Abstract
BackgroundHepatitis 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.MethodsWe 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.ResultsHCV 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.ConclusionFinally, we report that our persistent infection cell culture model consistently replicate HCV genotype 3a for more than 1 month.
Highlights
Hepatitis C virus (HCV) is a causal agent of both acute and chronic hepatitis [1] and is one of the foremost health problems affecting nearly 350 million people worldwide [2]
Effect of core specific small interfering RNA (siRNA) Csi476 on expression of HCV core gene in serum infected huh-7 cells We transfected core specific siRNA named Csi476 (100 μM) in our persistent HCV infection model and Development of persistent HCV genotype 3a huh-7 infectious model Huh-7 cells were infected with HCV genotype 3a serum of high titer (> 1 × 108 copies/μl)
Detection of HCV RNA was done through HCV genotype 3a core gene specific primers by semi quantitative RT-PCR which showed continuous core gene expressions in media supernatant; showed that HCV RNA was detected on the 10th day onward till 40th day post infection
Summary
HCV is a causal agent of both acute and chronic hepatitis [1] and is one of the foremost health problems affecting nearly 350 million people worldwide [2]. In 2005 Wakita et al successfully cloned HCV genotype 2a JFH1 and transfected it in Huh-7 cell line leading to successful replication and virions production [18], while Zhong et al achieved a very robust and efficient system for infectious virions in Huh-7 cell line [19]. Yi et al were able to achieve efficient HCV virions production with HCV genotype 1a J77-S virus in Huh-7 cell line [20] Despite of their effectiveness the Huh-7 derived HCV virions producing systems have several draw backs like utilization of unusual and rare cloned HCV genotype 2a JFH1 [18] and use of cloned HCV genotype 1a H77-S having five non-natural adaptive mutations [20]. 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
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