Association of RIG-I with innate immunity of ducks to influenza

Abstract

Ducks and wild waterfowl perpetuate all strains of influenza viruses in nature. In their natural host, influenza viruses typically cause asymptomatic infection and little pathology. Ducks are often resistant to influenza viruses capable of killing chickens. Here, we show that the influenza virus sensor, RIG-I, is present in ducks and plays a role in clearing an influenza infection. We show evidence suggesting that RIG-I may be absent in chickens, providing a plausible explanation for their increased susceptibility to influenza viruses compared with ducks. RIG-I detects RNA ligands derived from uncapped viral transcripts and initiates the IFN response. In this study, we show that the chicken embryonic fibroblast cell line, DF-1, cannot respond to a RIG-I ligand. However, transfection of duck RIG-I into DF-1 cells rescues the detection of ligand and induces IFN-beta promoter activity. Additionally, DF-1 cells expressing duck RIG-I have an augmented IFN response resulting in decreased influenza replication after challenge with either low or highly pathogenic avian influenza virus. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression is induced 200 fold, early in an innate immune response in ducks challenged with the H5N1 virus A/Vietnam/1203/04. Finding this natural disease resistance gene in ducks opens the possibility of increasing influenza resistance through creation of a transgenic chicken.

Fig. 1.

Amino acid alignment of duck, zebra finch, and human RIG-I. Alignment of duck RIG-I (accession no. EU363349), zebra finch (accession no. XM_002194524), and human RIG-I (accession no. AF038963) was performed using the ClustalW program and edited with Boxshade. Black shading indicates amino acid identity and gray shading indicates similarity (50% threshold). Plus signs indicate human residues involved in polyubiquitination and asterisks indicate residues involved in ligand binding. The ATP binding motif is boxed.

Fig. 2.

RIG-I is present in ducks and pigeons, but apparently absent in chickens. (A) Hybridization of a multiple exon duck RIG-I probe to HindIII and XbaI-digested genomic DNA from four White Pekin ducks and two White Leghorn chickens. (B) Hybridization of a single exon duck RIG-I probe to PstI, NdeI, and SacI digested genomic DNA from duck, chicken, and pigeon. (C) Hybridization of a single exon duck MDA5 probe to same blot.

Fig. 3.

Duck RIG-I rescues detection of 5′ppp RNA and induces an antiviral response in DF-1 chicken embryonic fibroblast cells. (A) IFN-β promoter activity in RIG-I or empty vector–transfected DF-1 cells following 15 h of ligand stimulation compared to mock-treated cells, shown as mean fold induction (±SD). Results are representative of three independent experiments and were analyzed using a single-factor ANOVA and Tukey post hoc test (different letters, P < 0.05). (B) RIG-I–transfected DF-1 cells respond to BC500 infection (MOI, 1) with increased expression of chicken IFN-β and the IFN-stimulated genes Mx1 and PKR, and decreased influenza matrix gene expression, relative to empty vector–transfected cells. RNA was extracted from cells for qRT-PCR 15 h PI, and fold difference in gene expression calculated for RIG-I and vector-only–transfected DF-1 cells. Results are representative of three independent experiments and error bars show RQ_Min/Max at a 95% confidence level and represent SE (n = 3). (C and D) RIG-I–transfected DF-1 cells had significantly lower influenza virus titers compared with empty vector–transfected cells for BC500 (C) or VN1203 (D). Both infections were performed 24 h after transfection at an MOI of 1. After 15 h, titer was determined by plaque assay from triplicate wells and results were analyzed with the two-tailed Student‘s t test (n = 3; P = 0.002).

Fig. 4.

RIG-I is dramatically up-regulated in duck lung infected with VN1203 but not BC500. Lung and intestinal RNA was extracted d 1 or d 3 PI and analyzed by qRT-PCR for RIG-I expression compared with a mock-infected animal. Fold expression of RIG-I mRNA in duck lung tissue infected with BC500 or VN1203 at d 1 PI (A) or d 3 PI (B). Fold expression of RIG-I mRNA in duck intestine infected with BC500 at d 1 PI (C) or d 3 PI (D). Error bars show RQ_Min/Max at a 95% confidence level and represent SE (n = 4).