Our overall hypothesis is that host population immunity directed at multiple antigens will influence the prevalence, diversity and evolution of influenza A virus (IAV) in avian populations where the vast subtype diversity is maintained. To investigate how initial infection influences the outcome of later infections with homologous or heterologous IAV subtypes and how viruses interact through host immune responses, we carried out experimental infections in mallard ducks (Anas platyrhynchos). Mallards were pre-challenged with an H3N8 low-pathogenic IAV and were divided into six groups. At five weeks post H3N8 inoculation, each group was challenged with a different IAV subtype (H4N5, H10N7, H6N2, H12N5) or the same H3N8. Two additional pre-challenged groups were inoculated with the homologous H3N8 virus at weeks 11 and 15 after pre-challenge to evaluate the duration of protection. The results showed that mallards were still resistant to re-infection after 15 weeks. There was a significant reduction in shedding for all pre-challenged groups compared to controls and the outcome of the heterologous challenges varied according to hemagglutinin (HA) phylogenetic relatedness between the viruses used. There was a boost in the H3 antibody titer after re-infection with H4N5, which is consistent with original antigenic sin or antigenic seniority and suggest a putative strategy of virus evasion. These results imply competition between related subtypes that could regulate IAV subtype population dynamics in nature. Collectively, we provide new insights into within-host IAV complex interactions as drivers of IAV antigenic diversity that could allow the circulation of multiple subtypes in wild ducks.

Wild waterfowl in the order Anseriformes are recognized reservoirs for influenza A viruses (IAVs); however, prevalence of infection can vary greatly by species. Few isolates of IAVs have been reported from snow geese (Chen caerulescens), and generally they have not been regarded as an important component of this reservoir. In February 2013, 151 combined cloacal and oropharangeal swabs and 147 serum samples were collected from snow geese wintering on the Gulf coast of Texas. None of the swab samples tested positive by virus isolation, but antibodies to IAVs were detected in 87 (59%) birds tested by competitive blocking ELISA (bELISA). To further characterize these detected antibodies, positive samples were tested by virus microneutralization (MN) for antibodies to viruses representing 14 hemagglutinin subtypes (HA1-HA12, H14, and H15). By MN, antibodies to H1 (n = 41; 47%), H5 (n = 32; 37%), H6 (n = 49; 56%), H9 (n = 50; 57%), and H12 (n = 24; 28%) were detected. Snow goose populations have increased in North America since the 1960s, and their association with agricultural lands provides a potential indirect source of IAV infection for domestic poultry. This potential, as well as the detection of antibodies to HA subtypes H5, H9, and H12 that are not well represented in other waterfowl species, suggests that further snow geese surveillance is indicated.

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Gulls are the known reservoir for H13 and H16 influenza A viruses (IAV) but also host a diversity of other IAV subtypes. Gulls also share habitats with both ducks and shorebirds, increasing the potential for cross-species IAV transmission. We serologically tested laughing gulls (Leucophaeus atricilla) collected at Delaware Bay during May when they were in direct contact with IAV-infected shorebirds; both species feed on horseshoe crab (Limulus polyphemus) eggs on beaches during this month. From 2010 to 2014, antibody prevalence as determined by competitive blocking enzyme-linked immunosorbent assay ranged from 25%-72%. Antibodies to H13 and H16 were detected by hemagglutination inhibition (HI) tests in 12% and 24% of tested gulls, respectively. Results from virus microneutralization (MN) tests for antibodies to H1-H12, H14, and H15 varied among years but the highest prevalence of neutralizing antibodies was detected against H1 (24%), H5 (25%), H6 (35%), H9 (33%), and H11 (42%) IAV. The subtype diversity identified by serology in gulls was dominated by Group 1 HA subtypes and only partially reflected the diversity of IAV subtypes isolated from shorebirds.

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Our overall hypothesis is that host population immunity directed at multiple antigens will influence the prevalence, diversity and evolution of influenza A virus (IAV) in avian populations where the vast subtype diversity is maintained. To investigate how initial infection influences the outcome of later infections with homologous or heterologous IAV subtypes and how viruses interact through host immune responses; we carried out experimental infections in mallard ducks (Anas platyrhynchos). Mallards were pre-challenged with an H3N8 low-pathogenic IAV and were divided into six groups. At five weeks post H3N8 inoculation, each group was challenged with a different IAV subtype or the same H3N8. Two additional pre-challenged groups were inoculated with the homologous H3N8 virus at weeks 11 and 15 after pre-challenge to evaluate the duration of protection, which showed that mallards were still resistant to re-infection after 15weeks. There was a significant reduction in shedding for all pre-challenged groups compared to controls and the outcome of the heterologous challenges varied according to hemagglutinin (HA) phylogenetic relatedness between the viruses used. There was a boost in the H3 antibody titer after re-infection with H4N5, which is consistent with original antigenic sin or antigenic seniority and suggest a putative strategy of virus evasion. These results imply strong competition between related subtypes that could regulate IAV subtype population dynamics in nature.Collectively, we provide new insights into within-host IAV complex interactions as drivers of IAV antigenic diversity that could allow the circulation of multiple subtypes in wild ducks. Author summaryMany features of pathogen diversification remain poorly explored although host immunity is recognized as a major driver of pathogen evolution. Influenza A viruses (IAVs) can infect many avian and mammalian hosts, but while few IAV subtypes circulate in human populations, subtype diversity is extensive in wild bird populations. How do these subtypes coexist in wild avian populations and do they compete within these natural host populations?Here we experimentally challenged mallard ducks with different IAVs to study how an initial infection with H3N8 determines the outcome of later infections (duration of infection and virus load) and antibody responses. There was complete protection to re-infection with the same H3N8 virus based on virus isolation. In addition, there was partial protection induced by H3N8 pre-challenge 3 to other subtypes and development of heterosubtypic immunity indicated by shorter infections and reduction in viral load compared to controls. This indicates that subtype dynamics in the host population are not independent. Amongst H3N8 pre-challenged groups, the highest protection was conferred to the H4N5 subtype which was most genetically related to H3N8. The H4N5challenge also induced an increase in H3 antibody levels and evidence for antigenic seniority.Thus, previous infections with IAV can influence the outcome of subsequent ...

Although waterfowl are the primary reservoir for multiple subtypes of influenza A virus (IAV), our understanding of population immunity in naturally infected waterfowl is poorly understood. Population immunity may be an important driver of seasonal subtype predominance in waterfowl populations and may affect the potential for establishment of introduced IAV such as the Eurasian-like A/Goose/Guangdong/1/1996 lineage in these populations. Here, we examine the prevalence of naturally acquired antibodies to nucleoprotein (NP), hemagglutinin (H3, H4, H5), and neuraminidase (N1, N2, N6, N8) in early migrating mallards (Anas platyrhynchos) sampled in Northwest Minnesota during staging and early fall migration in September 2014, 2015, 2017, and 2018. Serologic results were compared to historic and contemporary virus isolation results from these same study sites. The prevalence of antibodies to NP ranged from 60.8–76.1% in hatch-year (HY) birds and from 86.0–92.7% in after-hatch-year (AHY, >1-year-old) mallards indicating a high level of previous infection with IAV early in the fall migration season. Neutralizing antibodies were detected against H3, H4, and H5 in all years as were antibodies to N1, N2, N6, and N8. A high proportion of NP seropositive ducks tested positive for antibodies to multiple HA and NA subtypes, and this was more common in the AHY age class. Antibody prevalence to the HA and NA subtypes included in this study were consistent with the predominance of H4N6 in these populations during all years and reflected a broadening of the antibody response with age. Additional work is needed to document the longevity of these immune responses, if and how they correlate with protection against IAV transmission, infection, and disease, and if, as detected in this study, they adequately describe the true extent of exposure to IAV or specific HA or NA subtypes.