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

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.

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.

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 [PL^pro]), nsp5 (putative chymotrypsin-like protease [3CL^pro]), nsp12 (putative RNA-dependent RNA Pol), nsp13 (putative helicase), and other proteins of unknown functions.

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 PL^pro proteins, which are homologous to PL2^pro of group 1 and group 2a CoVs and PL^pro 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) K_a/K_s 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).

Phylogenetic analyses.

The phylogenetic trees constructed using the amino acid sequences of the 3CL^pro, 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 3CL^pro, 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).