Screening Nature Compounds With Antiviral Activities Against Infection Bursal Disease Virus (IBDV) And Investigation Their Antiviral Mechanisms

Objectives:To screen the antiviral activities of eighty-eight nature compounds extracted from traditional Chinese medicines (TCMs) on Infectious Bursal Disease Virus (IBDV) in vitro.The effect of compounds on IBDV VP1 gene/protein and VP2 gene were investigated using real-time PCR and Western blot assay. In addition, the p-JNK expressions of the compounds were analyzed by Western blot, and explore their antiviral mechanisms.Method:1. Visualization of cytopathologic effect (CPE) assay and (3-(4,5-dimethyithiazol-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 ration (MIR) on CEF cells. The antiviral mechanisms of the screened compounds, which possessed both SI value was large than 3 and MIR was higher than 50%.2. Real-time PCR was used to quantify the medicine for IBDV VP1,VP2 gene copy-number, at different time points of and at maximum safe concentration,respective. In addition, the expression of VP1, JNK and the p-JNK at different time points by Western blot.Results:1. Berberine derivatives showed potent anti-IBDV activity at screening model in vitro,and the maximum inhibition rate was 64.9%, And the maxium inhibition ratio of Ribavin (positive compound) was 70%.2. Real-time PCR was used to quantify IBDV copy-number base on the VP1,VP2 gene. BD could inhibit the expression of VP1 gene at 24,48,72 and 96 h post infection, and the inhibition rate at 72 h was the same with Ribavin (P>0.05).BD could inhibit the expression of VP2 gene at 24,48,72 and 96 h post infection, but the inhibition effect was weaker than Ribavin (P<0.05).3. Expression of viral VP1 Protein in BD treated cells and JNK and p-JNK Protein in BD, DG, LH, R, and JNK inhibitor treated cells by Western blot. BD possessed the inhibition effect on VP1 protein at 72 hpi, whereas Ribavin could inhibit the expression of VP1 protein at all the test time points expect for 96 h. and addition of compounds resulted in the expression of p-JNK protein was decreased at 24hpi.Conclutiom:1. BD inhibited IBDV via persistent inhibition IBDV replication and VP1 gene/protein and VP2 gene expression.2. BD, R, and SP600125 inhibition the expression of p-JNK.