Effects Of Cellular Apoptosis And Autophagy During Porcine Deltacoronavirus Infection

Porcine deltacoronaviruses(PDCoV),a newly emerging swine intestinal corona virus,which can infect pigs of all ages and cause a series of gastrointestinal symptoms that can lead to pig death.In recent years,outbreaks and epidemics of PDCoV in North America have caused economic losses to local aquaculture.Nowadays,reports on PDCoV are mainly focused on epidemiological investigations,little is known about the pathogenic mechanism of PDCoV.Apoptosis is a genetically regulated suicidal behavior in cells;Autophagy is a conserved protein degradation mechanism present in eukaryotic cells.Apoptosis and autophagy are related to each other and regulate the pathological and physiological processes of cells together.Viral infection can induce a series of reactions such as apoptosis and autophagy to protect cells from damage.However,some viruses may evolve strategies to achieve immune evasion,or hijack the mechanism of apoptosis or autophagy to facilitate viral replication.PDCoV can cause vacuolar degeneration and necrosis of IPEC-J2 cells in vivo,but not apoptosis.PDCoV can only cause apoptosis of LLC-PK cells and ST cells in vitro.In the study of PDCoV-induced apoptosis,cell shedding was observed in PDCoV infected porcine testicular cells,suggesting that this phenomenon is mediated by apoptosis induced by viral infection.In ST cells,caspase activity assay showed that the activity of caspase 3,caspase 8 and caspase 9 increased significantly with the infection of PDCoV,but not observed in UV irradiated PDCoV-infected cells,this indicates that PDCoV infection activates both endogenous and exogenous apoptotic pathways in ST cells,and the induction of apoptosis is dependent on viral infection.To further investigate the endogenous apoptosis induced by PDCoV,cytochrome C and apoptosis-inducing factors in cytoplasm and mitochondria were detected.The results showed that compared with normal cells,the amount of cytochrome C released from mitochondria to cytoplasm increased significantly in PDCoV-infected cells,and the release increased with the prolongation of infection,while the apoptosis-inducing factor was always localized to mitochondria,suggesting that PDCoV induced apoptosis is initiated through caspase-dependent mitochondrial apoptosis pathway by promoting cytochrome C in the mitochondrial membrane gap into the cytosol.In the study of PDCoV-induced autophagy,we first detected the punctate aggregation of GFP-LC3 in cells by laser confocal technique.The colocalization of autophagosomes and lysosomes was observed by laser confocal microscopy combined with immunoblotting to detect the degradation of p62.By monitoring the autophagy flux,it was proved that PDCoV induced complete autophagy in ST cells.In addition,the autophagy level of ST cells was regulated by the autophagy inducing agent rapamycin and the autophagy inhibitor chloroquine,and the expression of PDCoV and viral protein were detected.It was confirmed that autophagy induced by viral infection was beneficial to virus replication.These above results indicate that PDCoV infection requires an autophagy pathway to enhance viral replication and maturation in host cells.By studying the potential relationship between PDCoV infection and ST cell apoptosis and autophagy,this article revealed the infection characteristics of PDCoV,provided ideas for further study of the interaction between pathogens and host cells,and laid a foundation for the research and application of antiviral.