Understanding the ecologic interactions among wild birds that facilitate the maintenance, reassortment, and dispersal of influenza A viruses (IAV) is key to forecasting global spread (Hall et al., 2014; Hill et al., 2017; Hoye et al., 2011; Lang et al., 2016; The Global Consortium for H5N8 and Related Influenza Viruses, 2016). IAVs circulate endemically among wild aquatic migratory bird populations, includingAnseriformes (waterfowl, including ducks, geese, and swans) and Charadriiformes (seabirds, including gulls, shorebirds, terns, and auks), many of which migrate between breeding ranges in arctic and subarctic regions (ASRs) to wintering grounds in southern latitudes (Dusek et al., 2014; G. A. Gudmundsson, 1993; L. A. Gudmundsson, 1992; Hall et al., 2014; Hall et al., 2015; Hall et al., 2013; Hénaux, Parmley, Soos, & Samuel, 2013; Lang et al., 2016; Runstadler, Hill, Hussein, Puryear, & Keogh, 2013; van Dijk et al., 2014). During migration, birds use staging and breeding grounds within the circumpolar perimeter of the arctic circle, where populations from different geographic origins aggregate at high densities, enabling direct and environmental fecal-oral transmission of IAVs within and between species (Causey & Edwards, 2008; R. Chen & Holmes, 2009; Hill et al., 2016; Ito et al., 1995). The mass migration of birds to ASRs each spring is considered an important mechanism for inter-species transmission and global spread of IAVs, although empirical data are often lacking from these remote, high latitude regions where human populations are sparse (Causey & Edwards, 2008; Hoye, Munster, Nishiura, Klaassen, & Fouchier, 2010; Olsen et al., 2006).

Previous studies support that migratory connectivity throughout ASRs dictate the ecology and evolution of IAVs between hemispheres (Webster, Marra, Haig, Bensch, & Holmes, 2002). Northern latitudes are predicted to have a high risk of IAV outbreaks among wild birds due to the increased density of disparate bird populations during spring migration and the persistence of IAVs in low temperature climate regimes (Herrick, Huettmann, & Lindgren, 2013; A. M. Ramey et al., 2020; Runstadler et al., 2013). Host movement patterns within ASRs also determine how viruses disperse within the arctic circumpolar region and onward transport to southern latitudes globally. During post-breeding migration, wild birds such as shorebirds, gulls, skuas, and terns prioritize short distance flights around the perimeter of the greater polar arctic circle to avoid navigation challenges (Alerstam et al., 2007), exemplifying the ecologic importance of stopover and staging locations for migratory seabirds within ASRs. This great-circle orientation of seabird migration through ASRs is likely to have important but currently under-studied impacts on the directionality and rate of movement of IAVs in space and time across the globe. ASRs are also disproportionately affected by global climate change, where observed and predicted annual mean warming is more than twice the global mean (Arctic Monitoring and Assessment Programme (AMAP), 2019). Alterations to climate regimes may impact the distribution of wild birds and IAVs throughout the circumpolar north due to geographic shifts in habitat use and trophic food webs, changes in timing of migrations and breeding range locations, and declines in reproductive success (Mckinney et al., 2015). Increased surveillance in northern latitudes will enable researchers to gather baseline data with which to measure future shifts in IAV dynamics because of the rapidly changing arctic climate (Gilbert, Slingenbergh, & Xiao, 2008; Jensen et al., 2008).

The migratory connectivity of ASRs with the rest of the globe is particularly relevant given the expanding geographic range of highly pathogenic avian influenza (HPAI) virus subtype H5Nx of clade 2.3.4.4 which was first detected outside of China in 2014 (Antigua, Choi, Baek, & Song, 2019; Lee, Bertran, Kwon, & Swayne, 2017). HPAI clade 2.3.4.4 viruses have evolved by reassorting with low pathogenic viruses throughout Eurasia and North America, and evidence of intercontinental dissemination events through ASRs have been documented (Hill et al., 2017; Lee et al., 2017). In 2014, HPAI H5N8 of clade 2.3.4.4 was introduced by wild migratory birds from East Asia to North America via the trans-Beringian route through Alaska, from which H5N2 and H5N1 reassortant viruses emerged (Hill et al., 2017; Lee et al., 2015; A. Ramey et al., 2016). H5N2 reassortant viruses ultimately spread among domestic poultry throughout 15 states in the US, which precipitated the culling of approximately 49 million chickens and turkeys (Hill et al., 2017; Lee et al., 2016). Future introduction events are anticipated in view of the large number of clade 2.3.4.4 outbreaks in areas adjacent to North America, although the exact route of introduction remains elusive (Ramey et al., 2021).

Transcontinental virus exchange is often detected in oceanic and coastal regions at continental margins along migratory flyways (Alerstam et al., 2007; Huang et al., 2014; Pearce et al., 2009). In the Pacific Rim, Alaska has emerged as an ecologically important ‘hot spot’ through which IAV carrier species migrate, connecting East Asia and North America for virus exchange (Hill et al., 2017; Huang et al., 2014; Lee et al., 2015; A. Ramey et al., 2015; A. Ramey et al., 2016). Though significant westward virus migration from Asia to Europe has been documented, far less is known about the onward viral connectivity of mainland Europe with staging locations within the North Atlantic and North America (Kilpatrick et al., 2006; Olsen et al., 2006). Addressing how IAV dynamics may differ between the North Atlantic and Pacific coastal regions relative to global patterns of viral flow is critical for developing a well-rounded understanding of influenza ecology. The current knowledge gap highlights the potential for undetected virus spread between Europe and North America, especially for HPAI clade 2.3.4.4 and other highly consequential influenza viruses.

Iceland - at the confluence of the North Atlantic and Arctic oceans – provides a unique study system for characterizing inter-hemispheric viral flow between eastern and western hemispheres. The island is situated within the East Atlantic flyway between Europe and North America where resident and long-distance migratory birds interact (Butler, 2012; Dusek et al., 2014; Hall et al., 2014; Hall et al., 2015; Newton, 2008). Viruses of North American and Eurasian ancestral lineages, as well as reassortant strains between these lineages, have been isolated in Iceland, demonstrating the North Atlantic as a potential corridor for the inter-hemispheric movement of IAVs (Dusek et al., 2014). Previous research has described (a) high seroprevalence of IAVs among North Atlantic sea ducks (Hall et al., 2015) and (b) genes from HPAI virus lineages in low pathogenic IAVs among migrant birds in Iceland, revealing the importance of surveillance in the North Atlantic for viruses with pandemic potential in humans (Hall et al., 2014). There is very little evidence, however, describing the directionality of virus movement and avian host transmission dynamics within Iceland and throughout the North Atlantic region, which may influence the spread of IAV globally (Dusek et al., 2014; Hall et al., 2014; Hall et al., 2015; Kilpatrick et al., 2006; The Global Consortium for H5N8 and Related Influenza Viruses, 2016).

We hypothesize that IAVs are transported via wild bird migration between Europe and North America through Iceland and that Iceland plays an important role in the inter-hemispheric transmission and reassortment of IAVs in the North Atlantic, particularly with virus moving into North America. This study examines (i) viral connectivity of Iceland to other global regions, (ii) inter-species transmission dynamics among wild aquatic birds in this system, (iii) viral diffusion by wild aquatic birds within Iceland compared to other global regions, and (iv) whether local reassortment patterns may result in novel or HPAI reassortants with the potential for inter-hemispheric transport, warranting public health concern. These data reveal patterns of inter-hemispheric virus movement and inform surveillance efforts related to seasonal and emergent IAVs, in northern latitudes, particularly throughout the North Atlantic region.

Materials and Methods Field sample collection

From May 2010 through February 2018, we obtained IAV isolates from various species of seabirds, shorebirds, and waterfowl as well as environmental sampling of avian fecal material from locations throughout Iceland (Dusek et al., 2022). Live sampled birds were captured using a 18m x 12m cannon-propelled capture net, noose pole, or hand capture. Birds found dead or moribund were also sampled. Hunter-harvested waterfowl and fisheries-bycatch seabirds were sampled as available. All birds were identified to species and, for live birds, the majority were individually marked with metal bands. Age characteristics were determined and age was documented for each bird according to the following schemes adapted from U.S. Geological Survey (USGS) year classification codes: hatched in same calendar year as sampling (1CY), hatched previous calendar year (2CY), hatched previous calendar year or older exact age unknown (2CY+; i.e. at least 2CY but could be any age older then 2CY as well), hatched three calendar years prior to sampling (3CY), hatched four calendar years prior to sampling (4CY), hatched more than four calendar years prior to sampling (4CY+), or unknown if age could not be determined (U) (Olsen KM, 2004; Prater, Marchant, & Vuorinen, 1977; USGS, 2020). Due to species specific differences, not all aging categories could be applied to all species sampled. All live birds were immediately released following completion of sampling.

To sample for IAV, a single polyester-tipped swab was used to swab the cloaca only (2010-2013) or to first swab the oral cavity then the cloaca (2014-2017). Opportunistic environmental sampling of fecal material was also conducted using a direct swabbing method (2018). Each swab sample was immediately placed in individual cryovials containing 1.25 ml viral transport media (Docherty & Slota, 1988). Vials were held on ice for up to 5 hours prior to being stored in liquid nitrogen or liquid nitrogen vapor. Samples were shipped on dry ice from Iceland to Madison, Wisconsin, USA by private courier with dry ice replenishment during shipping. Once received in the laboratory, samples were stored at -80^o C until analysis.