HCV Genome 1b Asia Replicon Cell Model And Drug Screening

The hepatitis C virus (HCV) was the major causative agent of hepatitis. Globally, HCV is estimated to infect 170 million people (3% of the world's population). In most cases, the immune response is unable to clear the virus, resulting in its persistence. Patients with persistent infection are at high risk to develop chronic liver diseases including cirrhosis and hepatocellular carcinoma (HCC). Current options for prophylaxis and therapy of chronic hepatitis C are very limited. Thus far, there is no vaccine that prevents infection with HCV. Current therapy is based on the combination of a conjugated form of interferon-alpha (IFN-α) and ribavirin. The rate of sustained viral response deponds on the genotype. This therapy is costly and associated with significant side-effects. Therefore, more effective and better tolerated therapeutic strategies are urgently needed.A major step forward was the development of the replicon system in 1999. This system allowed for the first time the efficient propagation of genetically modified HCV RNAs in a human hepatoma cell line and since then, became a well established tool in most laboratories working with this virus.In this study, plasmid containing HCV 1b subgenomic replicon of Chinese population was constructed. HCV subgenomic replicon RNA was obtained by in vitro transcription, and then delivered into Huh-7.5.1 cell by electroporation. After screening the cells in the medium containing G418, we got the cell colonies. In order to get the cell clone that replicate HCV RNA effectively, we selected 90 cell colonies and cultured them. In the end, we got three cell clones. We detected the HCV RNA in the obtained cell colonies by RT-PCR and the HCV NS proteins by Western blot.The cell colonies which contain HCV RNA were treated by IFN in order to observe the diminished level of HCV RNA. This experiment imitates the process of drug screening and provides a good tool for anti-virus drug screening and evaluation.At the same time, we express HCV non-structural proteins in Escherichia coli and Bac-to-Bac baculovirus system in order to get the NS proteins for HCV study.We have successfully established a cell model for supporting HCV 1b subgenomic replicon of Chinese population replication in vitro. This cell model is a useful tool for the study on HCV pathogenesis, the screening of antiviral drugs and the development of vaccines.