摘要Severe fever with thrombocytopenia syndrome virus(SFTSV)is a tick-borne virus with a high fatality rate.Currently no approved drugs or vaccines are available against it.Sharing a common replication mechanism with negative-stranded,segmented viruses(NSVs),SFTSV utilizes a cap-dependent endonuclease(CEN)domain of the L segment to execute the cap-snatching process upon genome transcription initiation.Given the crucial role of CEN in the life cycle of NSVs,it is considered a promising target for discovery of antiviral agents against SFTSV.In this study,we established a high-throughput FRET-based enzymatic screening system to discover inhibitors of SFTSV CEN from a chemical library containing 3467 natural compounds.Finally,three compounds,i.e.,Licoflavone C,3,4-dicaffeoylquinic acid,and oleanolic acid displayed exceptional antiviral effects and minimal cytotoxicity.Licoflavone C(EC50=1.85 μM)was selected for further investigation.Administration of Licoflavone C(20mg/kg,i.v.)significantly reduced tissue viral loads in SFTSV-challenged mouse model.We demonstrated that Licoflavone C did not directly bind to the active pocket of SFTSV CEN but disrupted its active conformation,resulting in substrate non-competitive inhibition.Licoflavone C also exhibited broad-spectrum inhibition on several NSV CENs(HRTV,GTV,and LCMV)besides SFTSV.Furthermore,15 analogs of Licoflavone C sharing a typical flavonoid structure were verified for targeting SFTSV CEN and exhibiting antiviral activities.In conclusion,Licoflavone C is a promising inhibitor of SFTSV,offering insights into targeting CEN with flavonoids in drug discovery.