The highly pathogenic H5N1 avian influenza virus induces the mitogen-activated protein kinase signaling pathway in the trachea of two Ri chicken lines

Abstract

Objective: The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry and economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for studies on HPAIV resistance. Therefore, in this study, we investigated gene expression related to the mitogen-activated protein kinase (MAPK) signaling pathway by comparing non-infected, HPAI-infected resistant, and susceptible Ri chicken lines.

Methods: Resistant (Mx/A; BF2/B21) and susceptible Ri chickens (Mx/G; BF2/B13) were selected by genotyping the Mx and BF2 genes. Then, the tracheal tissues of non-infected and HPAIV H5N1 infected chickens were collected for RNA sequencing.

Results: A gene set overlapping test between the analyzed differentially expressed genes (DEGs) and functionally categorized genes was performed, including biological processes of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. A total of 1,794 DEGs were observed between control and H5N1-infected resistant Ri chickens, 432 DEGs between control and infected susceptible Ri chickens, and 1,202 DEGs between infected susceptible and infected resistant Ri chickens. The expression levels of MAPK signaling pathway-related genes (including MyD88, NF-κB, AP-1, c-fos, Jun, JunD, MAX, c-Myc), cytokines (IL-1β, IL-6, IL-8), type I interferons (IFN-α, IFN-β), and IFN-stimulated genes (Mx1, CCL19, OASL, and PRK) were higher in H5N1-infected than in non-infected resistant Ri chickens. MyD88, Jun, JunD, MAX, cytokines, chemokines, IFNs, and IFN-stimulated expressed genes were higher in resistant-infected than in susceptible-infected Ri chickens.

Conclusion: Resistant Ri chickens showed higher antiviral activity compared to susceptible Ri chickens, and H5N1-infected resistant Ri chickens had immune responses and antiviral activity (cytokines, chemokines, interferons, and IFN-stimulated genes), which may have been induced through the MAPK signaling pathway in response to H5N1 infection.

Keywords: H5N1; Mitogen-activated Protein Kinase (MAPK) Signaling Pathway; RNA Sequencing; Ri Chicken.

Figure 1

Distribution of DEGs represented in a Volcano plot. (A) D3-CTRL vs D3-HPAI infected resistant, (B) D3-CTRL vs D3-HPAI infected susceptible chickens, (C) D3-HPAI infected resistant vs D3-HPAI infected susceptible chickens for |log₂| (fold-change) and –log₁₀ (p-value) to show expression change and its significance. The blue dots indicate DEGs that have significantly downregulated; red dots indicate DEGs that have significantly upregulated; the gray dots indicate no significant differences between the two groups. DEGs, differentially expressed genes; HPAI, highly pathogenic avian influenza.

Figure 2

Gene ontology (GO) functional analysis for HPAI-infected resistant and HPAI-infected susceptible Ri chicken lines. The enriched biological terms include: (A) Top 10 singular enrichment analysis terms (SEA): GO biological process, (B) GO cellular component, and (C) GO molecular function (D) Kyoto encyclopedia of genes and genomes functional pathways from the 1,202 DEGs in HPAI infected resistant and HPAI infected susceptible Ri chicken lines obtained by criteria (|fold-change|≥2)∩ (p<0.05). GO, gene ontology; DEGs, differentially expressed genes; HPAI, highly pathogenic avian influenza.

Figure 3

Heat map of the individual MAPK signaling pathway genes from the tracheal tissue of three comparisons of Ri chickens, three days post-infection: Control resistant vs infected resistant chickens, Control susceptible vs infected susceptible chickens, infected susceptible vs infected resistant chickens. A green color indicates DEGs that have higher expression levels while a red color indicates DEGs that have lower expression levels, as calculated from the expression values in |log₂| (fold change) units. MAPK, mitogen-activated protein kinase; DEGs, differentially expressed genes.

Figure 4

Interactions of the 68 differentially expressed genes related to the MAPK signaling pathway in the tracheas of Ri chicken lines. This interaction analysis was conducted using the STRING version 11.0 (http://string-db.org/). MAPK, mitogen-activated protein kinase.

Figure 5

The qPCR analysis of the expression of genes associated with MAPK signaling pathway in (A) control and H5N1-infected resistant Ri chickens at 3 days post infection (dpi); (B) control and H5N1-infected susceptible Ri chickens; (C) H5N1-infected susceptible and H5N1-infected resistant Ri chickens. Relative quantitation data of qRT-PCR are represented as mean±SEM, normalized to GAPDH using the 2^−ΔΔCt method. Data are expressed as mean±SEM of three independent experiments: *** p<0.001. qPCR, real-time quantitative polymerase chain reaction; MAPK, mitogen-activated protein kinase; SEM, standard error of mean; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

The expression patterns of these genes were compared between qPCR and RNA sequencing data (|log₂|-fold change). The qRT-PCR data are expressed as mean±SEM of three independent experiments. qRT-PCR, real-time quantitative polymerase chain reaction; SEM, standard error of mean.