Influenza subtype reassortment
Reassortment primarily occurred among a limited number of subtypes, despite high subtype diversity in Iceland, including detection of 10 of 16 HA and eight of nine NA avian subtypes over an eight-year period. Phylodynamic analyses of HA subtype reassortment within Iceland demonstrated significant reassortment among donor H16 subtypes to recipient H2 subtypes (BF= 870.62), followed by H3 to H10 (BF= 21.35) and H11 (BF= 42.93), and H2 to H10 (BF= 19.08). Reassortment from donor H3 subtypes to recipient H10 and H11 subtypes was only detected in the Iceland-focused analysis and not detected in global analyses (Figure 5a and 5b). HA subtypes were largely host-specific, and HA reassortment patterns reflected inter-species transmission patterns described above (Figure 5c). IAV NA subtype reassortment in Iceland included N8 to N7 (BF= 6.97), N8 to N9 (BF= 337.71), N5 to N7 (BF= 3.47), and N3 to N7 (BF= 1358.31). Donor N5 subtypes to recipient N7 subtypes was the only reassortment pattern in Iceland that was not recapitulated in global analyses. H1 and N8 subtypes predominate across the global reassortment landscape as sources to almost all HA and NA subtypes (Figure 5). No low pathogenic to HPAI HA reassortment patterns were detected among Iceland isolates.
Discussion Our study is among the first to quantify the migratory connectivity of Iceland within the global context and the transmission dynamics that govern the spread of IAVs within Iceland and between adjacent geographic regions. Using Bayesian phylogeographic and phylodynamic inference, enabling the analysis of viral evolution simultaneously with ecological and population-level traits (Kühnert, Wu, & Drummond, 2011; Pybus & Rambaut, 2009), we add insights into inter-hemispheric virus movement through, and inter-species transmission dynamics within, the North Atlantic subarctic, and Iceland specifically, a region that has largely been neglected in global phylogenetic analyses of IAVs (Hall et al., 2015; Herrick et al., 2013; The Global Consortium for H5N8 and Related Influenza Viruses, 2016).
Our findings reveal that 18% and five percent of global IAV transitions between regional states occur between a) mainland Europe and Iceland and b) North America and Iceland, respectively (Figure 2b). This demonstrates the importance of geographic proximity for inter-hemispheric movement of IAVs through the North Atlantic. This almost-fourfold difference is likely due to Iceland’s geographical position along the East Atlantic flyway, which links western Africa and Europe and extends into Greenland and northeastern Canada via wild bird migration (Dusek et al., 2014). Additionally, previous research on avian migration in ASRs has suggested that birds avoid long-distance movements across the central Arctic Ocean and favor short distance flights along the great-circle orientation of the circumpolar north (Alerstam et al., 2007). Our findings further support this migration phenotype in the North Atlantic subarctic corridor, especially for the migratory transport of IAVs from mainland Europe to North America through intermediate staging locations in Iceland. Given the highly significant flow of IAVs from Asia to Europe, and mainland Europe to Iceland, many viruses are likely moving from Eurasia into Iceland along the east-west continuum into North America. This directional movement implies a higher risk than has been previously demonstrated for the flow of HPAI clade 2.3.4.4 viruses westward from Eurasia into North America.
Iceland serves as a highly transitional geography for migratory birds, converse to the North American system where birds tend to circulate for relatively longer periods of time (Supplementary table 3a). IAVs circulate much longer in North America (as compared with most other global regions aside from Asia) before transitioning to other global regions based on our Markov rewards analysis by global region (Supplementary table 3a). Long-term or endemic circulation in North America may be due to higher viral fitness relative to Eurasian origin clades (Bahl, Vijaykrishna, Holmes, Smith, & Guan, 2009), or the geographic size of the continent, which may encompass the entire annual cycle for many species of birds (Hill et al., 2017). North Atlantic intraregional phylogeographic analyses demonstrate that northeastern Canada and Greenland are the most significant sink regions for IAVs following state transitions from Iceland (Figure 2). These data describe significant viral flow between Iceland and proximal regions in North America. Our findings reveal and quantify the inter-hemispheric movement of IAVs between mainland Europe and North America and the role that Iceland serves as an ecological ‘land bridge’ for wild birds and IAVs in space and time. The vast migratory range of gulls in the North Atlantic and their common routing through Iceland, suggests these avian hosts as particularly important for the inter-hemispheric transport of IAVs, though further research is necessary to describe the migratory connectivity of northern regions (Figure 3, Supplementary figure 7).
Iceland provides a staging ground for many migratory birds between mainland Europe and proximal regions, and therefore inter-species interactions during stopovers likely increase IAV transmission (Dusek et al., 2014). The importance of subarctic regions as sources of IAVs to southward latitudes was demonstrated through our discrete phylogeographic intracontinental analyses which not only determined Iceland as a strongly supported source to northeastern Canada, but also revealed a similarly strong relationship between Alaska and the western United States (Figure 1b, Supplementary Table 4). Alaska has been previously described as a significant stopover region for wild birds and thus a site for IAV incursion between Asia and North America (Hill et al., 2017; Wilson et al., 2013; Winker et al., 2007). Iceland and Alaska, therefore, serve as east-to-west and west-to-east incursion points for IAVs through northern latitudes into North America, respectively. Given that both Iceland and Alaska stopover locations serve as summer breeding grounds and inter-hemispheric bridges for long-distance migratory birds, increased surveillance in ASRs along migratory flyways is imperative to increase knowledge of IAV incursion into southward latitudes (Hill et al., 2017; Winker et al., 2007). Connections between Iceland and Alaska, however, based on our analyses are uncommon.
While Anseriformes drive transmission dynamics globally, host transmission phylodynamics in Iceland demonstrate the unique importance of gulls in this localized system. Gulls act as sinks and not sources of viruses to other host taxonomic groups in Iceland, and IAVs circulate within these seabirds for a far greater proportion of time than any of the other avian species we studied. Though Charadriiformes predominated in sequence count and hosted the highest genetic diversity of IAVs in both the original and downsampled datasets, our downsampling strategy reduced the number of sequence taxa for computation while mitigating sampling bias to maintain viral genetic diversity in the dataset. As such, IAV circulation time within gull populations may be due to the number of gulls in the dataset or host and virus ecological factors dictating continuous within-gull transmission patterns. Charadriiformes host a diversity of IAV subtypes, though two HA subtypes, H13 and H16, are primarily associated with Charadriiformes globally (Fouchier et al., 2005; Guinn et al., 2016; Hanson et al., 2008; Van Borm et al., 2012). In contrast, gulls in Iceland, hosted a wide range of IAV subtypes - several more than Anseriformes (Supplementary Table 1a). A similar diversity of IAV lineages and geographically reassorted viruses have been recorded in gulls at other high latitude locations (Huang et al., 2014; Wille et al., 2011), which may be due to the presence of large gull populations in ASRs, their pelagic long-distance migration between hemispheres (Figure 3), and opportunistic foraging strategies that characterize Charadriiformes generally (Callaghan, 2021; Van Borm et al., 2012; Wille et al., 2011; Wong, 2014). More research is necessary to describe the contribution of gulls to the global IAV reassortment landscape.
Our data demonstrate that gulls serve as recipient hosts of IAVs from ducks and shorebirds and there is limited onward transmission to other species, suggesting that gulls are permissive hosts but rarely amplify viruses from other hosts. Such transmission dynamics are likely guided by several species-specific behavioral and ecological factors. First, though many gull species inhabit oceanic coastlines, their scavenging behavior often takes them to diverse habitats, such as wetlands where ducks predominate, increasing opportunities for reservoir host duck to gull transmission of IAVs (Huang et al., 2014; Kubetzki & Garthe, 2003). A large proportion of gulls staging in Iceland may be immuno-naïve juveniles following the spring breeding season, which may explain the high susceptibility of gulls as recipients of duck-transmitted IAVs at these interfaces (Altizer, Bartel, & Han, 2011; Hill & Runstadler, 2016). Second, gulls can fly great distances over vast marine ecosystems and facilitate migratory connectivity between global regions (Dugan et al., 2008; Dusek et al., 2014; Van Borm et al., 2012). Many gull species transiently stage in Iceland before onward short- and long-distance migrations to adjacent locations in the region (Hallgrimsson, Gunnarsson, Torfason, Buijs, & Camphuysen, 2012; Olsen KM, 2003; Wille et al., 2011), which may limit inter-species transmission opportunities from gulls to ducks and other avian taxa within Iceland.
Reassortment of IAV gene segments encoding HA surface glycoproteins is a mechanism for jumping between taxonomically distinct hosts (Ma, Hill, Zabilansky, Yuan, & Runstadler, 2016) and has been implicated in the emergence of pandemic influenza viruses in humans (Ward, Lycett, Avila, Bollback, & Leigh Brown, 2013). Though no reassortment between low pathogenic and HPAI HA subtypes was detected in Iceland during our eight-year sampling timeframe, we detected strong support for reassortment between gull-associated H16 to H2 in our sample (Figure 5). Both H16 and H2 subtypes were found at high prevalence among gulls in the study sample, demonstrating a reassortment pattern which may be due to the high frequency of both subtypes within this system and reflect a trend of sustained gull-to-gull transmission in Iceland. Though H2 subtype IAVs are diverse and distributed among two primary lineages (Zhuang et al., 2019), H2N2, the virus responsible for the 1957 pandemic, persists in wild migratory birds and commercial poultry in areas adjacent to the North Atlantic and other global regions (Glaser et al., 2006; Mo et al., 2021; Schäfer et al., 1993; Shortridge, 1979). Historically, avian-origin H2 subtype IAVs have demonstrated spillover followed by sustained transmission among humans, therefore monitoring of H2 viruses in wild birds is considered important for evaluating spillover events into humans (Jones et al., 2014). We also detected significant levels of H2 and H3 to H10 subtype reassortment. H10 viruses have been frequently isolated from wild and domestic avian species in recent years, with the first human cases of H10N7 confirmed in Egypt in 2004 (El-Shesheny et al., 2018). An H10N7 subtype has also been associated with mass mortality events in harbor seals (Phoca vitulina ) throughout northern Europe (Bodewes et al., 2015). Active reassortment and circulation of H10 subtypes in the North Atlantic pose a risk to mammalian hosts, especially given native harbor seals inhabit temperate coastal regions in Iceland, southern Greenland, and northeastern North America (Teilmann & Galatius, 2018).
We demonstrate novel significant findings describing IAV geographic movement, inter-species transmission, and reassortment in the North Atlantic region, although there are a few limitations to our analysis. First, despite eight consecutive years of sampling wild birds for IAVs in Iceland, the total number of fully and partially sequenced viruses to-date remain moderately low at 93 PB2 sequences from 92 individual birds. While our sampling effort was focused on those species groups most often associated with IAVs, sampling bias may have been introduced and other constraints may have impacted the detection, successful isolation, and sequencing of IAV gene segments. Potential sampling bias may have impacted findings related to the proportion of time IAVs spend within each host category. More systematic sampling across years may have provided an increased understanding of IAV dynamics in the North Atlantic region. Our downsampling strategy sought to limit the inclusion of such biases into our analyses. Second, while this analysis relied on one internal gene segment (PB2) to maximize the number of nucleotides in the analysis and investigate transmission dynamics without targeting a specific subtype, inclusion of multiple internal gene segments may have increased the robustness of and identified phylogenetic incongruence in the analysis, whereby different genes exhibit distinct phylogenies. Additionally, phylogenetic histories inferred from internal segments like PB2 may result in different conclusions to those from antigenic segments. Third, heterogenous sampling of IAVs across the globe and the computational limitations of discrete-trait Bayesian phylodynamics require that viruses from each region are downsampled to similarly sized groupings to avoid overrepresentation of certain traits (i.e., geographic region, host species, subtype) which can bias ancestral state reconstructions in the phylogenies. While we are confident our downsampling strategy evened groupings as much as possible while preserving viral, host, and temporal diversity across all regions, our approach does not remove the potential for sampling bias altogether. For example, we did not account for the geographic area nor population sizes of sampled regions, which could limit the inclusion of subpopulations and/or attenuate within-region migration dynamics (Kühnert, Stadler, Vaughan, & Drummond, 2016; Lemey et al., 2014).
Our findings reveal that IAV movement in arctic and subarctic regions follows wild bird migration around the perimeter of the circumpolar north which favors short-distance flights between proximal regions rather than long distance flights over the polar interior. Iceland connects mainland Europe and North America for the inter-hemispheric movement of IAVs, particularly due to the westward migration of wild birds from mainland Europe to Northeastern Canada and Greenland. We demonstrate that both Iceland and Alaska are incursion points of IAVs into North America and add evidence that northern regions in the subarctic are ecologically significant, where inter-continental mixing of disparate bird populations occurs and thus play an important role for inter-species transmission and onward dispersal of IAVs globally. Given that Iceland is situated along overlapping flyways that connect eastern and western hemispheres, its breeding and staging locations for migratory aquatic avian species are important sites for monitoring and surveillance of IAVs. Increased IAV surveillance in northern circumpolar regions may further reveal the source-sink dynamics between Eurasian and North American lineages. Further research could address ecologic and anthropogenic drivers of low and highly pathogenic IAV diffusion between hemispheres, and within the North Atlantic region specifically to support early warning for potential intercontinental incursion events into North America. As climate regimes in northern latitudes continue to shift, further monitoring of regional IAV dynamics may uncover important predictors of viral incursion of low and HPAI subtypes globally.