In this study, scientists from the U.S. Geological Survey and U.S. Fish and Wildlife Service harnessed a new type of DNA technology to investigate avian influenza viruses in Alaska. Using a “next generation” sequencing approach, which identifies gene sequences of interest more rapidly and more completely than by traditional techniques, scientists identified low pathogenic avian influenza viruses in Alaska that are nearly identical to viruses found in China and South Korea. The viruses were found in an area of western Alaska that is known to be a hot spot for both American and Eurasian forms of avian influenza. “Our past research in western Alaska has shown that 70 percent of avian influenza viruses isolated in this area were found to contain genetic material from Eurasia, providing evidence for high levels of intercontinental viral exchange,” said Andy Ramey, a scientist with the USGS Alaska Science Center and lead author of the study. “This is because Asian and North American migratory flyways overlap in western Alaska.” The new study, led by the USGS, found low pathogenic H9N2 viruses in an Emperor Goose and a Northern Pintail. Both of the H9N2 viruses were nearly identical genetically to viruses found in wild bird samples from Lake Dongting, China and Cheon-su Bay, South Korea. “These H9N2 viruses are low pathogenic and not known to infect humans, but similar viruses have been implicated in disease outbreaks in domestic poultry in Asia,” said Ramey. There is no commercial poultry production in western Alaska and highly similar H9N2 virus strains have not been reported in poultry in East Asia or North America, so it is unlikely that agricultural imports influenced this result. The finding provides evidence for intercontinental movement of intact avian influenza viruses by migratory birds. The USGS recently released a publication about the detection of a novel highly pathogenic H5N8 virus in the U.S. that is highly similar to the Eurasian H5N8 viruses. This suggests that the novel re-assortment may be adapted to certain waterfowl species, enabling it to survive long migrations. That virus, and associated strains, have now spread from early detections in wild and domestic birds in Pacific states to poultry outbreaks in Minnesota, Missouri and Arkansas. “The frequency of inter-hemispheric dispersal events of avian influenza viruses by migratory birds may be higher than previously recognized,” said Ramey. While some of the samples for the project came from bird fecal samples collected from beaches at Izembek National Wildlife Refuge, most of the samples came from sport hunters. “For the past several years, we’ve worked closely with sport hunters in the fall to obtain swab samples from birds and that has really informed our understanding of wildlife disease in this area,” said Bruce Casler, formerly a biologist with the USFWS Izembek National Wildlife Refuge and a co-author of the study. Non

1. Dispersal of H9N2 influenza A viruses between East Asia
and 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, USA
b
US Geological Survey, National Wildlife Health Center, 6006 Schroeder Road, Madison, WI, USA
c
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 2015
Returned to author for revisions
9 March 2015
Accepted 10 March 2015
Keywords:
Alaska
Anas acuta
Avian influenza
Chen canagica
China
East Asia
Emperor goose
Influenza A virus
North America
Northern pintail
South Korea
a b s t r a c t
Samples were collected from wild birds in western Alaska to assess dispersal of influenza A viruses
between East Asia and North America. Two isolates shared nearly identical nucleotide identity at eight
genomic segments with H9N2 viruses isolated from China and South Korea providing evidence for
intercontinental dispersal by migratory birds.
Published by Elsevier Inc.
Introduction
Wild birds play an important role in the global epidemiology of
influenza A virus (IAV) infections, but despite extensive research
and surveillance efforts, the role of migratory species in the
intercontinental dispersal of viruses remains unclear. Previous
research on IAVs in wild birds provides evidence that sampling
birds at the margins of North America where migratory flyways of
birds from different continents overlap may be a useful strategy
for maximizing the detection probability for foreign origin IAV
genomic segments (Pearce et al., 2009). Therefore, it is plausible
that the detection probability for foreign origin viruses may also be
high at these same locations. Indeed, the only detection of a
completely Eurasian lineage IAV in North America to date occurred
in the maritime province of Newfoundland, Canada, an area with
extensive evidence for interhemispheric viral gene flow (Huang
et al., 2014). In previous research conducted in western Alaska at
Izembek National Wildlife Refuge, 70% of IAV isolates contained
Eurasian origin genomic segments providing evidence for high
levels of intercontinental viral genetic exchange (Ramey et al.,
2010). Therefore, we sampled wild birds for IAVs at this site to gain
further inference on the intercontinental exchange of viruses
between 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 a
northern pintail (Anas acuta) cloacal swab collected on 23 and 30
September 2011, respectively. BLAST results and phylogenetic
analyses suggested that these IAVs were only distantly related to
H9N2 viruses previously isolated in poultry and other IAV strains
of public health concern. Additionally, phylogenetic analyses
provided evidence that genomic segments shared genetic ancestry
with viruses originating from both North America (NS, PA) and
Eurasia (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.2015.03.028
0042-6822/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

2. (Fig. 1). Both H9N2 subtype viruses isolated in Alaska shared
nearly identical nucleotide identity (i.e. Z99.4%) at all eight
genomic segments with viruses previously isolated from wild
birds samples from Lake Dongting, China (n¼23; Wang et al.,
2012; Zhu et al., 2014) and Cheon-su Bay, South Korea (n¼1; Lee
et al., 2014; Table 1; Fig. 2).
Izembek National Wildlife Refuge provides staging habitat for
hundreds of thousands of migratory birds during autumn migration
including several species with intercontinental migratory tendencies
such as emperor geese and northern pintails (Miller et al., 2005; Hupp
et al., 2007, 2011); Fig. 2. The novel finding of nearly identical viruses
in Alaska, China, and South Korea provides direct evidence for the
dispersal of influenza A viruses between East Asia and North America
by wild birds. As there is no commercial poultry production in western
Alaska and highly similar H9N2 IAV strains have not been reported in
poultry in East Asia or North America, it is unlikely that agricultural
imports influenced this result. Furthermore, highly similar H9N2
viruses were isolated from different labs in three countries providing
no evidence that our results could be explained by laboratory artifacts.
Our results, therefore, provide evidence that IAVs associated with
disease in humans and poultry in East Asia (e.g., H5N1 and H5N8) may
be introduced to North America via migratory birds provided that such
viruses 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 in
Alaska may have been dispersed between East Asia and North
America. Thus, the frequency of inter-hemispheric dispersal events
of IAVs by migratory birds may be higher than previously recognized.
Materials and methods
During September and October of 2011–2013, cloacal swabs and
fecal samples were collected from wild birds at Izembek National
Wildlife Refuge on the Alaska Peninsula. A total of 2924 samples were
collected from 24 species over three years (Table 2). All samples were
screened using real time RT-PCR (Spackman et al., 2002) and those
providing cycle threshold values r45 were inoculated into embryo-
nated eggs for virus isolation (Woolcock, 2008). A total of 90 influenza
A virus isolates were recovered (Table 2).
Genomes (ca., 13.5 kb) of resultant isolates were amplified in a
multiplex RT-PCR following Zhou et al. (2009). Excess dNTPs and
primers were removed using ExoSAP-ITs
(USB Corporation). PCR
products were quantified with fluorometry using a high sensitivity
Quant-iT dsDNA Assay Kit (Invitrogen) and prepared for sequencing
following the Nextera XT DNA sample preparation kit protocol
(Illumina, Inc.). Indexed libraries were pooled and sequenced on the
Illumina 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. Sequences
for isolates characterized as part of this study are highlighted in red.
A.M. Ramey et al. / Virology 482 (2015) 79–8380

3. (Langmead and Salzberg, 2012) using IAV data obtained from GenBank
as 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 H9N2
subtype (124,129–247,877 reads aligned per isolate). Given the role of
H9N2 IAVs in the evolution of H5N1 and H7N9 subtype viruses
associated 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 for
further genetic characterization. GenBank accession numbers for
H9N2 viruses isolated as part of this study are: KP336376–
KP336391. Nucleotide sequences were compared to IAV lineages
available on the NCBI website using the nucleotide BLAST function.
Sequence data for the top 100 BLAST hits per genomic segment were
downloaded, aligned with sequences for H9N2 subtype isolates
identified as part of this study, and trimmed to common length
(PB2: 2154bp, PB1: 2185bp, PA: 2083bp, HA: 1519bp, NP: 1335bp, N2:
Table 1
Nucleotide 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 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
A/emperor goose/Alaska/2011-0713/2011 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
South Korea
A/bean goose/Korea/220/2011a
99.8 99.8 99.9 99.7 99.8 99.8 99.9 100.0
China
A/wild waterfowl/Dongting/C2032/2011b
99.7 99.6 99.9 99.6 99.7 99.6 99.9 99.9
A/wild waterfowl/Dongting/C2123/2011b
99.8 99.7 100.0 99.8 99.5 99.5 100.0 99.7
A/wild waterfowl/Dongting/C2148/2011b
99.8 99.7 99.9 99.8 99.5 99.5 100.0 99.9
A/wild waterfowl/Dongting/C2149/2011b
99.7 99.8 99.9 99.8 99.5 99.5 100.0 99.9
A/wild waterfowl/Dongting/C2150/2011b
99.7 99.7 100.0 99.8 99.5 99.5 100.0 99.9
A/wild waterfowl/Dongting/C2203/2011b
99.8 99.7 100.0 99.9 99.6 99.6 100.0 99.9
A/wild waterfowl/Dongting/C3109/2011b
99.7 99.8 99.9 99.6 99.6 99.6 99.9 99.7
A/wild waterfowl/Dongting/PC2539/2012b
99.5 99.6 99.9 99.6 99.5 99.5 100.0 99.9
A/wild waterfowl/Dongting/PC2540/2012b
99.5 99.8 99.9 99.6 99.6 99.6 100.0 99.9
A/wild waterfowl/Dongting/PC2553/2012b
99.5 99.6 99.9 99.7 99.6 99.6 100.0 99.9
A/wild waterfowl/Dongting/PC2559/2012b
99.5 99.7 99.9 99.6 99.6 99.6 100.0 99.9
A/wild waterfowl/Dongting/PC2560/2012b
99.7 99.7 99.8 99.6 99.6 99.6 100.0 99.7
A/wild waterfowl/Dongting/PC2562/2012b
99.6 99.7 99.8 99.8 99.6 99.6 99.9 99.6
A/wild waterfowl/Dongting/PC2574/2012b
99.5 99.6 99.9 99.6 99.6 99.6 100.0 99.9
A/wild waterfowl/Dongting/C4296/2012b
99.7 99.6 99.8 99.7 99.6 99.6 100.0 99.7
A/wild waterfowl/Dongting/C4316/2012b
99.7 99.6 99.9 99.7 99.5 99.4 100.0 99.7
A/wild waterfowl/Dongting/C4317/2012b
99.5 99.7 99.9 99.6 99.6 99.6 100.0 99.9
A/wild waterfowl/Dongting/C4326/2012b
99.4 99.7 99.9 99.7 99.6 99.6 100.0 99.9
A/wild waterfowl/Dongting/C4327/2012b
99.4 99.7 99.9 99.7 99.6 99.6 100.0 99.7
A/wild waterfowl/Dongting/C4329/2012b
99.6 99.7 99.8 99.8 99.6 99.6 99.9 99.6
A/wild waterfowl/Dongting/C4429/2012b
99.7 99.7 99.8 99.8 99.6 99.6 100.0 99.7
A/wild waterfowl/Dongting/C4430/2012b
99.4 99.7 99.9 99.7 99.6 99.6 100.0 99.9
A/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 in
Alaska, 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

4. 1261bp, M: 888bp, and NS 775bp). A maximum likelihood phylogeny
was reconstructed for each genomic segment to provide inference on
genetic ancestry in MEGA version 5.1 (Tamura et al., 2011) using the
Nucleotide: Nearest-Neighbor-Interchange method with 1000 boot-
strap replicates. Additionally, sequences for all previously characterized
IAV isolates that shared Z99% nucleotide similarity at all eight
genomic segments with sequences for H9N2 subtype viruses identi-
fied as part of this study were downloaded, aligned, and trimmed to a
common length per genomic segment (PB2: 2232bp, PB1: 2241bp, PA:
2127bp, HA: 1611bp, NP: 1459bp, N2: 1334bp, M: 925bp, and NS
778bp), and compared Alaska H9N2 viruses via a pairwise distance
matrix using MEGA (Tamura et al., 2011).
Acknowledgments
We appreciate support provided by current and former U.S. Fish
and Wildlife Service staff at Izembek National Wildlife Refuge includ-
ing Doug Damberg, Nancy Hoffman, Leticia Melendez, and Stacey
Lowe. 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 thank
Kyle Hogrefe (U.S. Geological Survey; USGS), Mary Whalen (USGS),
John Takekawa (USGS), and Kyle Spragens (USGS) for assistance with
Fig. 2. We appreciate critical reviews provided by John Pearce (USGS),
Craig Ely (USGS), and two anonymous reviewers. This work was
funded by the U.S. Geological Survey through the Wildlife Program of
the Ecosystem Mission Area. 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.
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Table 2
Number 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 10
American wigeon (Anas americana) 17 0 0 13 0 0 4 0 0 34 0 0
Black brant (Branta bernicla) 191 0 0 0 0 0 1 0 0 192 0 0
Black scoter (Melanitta americana) 0 0 0 2 0 0 7 0 0 9 0 0
Bufflehead (Bucephala albeola) 0 0 0 11 1 1 4 2 0 15 3 1
Cackling goose (Branta hutchinsii) 220 5 0 0 0 0 1 0 0 221 5 0
Common eider (Somateria mollissima) 0 0 0 3 0 0 17 0 0 20 0 0
Common goldeneye (Bucephala clangula) 0 0 0 0 0 0 2 0 0 2 0 0
Emperor goose (Chen canagica)b
99 10 2 299 11 3 265 5 1 663 26 6
Eurasian wigeon (Anas penelope) 3 0 0 8 0 0 7 0 0 18 0 0
Gadwall (Anas strepera) 4 0 0 1 0 0 9 0 0 14 0 0
Greater scaup (Aythya marila) 15 0 0 13 0 0 8 1 0 36 1 0
Glaucous-winged gull (Larus glaucescens)b
152 6 3 302 6 1 256 22 12 710 34 16
Greater white-fronted goose (Anser albifrons) 2 0 0 0 0 0 0 0 0 2 0 0
Harlequin duck (Histrionicus histrionicus) 0 0 0 14 0 0 34 0 0 48 0 0
King eider (Somateria spectabilis) 0 0 0 0 0 0 5 2 1 5 2 1
Lesser scaup (Aythya affinis) 0 0 0 2 0 0 0 0 0 2 0 0
Long-tailed duck (Clangula hyemalis) 0 0 0 3 0 0 13 1 0 16 1 0
Mallard (Anas platyrhynchos) 18 1 0 22 3 2 32 3 2 72 7 4
Northern pintail (Anser acuta) 226 38 25 245 34 16 238 32 11 709 104 52
Northern shoveler (Anas clypeata) 1 1 0 2 0 0 2 0 0 5 1 0
Red-breasted merganser (Mergus serrator) 0 0 0 3 0 0 1 0 0 4 0 0
Ring-necked duck (Aythya collaris) 0 0 0 1 0 0 0 0 0 1 0 0
White-winged scoter (Melanitta deglandi) 0 0 0 5 0 0 10 0 0 15 0 0
Total 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.
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