Effects Of Chemokines In Porcine Epidemic Diarrhea Virus Infection

Porcine epidemic diarrhea?PED?,caused by porcine epidemic diarrhea virus?PEDV?,is an acute and highly contagious intestina disease of swine characterized by diarrea,vomiting,acute enteritis.In recent years,PEDV has caused a massive death of piglets and caused economic losses to the pig industry.However,an insight into immune mechanism on this diease is not enough.Chemokines are the low molecular weight proteins that recruit leukocytes to the site of infection and play an important role in the initial step of inflammatory response and in innate immunity.This study focused on the topic of PEDV infection immunity to explore the role of chemokines in PEDV infection through in vitro and in vivo experiments,to provide a scientific basis for further elucidating the immune mechanisms of PEDV infection.Six 21-day-old healthy piglets were divided into virulent PEDV infection group and negative control group randomly.Each piglet in the infected group was inoculated orally with 1.5×10^6.6 TCID₅₀ of virulent PEDV;the control group was inoculated with the same volume of cell maintenance mediums.Then on day post infection?dpi?3,microarray analysis and FQ-PCR were used to detect mRNA expression levels of chemokines in the ileal tissue.The results showed that the mRNA expression levels of chemokines MCP-1,MIP-1?,IL-8,CXCL9,CXCL10 and CXCL13 in the ileal tissue of infected pigs were significantly upregulated?P<0.05?.In order to verify the change of chemokines expression level in the host cells infected with PEDV,IPEC-J2 cells were divided into HBQY2016 virulent strain infection group,PEDV attenuated strain CV777 infection group,LPS positive control group and negative control group.Two viral infection groups were inoculated with 0.5 MOI PEDV of the HBQY2016 virulent strain and the attenuated strain CV777,respectively,the positive control group was stimulated with 10?g/mL LPS,and the negative control group was added with same volume of maintenance solution.The cells were collected 18 to 20 h after infection.The mRNA expressions levels of MCP-1,MIP-1?,IL-8,CXCL9 and CXCL13in the two infection groups were significantly increased?P<0.05?.The levels of MCP-1,MIP-1?,IL-8,CXCL9,CXCL10 and CXCL13 in the cell supernatants were significantly increased?P<0.05?.To further elucidate the role of chemokines in PEDV infection,the inhibitory activity siRNAs to chemokine MCP-1?CXCL9?CXCL13 were synthesized and sieved.IPEC-J2cells were divided into PEDV infection?strain virulent HBQY2016 or attenuated CV777?group,siRNA interference-PEDV?strain virulent HBQY2016 or attenuated CV777?infection group,siRNA-NC-PEDV?strain virulent HBQY2016 or attenuated CV777?infection control group and negative control group.Compared with PEDV strain virulent HBQY2016 and attenuated CV777 infection groups,the numbers of CD14⁺monocytes in the transwell underlayer in the MCP-1-siRNA interference-PEDV strain HBQY2016 and attenuated strain CV777 infection group decreased significantly by an average 400 cells and 1152 cells,respectively,through transwell chemotaxis assay and flow cytometry?FCM?detection;significant reductions of CD3⁺CD4^-CD8^-??T,CD3⁺CD4^-CD8⁺Tc,CD3⁺CD4⁺CD8^-Th and CD3⁺CD4⁺CD8⁺Tm subsets exhibited at 677,258,718,207 and2605,533,2729,949 cells,respectively,in the CXCL9-siRNA interference-PEDV strain HBQY2016 and attenuated CV777 infection groups;and the numbers of CD19⁺B cells droped remarkably in the CXCL13-siRNA interference-PEDV strain HBQY2016 and attenuated CV777 infection groups decreased by 279 cells and 164 cells,respectively.In summary,this study demonstrated that PEDV virulent strain infection caused a significant up-regulation of the expression levels of chemokines MCP-1,MIP-1?,IL-8,CXCL9,CXCL10,CXCL13.MCP-1 induced by strain HBQY2016 or attenuated CV777played a chemotactic effect on CD14⁺monocytes,CXCL9 had a chemotaxis to CD3⁺CD4^-CD8^-??T,CD3⁺CD4^-CD8⁺Tc,CD3⁺CD4⁺CD8^-Th and CD3⁺CD4⁺CD8⁺Tm subsets,and CXCL13 to CD19⁺B cells.