Study On The Mechanism Of The Apoptosis Of Vero Cells Induced By Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV)is the pathogen causing porcine epidemic diarrhea(PED).PEDV infection mainly causes severe diarrhea,vomiting and dehydration in pigs,resulting in extremely high mortality.The mortality of piglets less than six days infected with PEDV is up to 100%,which has caused great economic losses to the pig industry in the world and has significantly hampered the healthy development of the pig industry.Currently the effective and specific vaccines against PED are unavailable,so research on the pathogenesis of PEDV is particularly important.To fully understand the mechanism of pathogenesis of PEDV,we analyzed the gene expression profile of Vero cells infected with PEDV at different time points by transcriptome sequencing technology to provide a basis for the discovery of novel key proteins involved in the pathogenesis of PEDV.First,the Illumina transcriptome sequencing technology was used to sequence Vero cells that were not infected with PEDV and Vero cells infected with PEDV(MOI=0.5)for 12 h and 24 h,and the original sequencing data was quality controlled to obtain quality control data(Clean reads).Analysis of Clean reads data showed that compared with the uninfected blank control group,there were 4 significantly differentially expressed genes in the group infected with PEDV for 12 h(|log2FC|?1 and p-adjust<0.05),and 1085 significantly differentially expressed genes in the group infected with PEDV for 24 h(|log2FC|?1 and padjust<0.05);compared with the group infected with PEDV for 12 h,1643 significantly differentially expressed genes in the group infected with PEDV for 24 h were selected(|log2FC|?1 and p-adjust< 0.05).Gene ontology(GO)functional annotation analysis was performed on the differentially expressed genes screened above.The GO annotation of differentially expressed genes includes three aspects: biological process,cellular composition and molecular function,and is associated with cellular process,biological regulation,metabolic process,cellular composition,organelle composition and catalytic activity.Further analysis of the differentially expressed genes in the Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis showed that four differentially expressed genes in target gene annotation of the African green monkey genome is only enriched in the NOD-like receptor signaling pathway and Micro RNAs in cancer signaling pathway when Vero cells were infected with PEDV for 12h;when infected with PEDV for 24 h,some cells showed severe pathological effects while the cells are not broken and shed,differentially expressed genes in target gene annotation of the African green monkey genome is mainly enriched in TNF signaling pathway,p53 signaling pathway,JAK-STAT signaling pathway,immune inflammation and tumor transcriptional regulation.P53 is a sequence-specific DNA binding protein that acts as a transcription factor to regulate cell apoptosis.Puma can promote apoptosis and is a key mediator of p53-dependent and nondependent apoptosis pathways.At present,there is no report on the induction of apoptosis by activating Puma and p53-dependent pathway after PEDV infection.Based on the above analysis,this study analyzed the differentially expressed genes related to p53 and found that the Puma gene is involved in the regulation of the p53 signaling pathway;further KEGG analysis found that the Puma gene can target the BAX and Bcl-2 genes on the p53 signaling pathway.After inoculated with PEDV at an MOIof0.1,0.5 and 1,respectively,flow cytometry was used to detect the apoptosis rate of Vero cells,and Vero cells without PEDV inoculation were used as negative controls.The results showed that,compared with the non-infected PEDV group,the experimental groups with different multiplicity of infections showed different degrees of apoptosis,and PEDV can induce apoptosis in a dose-dependent manner.QRT-PCR was used to detect the p53,Puma,BAX and Bcl-2 genes on the p53 signal pathway,and the results were consistent with the transcriptome sequencing results.Subsequently,Western blotting was used to further detect the expression of these four genes at the protein level.The results showed that PEDV infection upregulated the expression of p53,Puma and BAX proteins(P<0.05),while down-regulating the expression of Bcl-2 protein(P<0.05),therefore,we speculated that PEDV infection can regulate apoptosis by activating p53 signaling pathway.Finally,by adding the p53 inhibitor Pifithrin-? to Vero cells infected with PEDV,it was further confirmed that after p53 was inhibited,the expression of BAX protein was further inhibited by inhibiting Puma to finally suppress the occurrence of apoptosis.In summary,it can activate Puma and induce apoptosis in a p53-dependent pathway after PEDV infects Vero cells.By activating the expression of p53 protein and Puma protein,and further regulating the expression of protein BAX and Bcl-2 to mediate apoptosis.The results provide the basis for further elucidating the the mechanism of pathogenesisof PEDV infection and developing an effective vaccine against PEDV.