Establishment Of A Model Of Porcine Intestinal Enteroids And The Role And Mechanism Of Interferon Lambda In Enteric Mucosal Immunity Against Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus（PEDV）,a member of the group of alphacoronaviruses,is the pathogen of a highly contagious gastrointestinal swine disease,which pose significant economic burdens.Porcine intestinal epithelia are the primary target cells of PEDV infection and are composed of the villus region and the crypt region.At present,PEDV is mainly performed in a single porcine cell line lacking the complex structure of the small intestinal epithelium,which restricts the study for pathogenic mechanism of PEDV and innate immune response to the host.Therefore,the development of models in vitro that can closely recapitulate the porcine intestinal epithelia is crucial for expanding the current knowledge of PEDV pathogenesis and facilitating further biological investigations of host-PEDV interactions.The human or mouse enteroids derived from intestinal crypt stem cells recently have been reported to well recapitulate in vivo intestinal epithelia and applied for the study of enteric development and infection.Here,we isolated swine intestinal crypt stem cells from different intestinal segments,optimized the culture conditions and successfully differentiated them into enteroids in matrigel 3D culture after 7days in vitro.Enteroids can reproduce the complex structure of intestinal epithelial multicellular types in vivo,and can be continuously passaged and cryopreserved.Enteroids were susceptible to PEDV and produced infectious virus particles(TCID₅₀ up to 10⁴/mL)measured by RT-qPCR and IFA,respectively.PEDV infected multiple types of cells including enterocytes,stem cells,and goblet cells.Enteroids exhibited segmental infection discrepancies compared with ileal enteroids and colonoids by RT-qPCR and IFA,and this finding was verified in vivo.Moreover,the clinical isolate PEDV-JMS propagated better than the cell-adapted PEDV CV777 in ileal enteroids.Finally,we found that PEDV inhibited IFN production in ileal enteroids during early infection,and IFN-λ1 induced stronger antiviral gene expression than IFN-α（ISG15,MxA,OASL and IFITM1）,and more effectively inhibited PEDV infection in ileal enteroids detected by RT-qPCR compared with IFN-α.Type Ⅲ interferon（IFN-λ）belong to the IL-10 family and mainly act on mucosal epithelia.Studies have shown that porcine IFN-λ3 inhibits PEDV infection more effectively than IFN-α.The molecular mechanism of the difference of antiviral activity of IFN-λ3 and IFN-αin intestinal epithelia is elusive,especially the expression profile induced by IFN-λ3 has not been reported.In order to analyze the molecular mechanism of porcine IFN-λ3 anti-mucosal virus and the difference in anti-PEDV activity compared with IFN-α,we compared the genes transcriptional profile induced by two IFNs in porcine intestinal epithelial cells（IPEC-J2）by RNA-seq.We found that after pretreatment of IPEC-J2 cells with IFN-λ3,983 genes were differentially expressed and IFN-αhad only 134 genes,of which 110 were common genes.Transcriptional enrichment analysis indicated that IFN-λ3 or IFN-αinvolved in the regulation of cellular processes such as cellular components and molecular functions of IPEC-J2,and IFN-λ3 activates more robust signaling pathways than IFN-α,especially antiviral JAK-STAT signaling pathway.Further more,we verified the RNA-seq results on IPEC-J2 and enteroids by qPCR,and we found IFN-λ3 up-regulates ISGs expression（MX2,IFIT3,OASL,OAS1,RSAD2,and PLAC8）more efficiently than IFN-α.Finally,we cloned and expressed rsad2 and mx2 genes in the first 10 genes induced by IFN-λ3.Overexpression confirmed that porcine RASD2 and MX2 significantly inhibited PEDV infection with a dose response.Collectively,we established swine enteroids that provide a novel in vitro model for exploring the pathogenesis of PEDV and for the in vitro study of the interplay between host and a variety of swine enteric viruses,as well as to lay the foundation for elucidating the mechanism of action of IFN-λagainst PEDV.Moreover,we found that IFN-λ3 induces a transcriptional profile that does not completely overlap with IFN-α,initially exploring the antiviral mechanism of IFN-λin mucosal immunity.