Screening and Action Pathway of Compounds Antagonistic Senacavirus A in vitro from Natural Product Library
[Objective]The experiment was aimed to search for natural products with inhibitory activity against Senecavirus A(SVA)and further investigate its antagonistic pathway.[Method]The concentration of the natural products were uniformly diluted to 10 μmol/L.Combined with the recombinant Senecavirus A with luciferase(rSVA-NLuc),compounds with luciferase inhibitory activity were screened from the natural products library,and their activity was further verified using Real-time quantitative RT-PCR.The maximum non-toxic concentration was determined by cytotoxicity testing using lactate dehydrogenase(LDH)method.Virus titer determination,Real-time quantitative RT-PCR,Western blotting,IFA and other techniques were used to study the effect and pathway of antagonism against SVA.[Result]Five compounds,monensin sodium salt,progesterone,hypophyllanthin,4-hydroxyderricin and 2-methoxyestrone,which could antagonize SVA in vitro,were screened from 136 natural products.Progesterone,a natural product that could antagonize SVA in vitro,was screened by Real-time quantitative RT-PCR and cytotoxicity detection.Compared with blank control group,the mRNA expression level of SVA was extremely significantly decreased in vitro(P＜0.01),and the maximum non-toxic concentration reached 50 μmol/L.Further study showed that progesterone could continuously inhibit the proliferation of SVA at 8,12,24 and 36 h after infection,and the virus titer was significantly decreased at 24 and 36 h after infection compared with control group(P＜50.05).Compared with control group,different concentrations of progesterone(10,20 and 50 μmol/L)extremely significantly decreased the adsorption level of SVA at the adsorption stage(P＜0.01).In the replication stage,the level of SVA VP3 and virus titer were extremely significantly decreased(P＜0.01).The proportion of released extracellular virus extremely significantly decreased(P＜0.01).But there was no significant effect on other approaches.These results indicated that progesterone could inhibit the adsorption,replication and release of SVA in vitro,but it had no obvious effect on its induction and assembly pathway.[Conclusion]In this study,progesterone,a natural product with low cytotoxicity and excellent antagonistic effect against SVA,was screened from the small molecule library of natural products.The product could inhibit the adsorption,replication and release of SVA,thus preventing the infection of SVA.This study provided a scientific reference for promoting the development of anti-SVA drugs and studying the infection bias of SVA.
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