A comparative analysis of host responses to avian influenza infection in ducks and chickens highlights a role for the interferon-induced transmembrane proteins in viral resistance

Abstract

Background: Chickens are susceptible to infection with a limited number of Influenza A viruses and are a potential source of a human influenza pandemic. In particular, H5 and H7 haemagglutinin subtypes can evolve from low to highly pathogenic strains in gallinaceous poultry. Ducks on the other hand are a natural reservoir for these viruses and are able to withstand most avian influenza strains.

Results: Transcriptomic sequencing of lung and ileum tissue samples from birds infected with high (H5N1) and low (H5N2) pathogenic influenza viruses has allowed us to compare the early host response to these infections in both these species. Chickens (but not ducks) lack the intracellular receptor for viral ssRNA, RIG-I and the gene for an important RIG-I binding protein, RNF135. These differences in gene content partly explain the differences in host responses to low pathogenic and highly pathogenic avian influenza virus in chicken and ducks. We reveal very different patterns of expression of members of the interferon-induced transmembrane protein (IFITM) gene family in ducks and chickens. In ducks, IFITM1, 2 and 3 are strongly up regulated in response to highly pathogenic avian influenza, where little response is seen in chickens. Clustering of gene expression profiles suggests IFITM1 and 2 have an anti-viral response and IFITM3 may restrict avian influenza virus through cell membrane fusion. We also show, through molecular phylogenetic analyses, that avian IFITM1 and IFITM3 genes have been subject to both episodic and pervasive positive selection at specific codons. In particular, avian IFITM1 showed evidence of positive selection in the duck lineage at sites known to restrict influenza virus infection.

Conclusions: Taken together these results support a model where the IFITM123 protein family and RIG-I all play a crucial role in the tolerance of ducks to highly pathogenic and low pathogenic strains of avian influenza viruses when compared to the chicken.

Fig. 1

Bayesian tree of the vertebrate IFITM1, 2 and 3-like gene family. A Bayesian tree was constructed using MrBayes (v3.2.2) with 1 million generations. Branch confidence values are shown at the nodes and coloured using the probability scale on the left. Scale bar corresponds to 0.3 substitutions per site. IFITM123 represent the mammalian IFITM1, 2 and 3-like genes and for simplicity are shown as a triangle. Details of the mammalian tree are shown in Additional file 7: Figure S5D. All the other IFITM genes are derived from avian, non-avian reptiles and amphibians. The species abbreviations are shown on Additional file 3: Table S2. The dotted circle indicates uncertainty in the split near the root of the tree

Fig. 2

Ingenuity Pathway Analysis of the chicken response to HPAI infection in the ileum at 1 dpi and LPAI infection in the lung at 3 dpi. a Molecular functions of genes responding to HPAI in the ileum at 1dpi. b Biological pathways which are significantly altered during the host response to LPAI in the lung at 3dpi. In each case p < 0.05. c Differential gene regulation in a biological network concerned with lipid metabolism (ileum). d Genes differentially expressed in the cell death, cell signalling and inflammatory response network (lung). Up-regulated genes (red) and down-regulated genes (green). The deeper the colour, the higher the level of differential expression

Fig. 3

Ingenuity Pathway Analysis of the duck response to HPAI infection in the ileum at 1 dpi and in the lung at 3 dpi. a Biological pathways which are significantly altered during the host response to HPAI in the ileum at 1dpi. b Biological pathways which are significantly altered during the host response to HPAI in the lung at 3dpi. In each case p < 0.05. c Differential gene regulation in a biological network concerned with the inflammatory response network (ileum). d Differential expression of genes involved in haematopoiesis (lung). Red represents up-regulated genes and green down-regulated genes. The deeper the colour, the higher the level of differential expression

Fig. 4

Venn diagram showing shared and unique responses to AI infection in duck and chicken. a Chicken response to H5N1 infection in the ileum at 1 dpi is compared to that of the duck. b Chicken response to H5N2 infection in the lung at 3 dpi is compared with the duck response to high path infection in the lung at 3 dpi

Fig. 5

QRT-PCR analysis of lung RNA at 1dpi (H5N1) in chicken and duck. IFITM gene expression in lung tissue measured by qRT-PCR in control and HPAI H5N1 (A/Vietnam/1203/04) infected chicken and duck samples 1 dpi. IFITM gene expression was measured in three control and three infected birds. Data are expressed as the mean fold change in infected birds relative to the uninfected controls. Error bars represent the standard deviation

Fig. 6

Co-expression clusters associated with the duck IFITM1, 2 and 3 genes. a IFITM1 and 2 gene expression cluster, b IFITM1 and 2 network, c IFITM3 gene expression cluster, d IFITM3 network. The gene expression clusters were calculated using CLUSTERGRAM and normalised data from all the RNAseq datasets. The gene networks were calculated using IPA analysis of the genes that were clustered with expression of IFITM genes in a and c

Fig. 7

Domain analysis and sequence characteristics of the IFITM1, 2 and 3 gene family. The sequence alignment of chicken (GALGA), duck (ANAPL) and human (HOMSA) IFITM peptides was created using T-coffee and displayed using Jalview, which was also used to create secondary structure (JNETHMM; red, helices; green, beta sheets) and conservation tracks. Sites predicted to be under positive selection (P_SITES, Table 1) are shown as triangles (red, duck and yellow, other birds). The transmembrane (TM1 and TM2), conserved intracellular loop (CIL) and other domains were predicted using SMART, SOSUI and ExPasy. MOTIFS, mark amino acid residues as triangles and are discussed in the main text: yellow Cysteine, green Phenylalanine, purple Lysine. Sequence logos were generated using Weblogo and based on the alignment of the duck, chicken and human IFITM sequences. For details of methods see Materials and Methods