抗A型塞内卡病毒天然产物筛选及颉颃作用机制研究  

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

作　　者：蒙英雯 李月华[2] 沙洲 李晨钰 巩有权 董雅琴[2,3] 魏荣 邹丰才[1] 尼博 MENG Yingwen;LI Yuehua;SHA Zhou;LI Chenyu;GONG Youquan;DONG Yaqin;WEI Rong;ZOU Fengcai;NI Bo(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;China Animal Health and Epidemiology Center,Qingdao 266032,China;Key Laboratory of Animal Biosafety Risk Prevention and Control(South),Ministry of Agriculture and Rural Affairs,Qingdao 266032,China;College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030800,China;College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163711,China)

机构地区：[1]云南农业大学动物医学院,昆明650201 [2]中国动物卫生与流行病学中心,青岛266032 [3]农业农村部动物生物安全风险预警及防控重点实验室(南方),青岛266032 [4]山西农业大学动物医学学院,太谷030800 [5]黑龙江八一农垦大学动物科技学院,大庆163711

出　　处：《中国畜牧兽医》2023年第2期734-744,共11页China Animal Husbandry & Veterinary Medicine

基　　金：中国动物卫生与流行病学中心创新基金(DW2021009);“十四五”国家重点研发计划(2021YFD180030)。

摘　　要：【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(TCID 50)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感染过程中的吸附、复制阶段;蕈青霉素在SVA的吸附、入胞、复制、组装释放阶段即整个病毒感染周期均发挥抑制作用;方胆碱能抑制SVA的入胞、复制;竹红菌乙素能抑制SVA的吸附、入胞、复制及组装释放阶段。【结论】本试验从天然产物库中筛选出了4种具有抗SVA活性的天然产物,这些化合物抗病毒效果良好,且作用机制各有不同。本研究为抗SVA药物的进一步研发提供重要参考。【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 10μmol/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(TCID 50).【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.

关 键 词：A型塞内卡病毒(SVA) 天然产物 高通量筛选 颉颃机制 

分 类 号：S853.74[农业科学—临床兽医学]