The Mechanism Of PEDV Entering The Blood Through Endothelium And Infecting Neonatal Piglets Through Exploiting Red Blood Cells

Porcine epidemic diarrhea(PED)is a highly contagious infectious disease.As the major pathogen of PED,porcine epidemic diarrhea virus(PEDV)infection causes acute atrophic enteritis and high mortality in piglets.Moreover,PEDV infection causes severe viremia in piglets,especially in neonatal piglets.However,the mechanism of PEDV-induced viremia and the role of viremia in the pathogenesis of PEDV are still unclear.Our research conducted an epidemiological investigation of PEDV in diarrheal pig farms in Jiangsu province.We found that PEDV was present in the red blood cells of PEDV-infected pigs.To investigate the role of the virus in red blood cells on the pathogenesis of PEDV,we transfused PEDV-inoculated neonatal red blood cells into neonatal piglets’blood.Transfusion with PEDV-inoculated red blood cells induced typical symptoms of PED in neonatal piglets.Moreover,we found PEDV invaded neonatal red blood cells through transferrin receptor(TFR)and clathrin-mediated endocytosis(CME).To investigate how PEDV enters the blood from the infected site through the endothelium,neonatal piglets were infected with PEDV through intranasal spraying.We found that intranasal infection with PEDV could destroy the endothelial integrity in the nasal mucosa,and observed that PEDV was present in red blood cells and peripheral blood mononuclear cells(PBMCs)in the blood.We further found PEDV infection enhanced the expression of matrix metalloproteinase-7(MMP-7)and pro-inflammatory factors in nasal epithelial cells(NECs).The released MMP-7 could degrade the tight junctions of endothelial cells,destroying the endothelial integrity.The released factors could promote the endothelial cells adhering to red blood cells,which might facilitate PEDV entering the blood.In addition,the released pro-inflammatory factors promoted transendothelial migration of PBMCs by enhancing the expression of intercellular adhesion molecule-1(ICAM-1).PEDV could enter the blood by exploiting PBMCs.This research is mainly divided into the following four parts:1.The study of PEDV infecting neonatal piglets through invading red blood cellsThrough epidemiological investigation of PEDV in several diarrhea pig farms in Jiangsu province,we found PEDV was present in the red blood cells of the sick pig,with a positive rate of 13.04%.To explore the role of PEDV in red blood cells on viral pathogenesis,neonatal red blood cells were inoculated with PEDV.Then,the PEDV-inoculated red blood cells(3.2×10~6 PFU/10~9 cells)were transfused into neonatal piglet’s blood.The results showed that after 48 h of transfusion with PEDV-loaded red blood cells,the neonatal piglets exhibited obvious symptoms of diarrhea.Through immunohistochemistry and immunofluorescence assays,we found transfusion with PEDV-inoculated red blood cells caused severe atrophy of the small intestinal villi and sloughing of the intestinal epithelium.Moreover,a large amount of PEDV was present in the intestinal epithelium.However,in the control group,neonatal piglets exhibited no symptoms,and no PEDV was detected in the intestine.This study demonstrated that autologous transfusion with PEDV-inoculated red blood cells could cause intestinal infection in neonatal piglets.2.The mechanism of PEDV invading neonatal red blood cellsTo explore the mechanism of PEDV invading neonatal red blood cells,neonatal red blood cells were inoculated with PEDV.We confirmed that PEDV could survive in red blood cells for 12 h by plaque formation assay,and found PEDV could be embedded in neonatal red blood cells by transmission electron microscope(TEM)observation.To further compared the PEDV invasion to red blood cells from piglets of different ages,we isolated red blood cells from piglets of different ages.We found PEDV was more likely to invade neonatal red blood cells.In addition,we found neonatal red blood cells exhibited high expression of TFR.TFR is a type II transmembrane glycoprotein that plays an important role in mediating viral adhesion and invasion through the participation of CME.Through antibody blocking TFR and chlorpromazine(CPZ)inhibiting CME,we found that blocking TFR expression and inhibiting CME significantly reduced the PEDV invading neonatal red blood cells.Under the condition of an intact endothelial barrier,red blood cells cannot directly contact intestinal epithelial cells.To explore the transmission of PEDV in red blood cells,we established a co-culture model between PBMCs and red blood cells.Through flow cytometry and TEM observation,we found that red blood cells could interact with T lymphocytes in PBMCs,transmitting PEDV to T lymphocytes.PEDV-loaded T lymphocytes could enter the small intestinal mucosa through the blood circulation,and transmit the virus to the small intestinal epithelial cells,causing infection.3.The mechanism of PEDV infection destroying the vascular endothelial barrier of the nasal mucosaAfter neonatal piglets intranasally infecting with PEDV(1×10~7PFU/m L),we found the permeability of vascular endothelial in the nasal mucosa was significantly increased by Western blot and Immunofluorescence assays.To further explore the role of PEDV infection on the endothelial barrier,we established an in vitro endothelial barrier model by using swine umbilicus veins endothelial cells(SUVECs).We found the destruction of endothelial integrity was caused by biomolecules released from PEDV-infected NECs,while direct PEDV infection had no effect on the endothelial integrity.Further studies confirmed that these released biomolecules could degrade the endothelial tight junctions,including ZO-1,occludin,and claudin-1.Moreover,these released biomolecules inhibited the repair of the endothelial barrier.Through RT-q PCR,we found that PEDV infection could promote the expression of MMP-7 and pro-inflammatory factors in NECs.Then,an inhibitor GM6001was used to inhibit metalloproteinases expression,and si RNA was exploited to interfere with MMP-7 expression.We found that inhibiting the release of MMP-7 significantly alleviated the degradation of tight junctions and destruction of the endothelial integrity.While overexpression of MMP-7 in NECs resulted in more severe degradation of tight junctions and destruction of the endothelial integrity.This study showed that PEDV infection of NECs enhanced the expression of MMP-7,and the released MMP-7 degraded the tight junctions,leading to the destruction of the endothelial integrity,which might facilitate PEDV entering the blood.4.The mechanism of PEDV entering the blood through the endothelial barrierAfter intranasal infection with PEDV,we found PEDV was present in red blood cells and PBMCs in neonatal piglet blood.To explore how PEDV enters the red blood cells and PBMCs through the endothelial barrier,we established an adhesion model between endothelial cells and red blood cells as well as a migration model between endothelial cells and PBMCs.Through the adhesion model,we found that the viral microenvironment from PEDV-infected NECs could promote the adhesion of endothelial cells to red blood cells,which might promote PEDV exploiting red blood cells to spread in the blood.Through a migration model,we found that the viral microenvironment from PEDV-infected NECs enhanced the transendothelial migration of PBMCs,and detected infectious PEDV in the migrated PBMCs.To explore the mechanism of PEDV infection affecting the transendothelial migration of PBMCs,we investigated ICAM-1 expression and NF-κB signaling activation by RT-q PCR,Western blot,and Immunofluorescence assays.The results showed that the viral microenvironment from PEDV-infected NECs enhanced the expression of ICAM-1 and the activation of NF-κB signaling in endothelial cells.Treatment with an inhibitor of ICAM-1 significantly hindered the transendothelial migration of PBMCs.Moreover,treatment with an inhibitor of NF-κB signaling significantly reduced the expression of ICAM-1 and the transendothelial migration of PBMCs.However,inhibiting ICAM-1 expression and NF-κB signaling activation did not affect endothelial cells adhering to red blood cells.This study confirmed that intranasal infection with PEDV could promote endothelial cells adhering to red blood cells,which might facilitate PEDV exploiting red blood cells to spread in the blood.PEDV infection could activate the NF-κB signaling in endothelial cells and induce ICAM-1 up-regulation,promoting the transendothelial migration of PBMCs.PEDV could exploit PBMCs to enter the blood.These studies clarified the mechanism of PEDV infecting neonatal piglets by exploiting red blood cells,as well as the mechanism of PEDV destroying the vascular endothelial barrier and entering the blood,providing a theoretical basis for the prevention and control of PED.