ABSTRACT
While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 103 to 1 × 105 copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks ) to the number of nonsynonymous substitutions per nonsynonymous site (Ka ) in multiple regions of the genome, especially the S gene (Ka /Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus.
INTRODUCTION
Coronaviruses (CoVs) are found in a wide variety of animals, in which they can cause respiratory, enteric, hepatic, and neurological diseases of various severities. On the basis of genotypic and serological characterization, CoVs were traditionally divided into three distinct groups (1–3). Recently, the Coronavirus Study Group of the International Committee for the Taxonomy of Viruses has proposed three genera, Alphacoronavirus, Betacoronavirus, and Gammacoronavirus, to replace the traditional group 1, 2, and 3 CoVs, respectively. As a result of the unique mechanism of viral replication, CoVs have a high frequency of recombination (2). Their tendency for recombination and high mutation rates may allow them to adapt to new hosts and ecological niches (4, 5).
The severe acute respiratory syndrome (SARS) epidemic, the discovery of SARS coronavirus (SARS-CoV), and the identification of SARS-CoV-like viruses from Himalayan palm civets and a raccoon dog from wild live markets in China in 2003 have boosted interest in the discovery of novel CoVs in both humans and animals (6–11). A novel human CoV (HCoV) of the genus Alphacoronavirus, HCoV-NL63, was reported in 2004 (12, 13). In 2005, we also described the discovery, complete genome sequence, clinical features, and molecular epidemiology of another novel HCoV, HCoV-HKU1, in the genus Betacoronavirus (14–16). As for animal CoVs, we and others have described the discovery of SARS-CoV-like viruses in horseshoe bats in the Hong Kong Special Administrative Region (HKSAR) and other provinces of China (17, 18). In addition, we have also discovered 19 other animal CoVs, which include two novel lineages in Betacoronavirus and a novel genus, Deltacoronavirus (19–27). From our studies, it was shown that bats are the gene source for Alphacoronavirus and Betacoronavirus and birds are the gene source for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination (26). Recently, a novel CoV, named Middle East respiratory coronavirus (MERS-CoV), that is closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5 has emerged as a cause of severe respiratory infections associated with high rates of mortality (28–30). Its isolation supported the suggestion that CoVs are important causes of major epidemics; and therefore, continuous discovery of novel CoVs and genomic and phylogenetic studies of these viruses are of crucial importance.
In the genus Deltacoronavirus, it was shown that, in addition to bird CoVs, it also comprises porcine coronavirus HKU15 from pigs (26). Similarly, in the genus Gammacoronavirus, in addition to Avian coronavirus, which consists of infectious bronchitis virus (IBV) and its closely related bird CoVs, a novel CoV was discovered from a white beluga whale in 2008 (31). As a result of this discovery, we hypothesized that there is a distinct group or species of marine mammal CoVs in Gammacoronavirus. To test this hypothesis, we carried out a molecular epidemiology study in marine mammals of the Ocean Park in HKSAR. Based on the results of comparative genome and phylogenetic analyses in the present study, we propose a novel bottlenose dolphin CoV in Gammacoronavirus. This distinct species of marine mammal CoV in Gammacoronavirus is also discussed.
MATERIALS AND METHODS
Marine mammal surveillance and sample collection.All respiratory, fecal, and blood samples were collected from the marine mammals by veterinary surgeons of the Ocean Park in HKSAR from August 2008 to July 2010 using standard procedures approved by the Animal Welfare Committee of Ocean Park (5, 25). A total of 18 Indo-Pacific bottlenose dolphins (Tursiops aduncus), 20 California sea lions (Zalophus californianus), and 7 harbor seals (Phoca vitulina) in the Ocean Park were tested in this study.
RNA extraction.Viral RNA was extracted from the respiratory, fecal, and blood samples using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany). The RNA was eluted in 60 μl of AVE buffer (Qiagen, Hilden, Germany) and was used as the template for reverse transcription-PCR (RT-PCR).
RT-PCR of the RdRp gene of CoVs using conserved primers and DNA sequencing.Initial CoV screening was performed by amplifying a 440-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene of CoVs using conserved primers (5′-GGTTGGGACTATCCTAAGTGTGA-3′ and 5′-ACCATCATCNGANARDATCATNA-3′) designed by multiple alignments of the nucleotide sequences of available RdRp genes of known CoVs. After the detection of the novel CoV from the bottlenose dolphin sample, subsequent screening was performed by amplifying the same 440-bp fragment of the RdRp gene using specific primers (5′-GGTTGGGACTATCCTAAGTGTGA-3′ and 5′-CCATCATCGCTCAATATCATGAGA-3′). Reverse transcription was performed 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, 0.01% gelatin), 200 μM each deoxynucleoside triphosphate, and 1.0 U Taq polymerase (Applied Biosystems, Foster City, CA). The mixtures were amplified by 60 cycles of 94°C for 1 min, 48°C for 1 min, and 72°C for 1 min with 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 results were observed for the negative controls.
The PCR products were gel purified 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 RdRp genes of CoVs in the GenBank database.
Viral culture.Original fecal samples from the three bottlenose dolphins that tested positive for CoV were cultured in cells of the HRT-18G (human colorectal adenocarcinoma; ATCC CRL-11663), Vero E6 (African green monkey kidney; ATCC CRL-1586), Caco-2 (human colorectal adenocarcinoma; ATCC HTB-37), and LLC-MK2 (rhesus monkey kidney; ATCC CCL-7) cell lines.
Complete genome sequencing.Three complete genomes of bottlenose dolphin coronavirus (BdCoV) HKU22 were amplified and sequenced using the RNA extracted from the fecal specimens as the 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 (15, 20) and the CoV database, CoVDB (32), for sequence retrieval. Additional primers were designed from the results of the first and subsequent rounds of sequencing. The 5′ ends of the viral genomes were confirmed by rapid amplification of cDNA ends (RACE) using a 5′/3′ RACE kit (Roche Diagnostics GmbH, Mannheim, Germany). 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 using the EMBOSS needle program (http://www.ebi.ac.uk). Phylogenetic tree construction was performed using the neighbor-joining method with 1,000 bootstraps and the Jones-Taylor-Thornton (JTT) substitution model with a gamma distribution among sites conducted in the MEGA (v5) program (33). Protein family analysis was performed using the PFAM and InterProScan programs (34, 35). Prediction of transmembrane domains was performed using the TMHMM program (36).
Genomes of other CoVs (and their GenBank accession numbers) included in the comparative analysis were as follows: porcine epidemic diarrhea virus (PEDV; NC_003436), Scotophilus bat coronavirus 512 (Sc-BatCoV-512; NC_009657), transmissible gastroenteritis virus (TGEV; DQ811789), feline infectious peritonitis virus (FIPV; AY994055), canine coronavirus (CCoV; GQ477367), porcine respiratory coronavirus (PRCV; DQ811787), mink coronavirus (MCoV; HM245925), Rhinolophus bat coronavirus HKU2 (RhBatCoV HKU2; EF203064), Miniopterus bat coronavirus 1A (Mi-BatCoV 1A; NC_010437), Miniopterus bat coronavirus 1B (Mi-BatCoV 1B; NC_010436), Miniopterus bat coronavirus HKU8 (Mi-BatCoV HKU8; NC_010438), human coronavirus 229E (HCoV-229E; NC_002645), human coronavirus NL63 (HCoV-NL63; NC_005831), Rousettus bat coronavirus HKU10 (Ro-BatCoV HKU10; JQ989270), Hipposideros bat coronavirus HKU10 (Hi-BatCoV HKU10; JQ989266), human coronavirus OC43 (HCoV OC43; NC_005147), bovine coronavirus (BCoV; NC_003045), sable antelope CoV (antelope CoV; EF424621), giraffe coronavirus (GiCoV; EF424622), equine coronavirus (ECoV; NC_010327), porcine hemagglutinating encephalomyelitis virus (PHEV; NC_007732), murine hepatitis virus (MHV; NC_001846), canine respiratory coronavirus (CRCoV; JX860640), rat coronavirus (RCoV; NC_012936), human coronavirus HKU1 (HCoV-HKU1; NC_006577), rabbit coronavirus HKU14 (RbCoV HKU14; JN874559), Tylonycteris bat coronavirus HKU4 (Ty-BatCoV HKU4; NC_009019), Pipistrellus bat coronavirus HKU5 (Pi-BatCoV HKU5; NC_009020), Middle East respiratory syndrome coronavirus (MERS-CoV; JX869059), SARS-related human coronavirus (SARS-CoV; NC_004718), SARS-related Rhinolophus bat coronavirus HKU3 (SARSr-Rh-BatCoV HKU3; DQ022305), SARS-related Chinese ferret badger coronavirus (SARSr CoV CFB; AY545919), SARS-related palm civet coronavirus (SARSr-CiCoV; AY304488), Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9; NC_009021), infectious bronchitis virus (IBV; NC_001451), partridge coronavirus (IBV-partridge; AY646283), turkey coronavirus (TCoV; NC_010800), peafowl coronavirus (IBV-peafowl; AY641576), Beluga whale coronavirus SW1 (BWCoV SW1; NC_010646), bulbul coronavirus HKU11 (BuCoV HKU11; FJ376619), thrush coronavirus HKU12 (ThCoV HKU12; FJ376621), munia coronavirus HKU13 (MunCoV HKU13; FJ376622), porcine coronavirus HKU15 (PorCoV HKU15; JQ065042), white-eye coronavirus HKU16 (WECoV HKU16; JQ065044), sparrow coronavirus HKU17 (SpCoV HKU17; JQ065045), magpie-robin coronavirus HKU18 (MRCoV HKU18; JQ065046), night-heron coronavirus HKU19 (NHCoV HKU19; JQ065047), wigeon coronavirus HKU20 (WiCoV HKU20; JQ065048), and common moorhen coronavirus HKU21 (CMCoV HKU21; JQ065049).
Real-time quantitative RT-PCR.For real-time quantitative RT-PCR assays, cDNA was amplified with a FastStart DNA Master SYBR green I mix reagent kit (Roche Diagnostics GmbH, Mannheim, Germany). Briefly, 20 μl of reaction mixtures containing 2 μl cDNA, 3 mM MgCl2, and 0.25 M forward and reverse primers (5′-CTGCTTATGCCAACAGTGCTT-3′ and 5′-AAGTCCATATCGGGCTTAT-3′) was thermal cycled at 95°C for 10 min, followed by 50 cycles of 95°C for 10 s, 55°C for 5 s, and 72°C for 7 s, using a LightCycler apparatus (Roche Diagnostics GmbH, Mannheim, Germany). A plasmid with the target sequence was used for generating the standard curve. At the end of the assay, PCR products (a 165-bp fragment of the RdRp gene) were subjected to a melting curve analysis (65 to 95°C, 0.1°C/s) to confirm the specificity of the assay.
Cloning and purification of His6-tagged recombinant N protein of BdCoV HKU22.To produce a plasmid for protein purification, primers 5′-TACAGCTAGCATGGCCTCTACATCGGGAAAG-3′ and 5′-GCATGCTAGCTTAAGCTTCAGACCATTCAAG-3′ were used to amplify the gene encoding the nucleocapsid (N) protein of BdCoV HKU22 by RT-PCR. The sequence coding for amino acid residues 1 to 380 of the N protein was amplified and cloned into the NheI sites of expression vector pET-28b(+) (Novagen, Madison, WI) in frame and downstream of the series of six histidine residues. The recombinant N protein was expressed and purified using an Ni2+-loaded HiTrap chelating system (GE Healthcare, Buckinghamshire, United Kingdom) according to the manufacturer's instructions.
Western blot analysis.Western blot analysis was performed according to our published protocol (15). Briefly, 600 ng of purified His6-tagged recombinant N protein of BdCoV HKU22 was loaded into each well of a sodium dodecyl sulfate (SDS)–10% polyacrylamide gel and subsequently electroblotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA). The blot was cut into strips, and the strips were incubated separately with 1:1,000 dilutions of serial serum samples obtained from the three bottlenose dolphins that were RT-PCR positive for BdCoV HKU22 as well as nine bottlenose dolphins that were RT-PCR negative for BdCoV HKU22. Antigen-antibody interaction was detected with 1:8,000-diluted horseradish peroxidase-conjugated anti-bottlenose dolphin IgG (Abcam) and an ECL fluorescence system (GE Healthcare, Buckinghamshire, United Kingdom).
Estimation of synonymous and nonsynonymous substitution rates.The number of synonymous substitutions per synonymous site (Ks) and the number of nonsynonymous substitutions per nonsynonymous site (Ka) for each coding region between each pair of strains were calculated using the Nei-Gojobori method (Jukes-Cantor) in MEGA (v5) (33).
Estimation of divergence dates.A maximum likelihood tree under the GTR+ gamma model of substitution was constructed using the PhyML program (37). A root-to-tip genetic distance determined from a maximum likelihood tree was plotted against the year of sampling using the Path-O-Gen (v1.4) tool (http://tree.bio.ed.ac.uk/software/pathogen/). The complete genome sequences of three strains of BdCoV HKU22 and one strain of BWCoV SW1 were used. The crossing point was taken as the time of the most recent common ancestor (tMRCA) for the four viruses under analysis.
RESULTS
Marine mammal surveillance and identification of CoV in Indo-Pacific bottlenose dolphin.A total of 193 samples, including 58 respiratory, 31 blood, and 104 fecal samples, were obtained from Indo-Pacific bottlenose dolphins, California sea lions, and harbor seals (Table 1). RT-PCR was positive for a 440-bp fragment in the RdRp genes of CoVs in specimens from the fecal samples of three Indo-Pacific bottlenose dolphins (samples CF090325, CF090327, and CF090331). Sequencing results suggested the presence of a CoV with 98% nucleotide sequence identity to BWCoV SW1 (Table 1). No positive results were obtained from any of the California sea lions and harbor seals tested (Table 1).
Marine mammals screened in the present study
Viral culture.Attempts to stably passage BdCoV HKU22 in cell cultures were unsuccessful, with no cytopathic effect or viral replication being detected.
Viral load.Quantitative real-time RT-PCR showed that the amounts of BdCoV HKU22 RNA ranged from 1 × 103 to 1 × 105 copies per ml in the three fecal samples positive for BdCoV HKU22.
Genome organization and coding potential of BdCoV HKU22.Complete genome sequence data for the three strains of BdCoV HKU22 from the three bottlenose dolphins were obtained by assembly of the sequences of the RT-PCR products from the RNA extracted from the corresponding individual specimens. The sizes of the three genomes of BdCoV HKU22 were 31,750 to 31,759 bases, and their G+C contents were 39% (Table 2).
Comparison of genomic features and amino acid identities of BdCoV HKU22 and other CoVs with complete genome sequences availablea
BdCoV HKU22 and BWCoV SW1 possess the same putative transcription regulatory sequence (TRS) motif, 5′-AAACA-3′, at the 3′ end of the leader sequence and preceding most ORFs (Table 3). This TRS has not been found to be the TRS for any CoVs for which complete genome sequences are available other than BdCoV HKU22 and BWCoV SW1. Interestingly, this putative TRS overlapped with the initiation codon AUG by 1 base in ORFs NS5a, NS7, NS9, and NS10. This has never been observed in any other CoVs for which complete genome sequences are available. Notably, the TRS of the N protein in the genomes of BdCoV HKU22 was separated from the corresponding AUG by 108 bases (Table 3). This long stretch of nucleotides between the TRS of N and the corresponding AUG was also observed in the genomes of other gammacoronaviruses (90 bases for IBV, 93 bases for TCoV, and 105 bases for BWCoV SW1), which is in contrast to the relatively small number of bases (≤16 bases) between the TRS for the N protein and the corresponding AUG in other genera of CoVs. Upstream of the initiation codons of NS5a, a short stretch of 13 nucleotides, CUUUAUUCUGUUU in BdCoV HKU22 and CUUUAUUCCGUUU in BWCoV SW1, was observed. These short stretches of 13 nucleotides were homologous to the known internal ribosome entry site (IRES) element UUUUAUUCUUUUU of the envelope (E) gene in murine hepatitis virus and, hence, could be potential IRES elements for NS5b and/or NS5c of BdCoV HKU22 and BWCoV SW1, as these two ORFs do not possess the putative TRS upstream of their initiation codons.
Coding potential and putative TRSs of the genomes of BdCoV HKU22
In addition to the same putative TRS, BdCoV HKU22 also possesses the same genome structure as BWCoV SW1 (Fig. 1). The replicase ORF1ab occupies 19.991 kb of the BdCoV HKU22 genome (Table 3). This ORF encodes a number of putative nonstructural proteins (nsp's), including nsp3 (which contains the putative papain-like protease [PLpro]), nsp5 (a putative chymotrypsin [3C]-like protease [3CLpro]), nsp12 (a putative RdRp), nsp13 (a putative helicase [Hel]), and other proteins of unknown functions. The proteolytic cleavage sites between the nonstructural proteins of ORF1ab in the genomes of BdCoV HKU22 and BWCoV SW1 were identical (Table 4). Similar to BWCoV SW1, the genome of BdCoV HKU22 contained one putative PLpro, which is homologous to the PL2pro enzymes of Alphacoronavirus and Betacoronavirus lineage A and the PLpro enzymes of Betacoronavirus lineages B, C, and D, Avian coronavirus of Gammacoronavirus, and Deltacoronavirus (Fig. 1).
Genome organizations of BdCoV HKU22 and representative CoVs from each genus. Orange boxes, papain-like proteases (PL1pro, PL2pro, and PLpro), chymotrypsin-like protease (3CLpro), and RNA-dependent RNA polymerase (RdRp); green boxes, hemagglutinin esterase (HE), spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins; blue boxes, putative accessory proteins.
Characteristics of putative nonstructural proteins of ORF1ab in BdCoV HKU22, BWCoV SW1, and IBV
The major difference between the genomes of BWCoV SW1 and BdCoV HKU22 was observed in their spike (S) genes, with only 74.3 to 74.7% amino acid identities between the protein encoded by the S gene of BdCoV HKU22 and that encoded by the S gene of BWCoV SW1. Most of the difference occurred in the N-terminal half of the S protein (Fig. 2). In addition to nonsynonymous substitutions, a number of deletions/insertions were also observed in the BdCoV HKU22 genomes compared to the sequence of the BWCoV SW1 genome (Fig. 2A). Moreover, most of the positive selection sites were also observed in the N-terminal half of the S protein (Fig. 2B). In addition to the S protein, the amino acid sequence of the protein encoded by the membrane (M) gene in BdCoV HKU22 also showed a significant difference (14.5%) compared to that of the protein encoded by the M gene in BWCoV SW1, which was greater than the differences detected in most other parts of the genome. Uniquely, BdCoV HKU22 and BWCoV SW1 possessed a number of ORFs (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their M and N genes. These ORFs lead BdCoV HKU22 and BWCoV SW1 to have the largest genome sizes among all CoVs. Among these ORFs, the putative amino acid sequence of NS5a in BdCoV HKU22 showed the largest number of differences from the amino acid sequence of NS5a in BWCoV SW1 (9.4% amino acid differences in NS5a and 0 to 8.5% amino acid differences in the proteins encoded by other ORFs). Similar to BWCoV SW1, a putative transmembrane domain (residues 45 to 65) was detected in NS5b of BdCoV HKU22. The putative protein encoded by NS6 was homologous to the capsid proteins of human astroviruses encoded by ORF2. Similar to BWCoV SW1, putative signal peptide sequences were detected in NS7 of BdCoV HKU22. NS7 of two of the three strains of BdCoV HKU22 (CF090325 and CF090331) contained a premature stop codon, resulting in NS7a and NS7b. Interestingly, a putative signal peptide sequence was detected in NS8 of BWCoV SW1 but not BdCoV HKU22 by the Signal IP (v4) program, but transmembrane domains were detected in the corresponding regions in NS8 of BdCoV HKU22 but not BWCoV SW1 by TMHMM, although there were just four amino acid differences between this signal peptide and the transmembrane region in BWCoV SW1 and BdCoV HKU22. This may reflect inherent limitations of these bioinformatics analysis programs for signal peptide and transmembrane domain detection. Similar to BWCoV SW1, a putative transmembrane domain (residues 7 to 27) was detected in NS9 of BdCoV HKU22. Unlike Avian coronavirus of Gammacoronavirus, most deltacoronaviruses, SARS-related Rhinolophus bat coronavirus (SARSr-Rh-BatCoV), and SARS-CoV, no stem-loop II motif (s2m), a conserved RNA element downstream of the gene for the N protein and upstream of the poly(A) tail, was observed in BdCoV HKU22 or BWCoV SW1.
Amino acid differences and selection pressure analysis of S proteins of BdCoV HKU22 and BWCoV SW1. (A) Distribution of amino acid changes in the S proteins of BdCoV HKU22 and BWCoV SW1. The positions of the amino acid changes are depicted by vertical lines, and deletions are marked by asterisks. (B) Distribution of positively selected sites in S protein identified using random effects likelihood (REL) (39). dN > dS, more nonsynonymous than synonymous substitutions.
Phylogenetic analyses.The phylogenetic trees constructed using the amino acid sequences of the ORF1b polyprotein and the S and N proteins of BdCoV HKU22 and other CoVs are shown in Fig. 3, and the pairwise amino acid identities in 3CLpro, RdRp, Hel, S, and N are shown in Table 2. In all three phylogenetic trees, BdCoV HKU22 was clustered with BWCoV SW1 (Fig. 3). On the basis of both phylogenetic tree analyses and amino acid sequence differences, BdCoV HKU22 and BWCoV SW1 should belong to the same clade in the genus Gammacoronavirus.
Phylogenetic analyses of the ORF1b polyprotein and the S and N proteins of BdCoV HKU22. The trees were constructed by using the neighbor-joining method in the JTT substitution model with a gamma-distributed rate variation and bootstrap values calculated from 1,000 trees. Bootstrap values below 70% are not shown. A total of 2,716, 1,495, and 379 amino acid positions in ORF1b polyprotein, S protein, and N protein, respectively, were included in the analyses. The tree was rooted to Breda virus (AY_427798). For the ORF1b polyprotein, the scale bars indicate the estimated number of substitutions per 10 amino acids. For the S and N proteins, the scale bars indicate the estimated number of substitutions per 5 amino acids. The three strains of BdCoV HKU22 characterized in this study are in boldface.
Western blot analysis.Prominent immunoreactive bands were visible for serum samples collected from the three bottlenose dolphins 4 to 8 weeks after their fecal samples tested positive for BdCoV HKU22 (Fig. 4, lanes 3 and 4, 7 and 8, and 11 and 12). The sizes of the bands were about 42 kDa, consistent with the expected size of 41.7 kDa for the full-length His6-tagged recombinant N protein. Only very faint bands were observed for serum samples obtained before and within a few days after their fecal samples tested positive for BdCoV HKU22 (Fig. 4, lanes 1 and 2, 5 and 6, and 9 and 10), as well as for serum samples obtained from the nine bottlenose dolphins that were RT-PCR negative for BdCoV HKU22 (data not shown).
Western blot analysis of purified recombinant BdCoV HKU22 N-protein antigen. Lanes 1 to 4, 5 to 8, and 9 to 12, serial serum samples collected from the three bottlenose dolphins RT-PCR positive for BdCoV HKU22, respectively. Prominent immunoreactive protein bands of about 42 kDa (arrowheads) were visible 4 to 8 weeks after the fecal samples of the three bottlenose dolphins tested positive for BdCoV HKU22 (lanes 3 and 4, 7 and 8, and 11 and 12). Only very faint bands were observed for serum samples obtained before and within a few days after their fecal samples tested positive for BdCoV HKU22 (lanes 1 and 2, 5 and 6, and 9 and 10). The dates of serum collection are indicated below each lane. The dates that the dolphins were RT-PCR positive and RT-PCR negative for BdCoV HKU22 are also shown.
Estimation of synonymous and nonsynonymous substitution rates.The Ka, Ks, and Ka/Ks ratio for the various coding regions in BdCoV HKU22 are shown in Table 5. Although the numbers of synonymous and nonsynonymous mutations were small, it was notable that the Ka/Ks ratios for the nsp8, S, and NS10 genes were >1, with the nsp8 and S genes having the highest Ka/Ks ratio of 2.5.
Estimation of nonsynonymous and synonymous substitution rates in the genomes of BdCoV HKU22
Estimation of divergence dates.The rate of nucleotide substitution for BWCoV SW1 and BdCoV HKU22 was estimated to be 6.19 × 10−4 from the root-to-tip regression. The estimated tMRCA for BWCoV SW1 and BdCoV HKU22 was approximately in 1959 (correlation coefficient = 0.96) (Fig. 5).
Estimation of date of divergence of BdCoV HKU22 and BWCoV SW1. (A) Regression of root-to-tip distances against the date of sampling of four genomic sequences to estimate the rate of evolution and the time of the most recent common ancestor. (B) Maximum likelihood tree with time scale using the estimated rate of evolution.
DISCUSSION
In this study, we discovered a novel CoV in Indo-Pacific bottlenose dolphins. A unique advantage of the present study was the invaluable opportunity that we had to collect serial fecal and serum samples from marine animals in an oceanarium. Among the serial fecal samples collected from the three bottlenose dolphins from which BdCoV HKU22 was detected, only one fecal sample from each bottlenose dolphin was positive for BdCoV HKU22, while the samples collected 7 months before and 13 months after the date of positive detection were negative for BdCoV HKU22 (data not shown). No persistent shedding of BdCoV HKU22 was observed in the same bottlenose dolphin. For the serial serum samples, only very faint bands were observed by Western blotting of the serum samples collected from the three bottlenose dolphins before and during the time that the fecal samples tested positive for BdCoV HKU22, but very high BdCoV HKU22 N-protein-specific antibody levels were detected in their serum samples at 4 to 8 weeks postinfection (Fig. 4). Moreover, an antibody response was not detected in bottlenose dolphins negative for BdCoV HKU22. These findings indicate that BdCoV HKU22 is associated with acute infections in bottlenose dolphins, where viral clearance was associated with a specific adaptive antibody response when the bottlenose dolphins recovered from the infections. In contrast to the previous report that described the detection of BWCoV SW1 in a 13-year-old beluga whale that died after generalized pulmonary disease and terminal acute liver failure (31), BdCoV HKU22 was probably associated with an asymptomatic or mild infection in these three bottlenose dolphins, as none of the three bottlenose dolphins positive for BdCoV HKU22 developed any notable symptoms.
BdCoV HKU22 and BWCoV SW1 represent a distinct species in Gammacoronavirus. Comparison of the amino acid identities of the seven conserved replicase domains for species demarcation (ADP-ribose-1″-phosphatase, nsp5 [3CLpro], nsp12 [RdRp], nsp13 [Hel], nsp14 [ExoN], nsp15 [NendoU], and nsp16 [2′-O-ribose methyltransferase]) (38) between BdCoV HKU22 and BWCoV SW1 showed that the sequences of all seven domains of the two CoVs had more than 90% identity to each other, indicating that these two CoVs should belong to the same CoV species. In addition, the genomes of BdCoV HKU22 and BWCoV SW1 possess unique characteristics distinct from those of other CoVs (Table 6). The size of their genomes is about 32,000 nucleotides, making their genomes the largest among those of all CoVs for which complete genome sequences are available. The putative TRS for most of their ORFs is AAACA. A large number of ORFs which span more than 4 kb and encode putative nonstructural proteins were observed between their M and N genes. In addition to these unique characteristics, the genomes of BdCoV HKU22 and BWCoV SW1 also differ from those of members of Avian coronavirus by the absence of conserved S1/S2 cleavage sites, ORFs between the S and E genes, and s2m downstream of the N gene (Table 6). Phylogenetically, BdCoV HKU22 is closely related to BWCoV SW1, with high bootstrap support in all phylogenetic trees constructed, while these two CoVs are, in turn, closely related to members of Avian coronavirus (Fig. 3). The close relatedness of BdCoV HKU22 and BWCoV SW1 is in line with the similar close relatedness of their corresponding hosts, as dolphins and whales both belong to the cetaceans, one of the four groups of marine mammals, namely, cetaceans (whales, dolphins, and porpoises), pinnipeds (seals, sea lions, and walruses), sirenians (manatees and dugongs), and fissipeds (polar bear and otters). Based on all these pieces of evidence, we propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, which includes BdCoV HKU22 and BWCoV SW1 in the model of CoV evolution (Fig. 6). In this model, BdCoV HKU22 and BWCoV SW1 originated from a common ancestral CoV approximately 54 years ago (Fig. 5). Gammacoronavirus also contains the species Avian coronavirus, which includes IBV and its closely related bird CoVs.
Comparison of genome characteristics among alphacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronavirusesa
Model of CoV evolution with a distinct species, Cetacean coronavirus, in Gammacoronavirus.
Despite their close relationships, BdCoV HKU22 and BWCoV SW1 represent two CoVs that probably use different receptors in Indo-Pacific bottlenose dolphins and white beluga whales, respectively. Although they belong to the same CoV species, there is a more than 25% difference between the amino acid sequences of the S protein in BdCoV HKU22 and that in BWCoV SW1. Most of the difference is observed in the N-terminal half of their S proteins, where the receptor binding domains should be located (Fig. 2). No recombination between the S gene of BdCoV HKU22 and that of BWCoV SW1 was observed by Simplot analysis (data not shown). Since all the three bottlenose dolphins were RT-PCR positive for BdCoV HKU22 on the same date and they were housed in the same oceanarium, their infections likely originated from a single source. However, the high Ka/Ks ratios in multiple regions of the genome, with the S gene having a very high Ka/Ks ratio of 2.5, indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to a new host. Epidemiological studies in bottlenose dolphins in other parts of the world as well as other groups of marine mammals will not only ascertain whether this CoV recently jumped from birds to marine mammals but also further delineate the phylogenetic map of CoVs in these animals.
ACKNOWLEDGMENTS
We thank Wing-Man Ko, secretary for food and health, and Constance Chan, director of the Department of Health, HKSAR, The Peoples' Republic of China. We also thank the Ocean Park Corporation for access to the specimen collection.
This work was partly supported by a Research Grant Council grant, University Grant Council; the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the HKSAR Department of Health; the HKSAR Health and Medical Research Fund; and the Strategic Research Theme Fund, The University of Hong Kong.
FOOTNOTES
- Received 20 August 2013.
- Accepted 5 November 2013.
- Accepted manuscript posted online 13 November 2013.
- Copyright © 2014, American Society for Microbiology. All Rights Reserved.
