Effect Of Porcine Epidemic Diarrhea Virus 3CLpro On RLRs-Mediated Interferon Signaling Pathway And The Evaluation Of Antiviral Effect Of Baicalin In Vitro

Porcine epidemic diarrhea virus（PEDV）is a member of the coronavirus family.It is the main cause of death from watery diarrhea in newborn pigs in recent years,posing a great threat to the swine industry.After infection,innate immune responses are activated through host pattern recognition receptors（PRRs）,which recognize the pathogen-associated molecular patterns（PAMPs）,triggering signaling cascades that lead to production of cytokines like type I interferon that suppress virus replication.However,recent studies have shown that PEDV can regulate the innate immune response of host cells by its encoded protein.For example,3C-like protease(3CL^pro)encoded by Nsp5 plays an important role in the process of virus proliferation and pathogenicity,but it’s still unclear how to antagonize the host’s innate immunity by 3CL^pro.Thus,the research expected to explore the effect of PEDV and 3CL^proon the innate immune signal pathway and find its active site.Meanwhile,we try to find the method to control PEDV infection,and explore the effect of anti-virus of baicalin.They provide theoretical support for in-depth understanding the pathogenic mechanism of PEDV and the development of antiviral drugs.1 Effect of PEDV on interferon signaling pathway mediated by RLRsPorcine small intestinal epithelial cells（IPEC-J2）were infected by the PEDV JS2013with MOI=1.The transcription levels of type I interferon IFN-βand type III interferon IFN-λwere detected by Q-PCR,which showed that they were significantly inhibited.The mRNA transcription levels of RLRs pathway related genes showed that PEDV JS2013 can significantly inhibit RIG-I,MDA5,MAVS,TRAF3,TBK1,IKKεand IRF3（P<0.05）,and inhibit Poly（I:C）-induced interferon pathway activation（P<0.05）.It showed that PEDV infection can antagonize the activation of interferon signaling pathway in host cells.2 Effect of PEDV 3C-like protease on interferon signaling pathway mediated by RLRsGiven pivotal role of 3C-like protease(3CL^pro)in virus replication and pathogenesis,we transfected 3CL^prointo IPEC-J2 or HEK293T cells to study.After Poly（I:C）stimulation,the transcription levels of RLRs pathway related genes were detected by Q-PCR,and it was found that 3CL^procan inhibit the transcription of interferon,MAVS and TRAF3.The wild-type 3CL^pro,enzyme active site mutants H41A and C144A,were further transfected to detect the changes of interferon and RLRs pathway related genes by Q-PCR,Western blot and dual luciferase reporter gene system.The results showed that 3CL^procan inhibit the gene and protein expression of RIG-I and TRAF3,thereby inhibiting the IFN-β pathway mediated by RLRs in a protease-dependent manner.3 Antiviral effect of baicalin on PEDV JS2013 and its mechanismSo far,there are no effective therapeutic drugs for PEDV.It is of great practical significance to find an antiviral drug with low side effects,low cost and good effect.The main component of baicalin is flavonoids,which have obvious effects in anti-virus,anti-inflammatory and other aspects.In the study,we detected cells viability by CCK-8 and found that 250μg/mL is the safe concentration,which can promote cell growth.Further Q-PCR detection found that baicalin could effectively inhibit virus replication by up-regulating the mRNA levels of antiviral genes and down-regulating the levels of inflammatory factor genes.Molecular docking technology predicted that baicalin may bind to the active site of PEDV 3CL^pro,which provides a theoretical basis for the development of antiviral drugs for PEDV.