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.

iTRAQ-based proteome analysis of porcine group A rotavirus-infected porcine IPEC-J2 intestinal epithelial cells.

Porcine rotavirus (PoRV), particularly group A, is one of the most important swine pathogens, causing substantial economic losses in the animal husbandry industry. To improve understanding of host responses to PoRV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitatively identify the differentially expressed proteins in PoRV-infected IPEC-J2 cells and confirmed the differentially accumulated proteins (DAPs) expression differences by performing RT-qPCR and Western blot analysis. Herein, in PoRV- and mock-infected IPEC-J2 cells, relative quantitative data were identified for 4724 proteins, 223 of which were DAPs (125 up-accumulated and 98 down-accumulated). Bioinformatics analyses further revealed that a majority of the DAPs are involved in numerous crucial biological processes and signaling pathways, such as metabolic process, immune system process, amino acid metabolism, energy metabolism, immune system, MHC class I peptide loading complex, Hippo signaling pathway, Th1 and Th2 cell differentiation, antigen processing and presentation, and tubule bicarbonate reclamation. The cellular localization prediction analysis indicated that these DAPs may be located in the Golgi apparatus, nucleus, peroxisomal, cytoplasm, mitochondria, extracellular, plasma membrane, and endoplasmic reticulum (ER). Expression levels of three up-accumulated (VAMP4, IKBKE, and TJP3) or two down-accumulated (SOD3 and DHX9) DAPs upon PoRV infection, were further validated by RT-qPCR and Western blot analysis. Collectively, this work is the first time to investigate the protein profile of PoRV-infected IPEC-J2 cells using quantitative proteomics; these findings provide valuable information to better understand the mechanisms underlying the host responses to PoRV infection in piglets. SIGNIFICANCE: The proteomics analysis of this study uncovered the target associated with PoRV-induced innate immune response or cellular damage, and provided relevant insights into the molecular functions, biological processes, and signaling pathway in these targets. Out of these 223 DAPs, the expression levels of three up-accumulated (VAMP4, IKBKE, and TJP3) and two down-accumulated (SOD3 and DHX9) DAPs upon PoRV infection, have been further validated using RT-qPCR and Western blot analysis. These outcomes could uncover how PoRV manipulated the cellular machinery, which could further our understanding of PoRV pathogenesis in piglets.

Quantitative Proteomic Analysis of Porcine Intestinal Epithelial Cells Infected with Porcine Deltacoronavirus Using iTRAQ-Coupled LC-MS/MS.

Porcine deltacoronavirus (PDCoV) is an emergent enteropathogenic coronavirus associated with swine diarrhea. Porcine small intestinal epithelial cells (IPEC) are the primary target cells of PDCoV infection in vivo. Here, isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantitatively identify differentially expressed proteins (DEPs) in PDCoV-infected IPEC-J2 cells. A total of 78 DEPs, including 23 upregulated and 55 downregulated proteins, were identified at 24 h postinfection. The data are available via ProteomeXchange with identifier PXD019975. To ensure reliability of the proteomics data, two randomly selected DEPs, the downregulated anaphase-promoting complex subunit 7 (ANAPC7) and upregulated interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), were verified by real-time PCR and Western blot, and the results of which indicate that the proteomics data were reliable and valid. Bioinformatics analyses, including GO, COG, KEGG, and STRING, further demonstrated that a majority of the DEPs are involved in numerous crucial biological processes and signaling pathways, such as immune system, digestive system, signal transduction, RIG-I-like receptor, mTOR, PI3K-AKT, autophagy, and cell cycle signaling pathways. Altogether, this is the first study on proteomes of PDCoV-infected host cells, which shall provide valuable clues for further investigation of PDCoV pathogenesis.