Differential Transcriptomics Analysis of IPEC-J2 Cells Single or Coinfected With Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus

Abstract

Porcine epidemic diarrhea (PED) and transmissible gastroenteritis (TGE) caused by porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are two highly contagious intestinal diseases in the swine industry worldwide. Notably, coinfection of TGEV and PEDV is common in piglets with diarrhea-related diseases. In this study, intestinal porcine epithelial cells (IPEC-J2) were single or coinfected with PEDV and/or TGEV, followed by the comparison of differentially expressed genes (DEGs), especially interferon-stimulated genes (ISGs), between different groups via transcriptomics analysis and real-time qPCR. The antiviral activity of swine interferon-induced transmembrane protein 3 (sIFITM3) on PEDV and TGEV infection was also evaluated. The results showed that DEGs can be detected in the cells infected with PEDV, TGEV, and PEDV+TGEV at 12, 24, and 48 hpi, and the number of DEGs was the highest at 24 hpi. The DEGs are mainly annotated to the GO terms of protein binding, immune system process, organelle part, and intracellular organelle part. Furthermore, 90 ISGs were upregulated during PEDV or TGEV infection, 27 of which were associated with antiviral activity, including ISG15, OASL, IFITM1, and IFITM3. Furthermore, sIFITM3 can significantly inhibit PEDV and TGEV infection in porcine IPEC-J2 cells and/or monkey Vero cells. Besides, sIFITM3 can also inhibit vesicular stomatitis virus (VSV) replication in Vero cells. These results indicate that sIFITM3 has broad-spectrum antiviral activity.

Keywords: coinfection; differential transcriptomics; interferon-induced transmembrane protein (IFITM); interferon-stimulated genes (ISGs); porcine epidemic diarrhea virus (PEDV); transmissible gastroenteritis virus (TGEV).

Figure 1

Phylogenetic analysis and proliferation kinetics of PEDV and TGEV in IPEC-J2 cells. (A) Phylogenetic trees of PEDV based on the S gene. (B) Phylogenetic trees of TGEV based on the S gene. (C) qPCR analysis of PEDV and TGEV infection in IPEC-J2 cells. IPEC-J2 cells were infected (MOI = 1) of PEDV or TGEV. The cells were collected at 0, 12, 24, 36, and 48 hpi respectively, followed by RT-PCR analysis. *, p-value <0.05; **, p-value <0.01; ***, p-value <0.001. (D) Western blot analysis of PEDV and TGEV replication in IPEC-J2 cells at 48 and 60 hpi. Unprocessed original images is found in Supplementary Figure S1 . (E) One-step growth curve of PEDV and TGEV in Vero or ST cells, respectively. Viruses were collected from IPEC-J2 cells, followed by TCID₅₀ evaluation.

Figure 2

The differentially expressed genes in all transcriptome groups. (A) Schematic diagram of sampling. (B) The number of DEGs at different times. (C, E) DEGs of different groups at different times. (D, F) DEGs of the coinfected group at different times.

Figure 3

Top 20 pathways of the shared DEGs. (A, B) GO annotation of up- (A) and downregulated (B) DEGs. (C, D) KEGG analysis of up- (C) and downregulated (D) DEGs.

Figure 4

Analysis of 90 ISGs. (A) Subcellular localization. (B) KEGG classification. (C) Antiviral activities of ISGs.

Figure 5

Protein–protein interaction networks.

Figure 6

Differentially expressed key genes. IPEC-J2 cells were infected with 1 MOI of PEDV, TGEV, and PEDV+TGEV for 24 hpi. The expression levels of IFITM1 (A), IFITM3 (B), IRF1 (C), and IRF7 (D) were compared at 24 hpi with RT-PCR. GAPDH was used as the internal control. *, p-value <0.05; **, p-value <0.01; ***, p-value <0.001; ****, p-value <0.0001. NS, no significant.

Figure 7

Evaluation of PEDV and TGEV in IFITM3-overexpressed and knocked-down cells. Porcine IPEC-J2 cells were transfected with si-ssc-IFITM3s or pLV-sIFITM3-Flag for 48 h, followed by infection with PEDV or TEGV (MOI = 1). The expressions levels of sIFITM3 were evaluated by Western blot at 48 h post-transfection (A, B), and the levels of viral genes were quantified at 24 h postinfection using real-time PCR (C–F). (A, C, D) IPEC-J2 cells transfected with siIFITM3s. (B, E, F) IPEC-J2 cells transfected with pLV-sIFITM3-Flag. Unprocessed original images are found in Supplementary Figure S2 . *, p-value <0.05; **, p-value <0.01; ****, p-value <0.0001.

Figure 8

Proliferation of PEDV in IFITM3-overexpressed and knocked-down Vero cells. Vero cells were transfected with pLV-sIFITM3-Flag or si-csa-IFITM3s for 48 h, followed by infection with PEDV (MOI = 1) for 48 h. The proliferation of PEDV in IFITM3-overexpressed and knocked-down Vero cells was evaluated at 24 h postinfection using real-time PCR, flow cytometry assay, and crystal violet staining assay. (A, B) Western blot. (C, D) Real-time PCR. (E, F) flow cytometry assay. (G, H) Crystal violet assay. Unprocessed original images are found in Supplementary Figure S3 . *, p-value <0.05; **, p-value <0.01; ***, p-value <0.001.

Figure 9

sIFITM3 antagonizes rVSV-GFP proliferation in different cells. A549, BHK21, and DF-1 cells were transfected with pCAGGS-sIFITM3-Flag using Lipofectamine 3000 reagent. 24 h post-transfection, the expression of IFITM3 was examined by Western blot (A) with anti-FLAG antibody. Then the cells were infected with rVSV-GFP at 0.1 MOI and the replication of rVSV-GFP was analyzed by examining via fluorescence microscopy (B) and flow cytometry (C) at 24 hpi. *, p-value <0.05.