抗A型塞内卡病毒天然产物筛选及颉颃作用机制研究

【目的】从天然产物库中筛选具有颉颃A型塞内卡病毒(Senecavirus A,SVA)活性的天然产物并研究其颉颃作用机制。【方法】利用荧光素酶重组塞内卡病毒(rSVA-NLuc)与BHK-21细胞，结合荧光素酶高通量筛选技术，建立抗SVA药物体外筛选平台。从天然产物库中筛选浓度为10μmol/L时具有抑制荧光素酶活性效应的天然产物，并进一步利用实时荧光定量RT-PCR验证其抑制活性，通过细胞毒性试验确定其最大无毒浓度。选择病毒感染周期的吸附、入胞、复制、组装释放4个主要过程，利用实时荧光定量RT-PCR、50%组织细胞感染量(TCID₅₀)测定等进行天然产物分子颉颃机制研究。【结果】从包含560种天然产物的分子库中筛选出16种候选抗SVA活性分子，通过实时荧光定量RT-PCR及细胞毒性检测，鉴定出4种安全、有效的天然产物，分别为20S-原人参三醇((20S)-protopanaxatriol)、蕈青霉素(paxilline)、方胆碱(fangchinoline)、竹红菌乙素(hypocrellin B)。作用机制研究显示，20S-原人参三醇能抑制SVA感染过程中的...

Screening of the Natural Products Against Senecavirus A and Study on Its Mechanism of Antagonism

【Objective】 The purpose of this study was to screen the natural products with anti-SVA activity from natural products library and to discover the mechanism of their antagonism.【Method】 Incorporating the use of the luciferase report technology,the high throughput platform for in vitro screening of anti-SVA natural products was established.In this platform,recombinant Luciferase reporter virus (rSVA-NLuc) system and BHK-21 cell were used to rapidly screen natural products against SVA from natural products library.The natural products with inhibitory effect on luciferase activity at the concentration of 10 μmol/L were screened from the natural product library,and their inhibitory activity was further verified by quantitative Real-time RT-PCR,at the same time,maximal nontoxic concentrations were determined by cytotoxicity test.The anti-viral mechanisms,which covering 4 main processes of the virus infection cycle including adsorption,entry,replication,assembly and release,were further studied with quantitative Real-time RT-PCR and 50% tissue culture infective dose(TCID₅₀).【Result】 16 natural products against SVA were screened from a library containing 560 natural products.Four safe and effective molecules were identified by quantitative Real-time RT-PCR and cytotoxicity test,namely (20S)-protopanaxatriol,paxilline,fangchinoline and hypocrellin B.Among them,(20S)-protopanaxatriol could inhibit the adsorption,replication of SVA.Paxilline could inhibit the adsorption,entry,replication,assembly and release of SVA.Fangchinoline mainly inhibited the entry and replication of SVA.Hypocrellin B could inhibit the adsorption,entry,replication,assembly and release of SVA.【Conclusion】 A total of 4 natural products against SVA were screened out from the natural products library.These compounds had good antiviral activity and different anti-viral mechanism.This study provided an important reference for the further development of anti SVA drugs.