Establishment Of Anti-HCV Compounds Screening System Based On Report Gene Recombinanted HCV Culture System And Its Preliminary Application

HCV(hepatitis C virus,HCV)is a major pathogens lead to chronic hepatitis,it was estimated that approximately 170 million people worldwide(approximately 3%)was infected with HCV.The increasing of infection case indicated the more severe prevalence of this disease.Once the occurrence of HCV infection,about 80%infected individuals will progressed to chronic disease.Among them,about 20%developed to liver failure and hepatocellular carcinoma.The current standard therapy for hepatitis C is pegylated interferon-α and ribavirin combined treatment.However,the low effection rate,the obvious side effects and high cost had hampered its clinical application.It was urgent to develop the new treatment using the more convincing anti-HCV drug screening system.Due to the absence of an efficient system of cell culture in vitro,the life cycle and functional mechanism of HCV are still poorly understood,and the development of vaccine and antiviral drugs are seriously hampered.Since the establishment of JFH1/Huh7 HCV culture system in 2005,a useful tool for HCV research is partial avaliable.Furthermore,it is possible to insert the reporter genes,such as Green Fluorescent Protein(GFP)or humanized Renilla luciferase(hRLuc),into recombinant plasmid to establish the high-throughput Drug screening systems.In this study,we obtain the Renilla luciferase reporter gene with PCR.Then,this reporter gene was introduced into the C-terminus of non-structural protein 5A(NS5A)of J6/JFH1 viral,the vectors construct comprising a reporter gene recombinant chimeric virus PFL-J6/JFH1-Rluc.The insertion was confirmed with gene squencing and double digestion.Using liposome transfection,the in vivo transcripted RNA was transduced into Huh7.5.1.The qualitative PCR was used to detetion the level of released HCV particle.It was shown that HCV viral load in the supernatant of cultured transfected cells is up to 105 copies/ml.Under the the different multiplicity of HCV infection,the virus proliferation character the recombinanted HCV in Huh7.5.1 cells was determined.The optimal host cell density for the following drug screening is 105cell/ml,and the best multiplicity of HCV infection is 3-3.5.In addition,virus infection experiments to test whether the Rluc gene-tagged HCV RNA can produce infection progeny virus.The results showed that Rluc activity assay showed that the changed of Rluc activity in cell can rescetively relect HCV RNA level.Hence,HCVcc system provides an effcient tool to search novel antiviral targets and strategies.Based on the successful establishment of 2a chimeric strains(J6/JFH1)HCV cell culture(Huh 7.5.1)system(HCVcc),IFNα-1b and Ribavirin was used as positive control to established MTT cytotoxic assay and real-time PCR viral load quantitaion method.These methods were combinated to evaluate the antiviral effect of compounds.Using this screening system,the anti-HCV effects of 25 candidates were evaluated.Among them,the obvious inhibition effects of 11 compounds with higher activity than RBV were discovered.It was suggested that they are effective for anti-HCV.However,the HCVcc infection system themselves has some limitation.There are obvious drawbacks such as the tedious and time-consumin quantitative PCR.As the described above,reporter geng-Rluc was introduced into HCV 2a chimeric to construct a series of HCV reporter plasmids.Rluc expression level could be taken as the index to evalate the preliferation of HCV.The half inhibitory concentration(EC50)could calculate under the intervention of compounds.MTT assay was used to determine the IC50 values.We selected the better anti-HCV activity of drugs(GQ-05,wbas7,wbas17)under the traditional screening system to have the rescreening with this new technology.It was shown that wbas7 with cytotoxicity IC50 of 14.90μg/ml,were dtermined to have EC50 of 0.109μg/ml with Renilla luciferase luminescence detection.Its SI value is 136,significantly highter than the positive control RBV.Moreover,IC50,EC50 and SI values were 13.44μg/ml,0.0967μg/ml and 139.99 for GQ-05;4.417μg/m,0.1615μg/ml and 27.34 for wbas.Three drug selection index selection indexes are higher than the positive control RBV,and it is consistent with that of traditional screening system.In comparing with replicon system and HCVcc system,this method is more sensitive,convenient to detect HCV RNA replieation level and progeny virus produetion by the luciferase activity assay.It has provided a more efficient tool for evaluation of HCVreplieation and antiviral activity.