Screening Of Porcine Epidemic Diarrhea Virus Non-Structural Proteins That Induce Autophagy In IPEC-J2 Cells

Porcine epidemic diarrhea virus(PEDV)can cause acute and highly contagious enteric disease,porcine epidemic diarrhea(PED),which has caused huge economic losses to pig industry in recent years.PEDV can infect all stages of pigs,which has the most serious damage to suckling piglets,and the mortality rate can reach 90%~100%.Since the end of 2010,PEDV had been prevalent in southern China.By 2014,PEDV could be detected in pig farms in most parts of China.Although the usage of inactivated vaccines has inhibited the further spread of PEDV to a certain extent,the protection effect of traditional vaccines is not satisfactory due to the emergence of PEDV variant strains in the clinic practice.PEDV is still one of the main causes of acute diarrhea in piglets.Autophagy is a highly conserved lysosomal-dependent degradation pathway widely present in eukaryotic cells.It is essential for maintain intracellular homeostasis and clear damage organelles.Under the exogenous stimulation,autophagy can also act as a defense mechanism.Studies have shown that autophagy has a close relationship with the occurrence of cancer,pathogen infection and other biological processes.Many viruses induce autophagy in the process of infecting host cells.In order to explore the mechanism by which PEDV CV777 strain infect ion of IPEC-J2 cells to induce autophagy and screen PEDV key non-structural proteins,the following studies were conducted.Firstly,PEDV CV777 strain was adaptively passaged in IPEC-J2 cells,and then analyzed the PEDV-induced autophagy by transmission electron microscopy,Western blot and laser confocal immunofluorescence.The results showed that PEDV infection could increase the formation of autophagic bilayer membrane vesicles in IPEC-J2 cells,and induced significantly GFP-LC3 dot-like aggregation in cells.Western blot analysis indicated that the expression of autophagy-associated marker proteins LC3-II and Beclin-1 were up-regulated,and the autophagy substrate marker protein p62 showed degradation trend.This indicates that PEDV infection of IPEC-J2 cells can induce the occurrence of complete autophagy.In order to further explore the effects of PEDV non-structural proteins on autophagy,we constructed eukaryotic expression plasmids for each PEDV non-structural proteins,and transfected into IPEC-J2 cells.The results showed that the non-structural proteins Nsp4,Nsp5,Nsp6,Nsp8,Nsp10 and Nsp14 of PEDV could up-regulate the expression of autophagy-related gene LC3-II,and the accumulate effect of LC3-II caused by Nsp5 and Nsp8 was extremely significant.We selected Nsp5,Nsp6 and Nsp8 to further study PEDV non-structural proteins-induced autophagy.We interfered the expression of Nsp5,Nsp6 and Nsp8 with si RNAs on the basis of transfection of p CMV-Nsp5,p CMV-Nsp6 and p CMV-Nsp8 plasmids.The results showed that si RNA could diminish the autophagy induced by PEDV Nsp5,Nsp6 and Nsp8 remarkably.In addition,we found that induction of autophagy promoted PEDV proliferation,while inhibition of autophagy could hinder PEDV proliferation.This indicates that autophagy has closely relationship with the proliferation of PEDV.This study demonstrates that PEDV can induce autophagy in IPEC-J2 cells,and autophagy facilitates the proliferation of PEDV,Nsp5 and Nsp8 are the 2 key PDEV non-structural proteins that can induce autophagy.This study provides theoretical and material basis for further study of the mechanism of PEDV non-structural proteins-induced autophagy.