Establishment Of Reporter HCV Replication/Infectious Models

Hepatitis caused by hepatitis C virus (HCV) presents a contagious disease threatening public health. HCV is a major cause of chronic liver disease, such as chronic hepatitis, hepatic cirrhosis, hepatocellular carcinoma, with over 170 million persistently infected individuals worldwide. So far, treatment options for chronic HCV infection are limited to a combination of alpha interferon(IFN-α) and ribavirin (RB),which has a low efficiency of only 45% and severe adverse effects. There are not yet effective vaccines and anti-HCV drugs.At present some HCV subgenomic replicons permitting HCV replication in cells, provide smart tools for study on HCV replication in vitro and screening on anti-HCV drugs. But they can neither be used to predict the efficiency of antiviral compounds in vivo nor produce infectious HCV viral particles,so that they can't satisfy the needs of researches on HCV entry into cells. The lack of a robust small-animal model has hindered unveiling pathogenicity of HCV and developing vaccines and antiviral drugs. So the efforts to establish efficient HCV models, including HCV replication small-animal models as well as infectious cell or animal models, are of great significances.Based on HCV subgenomic replicon, here we reported one type of firefly luciferase (Fluc) reporter subgenomic HCV replicon subcutaneously implanted nude mouse model.Meanwhile we are trying to establish green fluorescence protein(GFP) and Fluc reporter full-length genome HCV infectious cell system as well as JFH1 full-length genome HCV infectious cell model on the basis of JFH1 strain. The contents and results are as following:1. The Fluc-reporter subgenomic HCV replicon cell model was established by transfecting pFKI389LucUbiNeoNS3-3'/5.1 RNA obtained via in vitro transcription into Huh7 cells and selecting for 3-4 weeks in G418-containing medium. Fluc activity is much higher in selected Huh7 cells harboring pFKI389LucUbiNeoNS3-3'/ 5.1 replicon RNA than naive Huh7 cells as control. HCV NS5A protein was also detected by NS5A-specific indirect immunofluorescence(IF). Fluc activity declines in dose-dependent manner afterα-IFN-2b treatment for 72h.Then Fluc-reporter subgenomic HCV replicon mice model was established by subcutaneouly implanting Huh7 cells containing HCV replicon into Balb/c nude mice. HCV can efficiently replicate in nude mice for at least one month. The inhibition of HCV replication byα-IFN-2b was observed in vivo by noninvasive whole-body imaging, demonstrating the validation of this mouse model.2. Green fluorescence protein(GFP)was inserted into pFL-J6JFH1 at 2394 aa (amino acid) to construct pFL-J6JFH1-GFP. GFP reporter full-length genome HCV infectious cell model was constructed by transfection of in vitro transcribed J6JFH1-GFP RNA into Huh7.5 cells. GFP was visualized by fluorescence microscopy and HCV NS5A was monitored by NS5A-specific indirect IF in Huh7.5 cells transfected with J6JFH1-GFP RNA. Viral RNA of 3.5×107 copies/ml was detected in supernatant of this cell culture system. Afterα-IFN-2b treatment for 72h, green fluorescence almost disappeared, demonstrating the validation of this cell model.3. Luc reporter full-length genome HCV infectious cell model was established by transfection of in vitro transcribed Luc-JC1 RNA into Huh7.5 cells. HCV NS5A was detected by NS5A-specific indirect IF in Huh7.5 cells harboring Luc-JC1 RNA. Bioluminescence intensity was much higher in Huh7.5 cells harboring Luc-JC1 RNA by noninvasive imaging system than that in naive Huh7.5 cells, implying the expression of luciferase. Viral RNA of 3.3×106 copies/ml was detected in supernatant of this cell culture system.4. JFH1 full-length genome HCV cell model was established by transfecting plasmid pJFH1 into Huh7.5 cells and selecting with Blasticidin. HCV NS5A was detected by NS5A-specific indirect IF in Huh7.5 cells harboring JFH1 while HCV core protein was detected by western blotting. HCV RNA in supernatant after 3 days postransfection was up to 5×108 copies/ml ,which regularly declined to 106 copies/ml in the following 2-3 weeks. But RNA copies in supernatant frequently fluctuated around 106copies/ml during the next two months,implying the stability of this model . In conclusion, based on the published HCV replicon and JFH1-based infectious constructs, we established Fluc reporter subgenomic HCV replicon mouse model, JFH1 and reporter(GFP and Luc ) full-length genome HCV infectious cell model , which would facilitate unveiling pathogenicity of HCV and developing vaccines and anti-HCV drugs.