Virus Research 141 (2009) 96–100
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journal homepage: www.elsevier.com/locate/virusres
Molecular characterization of a canine respiratory coronavirus strain detected
Alessio Lorusso, Costantina Desario, Viviana Mari, Marco Campolo, Eleonora Lorusso,
Gabriella Elia, Vito Martella, Canio Buonavoglia, Nicola Decaro∗
Department of Public Health and Animal Sciences, Faculty of Veterinary Medicine of Bari, Strada per Casamassima km 3, 70010 Valenzano (Bari), Italy
a r t i c l ei n f o
Received 12 September 2008
Received in revised form 11 December 2008
Accepted 22 December 2008
Available online 20 January 2009
Canine respiratory coronavirus
a b s t r a c t
Coronaviruses (CoVs) are positive-stranded, non-segmented RNA viruses generally responsible for the
to evolve by genetic recombination and/or point mutation is recognized, thus giving rise to new viral
genotypes and mutants with different tissues or host tropism. In particular, a probable origin from the
strictly related bovine coronavirus (BCoV) or, alternatively, from a common ancestor has been suggested
for some group 2a CoVs, including canine respiratory coronavirus (CRCoV). In this study, we report the
sequence analysis of the viral RNA 3?-end of an Italian CRCoV, strain 240/05, together with the sequence
comparison with extant bovine-like viruses including the sole CRCoV strain 4182 previously described.
Interestingly, although the structural proteins show the same features of CRCoV 4182, the genomic region
between the spike and the envelope protein genes of CRCoV 240/05 encodes for three distinct products,
whereas CRCoV 4182 has a unique 8.8kDa protein.
© 2008 Elsevier B.V. All rights reserved.
Coronaviruses (CoVs) are enveloped (+) strand RNA viruses and
ing mammals and birds. With genome sizes ranging from 27.6kb
to 31.6kb, they represent the largest RNA viruses known to date
(Gorbalenya et al., 2006; Siddell and Snijder, 2008). After the emer-
in the scientific community, since animal CoVs can be assumed as
mented epidemiological survey programs, new members of the
family Coronaviridae have been detected in animals and humans.
Currently, CoVs are organized into three antigenic groups with
group 2 including bovine-like (group 2a) and SARS-like (group
2b) viruses (Gorbalenya, 2008). It has been suggested that human
coronavirus (HCoV) OC43 has arisen as consequence of a trans-
species infection caused by bovine coronavirus (BCoV) (Vijgen et
al., 2005), and this may be also true for some animal group 2a
CoVs, including porcine hemagglutinating encephalomyelitis virus
(PHEV), buffalo coronavirus (BuCoV), giraffe coronavirus (GiCoV),
alpaca coronavirus (ApCoV), and canine respiratory coronavirus
?The GenBank accession number of the sequence of CRCoV 240/05 reported in
this paper is EU999954.
∗Corresponding author. Tel.: +39 0804679832; fax: +39 0804679843.
E-mail address: email@example.com (N. Decaro).
Jin et al., 2007; Decaro et al., 2008; Genova et al., 2008). Alterna-
tively, BCoV and bovine-like CoVs may have arisen from a common
ancestor (Vijgen et al., 2006). Unlike the group 1a enteric canine
coronaviruses (CCoVs) types I and II (for a review, see Decaro and
Buonavoglia, 2008) CRCoV has been associated with mild respira-
tory signs and has been proposed as an etiological agent of canine
infectious respiratory disease (CIRD) together with Bordetella bron-
chiseptica, canine adenovirus types 1 and 2, canine parainfluenza
virus, canine herpesvirus, reoviruses and influenza viruses (Erles
et al., 2004; Buonavoglia and Martella, 2007; Erles and Brownlie,
2008). CRCoV has been detected firstly in UK in 2003 (Erles et al.,
2003) and subsequently in Italy (Decaro et al., 2007). A group 2a
CoV has also been identified in dogs in Canada (Ellis et al., 2005)
and Japan (Kaneshima et al., 2006), and there is serological evi-
dence that a bovine-like CoV is circulating in the canine population
in other countries, including USA, Ireland and Greece (Priestnall et
al., 2006). CRCoV possesses the canonical genome organization of
and for the structural proteins (hemagglutinin-esterase, HE; spike,
S; envelope, E; membrane, M; nucleocapsid, N), expressed through
spersed among the structural genes, CRCoV, analogously to BCoV,
carries some accessory genes, encoding for non-structural proteins
(nsp), located between the replicase and the HE protein genes and
0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
A. Lorusso et al. / Virus Research 141 (2009) 96–100
between the S and E protein genes that are also expressed from
sg mRNAs (Narayanan et al., 2008). These sg mRNAs each contain
a short leader sequence at their 5?-end, followed by a transcrip-
tion regulatory sequence (TRS). TRS sequences are also present in
the genome upstream of each gene and they serve as signals for the
2008). Despite the serological evidence that BCoV-like particles are
circulating in dogs, few partial genomic sequences of CRCoVs are
publicly available, mostly regarding the S and HE proteins, except
the English isolate CRCoV 4182 whose entire genomic 3?-end has
been determined and analyzed (Erles et al., 2007).
In the present study, we report the sequence and phylogenetic
analyses of the entire genomic 3?-end of the CRCoV 240/05 strain
A 9.6-kb region encompassing the complete 3?-end of the viral
genome of CRCoV 240/05 was determined through RT-PCR ampli-
fications of overlapping fragments by using primers previously
published (Decaro et al., 2008; Decaro and Buonavoglia, 2008)
and the kit SuperScriptTMOne-Step RT-PCR for Long Templates
(Life Technologies, Invitrogen, Milan, Italy) which contains a high-
fidelity DNA polymerase (Platinum®Taq Hi Fi). The RNA was
extracted from a fragment of the original lung sample stored in
RNAlater RNA Stabilization Reagent (Qiagen S.p.A., Milan, Italy) by
using the QIAamp®RNeasy Mini Kit (Qiagen S.p.A.), following the
manufacturer’s instructions, with the template RNA being eluted
in 50?l of RNase-free water. DNA samples generated from two dif-
ferent RT-PCR runs were sequenced in both directions by Cogenics
Europe (Meylan, France). Additional RT-PCR assays and sequencing
attempts were performed to close gaps between assembled con-
tigs using strain-specific primers. The analysis of the 240/05 3?-end
genomic sequence by means of the NCBI graphical analysis ORF
Finder tool (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) identi-
fied nine ORFs that can be deduced to encode the non-structural
and structural proteins of the virus (Fig. 1). All the predicted ORFs
but the 2.7kDa gene were preceded by a TRS sequence (CUAAAC
or CCAAAC) which is assumed to interact with the viral poly-
merase during the discontinuous transcription of the negative sg
RNAs (for a review, see Britton and Cavanagh, 2008). The ORFs
encoding structural proteins are ORF 2b (nt 1017–2291), ORF 3 (nt
2306–6397), ORF 5 (nt 7087–7341), ORF 6 (nt 7356–8048), ORF
7 (nt 8058–9404). The predicted translation products are the HE
(47.7kDa), S (151.1kDa), E (9.2kDa), M (26.4kDa) and N (49.3kDa)
proteins, respectively. Analogously to other bovine-like CoVs, the
genome of strain 240/05 contained three accessory genes encod-
ing for four group-specific non-structural proteins, namely ORF 2a,
located between pol1b and HE genes and encoding for the 32kDa,
the 2.7kDa proteins and ORF 4b encoding for the 12.8kDa protein.
Additionally, the N protein gene displayed an accompanying inter-
nal ORF 8 encoding for the I protein of 23.3kDa. CRCoV 240/05 and
4182 differed in 88 positions (excluding the putative 4.9kDa and
and scattered over all the proteins apart from the E protein.
The group 2a-specific accessory structural HE protein was 424
amino acids long with its TRS located nine nucleotides upstream
of the AUG protein initiation codon. The HE protein gene showed
eight synonymous and two non-synonymous nucleotide variations
when compared with that of CRCoV 4182. Interestingly, strain
and derivatives described to date, so therefore these mutations
are unique to CRCoV 4182. Amino acid sequence identity was
99.5% between 240/05 and 4182, whereas it ranged from 93.4% to
97.7% between 240/05 and other BCoV/bovine-like CoVs (Table 1).
By analysis with the NetNglyc server (http://www.cbs.dtu.dk/
services/NetNGlyc/), the HE of 240/05 was found to contain eight
potential glycosylation sites analogously to CRCoV 4182. Accord-
ingly, a potential signal peptide was identified by SignalP 3.0
Server (http://www.cbs.dtu.dk/services/SignalP/) at amino acids
1–18 with a potential cleavage site between amino acids 18 and 19
and the predicted site for neuraminidase-O-acetylesterase activity,
FGDS, was detected at the N-terminus.
The S protein was long 1363 amino acids and its AUG codon
was directly preceded by the TRS sequence. Amino acid identity in
comparison with the S protein of CRCoV 4182 was 98.4% (Table 1)
showing 22 amino acid substitutions, of which nine were found
to be unique to 240/05 strain whereas 10 were present in other
BCoV and derivatives. Twenty-one synonymous nucleotide varia-
tions were encountered in the S gene. Amino acid identity between
240/05 and BCoV/bovine-like viruses S protein ranged from 91.2%
to 95.7% (Table 1). A potential N-terminal signal peptide was iden-
tified at amino acids 1–17 and 21 potential glycosylation sites were
detected throughout the protein. No deletions or insertions were
observed in strain 240/05 compared with bovine-like CoVs. More-
The E protein was 84 amino acids long and identical, in terms of
amino acid sequence, to that of CRCoV 4182. Only one synonymous
nucleotide change differentiated the E protein encoding gene of
strains 240/05 and 4182. Two residues were found to be different
from the same protein of BCoV-Mebus, only one if compared with
the extant BCoVs. The TRS sequence was located 123 nucleotides
upstream the AUG codon as already shown in 4182 strain. SignalP
assigned a signal anchor at position 1–34 and no glycosylation sites
lation initiation codon was separated from the TRS stretch by three
variations were found with respect to the M gene of CRCoV 4182,
three of which were unique to 240/05 strain while the remaining
Fig. 1. Schematic comparison of the CRCoV 240/05 and 4182 genomes. Below the diagram, the length in amino acids is reported for the encoded structural and the putative
A. Lorusso et al. / Virus Research 141 (2009) 96–100
Amino acid identity (%) of CRCoV 240/05 to group 2 CoV reference strains in non-structural and structural proteins.
CoV strainAmino acid identity (%) to CRCoV 240/05
32kDa HES 4.9kDa 2.7kDaa
NA, not available; NP, not present.
aFor this comparison, the correspondent region of the 4.8kDa protein of the bovine-like CoVs was considered.
two changes were shared with extant BCoV strains. Moreover, four
potential O-glycosylation sites (http://www.cbs.dtu.dk/services/
NetOGlyc/) and one N-glycosylation site were identified at the N-
terminus of the protein as previously described for all bovine-like
The N protein of strain 240/05 had a length of 448 amino
acids and was highly conserved among the bovine and bovine-like
viruses. Six synonymous and two non-synonymous changes were
present with respect to strain 4182. The same amino acid changes
were present in extant BCoV-like viruses.
The putative 32kDa nsp showed the same length (278 amino
acids) as most other bovine-like CoVs, including GiCoV, ApCoV and
sable antelope coronavirus (SACoV), as well as HCoV-OC43 and
CRCoV 4182. Three amino acid changes were found in compari-
son with 4182 strain, of which one was unique to 240/05 whereas
the remaining two substitutions were shared with other bovine-
like strains. Five were the synonymous nucleotide substitutions.
The corresponding TRS sequence was located seven nucleotides
upstream of the AUG codon confirming the genome organization
of CRCoV 4182.
In the majority of their genome, CRCoV 240/05 and 4182 pos-
genome structure in the accessory genes encoding for nsp located
between the S and E genes. In this region, most BCoV-like CoVs
display three accessory genes, namely the 4.9kDa, 4.8kDa and
12.8kDa protein genes. In CRCoV 4182 only two accessory genes
were detected (Erles et al., 2007), whereas the genomic arrange-
ment of other bovine-like CoVs was partially conserved in strain
240/05. Indeed, CRCoV 4182 showed a unique 8.8kDa protein fash-
ioned by the fusion of the 4.9kDa protein and a truncated form
of the 4.8kDa protein. This was the result of a two nucleotide
deletion that inactivated the stop codon of the 4.9kDa protein
encoding sequence and introduced additional 12 amino acids not
found in any BCoV strains (Erles et al., 2007). In contrast, in the
240/05 genome the terminating codon of the 4.9kDa encoding
sequence is conserved exactly as in most group 2a CoVs, including
BCoVs-Mebus, Quebec and DB2, HECV-4408 and BuCoV. The cor-
responding TRS sequence is located 317 nucleotides upstream of
the initiation codon according to previous observations for other
ruminant CoVs. However, the 4.8kDa protein of most BCoV and
bovine-like CoVs is replaced in strain 240/05 by a 2.7kDa protein
(25 amino acids in length) due to the presence of an early stop
codon. An identical truncated protein has been already described
truncated protein is present in GiCoV and SACoV (Hasoksuz et al.,
1999). The corresponding TRS sequence was not detected in any
bovine-like viruses with the exception of CRCoV G9142 (Erles et al.,
2007). The 12.8kDa protein was 109 amino acids in length accord-
ing to the extant BCoV and bovine-like CoVs except BCoV Quebec
that exhibits a truncated form of the protein. The related TRS was
located 73 nucleotides upstream of the AUG codon. Two synony-
mous and three non-synonymous nucleotide substitutions with
respect to strain 4182 were detected in the coding sequence, with
one change being unique if compared to other bovine-like CoVs
previously described. The I protein was 207 amino acids in length
according to CRCoV 4182. Only one amino acid change and two
synonymous nucleotide variations differentiated the two CRCoV
strains and strain 240/05 showed almost 95% average amino acid
identity with most of the bovine-derivative viruses.
The rooted phylogenetic analysis (Fig. 2) performed on the S (a)
and M/N (b) proteins confirmed the high genomic relatedness of
previously (Decaro et al., 2007, 2008; Erles et al., 2007). Notewor-
thy, in the S and M/N phylogenetic trees, CRCoV 240/05 and 4182
formed a separate bunch among the group 2a CoVs, most likely as
a consequence of the speciation of BCoV in the canine host.
This study provides useful data for the molecular comprehen-
sion of bovine-like CoVs in the canine host. The CoV ecology is
intricate due to the number of apparently frequent cross-species
jumps that entail its evolution. Indeed, the presence of bovine-
like CoVs in dogs was clearly demonstrated in previous studies by
serological methods, sequence comparison and phylogenetic anal-
ysis, but molecular data available to date is incomplete and limited
in number. Sequence analysis of the 3?-end of the viral genome
showed that strain 240/05 has a genomic organization similar to
BCoV, including the presence of the ORFs encoding for the nsp
between the S and E protein genes. Although the structural genes
are highly conserved among CRCoV strains 240/05 and 4182, the
location and number of the accessory genes differ between the two
viruses. Moreover, their function in the viral life cycle has not been
established yet. In general, as already demonstrated for other CoVs,
accessory genes are not essential for replication and their expres-
sion could even decrease viral fitness in vitro (Schwarz et al., 1990;
A. Lorusso et al. / Virus Research 141 (2009) 96–100
Fig. 2. Rooted neighbor-joining tree inferred from multiple amino acid sequence alignment of the S (a) and M/N (b) protein, illustrating the relationship of CRCoV in the group
2a. For the analysis, CCoV-II CB/05 (DQ112226) served as outgroup and the following CoVs strain were used: BCoV-Mebus (U00735), Quebec (AF220295), DB 2 (DQ811784),
ENT (AF391541), LUN (AF391542), E-AH65 (EF424615), R-AH65 (EF424617), E-AH65-TC (EF424616), R-AH65-TC (EF424618), E-AH187 (EF424619), R-AH187 (EF424620);
GiCoV (EF424623); ApCoV (DQ915164); SACoV (EF424621); CRCoV-4182 (DQ682406); HCoV-OC43 (NC 005147); PHEV-VW572 (DQ011855); MHV-A59 (AY700211); SDAV
(AF207551); HCoV-HKU1 (NC 006577); BuCoV-179/07-11 (EU019216). A statistical support was provided by bootstrapping over 1000 replicates and bootstrap values >70 are
indicated at the correspondent node. The scale bars indicate the estimated numbers of amino acid substitutions per site.
al., 2005; Yount et al., 2005). Nevertheless, in field conditions those
genes are constantly maintained (Herrewegh et al., 1995; Smits et
al., 2005; Dijkman et al., 2006) and their loss is often accompa-
nied by the decline of virulence in the natural host (de Haan et
al., 2002; Ortego et al., 2003; Haijema et al., 2004). CRCoV 240/05
was directly sequenced from the lung sample of a dog allowing
the molecular characterization of viral RNA coming straight from
field conditions with no adaptation to cell culture. Apparently, in
natural conditions the 4.8kDa protein, strictly maintained in other
bovine-like CoVs, was truncated in CRCoV 240/05, and this may be
potentially associated to the cross-species transmission and subse-
quent adaptation of the ancestor BCoV to a different host. However,
directly downstream of the S protein gene, CRCoV 4182 possessed a
unique 8.8kDa protein gene, whereas CRCoV G9142 displayed the
canonical set of BCoV accessory genes but with the equal truncated
form of the 4.8kDa protein gene as in strain 240/05. Noteworthy,
the identical truncated 4.8kDa terminating codon is present in
240/05, G9142 and in the unique 8.8kDa corresponding nucleotide
than one BCoV strain or ancestor virus was likely involved in the
origin of CRCoV, thus leading to the emergence of different canine
strains with a different organizations of the accessory genes. It has
been hypothesized that the 4.9kDa and 4.8kDa proteins of BCoV
may have arisen through mutation from a bovine 11kDa protein
(Abraham et al., 1990). According to this scenario, CRCoV 4182 may
descent from a mutation of an ancestral bovine-like strain that
exhibited the full-length 11kDa protein, whereas CRCoV 240/05
and G9142 presumably descended from a different ancestor that
showed the two distinct non-structural proteins. Furthermore, nsp
4.9 and 4.8 are not present in the bovine-derivative HCoV-OC43
(Vijgen et al., 2005) and their function is yet unknown in BCoV
itself where the 4.9kDa protein could not been expressed due to
A. Lorusso et al. / Virus Research 141 (2009) 96–100
the absence of a start codon in its mRNA (Hofmann et al., 1993).
Obviously, it cannot be ruled out that the elevated level of genomic
differences among the accessory genes is due to the high frequency
to RNA recombination events that characterize CoV ecology (Lai et
level with extant BCoV strains (Fig. 2), reinforcing previous sugges-
tions that CRCoV apparently originated as a host variant of BCoV
or both viruses descended from a common ancestor. Nevertheless,
the elevated level of amino acid similarity with extant bovine-like
role of this protein in viral pathobiology (Erles et al., 2007). Unfor-
tunately, CRCoV 240/05 could not be propagated in tissue culture
probably as a consequence of the long storage of the tissue sample,
thus preventing further analysis of viral mRNAs in the context of
the infected cells. However, several studies are warranted in order
to investigate the effective functionality of the accessory genes in
their expression and the interconnected evolution of highly similar
viruses in different hosts.
This work was supported by grants from the Italian Min-
istry of Health, Ricerca finalizzata 2008, project “Mammalian
coronaviruses: molecular epidemiology, vaccine development and
implications for animal and human health”. The authors are grate-
ful to P.J. Collins (CIT, Department of Biology, Cork, Ireland) for the
English revision of the manuscript.
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