Avian influenza rapidly induces antiviral genes in duck lung and intestine

Abstract

Ducks are the natural reservoir of influenza A and survive infection by most strains. To characterize the duck immune response to influenza, we sought to identify innate immune genes expressed early in an infection. We used suppressive subtractive hybridization (SSH) to construct 3 libraries enriched in differentially expressed genes from lung RNA of a duck infected with highly pathogenic avian influenza virus A/Vietnam/1203/04 (H5N1), or lung and intestine RNA of a duck infected with low pathogenic avian influenza A/mallard/BC/500/05 (H5N2) compared to a mock-infected duck. Sequencing of 1687 clones identified a transcription profile enriched in genes involved in antiviral defense and other cellular processes. Major histocompatibility complex class I (MHC I), interferon induced protein with tricopeptide repeats 5 (IFIT5), and 2'-5' oligoadenylate synthetase-like gene (OASL) were increased more than 1000-fold in relative transcript abundance in duck lung at 1dpi with highly pathogenic VN1203. These genes were induced much less in lung or intestine following infection with low pathogenic BC500. The expression of these genes following infection suggests that ducks initiate an immediate and robust response to a potentially lethal influenza strain, and a minimal response to a low pathogenic strain.

Fig. 1

IFIT5 was enriched in lung and intestine SSH libraries from influenza-infected ducks. Venn diagram of sequences identified in forward-subtracted SSH cDNA libraries prepared from lung RNA from a duck infected with VN1203, and lung and intestine RNA of a duck infected with BC500.

Fig. 2

MHC class I UAA is upregulated in lung and intestine tissues from influenza-infected ducks. Lung RNA was extracted from ducks 1 dpi and 3 dpi with BC500 or VN1203 and analyzed in comparison to mock-infected ducks by qRT-PCR. Fold expression of UAA mRNA in duck lung tissues at 1 dpi and 3 dpi (A) and in duck intestine tissue at 1 dpi or 3 dpi (B) is shown relative to a mock-infected animal from each day. Dots represent individual ducks (n=3) and the mean is indicated. qRT-PCR was performed twice and data from one replicate plotted.

Fig. 3

Interferon-stimulated genes are vastly upregulated in duck lung at 1 dpi with VN1203 virus. Lung RNA extracted 1 dpi and 3 dpi with BC500 or VN1203, was analyzed for expression of ISGs in comparison to mock-infected ducks by qRT-PCR. Fold expression of mRNA transcripts for (A) ISG12-2, (B) IFIT5, (C) OASL and (D) IFITM1 is shown relative to a mock infected duck from each day. Dots represent individual ducks (n=3) and the mean is indicated. qRT-PCR was performed twice and data from one replicate plotted.

Fig. 4

Interferon-stimulated genes are upregulated in intestine tissue of BC500 infected ducks relative to mock-infected animals. Intestine RNA extracted 1 and 3 dpi with BC500 was analyzed for expression of ISGs relative to mock-infected ducks by qRT-PCR. Fold expression of mRNA transcripts for A) ISG12-2 and B) IFIT5 are shown relative to a mock-infected RNA sample. Bars represent individual ducks (n=3) and the mean is indicated. qRT-PCR was performed twice and data from one replicate plotted.