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 gammacoronaviruses 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

75

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

85

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

97

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

101

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

deltacoronaviruses. 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

138

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

182

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


Pintail CoV PBA-124

J0579/Anas crecca/091127Rock sandpiper CoV CIR-65828


Pintail CoV PBA-250.673

Gammacoronavirusesyp

J0807/Anas clypeata/091217

Glaucous-winged gull CoV CIR-66002Black-headed gull CoV CIR-66187


Glaucus-gull CoV PBA-173

J1435/Anas acuta/091230J1616/Anas acuta/100112


J0559/Anas crecca/091127




J0554/Anas clypeata/091127K589/Anas clypeata/091223


J0569/Platalea minor/091127


J1430/091230J1420/Anas crecca/091230

0.884

0.789

0.991

Thrush HKU12


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


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



Rock sandpiper



Common teal-J0589, Anseriformes

Northern Shoveler-J1362, Anseriformes

Northern Shoveler-K537, 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-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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