title: Development of persistent HCV genotype 3a infection cell culture model in huh-7 cell.
abstract: 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 40th 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 40th day, while HCV core protein was detected from the second day up to 40th 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 24th hour post transfection both at RNA and protein level with progressive increase in the expression of core gene after 3rd day. It clearly depicts that the Huh-7 successfully retained the HCV replication after degradation of siRNA.
  
  
  CONCLUSIONS
  Finally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.
authors.affiliation: Applied and Functional Genomics Lab, Centre of Excellence in Molecular Biology, 87-West Canal Bank Road, 53700 Lahore, Pakistan.
authors.forename: Sultan
authors.initials: S
authors.lastname: Asad
authors.forename: Bushra
authors.initials: B
authors.lastname: Ijaz
authors.forename: Waqar
authors.initials: W
authors.lastname: Ahmad
authors.forename: Humera
authors.initials: H
authors.lastname: Kausar
authors.forename: Muhammad Tahir
authors.initials: MT
authors.lastname: Sarwar
authors.forename: Sana
authors.initials: S
authors.lastname: Gull
authors.forename: Imran
authors.initials: I
authors.lastname: Shahid
authors.forename: Muhammad Kazim
authors.initials: MK
authors.lastname: Khan
authors.forename: Sajida
authors.initials: S
authors.lastname: Hassan
mesh_terms: D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity*; D022781:Hepatocytes / Q000502:physiology* / Q000821:virology*; D006801:Humans; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014764:Viral Proteins / Q000096:biosynthesis
chemical_list: D012367:RNA, Viral; D014764:Viral Proteins