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Veterinary Microbiology 132 (2008) 221–234

www.elsevier.com/locate/vetmic

Review

An update on canine coronaviruses: Viral evolution and

pathobiology
Nicola Decaro *, Canio Buonavoglia
Department of Public Health and Animal Sciences, Faculty of Veterinary Medicine of Bari,
Strada per Casamassima km 3, 70010 Valenzano, Bari, Italy

Received 30 January 2008; received in revised form 30 May 2008; accepted 6 June 2008

Abstract

The emergence of human severe acute respiratory syndrome incited renewed interest in animal coronaviruses (CoVs) as
potential agents of direct and indirect zoonoses. The reinforced epidemiological surveillance on CoVs has led to the
identification of new viruses, genotypes, pathotypes and host variants in animals and humans. In dogs, a CoV associated
with mild enteritis, canine coronavirus (CCoV), has been known since 1970s. CoV strains with different biological and
genetic properties with respect to classical CCoV strains have been identified in dogs in the last few years, leading to a full
reconsideration of the CoV-induced canine diseases. The genetic evolution of dog CoVs is paradigmatic of how CoVs
evolve through accumulation of point mutations, insertions or deletions in the viral genome, that led to the emergence of
new genotypes (CCoV type I), biotypes (pantropic CCoV) and host variants (canine respiratory coronavirus). This paper is
a review of the current literature on the recent genetic evolution of CCoV and emergence of new CoVs in the dog. The
significances of the newly acquired information for the canine health status and prophylaxis programmes are also
discussed.
# 2008 Elsevier B.V. All rights reserved.

Keywords: Dog; Coronaviruses; Genetic evolution; New genotypes/pathotypes

Contents

1. Coronavirus taxonomy and genomic organisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

2. Canine enteric coronavirus (CCoV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.1. History and pathobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.2. CCoV genotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
2.3. Virulent/divergent strains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

* Corresponding author. Tel.: +39 0804679832; fax: +39 0804679843.

E-mail address: [email protected] (N. Decaro).

0378-1135/$ – see front matter # 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2008.06.007
222 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234

3. Canine pantropic coronavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

4. Canine respiratory coronavirus (CRCoV) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
5. Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

1. Coronavirus taxonomy and genomic antigenic group 2 along with BCoV (Dea et al., 1990;
organisation Verbeek and Tijssen, 1991).
The 50 two-thirds of the 27.6–31-kb CoV genome
Coronaviruses (family Coronaviridae, order Nido- consists of two overlapping open reading frames
virales) are large, single-stranded, positive-sense RNA (ORFs) that encode non-structural proteins including
viruses, which are responsible for enteric and/or the viral RNA-dependent RNA polymerase and
respiratory disease in mammals and birds (Enjuanes proteases. Another one-third nucleotide sequences
et al., 2000). Currently, CoVs are classified into three from the 30 end contain ORFs encoding for the major
different antigenic groups, although divergent CoVs structural spike, envelope, membrane, and nucleo-
have been identified in bats and wild carnivores in capsid proteins. The trimeric spike (S) protein, the
recent years, thus suggesting revision of CoV main inducer of virus-neutralising antibodies
taxonomy (Tang et al., 2006; Dong et al., 2007). (Gebauer et al., 1991), forms characteristic viral
Phylogenetic relationship of CoVs of the different peplomers which mediate viral attachment to specific
groups is represented in Fig. 1. Group 1 CoVs include cell receptors and fusion between the envelope and
canine coronavirus (CCoV), feline coronaviruses plasma membrane (Enjuanes et al., 2000). The small
(FCoVs) type I and type II, transmissible gastro- membrane (E) protein, recently recognised as a
enteritis virus (TGEV) of swine, porcine respiratory structural component of the coronavirions, is thought
coronavirus (PRCoV), porcine epidemic diarrhoea to be important for viral envelope assembling
virus (PEDV) and human coronaviruses 229E (HCoV- (Vennema et al., 1996). The membrane (M) protein,
229E) and NL63 (HCoV-NL63). Recently, a ferret the most abundant structural component, is a type III
coronavirus has been identified as a member of group glycoprotein consisting of a short amino-terminal
1 (Wise et al., 2006). Currently, group 2 CoVs are ectodomain, a triple-spanning transmembrane
organised into bovine-like (subgroup 2a) and severe domain, and a long carboxyl-terminal inner domain
acute respiratory syndrome (SARS)-like (subgroup (Rottier, 1995). Antibodies to the M protein of MHV
2b) viruses. Members of subgroup 2a are bovine can neutralise viral infectivity, but only in the presence
coronavirus (BCoV), mouse hepatitis virus (MHV), of complement (Collins et al., 1982). The nucleo-
rat coronaviruses, porcine haemagglutinating ence- capsid (N) protein is a highly basic phosphoprotein
phalomyelitis virus (PHEV), human coronavirus that modulates viral RNA synthesis, binds to the viral
(HCoV) OC43, human enteric coronavirus (HECV) RNA and forms a helical nucleocapsid (Enjuanes
4408 (Enjuanes et al., 2000), and the newly recognised et al., 2000). Additional ORFs encoding non-structural
equine coronavirus (ECoV) (Guy et al., 2000), HCoV- proteins have been recognised in CoV genomes and
HKU1 (Woo et al., 2005) and canine respiratory their number, nucleotide sequence and gene order can
coronavirus (CRCoV) (Erles et al., 2003). SARS-CoV, vary remarkably among different CoVs (Boursnell
initially defined as prototype of a new group 4, has et al., 1987; Lee et al., 1991; Herold et al., 1993;
been placed more recently within group 2 CoVs, in a Eleouet et al., 1995). The functions of such genes are
subgroup 2b, together with SARS-like CoVs isolated in most cases unknown and most of them are not
from bats and wild carnivores (Gorbalenya et al., essential for virus replication but may play a part in
2004; Weiss and Navas-Martin, 2005). Group 3 virulence and host range (Yamanaka et al., 1998;
comprises CoVs of avian origin, whose prototype is Haijema et al., 2004). Group 2 CoV genomes contain
represented by avian bronchitis virus, although the an additional structural protein (HE) with haemag-
turkey coronavirus had been placed previously in glutinin-esterase activity, which shares up to 30%
 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234 223

amino acid identity to the analogous protein of FCoV-like CCoVs (Pratelli et al., 2001). A genetic
influenza C viruses (Enjuanes et al., 2000). drift to FCoV type II was also observed in the
sequence of CCoVs detected in the faeces of two
naturally infected pups during the late stages of long-
2. Canine enteric coronavirus (CCoV) term viral shedding (Pratelli et al., 2002). Subse-
quently, extensive sequence analysis on multiple
2.1. History and pathobiology regions of the viral genome, including ORF1a, ORF1b
and ORF5, of several CCoV positive faecal samples
The first report on CCoV infection is dated 1971, provided strong evidence for the existence of two
when Binn and colleagues first isolated a coronavirus separate genetic clusters of CCoV. The first cluster
(strain 1-71) from dogs with acute enteritis in a canine includes CCoVs intermingled with reference CCoV
military unit in Germany (Binn et al., 1974). The strains, such as Insavc-1 and K378, while the second
experimental administration of strain 1-71 to young cluster segregates separately from CCoVs and,
dogs was able to reproduce the gastroenteric disease presumably, represents a genetic outlier referred to
(Keenan et al., 1976). Since then, several CCoV as FCoV-like CCoV (Pratelli et al., 2003b).
outbreaks have been reported worldwide, showing that Finally, the nucleotide sequence of a region
CCoV is an important enteropathogen of the dog. encompassing about 80% of the S gene of one of
Serological and virological investigations have these FCoV-like CCoVs (strain Elmo/02) was
demonstrated that CCoV is widespread in dog determined (Pratelli et al., 2003a). Phylogenetic
population, mainly in kennels and animal shelters analysis on the inferred amino acid sequence
(Carmichael, 1978; Rimmelzwaan et al., 1991; (Fig. 1) clearly showed that strain Elmo/02 segregates
Tennant et al., 1993; Möstl et al., 1994; Bandai with FCoVs type I (81% identity) rather than
et al., 1999; Naylor et al., 2001b; Yeşilbağ et al., 2004; reference CCoVs and FCoVs type II (54% identity).
Schulz et al., 2008). CCoV infection is characterised On the basis of the significant genetic similarity
by high morbidity and low mortality, as well as by a between Elmo/02 and FCoVs type I, this strain has
typical faecal–oral route of transmission (Tennant been designated as the prototype of the newly
et al., 1991). CCoV is shed at high titres with the recognised CCoV type I, whereas reference CCoVs
faeces of the infected dogs and its infection is have been referred to as CCoV type II (Pratelli et al.,
restricted to the alimentary tract, leading to the onset 2003a). Unlike group 1 CoVs, CCoV type I shares
of clinical signs typical of the gastroenteric involve- with members of groups 2 and 3 a potential cleavage
ment including loss of appetite, vomiting, fluid site in the S protein (Pratelli et al., 2003a). Moreover,
diarrhoea, dehydration and, only occasionally, death. the genome of this genotype contains an additional
Usually, systemic disease is not observed during ORF, 624 nt in length, which has not been detected in
CCoV infection, although the virus has been isolated CCoV type II and other group 1 CoVs (Lorusso et al.,
from several tissues (tonsils, lungs and liver) of pups 2007, Fig. 2). Computer-aided analysis of this
infected experimentally (Tennant et al., 1991). Fatal additional ORF, which was referred to as ORF3,
disease commonly occurs as a consequence of mixed showed that the putative encoded protein is 207 aa
infections with CCoV together with canine parvovirus long, has a predicted molecular weight of about
type 2 (CPV-2) (Decaro et al., 2006, 2007b), canine 24 kDa and an isoelectric point of 7.02. Analysis of
adenovirus type 1 (Decaro et al., 2007a) or canine hydrophobic profile showed a neutral median hydro-
distemper virus (Decaro et al., 2004a). phaty pattern with a highly hydrophobic region
localised at the N-terminus due to the presence of a
2.2. CCoV genotypes leucin- and isoleucin-rich region. This region also
contains a signal peptide with the aa cleavage site at
Genetic analysis of several CCoVs detected in pups position 15 (12VAAKD16). This finding and the
with diarrhoea in Italy revealed a number of point observation that no transmembrane region was found
mutations affecting a fragment of the M gene, which suggest that the protein is secreted from the infected
has led to the designation of these atypical CCoVs as cells.
224 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234

Fig. 1. Phylogenetic relationship of the S proteins of animal and human coronaviruses. The tree was generated by the neighbor-joining method in
the Mega3 program (Kumar et al., 2004). For phylogenetic tree construction, the following CoV strains were used (GenBank accession numbers
are reported in parentheses): group 1: canine coronavirus type II (CCoVII) Insavc1 (D13096), CCoVII-BGF10 (AY342160), CCoVII-CB/05
(DQ112226), canine coronavirus type I (CCoVI) Elmo/02 (AY307020), CCoVI-23/03 (AY307021), feline coronavirus type II (FCoVII) 79-1146
(NC_007025), FCoVII-79-1683 (X80799), feline coronavirus type I (FCoVI) KU-2 (D32044), FCoVI-Black (AB088223), FCoVI-UCD1
(AB088222), transmissible gastroenteritis virus (TGEV) Purdue (NC_002306), Chinese ferret badger coronavirus (CFBCoV) CFB/GD/DM95/
03 (EF192156), human coronavirus (HCoV) NL63 (NC_005831); group 2: human severe acute respiratory syndrome coronavirus (SARS-CoV)
 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234 225

CCoV type I is distinguishable from CCoV type II 2.3. Virulent/divergent strains

by means of conventional RT-PCR assays, which are
able to selectively amplify fragments of the ORF2 Analogously to other CoVs, CCoV can mutate
and ORF5, but that genotype has not been adapted to readily and new potentially virulent or genetically
grow in vitro (Pratelli et al., 2004a). Recently, divergent strains have been reported in the last few
TaqMan-based real-time RT-PCR assays have been years. Sequence analysis of the S gene sequences
established for detection and quantification of CCoV showed that some CCoV type II reference and field
RNA in the faeces of dogs with diarrhoea (Decaro strains are more closely related to TGEV than to FCoV
et al., 2004b) and for discrimination between the two (Wesseling et al., 1994; Horsburgh and Brown, 1995;
CCoV genotypes (Decaro et al., 2005). Extensive Wesley, 1999). Naylor et al. (2001a) identified a
molecular analysis of faecal samples collected from virulent strain (UWSMN-1) from an outbreak of fatal
the Italian dog population revealed that CCoV gastroenteritis in a breeding colony in Australia, that
infection in dogs is frequently characterised by the appeared to be divergent from type II CCoVs
simultaneous presence of both genotypes (Decaro circulating in other countries. Sequence analysis of
et al., 2005). The significance of the simultaneous short genomic fragments showed nucleotide identities
infection by both CCoV genotypes has to be to reference type II CCoVs up to 96.1%, 86.1% and
determined, particularly with respect to the patho- 93.0% in ORF1b and in 50 and 30 ends of the spike
biology of CCoV infection, although failure to gene, respectively. Moreover, by phylogenetic analy-
isolate CCoV type I on cell cultures hinders the sis, strain UWSMN-1 was found to cluster separately
acquisition of key information on its pathogenic role from typical canine and feline CoVs, indicating a
in dogs. gradual accumulation of mutations throughout its
Epidemiological investigations revealed that CCoV genome rather than recombination events between
type I is now widespread in dogs in Turkey (Yeşilbağ CCoV and FCoV (Naylor et al., 2001a, 2002).
et al., 2004), Austria (Benetka et al., 2006) and China An epizootic outbreak caused by a hypervirulent
(Wang et al., 2006). In Austria, CCoV type I-like M strain of CCoV type II occurred in a beagle colony in
sequences were reported in cats (Benetka et al., 2006). the United Kingdom (Sanchez-Morgado et al., 2004).
In China, both CCoV genotypes were also found in the The strain, isolate BGF10, was characterised at
faeces of healthy foxes and raccoon dogs, showing molecular level, displaying an exceptionally long
high genetic relatedness with Italian canine isolates in non-structural protein 3b (250 amino acids, Fig. 2) and
the M gene (Ma and Lu, 2005; Wang et al., 2006). As a highly divergent N-terminus of the M protein.
for the pathogenic potential of CCoV type I, the Two cases of fatal CoV disease in pups without
limited data available so far account for its involve- evidence of co-infection by CPV-2 were reported by
ment in canine acute gastroenteritis as reported for Evermann et al. (2005). CCoV infection was demon-
CCoV type II. In fact, type I CCoVs were detected in strated by immunohistochemistry on gut sections and
the faeces of dogs with diarrhoea after natural or electron microscopy of intestinal contents. Histo-
experimental infection (Decaro et al., 2005). More- pathology showed moderate depletion and necrosis of
over, long-term viral shedding, up to 6 months, was lymphoid tissues, including thymus, spleen, lymph
reported in dogs naturally infected (Pratelli et al., nodes and gut-associated lymphoid tissues, in both
2002). pups. However, the authors did not conduct the genetic

Tor2 (NC 004718), bovine coronavirus (BCoV) Mebus (U00735), giraffe coronavirus (GiCoV) US/OH3/2003 (EF424623), alpaca coronavirus
(ACoV) (DQ915164), sable antelope coronavirus (SACoV) US/OH1/2003 (EF424621), bubaline coronavirus (BuCoV) 179/07-11 (EU019216),
canine respiratory coronavirus (CRCoV) 4182 (DQ682406), HCoV-OC43 ATCC VR-759 (NC_005147), human enteric coronavirus (HECoV)
4408 (L07748), porcine haemagglutinating encephalomyelitis virus (PHEV) VW572 (DQ011855), mouse hepatitis virus (MHV) A59
(AY700211), rat sialodacryoadenitis virus (SDAV) 681 (AF207551), HCoV-HKU1 (NC_006577), equine coronavirus (ECoV) NC99 (NC
010327); group 3: avian infectious bronchitis virus (IBV) Beaudette (NP 040831); turkey coronavirus (TCoV) G1 (AY342357). Coronaviruses of
dogs are grey shaded. A statistical support was provided by bootstrapping over 1000 replicates. The scale bar indicates the estimated numbers of
amino acid substitutions per site.
226 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234

Fig. 2. Schematic representation of the genomes of CCoVs and FCoVs depicting the genetic differences among the CCoV genotypes/biotypes.
Genes encoding for structural and non-structural proteins are shown in grey and white, respectively. ORF sizes are not drawn to scale. The arrows
indicate the transcription regulating sequences preceding each CoV gene. The length in amino acids of the nsp 3b of strains BGF10 and CB/05
and the 38-nt deletion in ORF3b of strain CB/05 are reported.
 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234 227

characterisation of the CCoV strains detected, thus which was responsible for a predicted truncated non-
preventing any possible speculations on the molecular structural protein 3b (Decaro et al., 2007d, Fig. 2).
mechanisms responsible for the exceptional strain Experimental infection of seronegative pups with
virulence. strain CB/05 reproduced the disease with occurrence
Further CCoV strains with high virulence were of severe clinical signs, including pyrexia, anorexia,
associated to a fatal outbreak of canine gastroenteritis depression, vomiting, diarrhoea and leukopenia
in Sweden (Escutenaire et al., 2007). The identifica- (Decaro et al., 2008a). A different clinical course
tion of different CCoVs was highly suggestive of was observed according to the age of the infected
strains already circulating in the Swedish dog pups. The older dogs, 6 months of age, slowly
population rather than of new emerging or imported recovered from the disease, whereas two out of three
variants. Importantly, some Swedish strains displayed 2.5-month-old dogs were sacrificed due to the severity
an S gene with the 50 and 30 ends closely related to of the CB/05-induced disease. The pantropism of the
CCoV type I and type II, respectively, thus indicating virus was confirmed by the presence of gross lesions in
their possible origin from recombination events the internal organs of the dead dogs, as well as by the
between the two CCoV genotypes. detection of viral RNA in those tissues, including
brains, albeit at lower titres with respect to those
detected in dogs succumbed to natural infection
3. Canine pantropic coronavirus (Decaro et al., 2007d). Traces of viral RNA were
detected in the blood of a single dog, although further
In 2005, a highly virulent variant of CCoV type II unpublished studies have demonstrated that detectable
(strain CB/05) was reported in Italy which caused a RNemia (viral RNA in white blood cells) can occur
systemic disease followed by a fatal outcome in pups easily during CB/05 experimental infection (Decaro
(Buonavoglia et al., 2006). Clinical signs consisted of et al., unpublished).
fever (39.5–40 8C), lethargy, loss of appetite, vomit- In a subsequent experiment, strain CB/05 was
ing, haemorrhagic diarrhoea, severe leukopenia and proven to be able to infect even dogs with a recent
neurological signs (ataxia, seizures) followed by death infection caused by an enteric CCoV strain, inducing
within 2 days after the onset of the symptoms. the occurrence of mild clinical signs (Decaro et al.,
Necropsy examination revealed severe gross lesions in manuscript in preparation). Although the dogs used in
lungs, liver, spleen, and kidneys. Virological and that study had a strong humoral immunity to enteric
bacteriological investigations on the parenchymatous CCoV at the time of challenge, experimental infection
organs failed to detect common canine pathogens, with strain CB/05 was successful in all pups
whereas CCoV type I and type II were identified in the irrespective of the viral dose administered. Exposure
intestinal content of all pups by genotype-specific to even low amounts of virus would have similar
real-time RT-PCR assays. Unexpectedly, CCoV type infectivity on seropositive animals, since dogs
II RNA was also detected at high titres in lungs, spleen, inoculated with different viral loads displayed the
liver, kidney and brain. A CCoV type II strain (CB/05) same duration of the viral shedding and not so very
was isolated on A-72 cells from all the examined different viral titres in the faeces. The duration of viral
tissues but brain. Immunohistochemistry using a shedding was shorter and the clinical signs milder with
CCoV-specific monoclonal antibody detected CCoV respect to previous observations in seronegative dogs
antigen in all tissues. Sequence analysis of the 30 end (Decaro et al., 2007d), attributed mainly to the cross-
of the genome of the pantropic CCoV strain, including protection induced by antibodies against enteric
ORFs 2 (S gene), 3a, 3b, 3c, 4 (E gene), 5 (M gene), 6 CCoV. Lymphotropism of the strain CB/05 was
(N gene), 7a and 7b, showed that strain CB/05 has a clearly demonstrated by the occurrence of moderate
high degree of amino acid identity to the cognate lymphopenia in several infected pups. However,
ORFs of CCoV type II, although the S protein despite the moderate lymphopenia and the presence
displayed the highest identity to FCoV type II strain of the virus in the lymphoid tissues, the viral RNA was
79-1683. A genetic marker was identified in the CB/05 not detected in the blood at any time. The association
genome, consisting of a 38-nt deletion in ORF3b of strain CB/05 to a severe, sometimes fatal, disease of
228 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234

dogs, together with the isolation of the virus from in-vitro growth, preliminary epidemiological surveys
organs with severe lesions, strongly suggests that were carried out in the United Kingdom, North
CCoV has changed its tropism, acquiring the ability to America, Japan and Italy, by means of serological
spread from the enteric tract to the internal organs assays using the strictly genetically and antigenically
(Decaro et al., 2007d). The molecular basis of the related BCoV as antigen (Erles and Brownlie, 2005;
change of virulence and tropism is being investigated Priestnall et al., 2006; Kaneshima et al., 2006; Decaro
through the assessment of a reverse genetics system et al., 2007c). Those studies detected seropositivity
similar to that established for feline infectious rates comprised between 17.8% (Kaneshima et al.,
peritonitis virus (Haijema et al., 2003). 2006) and 54.7% (Erles and Brownlie, 2005).
Serological evidence was obtained that CRCoV has
been circulating also in other countries, including
4. Canine respiratory coronavirus (CRCoV) Ireland and Greece (Priestnall et al., 2006). Virolo-
gical evidence for the CRCoV presence was provided
As a consequence of the recent emergence of for Canada, Japan and Italy. In Canada, the CRCoV
SARS-CoVand SARS-like viruses (Guan et al., 2003), RNA was identified in archival tissue samples (both
the role of CoVs as aetiological agents of novel collected in 1996) from 2/126 cases of CIRD, but no
diseases in the dog has been investigated. In 2003, a genetic characterisation of the detected strains was
group 2 CoV was identified in the respiratory tract of performed (Ellis et al., 2005). Further CRCoV strains
dogs housed in a rehoming kennel in the United were detected in the nasal (02/005) and rectal (04-009)
Kingdom with a history of endemic respiratory disease swabs of two Japanese dogs (Kaneshima et al., 2006).
(Erles et al., 2003). The viral RNA was detected by RT- The nucleotide sequence identity of the Japanese
PCR in 32/119 tracheal and 20/119 lung samples, CRCoVs to reference T101 strain was 98.0–99.7% in
showing the highest prevalence in dogs with mild the HE protein gene. The S gene was analysed only for
clinical signs. The virus, referred to as canine strain 02/005, showing a 99.1% identity to CRCoV-
respiratory coronavirus (CRCoV), showed a close T101. The Italian survey found the CRCoV RNA in a
genetic relatedness to the bovine subgroup in the single lung sample out of 109 tested by RT-PCR, with
replicase and spike proteins (Fig. 1). Sequence a 98.0% sequence identity to strain T101 in the S gene
analysis of the S gene of CRCoV strain T101 revealed (Decaro et al., 2007c).
a nucleotide identity of 97.3% and 96.9% to the group Although CRCoV has been detected in tissue
2 CoVs BCoV and HCoV-OC43, respectively, samples of several dogs by RT-PCR, isolation of the
suggesting a recent common ancestor for the three virus on canine cell lines as well as on the human
viruses and demonstrating the occurrence of repeated adenocarcinoma cell line HRT-18 was at first
host-species shifts (Vijgen et al., 2005, 2006). An unsuccessful (Erles et al., 2003; Kaneshima et al.,
additional suggestion for the bovine origin of CRCoV 2006). Only recently, Erles et al. (2006) were able to
was provided by the successful experimental infection propagate the CRCoV strain 4182 from a canine
of pups with a typical BCoV strain (Kaneshima et al., respiratory sample on HRT-18 cells and to determine
2007). Conversely, CRCoV was found to be geneti- the full-length sequence of the 30 end of its genomic
cally unrelated to CCoV, displaying only a 21.2% RNA (9.8 kb). By sequence analysis, strain 4182 was
amino acid identity to the enteric virus in the S protein found to have a genomic organisation similar to BCoV
(Erles et al., 2003). Unlike the enteric coronaviruses with a close genetic relatedness to the bovine CoV
CCoVs type I and II, CRCoV is responsible for mild subgroup in the major structural and non-structural
respiratory signs and is recognised as aetiological proteins, excepting for the ORFs encoding for small
agent of canine infectious respiratory disease (CIRD) non-structural proteins between the S and E genes. In
together with Bordetella bronchiseptica, canine that region, three different ORFs were identified in the
adenoviruses type 1 and type 2, canine parainfluenza- BCoV genome, encoding for the non-structural 4.9-
virus, canine herpesvirus, reoviruses and influenza kDa, 4.8-kDa and 12.7-kDa proteins, whereas only
viruses (Erles et al., 2004; Buonavoglia and Martella, two ORFs, which encode for the non-structural 8.8-
2007). Due to the difficult adaptation of CRCoV to the kDa and 12.8-kDa proteins, were present in the
 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234 229

CRCoV genome. This mutation, due to a 2-nt deletion group-specific genes 3c, 7b (Vennema et al., 1998) or
prior to the stop codon that terminates the 4.9-kDa 7a (Kennedy et al., 2001). Those mutations have been
protein of BCoV leading to the translation of a single suggested to be involved in changes in the tropism of
8.8-kDa joint protein, was found in all British the virus, which may acquire the ability to infect
CRCoVs but strain G9142. Despite the multiple monocytes/macrophages and to cause a systemic, fatal
reports of CRCoV in different areas of the world, the disease of cats known as feline infectious peritonitis
role of CRCoV in CIRD is not completely clear. (FIP). Similar drastic shifts of tissue tropism have
Analogously to other canine respiratory pathogens, it been observed with murine coronaviruses (Haspel
is likely that single infections with CRCoV determine et al., 1978). Adaptation to humans of the recently
only a subclinical or asymptomatic course. However, recognised SARS-associated coronavirus (SARS-
CRCoV replication in the respiratory epithelium may CoV) appears to be related to minor genome
damage the mucociliar system, leading to a more mutations, consisting of a 29-nt deletion in the
severe clinical course of infections caused by other genome of a wild-mammal coronavirus, that resulted
respiratory pathogens (Buonavoglia and Martella, in the translation of two different ORFs, 10 and 11,
2007). instead of the single ORF10 (Guan et al., 2003). There
is multiple genetic and antigenic evidence that several
subgroup 2a CoVs, such as HCoV-OC43, HECV-4408
5. Epilogue and PHEV, have arisen as consequence of trans-
species infections caused by BCoV (Zhang et al.,
Accumulation of point mutations, as well as small 1994; Vijgen et al., 2005, 2006; Erles et al., 2006).
insertions and deletions in coding and non-coding Recently, bovine-like CoVs were identified in wild or
sequences, are the dominant forces in the micro- domesticated ruminants, including several species of
evolution of positive-sense RNA viruses, resulting in deer, waterbuck antelope (Tsunemitsu et al., 1995),
proliferation of virus strains, serotypes and subtypes giraffe (Giraffa camelopardalis) (Hasoksuz et al.,
(Dolja and Carrington, 1992). Extremely large (+) 2007), alpaca (Lama pacos) (Jin et al., 2007), sable
RNA virus genomes, such as those of CoVs, are antelope (Hippotragus niger) (Spiro et al., unpub-
thought to mutate at high frequency as a consequence lished) and water buffalo (Bubalus bubalis) (Decaro
of high error rates of the RNA polymerase that are et al., 2008b) (Fig. 1), but the genetic determinants that
predicted to accumulate several base substitutions per may have caused the interspecies transmission from
round of replication (Jarvis and Kirkegaard, 1991; Lai cattle have not been identified so far.
and Holmes, 2001). Changes in virulence, tissue Another important mechanism for CoV genetic
tropisms and/or interspecies transmission of CoVs evolution is the high frequency of homologous RNA
occur through genetic variations in structural and/or recombination (Lai et al., 1985; Makino et al., 1986).
non-structural proteins (Laude et al., 1993; Vennema This process is believed to be mediated by a ‘‘copy-
et al., 1998; Guan et al., 2003; Rottier et al., 2005; choice’’ mechanism (Cooper et al., 1974; Kirkegaard
Song et al., 2005; Vijgen et al., 2005; Decaro et al., and Baltimore, 1986; Makino et al., 1986). Recombi-
2007d). The ORF2 of PRCoV has a 200-aa deletion in nation of CoV genomes has been observed during
the N-terminus with respect to TGEV, from which it growth in tissue cultures (Lai et al., 1985; Makino
presumably had arisen. Most likely, this deletion is et al., 1986; Sanchez et al., 1999; Kuo et al., 2000), in
responsible for the change in the viral pathobiology experimentally infected animals (Keck et al., 1988),
(Vaughn et al., 1995). Nevertheless, minor amino acid and in embryonated eggs (Kottier et al., 1995). There
differences in the sequence of the spike protein have is also evidence for homologous recombination in IBV
been shown to change the virulence of even very in the field (Jia et al., 1995). Recent findings suggest
closely related TGEV isolates (Sanchez et al., 1999). that this mechanism also may be an important factor in
The enteric biotype of FCoV, feline enteric corona- the evolution of FCoVs (Vennema et al., 1995;
virus (FECV), causes persistent infections of the Herrewegh et al., 1998).
intestinal mucosa that may lead to point mutations in In the few last years, CoVs of the dog have
the S gene (Rottier et al., 2005) and/or deletions in the undergone a genetic evolution mainly through
230 N. Decaro, C. Buonavoglia / Veterinary Microbiology 132 (2008) 221–234

accumulation of point mutations and deletions in some challenge with pantropic CCoV (Decaro et al.,
genomic regions rather than through recombination manuscript in preparation), the efficacy of currently
events. Changes in the group-specific genes located used vaccines prepared with enteric CCoV strains is
between ORFs 2 and 4 have been postulated to be likely to be much poorer against pantropic CB/05-like
responsible for increased virulence of CCoV type II viruses. As for CRCoV, experimental vaccines should
strains (Sanchez-Morgado et al., 2004; Decaro et al., be developed only if a clear pathogenic role in the
2007d). Point mutations in the S gene have been also occurrence of CIRD is demonstrated by future
suggested to be involved in the emergence of studies.
pantropic CCoV (Decaro et al., 2007d). CRCoV Further investigations would provide new insights
emerged likely as host variant of a BCoV strain that into the molecular mechanisms responsible for the
was able to spread from cattle to dogs (Erles et al., change in viral pathobiology and into the pathogenic
2006). A different origin could be hypothesised for and immunological aspects of the canine CoVs. At the
CCoV type I. Based on the close genetic relatedness to same time, enduring epidemiological surveillance will
FCoV type I in the S gene, a potential recombinant help a timely identification in dogs of further CoV
origin of this CCoV genotype had been suggested strains with different genetic and biological properties
(Pratelli et al., 2003a). However, the recent identifica- and a more in-depth comprehension of the pathogenic
tion of ORF3 in the genome of CCoV type I should potential of these animal CoVs.
lead to reconsider its origin. In fact, since remnants of
ORF3 were found in some type II CCoVs, the most
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