Mallard ducks are important natural hosts of low pathogenic avian influenza (LPAI) viruses and many strains circulate in this reservoir and cause little harm. Some strains can be transmitted to other hosts, including chickens, and cause respiratory and systemic disease. Rarely, these highly pathogenic avian influenza (HPAI) viruses cause disease in mallards, while chickens are highly susceptible. The long co-evolution of mallard ducks with influenza viruses has undoubtedly fine-tuned many immunological host–pathogen interactions to confer resistance to disease, which are poorly understood. Here, we compare innate responses to different avian influenza viruses in ducks and chickens to reveal differences that point to potential mechanisms of disease resistance. Mallard ducks are permissive to LPAI replication in their intestinal tissues without overtly compromising their fitness. In contrast, the mallard response to HPAI infection reflects an immediate and robust induction of type I interferon and antiviral interferon stimulated genes, highlighting the importance of the RIG-I pathway. Ducks also appear to limit the duration of the response, particularly of pro-inflammatory cytokine expression. Chickens lack RIG-I, and some modulators of the signaling pathway and may be compromised in initiating an early interferon response, allowing more viral replication and consequent damage. We review current knowledge about innate response mediators to influenza infection in mallard ducks compared to chickens to gain insight into protective immune responses, and open questions for future research.

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.

Zika virus (ZIKV) establishes persistent infections in multiple human tissues, a phenomenon that likely plays a role in its ability to cause congenital birth defects and neurological disease. Multiple nonstructural proteins encoded by ZIKV, in particular NS5, are known to suppress the interferon (IFN) response by attacking different steps in this critical antiviral pathway. Less well known are the potential roles of structural proteins in affecting the host immune response during ZIKV infection. Capsid proteins of flaviviruses are of particular interest because a pool of these viral proteins is targeted to the nuclei during infection and, as such, they have the potential to affect host cell gene expression. In this study, RNA-seq analyses revealed that capsid proteins from six different flaviviruses suppress expression of type I IFN and IFN-stimulated genes. Subsequent interactome and in vitro ubiquitination assays showed that ZIKV capsid protein binds to and prevents activating ubiquitination of RIG-I CARD domains by TRIM25, a host factor that is important for the induction arm of the IFN response. The other flavivirus capsid proteins also interacted with TRIM25, suggesting that these viral proteins may attenuate antiviral signaling pathways at very early stages of infection, potentially even before nonstructural proteins are produced.

RIG-I plays an essential role in the duck innate immune response to influenza infection. RIG-I engages the critical adaptor protein mitochondrial antiviral signaling (MAVS) to activate the downstream signaling pathway. The influenza A virus non-structural protein PB1-F2 interacts with MAVS in human cells to inhibit interferon production. As duck and human MAVS share only 28% amino acid similarity, it is not known whether the influenza virus can similarly inhibit MAVS signaling in avian cells. Using confocal microscopy we show that MAVS and the constitutively active N-terminal end of duck RIG-I (2CARD) co-localize in DF-1 cells, and duck MAVS is pulled down with GST-2CARD. We establish that either GST-2CARD, or duck MAVS can initiate innate signaling in chicken cells and their co-transfection augments interferon-beta promoter activity. Demonstrating the limits of cross-species interactions, duck RIG-I 2CARD initiates MAVS signaling in chicken cells, but works poorly in human cells. The D122A mutation of human 2CARD abrogates signaling by affecting MAVS engagement, and the reciprocal A120D mutation in duck 2CARD improves signaling in human cells. We show mitochondrial localization of PB1-F2 from influenza A virus strain A/Puerto Rico/8/1934 (H1N1; PR8), and its co-localization and co-immunoprecipitation with duck MAVS. PB1-F2 inhibits interferon-beta promoter activity induced by overexpression of either duck RIG-I 2CARD, full-length duck RIG-I, or duck MAVS. Finally, we show that the effect of PB1-F2 on mitochondria abrogates TRIM25-mediated ubiquitination of RIG-I CARD in both human and avian cells, while an NS1 variant from the PR8 influenza virus strain does not.

MHC class I is critically involved in defense against viruses, and diversity from polygeny and polymorphism contributes to the breadth of the immune response and health of the population. In this article, we examine MHC class I diversity in wild mallard ducks, the natural host and reservoir of influenza A viruses. We previously showed domestic ducks predominantly use UAA, one of five MHC class I genes, but whether biased expression is also true for wild mallards is unknown. Using RT-PCR from blood, we examined expressed MHC class I alleles from 38 wild mallards (Anas platyrhynchos) and identified 61 unique alleles, typically 1 or 2 expressed alleles in each individual. To determine whether expressed alleles correspond to UAA adjacent to TAP2 as in domestic ducks, we cloned and sequenced genomic UAA-TAP2 fragments from all mallards, which matched transcripts recovered and allowed us to assign most alleles as UAA. Allelic differences are primarily located in α1 and α2 domains in the residues known to interact with peptide in mammalian MHC class I, suggesting the diversity is functional. Most UAA alleles have unique residues in the cleft predicting distinct specificity; however, six alleles have an unusual conserved cleft with two cysteine residues. Residues that influence peptide-loading properties and tapasin involvement in chicken are fixed in duck alleles and suggest tapasin independence. Biased expression of one MHC class I gene may make viral escape within an individual easy, but high diversity in the population places continual pressure on the virus in the reservoir species.

Influenza A viruses are a major cause of human and animal disease. Periodically, avian influenza viruses from wild waterfowl, such as ducks, pass through intermediate agricultural hosts and emerge into the human population as zoonotic diseases with high mortality rates and epidemic potential.