Mechanisms Of Porcine Epidemic Diarrhea Virus Down-regulating The Expression Of SLA-DR In Porcine Bone Marrow-derived Dendritic Cells

Porcine epidemic diarrhea(PED)is an acute,highly contagious intestinal disease in which infected pigs are characterized by anorexia,vomiting,watery diarrhea and severe dehydration.Pigs of all ages can be infected and show varying degrees of symptoms,and neonatal mortality can be up to 100%.Since 2010,PED has gradually swept the world,and bringing huge economic losses to the pig industry.Vaccination has always been the main strategy for the prevention of porcine epidemic diarrhea virus(PEDV)infection,but the persistent variation of PEDV and its frequent co-infection with other enteroviruses make it difficult to prevent.PEDV mainly infects small intestinal epithelial cells and can also infect dendritic cells.As professional antigen-presenting cells,dendritic cells can capture internal and exogenous antigens and further present antigenic peptides to MHC-Ⅰ or Ⅱ molecules on the cell surface after intracellular processing,thus activating antigen-specific T cells to activate humoral and cellular immune responses.Thus,the efficiency of the immune response and host defense against pathogens largely depend on the proper functioning of dendritic cells.Currently,there are few reports on the impact of PEDV infected DC on its MHC-Ⅱ molecule-mediated antigen processing and presentation function.This study reveals the molecular mechanism of PEDV-mediated down-regulation of SLA-DR expression on antigen-presenting cells(DC),which will help to understand the mechanism of PEDV escaping the acquired immune response.The main works of this study are as follows:1.Preparation and activity identification of polyclonal antibody against PEDV-N protein.The recombinant PEDV-N protein was successfully obtained by Escherichia coli prokaryotic expression system.The PEDV-N protein was expressed in the supernatant with good biological activity and no need for protein renaturation and the higher purity of recombinant protein was obtained by Ni column purification.Polyclonal antibodies were obtained by immunizing Balb/c mice with PEDV-N protein.Western blot identification showed that the polyclonal antibody had good activity and could specifically detect the expression of N protein in cells after PEDV infection,which could be used for subsequent experiments.2.Effect of PEDV on BM-DC infection and SLA-Ⅱ molecule expression oncells.In this study,Western blot and IFA were used to verify that PEDV could infect BM-DC.Further studies found that after PEDV was infected with BM-DC,SLA-DRα and SLA-DRβ were down-regulated in both transcriptional and protein levels,and flow cytometry detection showed that SLA-DR expression on the surface of PEDV-infected BM-DCs was significantly down-regulated.Meanwhile,the transcriptional and protein levels of SLA-DQ were down-regulated after PEDV infection with BM-DC.It is revealed that PEDV can down-regulate the expression of SLA-DR and SLA-DQ at the same time,indicating that the virus has a comprehensive inhibitory effect on functional antigen presentation molecules on BM-DC.We speculated that PEDV might antagonize the presentation of pathogenic antigens by down-regulating the expression of class SLA-Ⅱ molecules,and finally achieve the escape of immune response.In order to confirm that the downregulation of SLA-DR caused by PEDV infection with BM-DC was caused by structural or non-structural proteins,we used ultraviolet light to inactivate PEDV and confirm that the virions were completely inactivated.Inactivated PEDV can also down-regulate SLA-DR expression at transcription and protein levels,indicating that the down-regulation of SLA-DR caused by PEDV infection with BM-DC is mediated by structural proteins.3.Molecular mechanism of down-regulation of SLA-DR molecules mediated by PEDV.The expression level of the protein depends on the balance between the synthesis and degradation rates.Therefore,the possible reasons for the down-regulation of SLA-DR on BM-DC mediated by structural proteins of PEDV include the decrease of protein expression caused by the inhibition of SLA-DR transcription levels or the acceleration of protein degradation,or both.In this study,proteasome inhibitors(MG-132)were used to treat BM-DCs infected with PEDV to investigate whether PEDV could down-regulate the expression of SLA-DR protein by accelerating ubiquitination degradation.Furthermore,the effects of structural protein and ORF3 encoded by PEDV on the activity of SLA-DR α and SLA-DR β promoters and protein expression were investigated by double luciferase reporter system and self-constructed HEK 293 T SLA-DRα Zsgreen /βm Cherry stable cell lines.The results showed that PEDV E and ORF3 inhibited SLA-DR m RNA expression by inhibiting SLA-DR promoter activity.PEDV does not mediate the protein degradation of SLA-DR through the ubiquitin-proteasome system,and the structural proteins encoded by PEDV do not directly affect the expression of SLA-DR at the protein level.The completion of this study reveals the possible mechanism of insufficient humoral immune response caused by PEDV vaccine immunization,which lays a theoretical foundation for the development of more effective vaccines in the future.