The Role Of MDA5 In Chicken Embryo Fibroblast Autophagy Induced By IBDV Infection

Infectious Bursal Disease Virus(IBDV)is a double-stranded RNA virus belonging to the Birnaviridae family.Autophagy is one of the ways in which eukaryotic cells die.Studies have shown that IBDV could cause autophagy in DF-1 cells,but its specific mechanism remains to be studied.Melanoma differentiation associated gene 5(MDA5)is a member of the pattern recognition receptor family and capable of specifically recognizing double-stranded RNA viruses.Previous studies by the research group showed that IBDV c ould activate the chicken MDA5(ch MDA5)signaling pathway in the bursa of chickens and participate d in the process of IBDV infection in chickens.Therefore,this study intends to use IBDV as the pathoge n,chicken embryo fibroblasts(CEF)as the research object,the relationship between MDA5 and IBDV-induced autophagy,further reveal the pathogenesis of IBDV.First,the double-stranded RNA virus mimic poly(I:C)was transfected into CEF,and the expression of ch MDA5 protein was detected by Western blot.It was found that poly(I:C)transfection could cause an increase in the expression of ch MDA5 in CEF.Then,CEF was divided into control group,poly(I:C)group,ch MDA5 si RNA+poly(I:C)group,Rapa+poly(I:C)gr oup,3-MA+poly(I:C)group,The expression of autophagy marker protein LC3 was detected by Western blot.The results showed that ch MDA5 mediates polymorphism of CEF induced by poly(I:C),and autophagy activator Rapa could increase autophagy induced by poly(I:C).The autophagy inhibitor 3-MA was capable of inhibiting autophagy caused by poly(I:C).On this basis,in order to study the role of ch MDA5 in CEF autophagy induced by IBDV infection,CEF was divided into normal control group,IBDV infection group,ch M DA5 si RNA+IBDV group,Rapa+IBDV group and 3-MA+IBDV group.Western blot,indirect ELISA,real-time fluorescent quantitative PCR and transmission electron microscopy were used to detect the occurrence of autophagy,activation of MDA5 and its signaling pathw ay,replication of IBDV and damage of CEF.The results showed that:(1)Compared with the normal group,the expression levels of ch MDA5 and its signaling pathway key molecules ch IPS-1,ch NF-?B,ch IRF3 and downstream products ch IL-1?,ch IL-6,ch TNF-?,and ch IFN-? were significantly increased in the IBDV group(P<0.01).Compared with the IBDV group,with the silencing of ch MDA5,the expression levels of ch IPS-1,ch NF-?B,ch IRF3,and ch IL-1?,ch IL-6,ch TNF-?,and ch IFN-? were also significantly or extremely significantly decreased(P<0.05 or P<0.01);the expression of ch IL-1? and ch TNF-? m RNA in Rapa+IBDV group was significantly decreased(P<0.05);the expressions of ch MDA5 and ch IPS-1,ch IRF3,ch IL-1?,ch IL-6,ch TNF-? and ch IFN-? were significantly decreased in the 3-MA+IBDV group(P<0.05).(2)Compared with the normal group,the expressions of autophagy-related proteins LC3 and Beclin1 in the IBDV group were significantly increased(P<0.01),and the phosphorylation levels of AKT and m TOR were significantly decreased(P<0.05).Compared with the IBDV group,the expression of LC3 and Beclin1 was significantly increased after ch MDA5 specific silencing(P<0.05),and the phosphorylation levels of AKT and m TOR were significantly decreased(P<0.05);the expression level of LC3 and the phosphorylation level of AKT and m TOR in Rapa+IBDV group were significantly or extremely significantly increased(P<0.05 or P<0.01);there was a significant or extremely significant difference in the expression of LC3 and Beclin1 between t he 3-MA+IBDV group and the IBDV group(P<0.05 or P<0.01).In the IBDV group,the mitochondria and endoplasmic reticulum were swollen,and autophagosomes were observed.In the ch MDA5 si RNA+IBDV group,mitochondrial cristae ruptured and dissolved,the endopl asmic reticulum swelled,the perinuclear space became wider,and the autophagosome structure increased.(3)The virus titer,load and VP2 expression in ch MDA5 si RNA+IBDV group and Rapa+IBDV group were significantly higher than those in IBDV infection group,and the titer and VP2 expression of 3-MA+IBDV group were significantly decreased(P<0.01).The above results indicated that IBDV infection could not only activate the intracellular MDA5 signaling pathway,but also induced autophagy in CEF by inactivation of AKT/m TOR pathway.At the same time,autophagy could promote the proliferation of IBDV,while MDA5 could attenuate CEF autophagy caused by IBDV and protected CEF.This study laid a foundation for further investigation of the relationship between virus a nd autophagy and the involvement of MDA5 pathway in the pathogenesis of IBDV,and provide d new ideas for the prevention and treatment of viral diseases.