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.