Molecular Mechanisms Underlying Porcine Epidemic Diarrhea Virus-Induced Apoptosis In IPEC-J2 Cells

Porcine epidemic diarrhoea virus(PEDV)is an alpha coronavirus of the Coronaviridae family.PEDV can infect lactating pigs and piglets,causing porcine epidemic diarrhoea(PED),mainly manifested by vomiting,diarrhoea and loss of body fluids,with high mortality in infected piglets.Since 2010,PED outbreaks have started in China,causing serious health hazards and economic losses to the domestic pig industry.Currently,inactivated or attenuated vaccines play an important role in the prevention of PED,but due to the genotypic changes caused by the constant mutation and recombination of the virus,and the ineffectiveness of traditional vaccines in protecting against the prevalent strains,the prevention and control of PED is not satisfactory.Apoptosis is one of the many forms of cell death,and is an intrinsically genetically encoded programmed cell death that occurs throughout the life cycle of the organism and plays an important role in the homeostasis of the internal environment and the development of the living organism.Many viruses can affect apoptosis in direct or indirect ways to achieve their replication and infection.By studying the interaction between different genotypes of PEDV strains and host cells,we could deepen our understanding of the cell apoptosis process in PEDV infection,provide an empirical basis for an in-depth discussion of the PEDV pathogenesis,and provention and control measures for PED.To investigate the mechanism of apoptosis induction by infection of IPEC-J2 cells with PEDV CV777 and HLJ strains,this paper carried out the following studies:First,PEDV CV777 and HLJ strains were adapted for passaging in IPEC-J2 cells.Under the scanning and transmission electron microscopy,the PEDV-infected IPEC-J2 cells appeared significantly deformed and shrunken,mitochondria swelled,cell nuclei shrunk and fragmentated,and apoptotic bodies appeared in the late stage.AO/EB staining of PEDV CV777-and HLJ-infected IPEC-J2 cells showed significant augments in apoptotic fluorescence.Flow cytometry was used to detect the apoptotic rate of PEDV-infected cells by time point,and the results showed that the apoptotic rates of PEDV CV777 and HLJ strains were 88.1% and 48.9% at 72 h post infection(PI),respectively.Furthermore,the induced apoptosis appeared in a time-dependent manner.The mitochondrial membrane potential was detected by JC-1 fluorescent probe,and the results showed that the mitochondrial membrane potential was depolarized in IPEC-J2 cells at 24 h PI,further validating the ultrastructural changes in mitochondria after virus infection.To investigate the pathway of PEDV-induced apoptosis in IPEC-J2 cells,real-time fluorescence quantitative PCR and Western Blot were used to detect relevant apoptotic factors and proteins,and it was found that Caspase-3/8/9,Fasl,AIF,P53,Bax,Cytc gene expression was up-regulated and Bcl-2 gene expression decreased.Western Blot showed that Caspase-3/8/9,Fasl Fas protein expression was up-regulated and Bcl-2 protein expression decreased after PEDV infection.In addition,with the transfection of PEDV CV777 non-structural protein gene into IPEC-J2 cells,we screened and identified that PEDV NSP1 and NSP13 could significantly cause apoptosis,and the use of apoptosis inhibitor(Z-VAD-FMK)was found to inhibit Caspase-3cleavage and viral N protein expression,and the viral titer also decreased significantly.In summary,PEDV CV777 and HLJ strains induced apoptosis in IPEC-J2 cells by inducing the caspase cascade through both mitochondrial-mediated endogenous and death receptor-mediated exogenous pathways.Among them,PEDV NSP1 and NSP13 significantly induced apoptosis in IPEC-J2 cells.And inhibition of apoptosis inhibited viral replication.PEDV CV777 induced more apoptosis than those of HLJ strain at each time point,and the apoptotic morphological changes was more obvious,and the expression of apoptotic factors and apoptotic proteins increased remarked.