Effect and Mechanism of Interferon Stimulated Gene ISG15 on Porcine Epidemic Diarrhea Virus Replication
[Objective]The aim of this study was to investigate the role and mechanism of interferon-stimulated gene ISG15 in the replication of Porcine epidemic diarrhea virus(PEDV).[Method]Real-time quantitative PCR was used to detect the tissue expression profile of ISG15 gene and the differential expression in intestinal tissues.Meanwhile,porcine small intestine epithelial cells(IPEC-J2)were used as the study model to detect the expression of PEDV M gene mRNA and N protein of PEDV CV777 infected cells at different time points.The expression of ISG15 was detected at RNA and protein levels.Porcine ISG15 gene interference and overexpression cells were constructed,and the influence of ISG15 gene expression on PEDV replication level was detected by Real-time quantitative PCR,Western blotting and indirect immunofluorescence assay.Transcriptome sequencing was performed before and after the overexpression of ISG15 gene to screen its downstream regulatory genes and signaling pathways.[Result]ISG15 gene was specifically highly expressed in intestinal tissue of piglets,with significantly higher expression in jejunum and ileum compared with other tissues(P＜0.01).The expression of ISG15 gene in duodenum,jejunum and ileum of PEDV infected group was significantly or extremely significantly higher than that of healthy group(P＜0.05 or P＜0.01).The expression of PEDV M gene mRNA and N protein showed an upward trend,and compared with 0 h,the expression of ISG15 gene was extremely significantly upregulated at 24 h(P＜0.01).After overexpression of ISG15 gene,there was a significant or extremely significant decrease in PEDV replication(P＜0.05 or P＜0.01),while after interference with ISG15 gene,PEDV replication was extremely significantly upregulated(P＜0.01).Transcriptome sequencing revealed 1 532 differentially expressed genes before and after overexpression of the ISG15 gene,which were mainly enriched in signaling pathways such as autophagy,MAPK signaling,and endocytosis.[Conclusion]This study revealed the regulatory function and mechanism of ISG15 during PEDV infection and the increase of ISG15 gene expression could significantly inhibit PEDV replication,which improved the understanding of the molecular mechanism of the interaction between PEDV and host cells.
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