Study On Anti-PEDV Mediated By Adaptor Proteins Of Porcine Innate Immune Signaling Pathways

Porcine epidemic diarrhea(PED)is an acute contagious intestinal disease caused by pathogenic porcine epidemic diarrhea virus(PEDV).The typical symptoms of PED are vomiting,diarrhea and dehydration.In severe cases,a large number of piglets may die from infection.PEDV is a single-stranded plus strand RNA virus with envelope membrane that belongs to alpha coronavirus family.As a worldwide swine epidemic virus,PEDV has brought great damage to the global pig industry scince it emerged.However,PEDV has a fast mutation rate,it is difficult for traditional vaccines to achieve efficient prevention and control,with the competition and co-evolution of PEDV and host.Therefore,deep research on PEDV infection and immunity is needed to provide new idea and theoretical support for prevention and control of PED.Innate immunity,also called the natural immunity,is the first line of host defense.It recognizes the conserved pathogen associated molecular patterns(PAMPs)by its pattern recognition receptors(PRRs),triggers intracellular signaling cascades,and produces a series of effector molecules to fight against infections including viral infections.The innate immunity plays a key role in the PEDV infection,however,the detailed roles of innate immunity in PEDV has not been fully elucidated.In this study,various porcine innate immune signaling pathways were investigated via signaing adaptor proteins for anti-PEDV function,and multiple innate signaling pathways were found to exert anti-PEDV function.In addition,the monclonal antibodies against PEDVN and S1 proteins were developed to provide useful tools for PEDV research.Specifically,the 9 signaling adaptors reflecting all currently known innate immune signaling pathways were systematically screened and analyzied in transfected Vero cells.The results showed that most of the 9 adaptors exhibited anti-PEDV activity to varied degrees,with the TRIF and MAVS better than others.Based on these results and considering the importance of nucleic acid sensing in antiviral immunity,we chosed the adaptors TIRF,MAVS and STING for further and detailed analysis of anti-PEDV function.Agonist stimulation experiments showed that endogenous TRIF,MAVS and STING possessed obvious anti-PEDV activity.The siRNA knockdown experiment also showed that TRIF,MAVS and STING all participated in anti PEDV innate immune response.The infections of TRIF-/-,MAVS-/-and STING-/-Vero cells made by CRISPR approach further verified the significant anti-PEDV effects of TRIF,MAVS and STING.At last,the anti-PEDV function by TRIF,MAVS and STING were recapitulated in the relevant porcine intestinal epithelial cells,IPEC-DQ cells,treated with different siRNAs.In addition,the PEDV N and S1 genes were cloned and expressed in bacteria,and the proteins were purified bacteira.The pure proteins were immunized into BALB/c mice and hybridoma were obtained by cell fusion.By ELISA screening,five N positive hybridoma(2B10,1C7,1G7,3C11,2F2)and three S1 positive hybridoma(2G9,4G9,1G7)were acquired.The mouse ascitic fluids were prepared from three N positive cell clones(2B10,1C7,3C11)and three S1 positve cell clones(2G9,4G9,1G7).The prepared ascetic monoclonal antibodies against N and S1 could specifically recognize PEDV in Western blotting and immune fluorescence assay(IFA),respectively.In conclusion,we developed monoclonal antibodies as detection tools for PEDV,and revealed that multiple porcine innate signaling pathways mediated by a variety of PRRs play an important role in anti-PEDV infection.It thus provides new ideas and new mateirals for treatment of PEDV infection.