Development of Cell Culture Replication Systems for Hepatitis Rirus Genotype 5A

Abstract

The hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma with an estimated 170 million people chronically infected with the virus. Among the genotypes of HCV, genotype 5a (GT 5a) was first identified in a cohort of South African patients with HCV-induced hepatocellular carcinoma accounting for more than 30-50% of HCV infections in South Africa. The goal of my research was to establish functional in-vitro replication systems for HCV GT 5a as a preclinical tool for better screening and optimization of new and current viral inhibitors. To this end, sub-genomic replicons were generated and RNA transcripts electroporated into Huh-7.5 cells and selected with geneticin (G418 final concentration of 500µg/mL at 48 hours post-electroporation) to test their replication efficiencies. Production of G418- resistant colonies in Huh-7.5 cells was dependent on an NS5A S22051 amino acid substitution, a cell culture adaptive mutation originally reported for genotype lb replicons. Further, electroporation of naïve Huh-7.5 cells with total cellular RNA isolated from replicon cells transmitted G418 resistance. RNA quantification and NS5A staining of selected colonies revealed high detectable levels of HCV RNA and viral NS5A protein. Sequence analysis revealed potential adaptive mutations, which when introduced back into the original constructs, substantially increased colony formation efficiency. In conclusion, we have established the first functional sub-genomic replicon system for HCV genotype 5a, which together with the recently published system for genotype 6a completes the panel of replicon systems for the clinical significant HCV genotypes 1-6.