The Study Of Canine Distemper Virus Infection Induced Apoptosis Via Autophagy Inhibition

Canine distemper is an acute,highly contagious infectious disease caused by canine distemper virus(CDV)infection.CDV quickly infects target cells,such as T and B lymphocytes,and causes a sharp decrease in lymphocytes,which in turn leads to severe immunosuppression.Autophagy is a mechanism by which lysosomes achieve cytoplasmic component clearance and recycling,and is a cyclical mechanism that maintains cell homeostasis,morphogenesis,senescence,cell differentiation and cellular immunity.Autophagy also plays an important role in the pathogenesis of various diseases including cancer,neurodegenerative diseases and infectious diseases.In recent years,the regulation of autophagy has become the focus of research in the process of cell damage and necrosis in various diseases.In this study,the mechanism of cell damage caused by the CDV replication in Vero-E6 cells is investigated,aiming to study the relationship between autophagy and apoptosis in the process of CDV replication,and to analysis the trend of CDV replication by using autophagy regulators and apoptosis regulators.The results confirmed that the replication of CDV in Vero-E6 cells could cause cells damage.The infected cells become round,detached and suspended with visible syncytia and other cytopathic effects,which are positively correlated with MOI and infection time.It was also observed by fluorescence microscopy that the NP red fluorescence of CDV has a increased tendency with the prolongation of infection time.The results obtained by western-blot showed that CDV could inhibit autophay at early infection stage.To further investigate the relationship between autophagy and CDV replication,the modulators Rapamycin(autophagy inducer),MRT68921(autophagy inhibitor),and Z-VAD-FMK(apoptosis inhibitor)were used prior to virus infection.It was found that autophagy inhibitors have benifical effect for CDV replication,whereas apoptosis inhibitors do the opposite.These results indicate that CDV infection inhibits autophagy in Vero-E6 and promotes virus replication in cells.In order to confirm whether the cytopathic effect caused by CDV infection is related to apoptosis,we examined a variety of apoptosis indicators of cells after CDV infection.The results demonstrated that mitochondrial membrane potential and mitochondrial ROS were increased,and Caspase-3 was robustly activated,meanwhile,the up-regulated Bax expression,mitochondrial division and release of contents after infection with CDV were also observed,suggesting that the CDV infected cell apoptosis may be related to mitochondria damage.