Differences in cytokines expression between Vero cells and IPEC-J2 cells infected with porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) primarily infects suckling piglets and causes severe economic losses to the swine industry. Cytokines, as part of the innate immune response, are important in PEDV infection. The cytokines secreted by cell infection models in vitro might reflect true response to viral infection of target cells in vivo. Vero cells and IPEC-J2 are commonly used as an in vitro model to investigate PEDV infection. However, it is not clear which type of cells is more beneficial to the study of PEDV. In our study, firstly, Vero cells and IPEC-J2 were successfully infected with PEDV virulent strains (HBQY2016) and attenuated vaccine strains (CV777) and were capable of supporting virus replication and progeny release. Moreover, cytokine differences expression by Vero cells and IPEC-J2 cells infected with two PEDV strains were analyzed. Compared with IPEC-J2 cells, only the mRNA levels of TGF-ß, MIP-1ß and MCP-1 were detected in Vero cells. ELISA assay indicated that compared to the control group, the PEDV-infected group had significantly induced expression levels of IL-1ß, MIP-1ß, MCP-1, IL-8, and CXCL10 in IPEC-J2 cells, while only secretion level of IL-1ß, MIP-1ß and IL-8 in Vero cells were higher in PEDV infected group. Finally, cytokines change of piglets infected PEDV-HBQY2016 strains were detected by cDNA microarray, and similar to those of IPEC-J2 cells infected PEDV. Collectively, these data determined that the IPEC-J2 could be more suitable used as a cell model for studying PEDV infection in vitro compared with Vero cells, based on the close approximation of cytokine expression profile to in vivo target cells.

Chemokines induced by PEDV infection and chemotactic effects on monocyte, T and B cells.

Porcine epidemic diarrhea virus (PEDV) is a re-emerging pathogen that causes severe economic loss in the pig industry. The host's innate immune system is the first line of defense on virus invasion of the small intestinal epithelial cells. Chemokines, as a part of the innate immune system, play an important role in host immunity against infection, however, and their expression and chemotactic effect on key immune cells in PEDV infection remains unclear. In this study, cDNA microarray was firstly performed to analyzed ileum tissue of piglets on the third day after PEDV infection. The differentially expressed genes mainly involved in multiple biological processes, chemokine signaling pathway and cytokine receptor interaction signaling pathway had the highest enrichment according to GO and KEGG enrichment analysis. The expression levels of chemokines MCP-1, MIP-1ß, IL-8, CXCL9, CXCL10 and CXCL13 in ileum of PEDV- infected piglets were significantly higher than those in the control group. The expression of chemokines in vivo experiment was further verified by RT-qPCR and ELISA using PEDV-infected IPEC-J2 cells. The results showed that the PEDV-infected IPEC-J2 cells had significantly induced protein expression of MCP-1, MIP-1ß, IL-8, CXCL9, CXCL-10 and CXCL13. These results indicated that the changes of chemokines expressed in the ileum of piglets (in vivo) were consistent with those in IPEC-J2 cells (in vitro) after PEDV infection. Finally, the role of chemokines in immune cell migration during PEDV infection was illustrated by siRNA-mediated knock down method and the co-culture model of IPEC-J2 cells with peripheral blood leukocyte cells (PBLCs). The FACS analysis showed that MCP-1 induced by PEDV infection played a chemotactic effect on CD14+ cells, CXCL9 on CD3+CD4-CD8-γδ T, CD3+CD4-CD8+ Tc, CD3+CD4+CD8- Th and CD3+CD4+CD8+ Tm subsets, and CXCL13 on CD19+ B cells. Collectively, our findings first indicate that PEDV-induced chemokines MCP-1, CXCL-9 and CXCL-13 attracted CD14+ cells, T cells and B cells, respectively. These results provide a theoretical basis for studying the mechanism of anti-PEDV infection in piglets.

Humoral immune responses in piglets experimentally infected with a field strain of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) causes severe clinical diarrhea in neonatal piglets, with reported mortality rates between 70-100%. The humoral immunity, especially the local intestinal IgA responses, plays an important role in the immune protection against PEDV infection. In this study, we evaluated the isotype antibody responses against the PEDV nucleocapsid (N) protein and the spike (S) protein subunits 1 (S1) and 2 (S2) in the serum and intestine of piglets. We also determined its serum neutralizing activity against the PEDV field strain HBMC2012 in 21-day-old piglets. Enzyme-linked immunosorbent assays (ELISA) revealed that the production of IgM against the N protein and S1 subunit was higher compared to the S2 subunit. The anti-S2 IgA antibodies were higher than the anti-N protein and anti-S1 IgA at 3 days post-infection (dpi). The specific IgA responses to the S2 subunit were higher than the responses observed in S1. The specific IgG responses against S1 and S2 subunits exceeded those of N protein. The serum neutralizing activities against PEDV were relatively low with a tendency to decline over time. No isotype-specific antibodies were found in the intestinal contents from infected pigs, except the one with weak IgA responses against N protein at 28 dpi. Immunohistochemical staining showed that a few IgM, IgA, and IgG antibody-secreting cells were mainly located in the mucosa of the duodenum and ileum of PEDV-infected pigs at 3 dpi. This study suggests poor systemic and intestinal isotype-specific antibody responses, especially those of IgA, and weak serum neutralizing activities against the field PEDV strain in piglets.