Comparative Investigation of Gene Regulatory Processes Underlying Avian Influenza Viruses in Chicken and Duck

Abstract

The avian influenza virus (AIV) mainly affects birds and not only causes animals' deaths, but also poses a great risk of zoonotically infecting humans. While ducks and wild waterfowl are seen as a natural reservoir for AIVs and can withstand most virus strains, chicken mostly succumb to infection with high pathogenic avian influenza (HPAI). To date, the mechanisms underlying the susceptibility of chicken and the effective immune response of duck have not been completely unraveled. In this study, we investigate the transcriptional gene regulation underlying disease progression in chicken and duck after AIV infection. For this purpose, we use a publicly available RNA-sequencing dataset from chicken and ducks infected with low-pathogenic avian influenza (LPAI) H5N2 and HPAI H5N1 (lung and ileum tissues, 1 and 3 days post-infection). Unlike previous studies, we performed a promoter analysis based on orthologous genes to detect important transcription factors (TFs) and their cooperation, based on which we apply a systems biology approach to identify common and species-specific master regulators. We found master regulators such as EGR1, FOS, and SP1, specifically for chicken and ETS1 and SMAD3/4, specifically for duck, which could be responsible for the duck's effective and the chicken's ineffective immune response.

Keywords: RNA sequencing; avian influenza; chicken; differentially expressed genes; duck; gene regulation; mallard; master regulators; transcription factor cooperation; upstream regulators.

Figure 1

Flow chart of the employed analyses. Differentially expressed genes (DEGs) were derived by comparing the gene expression rate of a specific condition against a mock infection for that condition (e.g., chicken lung at 1 dpi with H5N1 infection against chicken lung at 1 dpi with mock infection). TF and TFBS stand for transcription factor and transcription factor binding site, respectively. H5N1 is a high-pathogenic avian influenza virus (HPAIV), while H5N2 is a low-pathogenic avian influenza virus (LPAIV).

Figure 2

Venn diagrams of the DEGs (A) duck in ileum, (B) chicken in ileum, (C) duck in lung, and (D) chicken in lung with selected enriched Gene Ontology (GO) terms. The DEGs are obtained by comparing animals infected with AIV (H5N1 (HPAI) or H5N2 (LPAI)) with mock-infected animals. The colors within the venn diagram, as well as the colors of the GO-term boxes, stand for the respective condition: blue represents H5N1 infection 1 dpi, red represents H5N1 infection 3 dpi, green represents H5N2 infection 1 dpi, and yellow represents H5N2 infection 3 dpi for each species and tissue. Within the boxes, an arrow down indicates that the GO-term is enriched among the downregulated DEGs; otherwise, the terms are enriched among the upregulated DEGs. The GO-terms represent biological processes except, if stated differently, in the form of MF (molecular function). Venn diagrams are based on the data provided in Table S1.

Figure 3

Venn diagrams of TFBS enrichment to compare over- (OR) and underrepresented (UR) binding sites in chicken and duck. The promoter regions of DEGs (after infection with HPAIV H5N1) in duck and the corresponding orthologous genes in chicken were extracted to obtain the over- and underrepresented TFBSs. (A) shows the corresponding number of TFBSs for the ileum 1 dpi, (B) shows the ileum 3 dpi, (C) shows the lung 1 dpi and (D) shows the lung 3 dpi. Venn diagrams are based on the data provided in Table S3.

Figure 4

Differences in transcription factor (TF) cooperation networks found by the PC-TraFF algorithm for (A) ileum 1 dpi, (B) ileum 3 dpi, (C) lung 1 dpi and (D) lung 3 dpi with HPAIV H5N1. The difference in nodes in the networks of duck and chicken is denoted by the set difference sign (∖). The nodes are labeled by the PWM names representing TFs, as given by TRANSFAC [44]. They follow the structure V$factorname_version, where “V$” indicates that the PWM originates from a vertebrate TF, factorname specifies the name of the corresponding TF, and version is specified to uniquely identify the PWM. The networks were visualized with Cytoscape [74]. Full size image is provided in Figure S3.

Figure 5

Common and species-specific master regulators for the (A) ileum and (B) lung tissue regulating the TF-TF cooperations.