Antibodies to Influenza A(H5N1) Virus in Hunting Dogs Retrieving Wild Fowl, Washington, USA.

We detected antibodies to H5 and N1 subtype influenza A viruses in 4/194 (2%) dogs from Washington, USA, that hunted or engaged in hunt tests and training with wild birds. Historical data provided by dog owners showed seropositive dogs had high levels of exposure to waterfowl.

Field Research Is Essential to Counter Virological Threats.

The interface between humans and wildlife is changing and, with it, the potential for pathogen introduction into humans has increased. Avian influenza is a prominent example, with an ongoing outbreak showing the unprecedented expansion of both geographic and host ranges. Research in the field is essential to understand this and other zoonotic threats. Only by monitoring dynamic viral populations and defining their biology in situ can we gather the information needed to ensure effective pandemic preparation.

Maintenance and dissemination of avian-origin influenza A virus within the northern Atlantic Flyway of North America.

Wild waterbirds, the natural reservoirs for avian influenza viruses, undergo migratory movements each year, connecting breeding and wintering grounds within broad corridors known as flyways. In a continental or global view, the study of virus movements within and across flyways is important to understanding virus diversity, evolution, and movement. From 2015 to 2017, we sampled waterfowl from breeding (Maine) and wintering (Maryland) areas within the Atlantic Flyway (AF) along the east coast of North America to investigate the spatio-temporal trends in persistence and spread of influenza A viruses (IAV). We isolated 109 IAVs from 1,821 cloacal / oropharyngeal samples targeting mallards (Anas platyrhynchos) and American black ducks (Anas rubripes), two species having ecological and conservation importance in the flyway that are also host reservoirs of IAV. Isolates with >99% nucleotide similarity at all gene segments were found between eight pairs of birds in the northern site across years, indicating some degree of stability among genome constellations and the possibility of environmental persistence. No movement of whole genome constellations were identified between the two parts of the flyway, however, virus gene flow between the northern and southern study locations was evident. Examination of banding records indicate direct migratory waterfowl movements between the two locations within an annual season, providing a mechanism for the inferred viral gene flow. Bayesian phylogenetic analyses provided evidence for virus dissemination from other North American wild birds to AF dabbling ducks (Anatinae), shorebirds (Charidriformes), and poultry (Galliformes). Evidence was found for virus dissemination from shorebirds to gulls (Laridae), and dabbling ducks to shorebirds and poultry. The findings from this study contribute to the understanding of IAV ecology in waterfowl within the AF.

Putative Novel Avian Paramyxovirus (AMPV) and Reidentification of APMV-2 and APMV-6 to the Species Level Based on Wild Bird Surveillance (United States, 2016-2018).

Avian paramyxoviruses (APMVs) (subfamily Avulavirinae) have been isolated from over 200 species of wild and domestic birds around the world. The International Committee on Taxonomy of Viruses (ICTV) currently defines 22 different APMV species, with Avian orthoavulavirus 1 (whose viruses are designated APMV-1) being the most frequently studied due to its economic burden to the poultry industry. Less is known about other APMV species, including limited knowledge on the genetic diversity in wild birds, and there is a paucity of public whole-genome sequences for APMV-2 to -22. The goal of this study was to use MinION sequencing to genetically characterize APMVs isolated from wild bird swab samples collected during 2016 to 2018 in the United States. Multiplexed MinION libraries were prepared using a random strand-switching approach using 37 egg-cultured, influenza-negative, hemagglutination-positive samples. Forty-one APMVs were detected, with 37 APMVs having complete polymerase coding sequences allowing for species identification using ICTV's current Paramyxoviridae phylogenetic methodology. APMV-1, -4, -6, and -8 viruses were classified, one putative novel species (Avian orthoavulavirus 23) was identified from viruses isolated in this study, two putative new APMV species (Avian metaavulavirus 24 and 27) were identified from viruses isolated in this study and from retrospective GenBank sequences, and two putative new APMV species (Avian metaavulavirus 25 and 26) were identified solely from retrospective GenBank sequences. Furthermore, coinfections of APMVs were identified in four samples. The potential limitations of the branch length being the only species identification criterion and the potential benefit of a group pairwise distance analysis are discussed. IMPORTANCE Most species of APMVs are understudied and/or underreported, and many species were incidentally identified from asymptomatic wild birds; however, the disease significance of APMVs in wild birds is not fully determined. The rapid rise in high-throughput sequencing coupled with avian influenza surveillance programs have identified 12 different APMV species in the last decade and have challenged the resolution of classical serological methods to identify new viral species. Currently, ICTV's only criterion for Paramyxoviridae species classification is the requirement of a branch length of >0.03 using a phylogenetic tree constructed from polymerase (L) amino acid sequences. The results from this study identify one new APMV species, propose four additional new APMV species, and highlight that the criterion may have insufficient resolution for APMV species demarcation and that refinement or expansion of this criterion may need to be established for Paramyxoviridae species identification.

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.

Subtype Diversity of Influenza A Virus in North American Waterfowl: a Multidecade Study.

The discovery in 1976 of waterfowl as the primary reservoir of influenza A viruses (IAVs) has since spurred decades of waterfowl surveillance efforts by researchers dedicated to understanding the ecology of IAV and its subsequent threat to human and animal health. Here, we employed a multidecade, continental-scale approach of surveillance data to understand trends of seasonal IAV subtype diversity. Between 1976 and 2015, IAVs were detected in 8,427 (10.8%) of 77,969 samples from migratory waterfowl throughout the Central and Mississippi Migratory Flyways in the United States and Canada. A total of 96 hemagglutinin (HA)/neuraminidase (NA) subtype combinations were isolated, which included most HA (H1 to H14) and all 9 NA subtypes. We observed an annual trend of high influenza prevalence, involving a few dominant subtypes, on northern breeding grounds during summer with progressively lowered influenza prevalence, comprised of a highly diverse profile of subtypes, as waterfowl migrate toward southern wintering grounds. Isolates recovered during winter had the highest proportion of mixed and rare HA/NA combinations, indicating increased opportunity for reassortment of IAVs. In addition, 70% of H5 and 49% of H7 IAV isolates were recovered from samples collected during fall and spring, respectively; these are subtypes that can have significant implications for public health and agriculture sectors. Annual cyclical dominance of subtypes on northern breeding grounds is revealed through the longitudinal nature of this study. Our novel findings exhibit the unrealized potential for discovery using existing IAV surveillance data.IMPORTANCE Wild aquatic birds are the primary natural reservoir of influenza A viruses (IAVs) and are therefore responsible for the dispersal and maintenance of IAVs representing a broad range of antigenic and genetic diversity. The aims of IAV surveillance in waterfowl not only relate to understanding the risk of spillover risk to humans, but also to improving our understanding of basic questions related to IAV evolution and ecology. By evaluating several decades of surveillance data from wild aquatic birds sampled along North American migratory flyways, we discovered an annual trend of increasing subtype diversity during southbound migration, peaking on southern wintering grounds. Winter sampling revealed the highest proportion of mixed and rare infections that suggest higher opportunity for spillover. These findings allow improvements to surveillance efforts to robustly capture IAV diversity that will be used for vaccine development and cultivate a more thorough understanding of IAV evolution and persistence mechanisms.

Influenza A viruses remain infectious for more than seven months in northern wetlands of North America.

In this investigation, we used a combination of field- and laboratory-based approaches to assess if influenza A viruses (IAVs) shed by ducks could remain viable for extended periods in surface water within three wetland complexes of North America. In a field experiment, replicate filtered surface water samples inoculated with duck swabs were tested for IAVs upon collection and again after an overwintering period of approximately 6-7 months. Numerous IAVs were molecularly detected and isolated from these samples, including replicates maintained at wetland field sites in Alaska and Minnesota for 181-229 days. In a parallel laboratory experiment, we attempted to culture IAVs from filtered surface water samples inoculated with duck swabs from Minnesota each month during September 2018-April 2019 and found monthly declines in viral viability. In an experimental challenge study, we found that IAVs maintained in filtered surface water within wetlands of Alaska and Minnesota for 214 and 226 days, respectively, were infectious in a mallard model. Collectively, our results support surface waters of northern wetlands as a biologically important medium in which IAVs may be both transmitted and maintained, potentially serving as an environmental reservoir for infectious IAVs during the overwintering period of migratory birds.

The role of drought as a determinant of hemorrhagic disease in the eastern United States.

Bluetongue virus and epizootic hemorrhagic disease (HD) virus are globally distributed, vector-borne viruses that infect and cause disease in domestic and wild ruminant species. The forces driving increases in resulting HD may be linked to weather conditions and increasing severity has been noted in northerly latitudes. We evaluated the role of drought severity in both space and time on changes in HD reports across the eastern United States for a recent 15 year period. The objectives of this study were to: (a) develop a spatiotemporal model to evaluate if drought severity explains changing patterns of HD presence; and (b) determine whether this potential risk factor varies in importance over the present range of HD in the eastern United States. Historic data (2000-2014) from an annual HD presence-absence survey conducted by the Southeastern Cooperative Wildlife Disease Study and from the United States Drought Monitor were used for this analysis. For every county in 23 states and for each of 15 years, data were based on reported drought status for August, wetland cover, the physiographic region, and the status of HD in the previous year. We used a generalized linear mixed model to explain HD presence and evaluated spatiotemporal predictors across the region. We found that drought severity was a significant predictor of HD presence and the significance of this relationship was dependent on latitude. In more northerly latitudes, where immunological naivety is most likely, we demonstrated the increasing strength of drought severity as a determinant of reported HD and established the importance of variation in drought severity as a risk factor over the present range of HD in the eastern United States. Our research provides spatially explicit evidence for the link between climate forces and emerging disease patterns across latitude for a globally distributed disease.

Evaluation of 2012 US EHDV-2 outbreak isolates for genetic determinants of cattle infection.

Following a summer of severe drought and abnormally high temperatures, a major outbreak of EHDV occurred during 2012 in the USA. Although EHDV-1, -2 and -6 were isolated, EHDV-2 was the predominant virus serotype detected during the outbreak. In addition to large losses of white-tailed deer, the Midwest and northern Plains saw a significant amount of clinical disease in cattle. Phylogenetic analyses and sequence comparisons of newly sequenced whole genomes of 2012 EHDV-2 cattle isolates demonstrated that eight of ten EHDV-2 genomic segments show no genetic changes that separate the cattle outbreak sequences from other EHDV-2 isolates. Two segments, VP2 and VP6, did show several unique genetic changes specific to the 2012 cattle outbreak isolates, although the impact of the genetic changes on viral fitness is unknown. The placement of isolates from 2007 and 2011 as sister group to the outbreak isolates, and the similarity between cattle and deer isolates, point to environmental variables as having a greater influence on the severity of the 2012 EHDV outbreak than viral genetic changes.

Genetic Evidence Supports Sporadic and Independent Introductions of Subtype H5 Low-Pathogenic Avian Influenza A Viruses from Wild Birds to Domestic Poultry in North America.

Wild-bird origin influenza A viruses (IAVs or avian influenza) have led to sporadic outbreaks among domestic poultry in the United States and Canada, resulting in economic losses through the implementation of costly containment practices and destruction of birds. We used evolutionary analyses of virus sequence data to determine that 78 H5 low-pathogenic avian influenza viruses (LPAIVs) isolated from domestic poultry in the United States and Canada during 2001 to 2017 resulted from 18 independent virus introductions from wild birds. Within the wild-bird reservoir, the hemagglutinin gene segments of H5 LPAIVs exist primarily as two cocirculating genetic sublineages, and our findings suggest that the H5 gene segments flow within each migratory bird flyway and among adjacent flyways, with limited exchange between the nonadjacent Atlantic and Pacific Flyways. Phylogeographic analyses provided evidence that IAVs from dabbling ducks and swans/geese contributed to the emergence of viruses among domestic poultry. H5 LPAIVs isolated from commercial farm poultry (i.e., turkey) that were descended from a single introduction typically remained a single genotype, whereas those from live-bird markets sometimes led to multiple genotypes, reflecting the potential for reassortment with other IAVs circulating within live-bird markets. H5 LPAIVs introduced from wild birds to domestic poultry represent economic threats to the U.S. poultry industry, and our data suggest that such introductions have been sporadic, controlled effectively through production monitoring and a stamping-out policy, and are, therefore, unlikely to result in sustained detections in commercial poultry operations.IMPORTANCE Integration of viral genome sequencing into influenza surveillance for wild birds and domestic poultry can elucidate evolutionary pathways of economically costly poultry pathogens. Evolutionary analyses of H5 LPAIVs detected in domestic poultry in the United States and Canada during 2001 to 2017 suggest that these viruses originated from repeated introductions of IAVs from wild birds, followed by various degrees of reassortment. Reassortment was observed where biosecurity was low and where opportunities for more than one virus to circulate existed (e.g., congregations of birds from different premises, such as live-bird markets). None of the H5 lineages identified were maintained for the long term in domestic poultry, suggesting that management strategies have been effective in minimizing the impacts of virus introductions on U.S. poultry production.

Competition between influenza A virus subtypes through heterosubtypic immunity modulates re-infection and antibody dynamics in the mallard duck.

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.

The enigma of the apparent disappearance of Eurasian highly pathogenic H5 clade 2.3.4.4 influenza A viruses in North American waterfowl.

One of the major unresolved questions in influenza A virus (IAV) ecology is exemplified by the apparent disappearance of highly pathogenic (HP) H5N1, H5N2, and H5N8 (H5Nx) viruses containing the Eurasian hemagglutinin 2.3.4.4 clade from wild bird populations in North America. The introduction of Eurasian lineage HP H5 clade 2.3.4.4 H5N8 IAV and subsequent reassortment with low-pathogenic H?N2 and H?N1 North American wild bird-origin IAVs in late 2014 resulted in widespread HP H5Nx IAV infections and outbreaks in poultry and wild birds across two-thirds of North America starting in November 2014 and continuing through June 2015. Although the stamping out strategies adopted by the poultry industry and animal health authorities in Canada and the United States-which included culling, quarantining, increased biosecurity, and abstention from vaccine use-were successful in eradicating the HP H5Nx viruses from poultry, these activities do not explain the apparent disappearance of these viruses from migratory waterfowl. Here we examine current and historical aquatic bird IAV surveillance and outbreaks of HP H5Nx in poultry in the United States and Canada, providing additional evidence of unresolved mechanisms that restrict the emergence and perpetuation of HP avian influenza viruses in these natural reservoirs.

Introduction of Eurasian-Origin Influenza A(H8N4) Virus into North America by Migratory Birds.

We identified a Eurasian-origin influenza A(H8N4) virus in North America by sampling wild birds in western Alaska, USA. Evidence for repeated introductions of influenza A viruses into North America by migratory birds suggests that intercontinental dispersal might not be exceedingly rare and that our understanding of viral establishment is incomplete.

Potential for Low-Pathogenic Avian H7 Influenza A Viruses To Replicate and Cause Disease in a Mammalian Model.

H7 subtype influenza A viruses are widely distributed and have been responsible for human infections and numerous outbreaks in poultry with significant impact. Despite this, the disease-causing potential of the precursor low-pathogenic (LP) H7 viruses from the wild bird reservoir has not been investigated. Our objective was to assess the disease-causing potential of 30 LP H7 viruses isolated from wild avian species in the United States and Canada using the DBA/2J mouse model. Without prior mammalian adaptation, the majority of viruses, 27 (90%), caused mortality in mice. Of these, 17 (56.7%) caused 100% mortality and 24 were of pathogenicity similar to that of A/Anhui/1/2013 (H7N9), which is highly pathogenic in mice. Viruses of duck origin were more pathogenic than those of shorebird origin, as 13 of 18 (72.2%) duck origin viruses caused 100% mortality while 4 of 12 (33.3%) shorebird origin viruses caused 100% mortality, despite there being no difference in mean lung viral titers between the groups. Replication beyond the respiratory tract was also evident, particularly in the heart and brain. Of the 16 viruses studied for fecal shedding, 11 were detected in fecal samples. These viruses exhibited a strong preference for avian-type α2,3-linked sialic acids; however, binding to mammalian-type α2,6-linked sialic acids was also detected. These findings indicate that LP avian H7 influenza A viruses are able to infect and cause disease in mammals without prior adaptation and therefore pose a potential public health risk. IMPORTANCE: Low-pathogenic (LP) avian H7 influenza A viruses are widely distributed in the avian reservoir and are the precursors of numerous outbreaks of highly pathogenic avian influenza viruses in commercial poultry farms. However, unlike highly pathogenic H7 viruses, the disease-causing potential of LP H7 viruses from the wild bird reservoir has not been investigated. To address this, we studied 30 LP avian H7 viruses isolated from wild avian species in the United States and Canada using the DBA/2J mouse model. Surprisingly, the majority of these viruses, 90%, caused mortality in mice without prior mammalian adaptation, and 56.7% caused 100% mortality. There was also evidence of spread beyond the respiratory tract and fecal shedding. Therefore, the disease-causing potential of LP avian H7 influenza A viruses in mammals may be underestimated, and these viruses therefore pose a potential public health risk.

Low-Pathogenic Influenza A Viruses in North American Diving Ducks Contribute to the Emergence of a Novel Highly Pathogenic Influenza A(H7N8) Virus.

Introductions of low-pathogenic avian influenza (LPAI) viruses of subtypes H5 and H7 into poultry from wild birds have the potential to mutate to highly pathogenic avian influenza (HPAI) viruses, but such viruses' origins are often unclear. In January 2016, a novel H7N8 HPAI virus caused an outbreak in turkeys in Indiana, USA. To determine the virus's origin, we sequenced the genomes of 441 wild-bird origin influenza A viruses (IAVs) from North America and subjected them to evolutionary analyses. The results showed that the H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Preceding the outbreak, an isolate with six gene segments (PB2, PB1, PA, HA, NA, and NS) sharing >99% sequence identity with those of H7N8 turkey isolates was recovered from a diving duck sampled in Kentucky, USA. H4N8 IAVs from other diving ducks possessed five H7N8-like gene segments (PB2, PB1, NA, MP, and NS; >98% sequence identity). Our findings suggest that viral gene constellations circulating among diving ducks can contribute to the emergence of IAVs that affect poultry. Therefore, diving ducks may serve an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild-bird reservoir.IMPORTANCE In January 2016, a novel H7N8 HPAI virus caused a disease outbreak in turkeys in Indiana, USA. To determine the origin of this virus, we sequenced and analyzed 441 wild-bird origin influenza virus strains isolated from wild birds inhabiting North America. We found that the H7N8 LPAI virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkeys, in which it evolved high pathogenicity. Our results suggest that viral gene constellations circulating among diving ducks can contribute to the emergence of IAVs that affect poultry. Therefore, diving ducks may play an important and understudied role in the maintenance, diversification, and transmission of IAVs in the wild-bird reservoir. Our study also highlights the importance of a coordinated, systematic, and collaborative surveillance for IAVs in both poultry and wild-bird populations.

Pathogenicity and Transmission of H5 and H7 Highly Pathogenic Avian Influenza Viruses in Mallards.

Wild aquatic birds have been associated with the intercontinental spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the A/goose/Guangdong/1/96 (Gs/GD) lineage during 2005, 2010, and 2014, but dispersion by wild waterfowl has not been implicated with spread of other HPAI viruses. To better understand why Gs/GD H5 HPAI viruses infect and transmit more efficiently in waterfowl than other HPAI viruses, groups of mallard ducks were challenged with one of 14 different H5 and H7 HPAI viruses, including a Gs/GD lineage H5N1 (clade 2.2) virus from Mongolia, part of the 2005 dispersion, and the H5N8 and H5N2 index HPAI viruses (clade 2.3.4.4) from the United States, part of the 2014 dispersion. All virus-inoculated ducks and contact exposed ducks became infected and shed moderate to high titers of the viruses, with the exception that mallards were resistant to Ck/Pennsylvania/83 and Ck/Queretaro/95 H5N2 HPAI virus infection. Clinical signs were only observed in ducks challenged with the H5N1 2005 virus, which all died, and with the H5N8 and H5N2 2014 viruses, which had decreased weight gain and fever. These three viruses were also shed in higher titers by the ducks, which could facilitate virus transmission and spread. This study highlights the possible role of wild waterfowl in the spread of HPAI viruses. IMPORTANCE: The spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the Gs/GD lineage by migratory waterfowl is a serious concern for animal and public health. H5 and H7 HPAI viruses are considered to be adapted to gallinaceous species (chickens, turkeys, quail, etc.) and less likely to infect and transmit in wild ducks. In order to understand why this is different with certain Gs/GD lineage H5 HPAI viruses, we compared the pathogenicity and transmission of several H5 and H7 HPAI viruses from previous poultry outbreaks to Gs/GD lineage H5 viruses, including H5N1 (clade 2.2), H5N8 and H5N2 (clade 2.3.4.4) viruses, in mallards as a representative wild duck species. Surprisingly, most HPAI viruses examined in this study replicated well and transmitted among mallards; however, the three Gs/GD lineage H5 HPAI viruses replicated to higher titers, which could explain the transmission of these viruses in susceptible wild duck populations.

Influenza A virus on oceanic islands: host and viral diversity in seabirds in the Western Indian Ocean.

Ducks and seabirds are natural hosts for influenza A viruses (IAV). On oceanic islands, the ecology of IAV could be affected by the relative diversity, abundance and density of seabirds and ducks. Seabirds are the most abundant and widespread avifauna in the Western Indian Ocean and, in this region, oceanic islands represent major breeding sites for a large diversity of potential IAV host species. Based on serological assays, we assessed the host range of IAV and the virus subtype diversity in terns of the islands of the Western Indian Ocean. We further investigated the spatial variation in virus transmission patterns between islands and identified the origin of circulating viruses using a molecular approach. Our findings indicate that terns represent a major host for IAV on oceanic islands, not only for seabird-related virus subtypes such as H16, but also for those commonly isolated in wild and domestic ducks (H3, H6, H9, H12 subtypes). We also identified strong species-associated variation in virus exposure that may be associated to differences in the ecology and behaviour of terns. We discuss the role of tern migrations in the spread of viruses to and between oceanic islands, in particular for the H2 and H9 IAV subtypes.

Assessment of contemporary genetic diversity and inter-taxa/inter-region exchange of avian paramyxovirus serotype 1 in wild birds sampled in North America.

BACKGROUND: Avian paramyxovirus serotype 1 (APMV-1) viruses are globally distributed, infect wild, peridomestic, and domestic birds, and sometimes lead to outbreaks of disease. Thus, the maintenance, evolution, and spread of APMV-1 viruses are relevant to avian health. METHODS: In this study we sequenced the fusion gene from 58 APMV-1 isolates recovered from thirteen species of wild birds sampled throughout the USA during 2007-2014. We analyzed sequence information with previously reported data in order to assess contemporary genetic diversity and inter-taxa/inter-region exchange of APMV-1 in wild birds sampled in North America. RESULTS: Our results suggest that wild birds maintain previously undescribed genetic diversity of APMV-1; however, such diversity is unlikely to be pathogenic to domestic poultry. Phylogenetic analyses revealed that APMV-1 diversity detected in wild birds of North America has been found in birds belonging to numerous taxonomic host orders and within hosts inhabiting multiple geographic regions suggesting some level of viral exchange. However, our results also provide statistical support for associations between phylogenetic tree topology and host taxonomic order/region of sample origin which supports restricted exchange among taxa and geographical regions of North America for some APMV-1 sub-genotypes. CONCLUSIONS: We identify previously unrecognized genetic diversity of APMV-1 in wild birds in North America which is likely a function of continued viral evolution in reservoir hosts. We did not, however, find support for the emergence or maintenance of APMV-1 strains predicted to be pathogenic to poultry in wild birds of North America outside of the order Suliformes (i.e., cormorants). Furthermore, genetic evidence suggests that ecological drivers or other mechanisms may restrict viral exchange among taxa and regions of North America. Additional and more systematic sampling for APMV-1 in North America would likely provide further inference on viral dynamics for this infectious agent in wild bird populations.

Adaptive evolution and environmental durability jointly structure phylodynamic patterns in avian influenza viruses.

Avian influenza viruses (AIVs) have been pivotal to the origination of human pandemic strains. Despite their scientific and public health significance, however, there remains much to be understood about the ecology and evolution of AIVs in wild birds, where major pools of genetic diversity are generated and maintained. Here, we present comparative phylodynamic analyses of human and AIVs in North America, demonstrating (i) significantly higher standing genetic diversity and (ii) phylogenetic trees with a weaker signature of immune escape in AIVs than in human viruses. To explain these differences, we performed statistical analyses to quantify the relative contribution of several potential explanations. We found that HA genetic diversity in avian viruses is determined by a combination of factors, predominantly subtype-specific differences in host immune selective pressure and the ecology of transmission (in particular, the durability of subtypes in aquatic environments). Extending this analysis using a computational model demonstrated that virus durability may lead to long-term, indirect chains of transmission that, when coupled with a short host lifespan, can generate and maintain the observed high levels of genetic diversity. Further evidence in support of this novel finding was found by demonstrating an association between subtype-specific environmental durability and predicted phylogenetic signatures: genetic diversity, variation in phylogenetic tree branch lengths, and tree height. The conclusion that environmental transmission plays an important role in the evolutionary biology of avian influenza viruses-a manifestation of the "storage effect"-highlights the potentially unpredictable impact of wildlife reservoirs for future human pandemics and the need for improved understanding of the natural ecology of these viruses.

Surveillance for Eurasian-origin and intercontinental reassortant highly pathogenic influenza A viruses in Alaska, spring and 2015.

BACKGROUND: Eurasian-origin and intercontinental reassortant highly pathogenic (HP) influenza A viruses (IAVs) were first detected in North America in wild, captive, and domestic birds during November-December 2014. Detections of HP viruses in wild birds in the contiguous United States and southern Canadian provinces continued into winter and spring of 2015 raising concerns that migratory birds could potentially disperse viruses to more northerly breeding areas where they could be maintained to eventually seed future poultry outbreaks. RESULTS: We sampled 1,129 wild birds on the Yukon-Kuskokwim Delta, Alaska, one of the largest breeding areas for waterfowl in North America, during spring and summer of 2015 to test for Eurasian lineage and intercontinental reassortant HP H5 IAVs and potential progeny viruses. We did not detect HP IAVs in our sample collection from western Alaska; however, we isolated five low pathogenic (LP) viruses. Four isolates were of the H6N1 (n = 2), H6N2, and H9N2 combined subtypes whereas the fifth isolate was a mixed infection that included H3 and N7 gene segments. Genetic characterization of these five LP IAVs isolated from cackling (Branta hutchinsii; n = 2) and greater white-fronted geese (Anser albifrons; n = 3), revealed three viral gene segments sharing high nucleotide identity with HP H5 viruses recently detected in North America. Additionally, one of the five isolates was comprised of multiple Eurasian lineage gene segments. CONCLUSIONS: Our results did not provide direct evidence for circulation of HP IAVs in the Yukon-Kuskokwim Delta region of Alaska during spring and summer of 2015. Prevalence and genetic characteristics of LP IAVs during the sampling period are concordant with previous findings of relatively low viral prevalence in geese during spring, non-detection of IAVs in geese during summer, and evidence for intercontinental exchange of viruses in western Alaska.