Establishment Of Real-time PCR For PEDV Detection And Molecular Mechanism Of TARDBP Inhibiting PEDV Proliferation

Porcine epidemic diarrhea(PED)is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV),which is characterized by watery diarrhea,vomiting and dehydration.It is susceptible to pigs of all ages.Since 2010,mutated PEDV has broken out in China and even around the world,causing the mortality rate of suckling piglets under 7 days of age to reach 100%,seriously damaging the healthy development of pig industry.PEDV is a single-stranded plus strand RNA virus,which mainly encodes 4 kinds of structural proteins and 16 kinds of nonstructural proteins.PEDV N protein is the most abundant structural protein in the early stage of viral infection and plays an important role in viral replication,transcription and assembly.TARDBP(TAR DNA Binding protein)is an RNA binding protein,which has many functions such as transcription,m RNA pre-splicing,m RNA transport and stability.In the early stage,the host factor TARDBP interacting with PEDV N protein was screened out by mass spectrometry analysis.In order to further explore the influence of TARDBP on PEDV proliferation and its mechanism,the following experiments were carried out in this study:In order to detect the RNA of PEDV virus,a real-time fluorescence quantitative PCR detection method based on SYBR Green I was established by constructing the positive standard plasmid of PEDV N protein.A series of experiments showed that the linear correlation coefficient of the detection method was 0.99.The SYBR Green I real-time PCR method had strong specificity and high sensitivity,and the limit of detection was 2.23 copies/?L.It was about 100 times more sensitive than ordinary PCR.The coefficient of variation within groups was between 0.25% and 0.43% and between groups was between 0.67% and 0.97%,which showed that the method had good repeatability.For 96 clinical samples from various regions,the positive rate of PEDV was 25%.In this study,a real-time fluorescence quantitative PCR detection method for PEDV was successfully established,which laid a foundation for the detection of clinical samples of PEDV RNA and the detection test of basic research.The preliminary test of this study found that TARDBP could interact with PEDV N protein and inhibit the proliferation of PEDV.In order to explore the molecular mechanism of TARDBP inhibiting PEDV proliferation,this study started with the analysis of the transcription factors regulating TARDBP in PEDV-infected host cells and the inhibitory effect of TARDBP on PEDV proliferation.It was found that the expression of TARDBP could be down-regulated after PEDV infected LLC-PK1 cells.In order to explain this phenomenon,the promoter sequence of pig-derived TARDBP was amplified in this study,and its transcription factor binding site was analyzed.Fluorescence quantitative PCR,si RNA,Ch IP and other tests confirmed that PEDV infected host cells and down-regulated the expression of transcription factor KLF16,thus inhibiting the expression of TARDBP.TARDBP interacts with PEDV N protein and degrade PEDV N protein in a dose-dependent manner.In order to explore the mechanism of TARDBP degradation of PEDV N protein,it was found that TARDBP ubiquitinated PEDV N protein with E3 ubiquitin ligase MARCHF8 by blocking activator and inhibitor and interfering with autophagy pathway-related proteins.PEDV N protein was eventually degraded through autophagy-lysosome pathway and ubiquitination-proteasome pathway,exerting its inhibitory effect on PEDV proliferation.In order to further explore the molecular mechanism of host cells using TARDBP to inhibit the proliferation of PEDV,we detected the level of type I IFN by overexpressing TARDBP and found that TARDBP up-regulated type I IFN by double luciferase test.Further studies showed that TARDBP activated the protein level of TRAF3 and the phosphorylation of IRF3 by up regulating the protein level of My D88,the key molecule upstream of type I IFN,so as to up regulate type I IFN,stimulate innate immune response,and then inhibit the proliferation of PEDV.In conclusion,this study established a real-time PCR detection method for PEDV,revealing the molecular mechanism of host factor TARDBP degrading PEDV N protein through autophagylysosome pathway and ubiquitin proteasome pathway,and TARDBP promoting innate immunity and thus inhibiting PEDV proliferation,providing a new research idea for the prevention and control of PEDV.