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Avian coronavirus in wild aquatic birds - Journal of...
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Avian coronavirus in wild aquatic birds 1
Daniel K. W. Chu1, Connie Y. H. Leung1, Martin Gilbert2, Priscilla H. Joyner2, Erica M. Ng1, 2
Tsemay M Tse1, Yi Guan1, Joseph S. M. Peiris1,3*, Leo L. M. Poon1* 3
4
1 State Key Laboratory for Emerging Infectious Diseases, Department of Microbiology and 5
the Research Centre of Infection and Immunology, The University of Hong Kong, Hong 6
Kong Special Administrative Region, China; 7
2 Wildlife Conservation Society, Cambodia; 8
3 HKU-Pasteur Research Centre, Hong Kong Special Administrative Region, China. 9
*Corresponding authors: Prof JSM Peiris 10 Department of Microbiology, 11 University Pathology Building, 12 Queen Mary Hospital, Pokfulam, 13 Hong Kong SAR, 14 China 15 E-mail: [email protected] 16 17 Dr LLM Poon 18
Department of Microbiology, 19 University Pathology Building, 20 Queen Mary Hospital, Pokfulam, 21 Hong Kong SAR, 22 China 23 E-mail: [email protected] 24 25 26
Running title: Avian coronaviruses in wild birds 27
Word Count: 100 (Abstract) 28
1599 (Main Text)29
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Virol. doi:10.1128/JVI.05838-11 JVI Accepts, published online ahead of print on 28 September 2011
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Abstract 30
We detected a high prevalence (12.5%) of novel avian coronaviruses in aquatic wild birds. 31
Phylogenetic analyses of these coronaviruses suggest that there is a diversity of 32
gammacoronaviruses and deltacoronaviruses circulating in birds. Gammacoronaviruses were 33
predominantly found in Anseriformes, whereas deltacoronaviruses could be detected in 34
Ciconiiformes, Pelecaniformes and Anseriformes in this study. We observed that there are 35
frequent interspecies transmissions of gammacoronaviruses between duck species. By contrast, 36
deltacoronaviruses may have more stringent host specificities. Our analysis on these avian viral 37
and host mtDNA sequences also suggest that some, but not all, coronaviruses may have co-38
evolved with birds from the same order. 39
(100 words) 40
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Main Text 44
Coronaviruses are important pathogens of birds and mammals, including humans, and are 45
previously classified into 3 genera. Alphacoronaviruses and betacoronaviruses are found in 46
mammals, whereas gammacoronaviruses are primarily detected in birds. Surveillance of 47
coronaviruses in wild mammals has led to the discovery of a significant number of novel 48
alphacoronaviruses and betacoronaviruses in bats (18, 23, 27). By contrast, relatively little is 49
known about the biology of avian coronaviruses in wildlife. Gough and colleagues identified a 50
parrot coronavirus that is genetically distinct from alpha-, beta- and gamma- coronaviruses in 51
2006 (10). Additional novel coronaviruses that are genetically similar to the parrot coronavirus 52
were subsequently detected in mammals and terrestrial birds (9, 26). Viruses of this novel 53
lineage have been recently proposed to form a new genus, provisionally named 54
Deltacoronavirus 55
(http://talk.ictvonline.org/files/proposals/taxonomy_proposals_vertebrate1/m/vert02/3257.aspx) 56
(8). Besides, findings from other studies suggested that there is diversity of coronaviruses 57
circulating in wild birds (13, 17). These findings have prompted us to launch wild bird 58
surveillance in Hong Kong and Cambodia for avian coronaviruses. 59
60
From November 2009 to January 2010, site visits to Mai Po Marshes, Hong Kong were 61
organized bi-weekly. Isolated fresh bird droppings on pond shores were sampled and stored in 62
individual vials containing viral transport medium (VTM) (15). For samples collected in 63
Cambodia, cloacal swabs from captured free-ranging birds were collected in vials with VTM in 64
2008. RNA of these samples were extracted and reverse transcribed as described (5). 65
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Complementary DNA (cDNA) was subjected to a pan-coronavirus nested PCR (nPCR) for RdRp 66
sequence. Briefly, cDNA was amplified in a first-round PCR (forward primer: 5'-67
GGKTGGGAYTAYCCKAARTG-3' and reverse primer: 5'-GGKTGGGAYTAYCCKAARTG-68
3'; 40 cycles of 94 °C for 20 sec, 48°C for 30 sec and 72°C for 50 sec). PCR product was then 69
amplified in a second-round PCR with the amplification condition identical to the first-round 70
PCR, except that a new set of primers was used in the assay (forward primer 5'-71
GGTTGGGACTATCCTAAGTGTGA-3' and reverse primer 5'-72
CCATCATCAGATAGAATCATCAT-3'). The final PCR products (440 base pairs) were 73
analyzed by sequencing. 74
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The bird species studied in Cambodia were identified immediately upon capture by expert 76
ornithologists (20). A well validated nPCR assay targeting cDNA of mitochondrial COX1 77
sequence was used to identify the host species of representative Hong Kong samples (4, 24). 78
Furthermore, a similar nPCR assay using alternative primers for COX1 sequence was used for 79
result reconfirmations (First round: forward primer 5'-GAYATRGCKTTYCCKCGKATRAA-3' 80
and reverse primer 5'-ATKGCYCAKACYATKCCYATRTA-3'; Second round: forward primer 81
5'-GCKTTTCCKCGKATRAAYAAYAT-3' and reverse primer 5'-82
CCYATRTAKCCRAAKGGYTCYTT-3'). The deduced mtDNA sequences were blast against 83
database in GenBank and BOLD (19) for host identification. 84
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A total of 658 samples collected in Hong Kong were tested. Ninety-nine (15.0%) of these 86
samples were RT-PCR positive for coronavirus. Positive samples were detected in each visit, 87
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with positive rates ranging from 13.2% to 22.6%. Host mtDNA was used to determine the host 88
species of all the positive samples. In order to have an overview on the species diversity of our 89
sample collections, 94 randomly selected coronavirus negative specimens were also subjected to 90
the mtDNA barcoding assays. Comparable host mtDNA detection rates were found in 91
coronavirus positive (81.8%) and coronavirus negative (74.5%) samples. Species that were found 92
to be positive for coronaviruses are summarized in Table 1. The range of species that were 93
coronavirus positive was broadly similar to the one from coronavirus negative ones (Table 1) 94
(P>0.05, t-test). None of the mtDNA positive samples were found to have ambiguous results in 95
the barcoding assays. 96
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Cloacal swabs (N=263) were collected from pond herons, lesser whistling ducks and ruddy-98
breasted crakes in Cambodia. Coronavirus positive reactions were detected in 13.0% (16/123) of 99
pond herons and 3.0% (1/33) of lesser whistling ducks (Table 1). 100
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All the coronaviruses identified in this study could be phylogenetically classified as 102
gammacoronaviruses and deltacoronaviruses (Fig. 1; Supplementary Fig. 1). This observed 103
phylogeny is comparable with the split off between alphacoronaviruses and betacoronaviruses in 104
mammals. To roughly estimate the genetic difference between these partial RdRp sequences, we 105
selected 10 representative viruses from each genus to estimate the genetic distances between 106
these viruses (data not shown). The genetic distance between these two groups of avian 107
coronaviruses is similar to the inter-genus distances between coronaviruses from different genera, 108
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and this genetic distance is significantly higher than those observed within the same genus (Fig. 109
2, P <0.005). 110
111
Gammacoronaviruses found in this study were detected from wild waterfowl of orders 112
Anseriformes. These viruses include those from little whistling duck, tufted duck, common teal, 113
northern shoveler, Eurasian wigeon and northern pintail. Previously known representative 114
coronaviruses in this group include avian infectious bronchitis virus (AIBV), peafowl 115
coronavirus, turkey coronavirus, goose coronaviruses, pintail coronaviruses, gull coronavirus and 116
a virus collected from a sick beluga whale (2, 16, 17). By contrast, novel deltacoronaviruses 117
found in this study were detected in waterbirds from orders Ciconiiformes, Pelecaniformes and 118
Anseriformes. These viruses include those from grey herons, pond herons, great cormorants, 119
black-faced spoonbills and several duck species (Anas spp.). In addition, the prevalence of 120
deltacoronavirus was found to be much lower than that of gammacoronaviruses in the studied 121
duck samples. Of 63 avian coronaviruses detected from the Anas spp., only 6 of them (9.5%) are 122
deltacoronaviruses (Table 1). Previously known members in this group include CoV-HKU11 123
from bulbuls, CoV-HKU12 from thrushs, CoV-HKU13 from munias and Asian leopard cat 124
coronavirus (9, 26). 125
126
Our findings demonstrate that wild birds are major reservoirs for a wide range of gamma- and 127
delta- coronaviruses. Some of the studied species are found to have a high prevalence of 128
coronaviruses (Table 1). It is possible that these avian coronaviruses do not cause severe illness 129
to their hosts, thereby allowing themselves to be endemic in some avian populations. 130
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Interestingly, gammacoronaviruses which are similar to those discovered in Bering Strait area in 131
2005 (17) were also detected in our study. In particular, viruses that have identical partial RdRp 132
sequences were detected in northern pintails in both studied sites (Fig. 1, J1404 and Pintail CoV 133
PBA-25), indicating that some avian coronaviruses can persist in a specific bird species and are 134
carried by these migrating birds to other geographical locations 135
(http://www.werc.usgs.gov/ProjectSubWebPage.aspx?SubWebPageID=8&ProjectID=37&List=136
SubWebPages&Web=Project_37&Title=Movements) . 137
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We also noted that not all of our studied species were positive for coronaviruses. Many of these 139
species that were apparently negative may be a result of the small sample sizes. However, we 140
tested a substantial number of samples from ruddy-breasted crakes (N= 80) that were all RT-141
PCR negative for coronavirus. The ruddy-breasted crake is a permanent-resident bird in 142
Cambodia (20). By contrast, the positive rates of avian coronavirus in some of our studied 143
migratory species, such as ducks and great cormorant, are extremely high (Table 1). Previous 144
studies on migratory waterbirds and resident terrestrial birds have similar findings (17, 26). It is 145
not known whether these observations are due to the intrinsic biological differences between 146
coronaviruses circulating in these two distinct bird populations and/or the behavioral and 147
physiological differences between these two groups of birds (1). 148
149
The phylogenetic topology of gammacoronaviruses is very different from the one of 150
deltacoronaviruses. The gammacoronaviruses sampled from ducks, sandpipers and gulls form a 151
distinct clade (Fig. 1). The RdRp genes from this clade of viruses are genetically closely related, 152
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and the overall RdRp gene sequence homology differs by <7%. In this virus cluster, the same 153
avian species may contain viruses that are genetically heterogeneous (e.g. J1404, J1407, J1435 154
and J1451 from northern pintails from the same sampling day), whereas viruses that are 155
genetically very similar could be detected from different duck species (e.g. J0807 and J1616). 156
These findings suggest that there are frequent intergenus and interspecies transmissions of this 157
group of gammacoronaviruses amongst these avian species. By contrast, viruses in the 158
deltacoronavirus group were found to have distinct phylogenetic branches (e.g. J1517 and J0982 159
from great cormorants sampled at different dates), indicating that coronaviruses in this group 160
have a more stringent host specificity than those in the gamma group. Overall, these results 161
suggest that the ecology of gammacoronaviruses and deltacoronaviruses may be very different. 162
163
It was previously reported that there is a co-evolved virus-host relationship between 164
vespertilionid bats and their coronaviruses (7). We used some of our representative sequences to 165
perform a similar analysis with avian coronaviruses and their hosts (Fig. 4). It is observed that 166
both gammacoronaviruses and deltacoronaviruses could be detected in Superorder Galloanserae 167
and Neoaves (12), with the gamma group predominantly found in Galloanserae and delta group 168
predominantly found in Neoaves (Supplementary Fig. 1, highlighted in blue and green, 169
respectively). Some coronaviruses and their hosts even fall in the same monophyletic subclade 170
in the corresponding trees (e.g. gammacoronaviruses from ducks and deltacoronaviruses from 171
Passeriformes) (Fig. 4). We also observed that there are some exceptional cases in this study. For 172
example, gammacoronaviruses and deltacoronaviruses were detected in Charadriiformes and 173
Anseriformes, respectively (Supplementary Fig. 1, highlighted in red). As our sample set was 174
limited to just a few avian orders, additional surveillance work is needed to reveal the full picture 175
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of avian coronavirus ecology. Nonetheless, our findings suggest that at least some avian 176
coronaviruses may have circulated and co-evolved with specific orders or subsets of avian hosts. 177
To test this hypothesis, we have also examined additional coronaviruses reported to be detected 178
in different avian species (3, 6, 10, 13, 14, 17, 21, 26). Our preliminary analyses indicate that, 179
except ducks which can be infected by both gamma and delta groups, all the examined bird 180
orders are only positive for either gammacoronaviruses or deltacoronaviruses (data not shown). 181
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Our data suggest that circulation of coronaviruses in apparently healthy populations of wild 183
aquatic birds is more common than previously recognized. Additional coronavirus surveillance 184
in birds of different orders and full genome analysis of avian coronaviruses may help to better 185
understand the evolution of coronaviruses. 186
187
Acknowledgements 188
We would like to thank Kai-Chi Chow, Chuk-Kwan Ho and Yu-On Wu at the University of 189
Hong Kong for sample collection. This project was supported by National Institutes of Health 190
(NIAID contract HHSN266200700005C) and EMPERIE (grant no. EU FP7 223498). 191
192
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Table 1. Detection of coronavirus in avian samples 271
Location Species Total number
(Positive/Negative) Number positive for
gammacoronavirus (%) Number positive for deltacoronavirus (%)
Hong Kong*
Common teal (Anas crecca) 18 (10/8) 9 (50.0) 1 (5.6) Northern shoveler (Anas clypeata) 31(24/7) 22 (71.0) 2 (6.5) Eurasian wigeon (Anas penelope) 24 (10/14) 9 (37.5) 1 (4.2) Northern pintail (Anas acuta) 38 (17/21) 16 (42.1) 1 (2.6) Tufted duck (Aythya fuligula) 1 (1/0) 1 (100) 0 American wigeon (Anas americana) 1 (1/0) 0 1 (100) Grey heron (Ardea cinerea) 10 (4/6) 0 4 (40.0) Night heron (Nycticorax nycticorax) 1 (0/1) 0 0 Great cormorant (Phalacrocorax carbo) 24 (13/11) 0 13(54.2) Black-faced spoonbill (Platalea minor) 3 (1/2) 0 1 (33.3)
Cambodia Pond heron (Ardeola bacchus/speciosa) 123 (16/107) 0 16 (13.0) Lesser whistling duck (Dendrocygna javanica) 33 (1/32) 1 (3.0) 0 Ruddy-breasted crake (Porzana fusca) 80 (80/0) 0 0
*Only samples that were informative in the DNA barcoding analysis are listed. 272
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Figure legends 273
Figure 1. Phylogenetic analysis of alphacoronaviruses, betacoronaviruses, gammacoronaviruses 274
and deltacoronaviruses. Partial viral RdRp (360 bps) sequences were used in the analysis. 275
Sequences were aligned and edited manually by clustalW (25). The best evolution model 276
describing an alignment was determined by Mega5 (22). Phylogenetic trees were generated using 277
Phyml with the best substitution model selected, and with aLRT statistics SH-like branch 278
supports (11). All novel virus gene sequences generated in this study were deposited into 279
GenBank with accession numbers XXXXXX-XXXXXX. Novel avian coronaviruses identified 280
in this study are in bold. The host identities determined by DNA fingerprinting techniques are in 281
blankets. Sample collection dates are indicated (YYMMDD). aLRT statistics SH-like branch 282
supports are indicated on the nodes. *The dropping sample might derive from a hybrid between 283
Anas spp. or an American wigeon of captive origin. GenBank accession numbers of gene used: 284
229E (Human coronavirus 229E, AF304460); AIBV (Avian infectious bronchitis virus, 285
FJ904722); Asian leopard cat CoV (EF584908); BatCoV-61 (AY864196); BatCoV-HKU8 286
(DQ249228); Beluga Whale CoV SW1 (EU111742); Black-headed gull CoV CIR-66187 287
(GU396686); Brent goose CoV KR-70 (GU396676); Bulbul HKU11 (Bulbul coronavirus 288
HKU11, FJ376619); Glaucous-winged gull CIR-66002 (GU396682); Glaucus-gull CoV PBA-289
173 (GU396674); HKU1 (Human coronavirus HKU1, AY597011); MHV-A59 (Mouse hepatitis 290
virus, FJ647225); Munia HKU13 (Munia coronavirus HKU13, FJ376622); NL63 (Human 291
coronavirus NL63, AY567487); OC43 (Human coronavirus OC43, AY903460); PEDV (porcine 292
epidemic diarrhoea coronavirus, AF353511); Pintail CoV PBA-124 (GU396673); Pintail CoV 293
PBA-25 (GU396671); Rock sandpiper CoV CIR-65828 (GU396688); SARS CoV (Human 294
SARS coronavirus, JF292915); Snow goose CoV WIR-159 (GU396690); TGEV (Transmissible 295
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gastroenteritis coronavirus, DQ811789); Thrush HKU12 (Thrush coronavirus HKU12, 296
FJ376621); Turkey CoV (EU095850); Western sandpiper CoV KR-28 (GU396675). 297
298
Figure 2. Genetic distances between alphacoronaviruses, betacoronaviruses, 299
gammacoronaviruses and deltacoronaviruses. Partial ORF1b sequences of 10 representative 300
viruses from each genus were analyzed (data not shown). The genetic distances were estimated 301
by using Jukes-Cantor model. The median genetic distances of the studied viruses are shown. 302
*All intra-genus genetic distances were found to be significantly shorter than the relevant inter-303
genus genetic distances (P<0.005). 304
305
Figure 3. Phylogenetic correlation between avian coronaviruses and their hosts. Phylogenetic 306
tree of avian host mtDNA COX1 gene (450bps, left) and viral RdRp gene sequences (360 bps, 307
right) are shown. The bird order of each studied bird species is shown. Only viral and host 308
sequences from selected coronavirus positive bird dropping samples in our study were included. 309
Lines between the 2 trees were added to help visualize virus and host sequence congruence (solid 310
lines) or incongruence (broken lines). Host mtDNA sequences generated from this work were 311
deposited into GenBank with accession numbers XXXXXX-XXXXXX. Reference host 312
sequences used: brent goose (DQ433366); bulbul (FJ378536); chicken (GU261717); glaucous-313
winged gull (HM033523); kiwi (EU525317.1); munia (EF515788); rock sandpiper (GU571303); 314
snow goose (DQ434538); thrush (GQ482858); turkey (EF153719); western sandpiper 315
(AY666261). 316
317
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Figure 1.
TGEVNL63
0 998
B t C V KR 70Beluga Whale CoV SW1
HKU1
OC43SARS CoV
MHV-A590.887
0.993
0.864
BatCoV-61
PEDV
229E
BatCoV-HKU8
0.756
0.961
0.998 0.813
0.725
0.673Alphacoronaviruses
Betacoronaviruses
Snow goose CoV WIR-159
Turkey CoV
Brent goose CoV KR-70
J0588/Anas penelope/091127AIBV
KH08-0852/Dendrocygna javanica/080506J1482/Aythya fuligula/100112
J1404/Anas acuta/091230
K596/Anas penelope/091223J1561/Anas penelope/100112
0.972
0.956
0.833
K561/Anas clypeata/091223
J0126/Anas crecca/091106J1407/Anas acuta/091230
Western sandpiper CoV KR-28
J1404/Anas acuta/091230
Pintail CoV PBA-124
J0579/Anas crecca/091127Rock sandpiper CoV CIR-65828
K554/Anas clypeata/091223
Pintail CoV PBA-250.673
Gammacoronavirusesyp
J0807/Anas clypeata/091217
Glaucous-winged gull CoV CIR-66002Black-headed gull CoV CIR-66187
J1451/Anas acuta/091230
Glaucus-gull CoV PBA-173
J1435/Anas acuta/091230J1616/Anas acuta/100112
J1375/Anas acuta/091223
J0559/Anas crecca/091127
K547/Anas clypeata/091223
J0901/Anas clypeata/091217
J1491/Anas clypeata/100112
J0554/Anas clypeata/091127K589/Anas clypeata/091223
J1300/Anas clypeata/091223
J0569/Platalea minor/091127
J0590/Anas clypeata/091127
J1430/091230J1420/Anas crecca/091230
0.884
0.789
0.991
Thrush HKU12
J0590/Anas clypeata/091127
KH08-1475/Ardeola sp/081107
Munia HKU13Asian leopard cat CoV
Bulbul HKU11-934J1517/Phalacrocorax carbo/100112
K581/Ardea cinerea/091223K513/Ardea cinerea/091223
J0982/Phalacrocorax carbo/091217
0.997
0.8390.791
0.802
0.638
0.934
0.899
Deltacoronaviruses
0.3
KH08-1474/Ardeola sp/081107KH08 1475/Ardeola sp/081107
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Figure 2.
[0.627]α βα β[0.385]* [0.473]*
[0.605] [0.628]
[0.633]γ δγ δ
[0.201]* [0.418]*
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Figure 3.
Bird mtDNA COX1 genes Avian coronaviruses i l RdR
Snow goose
Lesser whistling duck-KH08-0852
229E
Chicken
Turkey
0 96
Kiwi, Struthioniformes
Snow goose, Anseriformes
Lesser whistling duck-KH08-0852, Anseriformes
Chicken, Galliformes
Turkey, Galliformes0.989
partial RdRp gene
rae
rus
Common teal-J0589
Snow goose
Brent goose
Northern Shoveler-J1118
Northern pintail-J1581
Eurasian wigeon-J0588
Tufted duck-J1482
Northern Shoveler-J1362
0.96
Tufted duck-J1482, Anseriformes
Snow goose, Anseriformes
Northern pintail-J1616, Anseriformes
Brent goose, Anseriformes
American wigeon-J1493, Anseriformes
Eurasian wigeon-J1497, Anseriformes
Eurasian wigeon-J0588, Anseriformes
Northern pintail J1581 Anseriformes
0.827
0.921
Galloan
ser
Gam
macoron
avir
N th Sh l K561
Western sandpiper
Glaucous winged gull
Northern pintail-J1616
Northern Shoveler-J1118
Rock sandpiper
Northern Shoveler-J0807
Northern pintail-J1542
Common teal-J0589, Anseriformes
Northern Shoveler-J1362, Anseriformes
Northern Shoveler-K537, Anseriformes
Northern pintail-J1542, Anseriformes
Common teal-J1420, Anseriformes
Common teal-J0559, Anseriformes
Northern pintail-J1581, Anseriformes
Northern Shoveler-K561
Common teal-J0559
Eurasian wigeon-J1497
Pond Heron-KH08-1475
American wigeon-J1493
Cormorant-J0982
Grey heron-K582 0.876
0.621
0.814
Glaucous winged gull, Charadriiformes
Western sandpiper, Charadriiformes
Cormorant-J0982, Pelecaniformes
Black-faced spoonbill-J0569, Ciconiiformes
0.79
0 964
Northern Shoveler-J0807, Anseriformes
Northern Shoveler-J1118, Anseriformes
Northern Shoveler-K561, Anseriformes
Neo
aves
Deltacorona
virus
Common teal-J1420
Black-faced spoonbill-J0569
Northern Shoveler-K537
Bulbul
Munia
Thrush
0.3
0.85
0.708
0.868
0.922
0.874
0.07
Thrush, Passeriformes
Pond Heron-KH08-1475, Ciconiiformes
Rock sandpiper, Charadriiformes
Bulbul, Passeriformes
Munia, Passeriformes
Grey heron-K582, Ciconiiformes
0.963
0.836
0.706
0.964
0.07
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