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Journal of Virology logoLink to Journal of Virology
. 2008 Oct 29;83(2):908–917. doi: 10.1128/JVI.01977-08

Comparative Analysis of Complete Genome Sequences of Three Avian Coronaviruses Reveals a Novel Group 3c Coronavirus▿

Patrick C Y Woo 1,2,3,4,†, Susanna K P Lau 1,2,3,4,†, Carol S F Lam 4, Kenneth K Y Lai 4, Yi Huang 4, Paul Lee 4, Geraldine S M Luk 5, Kitman C Dyrting 5, Kwok-Hung Chan 4, Kwok-Yung Yuen 1,2,3,4,*
PMCID: PMC2612373  PMID: 18971277

Abstract

In this territory-wide molecular epidemiology study of coronaviruses (CoVs) in Hong Kong involving 1,541 dead wild birds, three novel CoVs were identified in three different bird families (bulbul CoV HKU11 [BuCoV HKU11], thrush CoV HKU12 [ThCoV HKU12], and munia CoV HKU13 [MuCoV HKU13]). Four complete genomes of the three novel CoVs were sequenced. Their genomes (26,396 to 26,552 bases) represent the smallest known CoV genomes. In phylogenetic trees constructed using chymotrypsin-like protease (3CLpro), RNA-dependent RNA polymerase (Pol), helicase, spike, and nucleocapsid proteins, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 formed a cluster distantly related to infectious bronchitis virus and turkey CoV (group 3a CoVs). For helicase, spike, and nucleocapsid, they were also clustered with a CoV recently discovered in Asian leopard cats, for which the complete genome sequence was not available. The 3CLpro, Pol, helicase, and nucleocapsid of the three CoVs possessed higher amino acid identities to those of group 3a CoVs than to those of group 1 and group 2 CoVs. Unique genomic features distinguishing them from other group 3 CoVs include a distinct transcription regulatory sequence and coding potential for small open reading frames. Based on these results, we propose a novel CoV subgroup, group 3c, to describe this distinct subgroup of CoVs under the group 3 CoVs. Avian CoVs are genetically more diverse than previously thought and may be closely related to some newly identified mammalian CoVs. Further studies would be important to delineate whether the Asian leopard cat CoV was a result of interspecies jumping from birds, a situation analogous to that of bat and civet severe acute respiratory syndrome CoVs.

Coronaviruses (CoVs) are found in a wide variety of animals in which they can cause respiratory, enteric, hepatic, and neurological diseases of varying severity. Based on genotypic and serological characterization, CoVs have been divided into three distinct groups (3, 21, 50). As a result of the unique mechanism of viral replication, CoVs have a high frequency of recombination (21). Their tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches (17, 47).

The recent severe acute respiratory syndrome (SARS) epidemic, the discovery of SARS-CoV, and the identification of SARS-CoV-like viruses from Himalayan palm civets and a raccoon dog from wildlife markets in China have boosted interest in the discovery of novel CoVs in both humans and animals (5, 15, 29, 32, 35, 36, 45). For human CoVs (HCoVs), a novel group 1 HCoV, HCoV-NL63, was reported independently by two groups in 2004 (11, 40). In 2005, we also described the discovery, complete genome sequence, clinical features, and molecular epidemiology of another novel group 2 HCoV, HCoV-HKU1 (23, 41, 42, 46). As for animal CoVs, we and others have described the discovery of SARS-CoV-like viruses in horseshoe bats in Hong Kong Special Administrative Region (HKSAR) and other provinces of China (22, 26). Based on these findings, we conducted molecular surveillance studies to examine the diversity of CoVs in bats of our locality, as well as of the Guangdong province of southern China where the SARS epidemic originated and wet markets and game food restaurants serving bat dishes are commonly found. In these studies, at least nine other novel CoVs were discovered, including two novel subgroups of CoVs, groups 2c and 2d (24, 33, 43, 48). Other groups have also conducted molecular surveillance studies in bats and other animals, and additional novel CoVs were discovered and complete genomes sequenced (4, 6, 7, 8, 9, 12, 13, 14, 16, 20, 27, 28, 39, 49). Recently, beluga whale CoV (SW1), a novel CoV most closely related to infectious bronchitis virus (IBV), was discovered in a dead whale (30).

Birds are the reservoir of major emerging viruses, most notably, avian influenza viruses (25). Due to their ability to fly over long distances, birds have the potential to disseminate these emerging viruses efficiently. As for CoVs, the number of known CoVs in birds is relatively small in comparison to the number in bats. Therefore, we hypothesized that previously unrecognized CoVs may be present in birds. To test this hypothesis, we carried out a territory-wide molecular epidemiology study of CoVs in dead wild birds in HKSAR. In this study, three previously undescribed CoVs (bulbul CoV HKU11 [BuCoV HKU11], thrush CoV HKU12 [ThCoV HKU12], and munia CoV HKU13 [MuCoV HKU13]), which form a unique group of CoV distantly related to IBV, were discovered. In addition, we sequenced two complete genomes of BuCoV HKU11 and one complete genome each of ThCoV HKU12 and MuCoV HKU13. Based on the results of the present study, we propose a novel subgroup, group 3c, in the group 3 CoVs.

MATERIALS AND METHODS

Dead wild bird surveillance and sample collection.

The Department of Agriculture, Fisheries, and Conservation (AFCD), HKSAR, provided access to samples collected from various locations in HKSAR over a 7-month period (December 2006 to June 2007) as part of the AFCD avian influenza surveillance program on dead wild birds. Tracheal and cloacal swabs were collected from these birds by the Tai Lung Veterinary Laboratory, AFCD, using procedures described previously (10). A total of 1,548 samples from 1,541 dead wild birds of 77 different species in 32 families were tested.

RNA extraction.

Viral RNA was extracted from the tracheal and cloacal swabs by using an RNeasy Mini spin column (QIAgen, Hilden, Germany). The RNA was eluted in 50 μl of RNase-free water and was used as the template for reverse transcription-PCR (RT-PCR).

RT-PCR of the pol genes of CoVs using conserved primers and DNA sequencing.

Initial CoV screening was performed by amplifying a 440-bp fragment of the pol gene of CoVs using conserved primers (5′-GGTTGGGACTATCCTAAGTGTGA-3′ and 5′-CCATCATCAGATAGAATCATCATA-3′) designed by multiple alignments of the nucleotide sequences of available pol genes of known CoVs (42). After the complete pol gene sequence of the first BuCoV HKU11 was obtained, subsequent screening was performed by amplifying the same 440-bp fragment of the pol gene using primers 5′-GGTTGGGACTATCCTAAGTGTGA-3′ and 5′-CCATCATCAGATAGTATCATCAAC-3′, which were more sensitive for BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 than the primers used for initial screening. RT was performed by using a SuperScript III kit (Invitrogen, San Diego, CA). The PCR mixture (25 μl) contained cDNA, PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 3 mM MgCl2, and 0.01% gelatin), 200 μM of each deoxynucleoside triphosphate, and 1.0 U Taq polymerase (Pol) (Applied Biosystem, Foster City, CA). The mixtures were amplified in 60 cycles of 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min and a final extension at 72°C for 10 min in an automated thermal cycler (Applied Biosystems, Foster City, CA). Standard precautions were taken to avoid PCR contamination, and no false-positive result was observed in negative controls.

The PCR products were gel purified by using a QIAquick gel extraction kit (QIAgen, Hilden, Germany). Both strands of the PCR products were sequenced twice with an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, CA), using the two PCR primers. The sequences of the PCR products were compared with known sequences of the pol genes of CoVs in the GenBank database.

Complete genome sequencing.

Four complete genomes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 were amplified and sequenced, using the RNA extracted from the original swab specimens as templates. The RNA was converted to cDNA by a combined random-priming and oligo(dT)-priming strategy. The cDNA was amplified by degenerate primers designed by multiple alignments of the genomes of other CoVs with complete genomes available, using strategies described in our previous publications (42, 48) and our recently published CoV database, CoVDB (19), for sequence retrieval. Additional primers were designed from the results of the first and subsequent rounds of sequencing. These primer sequences are available on request. The 5′ ends of the viral genomes were confirmed by rapid amplification of cDNA ends using a 5′/3′ RACE kit (Roche, Germany). The sequences were assembled and manually edited to produce final sequences of the viral genomes.

Genome analysis.

The nucleotide sequences of the genomes and the deduced amino acid sequences of the open reading frames (ORFs) were compared to those of other CoVs by using EMBOSS needle (http://www.ebi.ac.uk). Phylogenetic tree construction was performed by using the neighbor joining method with ClustalX version 1.83. Protein family analysis was performed by using PFAM and InterProScan (1, 2). Prediction of transmembrane domains was performed by using TMpred and TMHMM (18, 37).

Nucleotide sequence accession numbers.

The nucleotide sequences of the four genomes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 have been lodged within the GenBank sequence database under accession nos. FJ376619 to FJ376622.

RESULTS

Dead wild bird surveillance and identification of three novel CoVs.

A total of 1,548 respiratory and alimentary specimens from 1,541 dead wild birds of 77 different species in 32 families were obtained from various locations in HKSAR (Table 1). RT-PCR for a 440-bp fragment in the pol genes of CoVs was positive in specimens from 21 dead wild birds. The sequencing results suggested the presence of three novel CoVs (Table 1 and Fig. 1). The first one (BuCoV HKU11) was positive in 15 bulbuls (10 Chinese bulbuls and 5 red-whiskered bulbuls), the second one (ThCoV HKU12) in four thrushes (three gray-backed thrushes and one blackbird), and the third one (MuCoV HKU13) in two munias (one white-rumped munia and one scaly-breasted munia). These three novel CoVs were most closely related to a CoV recently discovered in Asian leopard cats (9) but had <64% nucleotide identities to all other known CoVs (Fig. 1). No IBV or IBV-like CoVs were observed.

TABLE 1.

Bird species and associated CoVs in the present surveillance study

Bird family name Scientific name Common name No. of birds tested No. (%) of birds positive for CoVs CoV
Accipitridae 5 0 (0)
Alcedinidae 1 0 (0)
Anatidae 1 0 (0)
Ardeidae 11 0 (0)
Cacatuidae 5 0 (0)
Chloropseidae 1 0 (0)
Columbidae 253 0 (0)
Corvidae 19 0 (0)
Cuculidae 3 0 (0)
Emberizidae 4 0 (0)
Estrildidae Lonchura atricapilla Chestnut munia 10 0 (0)
Lonchura punctulata Scaly-breasted munia 35 1 (2.9) MuCoV HKU13
Lonchura striata White-rumped munia 82 1 (1.2) MuCoV HKU13
Fringillidae 3 0 (0)
Hirundinidae 1 0 (0)
Motacillidae 5 0 (0)
Muscicapidae 30 0 (0)
Nectariniidae 6 0 (0)
Passeridae 85 0 (0)
Phalacrocoracidae 1 0 (0)
Phasianidae 4 0 (0)
Phylloscopidae 1 0 (0)
Podicipedidae 1 0 (0)
Psittacidae 6 0 (0)
Pycnonotidae Hemixos castanonotus Chestnut bulbul 19 0 (0)
Pycnonotus jocosus Red-whiskered bulbul 178 5 (2.8) BuCoV HKU11
Pycnonotus melanicterus Black-crested bulbul 1 0 (0)
Pycnonotus sinensis Chinese bulbul 242 10 (4.1) BuCoV HKU11
Rallidae 7 0 (0)
Recurvirostridae 4 0 (0)
Scolopacidae 1 0 (0)
Strigidae 1 0 (0)
Sturnidae 15 0 (0)
Sylviidae 1 0 (0)
Timaliidae 14 0 (0)
Turdidae Myiophonus caeruleus Blue whistling thrush 4 0 (0)
Monticola solitarius Blue rock thrush 2 0 (0)
Turdus cardis Japanese thrush 30 0 (0)
Turdus hortulorum Gray-backed thrush 221 3 (1.4) ThCoV HKU12
Turdus obscurus Eyebrowed thrush 1 0 (0)
Turdus kessleri White-backed thrush 1 0 (0)
Turdus merula Blackbird 16 1 (6.3) ThCoV HKU12
Turdus pallidus Pale thrush 66 0 (0)
Zoothera dauma Scaly thrush 22 0 (0)
Zosteropidae 122 0 (0)
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FIG. 1.

FIG. 1.

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Phylogenetic analysis of amino acid sequences of the 393-bp fragment (excluding primer sequences) of RNA-dependent RNA Pol of CoVs identified from dead wild birds in the present study. The tree was constructed by the neighbor joining method using Kimura's two-parameter correction and bootstrap values calculated from 1,000 trees. The scale bar indicates the estimated number of substitutions per 50 amino acids. The three novel CoVs identified in the present study are shown in bold. The four genomes completely sequenced are highlighted in gray. HCoV-229E (NC_002645); PEDV, porcine epidemic diarrhea virus (NC_003436); TGEV, porcine transmissible gastroenteritis virus (NC_002306); FIPV, feline infectious peritonitis virus (AY994055); PRCV, porcine respiratory CoV (DQ811787); HCoV-NL63 (NC_005831); bat-CoV HKU2 (EF203064); bat-CoV HKU4 (NC_009019); bat-CoV HKU5 (NC_009020); bat-CoV HKU6 (DQ249224); bat-CoV HKU7 (DQ249226); bat-CoV HKU8 (NC_010438); bat-CoV HKU9 (NC_009021) BtCoV 1A, bat CoV 1A (NC_010437); BtCoV 1B, bat CoV 1B 1B (NC_010436); BtCoV/512/2005, bat CoV 512/2005 (NC_009657); HCoV-HKU1 (NC_006577); HCoV-OC43 (NC_005147); MHV (NC_006852); BCoV, bovine CoV (NC_003045); SDAV, rat sialodacryoadenitis CoV (AF124990); AntelopeCoV, sable antelope CoV (EF424621); GiCoV, giraffe CoV (EF424622); PHEV, porcine hemagglutinating encephalomyelitis virus (NC_007732); SARS-CoV (NC_004718); bat-SARS-CoV HKU3 (NC_009694); IBV (NC_001451); IBV-partridge, partridge CoV (AY646283); IBV-peafowl, peafowl CoV (AY641576); TCoV (NC_010800); ALC, Asian leopard cat CoV Guangxi/F230/2006 (EF584908); SW1 (NC_010646). BuCoV HKU11 (ChBu, chinese Bulbul, and RwBu, red-whiskered bulbul); ThCoV HKU12 (BlTh, blackbird, and GbTh, gray-backed thrush); MuCoV HKU13 (WrMu, white-rumped munia, and SbMu, scaly-breasted munia).

Genome organization and coding potential of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13.

Complete genome sequence data for two strains of BuCoV HKU11 (BuCoV HKU11-796 ChBu/Hong Kong/2007 and BuCoV HKU11-934 RwBu/Hong Kong/2007) and one strain each of ThCoV HKU12 (ThCoV HKU12-600 GbTh/Hong Kong/2007) and MuCoV HKU13 (MuCoV HKU13-3514 WrMu/Hong Kong/2007) were obtained by assembly of the sequences of the RT-PCR products from the RNA extracted from the corresponding individual swab specimens.

The sizes of the genomes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 are 26,476 to 26,487 bases, 26,396 bases, and 26,552 bases, respectively, which are the smallest known CoV genomes, and their G+C contents are 39%, 38%, and 43% (Table 2). Their genome organizations are similar to those of other CoVs, with the characteristic gene order 5′-replicase ORF1ab, spike (S), envelope (E), membrane (M), nucleocapsid (N)-3′ (Fig. 2 and Table 3). Both the 5′ and 3′ ends contain short untranslated regions. The replicase ORF1ab occupies 18.788 to 18.923 kb of the genomes (Table 3). This ORF encodes a number of putative proteins, including nsp3 (which contains the putative papain-like protease [PLpro]), nsp5 (putative chymotrypsin-like protease [3CLpro]), nsp12 (putative RNA-dependent RNA Pol), nsp13 (putative helicase), and other proteins of unknown functions.

TABLE 2.

Comparison of genomic features and amino acid identities between BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 and other CoVsa

CoV Genome features
Pairwise amino acid identity (%)
Size (bases) G+C content BuCoV HKU11
ThCoV HKU12
MuCoV HKU13
3CLpro Pol Hel S N 3CLpro Pol Hel S N 3CLpro Pol Hel S N
Group 1a
    PEDV 28033 0.42 36.7 49.1 47.8 35.5 21.3 35.8 48.8 48.1 35.8 21.6 36.4 49.6 47.9 37.4 22.4
    TGEV 28586 0.38 33.4 49.5 50.9 33.5 23.5 33.1 49.5 50.7 34.5 22.5 34.1 49.8 50.1 34.0 23.1
    FIPV 29355 0.38 34.9 49.6 50.6 34.5 23.7 35.3 49.3 50.4 34.8 22.4 34.7 50.4 49.8 34.2 22.9
Group 1b
    HCoV-229E 27317 0.38 33.8 48.8 49.7 41.4 19.0 34.2 48.8 48.8 40.8 21.4 34.5 49.6 49.7 40.5 20.3
    HCoV-NL63 27553 0.34 35.6 49.0 49.5 36.5 21.2 34.6 49.2 49.3 36.0 22.3 35.7 49.6 49.3 36.9 21.6
    Bat-CoV HKU2 27165 0.39 35.1 50.1 50.3 23.8 21.9 34.4 49.9 50.6 25.9 20.8 33.4 50.1 50.2 24.1 21.6
    BtCoV 1A 28326 0.38 33.8 49.2 51.5 34.2 21.8 34.2 48.6 51.0 33.6 23.9 34.1 49.5 51.1 34.4 23.6
    BtCoV 1B 28476 0.39 32.8 48.7 50.5 33.2 23.2 33.9 48.4 51.2 33.7 23.8 34.1 49.2 50.7 34.1 24.1
    Bat-CoV HKU8 28773 0.42 32.8 49.4 49.1 34.4 21.4 33.1 49.3 48.8 35.0 19.2 32.8 49.6 49.1 34.9 20.9
Group 2a
    HCoV-OC43 30738 0.37 38.1 51.3 48.3 20.9 22.0 38.1 51.3 47.8 23.9 19.2 37.5 52.1 49.3 24.8 21.0
    BCoV 31028 0.37 38.5 51.4 48.4 24.8 21.7 38.4 51.3 47.9 22.6 19.4 37.8 52.3 49.4 25.4 20.8
    PHEV 30480 0.37 38.5 51.4 48.3 25.5 21.9 38.4 51.3 47.8 24.3 20.7 37.8 52.1 49.3 25.2 19.6
    AntelopeCoV 30995 0.37 38.5 51.5 48.4 25.0 21.7 38.4 51.4 47.9 25.6 19.4 37.8 52.4 49.4 25.7 20.8
    GiCoV 30979 0.37 38.5 51.5 48.4 25.1 21.7 38.4 51.4 47.9 25.6 19.4 37.8 52.4 49.4 25.8 20.8
    MHV 31357 0.42 37.0 51.5 47.6 25.5 23.7 38.3 51.3 47.5 25.2 22.8 36.2 52.8 49.1 24.9 24.2
    HCoV-HKU1 29926 0.32 36.5 51.4 47.9 25.2 23.8 38.2 50.9 47.8 24.4 23.0 35.6 52.1 49.3 24.3 23.6
Group 2b
    SARS-CoV 29751 0.41 35.1 51.4 51.5 25.8 23.7 35.1 51.0 50.5 26.6 23.4 35.1 51.2 51.9 26.3 23.6
    Bat-SARS-CoV HKU3 29728 0.41 35.1 51.4 51.6 27.0 23.2 32.9 51.3 50.7 25.5 22.8 34.8 50.9 51.9 25.5 23.0
Group 2c
    Bat-CoV HKU4 30286 0.38 33.6 51.3 50.8 25.2 24.7 33.3 51.2 49.7 25.4 22.9 35.0 50.6 50.0 23.9 25.1
    Bat-CoV HKU5 30488 0.43 33.6 50.3 50.8 25.8 23.0 32.6 50.3 49.5 25.6 23.6 35.0 50.3 50.0 22.9 24.4
Group 2d
    Bat-CoV HKU9 29114 0.41 35.1 52.6 51.2 26.1 23.2 35.8 52.5 50.4 25.7 23.1 35.1 51.5 51.4 26.4 22.8
Group 3a
    IBV 27608 0.38 43.6 54.2 56.4 27.0 30.2 43.8 54.4 56.6 28.7 29.0 43.5 54.7 56.1 28.2 30.4
    TCoV 27657 0.38 43.3 54.0 57.4 28.6 30.2 44.1 54.7 57.5 30.0 30.0 43.1 54.6 56.8 29.7 29.6
Group 3b
    SW1 31686 0.39 40.3 52.3 52.8 26.2 31.4 39.0 52.8 52.8 24.7 30.6 37.1 52.5 52.8 25.9 30.1
Group 3c
    BuCoV HKU11 26476 0.39 88.3 92.5 96.7 46.3 74.6 79.8 88.4 93.6 68.1 72.5
    ThCoV HKU12 26396 0.38 88.3 92.5 96.7 46.3 74.6 80.5 86.9 93.1 48.1 75.4
    MuCoV HKU13 26552 0.43 79.8 88.4 93.6 68.1 72.5 80.5 86.9 93.1 48.1 75.4
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a

Comparison of genomic features of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 and other CoVs with complete genome sequences available and of amino acid identities between the predicted 3CLpro, RNA-dependent RNA Pol, helicase (Hel), S, and N proteins of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 and the corresponding proteins of other CoVs. PEDV, porcine epidemic diarrhea virus; TGEV, porcine transmissible gastroenteritis virus; FIPV, feline infectious peritonitis virus; BtCoV 1A, bat CoV 1A; BtCoV 1B, bat CoV 1B; BCoV, bovine CoV; AntelopeCoV, sable antelope CoV; GiCoV, giraffe CoV; PHEV, porcine hemagglutinating encephalomyelitis virus.

FIG. 2.

FIG. 2.

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Genome organizations of BuCoV HKU11, ThCoV HKU12, MuCoV HKU13, and representative CoVs from each group. Papain-like proteases (PL1, PL2, and PL), 3CLpro, RNA-dependent RNA Pol, hemagglutinin esterase (HE), spike (S), envelope (E), membrane (M), and nucleocapsid (N) are represented by gray boxes. Virus name abbreviations may be found in the Fig. 1 legend and the text.

TABLE 3.

Coding potential and putative transcription regulatory sequences of the genomes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13

CoV ORF Start-end (nucleotide position) No. of nucleotides No. of amino acids Frame Putative TRS
Nucleotide position in genome(s) TRS sequence(s) (distance in bases to AUG)
BuCoV HKU11 1ab 607-19394 18,788 6,262 +1, +3 72 ACACCA(529)AUG
S 19376-22867 3,492 1,163 +2 19230 or 19320 or 19368 ACACCA(140)AUG or AAACCA(50)AUG or CCACCA(2)AUG
E 22861-23109 249 82 +1 22835 ACACCA(20)AUG
M 23106-23807 702 233 +3 23079 ACACCA(21)AUG
NS6 23807-24094 288 95 +2
N 24115-25164 1,050 349 +1 24102 ACACCA(7)AUG
NS7a 25343-25714 372 123 +2
NS7b 25720-25974 255 84 +1 25689 ACACCA(25)AUG
NS7c 25971-26255 285 94 +3
ThCoV HKU12 1ab 592-19451 18,860 6,286 +3, +2 65 ACACCA(521)AUG
S 19433-23011 3,579 1,192 +1 19287 or 19377 or 19425 ACACCA(140)AUG or AAACCA(50)AUG or CCGCCA(2)AUG
E 23005-23253 249 82 +3 22979 ACACCA(20)AUG
M 23246-23899 654 217 +1 23223 ACACCA(17)AUG
NS6 23899-24174 276 91 +3
N 24192-25223 1,032 343 +2 24179 ACACCA(7)AUG
NS7a 25255-25626 372 123 +3
NS7b 25632-25883 252 83 +2 25604 UCACAA(22)AUG
NS7c 25940-26173 234 77 +1
MuCoV HKU13 1ab 595-19517 18,923 6,307 +3, +2 64 ACACCA(525)AUG
S 19499-22969 3,471 1,156 +1 19324 or 19443 or 19491 ACACCA(140)AUG or AAACCA(50)AUG or CCACCA(2)AUG
E 22963-23211 249 82 +3 22934 ACACCC(23)AUG
M 23204-23860 657 218 +1 23181 ACACCA(17)AUG
NS6 23860-24186 327 108 +3
N 24326-25384 1,059 352 +1 24311 ACACCA(9)AUG
NS7a 25417-25788 372 123 +3
NS7b 25794-26051 258 85 +2 25766 ACACCA(22)AUG
NS7c 26048-26329 282 93 +1
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BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 have the same genome structure (Fig. 2). They also possess the same putative transcription regulatory sequence (TRS) motif, 5′-ACACCA-3′, at the 3′ end of the leader sequence preceding each ORF except NS6, NS7a, and NS7c (Table 3). This TRS has not been found to be the TRS for any other CoVs with complete genomes available. Similar to other group 3 CoVs, the genomes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 have putative PLpro proteins, which are homologous to PL2pro of group 1 and group 2a CoVs and PLpro of group 2b, 2c, and 2d CoVs, IBV, turkey CoV (TCoV), and SW1 (Fig. 2). Interestingly, the perfect TRS's of S in the genomes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 were separated from the corresponding AUGs by 140 bases (Table 3). This is in contrast to the relatively small number of bases between the TRS for S and the corresponding AUG in all other CoVs (range, 0 bases in HCoV-NL63, bat CoV HKU2, HCoV-HKU1, bovine CoV, HCoV-OC43, mouse hepatitis virus [MHV], porcine hemagglutinating encephalomyelitis virus, SARS-CoV, and bat SARS-CoV to 52 bases in IBV). Alternatively, the S in these genomes may be using other potential imperfect TRS's located closer to the corresponding AUG (Table 3). In the genomes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13, one ORF (NS6) between M and N and three ORFs (NS7a, -7b, and -7c) downstream from N, which encode putative nonstructural proteins, were observed. Among these four ORFs, only NS7b was preceded by a TRS. The absence of a TRS preceding NS6 and the relatively high (0.500) Ka/Ks ratio (the ratio between the number of nonsynonymous substitutions per nonsynonymous site and the number of synonymous substitutions per synonymous site; an index of the action of selective forces) of NS6 in BuCoV HKU11 (data not shown) implied that this ORF may not be expressed.

BLAST search revealed no amino acid similarities between these four putative nonstructural proteins and other known proteins, and no functional domain was identified by PFAM and InterProScan. TMHMM and TMpred analyses showed one putative transmembrane domain in NS7b of BuCoV HKU11 (residues 42 to 64 by TMHMM and 41 to 62 by TMpred analysis), ThCoV HKU12 (residues 43 to 65 by TMHMM and 41 to 62 by TMpred analysis), and MuCoV HKU13 (residues 43 to 65 by TMHMM and 46 to 65 by TMpred analysis). TMHMM and TMpred analyses showed two putative transmembrane domains in NS7c of BuCoV HKU11 (residues 2 to 19 and 24 to 46 by TMHMM and 1 to 17 and 31 to 47 by TMpred analysis), one putative transmembrane domain in NS7c of ThCoV HKU12 (residues 10 to 32 by TMHMM and 10 to 30 by TMpred analysis) and two putative transmembrane domains in NS7c of MuCoV HKU13 (residues 2 to 19 and 29 to 48 by TMHMM and 1 to 19 and 31 to 47 by TMpred analysis). Each of the genomes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 contains a stem-loop II motif (s2m) (bases 26262 to 26304, 26175 to 26217, and 26390 to 26432, respectively) as a conserved RNA element downstream from N and upstream from the poly(A) tail, similar to those in IBV, TCoV, bat SARS-CoV, and SARS-CoV, as well as other in CoVs discovered in Asian leopard cats, graylag geese, feral pigeons, and mallards but without complete genomes available (Fig. 3) (13, 20, 34).

FIG. 3.

FIG. 3.

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Multiple alignment of conserved s2m's of BuCoV HKU11, ThCoV HKU12, MuCoV HKU13, Asian leopard cat CoV (ALC), IBV, SARS-CoV, and bat-SARS-CoV. The identical nucleotides are marked by asterisks.

Phylogenetic analyses.

The phylogenetic trees constructed using the amino acid sequences of the 3CLpro, Pol, helicase, S, and N proteins of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 and other CoVs are shown in Fig. 4, and the corresponding pairwise amino acid identities are shown in Table 2. For all five gene products, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 possessed higher amino acid identities to each other than to any other known CoVs with complete genomes available (Table 2). In all five trees, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 were clustered (Fig. 4). For the Hel, S, and N genes, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 were also clustered with a CoV recently discovered in Asian leopard cats (9) for which the sequences of these genes were available (Fig. 4). There were 14.5 to 18%, 37.4 to 47%, and 25.1 to 29.3% base differences between the helicase, S, and N genes of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 and that of the Asian leopard cat CoV. Notably, a short fragment (184 bases) of pol in MuCoV HKU13 possessed 92.5% nucleotide identity to a recently isolated CoV from a parrot (14). However, no sequence from other regions of the latter was available for further analysis. For 3CLpro, Pol, helicase, and N, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 possessed higher amino acid identities to the homologous gene products in IBV and TCoV than to those of group 1 and group 2 CoVs (Table 2). Based both on phylogenetic tree analyses and amino acid differences, we propose a novel subgroup, group 3c, under the group 3 CoVs to describe this distinct subgroup of CoVs which includes BuCoV HKU11, ThCoV HKU12, MuCoV HKU13, and probably, the recently discovered CoV from Asian leopard cats for which a complete genome sequence is not available (9). Interestingly, the S proteins of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 possessed higher amino acid identities to those of group 1 CoVs than those of IBV and TCoV. This is analogous to a phenomenon we recently described in a group 1 CoV, bat CoV HKU2, in which S is not closely related to those of any known CoVs (Fig. 4) (24).

FIG. 4.

FIG. 4.

Open in a new tab

Phylogenetic analyses of 3CLpro, RNA-dependent RNA Pol, helicase (Hel), S, and N proteins of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13. The trees were constructed by using the neighbor joining method using Kimura's two-parameter correction and bootstrap values calculated from 1,000 trees. Three hundred eight, 958, 609, 1,735, and 592 amino acid positions in 3CLpro, Pol, helicase, S, and N, respectively, were included in the analyses. The scale bar indicates the estimated number of substitutions per 10 amino acids. PEDV, porcine epidemic diarrhea virus; TGEV, porcine transmissible gastroenteritis virus; FIPV, feline infectious peritonitis virus; BtCoV 1A, bat CoV 1A; BtCoV 1B, bat CoV 1B; BtCoV/512/2005, bat CoV 512/2005; BCoV, bovine CoV; AntelopeCoV, sable antelope CoV; GiCoV, giraffe CoV; IBV-partridge, partridge CoV; IBV-peafowl, peafowl CoV; PHEV, porcine hemagglutinating encephalomyelitis virus; ALC, Asian leopard cat CoV Guangxi/F230/2006.

DISCUSSION

In this territory-wide surveillance study of dead wild birds for CoVs, we identified a novel putative subgroup, 3c, of CoVs. BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 formed three distinct branches within this putative subgroup 3c lineage in all five phylogenetic trees analyzed (Fig. 4). Moreover, all 15 strains of BuCoV HKU11 were found in bulbuls, the four strains of ThCoV HKU12 were found in thrushes, and the two strains of MuCoV HKU13 were found in munias. These data support the idea that BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 are three separate novel CoV species infecting three different families of birds. As the three novel CoVs possess the same genome organization and share the same putative TRS, we speculate that they originated from the same ancestor, which subsequently diverged to adapt to different hosts and ecological niches. Based on phylogenetic tree analyses, the three novel avian CoVs formed a unique cluster, most closely related to but distinct from other group 3 CoVs. These three novel avian CoVs were not cultivable using six different cell lines and specific-pathogen-free embryonated chicken eggs (data not shown). BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 of this new proposed subgroup possessed genomic features different from those of other group 3 CoVs (Table 4). For the coding potential of the genomes, IBV and TCoV but not SW1, BuCoV HKU11, ThCoV HKU12, or MuCoV HKU13, possess NS3a and -3b between S and E. IBV, TCoV, SW1, and the three novel putative subgroup 3c CoVs possess two, three, eight, and one small ORF between M and N, respectively. BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 but not IBV, TCoV, or SW1 possess NS7a, -7b, and -7c downstream from N. As for the TRS, the sequences for the TRS's of IBV and TCoV are CUUAACAA, that of SW1 is AAACA, and those of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 are ACACCA.

TABLE 4.

Comparison of characteristics in the genomes of group 3a, group 3b, and group 3c CoVs

Group (CoV[s]) Coding potential
TRS sequence
Small ORFs between S and E Small ORFs between M and N Small ORFs downstream from Na
3a (IBV, TCoV) NS3a, -3b NS5a, -5b (IBV), ORFx, NS5a, -5b (TCoV) CUUAACAA
3b (SW1) NS5a, -5b, -5c, -6, -7, -8, -9, -10 AAACA
3c NS6 NS7a, -7b, -7c ACACCA
Open in a new tab
a

Newly discovered CoVs from geese, ducks, and pigeons contain small ORFs (orfx and orfy) downstream from N.

Group 3 CoVs are found in both birds and mammals and probably contain at least three distinct lineages. Historically, group 1 and 2 CoVs were found in mammals and group 3 CoVs were found in birds. Although puffinosis virus, a group 2a CoV, had been found in birds, its passage in mouse brains had raised the suspicion that it could be a contaminant of MHV (31). Recently, SW1, with a genome most closely related to IBV, was discovered from a whale (30). Interestingly, BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 were also clustered with a CoV recently described in Asian leopard cats (9), for which a complete genome sequence is not available, in phylogenetic trees constructed using Hel, S, and N. This implied that this lineage of CoVs may be present not only in birds but also in some mammals. The clustering of IBV, TCoV, and IBV-like viruses into one lineage, SW1 as a second lineage, and BuCoV HKU11, ThCoV HKU12, MuCoV HKU13, and the Asian leopard cat CoV as a third lineage implies that group 3 CoVs probably contain at least three subgroups. Although the complete genome sequence is not available for the Asian leopard cat CoV, the same putative TRS for the ORFs available, the presence of NS6 between M and N, and the presence of s2m imply that it may possess a genome organization similar to those of BuCoV HKU11, ThCoV HKU12, and MuCoV HKU13 (Fig. 2 and 3). Moreover, the presence of s2m in the Asian leopard cat CoV genome could also suggest that bat SARS-CoV and SARS-CoV may well have acquired their s2m's by recombining with a group 3 mammalian CoV rather than an avian CoV (38). Of note is that 13 of the 21 dead wild birds were collected in urban areas of HKSAR. It would be of great importance to determine whether the Asian leopard cat CoV was a result of interspecies jumping from birds, a situation analogous to that of bat SARS-CoV and civet SARS-CoV. White-rumped Munias are commonly released or are found as wild birds around human dwellings, and bulbuls and thrushes are also resident birds found in most habitats in Hong Kong. If group 3c CoV can really adapt to infect mammals such as Asian leopard cats, a common wild mammal in southern China, the mixing of such birds and mammals in wildlife markets may provide the correct environment for interspecies jumping and could subsequently pose the risk of further genetic changes toward adapting to the human host, as in the case of SARS (44).

Similar to bats, birds also contain a wide diversity of CoVs. Before the SARS epidemic in 2003, 19 (2 human, 13 mammalian, and 4 avian) CoVs were known. After the SARS epidemic, two novel HCoVs, HCoV-NL63 and HCoV-HKU1, were discovered (11, 40, 42). In the most-recent 4 years, at least 10 previously unrecognized CoVs from bats have been described in Hong Kong and mainland China (22, 23, 26, 33, 39, 43, 48). In addition to the generation of a large number of CoV species, recombination has also resulted in different genotypes in certain CoV species, most notably the three genotypes in HCoV-HKU1 (47). In the present study and others, a wide diversity of CoVs are also observed in birds. The wide diversity of CoVs in bats and birds is probably a result of both a higher mutation rate of RNA viruses due to the infidelity of their Pols and a higher chance of recombination due to their unique replication mechanism. Further molecular epidemiological studies in bats and birds of other countries, as well as in other animals, and complete genome sequencing will shed more light on the diversity of CoVs and their evolutionary histories.

Acknowledgments

We thank York Y. N. Chow, Secretary of Health, Welfare, and Food, HKSAR, The Peoples' Republic of China; and Virology and Biotechnology Laboratory staff of the Tai Lung Veterinary Laboratory of the Agriculture, Fisheries, and Conservation Department of the HKSAR.

The views expressed in this paper are those of the authors only and may not represent the opinion of the Agriculture, Fisheries, and Conservation Department of the government of the HKSAR.

We are grateful for the generous support of Hui Hoy and Hui Ming in the genomic sequencing platform. This work is partly supported by a Research Grant Council grant; a University Development Fund and Outstanding Young Researcher Award from The University of Hong Kong; The Tung Wah Group of Hospitals Fund for Research in Infectious Diseases; the HKSAR Research Fund for the Control of Infectious Diseases of the Health, Welfare, and Food Bureau; and the Shaw Foundation.

Footnotes

▿

Published ahead of print on 29 October 2008.

REFERENCES

  • 1.Apweiler, R., T. K. Attwood, A. Bairoch, A. Bateman, E. Birney, M. Biswas, P. Bucher, L. Cerutti, F. Corpet, M. D. Croning, R. Durbin, L. Falquet, W. Fleischmann, J. Gouzy, H. Hermjakob, N. Hulo, I. Jonassen, D. Kahn, A. Kanapin, Y. Karavidopoulou, R. Lopez, B. Marx, N. J. Mulder, T. M. Oinn, M. Pagni, F. Servant, C. J. Sigrist, and E. M. Zdobnov. 2001. The InterPro database, an integrated documentation resource for protein families, domains and functional sites. Nucleic Acids Res. 2937-40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bateman, A., E. Birney, L. Cerruti, R. Durbin, L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall, and E. L. Sonnhammer. 2002. The Pfam protein families database. Nucleic Acids Res. 30276-280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Brian, D. A., and R. S. Baric. 2005. Coronavirus genome structure and replication. Curr. Top. Microbiol. Immunol. 2871-30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Cao, J., C. C. Wu, and T. L. Lin. 2008. Complete nucleotide sequence of polyprotein gene 1 and genome organization of turkey coronavirus. Virus Res. 13643-49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Cheng, V. C., S. K. Lau, P. C. Woo, and K. Y. Yuen. 2007. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin. Microbiol. Rev. 20660-694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chu, D. K., J. S. Peiris, H. Chen, Y. Guan, and L. L. Poon. 2008. Genomic characterizations of bat coronaviruses (1A, 1B and HKU8) and evidence for co-infections in Miniopterus bats. J. Gen. Virol. 891282-1287. [DOI] [PubMed] [Google Scholar]
  • 7.Circella, E., A. Camarda, V. Martella, G. Bruni, A. Lavazza, and C. Buonavoglia. 2007. Coronavirus associated with an enteric syndrome on a quail farm. Avian Pathol. 36251-258. [DOI] [PubMed] [Google Scholar]
  • 8.Dominguez, S. R., T. J. O'Shea, L. M. Oko, and K. V. Holmes. 2007. Detection of group 1 coronaviruses in bats in North America. Emerg. Infect. Dis. 131295-1300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Dong, B. Q., W. Liu, X. H. Fan, D. Vijaykrishna, X. C. Tang, F. Gao, L. F. Li, G. J. Li, J. X. Zhang, L. Q. Yang, L. L. Poon, S. Y. Zhang, J. S. Peiris, G. J. Smith, H. Chen, and Y. Guan. 2007. Detection of a novel and highly divergent coronavirus from Asian leopard cats and Chinese ferret badgers in Southern China. J. Virol. 816920-6926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Ellis, T. M., R. B. Bousfield, L. A. Bissett, K. C. Dyrting, G. S. Luk, S. T. Tsim, K. Sturm-Ramirez, R. G. Webster, Y. Guan, and J. S. Peiris. 2004. Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002. Avian Pathol. 33492-505. [DOI] [PubMed] [Google Scholar]
  • 11.Fouchier, R. A., N. G. Hartwig, T. M. Bestebroer, B. Niemeyer, J. C. de Jong, J. H. Simon, and A. D. Osterhaus. 2004. A previously undescribed coronavirus associated with respiratory disease in humans. Proc. Natl. Acad. Sci. USA 1016212-6216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Gloza-Rausch, F., A. Ipsen, A. Seebens, M. Göttsche, M. Panning, J. Felix Drexler, N. Petersen, A. Annan, K. Grywna, M. Müller, S. Pfefferle, and C. Drosten. 2008. Detection and prevalence patterns of group I coronaviruses in bats, northern Germany. Emerg. Infect. Dis. 14626-631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Gomaa, M. H., J. R. Barta, D. Ojkic, and D. Yoo. 2008. Complete genomic sequence of turkey coronavirus. Virus Res. 135237-246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Gough, R. E., S. E. Drury, F. Culver, P. Britton, and D. Cavanagh. 2006. Isolation of a coronavirus from a green-cheeked Amazon parrot (Amazon viridigenalis Cassin) Avian Pathol. 35122-126. [DOI] [PubMed] [Google Scholar]
  • 15.Guan, Y., B. J. Zheng, Y. Q. He, X. L. Liu, Z. X. Zhuang, C. L. Cheung, S. W. Luo, P. H. Li, L. J. Zhang, Y. J. Guan, K. M. Butt, K. L. Wong, K. W. Chan, W. Lim, K. F. Shortridge, K. Y. Yuen, J. S. Peiris, and L. L. Poon. 2003. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302276-278. [DOI] [PubMed] [Google Scholar]
  • 16.Hasoksuz, M., K. Alekseev, A. Vlasova, X. Zhang, D. Spiro, R. Halpin, S. Wang, E. Ghedin, and L. J. Saif. 2007. Biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe. J. Virol. 814981-4990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Herrewegh, A. A., I. Smeenk, M. C. Horzinek, P. J. Rottier, and R. J. de Groot. 1998. Feline coronavirus type II strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type I and canine coronavirus. J. Virol. 724508-4514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Hofmann, K. S. 1993. TMbase: a database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler 374166. [Google Scholar]
  • 19.Huang, Y., S. K. Lau, P. C. Woo, and K. Y. Yuen. 2008. CoVDB: a comprehensive database for comparative analysis of coronavirus genes and genomes. Nucleic Acids Res. 36D504-D511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Jonassen, C. M., T. Kofstad, I. L. Larsen, A. Lovland, K. Handeland, A. Follestad, and A. Lillehaug. 2005. Molecular identification and characterization of novel coronaviruses infecting graylag geese (Anser anser), feral pigeons (Columbia livia) and mallards (Anas platyrhynchos). J. Gen. Virol. 861597-1607. [DOI] [PubMed] [Google Scholar]
  • 21.Lai, M. M., and D. Cavanagh. 1997. The molecular biology of coronaviruses. Adv. Virus. Res. 481-100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Lau, S. K., P. C. Woo, K. S. Li, Y. Huang, H. W. Tsoi, B. H. Wong, S. S. Wong, S. Y. Leung, K. H. Chan, and K. Y. Yuen. 2005. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc. Natl. Acad. Sci. USA 10214040-14045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lau, S. K., P. C. Woo, C. C. Yip, H. Tse, H. W. Tsoi, V. C. Cheng, P. Lee, B. S. Tang, C. H. Cheung, R. A. Lee, L. Y. So, Y. L. Lau, K. H. Chan, and K. Y. Yuen. 2006. Coronavirus HKU1 and other coronavirus infections in Hong Kong. J. Clin. Microbiol. 442063-2071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Lau, S. K., P. C. Woo, K. S. Li, Y. Huang, M. Wang, C. S. Lam, H. Xu, R. Guo, K. H. Chan, B. J. Zheng, and K. Y. Yuen. 2007. Complete genome sequence of bat coronavirus HKU2 from Chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome. Virology 367428-439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Li, K. S., Y. Guan, J. Wang, G. J. Smith, K. M. Xu, L. Duan, A. P. Rahardjo, P. Puthavathana, C. Buranathai, T. D. Nguyen, A. T. Estoepangestie, A. Chaisingh, P. Auewarakul, H. T. Long, N. T. Hanh, R. J. Webby, L. L. Poon, H. Chen, K. F. Shortridge, K. Y. Yuen, R. G. Webster, and J. S. Peiris. 2004. Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430209-213. [DOI] [PubMed] [Google Scholar]
  • 26.Li, W., Z. Shi, M. Yu, W. Ren, C. Smith, J. H. Epstein, H. Wang, G. Crameri, Z. Hu, H. Zhang, J. Zhang, J. McEachern, H. Field, P. Daszak, B. T. Eaton, S. Zhang, and L. F. Wang. 2005. Bats are natural reservoirs of SARS-like coronaviruses. Science 310676-679. [DOI] [PubMed] [Google Scholar]
  • 27.Liu, S., J. Chen, J. Chen, X. Kong, Y. Shao, Z. Han, L. Feng, X. Cai, S. Gu, and M. Liu. 2005. Isolation of avian infectious bronchitis coronavirus from domestic peafowl (Pavo cristatus) and teal (Anas). J. Gen. Virol. 86719-725. [DOI] [PubMed] [Google Scholar]
  • 28.Mardani, K., A. H. Noormohammadi, P. Hooper, J. Ignjatovic, and G. F. Browning. 2008. Infectious bronchitis viruses with a novel genomic organization. J. Virol. 822013-2024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Marra, M. A., S. J. Jones, C. R. Astell, R. A. Holt, A. Brooks-Wilson, Y. S. Butterfield, J. Khattra, J. K. Asano, S. A. Barber, S. Y. Chan, A. Cloutier, S. M. Coughlin, D. Freeman, N. Girn, O. L. Griffith, S. R. Leach, M. Mayo, H. McDonald, S. B. Montgomery, P. K. Pandoh, A. S. Petrescu, A. G. Robertson, J. E. Schein, A. Siddiqui, D. E. Smailus, J. M. Stott, G. S. Yang, F. Plummer, A. Andonov, H. Artsob, N. Bastien, K. Bernard, T. F. Booth, D. Bowness, M. Czub, M. Drebot, L. Fernando, R. Flick, M. Garbutt, M. Gray, A. Grolla, S. Jones, H. Feldmann, A. Meyers, A. Kabani, Y. Li, S. Normand, U. Stroher, G. A. Tipples, S. Tyler, R. Vogrig, D. Ward, B. Watson, R. C. Brunham, M. Krajden, M. Petric, D. M. Skowronski, C. Upton, and R. L. Roper. 2003. The genome sequence of the SARS-associated coronavirus. Science 3001399-1404. [DOI] [PubMed] [Google Scholar]
  • 30.Mihindukulasuriya, K. A., G. Wu, J. St. Leger, R. W. Nordhausen, and D. Wang. 2008. Identification of a novel coronavirus from a beluga whale by using a panviral microarray. J. Virol. 825084-5088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Nuttall, P. A., and K. A. Harrap. 1982. Isolation of a coronavirus during studies on puffinosis, a disease of the Manx shearwater (Puffinus puffinus). Arch. Virol. 731-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Peiris, J. S., S. T. Lai, L. L. Poon, Y. Guan, L. Y. Yam, W. Lim, J. Nicholls, W. K. Yee, W. W. Yan, M. T. Cheung, V. C. Cheng, K. H. Chan, D. N. Tsang, R. W. Yung, T. K. Ng, and K. Y. Yuen. 2003. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 3611319-1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Poon, L. L., D. K. Chu, K. H. Chan, O. K. Wong, T. M. Ellis, Y. H. Leung, S. K. Lau, P. C. Woo, K. Y. Suen, K. Y. Yuen, Y. Guan, and J. S. Peiris. 2005. Identification of a novel coronavirus in bats. J. Virol. 792001-2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Robertson, M. P., H. Igel, R. Baertsch, D. Haussler, M. Ares, Jr., and W. G. Scott. 2005. The structure of a rigorously conserved RNA element within the SARS virus genome. PLoS Biol. 3e5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Rota, P. A., M. S. Oberste, S. S. Monroe, W. A. Nix, R. Campagnoli, J. P. Icenogle, S. Penaranda, B. Bankamp, K. Maher, M. H. Chen, S. Tong, A. Tamin, L. Lowe, M. Frace, J. L. DeRisi, Q. Chen, D. Wang, D. D. Erdman, T. C. Peret, C. Burns, T. G. Ksiazek, P. E. Rollin, A. Sanchez, S. Liffick, B. Holloway, J. Limor, K. McCaustland, M. Olsen-Rasmussen, R. Fouchier, S. Gunther, A. D. Osterhaus, C. Drosten, M. A. Pallansch, L. J. Anderson, and W. J. Bellini. 2003. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 3001394-1399. [DOI] [PubMed] [Google Scholar]
  • 36.Snijder, E. J., P. J. Bredenbeek, J. C. Dobbe, V. Thiel, J. Ziebuhr, L. L. Poon, Y. Guan, M. Rozanov, W. J. Spaan, and A. E. Gorbalenya. 2003. Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J. Mol. Biol. 331991-1004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Sonnhammer, E. L., G. von Heijne, and A. Krogh. 1998. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol. 6175-182. [PubMed] [Google Scholar]
  • 38.Stavrinides, J., and D. S. Guttman. 2004. Mosaic evolution of the severe acute respiratory syndrome coronavirus. J. Virol. 7876-82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Tang, X. C., J. X. Zhang, S. Y. Zhang, P. Wang, X. H. Fan, L. F. Li, G. Li, B. Q. Dong, W. Liu, C. L. Cheung, K. M. Xu, W. J. Song, D. Vijaykrishna, L. L. Poon, J. S. Peiris, G. J. Smith, H. Chen, and Y. Guan. 2006. Prevalence and genetic diversity of coronaviruses in bats from China. J. Virol. 807481-7490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.van der Hoek, L., K. Pyrc, M. F. Jebbink, W. Vermeulen-Oost, R. J. Berkhout, K. C. Wolthers, P. M. Wertheim-van Dillen, J. Kaandorp, J. Spaargaren, and B. Berkhout. 2004. Identification of a new human coronavirus. Nat. Med. 10368-373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Woo, P. C., Y. Huang, S. K. Lau, H. W. Tsoi, and K. Y. Yuen. 2005. In silico analysis of ORF1ab in coronavirus HKU1 genome reveals a unique putative cleavage site of coronavirus HKU1 3C-like protease. Microbiol. Immunol. 49899-908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Woo, P. C., S. K. Lau, C. M. Chu, K. H. Chan, H. W. Tsoi, Y. Huang, B. H. Wong, R. W. Poon, J. J. Cai, W. K. Luk, L. L. Poon, S. S. Wong, Y. Guan, J. S. Peiris, and K. Y. Yuen. 2005. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 79884-895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Woo, P. C., S. K. Lau, K. S. Li, R. W. Poon, B. H. Wong, H. W. Tsoi, B. C. Yip, Y. Huang, K. H. Chan, and K. Y. Yuen. 2006. Molecular diversity of coronaviruses in bats. Virology 351180-187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Woo, P. C., S. K. Lau, and K. Y. Yuen. 2006. Infectious diseases emerging from Chinese wet-markets: zoonotic origins of severe respiratory viral infections. Curr. Opin. Infect. Dis. 19401-407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Woo, P. C., S. K. Lau, H. W. Tsoi, K. H. Chan, B. H. Wong, X. Y. Che, V. K. Tam, S. C. Tam, V. C. Cheng, I. F. Hung, S. S. Wong, B. J. Zheng, Y. Guan, and K. Y. Yuen. 2004. Relative rates of non-pneumonic SARS coronavirus infection and SARS coronavirus pneumonia. Lancet 363841-845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Woo, P. C., S. K. Lau, H. W. Tsoi, Y. Huang, R. W. Poon, C. M. Chu, R. A. Lee, W. K. Luk, G. K. Wong, B. H. Wong, V. C. Cheng, B. S. Tang, A. K. Wu, R. W. Yung, H. Chen, Y. Guan, K. H. Chan, and K. Y. Yuen. 2005. Clinical and molecular epidemiological features of coronavirus HKU1-associated community-acquired pneumonia. J. Infect. Dis. 1921898-1907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Woo, P. C., S. K. Lau, C. C. Yip, Y. Huang, H. W. Tsoi, K. H. Chan, and K. Y. Yuen. 2006. Comparative analysis of 22 coronavirus HKU1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus HKU1. J. Virol. 807136-7145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Woo, P. C., M. Wang, S. K. Lau, H. Xu, R. W. Poon, R. Guo, B. H. Wong, K. Gao, H. W. Tsoi, Y. Huang, K. S. Li, C. S. Lam, K. H. Chan, B. J. Zheng, and K. Y. Yuen. 2007. Comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features. J. Virol. 811574-1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Zhang, J., J. S. Guy, E. J. Snijder, D. A. Denniston, P. J. Timoney, and U. B. Balasuriya. 2007. Genomic characterization of equine coronavirus. Virology 36992-104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Ziebuhr, J. 2004. Molecular biology of severe acute respiratory syndrome coronavirus. Curr. Opin. Microbiol. 7412-419. [DOI] [PMC free article] [PubMed] [Google Scholar]

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