Establishment and Viral Load Analysis HCVcc in Human hepatoma Cell line (Huh 7.5) via HCV- RNA Transfection

Background and Objectives: Hepatitis C virus (HCV), and positive-stranded RNA viruses cause chronic hepatitis and hepatocellular carcinoma (HCC). According to the WHO, about 3% of the world population is infected with hepatitis C virus. T7 RNA polymerase transcripts of a full-length genome plasmid of Hepatitis C virus (genotype 1b) were used to transfect a human hepatoma cell line, Huh7.5.We report here the establishment of a long-term, persistently infected Huh7 culture following RNA transfection. This method allows us to use regions of the virus genome that are important in HCV replication to study the inhibition or reduction of virus pathogenicity in human liver cells (such as novel therapeutic approaches e.g. microRNAs). Materials and Methods: cells. The Huh 7.5 cell line was cultured in complete growth medium at 37 ºC in a 5% CO2 incubation. Full-length infectious HCV RNA of the genotype 1b (pCon-1) was prepared by transcription using the MEGA script T7 kit in vitro. Liposome-mediated transfection was performed with Lipofectamine 2000 in cell suspensions containing 3×105 cells. In summary, one day before infection, 1× 105 Huh 7.5 cells were cultured in a 6-well plate as described in the previous section. The RNA copy number of Hepatitis C virus (HCV) in supernatant of infected cells was determined by using HCV quantitative high pure viral RNA kit. Results: HCV RNAs of positive polarity were transcribed from transcription vectors by T7 polymerase. Huh 7.5 cell lines transfected with HCV RNA. A viral load of 1.3 × 104 Copy/mL was detected compared to the standard concentrations. This indicates the high accuracy of the test. Conclusions: We have synthesized full-length HCV RNA from a pCon-1 vector transcribed in vitro, using T7 RNA polymerase, and have then transfected it into Huh7.5 cells. Several lines of evidence show that this synthetic RNA was infectious and replication competent. Cell media derived from transfected cells were capable of infecting fresh Huh7.5 cells suggesting that infectious virus was secreted from the transfected cells. Compared to other HCV strains, pCon-1 could produce higher titers of virus particles and its production of core antigen was 10 times higher than that of other strains.