Animal And Culture Models of Viral Hepatitis And Hepatocellular Carcinoma

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Previously, we showed that a unique genotype 2a 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 gml with an average diameter of about 55 nm. The secreted virus is infectious for Huh7 cells. The cell culture-generated HCV is infectious in chimpanzee. In addition, we showed the production of HCV particles by using a DNA expression plasmid containing full-length HCV cDNA flanked by self-cleaving ribozymes. We produced HCV particles of various genotypes including 1a (H77), 1b (CG1b) and 2a (J6 and JFH-1) in the HCV-ribozyme system. 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 the culture medium. HCV replication levels of strains H77, CG1b and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells were inoculated to naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. 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 systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.[unreadable] [unreadable] Current treatment of chronic hepatitis C based on combination of peginterferon and ribavirin is only effective in about half of the patients and is accompanied by substantial side effects. Developing new classes of drugs against HCV is crucial. Phosphorothioate oligonucleotides (PS-ONs) have a sequence-independent antiviral activity against HIV-1 by inhibiting virus-cell fusion. Because viral entry is a highly conserved mechanism, this antiviral action of PS-ONs may be effective against infection by other enveloped viruses with type I or II fusion mechanisms. By applying the cell culture system described above, we assessed whether PS-ONs inhibit HCV infection and to evaluate the antiviral mechanism of action of PS-ONs. Various forms of PS-ONs and the control phosphodiester oligonucleotides (PO-ONs) were synthesized and evaluated in infectious HCV cell culture system systems. To test the efficacy of PS-ON in vivo, human hepatocytes transplanted uPASCID mice were inoculated with infectious HCV and treated with the PS-ON. The PS-ONs exhibited potent inhibitory activities in both cell culture-generated HCV-JFH1 (HCVcc) and HCV pseudo-particles (HCVpp) systems. This inhibitory activity was size and phosphorothioation dependent but sequence independent. The control PO-ONs had no inhibitory activity against HCV infection. The PS-ONs had no effect on viral replication in the HCV replicon system and binding of HCV-LPs to cells, indicating that the target of inhibition by PS-ONs is at the post-binding, cell-entry step. In human hepatocyte-engrafted uPASCID mice, the PS-ONs also appeared to efficiently block de novo HCV infection. The PS-ONs (amphipathic DNA polymers) are a promising new class of antiviral compounds that inhibit HCV fusion and entry. [unreadable] [unreadable] Based on the infectious HCV cell culture system, we are also setting up a cell-based assay for high-throughput screening (HTS) of small molecule chemical library in collaboration with the NIH Chemical Genomics Center. The NCGC has a large collection of over 250,000 chemical compounds and has established the facility for HTS. By performing cell-based HTS, we hope to identify novel targets and lead compounds for HCV therapeutic development.[unreadable] [unreadable] The identification of the hepatitis C virus (HCV) strain JFH-1 enabled the successful development of infectious cell culture systems. Although this strain replicates efficiently and produces infectious virus in cell culture, the replication capacity and pathogenesis in vivo are still undefined. To assess the in vivo phenotype of the JFH-1 virus, cell culture-generated JFH-1 virus (JFH-1cc) and patient serum from which JFH-1 was isolated were inoculated into chimpanzees. Both animals became HCV RNA-positive 3 days after inoculation, but showed low-level viremia and no evidence of hepatitis. HCV viremia persisted 8 and 34 weeks in JFH-1cc and patient serum-infected chimpanzees, respectively. Immunological analysis revealed that HCV-specific immune responses were similarly induced in both animals. Sequencing of HCV at various time points of infection revealed more substitutions in the patient serum-inoculated chimpanzee and the higher level of sequence variations seemed to be associated with a prolonged infection in this animal. A common mutation G838R in the NS2 region emerged early in both chimpanzees. This mutation enhances viral assembly leading to an increase in viral production in transfected or infected cells.