Molecular Mechanism Of Influencing The Replication Of Porcine Epidemic Diarrhea Virus By Polyimmunoglobulin Receptor(pIgR)

Porcine epidemic diarrhea virus?PEDV?,a highly contacting intestinal Coronavirus,causes severe diarrhea or even death in suckling piglets,threatening the health of newborn piglets severely nowadays.PEDV disrupts the mucosal immune barrier by invading the epithelial cells of intestinal villi,developing clinical symptoms in piglets.Mucosal immune response mediated by the secreted immunoglobulins?sIgs?plays an important role in the control of PEDV infection,and the secretion of sIgs mainly depends on the polyimmunoglobulin receptor?pIgR?.Therefore,exploring the role of pIgR in the process of PEDV infection and replication will be of great value in analyzing the pathogenesis of PEDV.pIgR mediates the secretion of sIgs into enteric cavity to resist the invasion of intestinal pathogens.To understand the expression of pIgR after PEDV infection,different intestinal segments of both PEDV infected piglets and mock infected piglets were selected to explore the transcription of p IgR.The results showed that the mRNA of pIgR in intestinal tissues increased significantly after PEDV infection,indicating that PEDV infection may affect the expression of p IgR significantly in vivo.Simultaneously,the expression of p IgR was analyzed in vitro by infecting LLC-PK1 cells with PEDV FJzz1 strain.The results showed that pIgR mRNA increased significantly in 12 hours postinfection?hpi?,while FJzz1 infection significantly reduced the expression level of pIgR protein at 18 hpi.To investigate the effect of pIgR on PEDV proliferation,we used FJzz1 strain to infect pIgR overexpressing host cells,and monitored the virus proliferation by conducting RT-qPCR and progeny virus TCID₅₀0 measurement.It was found that the expression level of PEDV N protein,the viral mRNA copy number and the viral titer increased significantly,suggesting that the overexpression of pIgR promotes the proliferation of PEDV.At the same time,in the LLC-PK1 cells using RNA interference to knock down the expression of endogenous pIgR and the LLC-PK1^?pIgRpIgR cell line lacking endogenous pIgR,the N protein of PEDV,viral mRNA copy number and viral titer were significantly reduced.It shows that knockdown and deletion of pIgR expression can inhibit the proliferation of PEDV.Therefore,we speculate that pIgR may play an important role in PEDV infection and replication.To analyze the molecular pathways that regulate pIgR expression after PEDV infection,TNF-?mRNA was measured and found significantly up-regulated after PEDV infection.When LLC-PK1cells were treated with TNF-?,it was found that the expression of pIgR was significantly increased by increasing TNF-?dose;When treated with the NF-?B inhibitor BAY11-708,the expression of pIgR induced by TNF-?was significantly inhibited,indicating that NF-?B-mediated signaling pathway may be involved in regulating pIgR expression.We further analyze the impact of PEDV infection on key molecules in the NF-?B signaling pathway.The results showed that after 18 hours of PEDV infection,IkB-?,p65 in the NF-?B classic pathway and p100,P-p100,and p52 in the alternative pathway were all down-regulated.Based on this,we speculate that PEDV infection may down-regulate p IgR expression by inhibiting the NF-?B signaling pathway.Moreover,the change of pIgR expression was examined after treating cells with the protease inhibitor MG132.The results displayed that the expression level of p IgR protein was not restored effectively by MG132,indicating down-regulate p IgR by PEDV infection is not through the ubiquitin proteasome pathway-mediated degradation.This study focused on the role of pIgR proteinin PEDV infection,which plays an important role in mucosal immune response.We confirmed that pIgR may affect the proliferation of PEDV in vitro,and PEDV infection may regulate the expression of pIgR in host cells by affecting the NF-?B signaling pathway.This study provides a new theoretical basis for deeply understanding the pathogenesis of PEDV.