The Mechanism Of Intranasal Invasion PEDV Caused Intestinal Infection In Piglets

Porcine epidemic diarrhea(PED)is an acute and highly infectious gastrointestinal disease caused by porcine epidemic diarrhea virus(PEDV).The disease is characterized by rapid transmission and widely spread,which has catastrophic impacts on the global pig industry since 2011.Although the fecal-oral route is generally accepted,an increased number of reports indicate that airborne transmission may contribute to PEDV outbreak and virus can be detected in nasal cavity.Therefore,we propose the hypothesis that PEDV can cause intestinal diseases in piglets through the nasal cavity.To test this hypothesis,we performed the virus challenge experiment in 5 days piglet through nasal inoculation.The results showed that the piglets appeared typical PED diarrhea symptoms and a large number of viruses colonized the small intestine at 60 hours after nasal inoculation.According to this result,out study will study the specify mechanism by which PEDV cause intestine disease via the nasal cavity.According to the results in vivo and in vitro,we found PEDV could entry the nasal mucosa through the uptake ability of DCs,which can form transepithelial dendrites(TEDs)across the nasal epithelial barrier to capture luminal viruses.Subsequently,PEDV-carrying dendritic cells(DCs)allow the virus to be transferred to T lymphocytes(T cells)via the virological synapse.Finally,virus-loaded T cells reach the intestine through the blood circulation,leading to intestinal infection via cell to-cell contact.The results will provide a new perspective on the tropism of PEDV pathogenesis and could contribute the control or PED infection.Our research is divided into the following four parts in detail:1.Animal challenge experiment verify PEDV can cause typical diarrhea in piglets after intranasal inoculationAt 60 hpi,the intranasal inoculated piglets from began to exhibit classical PEDV symptoms and intestine lesions.Immunofluorescence analysis(IFA)showed a large amount of PEDV-positive cells in the small intestine of piglets with diarrhea.The results of FACS and RT-qPCR demonstrated the virus mainly colonized in the olfactory region and showed a certain degree of replication in the nasal epithelium within 24 h.We further used a nasal epithelial cells air-liquid culture model(NECAM)to verify the consequences of PEDV infection in vitro.PEDV infection cause no apparent visual cytopathic effect(CPE)in NECAM and infectious virus was recovered only from the apical side of the NECAM,not at the basolateral side of the NECAM.Altogether,these results demonstrated PEDV could cause typical PED symptoms intranasal inoculation and develop a transient nasal epithelium infection in piglets.2.Submucosal DCs may function as an important portal for PEDV entrySubmucosal DCs are characterized by the extension of cellular processes into the lumen for continuous surveillance.However,as a double-edged sword,the function of DCs is sometimes exploited by the virus to overcome the mucosal barrier.After 12 h post intranasal administration of PEDV,the capture of luminal PEDV by DCs was detected in the nasal associated lymphoid tissue(NALT)and lamina propria of the nasal mucosa in vivo.By NECAM/DCs coculture system,we found PEDV can be detected in the submucosal DCs without epithelial barrier damage.DCs were clearly visualized as enlarged TEDs across the tight juctions of NECs and had internalized luminal PEDV.To better understand the molecular cues required for DCs recruitment and TED formation in the nasal passage,we assessed the expression of chemokines in nasal epithelial associated with DCs migration.While activation of NF-?B in NECs by PEDV was associated with the release of the chemokine CCL20 and mobilized DCs to accumulate in the submucosal regions.Taken together,these results implyed that,although the virus can be released only from the apical side of the NECAM,submucosal DCs may function as an important portal for PEDV entry into the nasal mucosa.3.The study of virus transferred from DCs to T cellsThe virus-carrying DCs can enter nearby draining lymph nodes and interact with T cells.After intranasal inoculation,PEDV was detected in T cells in peripheral blood mononuclear cells(PBMCs)of piglets within 24 h.Virus in T cells may be acquired from DCs,which take up virus into the lumen across the nasal mucosa,migrate and engage with T cells.In our study,PEDV could be transferred from DCs to T cells at 2 h and the viral RNA expression level in T cells remained stable within 48 h.While PEDV can induced functional cross-talk within the conjugated structure between DCs and T cells,and virus was detected in these structures.This structure may play an important role in PEDV transmission.4.PEDV-loaded T cells reach the intestine and transfer the virus to intestinal epithelial cellsFrom the competitive T cell autotransfusion assay in piglets,we found that PEDV-loaded T cells can enter the peripheral blood and reach the intestinal mucosa through the blood circulation.Moreover,PEDV infection of Vero cells occurred efficiently by cell-to-cell contact with PEDV-infected T cells in the T/Vero cells coculture system.PEDV could be transferred within 2 h through T cell-Vero contact coculture,in turn large amounts of virus were transferred from infected Vero cells to T cells.Cell-to-cell spread of PEDV in trans or in cis may permit efficient ongoing replication and spreading despite a variety of defense strategies in the intestinal mucosa.Our study provides evidence for nasal invasion PEDV caused intestinal disease and illustrates the mechanism of its transport from the entry site to the pathogenic site.These findings are helpful in facilitating strategies for PEDV prevention in our country.