IFN and cytokine responses in ducks to genetically similar H5N1 influenza A viruses of varying pathogenicity

Abstract

Ducks, the reservoir host, are generally permissive to influenza A virus infection without disease symptoms. This natural ecology was upset by the emergence of H5N1 strains, which can kill ducks. To better understand host-virus interactions in the reservoir host, and influenza strain-specific molecular contributions to virulence, we infected White Pekin ducks with three similar H5N1 viruses, with known differences in pathogenicity and replication rate. We quantified viral replication and innate immune gene activation by qPCR, in lung and spleen tissues, isolated on each of the first 3 days of infection. The three viruses replicated well, as measured by accumulation of matrix gene transcript, and viral load declined over time in the spleen. The ducks produced rapid, but temporally limited, IFN and cytokine responses, peaking on the first day post-infection. IFN and proinflammatory cytokine gene induction were greater in response to infection with the more lethal viruses, compared to an attenuated strain. We conclude that a well-regulated IFN response, with the ability to overcome early viral immune inhibition, without hyperinflammation, contributes to the ability of ducks to survive H5N1 influenza replication in their airways, and yet clear systemic infection and limit disease.

Keywords: H5N1; cytokine; duck; inflammation; influenza A; interferon.

Fig. 1.

H5N1 influenza A viruses replicated to high levels in infected ducks. (a) Tracheal virus titres in ducks infected with different H5N1 strains. Tracheal and cloacal swabs were collected at 1, 2 and 3 days p.i. and neat swab material was tested for the presence of influenza virus by egg inoculation, and positive tracheal samples were titrated further by calculating EID₅₀. H5N1 replication in tissues was determined by amplification of the influenza A matrix 1 gene. RNA was extracted from lung (b) and spleen (c) tissues of ducks at 1, 2 and 3 days p.i. with rgVN1203, VN1203 or D4AT viruses. Influenza matrix gene copy number is determined against a known copy number of an influenza matrix M1 clone. Each dot represents one duck. Significant differences in mean viral titre each day were determined by two-way ANOVA (P<0.05). *P<0.05, ns not significant.

Fig. 2.

RIG-I is upregulated early in lung and spleen tissues of H5N1 influenza A-infected ducks. RNA was extracted from lung (a) and spleen (b) tissues of ducks at 1, 2 and 3 days p.i. with rgVN1203, VN1203 or D4AT viruses. DDX58 transcription was analysed by qPCR and normalized to GAPDH. Fold-induction compared to a mock-treated animal is shown (each dot represents one duck). Significant differences between the mean gene expression levels in tissues infected with different viruses were determined by two-way ANOVA (P<0.05), *P<0.05.

Fig. 3.

IFNs are upregulated in lung and spleen tissues of H5N1 influenza A-infected ducks at 1 day p.i. RNA was extracted from lung and spleen tissues of ducks at 1, 2 and 3 days p.i. with rgVN1203, VN1203 or D4AT viruses. IFN transcription was analysed by qPCR and normalized to GAPDH. Fold-induction compared to mock-infected animals is shown for genes encoding IFN-α in lung (a) and spleen (b); IFN-β in lung (c) and spleen (d); IFN-γ in lung (e) and spleen (f); IFN-λ in lung (g) and spleen (h). Each dot represents one duck. Significant differences between mean expression levels in tissues infected with different viruses were determined by two-way ANOVA (P<0.05). *P<0.05, ** P<0.01, ***P<0.001, ****P<0.0001, ns not significant.

Fig. 4.

ISGs are upregulated early in lung and spleen tissues of H5N1 influenza A-infected ducks. RNA was extracted from lung and spleen tissues of ducks at 1, 2 and 3 days p.i. with rgVN1203, VN1203 or D4AT viruses. ISG transcription was analysed by qPCR and normalized to GAPDH. Fold-induction compared to mock-infected animals is shown for OASL in lung (a) and spleen (b); IFIT5 in lung (c) and spleen (d); MX1 in lung (e) and spleen (f). Each dot represents one duck. Significant differences between mean expression levels in tissues infected with different viruses were determined by two-way ANOVA (P<0.05), *P<0.05, ns not significant.

Fig. 5.

ILs are upregulated in lung and spleen tissues of H5N1 influenza A-infected ducks at 1 day p.i. RNA was extracted from lung and spleen tissues of ducks at 1, 2 and 3 days p.i. with rgVN1203, VN1203 or D4AT viruses. IL transcription was analysed by qPCR and normalized to GAPDH. Fold-induction compared to mock-infected animals is shown for IL1B in lung (a) and spleen (b); IL6 in lung (c) and spleen (d); IL18 in lung (e) and spleen (f) and IL10 in lung (g) and spleen (h). Each dot represents one duck. Significant differences between mean expression levels in tissues infected with different viruses were determined by two-way ANOVA (P<0.05). *P<0.05, ** P<0.01, ns not significant.