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. 2018 Aug 8;19(8):2328.
doi: 10.3390/ijms19082328.

Comparative Transcriptomic Analysis of Immune-Related Gene Expression in Duck Embryo Fibroblasts Following Duck Tembusu Virus Infection

Guanliu Yu  1   2   3 , Yun Lin  4   5   6 , Yi Tang  7   8   9 , Youxiang Diao  10   11   12
Affiliations

Comparative Transcriptomic Analysis of Immune-Related Gene Expression in Duck Embryo Fibroblasts Following Duck Tembusu Virus Infection

Guanliu Yu et al. Int J Mol Sci. .

Abstract

Duck is a major waterfowl species in China, providing high-economic benefit with a population of up to 20⁻30 billion per year. Ducks are commonly affected by severe diseases, including egg-drop syndrome caused by duck Tembusu virus (DTMUV). The immune mechanisms against DTMUV invasion and infection remain poorly understood. In this study, duck embryo fibroblasts (DEFs) were infected with DTMUV and harvested at 12 and 24 h post-infection (hpi), and their genomes were sequenced. In total, 911 (764 upregulated and 147 downregulated genes) and 3008 (1791 upregulated and 1217 downregulated) differentially expressed genes (DEGs) were identified at 12 and 24 hpi, respectively. Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that DEGs were considerably enriched in immune-relevant pathways, including Toll-like receptor signaling pathway, Cytosolic DNA-sensing pathway, RIG-I-like receptor signaling pathway, Chemokine signaling pathway, NOD-like receptor signaling pathway, and Hematopoietic cell lineage at both time points. The key DEGs in immune system included those of the cytokines (IFN α2, IL-6, IL-8L, IL-12B, CCR7, CCL19, and CCL20), transcription factors or signaling molecules (IRF7, NF-κB, STAT1, TMEM173, and TNFAIP3), pattern recognition receptors (RIG-I and MDA5), and antigen-presenting proteins (CD44 and CD70). This suggests DTMUV infection induces strong proinflammatory/antiviral effects with enormous production of cytokines. However, these cytokines could not protect DEFs against viral attack. Our data revealed valuable transcriptional information regarding DTMUV-infected DEFs, thereby broadening our understanding of the immune response against DTMUV infection; this information might contribute in developing strategies for controlling the prevalence of DTMUV infection.

Keywords: DTMUV; RNA-seq; duck embryo fibroblast; transcriptome; virus infection.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Cytopathic effects following infection of duck embryo fibroblasts with duck Tembusu virus at 12, 24, and 48 h post-infection. (a,c,e) no CPEs were observed in mock-infected cells. (b,d,f) the CPEs (e.g., cell shrinkage, rounding, and suspension) were found in cells infected with DTMUV. The blue scale bar represents 200 μm.
Figure 2
Figure 2
Viability of duck embryo fibroblasts at 12 (a–c) and 24 h (d–f) after infection with duck Tembusu virus (DTMUV). CPEs (cell shrinkage and rounding, red arrow) were observed in Figure 2b,e. Cell viability is shown as the mean ± SD of eight replicates.** p < 0.01.
Figure 3
Figure 3
Principal component of samples (a) and visibility graph of differentially expressed genes (b) at 12 and 24 h post-infection (hpi). Note: (a): PC1 shows the differences among duck Tembusu virus (DTMUV)-infected samples; PC2 indicates differences between mock- and DTMUV-infected samples. 12D, mock-infected DEFs at 12 hpi; 24D, mock-infected DEFs at 24 hpi; 12S, DTMUV-infected DEFs at 12 hpi; 24S, DTMUV-infected DEFs at 24 hpi. (b): Upregulated differentially expressed genes (DEGs) are shown in red, whereas downregulated DEGs are shown in green. The green curve in the circle represents the common DEGs at 12 and 24 hpi.
Figure 4
Figure 4
Gene Ontology enrichment of differentially expressed genes (a): 12 h post-infection (hpi); (b): 24 h post-infection 24 hpi.
Figure 5
Figure 5
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis at 12 (a) and 24 h post-infection (b). The top six enriched KEGG terms at both the time points are marked in black bold font.
Figure 6
Figure 6
The immune system pathways enriched during duck Tembusu virus infections at 12 h (a) and 24 h (b) post-infection (hpi) (p < 0.05).
Figure 7
Figure 7
Differentially expressed genes (DEGs) in six common immune system-related signaling pathways at 12 and 24 h post-infection (hpi). (a) DEGs in Toll-like receptor signaling pathway at 12 and 24 hpi. (b) DEGs in Chemokine signaling pathway at 12 and 24 hpi. (c) DEGs in Nod-like receptor signaling pathway at 12 and 24 hpi. (d) DEGs in Hematopoietic cell lineage at 12 and 24 hpi. (e) DEGs in RIG-I-like receptor signaling pathway at 12 and 24 hpi. (f) DEGs in Cytosolic DNA-sensing signaling pathway at 12 and 24 hpi. The six DEGs with the largest fold changes in expression in each pathway are marked by black dots.
Figure 8
Figure 8
The top six differentially expressed genes in the Toll-like receptor signaling pathway, Chemokine signaling pathway, Nod-like receptor signaling pathway, Hematopoietic cell lineage, RIG-I-like receptor signaling pathway, and Cytosolic DNA-sensing signaling pathway. The colors in the grid represent the expression fold change as indicated by the color scale. Note: LOC101794331 (T-lymphocyte activation antigen CD86-like); LOC101795038 (fractalkine-like); LOC101800410 (T-cell surface glycoprotein CD1b-3-like).
Figure 9
Figure 9
Confirmation of the transcriptome sequencing data by quantitative real-time (qRT)-PCR. Six differentially expressed genes (DEGs) involved in immune-related pathways were selected for qRT-PCR, and expression was estimated using the 2−∆∆CT method. The qRT-PCR data are presented as the mean ± standard deviation (n = 5) (a–f). Pearson’s correlation analyses of the expression of above six DEGs between Seq-RNA and qRT-PCR (g).
Figure 10
Figure 10
Concentrations of IL-6 (a); IL-8 (b); IL-12 (c); and IFN-α (d) in the culture supernatant of the mock- and duck Tembusu virus (DTMUV)-infected groups. The data are presented as the mean ± standard deviation (n = 6). * p < 0.05, ** p < 0.01.
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