Date Published: July 7, 2011
Publisher: Hindawi Publishing Corporation
Author(s): Annamaria Pratelli.
Since the first identification of the virus in 1971, the disease caused by canine coronavirus (CCoV) has not been adequately investigated, and the role that the virus plays in canine enteric illness has not been well established. Only after the emergence in 2002 of SARS in human has new attention been focused on coronaviruses. As a consequence of the relatively high mutation frequency of RNA-positive stranded viruses, CCoV has evolved and, with the biomolecular techniques developed over the last two decades, new virus strains, serotypes, and subtypes have been identified in infected dogs. Considering the widespread nature of CCoV infections among dog populations, several studies have been carried out, focusing upon the epidemiological relevance of these viruses and underlining the need for further investigation into the biology of CCoVs and into the pathogenetic role of the infections. This paper reports the evolutionary processes of CCoVs with a note onto recent diagnostic methods.
Coronaviruses (CoVs), a genus in the Coronaviridae family, order Nidovirales, are large, enveloped, RNA viruses that cause highly prevalent diseases in humans and domestic animals. CoVs are spherical enveloped particles about 100–120 nm in diameter with a capped, polyadenylated single-stranded, positive-sense genomic RNA 27.6 to 31 kb in length, the largest known RNA virus genome. The 5′ end of the genome consists of a 65 to 98 nt sequence, termed the leader RNA, that is also present at the 5′ end of all subgenomic mRNAs. An untranslated region (UTR) of 200 to 400 nts follows this leader sequence. At the 3′ end of the RNA genome is another UTR of 200 to 500 nts followed by a poly(A) sequence of variable length. Both 5′- and 3′-UTRs are important for RNA replication and transcription. The remaining genomic sequence includes different open reading frames (ORFs) which differ markedly among coronaviruses in number, nt sequence, genes order, and in method of expression. At the 5′ end of each gene, all CoVs have a common intergenic sequence of about 7 bases which is essential for the formation of subgenomic RNAs .
CCoV was first described during an epizootic in a canine military unit in Germany in 1971 . Starting from this first report, CCoV was isolated repeatedly from affected dogs and today appears to be enzootic worldwide, and dogs of all breeds and ages seem to be susceptible to infection [20–26].
The factors regulating the course of the natural diseases caused by enteric CCoVs are not well understood. CCoVs are responsible for enteritis in dogs, and signs of infections may vary from mild to moderate, but they are more severe in young pups or in combination with other pathogens. Common signs include soft faeces or fluid diarrhoea, vomiting, dehydration, loss of appetite, and, occasionally, death. Dual infections by CCoV and canine parvovirus type 2 (CPV2) are especially severe when infections occur simultaneously , but CCoVs can also enhance the severity of a sequential CPV2 infection .
CCoVs, like other CoVs, show relatively high mutation frequency, and, in recent decades divergent strains have been described. One of the first observations was from Wesley,  who demonstrated that although most CCoV sequences are FCoV-like, the N-terminus of the S gene of CCoV strain UCD-1 is more closely related to TGEV.
Another example of the evident evolution of dog coronaviruses, as a consequence of the accumulation of point mutations, small insertions, and deletions in coding and non-coding regions of the genome, is the recent identification of a novel coronavirus, canine respiratory coronavirus CRCoV, in tissue samples collected from the respiratory tract of diseased dogs. During a survey to establish the causes of canine infectious respiratory disease in a large rehoming kennel in the United Kingdom, a CRCoV was isolated in tracheal and lung samples in dogs with mild clinical symptoms. The virus showed a close relationship to the betacoronavirus in the polymerase and S genes, with the highest amino acid identity with the corresponding BCoV proteins and proved to be only distantly related to enteric CCoVs. By sequence comparison of cDNA polymerase in the analyzed 251 bp sequence, the identity was 98.8% for BCoV and 98.4% for HCoV polymerase gene, whereas it was only 68.5% for CCoV, strain 1–71. When comparing the amino acid sequence obtained by translation of the cDNA sequence from CRCoV to the amino acid sequence of BCoV, HCoV-OC43, and enteric CCoV spike proteins in an overlap of 1093 amino acids, the identities were 96%, 95.2%, and 21.2%, respectively (Figure 3). Moreover, the presence of the HE gene, which is a characteristic protein gene of the members of the betacoronaviruses, has been demonstrated in the CRCoV genome [12, 52]. Since its detection in 2003, CRCoV has been found to be present in dogs in other European countries, as well as in Canada and in Japan [53–58]. The evolution of the betacoronaviruses is closely linked, and it has been suggested that these viruses share a recent common ancestor. CRCoV may also share this ancestor or may have originated from a transfer of BCoV to dogs. In order to conclusively answer the question of whether CRCoV has only recently emerged, a greater number of archived materials need to be tested. In addition, more sequence information from CRCoV strains will be required to perform phylogenetic analyses that may shed more light on their origins.
The clinical signs most frequently associated with enteric CCoVs are not easily differentiated from those associated with other enteric pathogens such as CPV2 or canine rotavirus and canine adenovirus. Consequently, CCoV diagnosis requires laboratory confirmation. The diagnostic techniques employed for the detection of CCoVs in fecal samples include electron microscopy (EM), virus isolation (VI) in cell cultures, and biomolecular analysis. EM examination of negatively stained fecal suspensions and immune electron microscopy are rapid procedures for detecting coronavirus and appear to be valuable diagnostic tools . However, coronavirus-like particles in intestinal contents often resemble coronaviruses , and EM examination required specialized laboratories and technicians. VI is the most commonly used technique for diagnosis of CCoVs infection , but is more complex, more time-consuming and less sensitive than other methods. CCoV type 2 grows on several cell lines of canine and feline origin, and the identification of an isolate requires neutralization of the cytopathic effects and/or immunofluorescence test with a reference serum or monoclonal antibodies. Failure to isolate CCoV type 1 in cell cultures  reduces the changes of identifying many forms of enteritis caused by this virus, so the frequency of CCoV type 1 disease is probably underestimated. Such difficulties and limitations prevent an authentic evaluation of the immunological characteristics of this new genotype and hinder the acquisition of key information on its pathogenetic role in dogs.
One of RNA’s most intriguing features is its ability to carry genetic information despite its labile nature. CoVs are unique among RNA viruses in many aspects of their biology. They are characterized by a extremely large genome, by a nested set of subgenomic mRNAs, by a discontinuous transcription mechanism, and by a high frequency of RNA recombination events because of the high error frequencies of RNA polymerase . Genetic recombination is an important mechanism for generating novel genomes that may have selective advantages over parental genomes. In the evolution of RNA viruses, RNA recombination is a widespread phenomenon that has shaped viruses by rearranging viral genomes or disseminating functional modules among different viruses . Although nonsegmented genomes of RNA viruses generally exhibit very low or undetectable recombination frequencies, the recombinations for the entire CoV genome have been calculated to be as high as 25% . The high frequency of RNA recombination in CoVs is probably the result of the unique mechanism of coronavirus synthesis, which involves discontinuous transcription and polymerase jumping. It is possible that the viral polymerase associated with the incomplete nascent RNAs, dissociates from its template at a random point, and switches to a homologous site on a different RNA template to complete RNA synthesis by a copy-choice mechanism . Depending on the precision of the repair mechanism, the repaired genome may be similar to the parental genome, or it may contain further mutations. This illustrates that sequence diversity in RNA sequences generated by genetic recombination can involve both gross changes and minor mutations. Genetic divergence within the alphacoronavirus is accounted for by linear evolution as well as by sudden dramatic shifts generated by RNA deletions or recombination .