Studies on proliferating mechanism of hepatitis C virus using cultured human liver cell lines by radialflowtype-bioreactor

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In order to carry out a construction of infectious clones to infect into the artificial bio-liver, we constructed a full length cDNA clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood sample was shown to be infectious to both a recipient and chimpanzees. Then we constructed a recombinant baculovirus containing the full length cDNA of HCV by using a charomid system. By immunoprecipitation analysis, properly processed HCV proteins were expressed in cells infected with the recombinant baculovirus.Cell lines were then assessed for their suitability in artificial models of HCV infection and replication. We constructed a replication-deficient recombinant adenovirus expressing bacteriophage T7 RNA polymerase under the control of CAG promoter (AdexCAT7). A high level of T7 RNA polymerase was detectable. Cells infected with AdexCAT7 were then transfected with plasmids carrying (i) the T7 promoter, the 5' untranslated region (UTR) of encephalomyocarditis virus and … More a luciferase (pT7EMCVLuc), or (ii) the T7 promoter, the 5'UTR of HCV and a luciferase gene (pT7HCVLuc). Most of the cell lines examined supported a higher expression of luciferase by transfection with pT7EMCVLuc than with p7F7HCVLuc. However, one cell line, FLC4, derived from a human hepatocellular carcinoma exhibited very high reporter gene expression with pT7HCVLuc. In this cell line, transfection with RNA synthesized in vitro from pT7HCVLuc induced a higher level of reporter gene expression than RNA from pT7EMCVLuc. It may be due to robust stability of the HCV RNA minigene transcription process in FLC4 owing to some as yet unknown factor. In monolayer culture, FLC4 cells showed continuous secretion of HCV RNA when experiments were carried out using plasma taken from a chronic Hepatitis C patient Taken together, these findings persuaded us to select FLC4 cells as the most suitable for further studies in artificial models of HCV infection and replication. Stable high-density cultivation in bioreactor environments was successfully carried out for more than 60 days, and furthermore, proliferation rates could be controlled by cultivating at lower temperatures.Using this artificial liver model, it was possible to confirm by RT-PCR the secretion of HCV RNA in response to Hepatitis C infected plasma HCV RNA tested positive for the first two days after infection, but tests were negative thereafter. Unfortunately, infection of the culture medium could not be confirmed.We are presently continuing studies using the infectious clones we have developed as well as those obtained from the NIH USA which have been shown to infect chimpanzees. Less