Journal Article

Development of persistent HCV genotype 3a infection cell culture model in huh-7 cell

Shahid, Imran, Hassan, Sajida, Asad, Sultan, Gull, Sana, Kausar, Humera, Ijaz, Bushra, Khan, Muhammad Kazim, Ahmad, Waqar, Sarwar, Muhammad Tahir

2012

Overview

Background Hepatitis C virus (HCV) is one of the major health concerns globally, with genotype 3a as the most prevalent in Pakistan. Lack of efficient HCV genotype 3a small animal models as well as genomic replicons has hampered the complete understanding of its life cycle, pathogenesis and therapeutic options. In this study we aimed to develop a persistent HCV genotype 3a infectious cell culture model. Methods We inoculated Huh-7 cells with HCV genotype 3a serum. Cells and media supernatant were collected at different time periods up to 40 th day post infection. Culture media supernatant was also collected to find out its ability to infect naive Huh-7 cells. Results HCV replication was confirmed at both RNA and protein level through Real Time RCR and western blot using HCV core as marker. In order to validate the persistence of our model for HCV genotype 3a replication we inhibited the HCV replication through core specific siRNAs. The HCV RNA was detected intracellularly from the day one post infection up till 40 th day, while HCV core protein was detected from the second day up to 40 th day consistently. In culture media supernatant HCV RNA was also actively detected conferring its ability to infect the naive Huh-7 cells. Furthermore, core specific siRNA showed significant inhibition at 24 th hour post transfection both at RNA and protein level with progressive increase in the expression of core gene after 3 rd day. It clearly depicts that the Huh-7 successfully retained the HCV replication after degradation of siRNA. Conclusion Finally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: animal models / Binding sites / Biomedical and Life Sciences / Biomedicine / blood serum / Blotting, Western / Care and treatment / cell culture / Cell Culture Techniques - methods / chronic diseases / Cloning / Colleges & universities / culture media / Demographic aspects / Gene expression / genes / Genetic aspects / Genotype / Genotype & phenotype / HCV Core / HCV models / Hepacivirus - genetics / Hepacivirus - growth & development / Hepacivirus - pathogenicity / Hepatitis / Hepatitis C / Hepatitis C virus / Hepatocytes - physiology / Hepatocytes - virology / Huh-7 cell line / Humans / Liver cancer / Pakistan / pathogenesis / Prevention / protein content / Real time / Real-Time Polymerase Chain Reaction / Risk factors / RNA, Viral - biosynthesis / RNA, Viral - genetics / Rodents / siRNA / small interfering RNA / therapeutics / transfection / Viral Proteins - biosynthesis / Virology / Western blotting