of chronic liver injury and are at risk for progressive hepatic fibrosis leading to cirrhosis and ultimately death due to liver failure or hepatocellular carcinoma. Approximately 10,000 individuals die each year with chronic liver disease associated with HCV infection in the United States alone (1), rendering this virus a very significant public health problem.HCV is classified within a distinct genus, Hepacivirus, in the family Flaviviridae (15). Its genome consists of positive sense, single-stranded RNA of approximately 9.5 kb in length (9). The genomic RNA contains a single open reading frame, from which one large polyprotein is expressed, and flanking 5Ј and 3Ј untranslated regions. Translation of the viral RNA occurs through a cap-independent mechanism via an internal ribosomal entry site (IRES) located in the 5Ј untranslated region. A combination of cellular and virally encoded protease activities leads to processing of the polyprotein into individual structural and nonstructural proteins. Three structural proteins have been identified with the core protein forming a poorly defined viral capsid, and E1 and E2, both glycosylated envelope proteins, forming heterodimers. These structural proteins are processed by host signal peptidases. C terminal to E2, further proteolytic processing by host peptidases yields a short hydrophobic peptide, p7, which may function as a membrane channel (44), followed by the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. NS2 and the amino-terminal protease domain of NS3 are responsible for proteolytic cleavage at the NS2-NS3 junction (20, 52), whereas other proteolytic cleavage events involved in the maturation of the NS proteins are carried out by the major viral serine protease, NS3, in association with its cofactor, NS4A (5,7,35). In addition to its function as a protease, NS3 also contains helicase and nucleoside triphosphatase activities within its C-terminal domain (28). Specific replicative functions have not been identified for NS4B and NS5A, but both are likely to contribute to the viral replicase complex. NS5B constitutes the HCV RNA-dependent RNA polymerase and is thus the catalytic core of the replicase.In addition to functions related to RNA replication, both NS3/4A and NS5A appear to play important roles in modulating antiviral responses within infected cells and in promoting long-term persistence of the virus. The NS3/4A protease blocks

H epatitis C virus (HCV) infection contributes significantly to human morbidity and mortality worldwide, and new and better therapeutics are urgently needed. 1 However, the evaluation of candidate antiviral compounds is hindered by the absence of readily available animal models. Chimpanzees (Pan troglodytes) are susceptible to HCV infection, but are endangered as a species and increasingly difficult to access for experimental studies. More recently developed murine models with chimeric human livers are technically challenging and, moreover, do not recapitulate pathogenesis because of underlying immunodeficiency. 2,3 GB virus B (GBV-B), a hepatotropic member of the Flaviviridae family with close phylogenetic relatedness to HCV (genus Hepacivirus), 4,5 thus provides a potentially useful surrogate for animal studies of hepatitis C. It is capable of infecting and causing hepatitis in several different nonendangered species of New World primates 6-9 and, like HCV, can establish a persistent infection associated with chronic liver disease. 10 The GBV-B and HCV genomes share many similarities and

Hepatitis C virus (HCV) infection is a major global health problem. Hepatic expression of immune costimulatory signaling molecules (e.g., B7) is known to be associated with ongoing liver injury in hepatitis C patients. However, due to the general lack of viral culture systems and adequate animal models, the function of these molecules in disease pathogenesis is poorly understood. To investigate the role of CD86 in HCV-related liver injury, we developed two transgenic mouse lineages with inducible expression of HCV structural proteins and constitutive expression of the costimulatory molecule CD86/B7.2 in the liver. Using a hydrodynamic-based, nonviral delivery protocol, we induced HCV transgene expression in the livers of HCV and CD86 single-and double-transgenic mice. We found that hepatic CD86 expression resulted in increased activation of and cytokine production (e.g., interleukin-2 and gamma interferon) by CD4؉ T cells and that the retention of these cells was associated with more pronounced necroinflammatory lesions in the liver. Taken together, these data suggest that augmented, parenchymal antigen presentation conferred by hepatocyte CD86 expression alters homeostasis and effector functions of CD4 ؉ T cells and contributes to liver injury. This study provides an additional rationale for exploring immunomodulation-based therapies that could reduce disease progression in individuals with chronic HCV infection.Liver disease due to hepatitis C virus (HCV) infection is an emerging public health problem, as persistent viral infection leads to liver cirrhosis and cancer in some patients (63a). No vaccine for prevention of HCV infection exists, and current interferon-based therapies result in a sustained antiviral response in only about 50% of patients (39). The mechanisms of pathogenesis are not fully understood. Since immune-based therapies that could improve viral clearance and reduce disease progression in chronically infected individuals would be of great benefit (21, 41, 65), a better understanding of the mechanisms contributing to liver injury and T-cell clearance of HCV infection are important goals for HCV research.The body maintains tolerance to many gut-derived antigens (54). Although the mechanisms underlying this process remain unclear, it is thought that immune responses within the liver are associated with tolerance. In the liver, hepatocytes express low levels of major histocompatibility complex (MHC) and virtually no immune costimulatory molecules (such as CD80/ B7.1, CD86/B7.2, and CD40). These conditions ensure that T cells "ignore" antigens expressed by the parenchymal cells. To mount an efficient immune response, costimulatory molecules on antigen-presenting cells need to engage their ligands on T cells, and this interaction provides a crucial signal permitting the activation and differentiation of T cells into effector cells.In human HCV infection, high levels of MHC class II and costimulatory molecules are expressed on the activated Kupffer cells and hepatocytes, and their levels are also co...