Study On Apoptosis Of Duck Embryo Fibroblasts Cells Induced By Hexon Protein Of Egg Drop Syndrome Virus

Egg drop syndrome virus(Egg drop syndrome virus,EDSV)originally isolated from the ducks and was named duck adenovirus Ⅰtype.EDSV mainly causes sudden drop in egg production in laying birds accompanied by a reduction in egg quality.In recent years,several studies revealed that EDSV caused serious respiratory symptoms,and even death in ducklings.Although extensive efforts have been made to understand adenovirus infection in human cells,little is known about the mechanism underlying infection of EDSV in poultry.This study demonstrated that EDSV and its Hexon protein could induce apoptosis in duck embryonic fibroblasts(DEF),which provide the relevant theoretical basis and scientific basis for studying the pathogenicies of the virus on poultry.The results are as follows:1.DEF was inoculated with EDSV,and then measured the cytopathic effect(CPE)and the level of virus proliferation.The results showed that with the prolongation of infection time,EDSV caused DEF cells to occur more obvious cytopathic effect(CPE).Cell rounding,and eventual degeneration and detachment of cells could be observed within the monolayer of infection.The quantitative real-time PCR was used to assess viral copy number in DEF cells infected with EDSV.The results showed that with the prolongation of infection time,the viral copy number marked increase during 24 to 60 hpi.At the same time we tested the virus titer in the cell supernatant,and the virus titer increased from 23 to 28.2.The process of EDSV-induced DEF apoptosis was assessed using Annexin V/PI flow cytometry.EDSV infection produced a significant high level of apoptosis in cells compared to mock infected cells at 24 hpi,reaching a maximum of 29.6% at 60 hpi.Interestingly,more TUNEL positive cells were observed in EDSV-infected groups at 36 and 60 hpi.The results of Caspase activity and Western blot analysis showed that EDSV infected DEF cells could significantly activate Caspase-8,Caspase-9 and Caspase-3.EDSV infection can upregulate the expression of Bax in the cell,and down-regulate the expression of Bcl-2.The ratio of Bax / Bcl-2 increased with the time of infection.3.We constructed eukaryotic vector pCDNA-Hexon of hexon protein in the present study..Western blot analysis showed expression level of hexon protein peaked at 24 h and 36 h in hmogenates for primary DEF cells after transfection compared to mock-infected cells.TUNEL detection also found that more TUNEL-positive cells were observed in Hexon-transfected cells as compared with that in pCDNA-C1-transfected control cells at any indicated time points.In particular,the numbers of TUNEL-positive cells were significantly higher in Hexon-transfected cells at 24 and 36 hpi than that at 48 and 60 hpi.The results of Caspase activity and Western blot showed that the activation of Caspase-3,-8,and-9,as well as Bcl-2 and Bax expression indeed correlated with the time-dependent decrease of hexon expression in pCDNA-3.1-Hexon transfected DEFs.Hexon expression can upregulate the expression of Bax in the cell,and down-regulate the expression of Bcl-2.The ratio of Bax / Bcl-2 increased with the time of infection.These data demonstrate that EDSV infection or EDSV hexon transfection can induce apoptosis in DEF cells via activating both the exogenous and the mitochondrial pathway.These results not only lay the foundations for revealing the molecular pathogenesis of EDSV infection,but also provide new ideas for the study of other pathogenic mechanism of poultry disease.