Dispersal of H9N2 influenza A viruses between East Asiaand North America by wild birds

Andrew M. Ramey a,n, Andrew B. Reeves a, Sarah A. Sonsthagen a, Joshua L. TeSlaa b,Sean Nashold b, Tyrone Donnelly a, Bruce Casler c, Jeffrey S. Hall b

a US Geological Survey, Alaska Science Center, 4210 University Drive, Anchorage, AK, USAb US Geological Survey, National Wildlife Health Center, 6006 Schroeder Road, Madison, WI, USAc US Fish and Wildlife Service, Izembek National Wildlife Refuge, P. O. Box 127, Cold Bay, AK, USA

a r t i c l e i n f o

Article history:Received 30 January 2015Returned to author for revisions9 March 2015Accepted 10 March 2015

Keywords:AlaskaAnas acutaAvian influenzaChen canagicaChinaEast AsiaEmperor gooseInfluenza A virusNorth AmericaNorthern pintailSouth Korea

a b s t r a c t

Samples were collected from wild birds in western Alaska to assess dispersal of influenza A virusesbetween East Asia and North America. Two isolates shared nearly identical nucleotide identity at eightgenomic segments with H9N2 viruses isolated from China and South Korea providing evidence forintercontinental dispersal by migratory birds.

Published by Elsevier Inc.

Introduction

Wild birds play an important role in the global epidemiology ofinfluenza A virus (IAV) infections, but despite extensive researchand surveillance efforts, the role of migratory species in theintercontinental dispersal of viruses remains unclear. Previousresearch on IAVs in wild birds provides evidence that samplingbirds at the margins of North America where migratory flyways ofbirds from different continents overlap may be a useful strategyfor maximizing the detection probability for foreign origin IAVgenomic segments (Pearce et al., 2009). Therefore, it is plausiblethat the detection probability for foreign origin viruses may also behigh at these same locations. Indeed, the only detection of acompletely Eurasian lineage IAV in North America to date occurredin the maritime province of Newfoundland, Canada, an area withextensive evidence for interhemispheric viral gene flow (Huanget al., 2014). In previous research conducted in western Alaska at

Izembek National Wildlife Refuge, 70% of IAV isolates containedEurasian origin genomic segments providing evidence for highlevels of intercontinental viral genetic exchange (Ramey et al.,2010). Therefore, we sampled wild birds for IAVs at this site to gainfurther inference on the intercontinental exchange of virusesbetween East Asia and North America via Alaska.

Results and discussion

Genetically indistinguishable H9N2 subtype IAVs were recov-ered from an emperor goose (Chen canagica) fecal sample and anorthern pintail (Anas acuta) cloacal swab collected on 23 and 30September 2011, respectively. BLAST results and phylogeneticanalyses suggested that these IAVs were only distantly related toH9N2 viruses previously isolated in poultry and other IAV strainsof public health concern. Additionally, phylogenetic analysesprovided evidence that genomic segments shared genetic ancestrywith viruses originating from both North America (NS, PA) andEurasia (M, NP, PB1, PB2) with more complicated, transhemi-spheric ancestry for the HA H9 and NA N2 genomic segments

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/yviro

Virology

http://dx.doi.org/10.1016/j.virol.2015.03.0280042-6822/Published by Elsevier Inc.

n Corresponding author. Tel.: þ1 907 786 7174; fax: þ1 907 786 7021.E-mail address: aramey@usgs.gov (A.M. Ramey).

Virology 482 (2015) 79–83

(Fig. 1). Both H9N2 subtype viruses isolated in Alaska sharednearly identical nucleotide identity (i.e. Z99.4%) at all eightgenomic segments with viruses previously isolated from wildbirds samples from Lake Dongting, China (n¼23; Wang et al.,2012; Zhu et al., 2014) and Cheon-su Bay, South Korea (n¼1; Leeet al., 2014; Table 1; Fig. 2).

Izembek National Wildlife Refuge provides staging habitat forhundreds of thousands of migratory birds during autumn migrationincluding several species with intercontinental migratory tendenciessuch as emperor geese and northern pintails (Miller et al., 2005; Huppet al., 2007, 2011); Fig. 2. The novel finding of nearly identical virusesin Alaska, China, and South Korea provides direct evidence for thedispersal of influenza A viruses between East Asia and North Americaby wild birds. As there is no commercial poultry production inwesternAlaska and highly similar H9N2 IAV strains have not been reported inpoultry in East Asia or North America, it is unlikely that agriculturalimports influenced this result. Furthermore, highly similar H9N2viruses were isolated from different labs in three countries providingno evidence that our results could be explained by laboratory artifacts.Our results, therefore, provide evidence that IAVs associated withdisease in humans and poultry in East Asia (e.g., H5N1 and H5N8) maybe introduced to North America via migratory birds provided that suchviruses can be maintained in free-ranging hosts.

Our phylogenetic analyses corroborate previous reports of inter-continental reassortant H9N2 IAVs (Zhu et al., 2014; Lee et al., 2014).The finding of nearly identical H9N2 viruses on two continents with

genomic segments representative of both North American and Eur-asian lineages suggests that other reassortant viruses detected inAlaska may have been dispersed between East Asia and NorthAmerica. Thus, the frequency of inter-hemispheric dispersal eventsof IAVs by migratory birds may be higher than previously recognized.

Materials and methods

During September and October of 2011–2013, cloacal swabs andfecal samples were collected from wild birds at Izembek NationalWildlife Refuge on the Alaska Peninsula. A total of 2924 samples werecollected from 24 species over three years (Table 2). All samples werescreened using real time RT-PCR (Spackman et al., 2002) and thoseproviding cycle threshold values r45 were inoculated into embryo-nated eggs for virus isolation (Woolcock, 2008). A total of 90 influenzaA virus isolates were recovered (Table 2).

Genomes (ca., 13.5 kb) of resultant isolates were amplified in amultiplex RT-PCR following Zhou et al. (2009). Excess dNTPs andprimers were removed using ExoSAP-ITs (USB Corporation). PCRproducts were quantified with fluorometry using a high sensitivityQuant-iT dsDNA Assay Kit (Invitrogen) and prepared for sequencingfollowing the Nextera XT DNA sample preparation kit protocol(Illumina, Inc.). Indexed libraries were pooled and sequenced on theIllumina MiSeq using a 500 cycle reagent kit with paired-end reads.Sequence reads were assembled using Bowtie 2 version 2.2.3

PB2 PB1 PA H9

NP N2 M NS

Fig. 1. Partial maximum likelihood phylogenies for influenza A virus genomic segments depicting inferred ancestry of highly similar H9N2 viruses detected in Alaska, China,and South Korea. Bootstrap support values Z70 are shown. Sequences for strains originating from North America (blue) and Eurasia (black) are indicated by color. Sequencesfor isolates characterized as part of this study are highlighted in red.

A.M. Ramey et al. / Virology 482 (2015) 79–8380

(Langmead and Salzberg, 2012) using IAV data obtained from GenBankas reference genomes for mapping reads to the eight segments.Consensus sequences were generated with SAMtools version 1.1(Li et al., 2009) and validated with FLuANotation (FLAN).

Among the 90 IAV isolates recovered were two viruses of the H9N2subtype (124,129–247,877 reads aligned per isolate). Given the role ofH9N2 IAVs in the evolution of H5N1 and H7N9 subtype virusesassociated with human disease in Asia (Li et al., 2004; Gao et al., 2013)and concerns regarding the pandemic potential of IAVs of this subtype

(Li et al., 2003), sequences for these two isolates were selected forfurther genetic characterization. GenBank accession numbers forH9N2 viruses isolated as part of this study are: KP336376–KP336391. Nucleotide sequences were compared to IAV lineagesavailable on the NCBI website using the nucleotide BLAST function.Sequence data for the top 100 BLAST hits per genomic segment weredownloaded, aligned with sequences for H9N2 subtype isolatesidentified as part of this study, and trimmed to common length(PB2: 2154bp, PB1: 2185bp, PA: 2083bp, HA: 1519bp, NP: 1335bp, N2:

Table 1Nucleotide similarity between H9N2 subtype influenza A virus strains isolated from wild birds samples collected in Alaska, Chinab,c, and South Koreaa.

Location of origin Shared nucleotide identity (%) to Alaska H9N2 isolates

PB2 PB1 PA H9 NP N2 M NS

Alaska (USA)A/northern pintail/Alaska/2011-0703/2011 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0A/emperor goose/Alaska/2011-0713/2011 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0South KoreaA/bean goose/Korea/220/2011a 99.8 99.8 99.9 99.7 99.8 99.8 99.9 100.0

ChinaA/wild waterfowl/Dongting/C2032/2011b 99.7 99.6 99.9 99.6 99.7 99.6 99.9 99.9A/wild waterfowl/Dongting/C2123/2011b 99.8 99.7 100.0 99.8 99.5 99.5 100.0 99.7A/wild waterfowl/Dongting/C2148/2011b 99.8 99.7 99.9 99.8 99.5 99.5 100.0 99.9A/wild waterfowl/Dongting/C2149/2011b 99.7 99.8 99.9 99.8 99.5 99.5 100.0 99.9A/wild waterfowl/Dongting/C2150/2011b 99.7 99.7 100.0 99.8 99.5 99.5 100.0 99.9A/wild waterfowl/Dongting/C2203/2011b 99.8 99.7 100.0 99.9 99.6 99.6 100.0 99.9A/wild waterfowl/Dongting/C3109/2011b 99.7 99.8 99.9 99.6 99.6 99.6 99.9 99.7A/wild waterfowl/Dongting/PC2539/2012b 99.5 99.6 99.9 99.6 99.5 99.5 100.0 99.9A/wild waterfowl/Dongting/PC2540/2012b 99.5 99.8 99.9 99.6 99.6 99.6 100.0 99.9A/wild waterfowl/Dongting/PC2553/2012b 99.5 99.6 99.9 99.7 99.6 99.6 100.0 99.9A/wild waterfowl/Dongting/PC2559/2012b 99.5 99.7 99.9 99.6 99.6 99.6 100.0 99.9A/wild waterfowl/Dongting/PC2560/2012b 99.7 99.7 99.8 99.6 99.6 99.6 100.0 99.7A/wild waterfowl/Dongting/PC2562/2012b 99.6 99.7 99.8 99.8 99.6 99.6 99.9 99.6A/wild waterfowl/Dongting/PC2574/2012b 99.5 99.6 99.9 99.6 99.6 99.6 100.0 99.9A/wild waterfowl/Dongting/C4296/2012b 99.7 99.6 99.8 99.7 99.6 99.6 100.0 99.7A/wild waterfowl/Dongting/C4316/2012b 99.7 99.6 99.9 99.7 99.5 99.4 100.0 99.7A/wild waterfowl/Dongting/C4317/2012b 99.5 99.7 99.9 99.6 99.6 99.6 100.0 99.9A/wild waterfowl/Dongting/C4326/2012b 99.4 99.7 99.9 99.7 99.6 99.6 100.0 99.9A/wild waterfowl/Dongting/C4327/2012b 99.4 99.7 99.9 99.7 99.6 99.6 100.0 99.7A/wild waterfowl/Dongting/C4329/2012b 99.6 99.7 99.8 99.8 99.6 99.6 99.9 99.6A/wild waterfowl/Dongting/C4429/2012b 99.7 99.7 99.8 99.8 99.6 99.6 100.0 99.7A/wild waterfowl/Dongting/C4430/2012b 99.4 99.7 99.9 99.7 99.6 99.6 100.0 99.9A/egret/Hunan/1/2012c 99.6 99.6 99.9 99.4 99.5 99.4 99.7 99.5

a Lee et al. (2014).b Zhu et al. (2014).c Wang et al. (2012).

Izembek NWR, Alaska (USA)

Cheon-su Bay,South Korea

Donting Lake,China

Fig. 2. Generalized migratory patterns of emperor geese (blue) and northern pintails (red) in the East Asian-Australasian and Pacific Americas flyways and locations inAlaska, China, and South Korea at which wild birds were infected with highly similar H9N2 influenza A viruses.

A.M. Ramey et al. / Virology 482 (2015) 79–83 81

1261bp, M: 888bp, and NS 775bp). A maximum likelihood phylogenywas reconstructed for each genomic segment to provide inference ongenetic ancestry in MEGA version 5.1 (Tamura et al., 2011) using theNucleotide: Nearest-Neighbor-Interchange method with 1000 boot-strap replicates. Additionally, sequences for all previously characterizedIAV isolates that shared Z99% nucleotide similarity at all eightgenomic segments with sequences for H9N2 subtype viruses identi-fied as part of this study were downloaded, aligned, and trimmed to acommon length per genomic segment (PB2: 2232bp, PB1: 2241bp, PA:2127bp, HA: 1611bp, NP: 1459bp, N2: 1334bp, M: 925bp, and NS778bp), and compared Alaska H9N2 viruses via a pairwise distancematrix using MEGA (Tamura et al., 2011).

Acknowledgments

We appreciate support provided by current and former U.S. Fishand Wildlife Service staff at Izembek National Wildlife Refuge includ-ing Doug Damberg, Nancy Hoffman, Leticia Melendez, and StaceyLowe. We are grateful to Srinand Sreevatsan (University of Minnesota;UMN) and Kamol Suwannakarn (UMN) for their assistance in devel-oping a next generation sequencing data analysis pipeline. We thankKyle Hogrefe (U.S. Geological Survey; USGS), Mary Whalen (USGS),John Takekawa (USGS), and Kyle Spragens (USGS) for assistance withFig. 2. We appreciate critical reviews provided by John Pearce (USGS),Craig Ely (USGS), and two anonymous reviewers. This work wasfunded by the U.S. Geological Survey through the Wildlife Program ofthe Ecosystem Mission Area. None of the authors have any financialinterests or conflict of interest with this article. Any use of trade namesis for descriptive purposes only and does not imply endorsement bythe U.S. Government.

References

Gao, R., Cao, B., Hu, Y., Feng, Z., Wang, D., Hu, W., et al., 2013. Human infection witha novel avian-origin influenza A (H7N9) virus. N. Engl. J. Med. 368, 1888–1897.

Huang, Y., Wille, M., Benkaroun, J., Munro, H., Bond, A.L., Fifield, D.A., et al., 2014.Perpetuation and reassortment of gull influenza A viruses in Atlantic NorthAmerica. Virology 456–457, 353–363.

Hupp, J.W., Schmutz, J.A., Ely, C.R., Syroechkovskiy, E.E., Kondratyev, A.V., Eldridge,W.D., et al., 2007. Moult migration of emperor geese Chen canagica betweenAlaska and Russia. J. Avian Biol. 38, 462–470.

Hupp, J.W., Yamaguchi, N., Flint, P.L., Pearce, J.M., Tokita, K., Shimada, T., Ramey,A.M., Kharitonov, S., Higuchi, H., 2011. Variation in spring migration routes andbreeding distribution of northern pintails Anas acuta that winter in Japan. J.Avian Biol. 42, 289–300.

Langmead, B., Salzberg, S.L., 2012. Fast gapped-read alignment with Bowtie 2. Nat.Methods 9, 357–359.

Lee, D.-H., Park, J.-K., Yuk, S.-S., Erdine-Ochir, T.-O., Kwon, J.-H., Lee, J.-B., et al., 2014.Complete genome sequence of a natural reassortant H9N2 avian influenza virusfound in bean goose (Anser fabalis): direct evidence for virus exchange betweenKorea and China via wild birds. Infect. Genet. Evol. 26, 250–254.

Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis,G., Durbin, R., 2009. 1000 genome project data processing subgroup. Thesequence alignment/map format and SAM tools. Bioinformatics 25, 2078–2079.

Li, K.S., Xu, K.M., Peiris, J.S.M., Poon, L.L.M., Yu, K.Z., Yuen, K.Y., et al., 2003.Characterization of H9 subtype influenza viruses from the ducks of southernChina: a candidate for the next influenza pandemic in humans? J. Virol. 77,6988–6994.

Li, K.S., Guan, Y., Wang, J., Smith, G.J.D., Xu, K.M., Duan, L., et al., 2004. Genesis of ahighly pathogenic and potentially pandemic H5N1 influenza virus in easternAsia. Nature 430, 209–213.

Miller, M.R., Takekawa, J.Y., Fleskes, J.P., Orthmeyer, D.L., Casazza, M.L., Perry, W.M.,2005. Spring migration of Northern Pintails from California's central valleywintering area tracked with satellite telemetry: routes, timing, and destina-tions. Can. J. Zool. 83, 1314–1332.

Pearce, J.M., Ramey, A.M., Flint, P.L., Koehler, A.V., Fleskes, J.P., Franson, J.C., et al.,2009. Avian influenza at both ends of a migratory flyway: characterizing viralgenomic diversity to optimize surveillance plans for North America. Evolut.Appl. 2, 457–468.

Ramey, A.M., Pearce, J.M., Flint, P.L., Ip, H.S., Derksen, D.V., Franson, J.C., et al., 2010.Intercontinental reassortment and genomic variation of low pathogenic avianinfluenza viruses isolated from northern pintails (Anas acuta) in Alaska:examining the evidence through space and time. Virology 401, 179–189.

Spackman, E., Senne, D.A., Myers, T.J., 2002. Development of a real-time reversetranscriptase PCR assay for type A influenza virus and the avian H5 and H7hemagglutinin subtypes. J. Clin. Microbiol. 40, 3256–3260.

Tamura, K, Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:molecular evolutionary genetics analysis using maximum likelihood, evolu-tionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28,2731–2739.

Table 2Number of wild bird samples collected at Izembek National Wildlife Refuge, Alaska by species and year and results of molecular screening (MAþ) and virus isolation (VIþ)a.

Species 2011 2012 2013 All years combined

n¼ MAþ VIþ n¼ MAþ VIþ n¼ MAþ VIþ n¼ MAþ VIþ

American green-winged teal (Anas crecca) 47 15 6 31 4 3 33 7 1 111 26 10American wigeon (Anas americana) 17 0 0 13 0 0 4 0 0 34 0 0Black brant (Branta bernicla) 191 0 0 0 0 0 1 0 0 192 0 0Black scoter (Melanitta americana) 0 0 0 2 0 0 7 0 0 9 0 0Bufflehead (Bucephala albeola) 0 0 0 11 1 1 4 2 0 15 3 1Cackling goose (Branta hutchinsii) 220 5 0 0 0 0 1 0 0 221 5 0Common eider (Somateria mollissima) 0 0 0 3 0 0 17 0 0 20 0 0Common goldeneye (Bucephala clangula) 0 0 0 0 0 0 2 0 0 2 0 0Emperor goose (Chen canagica)b 99 10 2 299 11 3 265 5 1 663 26 6Eurasian wigeon (Anas penelope) 3 0 0 8 0 0 7 0 0 18 0 0Gadwall (Anas strepera) 4 0 0 1 0 0 9 0 0 14 0 0Greater scaup (Aythya marila) 15 0 0 13 0 0 8 1 0 36 1 0Glaucous-winged gull (Larus glaucescens)b 152 6 3 302 6 1 256 22 12 710 34 16Greater white-fronted goose (Anser albifrons) 2 0 0 0 0 0 0 0 0 2 0 0Harlequin duck (Histrionicus histrionicus) 0 0 0 14 0 0 34 0 0 48 0 0King eider (Somateria spectabilis) 0 0 0 0 0 0 5 2 1 5 2 1Lesser scaup (Aythya affinis) 0 0 0 2 0 0 0 0 0 2 0 0Long-tailed duck (Clangula hyemalis) 0 0 0 3 0 0 13 1 0 16 1 0Mallard (Anas platyrhynchos) 18 1 0 22 3 2 32 3 2 72 7 4Northern pintail (Anser acuta) 226 38 25 245 34 16 238 32 11 709 104 52Northern shoveler (Anas clypeata) 1 1 0 2 0 0 2 0 0 5 1 0Red-breasted merganser (Mergus serrator) 0 0 0 3 0 0 1 0 0 4 0 0Ring-necked duck (Aythya collaris) 0 0 0 1 0 0 0 0 0 1 0 0White-winged scoter (Melanitta deglandi) 0 0 0 5 0 0 10 0 0 15 0 0Total 995 76 36 980 59 26 949 75 28 2924 210 90

a MAþ , cycle threshold value r45 using real-time reverse transcriptase PCR; VIþ , virus isolated in specific pathogen free eggs.b Fecal samples; all other samples were cloacal swabs.

A.M. Ramey et al. / Virology 482 (2015) 79–8382

Wang, B., Chen, Q., Chen, Z., 2012. Complete genome sequence of an H9N2 avianinfluenza virus isolated from egret in Lake Dongting wetland. J. Virol. 86, 11939.

Woolcock, P.R., 2008. Avian influenza virus isolation and propagation in chickeneggs Avian influenza virus. In: Spackman, E. (Ed.), Methods in MolecularBiology, Vol. 436. Humana Press, Totowa, pp. 35–46.

Zhou, B., Donnelly, M.E., Scholes St, D.T., George, K., Hatta, M., Kawaoka, Y.,Wentworth, D.E., 2009. Single-reaction genomic amplification accelerates

sequencing and vaccine production for classical and Swine origin humaninfluenza a viruses. J. Virol. 83, 10309–10313.

Zhu, Y., Hu, S., Bai, T., Yang, L., Zhao, X., Zhu, W., et al., 2014. Phylogenetic andantigenic characterization of reassortant H9N2 avian influenza viruses isolatedfrom wild waterfowl in the East Dongting Lake wetland in 2011–2012. Virol. J.11, 77.

A.M. Ramey et al. / Virology 482 (2015) 79–83 83