Isolation And Identification Of Senecavirus A And Its Molecular Mechanism Of Inducing Cellular Autophagy

Senecavirus A(SVA)is the only member of the genus Senecavirus of the Picornaviridae.The clinical symptoms of SVA are vesicular on the lesions,snout hoof and coronary bands,which is indistinguishable from the infection of FMDV,SVDV and VSV.Since the disease appeared in our country,SVA has spread and endemic in many provinces and cities,causing serious economic losses to the breeding industry.Therefore,exploring the relationship between the virus and host cells and understanding the body's cellular defense mechanism against SVA will provide a theoretical basis for the pathogenic mechanism of SVA.Autophagy is a self-protection and defense mechanism that plays an important role in maintaining cell homeostasis.Studies have shown that autophagy is closely related to pathogen infection,and has a dual effect on the proliferation of viruses in host cells.In this study,we investigated the relationship between SVA infection and autophagy,the launched and obtained work and results as follow:(1)In this study,the clinical sample was amplificated using PCR reaction,as a result,a positive SVA sample was successfully obtained.PK-15 cells were used to isolate SVA from the positive sample,and obvious cytopathic effects could be seen in blind transmission to the 10th generation.The TCID50 was calculated to be 10-5.776/0.1 mL according to the Reed-Muench method.The isolation was identified by transmission electron microscopy,indirect immunofluorescence,and RT-PCR methods,which showed that the virus particles and specific fluorescent spots were visible,as well as the target bands of different generations of viruses,which proved a strain of SVA had been successfully isolated and named SVA HB-BD.(2)To determine whether SVA can induce autophagy in cells,the methods of transmission electron microscope,laser confocal,Western blot,RT-qPCR and IFA were used.The results showed that SVA could induce the formation of autophagic double-layer membrane structure after infection.And the conversion of autophagosomes to autolysosomes were observed.The expression level of LC3 protein and genes of ATG5 and Beclin-1 were significantly increased(P<0.05).The p62 protein was significantly increased after SVA infection pretreated with CQ(P<0.05),indicating that SVA could induce a complete autophagy process.In addition,UV-inactivated SVA could not induce autophagy after infection cells,indicating that the induce of autophagy required the replication of SVA.(3)To determine the effect of SVA structural protein VP2 on autophagy,the eukaryotic expression vector of VP2 gene was constructed.The LC3 protein and autophagy genes in the transfected cells were significantly up-regulated detecting by Western blot,IFA and RT-qPCR methods(P<0.05),which preliminarily proved that SVA structural protein VP2 could induce the autophagy.(4)To determine the effect of autophagy on the proliferation of SVA,Rapamycin,3-MA and CQ were used to pretreat cells in this study.The proliferation of SVA was inhibited in PK-15 cells detecting by Western blot and RT-qPCR methods,while 3-MA and CQ could significantly promote the replication of the virus.Furthermore,the RNAi technology was used to interfere with the expression of LC3 and Beclin-1 genes,it significantly promotes the proliferation of the virus.These results indicated that autophagy inhibited the virus proliferation.(5)To further explore the molecular mechanism of SVA-induced autophagy,the expression of proteins was significant increased at different time points after SVA infection of cells.In order to further verify the effect of SVA infection on the signal pathway,AICAR was used to pretreat cells and then inoculated with SVA.The results showed that the pretreatment group significantly increased the expression level of signal protein,which proved that SVA induced the autophagy through AMPK-mTOR pathways.In summary,this study successfully isolated and identified a SVA strain.And it is proved that SVA infection of PK-15 cells could induce autophagy.The structural protein VP2 of SVA promoted the autophagy process.The autophagy negatively regulated the proliferation of SVA.SAV induced the autophagy process through AMPK-mTOR signaling pathways in PK-15 cells.