Serological Survey of Retrovirus and Coronavirus Infections, including SARS-CoV-2, in Rural Stray Cats in The Netherlands, 2020-2022.

Stray cats can host (zoonotic) viral pathogens and act as a source of infection for domestic cats or humans. In this cross-sectional (sero)prevalence study, sera from 580 stray cats living in 56 different cat groups in rural areas in The Netherlands were collected from October 2020 to July 2022. These were used to investigate the prevalence of the cat-specific feline leukemia virus (FeLV, n = 580), the seroprevalence of the cat-specific feline viruses feline immunodeficiency virus (FIV, n = 580) and feline coronavirus (FCoV, n = 407), and the zoonotic virus severe acute respiratory coronavirus-2 (SARS-CoV-2, n = 407) using enzyme-linked immunosorbent assays (ELISAs). ELISA-positive results were confirmed using Western blot (FIV) or pseudovirus neutralization test (SARS-CoV-2). The FIV seroprevalence was 5.0% (95% CI (Confidence Interval) 3.4-7.1) and ranged from 0-19.0% among groups. FIV-specific antibodies were more often detected in male cats, cats ≥ 3 years and cats with reported health problems. No FeLV-positive cats were found (95% CI 0.0-0.6). The FCoV seroprevalence was 33.7% (95% CI 29.1-38.5) and ranged from 4.7-85.7% among groups. FCoV-specific antibodies were more often detected in cats ≥ 3 years, cats with reported health problems and cats living in industrial areas or countryside residences compared to cats living at holiday parks or campsites. SARS-CoV-2 antibodies against the subunit 1 (S1) and receptor binding domain (RBD) protein were detected in 2.7% (95% CI 1.4-4.8) of stray cats, but sera were negative in the pseudovirus neutralization test and therefore were considered SARS-CoV-2 suspected. Our findings suggest that rural stray cats in The Netherlands can be a source of FIV and FCoV, indicating a potential risk for transmission to other cats, while the risk for FeLV is low. However, suspected SARS-CoV-2 infections in these cats were uncommon. We found no evidence of SARS-CoV-2 cat-to-cat spread in the studied stray cat groups and consider the likelihood of spillover to humans as low.

Highly Pathogenic Avian Influenza Viruses at the Wild-Domestic Bird Interface in Europe: Future Directions for Research and Surveillance.

Highly pathogenic avian influenza (HPAI) outbreaks in wild birds and poultry are no longer a rare phenomenon in Europe. In the past 15 years, HPAI outbreaks-in particular those caused by H5 viruses derived from the A/Goose/Guangdong/1/1996 lineage that emerged in southeast Asia in 1996-have been occuring with increasing frequency in Europe. Between 2005 and 2020, at least ten HPAI H5 incursions were identified in Europe resulting in mass mortalities among poultry and wild birds. Until 2009, the HPAI H5 virus outbreaks in Europe were caused by HPAI H5N1 clade 2.2 viruses, while from 2014 onwards HPAI H5 clade 2.3.4.4 viruses dominated outbreaks, with abundant genetic reassortments yielding subtypes H5N1, H5N2, H5N3, H5N4, H5N5, H5N6 and H5N8. The majority of HPAI H5 virus detections in wild and domestic birds within Europe coincide with southwest/westward fall migration and large local waterbird aggregations during wintering. In this review we provide an overview of HPAI H5 virus epidemiology, ecology and evolution at the interface between poultry and wild birds based on 15 years of avian influenza virus surveillance in Europe, and assess future directions for HPAI virus research and surveillance, including the integration of whole genome sequencing, host identification and avian ecology into risk-based surveillance and analyses.

Host Range of Influenza A Virus H1 to H16 in Eurasian Ducks Based on Tissue and Receptor Binding Studies.

Dabbling and diving ducks partly occupy shared habitats but have been reported to play different roles in wildlife infectious disease dynamics. Influenza A virus (IAV) epidemiology in wild birds has been based primarily on surveillance programs focused on dabbling duck species, particularly mallard (Anas platyrhynchos). Surveillance in Eurasia has shown that in mallards, some subtypes are commonly (H1 to H7 and H10), intermediately (H8, H9, H11, and H12), or rarely (H13 to H16) detected, contributing to discussions on virus host range and reservoir competence. An alternative to surveillance in determining IAV host range is to study virus attachment as a determinant for infection. Here, we investigated the attachment patterns of all avian IAV subtypes (H1 to H16) to the respiratory and intestinal tracts of four dabbling duck species (Mareca and Anas spp.), two diving duck species (Aythya spp.), and chicken, as well as to a panel of 65 synthetic glycan structures. We found that IAV subtypes generally showed abundant attachment to colon of the Anas duck species, mallard, and Eurasian teal (Anas crecca), supporting the fecal-oral transmission route in these species. The reported glycan attachment profile did not explain the virus attachment patterns to tissues but showed significant attachment of duck-originated viruses to fucosylated glycan structures and H7 virus tropism for Neu5Gc-LN. Our results suggest that Anas ducks play an important role in the ecology and epidemiology of IAV. Further knowledge on virus tissue attachment, receptor distribution, and receptor binding specificity is necessary to understand the mechanisms underlying host range and epidemiology of IAV.IMPORTANCE Influenza A viruses (IAVs) circulate in wild birds worldwide. From wild birds, the viruses can cause outbreaks in poultry and sporadically and indirectly infect humans. A high IAV diversity has been found in mallards (Anas platyrhynchos), which are most often sampled as part of surveillance programs; meanwhile, little is known about the role of other duck species in IAV ecology and epidemiology. In this study, we investigated the attachment of all avian IAV hemagglutinin (HA) subtypes (H1 to H16) to tissues of six different duck species and chicken as an indicator of virus host range. We demonstrated that the observed virus attachment patterns partially explained reported field prevalence. This study demonstrates that dabbling ducks of the Anas genus are potential hosts for most IAV subtypes, including those infecting poultry. This knowledge is useful to target the sampling of wild birds in nature and to further study the interaction between IAVs and birds.

A Comparative Study of the Innate Humoral Immune Response to Avian Influenza Virus in Wild and Domestic Mallards.

Domestic mallards (Anas platyrhynchos domesticus) are traditionally used as a model to investigate infection dynamics and immune responses to low pathogenic avian influenza viruses (LPAIVs) in free-living mallards. However, it is unclear whether the immune response of domestic birds reflects the response of their free-living counterparts naturally exposed to these viruses. We investigated the extent to which the innate humoral immune response was similar among (i) wild-type domestic mallards in primary and secondary infection with LPAIV H4N6 in a laboratory setting (laboratory mallards), (ii) wild-type domestic mallards naturally exposed to LPAIVs in a semi-natural setting (sentinel mallards), and (iii) free-living mallards naturally exposed to LPAIVs. We quantified innate humoral immune function by measuring non-specific natural antibodies (agglutination), complement activity (lysis), and the acute phase protein haptoglobin. We demonstrate that complement activity in the first 3 days after LPAIV exposure was higher in primary-exposed laboratory mallards than in sentinel and free-living mallards. LPAIV H4N6 likely activated the complement system and the acute phase response in primary-exposed laboratory mallards, as lysis was higher and haptoglobin lower at day 3 and 7 post-exposure compared to baseline immune function measured prior to exposure. There were no differences observed in natural antibody and haptoglobin concentrations among laboratory, sentinel, and free-living mallards in the first 3 days after LPAIV exposure. Our study demonstrates that, based on the three innate humoral immune parameters measured, domestic mallards seem an appropriate model to investigate innate immunology of their free-living counterparts, albeit the innate immune response of secondary-LPAIV exposed mallards is a better proxy for the innate immune response in pre-exposed free-living mallards than that of immunologically naïve mallards.

Phylogeography and Antigenic Diversity of Low-Pathogenic Avian Influenza H13 and H16 Viruses.

Low-pathogenic avian influenza viruses (LPAIVs) are genetically highly variable and have diversified into multiple evolutionary lineages that are primarily associated with wild-bird reservoirs. Antigenic variation has been described for mammalian influenza viruses and for highly pathogenic avian influenza viruses that circulate in poultry, but much less is known about antigenic variation of LPAIVs. In this study, we focused on H13 and H16 LPAIVs that circulate globally in gulls. We investigated the evolutionary history and intercontinental gene flow based on the hemagglutinin (HA) gene and used representative viruses from genetically distinct lineages to determine their antigenic properties by hemagglutination inhibition assays. For H13, at least three distinct genetic clades were evident, while for H16, at least two distinct genetic clades were evident. Twenty and ten events of intercontinental gene flow were identified for H13 and H16 viruses, respectively. At least two antigenic variants of H13 and at least one antigenic variant of H16 were identified. Amino acid positions in the HA protein that may be involved in the antigenic variation were inferred, and some of the positions were located near the receptor binding site of the HA protein, as they are in the HA protein of mammalian influenza A viruses. These findings suggest independent circulation of H13 and H16 subtypes in gull populations, as antigenic patterns do not overlap, and they contribute to the understanding of the genetic and antigenic variation of LPAIVs naturally circulating in wild birds.IMPORTANCE Wild birds play a major role in the epidemiology of low-pathogenic avian influenza viruses (LPAIVs), which are occasionally transmitted-directly or indirectly-from them to other species, including domestic animals, wild mammals, and humans, where they can cause subclinical to fatal disease. Despite a multitude of genetic studies, the antigenic variation of LPAIVs in wild birds is poorly understood. Here, we investigated the evolutionary history, intercontinental gene flow, and antigenic variation among H13 and H16 LPAIVs. The circulation of subtypes H13 and H16 seems to be maintained by a narrower host range, in particular gulls, than the majority of LPAIV subtypes and may therefore serve as a model for evolution and epidemiology of H1 to H12 LPAIVs in wild birds. The findings suggest that H13 and H16 LPAIVs circulate independently of each other and emphasize the need to investigate within-clade antigenic variation of LPAIVs in wild birds.

Global patterns of avian influenza A (H7): virus evolution and zoonotic threats.

Avian influenza viruses (AIVs) continue to impose a negative impact on animal and human health worldwide. In particular, the emergence of highly pathogenic AIV H5 and, more recently, the emergence of low pathogenic AIV H7N9 have led to enormous socioeconomical losses in the poultry industry and resulted in fatal human infections. While H5N1 remains infamous, the number of zoonotic infections with H7N9 has far surpassed those attributed to H5. Despite the clear public health concerns posed by AIV H7, it is unclear why specifically this virus subtype became endemic in poultry and emerged in humans. In this review, we bring together data on global patterns of H7 circulation, evolution and emergence in humans. Specifically, we discuss data from the wild bird reservoir, expansion and epidemiology in poultry, significant increase in their zoonotic potential since 2013 and genesis of highly pathogenic H7. In addition, we analysed available sequence data from an evolutionary perspective, demonstrating patterns of introductions into distinct geographic regions and reassortment dynamics. The integration of all aspects is crucial in the optimisation of surveillance efforts in wild birds, poultry and humans, and we emphasise the need for a One Health approach in controlling emerging viruses such as AIV H7.

Avian influenza viruses at the wild-domestic bird interface in Egypt.

Wild birds of the orders Anseriformes (mainly ducks, geese and swans) and Charadriiformes (mainly gulls, terns and waders) constitute the natural reservoir for low pathogenic avian influenza (LPAI) viruses. In Egypt, highly pathogenic avian influenza (HPAI) H5N1 and LPAI H9N2 viruses are endemic in domestic poultry, forming a threat to animal and human health and raising questions about the routes of introduction and mechanisms of persistence. Recently, HPAI H5N8 virus was also introduced into Egyptian domestic birds. Here we review the literature on the role of wild birds in the introduction and endemicity of avian influenza viruses in Egypt. Dabbling ducks in Egypt harbor an extensive LPAI virus diversity and may constitute the route of introduction for HPAI H5N1 and HPAI H5N8 viruses into Egypt through migration, however their role in the endemicity of HPAI H5N1, LPAI H9N2 and potentially other avian influenza virus (AIV) strains - by means of reassortment of viral genes - is less clear. Strengthened surveillance programs, in both domestic and wild birds, that include all LPAI virus subtypes and full genome sequencing are needed to better assess the wild-domestic bird interface and form a basis for evidence-based measures to limit and prevent AIV transmission between wild and domestic birds.

Wild ducks excrete highly pathogenic avian influenza virus H5N8 (2014-2015) without clinical or pathological evidence of disease.

Highly pathogenic avian influenza (HPAI) is essentially a poultry disease. Wild birds have traditionally not been involved in its spread, but the epidemiology of HPAI has changed in recent years. After its emergence in southeastern Asia in 1996, H5 HPAI virus of the Goose/Guangdong lineage has evolved into several sub-lineages, some of which have spread over thousands of kilometers via long-distance migration of wild waterbirds. In order to determine whether the virus is adapting to wild waterbirds, we experimentally inoculated the HPAI H5N8 virus clade 2.3.4.4 group A from 2014 into four key waterbird species-Eurasian wigeon (Anas penelope), common teal (Anas crecca), mallard (Anas platyrhynchos), and common pochard (Aythya ferina)-and compared virus excretion and disease severity with historical data of the HPAI H5N1 virus infection from 2005 in the same four species. Our results showed that excretion was highest in Eurasian wigeons for the 2014 virus, whereas excretion was highest in common pochards and mallards for the 2005 virus. The 2014 virus infection was subclinical in all four waterbird species, while the 2005 virus caused clinical disease and pathological changes in over 50% of the common pochards. In chickens, the 2014 virus infection caused systemic disease and high mortality, similar to the 2005 virus. In conclusion, the evidence was strongest for Eurasian wigeons as long-distance vectors for HPAI H5N8 virus from 2014. The implications of the switch in species-specific virus excretion and decreased disease severity may be that the HPAI H5 virus more easily spreads in the wild-waterbird population.

Host and virus ecology as determinants of influenza A virus transmission in wild birds.

Low pathogenic influenza A virus (LPIAV) prevalence and subtype distribution differs between and across bird taxa. A crucial factor in the epidemiology of these viruses and virus subtypes is the ability to transmit between and within different host taxa and individuals. Successful viral transmission depends on availability of susceptible hosts and exposure of host to virus. Exposure to viruses and susceptibility to virus infection and/or disease are shaped by both host and virus traits. In this review we have identified key host and virus traits that can affect LPIAV transmission, both in terms of exposure and susceptibility. Furthermore we highlight current challenges in assessment of these traits and identify methodological considerations for future studies.

No evidence that migratory geese disperse avian influenza viruses from breeding to wintering ground.

Low pathogenic avian influenza virus can mutate to a highly pathogenic strain that causes severe clinical signs in birds and humans. Migratory waterfowl, especially ducks, are considered the main hosts of low pathogenic avian influenza virus, but the role of geese in dispersing the virus over long-distances is still unclear. We collected throat and cloaca samples from three goose species, Bean goose (Anser fabalis), Barnacle goose (Branta leucopsis) and Greater white-fronted goose (Anser albifrons), from their breeding grounds, spring stopover sites, and wintering grounds. We tested if the geese were infected with low pathogenic avian influenza virus outside of their wintering grounds, and analysed the spatial and temporal patterns of infection prevalence on their wintering grounds. Our results show that geese were not infected before their arrival on wintering grounds. Barnacle geese and Greater white-fronted geese had low prevalence of infection just after their arrival on wintering grounds in the Netherlands, but the prevalence increased in successive months, and peaked after December. This suggests that migratory geese are exposed to the virus after their arrival on wintering grounds, indicating that migratory geese might not disperse low pathogenic avian influenza virus during autumn migration.

Discordant detection of avian influenza virus subtypes in time and space between poultry and wild birds; Towards improvement of surveillance programs.

Avian influenza viruses from wild birds can cause outbreaks in poultry, and occasionally infect humans upon exposure to infected poultry. Identification and characterization of viral reservoirs and transmission routes is important to develop strategies that prevent infection of poultry, and subsequently virus transmission between poultry holdings and to humans. Based on spatial, temporal and phylogenetic analyses of data generated as part of intense and large-scale influenza surveillance programs in wild birds and poultry in the Netherlands from 2006 to 2011, we demonstrate that LPAIV subtype distribution differed between wild birds and poultry, suggestive of host-range restrictions. LPAIV isolated from Dutch poultry were genetically most closely related to LPAIV isolated from wild birds in the Netherlands or occasionally elsewhere in Western Europe. However, a relatively long time interval was observed between the isolations of related viruses from wild birds and poultry. Spatial analyses provided evidence for mallards (Anas platyrhynchos) being more abundant near primary infected poultry farms. Detailed year-round investigation of virus prevalence and wild bird species distribution and behavior near poultry farms should be used to improve risk assessment in relation to avian influenza virus introduction and retarget avian influenza surveillance programs.

Lack of virological and serological evidence for continued circulation of highly pathogenic avian influenza H5N8 virus in wild birds in the Netherlands, 14 November 2014 to 31 January 2016.

In 2014, H5N8 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/Guangdong/1/1996 lineage emerged in poultry and wild birds in Asia, Europe and North America. Here, wild birds were extensively investigated in the Netherlands for HPAI H5N8 virus (real-time polymerase chain reaction targeting the matrix and H5 gene) and antibody detection (haemagglutination inhibition and virus neutralisation assays) before, during and after the first virus detection in Europe in late 2014. Between 21 February 2015 and 31 January 2016, 7,337 bird samples were tested for the virus. One HPAI H5N8 virus-infected Eurasian wigeon (Anas penelope) sampled on 25 February 2015 was detected. Serological assays were performed on 1,443 samples, including 149 collected between 2007 and 2013, 945 between 14 November 2014 and 13 May 2015, and 349 between 1 September and 31 December 2015. Antibodies specific for HPAI H5 clade 2.3.4.4 were absent in wild bird sera obtained before 2014 and present in sera collected during and after the HPAI H5N8 emergence in Europe, with antibody incidence declining after the 2014/15 winter. Our results indicate that the HPAI H5N8 virus has not continued to circulate extensively in wild bird populations since the 2014/15 winter and that independent maintenance of the virus in these populations appears unlikely.

Long-Term Effect of Serial Infections with H13 and H16 Low-Pathogenic Avian Influenza Viruses in Black-Headed Gulls.

UNLABELLED: Infections of domestic and wild birds with low-pathogenic avian influenza viruses (LPAIVs) have been associated with protective immunity to subsequent infection. However, the degree and duration of immunity in wild birds from previous LPAIV infection, by the same or a different subtype, are poorly understood. Therefore, we inoculated H13N2 (A/black-headed gull/Netherlands/7/2009) and H16N3 (A/black-headed gull/Netherlands/26/2009) LPAIVs into black-headed gulls (Chroicocephalus ridibundus), their natural host species, and measured the long-term immune response and protection against one or two reinfections over a period of >1 year. This is the typical interval between LPAIV epizootics in wild birds. Reinfection with the same virus resulted in progressively less virus excretion, with complete abrogation of virus excretion after two infections for H13 but not H16. However, reinfection with the other virus affected neither the level nor duration of virus excretion. Virus excretion by immunologically naive birds did not differ in total levels of excreted H13 or H16 virus between first- and second-year birds, but the duration of H13 excretion was shorter for second-year birds. Furthermore, serum antibody levels did not correlate with protection against LPAIV infection. LPAIV-infected gulls showed no clinical signs of disease. These results imply that the epidemiological cycles of H13 and H16 in black-headed gulls are relatively independent from each other and depend mainly on infection of first-year birds. IMPORTANCE: Low-pathogenic avian influenza viruses (LPAIVs) circulate mainly in wild water birds but are occasionally transmitted to other species, including humans, where they cause subclinical to fatal disease. To date, the effect of LPAIV-specific immunity on the epidemiology of LPAIV in wild birds is poorly understood. In this study, we investigated the effect of H13 and H16 LPAIV infection in black-headed gulls on susceptibility and virus excretion of subsequent infection with the same or the other virus within the same breeding season and between breeding seasons. These are the only two LPAIV hemagglutinin subtypes predominating in this species. The findings suggest that H13 and H16 LPAIV cycles in black-headed gull populations are independent of each other, indicate the importance of first-year birds in LPAIV epidemiology, and emphasize the need for alternatives to avian influenza virus (AIV)-specific serum antibodies as evidence of past LPAIV infection and correlates of protection against LPAIV infection in wild birds.

One health, multiple challenges: The inter-species transmission of influenza A virus.

Influenza A viruses are amongst the most challenging viruses that threaten both human and animal health. Influenza A viruses are unique in many ways. Firstly, they are unique in the diversity of host species that they infect. This includes waterfowl (the original reservoir), terrestrial and aquatic poultry, swine, humans, horses, dog, cats, whales, seals and several other mammalian species. Secondly, they are unique in their capacity to evolve and adapt, following crossing the species barrier, in order to replicate and spread to other individuals within the new species. Finally, they are unique in the frequency of inter-species transmission events that occur. Indeed, the consequences of novel influenza virus strain in an immunologically naïve population can be devastating. The problems that influenza A viruses present for human and animal health are numerous. For example, influenza A viruses in humans represent a major economic and disease burden, whilst the poultry industry has suffered colossal damage due to repeated outbreaks of highly pathogenic avian influenza viruses. This review aims to provide a comprehensive overview of influenza A viruses by shedding light on interspecies virus transmission and summarising the current knowledge regarding how influenza viruses can adapt to a new host.

Influenza A virus evolution and spatio-temporal dynamics in Eurasian wild birds: a phylogenetic and phylogeographical study of whole-genome sequence data.

Low pathogenic avian influenza A viruses (IAVs) have a natural host reservoir in wild waterbirds and the potential to spread to other host species. Here, we investigated the evolutionary, spatial and temporal dynamics of avian IAVs in Eurasian wild birds. We used whole-genome sequences collected as part of an intensive long-term Eurasian wild bird surveillance study, and combined this genetic data with temporal and spatial information to explore the virus evolutionary dynamics. Frequent reassortment and co-circulating lineages were observed for all eight genomic RNA segments over time. There was no apparent species-specific effect on the diversity of the avian IAVs. There was a spatial and temporal relationship between the Eurasian sequences and significant viral migration of avian IAVs from West Eurasia towards Central Eurasia. The observed viral migration patterns differed between segments. Furthermore, we discuss the challenges faced when analysing these surveillance and sequence data, and the caveats to be borne in mind when drawing conclusions from the apparent results of such analyses.

Migratory birds reinforce local circulation of avian influenza viruses.

Migratory and resident hosts have been hypothesized to fulfil distinct roles in infectious disease dynamics. However, the contribution of resident and migratory hosts to wildlife infectious disease epidemiology, including that of low pathogenic avian influenza virus (LPAIV) in wild birds, has largely remained unstudied. During an autumn H3 LPAIV epizootic in free-living mallards (Anas platyrhynchos) - a partially migratory species - we identified resident and migratory host populations using stable hydrogen isotope analysis of flight feathers. We investigated the role of migratory and resident hosts separately in the introduction and maintenance of H3 LPAIV during the epizootic. To test this we analysed (i) H3 virus kinship, (ii) temporal patterns in H3 virus prevalence and shedding and (iii) H3-specific antibody prevalence in relation to host migratory strategy. We demonstrate that the H3 LPAIV strain causing the epizootic most likely originated from a single introduction, followed by local clonal expansion. The H3 LPAIV strain was genetically unrelated to H3 LPAIV detected both before and after the epizootic at the study site. During the LPAIV epizootic, migratory mallards were more often infected with H3 LPAIV than residents. Low titres of H3-specific antibodies were detected in only a few residents and migrants. Our results suggest that in this LPAIV epizootic, a single H3 virus was present in resident mallards prior to arrival of migratory mallards followed by a period of virus amplification, importantly associated with the influx of migratory mallards. Thus migrants are suggested to act as local amplifiers rather than the often suggested role as vectors importing novel strains from afar. Our study exemplifies that a multifaceted interdisciplinary approach offers promising opportunities to elucidate the role of migratory and resident hosts in infectious disease dynamics in wildlife.

Epidemiology of influenza A virus among black-headed gulls, the Netherlands, 2006-2010.

We sampled 7,511 black-headed gulls for influenza virus in the Netherlands during 2006-2010 and found that subtypes H13 and H16 caused annual epidemics in fledglings on colony sites. Our findings validate targeted surveillance of wild waterbirds and clarify underlying factors for influenza virus emergence in other species.

Juveniles and migrants as drivers for seasonal epizootics of avian influenza virus.

Similar to other infectious diseases, the prevalence of low pathogenic avian influenza viruses (LPAIV) has been seen to exhibit marked seasonal variation. However, mechanisms driving this variation in wild birds have yet to be tested. We investigated the validity of three previously suggested drivers for the seasonal dynamics in LPAIV infections in wild birds: (i) host density, (ii) immunologically naïve young and (iii) increased susceptibility in migrants. To address these questions, we sampled a key LPAIV host species, the mallard Anas platyrhynchos, on a small spatial scale, comprehensively throughout a complete annual cycle, measuring both current and past infection (i.e. viral and seroprevalence, respectively). We demonstrate a minor peak in LPAIV prevalence in summer, a dominant peak in autumn, during which half of the sampled population was infected, and no infections in spring. Seroprevalence of antibodies to a conserved gene segment of avian influenza virus (AIV) peaked in winter and again in spring. The summer peak of LPAIV prevalence coincided with the entrance of unfledged naïve young in the population. Moreover, juveniles were more likely to be infected, shed higher quantities of virus and were less likely to have detectable antibodies to AIV than adult birds. The arrival of migratory birds, as identified by stable hydrogen isotope analysis, appeared to drive the autumn peak in LPAIV infection, with both temporal coincidence and higher infection prevalence in migrants. Remarkably, seroprevalence in migrants was substantially lower than viral prevalence throughout autumn migration, further indicating that each wave of migrants amplified local AIV circulation. Finally, while host abundance increased throughout autumn, it peaked in winter, showing no direct correspondence with either of the LPAIV infection peaks. At an epidemiologically relevant spatial scale, we provide strong evidence for the role of migratory birds as key drivers for seasonal epizootics of LPAIV, regardless of their role as vectors of these viruses. This study exemplifies the importance of understanding host demography and migratory behaviour when examining seasonal drivers of infection in wildlife populations.