Development Of Cell Model And Molecular Detection Method For Screening Drugs Against Grass Carp Reovirus

Grass carp (Ctenopharyngodon idellus) is one of the most popular commercial fish species cultured in China, its annual yield covers 20% in total output of fishery products. Grass carp is sensitive to the infection of a wide range of aquatic pathogens, especially, the grass carp hemorrhage caused by grass carp reovirus (GCRV) lasts for a long period of time, prevails in a wide range of areas and causes high mortality, and leads to huge economic loss in aquaculture industry. Therefore, the investigation of effective methods for preventing and treating grass carp hemorrhage has continuously been the critical points. At present, there is no treatment method available for controlling grass carp hemorrhage, immunization is the effective method to prevent the disease. Since immunization still has a long way to go to be applied in practice, to screen effective drugs is important for grass carp farming.By using the staining assay with tetratzole (MTT), the experiments for screening the drugs with antiviral characters against GCRV were conducted on grass carp kidney (CIK) cell line, therefore, a cell model for screening the anti-GCRV drugs has been established in grass carp kidney (CIK) cell culture system in vitro. Moreover, a real-time fluorescence quantitative PCR was applied to evaluate the effect on the reduction of viral nucleic copies mediated by antiviral drugs.In the group that drugs were administrated to cells before GCRV challenge, the results of MTT assay showed that Potassium Dehydroandrograpolide Succinate had the strongest effect in blocking the GCRV to attach and penetrate into the CIK cells, followed by Ribavirin and Radix Isatidis, Radix Scutellariae had the least effect among them. The therapeutic indexes of four selected drugs were 810±47,108±13,35.28±8.35 and 18.08±3.76, respectively. In the group that drugs were administrated to cells after GCRV infection, the results indicated that Ribavirin, Potassium Dehydroandrograpolide Succinate and Radix Scutellariae had significant effects on inhibiting the replication of GCRV, the therapeutic index were 358±26,555±48 and 74.2±12.53, respectively, while Radix Isatidis had no apparent effect in this test. In the assay of the selected drugs directly killing GCRV in vitro, the results demonstrated that Ribavirin had a significant effect in killing GCRV in vitro with a therapeutic index 102±16, while the other selected drugs had no impact in killing GCRV in vitro. The total RNA of viral genome in infected CIK cells was extracted and the GCRV-VP6 coding gene was used as templates for specific primer pairs design and for real-time PCR amplification to evaluate the effect of drugs on the reduction of GCRV genome RNA copies. The results showed that the GCRV-VP6 gene was not detected in the Potassium Dehydroandrograpolide Succinate treated group, and the relative copies of GCRV-VP6 coding gene in Ribavirin, Radix Scutellariae and Radix Isatidis treated groups accounted for 3.51%±0.62%,34.62%±4.18%,25.34%±2.48% of the positive control. The real-time PCR detection results were consistent with that of MTT assay.