RNA-Seq-Based Whole Transcriptome Analysis of IPEC-J2 Cells During Swine Acute Diarrhea Syndrome Coronavirus Infection

Abstract

The new emergence of swine acute diarrhea syndrome coronavirus (SADS-CoV) has resulted in high mortality in suckling pigs in China. To date, the transcriptional expression of host cells during SADS-CoV infection has not been documented. In this study, by means of RNA-Seq technology, we investigated the whole genomic expression profiles of intestinal porcine epithelial cells (IPEC-J2) infected with a SADS-CoV strain SADS-CoV-CH-FJWT-2018. A total of 24,676 genes were identified: 23,677 were known genes, and 999 were novel genes. A total of 1,897 differentially expressed genes (DEGs) were identified between SADS-CoV-infected and uninfected cells at 6, 24, and 48 h post infection (hpi). Of these, 1,260 genes were upregulated and 637 downregulated. A Gene Ontology enrichment analysis revealed that DEGs in samples from 6, 24, and 48 hpi were enriched in 79, 383, and 233 GO terms, respectively, which were mainly involved in immune system process, response to stimulus, signal transduction, and cytokine-cytokine receptor interactions. The 1,897 DEGs were mapped to 109 KEGG Ontology (KO) pathways classified into four main categories. Most of the DEGs annotated in the KEGG pathways were related to the immune system, infectious viral disease, and signal transduction. The mRNA of porcine serum amyloid A-3 protein (SAA3), an acute phase response protein, was significantly upregulated during the infection. Over-expressed SAA3 in IPEC-J2 cells drastically inhibited the replication of SADS-CoV, while under-expressed SAA3 promoted virus replication. To our knowledge, this is the first report on the profiles of gene expression of IPEC-J2 cells infected by SADS-CoV by means of RNA-Seq technology. Our results indicate that SADS-CoV infection significantly modified the host cell gene expression patterns, and the host cells responded in highly specific manners, including immune response, signal and cytokine transduction, and antiviral response. The findings provide important insights into the transcriptome of IPEC-J2 in SADS-CoV infection.

Keywords: RNA-Seq; SADS-CoV; gene expression; porcine serum amyloid A-3; transcriptome.

Figure 1

Cytopathic effects of SADS-CoV isolates in inoculated Vero 81 and IPEC-J2 cells (A) and One-step growth curve of SADS-CoV in IPEC-J2 cells (B) One-step growth curve of SADS-CoV in IPEC-J2 cells. Cells were inoculated with virus at an MOI of 1. Total virus titer was determined by qPCR-based infectivity assay at the indicated time intervals. Data shown are from experiments performed in triplicates, with error bars indicating standard deviations.

Figure 2

The differently expressed gene of volcano and Venn diagram analysis of up- and downregulated genes and shared genes. The vertical line of volcano diagram is the 2-fold expression difference threshold, and the horizontal line is the p_adj-value = 0.05 threshold; the red, green, and blue points represent the upregulated, downregulated, and non-significant differentially expressed genes. (A) Infected vs. negative control at 6 hpi, (B) Infected vs. negative control at 24 hpi, (C) Infected vs. negative control at 48 hpi. (D) Venn diagram of DEGs distributions at 6, 24, and 48 hpi.

Figure 3

Functional map of differentially expressed genes enriched for GO terms. All categories were statistically significant (p_adj < 0.05). The bars represent the –log₁₀p_adj-value of the comparison of respective GO terms from infected and uninfected samples. Red, green, and blue bars indicated GO terms clustered in the biological process, cellular component, and molecular function terms, respectively.

Figure 4

The directed acycline graph of the GO category in the biological process, indicating it is significantly enriched in infected samples at 6 hpi. Boxes and ellipses with colored background represent significantly enriched terms.

Figure 5

Different KEGG pathway gene enrichment statistics. The dot diameter indicates the number of differential genes; color depth indicates significance; abscissa indicates enrichment abundance; and the ordinate indicates different pathways.

Figure 6

Validation of gene expression. Heat-maps of log₂ transformed expression fold changes in SADS-CoV infected cells to time matched negative controls across the time points in hours using both RNA-Seq and quantitative real-time PCR (qPCR) methods. On the scale bar, red indicates upregulation, and green stands for downregulation of mRNA compared to mock-infected controls. Data are the medians of three independent biological replicates.

Figure 7

Measurement of SADS-CoV replication in recommended cells featured either overexpressed or silencing SAA3 protein gene. IPEC-J2 cells were transfected with an overexpressing/silencing plasmid pCAGGS-SAA3/pcDNA3.1-U6-shRNA or a mock vector and subsequently infected with SADS-CoV at an MOI of 1. The transfectants were harvested at 24 hpi and subjected to western blotting analysis (A) and a TCID₅₀ assay (B). (A) Expression/inhibition of SAA3 gene was detected by western blotting with an anti-SAA3 antibody. (B) SADS-CoV replication was titrated by a TCID₅₀ assay. ***indicates the p value ≤ 0.001.