Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the effects of viral gene expression on infected cells remain unclear in vivo. Efforts to develop culture and animal models for hepatitis C are critical for the understanding of the virus and the disease it causes. Our collaborators had previously shown that subgenomic replicon of the JFH-1 genotype 2a strain cloned from a patient with fulminant hepatitis replicates efficiently in cell culture. In a recent study, we showed that the full-length JFH-1 genome replicates efficiently and secrets viral particles after transfection into a human hepatoma cell line. The viral particles have a density of about 1.17 g/ml with an average diameter of about 55 nm. The secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies as well as immunoglobulins from chronically infected patients. The cell culture-generated HCV is infectious in chimpanzee. Recently, we also showed the production of HCV particles by using a DNA expression plasmid containing full-length HCV cDNA flanked by self-cleaving ribozymes. This construct also contains the secreted alkaline phosphatase gene to monitor the expression from this construct and to normalize transfection efficiency and to control for effects of culture conditions, such as anti-viral testing. Various strains of HCV genotype 1a (H77), 1b (CG1b) and 2a (J6 and JFH1) were constructed and tested. After transfection into Huh7 derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by detection of HCV RNA and core antigen in culture medium. HCV replication levels of strains H77, CG1b and J6 were comparable. However, HCV replication level of JFH1 was substantially higher as compared with other clones. Iodixanol density gradient centrifugation of the culture medium revealed co-localization of HCV RNA and structural proteins, and presence of HCV particles around the peak fraction was confirmed by electron microscope. The HCV generated by this system was infectious in naive Huh7.5.1 cells; HCV core and NS3 proteins were detected by immuno-fluorescence study 3 days after inoculation. The culture medium from CG1b (HCV genotype 1b)?transfected cells caused a typical course of HCV infection in chimpanzee. The HCV 1b propagated in vitro and in vivo had identical sequences as the HCV genomic cDNA used for cell culture transfection. The development of culture system for production of infectious HCV particles of various genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.? ? Based on the tetracycline regulatory system, we established a binary transgenic model in which the conditional expression of two transgenes, SV40 T antigen (TAg) and LacZ, can be tightly regulated in the liver by administration of tetracycline. Mice with tTA and TAg transgenes developed hepatocellular adenomas and hyperplasia that could be prevented by continuous tetracycline administration. We have adopted this transgenic system to the regulated expression of HCV proteins in the liver using this model system. Analysis of Alb-tTA/TRE-HCV double transgenic mice revealed that the HCV RNA and protein expression in the liver could be completely suppressed by tetracycline administration, and induced in a reversible fashion by tetracycline withdrawal. Mice with constitutive expression of HCV had no evidence of hepatic pathology until 11 months of age, when steatosis developed. In contrast, mice with tetracycline-mediated suppression and then withdrawal at 2 months of age developed hepatic inflammation with ALT elevation by 4 months of age. Hepatic steatosis also became evident at 5 months of age, which occurred much earlier than the mice with constitutive HCV expression. Neither hepatic inflammation nor steatosis was observed in mice that had been continuously subjected to tetracycline treatment. With a tightly regulatable HCV expression, this model resembles those events observed in natural infection and offers a valuable tool to study the pathogenesis of hepatitis C.
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