Optimization of HCV Cell Culture System for Evaluation of Antiviral Drugs against HCV Genotypes 2a and 4a

Evaluation of hepatitis C virus (HCV) infection in cell culture is important for development of new antiviral agents and for understanding the viral life cycle, as well as the correlation among the genotypes spreading worldwide. Development of HCV cell culture systems offers great advantage by enabling growth of the virus in cell culture, allowing for discovery of potential therapeutics and vaccines. The aim of this study was to introduce the steps of HCV infection in the cell culture system as a determinant for the competence of more potent antiviral compounds. Two plasmids, JFH1 (genotype 2a) full length and 4a (ED43/C-NS2/NS5A) were subjected to <i>in vitro</i> transcription for HCV RNA synthesis followed by transfection of Huh7.5 with the resulting RNA from each genotype processed to generate the virus. Later, Huh7.5 cells infected with the replicated virus resulted in the generated HCV infected cells. Immunostaining of the infected cells showed fluorescence of green color, indicating positive infection of both genotypes 2a and 4a. In addition, real-time polymerase chain reaction was performed to measure viral load from extracted cells previously infected with the RNAs of genotypes 2 and 4, which resulted in 32,500 and 107,375 copies/mL, respectively. In conclusion, introduction of HCV cell culture system is a convenient method to evaluate active antiviral drugs against HCV genotypes 2a and 4a using both qualitative and quantitative techniques in distinguishing the effectiveness of antiviral agents.