Establishment Of Anti-hcv Compounds Screening System And Its Preliminary Application

Hepatitis C virus (HCV), a small and enveloped RNA virus, was classified to Hepacivirus genus of Flaviviridae family. Its genome was a single- stranded positive-sense RNA of 9.6 kb length. It was estimated that 170 million people was infected with HCV worldwide. The increasing of infection case indicated the more severe prevalence. Once the occurrence of HCV infection, about 80% infected individuals will progressed to chronic disease. Among them, about 20% ended with liver failure and hepatocellular carcinoma. The current standard therapy was pegylated interferon-αand ribavirin combined treatment. However, less than 50% patients was shown with a sustained viral response (SVR), the effection of treatment is much poor in patients with HCV lb strains infection. In addition, 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.The successfully established 2a chimeric strains (J6/JFH1) HCV cell culture (Huh 7.5.1) system (HCVcc) had been introduced into our lab. With optimization of cell culture condition and analyze on Huh 7.5.1 cell growth curve, it was found that the cell grown exponentially between 2-6 culture days with started cell density of 9×104 cells/ml. And, the cultured No.3 day could be taken as the test time of cell toxicity. With the different infection multiplicity of infection and followd virus proliferation assay, the exponential growth phase was from 2 to 6 day after virus inoculation. The best determine time was No. 3 day with viral load of 6.5×106 IU/ml. The optimal multiplicity of infection of HCV strains was 1. Furthermore, IFNa-lb and Ribavirin was used as positive control to established MTT cytotoxic assay and real-time PCR viral load quantitaion method to evaluate the antiviral effect of drugs. It was shown that IFNa-lb have no obvious cell toxicity, which half effective concentration value was 1.452 IU/ml. For Ribavirin, half of the cell growth inhibition concentration (IC50) value was determined as 20.14μg/ml, whose half effective concentration (EC50) values was 2.553μg/ml, the calculated selective index (SI) value was 7.889. In parallel, IFNa-lb was shown no obvious cyto-toxicity with EC50 of 1.452 IU/ml. The different drug delivery time and patterns were performed furtherly, the 18 hours after virus inoculation and concurrent of inoculation was shown as the best start interrupte time for IFNa-lb and RBV respectively. The inhibite effect of combinate treatment was better than single using of IFNa-lb or Ribavirin.With the established screening system, the anti-HCV effects of 15 candidates were tested. Among them, the obvious inhibition effects of 6 compounds were proved. It was listed as shm-7 (IC50:1.089μg/ml, EC50:0.025μg/ml, SI,44), LDX22 (IC50:5.752μg/ml, EC50:0.180μg/ml, SI:32), LDX28a (IC50:20.390μg/ml, EC50:0.672μg/ml, SI:30), ZDH7(IC50:32.640μg/ml, EC50:0.561μg/ml, SI:58), ZDH22 (IC50:5.089μg/ml, EC50: 0.260μg/ml, SI:20) and ZDH26 (IC50:14.870μg/ml, EC50:0.127μg/ml, SI:116). LDX28a and ZDH7 hadn't any cytotoxity. It was notable that selective index of ZDH26 was up to 116, which was much higher than positive control RBV. The anti-HCV effects of remained 9 compounds (shm-4, shm-9a, shm-14, LDX24, ZCS3, ZCS4, ZCS11, ZCS17 and ZDH17) haven't shown here.In summary, based on the introduced HCV cell culture system, the anti-HCV drug screening system had been established with interferon and ribavirin as a positive control. Using this menthod, the anti-HCV effects of 6 compounds from 15 candidates was determined. This screening system will be taken as a useful tool to discover anti-HCV compound from the prolific drug resource of Yunnan. The promotion of this technique platform on drug industry could be expected.