In Vitro Screening Natural Compounds And Derivatives With Antiviral Activities Against Porcine Reproductive And Respiratory Syndrome Virus (PRRSV) And Investigating Their Molecular Mechanisms

Objectives:To screen the antiviral activities of drugs from thirty-six natural compound and derivatives on porcine reproductive and respiratory virus (PRRSV) in vitro and explore their underlying antiviral mechanisms.Methods:At cell levels, Visualization of cytopathologic effect (CPE) assay and 3-(4, 5-dimethyithlazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test were used to determine maximum no-cytotoxic concentration (MNTC),50% cytotoxic concentration (CC50).50% effective concentration (EC50) and maximum inhibition ratio (MIR) on Marc-145 cells also were determined in the safe scope by the same way. At last, the durg was curtained by SI>3 and MIR>50%. Then, virucidal assay, time-of-addition assay and adsorption inhibition assay were explore their antiviral mechanisms. At last, RT-PCR was used to test the copies of N gene and the expression of cell genes at different points with MNTC, and to quantify the expression of N protein with TS at different points by Western Blot. In addition, Annexin V/PI double staining was detected the changes of PRRSV-induced apoptosis.Results:Form thirty-six natural compound and derivatives, we find seven drugs (No.3、No.27、No.30、 No.32、No.33、No.34、No.35) showed potent anti-PRRSV activity on Marc-145 cells and they are in dose-dependent manner, including No.3 (Benzoic acid derivatives, trans olefinic bond has been saturated), No.27 (Steroid cholic acid derivatives), No.30 (TS, characterizated tea saponins), No.32, No.33, No.34 and No.35(Glycyrrhetinic acid compounds). The highest inhibition ratio of TS was 100%. The highest inhibition ratio was more than 76% at 1-14 h with virus replication. The highest inhibition ratio of blocking PRRSV attached to cells with 65% and kilingl virus with 80%. The result of RT-PCR suggested TS can effectively inhibit the copy number of N gene at 20 h-52 h (P<0.001). Compared with PRRSV control at 24 hpi, only the expression of PABP mRNA declined P<0.001).The result of Western Blot showed cell treated with TS can significantly inhibit the expression of N protein. The inhibition ratio is a dose-dependent manner. Besides, the number of late apoptotic with TS significantly was lower than PRRSV control group.Conclusions:TS inhibit PRRSV effectively via directly inactivating PRRSV, persistent inhibiting PRRSV replication, blocking virus attach cell and disturbing PRRSV-induced apoptosis. Its antiviral mechanisms were explored through inhibiting the expression of N gene/protein and down-regulating mRNA expression of PABP.