Molecular Mechanism Of Swine Acute Diarrhea Syndrome Coronavirus Induced Host Cell Apoptosis

Swine acute diarrhea syndrome coronavirus(SADS-Co V),a newly found porcine enteric coronavirus in China in 2017,which mainly causes watery diarrhea,vomiting,dehydration and even death of newborn piglets under 5 days old.The outbreak of SADS-Co V had great attention of researchers.However,it is unclear whether the infection of SADS-Co V induces apoptosis of host cells and the effect of apoptosis on the replication of SADS-Co V.Therefore,the purpose and significance of this study is to explore the phenimenon of SADS-Co V infection-induced host cell apoptosis and the effect of apoptosis on the replication of SADS-Co V.It will provide scientific evidence and theoretical basis for the prevention of SADS-Co V by analyzing the pathway of SADS-Co V-induced apoptosis and clarifing the mechanism of interaction between SADS-Co V and apoptosis.In order to reveal whether SADS-Co V can induce apoptosis in vivo and in vitro,SADS-Co V-infected Vero E6 cells were observed by transmission electron microscope.The results showed that the cells had obvious shrinkage,mitochondria swelling,nuclear atrophy and chromatin condensation.TUNEL assay showed that SADS-Co V induced obvious apoptosis in Vero E6 and IPI-2I cells.The DNA Ladder analysis showed SADS-Co V infection break up the DNA of the cell genome and appear obvious fragmentation,and the intensity of the ladder bands increased with time and dose of infection.To obtain the accurate apoptosis rate,flow analysis of apoptosis showed that the apoptotic rate increased with the time of SADS-Co V infection and reached of 25.7 % and 37.5 % in Vero E6 and IPI-2I cells at 48 hpi,respectively.These data demonstrated that SADS-Co V infection induced obvious apoptosis in Vero E6 and IPI-2I cells.Caspases are cysteine proteases which is the key enzyme inducing apoptosis.Western blot analysis showed that Caspase-8?Caspase-9 and Caspase-3 were activated in SADS-Co V-infection Vero E6 and IPI-2I cells.Activated Caspase-3 cleaved the downstream effector molecule PARP(poly ADP ribose polymer)and which maked PARP inactive.At the same time,Caspase-3 and PARP were cleaved in SADS-Co V-infected ileal tissues of piglets.These results indicated that SADS-Co V infection induced host cells apoptosis in vivo.To further determine the pathway of apoptosis induced by SADS-Co V,Western blot showed that Fas L expression was up-regulated in Vero E6 cells infected with SADS-Co V,indicating that SADS-Co V could activate Fas L-mediated extrinsic pathway.Furthermore,SADS-Co V infection resulted in cleavage of the Bid to 15 k Da t Bid.These results imply that apoptosis signals from Fas L are transmitted to activate Caspase-8,which in turn cleaves Bid.Cleaved Bid activates Caspase-9,which links the extrinsic and intrinsic pathways.Western blot,flow cytometry and immunofluorescence were used to detect the cell apoptosis and virus replication after using different apoptosis inhibitors.The results showed that Z-VADFMK,Z-IETD-FMK of Caspases-8 and Z-LEHD-FMK of Caspases-9 significantly decreased PARP cleavage and the expression of SADS-Co V N protein.And the virus titer exhibited a significant decrease in a dose-dependent manner.The results of confocal laser scanning and Western blot showed that the proapoptotic proteins Bax and Cytochrome c(Cyt c)relocalized to the mitochondria and cytoplasm,respectively,after infection by SADS-Co V.Further,the results revealed that SADS-Co V induced apoptosis through mitochondria mediated intrinsic pathway.Cyclophilin D(Cyp D)is located in the mitochondrial membrane matrix and is the main component in the process of mitochondrial permeability transition pore(MPTP)opening.Vero E6 and IPI-2I cells treated with cyclosporin A(Cs A),an inhibitor of Cyp D,were completely protected from SADS-Co V-induced apoptosis and viral replication,suggesting the involvement of cyclophilin D(Cyp D)in these processes.Taken together,our results indicate that the Fas/Fas L mediated death receptor pathway and mitochondrial-mediated intrinsic pathway are both activated in SADS-Co V-infected cells.These findings demonstrate mechanisms by which SADS-Co V induces apoptosis and improve our understanding of SADS-Co V pathogenesis.