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<ul><li><p>GENOMIC ANALYSIS OF AVIAN INFLUENZA VIRUSES FROM</p><p>WATERFOWL IN WESTERN ALASKA, USA</p><p>Andrew B. Reeves,1,4 John M. Pearce,1 Andrew M. Ramey,1 Craig R. Ely,1 Joel A. Schmutz,1</p><p>Paul L. Flint,1 Dirk V. Derksen,1 Hon S. Ip,2 and Kimberly A. Trust3</p><p>1 US Geological Survey, Alaska Science Center, 4210 University Drive, Anchorage, Alaska 99508, USA2 US Geological Survey, National Wildlife Health Center, 6006 Schroeder Road, Madison, Wisconsin 53711, USA3 US Fish and Wildlife Service, National Wildlife Refuge System, 4401 N. Fairfax Drive, Arlington, Virginia 22203, USA4 Corresponding author (email:</p><p>ABSTRACT: The Yukon-Kuskokwim Delta (Y-K Delta) in western Alaska is an immense andimportant breeding ground for waterfowl. Migratory birds from the Pacific Americas, CentralPacific, and East Asian-Australasian flyways converge in this region, providing opportunities forintermixing of North American- and Eurasian-origin hosts and infectious agents, such as avianinfluenza virus (AIV). We characterized the genomes of 90 low pathogenic (LP) AIV isolates from11 species of waterfowl sampled on the Y-K Delta between 2006 and 2009 as part of an interagencysurveillance program for the detection of the H5N1 highly pathogenic (HP) strain of AIV. Wefound evidence for subtype and genetic differences between viruses from swans and geese,dabbling ducks, and sea ducks. At least one gene segment in 39% of all isolates was Eurasian inorigin. Target species (those ranked as having a relatively high potential to introduce HP H5N1AIV to North America) were no more likely than nontarget species to carry viruses with genes ofEurasian origin. These findings provide evidence that the frequency at which viral gene segmentsof Eurasian origin are detected does not result from a strong species effect, but rather we suspect itis linked to the geographic location of the Y-K Delta in western Alaska where flyways fromdifferent continents overlap. This study provides support for retaining the Y-K Delta as a highpriority region for the surveillance of Asian avian pathogens such as HP H5N1 AIV.</p><p>Key words: Alaska, avian influenza virus, genome, migratory birds, surveillance, waterfowl.</p><p>INTRODUCTION</p><p>In response to potential introduction ofhighly pathogenic (HP) H5N1 avian influ-enza virus (AIV) from Eurasia to NorthAmerica by migratory birds, a US Inter-agency Strategic Plan (hereafter the Plan)was developed to guide surveillance ef-forts for early detection of the virus(Interagency Working Group, 2006). Sam-pling birds in Alaska was a priority of thePlan due to its location along migratoryflyways linking western and eastern hemi-spheres (Winker and Gibson, 2010). ThePlan also identified and ranked wild birdspecies to be sampled based on criteriathat assessed each species potential forcarrying HP H5N1 AIV to North America(Ip et al., 2008). The highest rankingspecies were identified as target species.Although HP H5N1 AIV has never beendetected in North America, genomic anal-yses of low pathogenic (LP) AIVs isolatedfrom surveillance sampling have enhancedmonitoring efforts by identifying sampling</p><p>locations and host species more likely to beassociated with intercontinental move-ments of viruses or viral gene segments(Pearce et al., 2009; Ramey et al., 2010b).Entirely Eurasian-origin LP AIV genomeshave not been documented in NorthAmerica, although gene segments descend-ed from Eurasian ancestors have (Krausset al., 2007; Dugan et al., 2008; Koehler etal., 2008; Wille et al., 2011). TheseEurasian-origin genes have been detectedmore frequently in western Alaska than inother locations in North America (Pearce etal., 2009; Ramey et al., 2010a). However,there has not been a study of viruses from acommunity of species that covers a largegeographic area in this region.</p><p>The Yukon-Kuskokwim Delta (Y-KDelta) in western Alaska (Fig. 1) is a vastbreeding ground of global significance formany species of migratory birds. Thediversity and abundance of birds, manywith migratory connectivity to Eurasia,combined with the feasibility of collectingsamples, made the Y-K Delta one of the</p><p>DOI: 10.7589/2012-04-108 Journal of Wildlife Diseases, 49(3), 2013, pp. 600610# Wildlife Disease Association 2013</p><p>600</p></li><li><p>most heavily sampled regions in theUnited States for HP H5N1 AIV surveil-lance (Ip et al., 2008). Therefore, molec-ular characterization of LP AIVs frombirds across the Y-K Delta provide aunique opportunity to examine virusdiversity among multiple hosts and inves-tigate exchange of AIV lineages across alarge community of migratory species. Weexamined subtype diversity from threetaxonomic groups of waterfowl: swansand geese, dabbling ducks, and sea ducks.We also characterized LP AIV genomesfrom target and nontarget species sampledon the Y-K Delta to test whether targetspecies were more likely to be infectedwith viruses with mixed continental line-ages. We interpret these results in thecontext of past surveillance and futuremonitoring programs in North America,Europe, and Asia.</p><p>MATERIALS AND METHODS</p><p>Sampling, virus isolation, and sequencing</p><p>Cloacal swab samples were collected fromwild birds between 2006 and 2009 duringspring and fall subsistence harvests and live-captures (MayAugust). Samples were storedin viral transport media and then screened forAIVs at the US Geological Survey (USGS),National Wildlife Health Center (NWHC)using methods of Ip et al. (2008). Weattempted to sequence AIVs isolated fromcloacal swab samples collected on the Y-KDelta (boroughs of Wade Hampton andBethel, and the geographically contiguouslocation of Stebbins within the Nome Bor-ough; Fig. 1) if harvested allantoic fluidagglutinated chicken red blood cells and waspositive for the influenza A matrix gene byrRT-PCR. Viral RNA was extracted fromallantoic fluid with the MagMAX AI/NDVRNA extraction kits (Ambion Inc., AustinTexas, USA) and amplified with the One-StepRT PCR kit (QIAGEN Inc., Valencia, Califor-nia, USA) using combinations of previouslypublished primers (Zou, 1997; Hoffmann et al.,2001; Phipps et al., 2004; Bragstad et al., 2005;Chan et al., 2006; Obenauer et al., 2006; Liet al., 2007; Koehler et al., 2008; Pearce et al.,2011) and primers designed by researchers atthe USGS Alaska Science Center (availableupon request from the authors). PCR productswere gel purified and extracted using theQIAquick Gel Extraction Kit (Qiagen) ortreated with ExoSap-IT (USB Inc., Cleveland,Ohio, USA) without additional purificationbefore sequencing. Cycle sequencing wasperformed with identical primers used forPCR along with BigDye Terminator version3.1 mix (Applied Biosystems, Foster City,California, USA). Samples were analyzed onan Applied Biosystems 3730xl automatedDNA sequencer. Sequences were alignedand edited using Sequencher 4.9 (Gene CodesCorporation, Ann Arbor, Michigan, USA).</p><p>Isolates with evidence of coinfection (i.e.,chromatograms with multiple peaks through-out a sequence) were excluded from furtheranalyses (n53) as were 16 isolates for whichcontamination concerns could not be ruledout. These included seven isolates collectedfrom nonwaterfowl species (Pectoral Sandpip-er, Calidris melanotos; and Sandhill Crane,Grus canadensis). As a result of their removal,our analyses focus exclusively on LP AIVs inwaterfowl. Ninety LP AIV isolates weresequenced, including 24 from previous Alaskastudies (Koehler et al., 2008; Ramey et al.,2010b; Pearce et al., 2011) and 66 obtained</p><p>FIGURE 1. Map of the Yukon-Kuskokwim Deltain western Alaska, USA (inset), where waterfowlsamples were collected 20062009 to characterizeavian influenza virus (AIV) isolates. Species groups(swans and geese5SG, dabbling ducks5DD, and seaducks5SD) and the number of avian influenza virusisolates characterized are provided for each of theapproximated collection sites (open circles). Loca-tions discussed in text and major geographicreferences are labeled.</p><p>REEVES ET AL.AVIAN INFLUENZA VIRUSES FROM WESTERN ALASKA WATERFOWL 601</p></li><li><p>specifically for this project. All eight genesegments were sequenced for 82 isolates; theremaining eight were missing data for one ortwo segments each. Thus, data from 709 of 720potential gene segments were used in analyses.GenBank accession numbers for gene seg-ments sequenced in this study are JX080722JX081243. Strain names and accession num-bers for sequences obtained in previousstudies are available upon request from theauthors. For phylogenetic analyses, sequencesfor each gene segment were cropped to thefollowing number of nucleotides: PB2 (2189),PB1 (2216), PA (2146), HA (14701656), NP(1411), NA (12221388), M (732), and NS(666669).</p><p>Subtype comparisons among waterfowl groups</p><p>Subtyping was determined using the NCBIBLAST tool (Altschul et al., 1990) for hemag-glutinin (HA) and neuraminidase (NA) sequen-ces and confirmed by phylogenetic comparisonsto reference sequences. We compared distribu-tions of HA and NA subtypes, and subtypecombinations for viruses isolated from water-fowl groups using chi-square (x2) tests ofhomogeneity and calculated the exact P valuesusing a randomized sampling distribution (1,000replicates) of our data.</p><p>Analyses were based on the followingorganization structure of three waterfowlgroups: swans and geese (Tundra Swan,Cygnus columbianus; Canada/Cackling goose,Branta canadensis/B. hutchinsii; Black Brant,B. bernicla; Greater White-fronted goose,Anser albifrons; Lesser Snow Goose, Chencaerulescens; and Emperor Goose, C. cana-gica), dabbling ducks (Mallard, Anas platy-rhynchos; Green-winged Teal, A. crecca; andNorthern Pintail, A. acuta), and sea ducks(King Eider, Somateria spectabilis; and Spec-tacled Eider, S. fischeri). We combinedTundra Swans and geese for analyses becauseboth taxa are large-bodied grazers utilizingsimilar habitats. Isolates were grouped intotwo periods for each year; spring samples werecollected in April and May, and fall samplesfrom JulyOctober.</p><p>Comparing genomic identities</p><p>To examine patterns of virus sharing, wequantified the similarity of AIV genomes bycalculating nucleotide pairwise distances(PWD) for all gene segments, thus comparingentire genome constellations for all viruses.Virus genomes were considered highly similarwhen all eight gene segments between twoisolates were .99% similar (PWD ,0.01;Reeves et al., 2011). To evaluate persistence</p><p>of highly similar genomes, we calculated thenumber of days between sample collections.The frequencies at which viruses were sharedwithin and between species were calculatedand observed differences between these weretested using a contingency table to compute x2.</p><p>Identifying intercontinental reassortment</p><p>Our phylogenetic methods for detectingEurasian-origin gene segments were similarto those of Koehler et al. (2008). All genesegments in this study were compared tosequences of viruses isolated from wildwaterfowl in Eurasia (internal segments,n53038; HA and NA segments, n5838)and North America (internal segments n53044; HA and NA segments, n51847) availablefrom GenBank for each RNA segment torepresent respective continental lineages (ac-cession numbers for reference sequences areavailable upon request from the authors).Reference sequences were selected to coverwide geographic distributions of collectionlocations from both continents and restrictedto samples collected from 20062009 with theexception of Eurasian references from 20002009 for H2, H8, H10, H11, H12, N2, N4, andN5 genes because few sequences of thesegenes were available from the more contem-porary time period. Neighbor-joining treeswere generated using MEGA Version 4.0.2(Tamura et al., 2007) with the maximumcomposite likelihood model for nucleotidesequences and 10,000 bootstrap replicates.Sequences isolated from Y-K Delta samplesphylogenetically embedded in continentalclades of Eurasian reference sequences wereconsidered evidence of intercontinental move-ment of influenza genes. NCBI BLAST resultswere used to assess independence of Eurasian-origin gene segments from our Y-K Deltaviruses from lineages representing introduc-tion events previously observed in NorthAmerica. Sequences from the Y-K Deltawhere the highest identity matches in Gen-Bank were to nonNorth American sequenceswere considered evidence of independentoutsider events and subject to further phylo-genetic analysis. We compared the frequenciesat which isolates had at least one gene ofEurasian origin for target and nontargetspecies using a x2 test of homogeneity.</p><p>RESULTS</p><p>Isolates and subtypes</p><p>In total, 24,170 field swab samples werecollected from 82 species of wild birds on</p><p>602 JOURNAL OF WILDLIFE DISEASES, VOL. 49, NO. 3, JULY 2013</p></li><li><p>the Y-K Delta between 2006 and 2009(Table 1). From these samples, we char-acterized the genomes of 90 LP AIVs from11 waterfowl species (Table 1). Althoughwe attempted to maximize the sample sizeof genomic sequence data from this regionand time, the 90 LP AIVs in this study donot represent all of the possible AIVpositive samples, and therefore the num-ber of genomes sequenced should not beused to infer prevalence and isolaterecovery rates. All viruses sequenced inthis study were collected on the mainlandof the Y-K Delta west of 161u0900Wlongitude (Fig. 1). Seven of the 11 speciesfrom which viruses were sequenced weretarget species (Interagency WorkingGroup, 2006). There were significantdifferences in the frequency of HA types(x2560.3, df518, P,0.001), NA types(x2577.01, df516, P,0.001), and subtypecombinations (x25131.5, df536, P,0.001)among waterfowl groups (Fig. 2). We</p><p>detected 10 HA and nine NA subtypespaired in 19 combinations (Fig. 2) from 86viruses for which both HA and NA weresequenced. H3N8 (n519) and H4N6(n518), the most common subtype combi-nations, and H11N9 (n53) were recoveredfrom species of geese and dabbling ducks.Only one isolate from sea ducks shared asubtype combination (H3N8) with speciesfrom other waterfowl groups.</p><p>Highly similar genomes</p><p>Thirty-eight of 4,005 isolate-to-isolategenomic comparisons were highly similarwith identities .99% (PWD,0.01) for allgene segments (Table 2). These highlysimilar genomes were observed more oftenwithin (68%) than between (32%) species(x2550.0, df51, P,0.001). Only one (3%)of the 38 comparisons with highly similargenomes occurred between viruses fromspecies of different waterfowl groups: A/Northern Pintail/Alaska/44340-441/2007</p><p>TABLE 1. Numbers of cloacal swab samples collected on the Yukon-Kuskokwim Delta, Alaska, USA, byspecies, 20062009, and numbers of avian influenza virus (AIV) genomes sequenced for each collection year.Numbers of species are given in parentheses for categories from which no isolates were sequenced. Thesedata are not to be interpreted as prevalence of infection/isolate recovery rates, but rather are presented hereto highlight extent of the surveillance effort on the Y-K Delta.</p><p>AIV genomes sequenced</p><p>Species Samples collected 2006 2007 2008 2009 Total</p><p>Target Species 15,497 28 20 4 15 67</p><p>Tundra Swan 728 4 0 0 0 4Lesser Snow Goose 1,571 1 1 0 0 2Emperor Goose 1,930 8 4 0 0 12Black Brant 3,388 1 4 1 0 6Northern Pintail 1...</p></li></ul>