JVI Accepted Manuscript Posted Online 8 April 2015 J. Virol. doi:10.1128/JVI.00728-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
Title
2
Intercontinental Spread of Asian-origin H5N8 to North America through Beringia by Migratory
3
Birds
4
Running Title
5
Intercontinental Spread of HPAI H5N8 viruses
6
Authors
7
Dong-Hun Lee1, Mia Kim Torchetti2, Kevin Winker3, Hon S Ip4, Chang-Seon Song5, David E
8
Swayne1*
9
Author affiliations: 1Exotic and Emerging Avian Viral Diseases Research Unit, Southeast
10
Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research
11
Service, U.S. Department of Agriculture, Athens, Georgia, USA; 2National Veterinary Services
12
Laboratories, Science, Technology and Analysis Services, Veterinary Services, Animal and Plant
13
Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa, USA; 3University of
14
Alaska Museum, University of Alaska Fairbanks, Fairbanks, Alaska, USA; 4US Geological
15
Survey, National Wildlife Health Center, Madison, Wisconsin, USA; 5College of Veterinary
16
Medicine, Konkuk University, Seoul, Republic of Korea
17
* Contact information for Corresponding Author: USDA ARS Southeast Poultry Research
18
Laboratory, 934 College Station Rd., Athens, GA 30605, 706-546-3433 (phone), 706-546-3161
19
(fax), [email protected] (e-mail)
20
(Word count for the abstract: 84, word count for the text: 1403)
1
21
Abstract
22
Phylogenetic network analysis and understanding of waterfowl migration patterns suggest the
23
Eurasian H5N8 clade 2.3.4.4 avian influenza virus emerged in late 2013 in China, spread in early
24
2014 to South Korea and Japan, and reached Siberia and Beringia by summer 2014 via migratory
25
birds. Three genetically distinct subgroups emerged and subsequently spread along different
26
flyways during fall 2014 into Europe, North America, and East Asia, respectively. All three
27
subgroups reappeared in Japan, a wintering site for waterfowl from Eurasia and parts of North
28
America.
29
2
30
Since 1996, the Asian-origin H5N1 A/goose/Guangdong/1/1996 (Gs/GD) lineage of
31
highly pathogenic avian influenza viruses (HPAIV) has caused outbreaks in poultry, infections in
32
wild birds, and clinical, often fatal, cases in humans in Eurasia and North Africa (1). Historically,
33
geographic barriers appeared to limit the spread of low pathogenicity avian influenza viruses
34
(LPAIV) through migratory aquatic birds between the Old and New worlds, allowing distinct
35
lineages of Eurasian and North American viruses to evolve, but such barriers were not complete
36
as occasional spillovers of gene segments occurred (2). Migratory birds have previously been
37
implicated in the spread of H5N1 HPAIV in Eurasian (3), but geographic barriers, such as the
38
Bering Strait and Chukchi Sea, prevented spread of the Gs/GD lineage virus to North America (4,
39
5).
40
In early 2014, outbreaks of novel reassortant H5N8 HPAIV of the Gs/GD lineage H5
41
clade 2.3.4.4 were reported in South Korea (6), and aquatic migratory birds were strongly
42
suspected to play a key role in the introduction of this strain from China and in the subsequent
43
spread during the outbreak (7). In late autumn 2014, H5N8 clade 2.3.4.4 was reported in Europe
44
and East Asia. Concurrently, this virus lineage was detected in Washington in captive falcons,
45
wild birds, and backyard poultry (8). Verhagen et al. hypothesized that the timing and direction
46
of intercontinental spread coincided with autumn bird migration out of Russia; the H5N8 virus
47
was identified in a long-distance migrant bird in Russia in September 2014 and subsequently in
48
Japan, Europe, and the west coast of North America (9). Similarly, wide geographic
49
dissemination of the Gs/GD lineage virus was seen between 2005 and 2006, as clade 2.2 viruses
50
spread from Qinghai Lake, China to Siberia and then to various countries of Asia, Europe, and
51
Africa (2, 10). In addition, another novel reassortant HPAIV of H5 clade 2.3.4.4 H5N2 (Eurasian
52
[EA] polymerase basic 2 [PB2], polymerase acidic [PA], hemagglutinin [HA], matrix [M], and
3
53
non-structural proteins [NS] gene segments) with North American [AM] lineage neuraminidase
54
(NA), polymerase basic 1 (PB1), and nucleoprotein (NP) gene segments was identified as the
55
cause of outbreaks in poultry farms in British Columbia and subsequently detected in wild
56
waterfowl in Oregon near the Washington state border (8, 11). Both the H5N8 and H5N2-
57
reassortant have been detected in wild waterfowl, as well as backyard and commercial poultry
58
along the Pacific Flyway (8, 12).
59
In this study, a molecular epidemiological approach based on genome sequences and
60
outbreak information from Asia, Europe, and North America during 2014 was used to describe
61
the pattern of global spread of this Asian-origin H5N8-reassortant clade 2.3.4.4 HPAIV.
62
For phylogenetic analysis, nucleotide sequences used in this study (Supplemental Table 1)
63
were deposited in GISAID (www.gisaid.org), as acknowledged in Supplemental Table 2, and
64
GenBank (www.ncbi.nlm.nih.gov/genomes/FLU). Neighbor-joining (NJ) phylogenetic trees
65
were constructed for each of the 8 gene segments. The median-joining phylogenetic network and
66
time-scaled phylogenetic tree of the HA gene were constructed using nucleotide sequences
67
genetically close to H5 viruses identified in 2014 based on the result of NJ phylogenetic
68
analyses. Detailed methods are described in the Supplemental Materials.
69
The South Korean H5N8 outbreak in January 2014 was the first reported case of the
70
H5N8 clade 2.3.4.4 outside of China. During this outbreak, two distinct H5N8 groups were
71
identified, Groups A (Buan2-like) and B (Gochang1-like) viruses (figures s1-s9) (6). Group A
72
viruses predominated and were identified in poultry farm outbreaks and subsequently along
73
migratory bird pathways (7). This group now comprises Chinese, Russian, South Korean,
74
Japanese, European, and North American H5N8 2.3.4.4 viruses representing an “Intercontinental
4
75
Group A (icA).” The icA-group H5 viruses further evolved into three distinct subgroups, icA1,
76
icA2, and icA3, by late 2014 (Figures 1 and s2). The icA1 subgroup is composed of H5N8
77
viruses from Europe and Russia from late 2014, and 3 viruses detected in Japan during
78
December 2014. The icA-2 subgroup is composed of H5N8 as well as H5 clade 2.3.4.4 North
79
American HPAIV reassortants (H5N2 and H5N1) detected in North America starting in late
80
2014 (12). A Japanese virus, A/crane/Kagoshima/KU1/2014 (H5N8), detected in November
81
2014, is also within the icA-2 subgroup. The icA3 subgroup is composed of H5N8 viruses from
82
Japan in December 2014 and Korea in January 2015 (Figure 1).
83
An important route of the East Asian Flyway goes from China to Japan (China–South
84
Korea–Russian Far East–Japan), and the timing of H5N1 outbreaks and virus movements have
85
been closely associated within this flyway (13). In addition, satellite-tracking of the movement
86
patterns of wild birds in East Asia showed that wild ducks followed the East Asian Flyway along
87
the coast to breeding areas in eastern Russia (14). The phylogenetic analyses of early and late
88
2014 icA-H5N8 (Figure 1 and 2) and aquatic bird migration patterns suggests that viruses likely
89
moved to parts of Siberia and Beringia via migratory birds in spring 2014, evolved into
90
subgroups during the breeding season, and subsequently spread along different flyways during
91
autumn migrations into Europe, North America, and East Asia (Figure 2). For example, the
92
earliest member of the icA1 viruses was isolated in September 2014 from Russia
93
[A/wigeon/Sakha/1/2014(H5N8)] prior to the November outbreaks in Europe (Figure 2).
94
Interestingly, viruses from all genetic subgroups have subsequently been detected in Japan
95
during late 2014, where some waterfowl from Eurasia and North America winter.
96
The relatively high incidence of icA isolates from migratory waterfowl suggests that they
97
are important hosts in both the maintenance and spread of these viruses. Areas such as the 5
98
Beringian Crucible (Alaska and the Russian Far East), where the New World and Old World
99
migration systems share common breeding grounds, provide ample opportunity for virus
100
reassortment due to the large-scale intercontinental associations of waterfowl; it is estimated that
101
1.5–2.9 million aquatic migratory birds move from Asia to Alaska annually (4, 5). While
102
influenza surveillance in Alaskan waterfowl species found predominantly LPAIV (15), frequent
103
reassortment was noted in one study of northern pintails, with nearly half (44.7%) of LPAIV
104
having at least one gene segment demonstrating closer relatedness to Eurasian than to North
105
American LPAIV genes (16). In contrast, relatively few aquatic birds move from Asia to North
106
America south of Alaska, and relatively few from Alaska are associated with the Atlantic flyway,
107
these being birds that winter in the eastern U.S. and come northwest in spring (17). Although the
108
North Atlantic area is a potential route for migratory birds to transport avian influenza viruses
109
between North America and Europe (18), the possibility of viral spread through Atlantic rim was
110
excluded based upon the timing of the detections, the very low level of viral gene flow between
111
Pacific and Atlantic flyways (19), and because the icA-H5 viruses identified in North America
112
were genetically closer to East Asian viruses than European viruses. Additionally, there have
113
been no detections of icA-H5 virus in poultry or wild aquatic bird in the Atlantic flyway, despite
114
ongoing routine surveillance. Detection of icA-H5N8 HPAIV from apparently healthy wild
115
waterfowl from multiple countries and high viral shedding in the absence of illness in
116
experimentally icA-H5N8 infected waterfowl (20) support the theory that the global spread of
117
icA-H5N8 HPAIV has been driven by migratory birds. While the closest relatives of the icA-
118
H5N2 viruses appear to be South Korean/Japanese H5N8 viruses, introduction of icA-H5N8 via
119
illegal importation of poultry or poultry products into either Europe or North America seems
120
unlikely as Koreans and Japanese have low cultural preference for live poultry market systems,
6
121
which in mainland Asia is the source of illegally moved poultry and poultry products, plus the
122
added difficulty of smuggling live birds or uncooked product across the Pacific Ocean with
123
current transportation security measures. Examination of the timing and genetic footprints of
124
these viruses through phylogenetic analysis and waterfowl migratory patterns support the
125
hypothesis that icA-H5N8 clade 2.3.4.4 was introduced into North America through the
126
Beringian Crucible via intercontinental associations of waterfowl (5, 6). In addition, reassortment
127
of the icA2-H5N8 with North American LPAIV has already occurred within the Pacific Flyway,
128
generating reassortant icA2-H5N2 and icA-H5N1 HPAIV. The icA2-H5N8 has been detected as
129
far south as California and Utah, the icA2-H5N2-reassortant in BC, northwestern US states and
130
more recently mid-western (Mississippi Flyway) states, and the icA2-H5N1-reassortant in a wild
131
bird in Washington and a backyard flock in BC (8). Enhanced active surveillance is required to
132
monitor the spread and potential for onward reassortment of icA-group 2.3.4.4; such efforts
133
could further the epidemiological understanding of HPAIV and the design of improved
134
prevention strategies.
135 136
Acknowledgments
137
Seung-Hee Lee is thanked for providing Figure 2. The authors would like to acknowledge the
138
contributions of dedicated staff at their respective institutions including Robert Dusek and David
139
Blehert (NWHC), Kira Moresco (SEPRL), Mary Lea Killian (NVSL), and Jung-Hoon Kwon
140
(Konkuk University).
141 142
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Kang HM, Lee EK, Song BM, Jeong J, Choi JG, Jeong JJ, Moon OK, Yoon H, Cho YM, Kang YM, Lee HS, Lee YJ. 2015. Novel Reassortant Influenza A(H5N8) Viruses
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11
215
Figure Legends
216
Figure 1. Median-joining phylogenetic network of HPAI H5 clade 2.3.4.4 viruses. The median-
217
joining network was constructed from the HA gene. This network includes all the most
218
parsimonious trees linking the sequences. Each unique sequence is represented by a circle sized
219
relative to its frequency in the dataset. Branch length is proportional to the number of mutations.
220
Isolates are colored according to the origin and season of the sample; red inner circle: poultry
221
farm isolates, purple inner circle: wild bird isolates, black outer circle: early 2014 isolates, and
222
blue outer circle: late 2014 isolates.
223 224
Figure 2. Geographic map showing movement of H5N8 HPAIV in Asia, Europe, and North
225
America in relation to regional waterfowl migration routes.
226 227
12
1
Title
2
Intercontinental Spread of Asian-origin H5N8 to North America through Beringia by Migratory
3
Birds
4
Running Title
5
Intercontinental Spread of HPAI H5N8 viruses
6
Authors
7
Dong-Hun Lee1, Mia Kim Torchetti2, Kevin Winker3, Hon S Ip4, Chang-Seon Song5, David E
8
Swayne1*
9
Author affiliations: 1Exotic and Emerging Avian Viral Diseases Research Unit, Southeast
10
Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research
11
Service, U.S. Department of Agriculture, Athens, Georgia, USA; 2National Veterinary Services
12
Laboratories, Science, Technology and Analysis Services, Veterinary Services, Animal and Plant
13
Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa, USA; 3University of
14
Alaska Museum, University of Alaska Fairbanks, Fairbanks, Alaska, USA; 4US Geological
15
Survey, National Wildlife Health Center, Madison, Wisconsin, USA; 5College of Veterinary
16
Medicine, Konkuk University, Seoul, Republic of Korea
17
* Contact information for Corresponding Author: USDA ARS Southeast Poultry Research
18
Laboratory, 934 College Station Rd., Athens, GA 30605, 706-546-3433 (phone), 706-546-3161
19
(fax), [email protected] (e-mail)
20
(Word count for the abstract: 84, word count for the text: 1403)
1
21
Abstract
22
Phylogenetic network analysis and understanding of waterfowl migration patterns suggest the
23
Eurasian H5N8 clade 2.3.4.4 avian influenza virus emerged in late 2013 in China, spread in early
24
2014 to South Korea and Japan, and reached Siberia and Beringia by summer 2014 via migratory
25
birds. Three genetically distinct subgroups emerged and subsequently spread along different
26
flyways during fall 2014 into Europe, North America, and East Asia, respectively. All three
27
subgroups reappeared in Japan, a wintering site for waterfowl from Eurasia and parts of North
28
America.
29
2
30
Since 1996, the Asian-origin H5N1 A/goose/Guangdong/1/1996 (Gs/GD) lineage of
31
highly pathogenic avian influenza viruses (HPAIV) has caused outbreaks in poultry, infections in
32
wild birds, and clinical, often fatal, cases in humans in Eurasia and North Africa (1). Historically,
33
geographic barriers appeared to limit the spread of low pathogenicity avian influenza viruses
34
(LPAIV) through migratory aquatic birds between the Old and New worlds, allowing distinct
35
lineages of Eurasian and North American viruses to evolve, but such barriers were not complete
36
as occasional spillovers of gene segments occurred (2). Migratory birds have previously been
37
implicated in the spread of H5N1 HPAIV in Eurasian (3), but geographic barriers, such as the
38
Bering Strait and Chukchi Sea, prevented spread of the Gs/GD lineage virus to North America (4,
39
5).
40
In early 2014, outbreaks of novel reassortant H5N8 HPAIV of the Gs/GD lineage H5
41
clade 2.3.4.4 were reported in South Korea (6), and aquatic migratory birds were strongly
42
suspected to play a key role in the introduction of this strain from China and in the subsequent
43
spread during the outbreak (7). In late autumn 2014, H5N8 clade 2.3.4.4 was reported in Europe
44
and East Asia. Concurrently, this virus lineage was detected in Washington in captive falcons,
45
wild birds, and backyard poultry (8). Verhagen et al. hypothesized that the timing and direction
46
of intercontinental spread coincided with autumn bird migration out of Russia; the H5N8 virus
47
was identified in a long-distance migrant bird in Russia in September 2014 and subsequently in
48
Japan, Europe, and the west coast of North America (9). Similarly, wide geographic
49
dissemination of the Gs/GD lineage virus was seen between 2005 and 2006, as clade 2.2 viruses
50
spread from Qinghai Lake, China to Siberia and then to various countries of Asia, Europe, and
51
Africa (2, 10). In addition, another novel reassortant HPAIV of H5 clade 2.3.4.4 H5N2 (Eurasian
52
[EA] polymerase basic 2 [PB2], polymerase acidic [PA], hemagglutinin [HA], matrix [M], and
3
53
non-structural proteins [NS] gene segments) with North American [AM] lineage neuraminidase
54
(NA), polymerase basic 1 (PB1), and nucleoprotein (NP) gene segments was identified as the
55
cause of outbreaks in poultry farms in British Columbia and subsequently detected in wild
56
waterfowl in Oregon near the Washington state border (8, 11). Both the H5N8 and H5N2-
57
reassortant have been detected in wild waterfowl, as well as backyard and commercial poultry
58
along the Pacific Flyway (8, 12).
59
In this study, a molecular epidemiological approach based on genome sequences and
60
outbreak information from Asia, Europe, and North America during 2014 was used to describe
61
the pattern of global spread of this Asian-origin H5N8-reassortant clade 2.3.4.4 HPAIV.
62
For phylogenetic analysis, nucleotide sequences used in this study (Supplemental Table 1)
63
were deposited in GISAID (www.gisaid.org), as acknowledged in Supplemental Table 2, and
64
GenBank (www.ncbi.nlm.nih.gov/genomes/FLU). Neighbor-joining (NJ) phylogenetic trees
65
were constructed for each of the 8 gene segments. The median-joining phylogenetic network and
66
time-scaled phylogenetic tree of the HA gene were constructed using nucleotide sequences
67
genetically close to H5 viruses identified in 2014 based on the result of NJ phylogenetic
68
analyses. Detailed methods are described in the Supplemental Materials.
69
The South Korean H5N8 outbreak in January 2014 was the first reported case of the
70
H5N8 clade 2.3.4.4 outside of China. During this outbreak, two distinct H5N8 groups were
71
identified, Groups A (Buan2-like) and B (Gochang1-like) viruses (figures s1-s9) (6). Group A
72
viruses predominated and were identified in poultry farm outbreaks and subsequently along
73
migratory bird pathways (7). This group now comprises Chinese, Russian, South Korean,
74
Japanese, European, and North American H5N8 2.3.4.4 viruses representing an “Intercontinental
4
75
Group A (icA).” The icA-group H5 viruses further evolved into three distinct subgroups, icA1,
76
icA2, and icA3, by late 2014 (Figures 1 and s2). The icA1 subgroup is composed of H5N8
77
viruses from Europe and Russia from late 2014, and 3 viruses detected in Japan during
78
December 2014. The icA-2 subgroup is composed of H5N8 as well as H5 clade 2.3.4.4 North
79
American HPAIV reassortants (H5N2 and H5N1) detected in North America starting in late
80
2014 (12). A Japanese virus, A/crane/Kagoshima/KU1/2014 (H5N8), detected in November
81
2014, is also within the icA-2 subgroup. The icA3 subgroup is composed of H5N8 viruses from
82
Japan in December 2014 and Korea in January 2015 (Figure 1).
83
An important route of the East Asian Flyway goes from China to Japan (China–South
84
Korea–Russian Far East–Japan), and the timing of H5N1 outbreaks and virus movements have
85
been closely associated within this flyway (13). In addition, satellite-tracking of the movement
86
patterns of wild birds in East Asia showed that wild ducks followed the East Asian Flyway along
87
the coast to breeding areas in eastern Russia (14). The phylogenetic analyses of early and late
88
2014 icA-H5N8 (Figure 1 and 2) and aquatic bird migration patterns suggests that viruses likely
89
moved to parts of Siberia and Beringia via migratory birds in spring 2014, evolved into
90
subgroups during the breeding season, and subsequently spread along different flyways during
91
autumn migrations into Europe, North America, and East Asia (Figure 2). For example, the
92
earliest member of the icA1 viruses was isolated in September 2014 from Russia
93
[A/wigeon/Sakha/1/2014(H5N8)] prior to the November outbreaks in Europe (Figure 2).
94
Interestingly, viruses from all genetic subgroups have subsequently been detected in Japan
95
during late 2014, where some waterfowl from Eurasia and North America winter.
96
The relatively high incidence of icA isolates from migratory waterfowl suggests that they
97
are important hosts in both the maintenance and spread of these viruses. Areas such as the 5
98
Beringian Crucible (Alaska and the Russian Far East), where the New World and Old World
99
migration systems share common breeding grounds, provide ample opportunity for virus
100
reassortment due to the large-scale intercontinental associations of waterfowl; it is estimated that
101
1.5–2.9 million aquatic migratory birds move from Asia to Alaska annually (4, 5). While
102
influenza surveillance in Alaskan waterfowl species found predominantly LPAIV (15), frequent
103
reassortment was noted in one study of northern pintails, with nearly half (44.7%) of LPAIV
104
having at least one gene segment demonstrating closer relatedness to Eurasian than to North
105
American LPAIV genes (16). In contrast, relatively few aquatic birds move from Asia to North
106
America south of Alaska, and relatively few from Alaska are associated with the Atlantic flyway,
107
these being birds that winter in the eastern U.S. and come northwest in spring (17). Although the
108
North Atlantic area is a potential route for migratory birds to transport avian influenza viruses
109
between North America and Europe (18), the possibility of viral spread through Atlantic rim was
110
excluded based upon the timing of the detections, the very low level of viral gene flow between
111
Pacific and Atlantic flyways (19), and because the icA-H5 viruses identified in North America
112
were genetically closer to East Asian viruses than European viruses. Additionally, there have
113
been no detections of icA-H5 virus in poultry or wild aquatic bird in the Atlantic flyway, despite
114
ongoing routine surveillance. Detection of icA-H5N8 HPAIV from apparently healthy wild
115
waterfowl from multiple countries and high viral shedding in the absence of illness in
116
experimentally icA-H5N8 infected waterfowl (20) support the theory that the global spread of
117
icA-H5N8 HPAIV has been driven by migratory birds. While the closest relatives of the icA-
118
H5N2 viruses appear to be South Korean/Japanese H5N8 viruses, introduction of icA-H5N8 via
119
illegal importation of poultry or poultry products into either Europe or North America seems
120
unlikely as Koreans and Japanese have low cultural preference for live poultry market systems,
6
121
which in mainland Asia is the source of illegally moved poultry and poultry products, plus the
122
added difficulty of smuggling live birds or uncooked product across the Pacific Ocean with
123
current transportation security measures. Examination of the timing and genetic footprints of
124
these viruses through phylogenetic analysis and waterfowl migratory patterns support the
125
hypothesis that icA-H5N8 clade 2.3.4.4 was introduced into North America through the
126
Beringian Crucible via intercontinental associations of waterfowl (5, 6). In addition, reassortment
127
of the icA2-H5N8 with North American LPAIV has already occurred within the Pacific Flyway,
128
generating reassortant icA2-H5N2 and icA-H5N1 HPAIV. The icA2-H5N8 has been detected as
129
far south as California and Utah, the icA2-H5N2-reassortant in BC, northwestern US states and
130
more recently mid-western (Mississippi Flyway) states, and the icA2-H5N1-reassortant in a wild
131
bird in Washington and a backyard flock in BC (8). Enhanced active surveillance is required to
132
monitor the spread and potential for onward reassortment of icA-group 2.3.4.4; such efforts
133
could further the epidemiological understanding of HPAIV and the design of improved
134
prevention strategies.
135 136
Acknowledgments
137
Seung-Hee Lee is thanked for providing Figure 2. The authors would like to acknowledge the
138
contributions of dedicated staff at their respective institutions including Robert Dusek and David
139
Blehert (NWHC), Kira Moresco (SEPRL), Mary Lea Killian (NVSL), and Jung-Hoon Kwon
140
(Konkuk University).
141 142
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Figure Legends
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Figure 1. Median-joining phylogenetic network of HPAI H5 clade 2.3.4.4 viruses. The median-
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joining network was constructed from the HA gene. This network includes all the most
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parsimonious trees linking the sequences. Each unique sequence is represented by a circle sized
219
relative to its frequency in the dataset. Branch length is proportional to the number of mutations.
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Isolates are colored according to the origin and season of the sample; red inner circle: poultry
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farm isolates, purple inner circle: wild bird isolates, black outer circle: early 2014 isolates, and
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blue outer circle: late 2014 isolates.
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Figure 2. Geographic map showing movement of H5N8 HPAIV in Asia, Europe, and North
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America in relation to regional waterfowl migration routes.
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