Virus Research 148 (2010) 44–50 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres Limited evidence of trans-hemispheric movement of avian influenza viruses among contemporary North American shorebird isolates John M. Pearce a,∗ , Andrew M. Ramey a , Hon S. Ip b , Robert E. Gill Jr. a a b Alaska Science Center, U.S. Geological Survey, Anchorage, AK, USA National Wildlife Health Center, U.S. Geological Survey, Madison, WI, USA a r t i c l e i n f o Article history: Received 14 September 2009 Received in revised form 30 November 2009 Accepted 2 December 2009 Available online 6 December 2009 Keywords: Avian influenza Gene segments Phylogenetic Shorebird Surveillance Wild bird a b s t r a c t Migratory routes of gulls, terns, and shorebirds (Charadriiformes) are known to cross hemispheric boundaries and intersect with outbreak areas of highly pathogenic avian influenza (HPAI). Prior assessments of low pathogenic avian influenza (LPAI) among species of this taxonomic order found some evidence for trans-hemispheric movement of virus genes. To specifically clarify the role of shorebird species in the trans-hemispheric movement of influenza viruses, assess the temporal variation of Eurasian lineages observed previously among North American shorebirds, and evaluate the necessity for continued sampling of these birds for HPAI in North America, we conducted a phylogenetic analysis of >700 contemporary sequences isolated between 2000 and 2008. Evidence for trans-hemispheric reassortment among North American shorebird LPAI gene segments was lower (0.88%) than previous assessments and occurred only among eastern North American isolates. Furthermore, half of the reassortment events occurred in just two isolates. Unique phylogenetic placement of these samples suggests secondary infection and or involvement of other migratory species, such as gulls. Eurasian lineages observed in North American shorebirds before 2000 were not detected among contemporary samples, suggesting temporal variation of LPAI lineages. Results suggest that additional bird migration ecology and virus phylogenetics research is needed to determine the exact mechanisms by which shorebirds in eastern North America become infected with LPAI that contain Eurasian lineage genes. Because of the low prevalence of avian influenza in non-eastern North America sites, thousands more shorebirds will need to be sampled to sufficiently examine genetic diversity and trans-hemispheric exchange of LPAI viruses in these areas. Alternatively, other avian taxa with higher virus prevalence could serve as surrogates to shorebirds for optimizing regional surveillance programs for HPAI through the LPAI phylogenetic approach. Published by Elsevier B.V. 1. Introduction Wild migratory birds, primarily in the avian orders of Anseriformes (ducks, geese, and swans) and Charadriiformes (gulls, terns, and shorebirds), are the natural reservoirs of a large diversity of low pathogenic avian influenza (LPAI) subtypes (Webster et al., 1992; Kim et al., 2009). Molecular surveys of LPAI isolates from water bird species have revealed substantial levels of sequence divergence between Eurasian and North American hemispheric virus populations (Gorman et al., 1990a,b; Ito et al., 1991; Kawaoka et al., 1998). However, there is also evidence of trans-hemispheric movement of LPAI viruses via wild birds, particularly for those with migratory pathways that span different continents and in geographic regions ∗ Corresponding author at: Alaska Science Center, U.S. Geological Survey, 4210 University Drive, Anchorage, AK 99508, USA. Tel.: +1 907 786 7094; fax: +1 907 786 7021. E-mail address: jpearce@usgs.gov (J.M. Pearce). 0168-1702/$ – see front matter Published by Elsevier B.V. doi:10.1016/j.virusres.2009.12.002 that are adjacent to divergent viral gene pools (Krauss et al., 2007; Koehler et al., 2008). For example, gene segments of LPAI viruses from Northern Pintails (Anas acuta) in Alaska contained a higher number of Asian lineages than observed elsewhere in North America (Koehler et al., 2008). This result is likely due to the geographic proximity of Alaska to Asian populations of avian influenza virus and the migratory movements of northern pintails between Asia and Alaska (Pearce et al., 2009). Some of the earliest evidence of Eurasian lineages in North American virus isolates via reassortment came from shorebird species in eastern North America based on analysis of the hemagglutinin (HA) gene (Marakova et al., 1999). Subsequent analysis of additional shorebird LPAI isolates from eastern North America for additional HA subtypes (Jackwood and Stallknecht, 2007), the matrix (M) gene (Widjaja et al., 2004) and the H6 gene from a single Alaskan shorebird (Wahlgren et al., 2008) also showed evidence for reassortment of North American and Eurasian gene segments. In a comparison of full genome LPAI viruses isolated from species of Anseriformes (ducks) and Charadriiformes (shore- J.M. Pearce et al. / Virus Research 148 (2010) 44–50 45 Table 1 Number of contemporary (2000–2008) shorebird and reference sequences examined for each avian influenza gene segment. Shorebird species Ruddy Turnstone Shorebird Red Knot Least Sandpiper Red-necked Stint Dunlin Sanderling Sharp-tailed Sandpiper Pacific Golden-Plover Semi-palmated Sandpiper Total shorebird Total referencea a Gene segment PB2 PB1 PA NP M NS H4 H6 77 15 6 4 3 2 1 2 1 0 110 25 76 16 6 4 3 2 2 2 1 0 112 25 77 19 6 4 3 2 2 2 1 0 116 23 35 19 3 4 3 2 2 2 1 0 71 23 44 17 3 4 3 2 2 2 1 1 79 23 48 19 4 4 3 2 2 2 1 0 85 23 19 3 2 1 3 4 H10 H12 N5 N6 N7 16 2 3 15 15 21 2 2 1 23 5 4 1 1 27 19 6 39 21 6 17 17 17 9 23 27 32 15 Includes samples from Europe, Asia, and North America (see Section 2). birds and gulls), Krauss et al. (2007) observed a higher frequency of trans-hemispheric reassortment events among shorebirds and gulls combined from eastern North America than among ducks. To date, there has been no comprehensive assessment of LPAI virus genetics in shorebirds specifically, although they are frequently targeted in live bird HPAI surveillance sampling in North America and Europe. Hindering a broader examination of LPAI in shorebirds is the low prevalence of these viruses in this taxonomic group across the globe, including Alaska (Ip et al., 2008; Iverson et al., 2008; Winker et al., 2008), the Pacific coast of North America (Iverson et al., 2008; Dusek et al., 2009), South America (D’Amico et al., 2007; Douglas et al., 2007; Escudero et al., 2008; Ghersi et al., 2009), Australia (Haynes et al., 2009), New Zealand (Langstaff et al., 2009) and Europe (Munster et al., 2007; Hesterberg et al., 2008). In an effort to broaden the geographic scale of shorebird LPAI virus genomics, we describe here two recently isolated shorebird LPAI viruses from Alaska and compare them phylogenetically to other contemporary genomic data from shorebird species sampled in Alaska, eastern North America, Australia, and Europe. Our main objective was to assess contemporary levels of trans-hemispheric reassortment of LPAI lineages in North American shorebirds through a comprehensive analysis of all gene segments isolated between 2000 and 2008. Our overall goal is to assist future surveillance plans by highlighting geographic regions and species that frequently demonstrate trans-hemispheric reassortment. Such locations and taxa may be informative for continued sampling to detect the arrival of highly pathogenic avian influenza in North America via the migratory movements of wild birds. 2. Materials and methods For the analyses presented here, we used full genome data for two shorebird isolates from Alaska that were characterized by our lab, plus a large number of sequences available on the National Center for Biotechnology Information (NCBI) Influenza Virus Resource (Appendix A). For the two Alaska isolates, we sequenced all eight gene segments from two LPAI positive samples: a hatching year Pacific Golden-Plover (Pluvialis fulva) collected on St. Lawrence Island, Alaska, in October 2006 and a hatching year Dunlin (Calidris alpina) sampled on the Yukon Delta, Alaska, in September 2008. Both samples were collected as part of a state-wide surveillance program (see Ip et al., 2008). The eight RNA segments of these two shorebird isolates were amplified with the one-step RT PCR kit (Qiagen, Inc., Valencia, CA) using a combination of previously published primers (see Pearce et al., 2009) or primers specifically designed for this study which are available from the authors upon request. PCR products were gel purified and extracted using the QIAquick gel extraction kit (Qiagen, Inc., Valencia, CA) or treated with ExoSap-IT (USB Inc., Cleveland, OH) without additional purification before sequencing. Cycle sequencing was performed with identical primers used for PCR along with BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA). Samples were analyzed on an Applied Biosystems 3730xl automated DNA sequencer (Applied Biosystems, Foster City, CA). Sequences were assembled and edited with Sequencher version 4.7 (Gene Codes Corp., Ann Arbor, MI). GenBank accession numbers for the Alaska isolates are GQ168607–GQ168614 (Pacific Golden-Plover) and GQ411889–GQ411896 (Dunlin). All other sequence data for contemporary (between 2000 and 2008) shorebird sequences (n = 716) and reference (n = 274) viral gene segments used in this analysis was obtained from NCBI (Table 1, Appendix A). Contemporary shorebird sequences downloaded from NCBI were isolated in Alaska (n = 5), Australia (n = 5), eastern North America (n > 80), and the Netherlands (n = 1) and included seven species of shorebirds, including Least Sandpiper (C. minutilla), Sharp-tailed Sandpiper (C. acuminata), Red Knot (C. canutus), Sanderling (C. alba), Red-necked Stint (C. ruficollis), Ruddy Turnstone (Arenaria interpres) and Dunlin. Additional sequences not identified to a specific host, but rather only “shorebird”, were also included. Reference samples came from migratory waterfowl and other species of Charadriiformes. We also included select sequences from phylogenetic surveys of Krauss et al. (2007) and Bahl et al. (2009) that represent specific clades of lineages to aid in interpretation of gene trees. Some of these were from shorebird species sampled prior to 2000 and are considered reference samples for data summaries. Not all gene segments were available for every isolate and sample sizes given here are maximums (see Table 1 for sample sizes according to each species and gene segment). We restricted our phylogenetic analysis to the six internal gene segments (PB2, PB1, PA, NP, M, and NS) and the six most frequent (each >15% of all isolates) HA and NA subtypes (H4, H10, H12, N5, N6, and N7). Because of the recent finding of introduction and establishment of Eurasian H6 lineages in North America (Bahl et al., 2009) we re-analyzed sequences of the H6 subtype, including a shorebird from Alaska (Wahlgren et al., 2008) and five from eastern North America. The total number of nucleotides included in the analysis of each RNA segment was: PB2 (2252), PB1 (2262), PA (2068), NP (1337), M (859), NS (866), HA (range 1650–1656), and NA (range 1346–1391). We used MEGA version 4.0 (Tamura et al., 2007) to generate neighbor-joining trees using 10,000 bootstrap replicates and MrBayes version 3.1.2 (Ronquist and Huelsenbeck, 2003) to determine posterior probabilities of clade support. In MrBayes, each analysis was run for ∼1 × 106 generations or until the average standard deviation of split frequencies was ≤1.00. We also verified that the potential scale reduction factor was ∼1.00 as another indicator of convergence (see Ronquist and Huelsenbeck, 2003). 46 J.M. Pearce et al. / Virus Research 148 (2010) 44–50 Fig. 1. Phylogram of the H6 subtype that includes the Alaskan isolate A/Dunlin/Barrow/65/2005/H6N1 of Wahlgren et al. (2008) in relation to low pathogenic avian influenza water bird lineages from North America (closed circles) and Eurasia (open circles). The Eurasian/Alaskan clade includes reassortment events observed in Alaskan northern pintails by Koehler et al. (2008). All taxa correspond to lineage A of Bahl et al. (2009) except for the three North American mallard lineages at the bottom of the figure, which are from lineage B (see text). Levels of neighbor-joining bootstrap support >70% are shown for major clades. Average posterior probabilities of the 50% majority rule consensus tree topologies were estimated by sampling likelihood parameters every 100 generations. Trees were visualized with TreeView (Page, 1996). Following the construction of phylograms, we determined that a viral reassortment event had occurred between Eurasian/Australian and North American viruses when a lineage from a shorebird sample in North America was most closely related to Eurasian/Australian sequences. We used BLASTN version 2.2.2 (Zhang et al., 2000) to examine the BLAST results (species and geographic regions with maximum identity scores ≥95%) for gene segments of two shorebird isolates that appeared as outlier lineages (see Section 3). 3. Results 3.1. Genetic variation We observed a broad range of HA and NA subtypes among the shorebird lineages included in this analysis (not shown). The subtypes of the two Alaska shorebird isolates (H6N1, H3N8) were similar to those observed among Northern Pintails in Alaska (Koehler et al., 2008). All viruses from Alaskan Least Sandpipers were the same subtype (H4N8). Eastern North American isolates were classified to a variety of subtypes, with H4, H10, H12, N5, N6, and N7 being the most frequent (each >10% of isolates). We observed no evidence for geographic segregation of isolates collected within North America (i.e., between Alaska and eastern North America). Sequences for Least Sandpipers and other Alaskan shorebirds were distributed throughout the larger collection of eastern North American shorebirds and did not cluster consistently, although sample sizes are small from Alaska. Additionally, isolates from the four Least Sandpipers may not represent truly independent samples as they all shared the same subtype (H4N8) and clustered together in every phylogram. In contrast, Australian shorebird isolates were highly divergent from North American lineages and clustered more closely with the Eurasian clade for internal gene segments (Fig. 1). J.M. Pearce et al. / Virus Research 148 (2010) 44–50 47 Fig. 2. Phylograms depicting genetic diversity among sequences from the six internal non-surface glycoprotein RNA segments of shorebird isolates from North America (closed circles), Australia (open triangles), and waterfowl reference samples from Eurasia (open circles). The turnstone isolate from the Netherlands clustered with Eurasian samples in every phylogram. Large circles show North American lineages of Eurasian origin (reassortment events). Arrows denote Eurasian lineage viruses isolated in shorebirds in eastern North America prior to 2000 that were not observed in this analysis. The placement of isolates A/shorebird/DE/68/2004 and A/shorebird/DE/168/2006 are shown on each phylogram (see text). Bayesian posterior probabilities and levels of neighbor-joining bootstrap support >70% are shown for major clades. 3.2. Trans-hemispheric reassortment Our phylogenetic analysis of 143 shorebird sequences from the seven HA and NA gene segments (Table 1) revealed a clear separation between North American and Eurasian clades and no evidence for trans-hemispheric reassortment within North American shorebirds. However, this analysis included the H6 subtype for which Wahlgren et al. (2008) determined that the HA gene of A/Dunlin/Barrow/65/2005 was of Asian-origin. In our analysis (Fig. 2), which incorporates a different set of reference sequences, this isolate clusters more closely with North American shorebird and waterfowl lineages within a larger mixed clade that also contained sequences from Eurasia. This mixed clade of Eurasian and North American sequences is now the predominant lineage throughout North American birds (Bahl et al., 2009), making quantifications of reassortment in the H6 subtype problematic (see Section 4). Among the 538 North American shorebird internal gene sequences (Table 1, Appendix A) we observed 12 (2.2%) cases where contemporary North American shorebird sequences were more closely related to Eurasian reference samples (Table 2, Fig. 2). When nearly identical and thus non-independent sequences were removed following the methodology of Krauss et al. (2007), a Table 2 Contemporary (isolated in 2000–2008) North American low pathogenic avian influenza virus isolates from shorebirds with one or more Eurasian lineage gene segment. Isolate Gene segment PB2 A/shorebird/Delaware/66/2003/H9N2 A/shorebird/Delaware/261/2003/H9N5 A/shorebird/Delaware/286/2003/H9N2 A/shorebird/Delaware/68/2004/H3N9 A/shorebird/Delaware/133/2006/H6N8 A/shorebird/Delaware/168/2006/H16N3 a PB1 PA 1 1 1 1 1 1 NP M NS 1 1 1a 1 1a 1 Occurs in a mixed lineage Eurasian/North American clade (Bahl et al., 2009). 48 J.M. Pearce et al. / Virus Research 148 (2010) 44–50 total of six (1.1%) reassortment events were determined to have occurred. All of these reassortment events were virus isolates obtained from shorebirds in eastern North America. We did not observe any contemporary shorebird sequences to be associated with the Eurasian lineages identified by Krauss et al. (2007) in shorebird isolates collected between 1986 and 1998 from eastern North America (Fig. 2). There was no isolate with completely Eurasian lineage gene segments, but two isolates (A/shorebird/Delaware/68/2004/H3N9 and A/shorebird/Delaware/168/2006/H16N3) had four gene sequences that were more closely related to Eurasian reference samples (Table 2). Furthermore, these two isolates were often associated with phylogenetic groupings that were distinct from the main Eurasian or North American clades (Fig. 2). BLAST results for the eight gene segments for A/shorebird/Delaware/68/2004/H3N9 yielded maximum identity scores of ≥95% for North American and Eurasian gull lineages except for PB1 and NA, which yielded North American waterfowl, gallinaceous poultry and shorebird lineages. For A/shorebird/Delaware/168/06/H16N3, maximum identity scores of ≥95% were obtained for Eurasian gull and waterfowl lineages for all segments except PB1, which more closely matched North American waterfowl, gallinaceous poultry and shorebird lineages. and A/shorebird/Delaware/168/2006/H16N3) that appeared as distinct lineages separate from either North American or Eurasian clades in the phylograms of NP, M, and NS genes (Fig. 2). These isolates exhibited close genetic relationships to European gull lineages via BLAST searches of the NCBI influenza database, which raises questions about the direct role that shorebirds in eastern North America play in the trans-hemispheric movement of avian influenza viruses versus secondary infection from other migratory species, such as gulls. Krauss et al. (2007) noted a higher relative frequency of trans-hemispheric reassortment of LPAI lineages in shorebirds than in ducks. However, that study combined gulls and shorebirds as a single group, when for some gene segments, LPAI viruses of North American and Eurasian gulls appear phylogenetically distinct (Olsen et al., 2006). Thus, the unique placement and BLAST results for the two outlier isolates from eastern North American shorebirds may be indicative of trans-hemispheric movement of viruses by gulls followed by interspecific transmission to shorebirds. Additional research on the migratory pathways of water birds that congregate in eastern North America during spring would help to clarify which species and mechanisms contribute to the continued observation of Eurasian LPAI lineages in this region. The overlap of Atlantic American and eastern Atlantic (European) migratory flyways in northeastern Canada may also serve as link between continental gene pools, similar to that observed between Pacific flyways (Flint et al., 2009). 4. Discussion 4.2. Temporal variation of Eurasian lineages 4.1. Genetic diversity and reassortment Subtypes observed among eastern North American shorebird samples differed in type and frequency from those most commonly found in shorebird isolates in Europe (H13, H16, N3, and N8) (Munster et al., 2007). We found no evidence for geographic subdivision of LPAI viruses between Alaska and eastern North America, similar to other studies that have examined geographic subdivision among North American LPAI viruses in migratory water birds (Spackman et al., 2005; Chen and Holmes, 2009). LPAI shorebird sequences from Australia and the Netherlands consistently clustered with other Eurasian lineages. We observed 1.8% of 681 North American shorebird LPAI sequences to phylogenetically be of Eurasian lineage. This percentage was reduced to 0.88% after excluding closely related LPAI sequences, considerably lower than the 9.8% reported by Krauss et al. (2007) which combined multiple species of Charadriiformes (gulls and shorebirds) and a broader temporal span of isolates (1970s to 2004). All reassortment events were observed among shorebird isolates from eastern North America, with none observed among the few isolates available from Alaska. Interestingly, the migratory routes of Ruddy Turnstones, Red Knots, Sanderlings, and other shorebird species that move north through eastern North America during spring originate from wintering areas in South and Central America (Nettleship, 2000) and not from areas associated with Eurasian avian influenza gene pools (Olsen et al., 2006). But, all three species nest in the High Arctic of eastern Canada where they do have direct contact – or occur in very close proximity – to subspecies of each that migrate down the Eastern Atlantic Flyway (Wymenga et al., 1990; Engelmoer and Roselaar, 1998). In addition, species such as the Ringed Plover (Charadrius hiaticula) nest in numbers on Baffin Island and Canadian archipelagoes further north, and then migrate to western Europe and western Africa for winter (Wymenga et al., 1990). Several gull species also spend the summer breeding season in northeastern Canada and migrate to nonbreeding areas in Europe and or eastern North America and South America (Burger and Gochfield, 1996). Half of the reassortment events we observed were restricted to two isolates (A/shorebird/Delaware/68/2004/H3N9 Among contemporary shorebird samples, we did not observe descendents of any of the Eurasian lineages identified by Krauss et al. (2007) in shorebird isolates collected prior to 2000 (arrows in Fig. 2). In that study, eight reassortment events in shorebirds were observed for the M gene, two for PB2 and one for PB1. The lack of these particular lineages in contemporary isolates suggests either extinction or insufficient sampling to detect these strains in the current population. Lineage extinction is a common feature of avian influenza and other viruses, which exhibit pronounced temporal variation due to reassortment and competition among different strains that infect a host and higher rates of mutation and selection pressures (Rambaut et al., 2008; Bahl et al., 2009). 4.3. Patterns of reassortment among different virus genes Our analysis of the H6 sequence for the A/Dunlin/Barrow/ 65/2005/H6N1 from Alaska suggests that the Asian lineage described by Wahlgren et al. (2008) may not be a contemporary reassortment event, but instead result from the historic introduction and establishment of H6 lineage A described by Bahl et al. (2009) and zu Dohna et al. (2009). This complex pattern of the H6 subtype has been noted in a number of studies (Webby et al., 2003; Spackman et al., 2005; Dugan et al., 2008; Pearce et al., 2009). Based on these collective observations, we suggest that accurately quantifying reassortment events for H6, and possibly other HA and NA subtypes, is problematic. Historic introduction events and higher rates of nucleotide substitution and selection pressures are common traits of these genes (Bahl et al., 2009), which in turn contributes to greater genetic diversity that likely assists in the evasion of host immunity (Dugan et al., 2008). Although our analysis of other HA and NA subtypes revealed monophyletic clades (North America or Eurasia), this result could be biased by the available sequences that formed our reference set. Many avian influenza sequences available on NCBI are either domestic species or not identified to species, especially those from Asia. Thus, genetic analyses of virus segments from sedentary species may sort into divergent clades in association with their continental origins because of the lack of long-distance gene flow, whereas lineages J.M. Pearce et al. / Virus Research 148 (2010) 44–50 from migratory species may be more prone to complicated phylogenetic trees. For example, in a recent meta-analysis of all LPAI HA and NA segments available on NCBI for avian taxa, Liu et al. (2009) confirmed distinct clades of Eurasian and North American lineages for several subtypes, including N8, whereas other HA and NA types demonstrated more complicated, mixed lineage patterns, such as that of H6. In contrast, a recent genomic assessment of a highly migratory species, the Northern Pintail, suggests that the phylogeny for the N8 segment is indeed complicated (Pearce et al., 2009) and not monophyletic. Thus, accurately assessing transhemispheric reassortment in regions and species that demonstrate connectivity to outbreak areas of highly pathogenic avian influenza requires analysis of multiple gene segments from isolated viruses and representative reference samples. 5. Conclusions Shorebirds have long been considered a reservoir of avian influenza viruses (Webster et al., 1992) and are intercontinental and trans-hemispheric migrants, often crossing large distances in a single flight (Gill et al., 2009). As many as 20 shorebird species have migratory routes that include countries in which outbreaks of the H5N1 highly pathogenic avian influenza (HPAI) have occurred, such as along the Pacific and East Asian/Australasian flyways (Hurt et al., 2006; Olsen et al., 2006). Thus, from a migratory perspective shorebirds constitute an important taxonomic group for continued surveillance sampling, especially in areas where intercontinental pathways overlap. However, the low prevalence of LPAI viruses in shorebird samples from areas away from eastern North America hampers robust conclusions about the ecology and evolution of avian influenza in this taxonomic group. Many thousands of field swab or fecal samples are likely needed to obtain a sufficient sample (>20) of virus isolates with recoverable RNA for sequencing and genomic characterization. Such large numbers may be obtained on southern wintering grounds when shorebirds are also aggregated, but a phylogenetic analysis of winter samples from north, central or South America is likely less informative for evaluating trans-hemispheric movement of LPAI viruses. In a recent analysis Pearce et al. (2009) found no internal gene segments of Asian lineage on wintering areas of the Northern Pintail in contrast to a larger number of Asian lineage segments on the breeding grounds in Alaska. These authors suggest that reassortment and rapid nucleotide evolution during southern migration likely influence viral genomics between breeding and wintering areas. Given the apparent high prevalence of LPAI viruses in shorebirds of Australia (Hurt et al., 2006) and eastern North America, plus the unique phylogenetic placement of lineages from these geographic areas, these regions are worthy of continued assessment. In other areas, such as the west coast of North America, the low prevalence of LPAI combined with the likelihood of temporal variation and lineage extinction of Eurasian gene segments as birds move to more southerly wintering areas, suggests that sampling live shorebird species in areas that are far removed from HPAI outbreak areas may be an inefficient HPAI surveillance strategy. An alternative to intensive monitoring needed to obtain sufficient virus strains for sequence analysis would be to first identify epidemiological patterns for influenza virus infection in shorebirds. In eastern North America, the peak prevalence during spring allows highly efficient virus recovery (Hanson et al., 2008). Along other migratory flyways, the peak prevalence of LPAI at different staging sites has not yet been identified. The identification of such sites is crucial for continued surveillance and would be more cost effective than continuing extensive sampling at areas with low virus prevalence. We conclude by urging biologists and virologists alike to identify the exact species from which viral samples are isolated, especially 49 when the sequences of these virus isolates are deposited on publicly available databases, such as NCBI. A similar plea was made previously by Yasué et al. (2006). All reassortment events identified in this analysis come from “shorebird” samples (Table 2), but the exact species of these isolates remains unknown. Species designations are important for unraveling the routes that viruses take between continents, the likely migratory pathways that are used by avian hosts, and host-specific phylogenetic patterns that may exist for this pathogen. Acknowledgments This research was funded by the U.S. Geological Survey (USGS) and the U.S. Fish and Wildlife Service (USFWS). We are grateful to S. Haseltine, R. Kearney, and P. Bright (USGS), D. Rocque and K. Trust (USFWS) for their support of field collections and laboratory analyses. D. Irons and L. Sheffield (USFWS), T. Donnelly (USGS), the native communities of St. Lawrence Island, Alaska, and the Yukon Delta National Wildlife Refuge (USFWS) assisted with the collection of the Dunlin and Pacific Golden-Plover isolates. Y. Gillies (USGS Alaska Science Center), D. Goldberg, Z. Najacht and R. Zane (USGS National Wildlife Health Center; NWHC) coordinated distribution of sampling materials, receipt of samples, and data verification. We thank past and current members of the Diagnostic Virology Laboratory at the USGS NWHC, including T. Egstad, K. Griffin, A. Hauser, M. Houfe, K. Kooiman, R. Long, A. Miyamoto, J. Montez, Z. Najacht, S. Nashold, J. TeSlaa, J. Tuscher and A. Ray. P. Flint and D. Derksen provided comments on an earlier version of this manuscript. None of the authors have any financial interests or conflict of interest with this article. Any use of trade names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2009.12.002. References Bahl, J., Vijaykrishna, D., Holmes, E.C., Smith, G.J.D., Guan, Y., 2009. 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