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.

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.

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.

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.

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.

Multi-locus reassessment of a striking discord between mtDNA gene trees and taxonomy across two congeneric species complexes.

Resolving relationships among members of the yellow and citrine wagtail species complexes is among the greatest challenges in avian systematics due to arguably the most dramatic disagreements between traditional taxonomy and mtDNA phylogeny. Each species complex is divided into three geographically cohesive mtDNA clades. Each clade from one species complex has a sister from the other complex. Furthermore, one cross-complex pair is more distantly related to the remaining two pairs than are several other wagtail species. To test mtDNA gene tree topology, we sequenced the mtDNA ND2 gene and 11 nuclear introns for seven wagtail species. Our mtDNA gene tree reconstruction supported the results of previous studies, thereby confirming the disagreement between mtDNA phylogeny and taxonomy. However, our multi-locus species tree which used mtDNA clades as "taxa" was consistent with traditional taxonomy regardless of whether mtDNA was included in the analysis or not. Our multi-locus data suggest that despite the presence of strongly supported, geographically structured mtDNA variation, the mtDNA gene tree misrepresents the evolutionary history of the yellow and citrine wagtail complexes. This mito-nuclear discord results from mtDNA representing the biogeographic, but not evolutionary history of these recently radiated Palearctic wagtails.

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.

Evidence for wild waterfowl origin of H7N3 influenza A virus detected in captive-reared New Jersey pheasants.

In August 2014, a low-pathogenic H7N3 influenza A virus was isolated from pheasants at a New Jersey gamebird farm and hunting preserve. In this study, we use phylogenetic analyses and calculations of genetic similarity to gain inference into the genetic ancestry of this virus and to identify potential routes of transmission. Results of maximum-likelihood (ML) and maximum-clade-credibility (MCC) phylogenetic analyses provide evidence that A/pheasant/New Jersey/26996-2/2014 (H7N3) had closely related H7 hemagglutinin (HA) and N3 neuraminidase (NA) gene segments as compared to influenza A viruses circulating among wild waterfowl in the central and eastern USA. The estimated time of the most recent common ancestry (TMRCA) between the pheasant virus and those most closely related from wild waterfowl was early 2013 for both the H7 HA and N3 NA gene segments. None of the viruses from waterfowl identified as being most closely related to A/pheasant/New Jersey/26996-2/2014 at the HA and NA gene segments in ML and MCC phylogenetic analyses shared ≥99 % nucleotide sequence identity for internal gene segment sequences. This result indicates that specific viral strains identified in this study as being closely related to the HA and NA gene segments of A/pheasant/New Jersey/26996-2/2014 were not the direct predecessors of the etiological agent identified during the New Jersey outbreak. However, the recent common ancestry of the H7 and N3 gene segments of waterfowl-origin viruses and the virus isolated from pheasants suggests that viral diversity maintained in wild waterfowl likely played an important role in the emergence of A/pheasant/New Jersey/26996-2/2014.

Infectivity of Wild-Bird Origin Influenza A Viruses in Minnesota Wetlands across Seasons.

The environmental tenacity of influenza A viruses (IAVs) in the environment likely plays a role in their transmission; IAVs are able to remain infectious in aquatic habitats and may have the capacity to seed outbreaks when susceptible wild bird hosts utilize these same environments months or even seasons later. Here, we aimed to assess the persistence of low-pathogenicity IAVs from naturally infected ducks in Northwestern Minnesota through a field experiment. Viral infectivity was measured using replicate samples maintained in distilled water in a laboratory setting as well as in filtered water from four natural water bodies maintained in steel perforated drums (hereafter, mesocosms) within the field from autumn 2020 to spring 2021. There was limited evidence for the extended persistence of IAVs held in mesocosms; from 65 initial IAV-positive samples, only six IAVs persisted to at least 202 days in the mesocosms compared to 17 viruses persisting at least this long when held under temperature-controlled laboratory settings in distilled water. When accounting for the initial titer of samples, viruses detected at a higher concentration at the initiation of the experiment persisted longer than those with a lower starting titer. A parallel experimental laboratory model was used to further explore the effects of water type on viral persistence, and the results supported the finding of reduced tenacity of IAVs held in mesocosms compared to distilled water. The results of this investigation provide evidence that many factors, including temperature and physicochemical properties, impact the duration of viral infectivity in natural settings, further extending our understanding of the potential and limitations of environmental-based methodologies to recover infectious IAVs.

Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread.

Avian paramyxovirus serotype 1 (APMV-1), or Newcastle disease virus, is the causative agent of Newcastle disease, one of the most economically important diseases for poultry production worldwide and a cause of periodic epizootics in wild birds in North America. In this study, we examined the genetic diversity of APMV-1 isolated from migratory birds sampled in Alaska, Japan, and Russia and assessed the evidence for intercontinental virus spread using phylogenetic methods. Additionally, we predicted viral virulence using deduced amino acid residues for the fusion protein cleavage site and estimated mutation rates for the fusion gene of class I and class II migratory bird isolates. All 73 isolates sequenced as part of this study were most closely related to virus genotypes previously reported for wild birds; however, five class II genotype I isolates formed a monophyletic clade exhibiting previously unreported genetic diversity, which met criteria for the designation of a new sub-genotype. Phylogenetic analysis of wild-bird isolates provided evidence for intercontinental virus spread, specifically viral lineages of APMV-1 class II genotype I sub-genotypes Ib and Ic. This result supports migratory bird movement as a possible mechanism for the redistribution of APMV-1. None of the predicted deduced amino acid motifs for the fusion protein cleavage site of APMV-1 strains isolated from migratory birds in Alaska, Japan, and Russia were consistent with those of previously identified virulent viruses. These data therefore provide no support for these strains contributing to the emergence of avian pathogens. The estimated mutation rates for fusion genes of class I and class II wild-bird isolates were faster than those reported previously for non-virulent APMV-1 strains. Collectively, these findings provide new insight into the diversity, spread, and evolution of APMV-1 in wild birds.

Evidence for interannual persistence of infectious influenza A viruses in Alaska wetlands.

Influenza A viruses (IAVs) deposited by wild birds into the environment may lead to sporadic mortality events and economically costly outbreaks among domestic birds. There is a paucity of information, however, regarding the persistence of infectious IAVs within the environment following deposition. In this investigation, we assessed the persistence of 12 IAVs that were present in cloacal and/or oropharyngeal swabs of naturally infected ducks. Infectivity of these IAVs was monitored over approximately one year with each virus tested in five water types: (1) distilled water held in the lab at 4 °C and (2-5) filtered surface water from each of four Alaska sites and maintained in the field at ambient temperature. By evaluating infectivity of IAVs in ovo following sample retrieval at four successive time points, we observed declines in IAV infectivity through time. Many viruses persisted for extended periods, as evidenced by ≥25% of IAVs remaining infectious in replicate samples for each treatment type through three sampling time points (144-155 days post-sample collection) and two viruses remaining viable in a single replicate sample each when tested upon collection at a fourth time point (361-377 days post-sample collection). The estimated probability of persistence of infectious IAVs in all five water types was estimated to be between 0.25 and 0.75 during days 50-200 post-sample collection as inferred through Kaplan-Meier survival analysis. Our results provide evidence that IAVs may remain infectious for extended periods, up to or even exceeding one year, when maintained in surface waters under ambient temperatures. Therefore, wetlands may represent an important medium in which infectious IAVs may reside outside of a biotic reservoir.

Interspecific exchange of avian influenza virus genes in Alaska: the influence of trans-hemispheric migratory tendency and breeding ground sympatry.

The movement and transmission of avian influenza viral strains via wild migratory birds may vary by host species as a result of migratory tendency and sympatry with other infected individuals. To examine the roles of host migratory tendency and species sympatry on the movement of Eurasian low-pathogenic avian influenza (LPAI) genes into North America, we characterized migratory patterns and LPAI viral genomic variation in mallards (Anas platyrhynchos) of Alaska in comparison with LPAI diversity of northern pintails (Anas acuta). A 50-year band-recovery data set suggests that unlike northern pintails, mallards rarely make trans-hemispheric migrations between Alaska and Eurasia. Concordantly, fewer (14.5%) of 62 LPAI isolates from mallards contained Eurasian gene segments compared to those from 97 northern pintails (35%), a species with greater inter-continental migratory tendency. Aerial survey and banding data suggest that mallards and northern pintails are largely sympatric throughout Alaska during the breeding season, promoting opportunities for interspecific transmission. Comparisons of full-genome isolates confirmed near-complete genetic homology (>99.5%) of seven viruses between mallards and northern pintails. This study found viral segments of Eurasian lineage at a higher frequency in mallards than previous studies, suggesting transmission from other avian species migrating inter-hemispherically or the common occurrence of endemic Alaskan viruses containing segments of Eurasian origin. We conclude that mallards are unlikely to transfer Asian-origin viruses directly to North America via Alaska but that they are likely infected with Asian-origin viruses via interspecific transfer from species with regular migrations to the Eastern Hemisphere.

Evidence for limited exchange of avian influenza viruses between seaducks and dabbling ducks at Alaska Peninsula coastal lagoons.

Avian influenza virus (AIV) prevalence and sequence data were analyzed for Steller's eiders (Polysticta stelleri) to assess the role of this species in transporting virus genes between continents and maintaining a regional viral reservoir with sympatric northern pintails (Anas acuta). AIV prevalence was 0.2% at Izembek Lagoon and 3.9% at Nelson Lagoon for Steller's eiders and 11.2% for northern pintails at Izembek Lagoon. Phylogenetic analysis of 13 AIVs from Steller's eiders revealed that 4.9% of genes were of Eurasian origin. Seven subtypes were detected, including two also observed in northern pintails. No AIV strains were highly similar (> 99%) at all gene segments between species; however, highly similar individual genes were detected. The proportion of highly similar genes was greater within rather than between species. Steller's eiders likely transport AIV genes between continents through long-distance migratory movements. Differences in AIV prevalence, subtype distribution, and the proportion of highly similar genes suggest limited AIV exchange between Steller's eiders and northern pintails at Alaska Peninsula coastal lagoons during autumn.

Coding-Complete Genome Sequence of Avian orthoavulavirus 16, Isolated from Emperor Goose (Anser canagicus) Feces, Alaska, USA.

We sequenced the coding-complete genome of an avian orthoavulavirus serotype 16 (AOAV-16) isolate recovered from emperor goose (Anser canagicus) feces collected in Alaska. The detection of AOAV-16 in North America and genomic sequencing of the resultant isolate confirms that the geographic distribution of this virus extends beyond Asia.

Evidence for continental-scale dispersal of antimicrobial resistant bacteria by landfill-foraging gulls.

Anthropogenic inputs into the environment may serve as sources of antimicrobial resistant bacteria and alter the ecology and population dynamics of synanthropic wild animals by providing supplemental forage. In this study, we used a combination of phenotypic and genomic approaches to characterize antimicrobial resistant indicator bacteria, animal telemetry to describe host movement patterns, and a novel modeling approach to combine information from these diverse data streams to investigate the acquisition and long-distance dispersal of antimicrobial resistant bacteria by landfill-foraging gulls. Our results provide evidence that gulls acquire antimicrobial resistant bacteria from anthropogenic sources, which they may subsequently disperse across and between continents via migratory movements. Furthermore, we introduce a flexible modeling framework to estimate the relative dispersal risk of antimicrobial resistant bacteria in western North America and adjacent areas within East Asia, which may be adapted to provide information on the risk of dissemination of other organisms and pathogens maintained by wildlife through space and time.

Ecology of Influenza A Viruses in Wild Birds and Wetlands of Alaska.

Alaska represents a globally important region for the ecology of avian-origin influenza A viruses (IAVs) given the expansive wetlands in this region, which serve as habitat for numerous hosts of IAVs that disperse among four continents during the annual cycle. Extensive sampling of wild birds for IAVs in Alaska since 1991 has greatly extended inference regarding intercontinental viral exchange between North America and East Asia and the importance of Beringian endemic species to IAV ecology within this region. Data on IAVs in aquatic birds inhabiting Alaska have also been useful for helping to establish global patterns of prevalence in wild birds and viral dispersal across the landscape. In this review, we summarize the main findings from investigations of IAVs in wild birds and wetlands of Alaska with the aim of providing readers with an understanding of viral ecology within this region. More specifically, we review viral detections, evidence of IAV exposure, and genetic characterization of isolates derived from wild bird samples collected in Alaska by host taxonomy. Additionally, we provide a short overview of wetland complexes within Alaska that may be important to IAV ecology at the continental scale.

Estudio recapitulativo - Ecología de los virus de la influenza A en aves silvestres y humedales de Alaska. Alaska representa una región de importancia mundial para la ecología de los virus de la influenza A de origen aviar (IAV) dados los humedales expansivos en esta región, que sirven como hábitat para numerosos huéspedes de los virus de influenza A que se dispersan entre los cuatro continentes durante el ciclo anual. El extenso muestreo de aves silvestres para virus de influenza en Alaska desde 1991 ha extendido enormemente la inferencia sobre el intercambio viral intercontinental entre América del Norte y Asia Oriental y la importancia de las especies endémicas de la zona Beringia para la ecología de los virus de influenza aviar dentro de esta región. Los datos sobre los virus de influenza aviar en aves acuáticas que habitan Alaska también han sido útiles para ayudar a establecer patrones globales de prevalencia en aves silvestres y dispersión viral a través del ecosistema. En esta revisión, se resumen los principales hallazgos de las investigaciones acerca de los virus de influenza en aves silvestres y humedales de Alaska con el objetivo de proporcionar a los lectores una comprensión de la ecología viral en esta región. Más específicamente, se revisan las detecciones virales, la evidencia de exposición a virus de influenza aviar y la caracterización genética de los aislamientos derivados de muestras de aves silvestres recolectadas en Alaska por la taxonomía del huésped. Además, se ofrece una breve descripción de los complejos de humedales dentro de Alaska que pueden ser importantes para la ecología de los virus de influenza en escala continental.

The Genome Sequence of an H6N5 Influenza A Virus Strain Isolated from a Northern Pintail (Anas acuta) Sampled in Alaska, USA, Shares High Identity with That of a South Korean Wild Bird Strain.

We report the genome sequence of an H6N5 influenza A virus isolated from a northern pintail sampled in Alaska in 2017. All segment sequences shared >99% nucleotide identity with those of a wild bird strain from South Korea. This finding supports viral dispersal between East Asia and North America by wild birds.

Field-based method for assessing duration of infectivity for influenza A viruses in the environment.

Understanding influenza A virus (IAV) persistence in wetlands is limited by a paucity of field studies relating to the maintenance of infectivity over time. The duration of IAV infectivity in water has been assessed under variable laboratory conditions, but results are difficult to translate to more complex field conditions. We tested a field-based method to assess the viability of IAVs in an Alaska wetland during fall and winter which incorporated physical and chemical properties of the waterbody in which samples were held. Filtered pond water was inoculated with avian fecal samples collected from the environment, aliquoted into a series of duplicate sealed vials and submerged back in the wetland for up to 132 days (October 2018-March 2019). Sample aliquots were sequentially recovered and tested for IAVs by rRT-PCR and virus isolation. One sample remained rRT-PCR positive for the duration of the study and virus isolation positive for 118 days. The surrounding water temperature was 1°-6 °C with near neutral pH (6.6-7.3) for the duration of the study. This proof of concept study demonstrates a protocol for testing the persistence of infectious IAV naturally shed from waterfowl under ambient environmental conditions.

Emperor geese (Anser canagicus) are exposed to a diversity of influenza A viruses, are infected during the non-breeding period and contribute to intercontinental viral dispersal.

Emperor geese (Anser canagicus) are endemic to coastal areas within Beringia and have previously been found to have antibodies to or to be infected with influenza A viruses (IAVs) in Alaska. In this study, we use virological, serological and tracking data to further elucidate the role of emperor geese in the ecology of IAVs in Beringia during the non-breeding period. Specifically, we assess evidence for: (a) active IAV infection during spring staging, autumn staging and wintering periods; (b) infection with novel Eurasian-origin or interhemispheric reassortant viruses; (c) contemporary movement of geese between East Asia and North America; (d) previous exposure to viruses of 14 haemagglutinin subtypes, including Eurasian lineage highly pathogenic (HP) H5 IAVs; and (e) subtype-specific antibody seroconversion and seroreversion. Emperor geese were found to shed IAVs, including interhemispheric reassortant viruses, throughout the non-breeding period; migrate between Alaska and the Russian Far East prior to and following remigial moult; have antibodies reactive to a diversity of IAVs including, in a few instances, Eurasian lineage HP H5 IAVs; and exhibit relatively broad and stable patterns of population immunity among breeding females. Results of this study suggest that emperor geese may play an important role in the maintenance and dispersal of IAVs within Beringia during the non-breeding period and provide information that may be used to further optimize surveillance activities focused on the early detection of Eurasian-origin IAVs in North America.

Influenza A virus recovery, diversity, and intercontinental exchange: A multi-year assessment of wild bird sampling at Izembek National Wildlife Refuge, Alaska.

Western Alaska is a potential point-of-entry for foreign-origin influenza A viruses (IAVs) into North America via migratory birds. We sampled waterfowl and gulls for IAVs at Izembek National Wildlife Refuge (NWR) in western Alaska, USA, during late summer and autumn months of 2011-2015, to evaluate the abundance and diversity of viruses at this site. We collected 4842 samples across five years from 25 species of wild birds resulting in the recovery, isolation, and sequencing of 172 IAVs. With the intent of optimizing sampling efficiencies, we used information derived from this multi-year effort to: 1) evaluate from which species we consistently recover viruses, 2) describe viral subtypes of isolates by host species and year, 3) characterize viral gene segment sequence diversity with respect to host species, and assess potential differences in the viral lineages among the host groups, and 4) examine how evidence of intercontinental exchange of IAVs relates to host species. We consistently recovered viruses from dabbling ducks (Anas spp.), emperor geese (Chen canagica) and glaucous-winged gulls (Larus glaucescens). There was little evidence for differences in viral subtypes and diversity from different waterfowl hosts, however subtypes and viral diversity varied between waterfowl host groups and glaucous-winged gulls. Furthermore, higher proportions of viral sequences from northern pintails (Anas acuta), emperor geese and glaucous-winged gulls were grouped in phylogenetic clades that included IAV sequences originating from wild birds sampled in Asia as compared to non-pintail dabbling ducks, a difference that may be related to intercontinental migratory tendencies of host species. Our summary of research and surveillance efforts at Izembek NWR will assist in future prioritization of which hosts to sample and swab types to collect in Alaska and elsewhere in order to maximize isolate recovery, subtype and sequence diversity for resultant viruses, and detection of evidence for intercontinental viral exchange.