Molecular Mechanism Of Apoptosis Induced By Duck Plague Virus

Duck plague virus?DPV?can cause apoptosis of duck embryo fibroblasts?DEF?and lymphocytes,and studies around the DPV-induced DEF apoptotic pathway and cell cycle,and obtained the following main results:1.Study on the relationship between DEF cell cycle,apoptosis and virus replication induced by DPV?1?DEF cells were detected by flow cytometry at 24h and 36h after DPV infection.The results showed that the proportion of G0/G1 phase cells was significantly lower in the group and the proportion of cells in the S phase was significantly higher than that of the control group,leading to cell cycle S phase arrest.?2?The DEF cells detected by DAPI and flow cytometry at 12h,24h,36h,48h and60h of DPV infection showed that there were more apoptotic cells at 12h,apoptotic cells decreased at 24h,36h and 48h;qRT-PCR and TCID₅₀ The virus content at the corresponding time point showed that the virus content peaked at 48 h and gradually decreased at 60 h.This indicates that DPV replication is not directly proportional to induction of DEF apoptosis.2.Role of host caspase family proteins in DPV-induced DEF cell apoptosisDEF cells infected with DPV at 12h,24h,36h,48h and 60h,and mRNA levels of caspase-3,caspase-7,caspase-8,and caspase-9 detected by qRT-PCR showed that the mRNA content of DPV-infected cells was 2 times more than the control group.Activity assays of caspase-3,caspase-8,and caspase-9 showed that caspase-8 activity was significantly higher at 24 h,36 h,and 48 h than that of the control group,and caspase-9 activity was significantly higher at 36 h,48 h,and 60 h.In the control group.The activity of caspase-9 at 60 h was significantly higher than that of the control group.Caspase-3,caspase-9 specific inhibitors and caspase protein family spectral inhibitors can inhibit DPV-induced apoptosis,whereas caspase-8-specific inhibitors cannot inhibit DPV-induced apoptosis.3.DPV induces apoptosis in DEF cells through mitochondria,death receptors and endoplasmic reticulum pathwaysDEF cells infected with DPV at 12h,24h,36h,48h and 60h:?1?JC-1 fluorescent probe detected mitochondrial membrane potential,and the result was that the infected cells had more fluorescence than the control;ROS detected cells infected at 12h,24h,36h and 48h.The red fluorescence values were 1.28,1.36,1.18,and 1.06 times higher than those of the control,and ROS scavenger pretreated intracellular ROS could be cleared to increase mitochondrial membrane potential and inhibit apoptosis.?2?qRT-PCR results showed that Mitochondrial pathway-associated genes?Bak,Cyt-c,Smac,PARP,Apaf-1,Bid,Bcl-2,XIAP,and AIF?,death-receptor pathway-associated genes?Fas,FasL,FADD,and TRAIL?,endoplasmic reticulum pathway The mRNA levels of related genes(PERK,IRE1,Ca²⁺,CHOP,eiF2?,and ATF4)were more than twice that of the control group.In summary,DPV-infected DEF cells cause cell cycle S-phase arrest and can induce apoptosis through mitochondria,death receptors and endoplasmic reticulum pathways.