Characterization of pantropic canine coronavirus from Brazil.

Characterization of canine coronavirus (CCoV) strains currently in circulation is essential for understanding viral evolution. The aim of this study was to determine the presence of pantropic CCoV type IIa in tissue samples from five puppies that died in Southern Brazil as a result of severe gastroenteritis. Reverse-transcriptase PCR was used to generate amplicons for sequence analysis. Phylogenetic analysis of the CCoV-IIa strains indicated that they were similar to those found in other countries, suggesting a common ancestor of these Brazilian isolates. This is the first report of pantropic CCoV-II in puppies from Latin America and our findings highlight that CCoV should be included as a differential diagnosis when dogs present with clinical signs and lesions typically seen with canine parvovirus infection.

Analysis of putative recombination hot sites in the S gene of canine coronaviruses.

The S gene sequence of Canine coronavirus strain 1-71 (CCoV 1-71) was cloned, sequenced, and compared to those of other CCoVs, Transmissible gastroenteritis virus (TGEV), and Feline coronavirus (FCoV). The sequence analysis showed that CCoV 1-71 displayed a 98.8-99.8% identity with CCoVs strains V1, K378, and GP. Four putative recombination sites were found at the 5'-end of the S gene, namely at nt 53, 75, 425, 991. Both sequences flanking each site were significantly different. Three recombination hot regions were found on the S gene, namely at nt 337-437, 1545-3405, and 4203-4356, which shared a common recombination signal with Group 2 coronaviruses. The G/CTAAAAA/GT sequence downstream of the recombination site may represent a specific recombination signal in CCoVs. The CCoV 1-71 S protein sequence was found to be similar to those of other CCoVs except for several N-glycosylation sites at the N-terminus of the S protein, which could be related to the differences in virulence and cell tropism in individual CCoVs. This study indicated that the similarity of CCoVs in virulence and tropism was mostly acquired by the homologous RNA recombination and not only by simple mutation and selection.

Gain, preservation, and loss of a group 1a coronavirus accessory glycoprotein.

Coronaviruses are positive-strand RNA viruses of extraordinary genetic complexity and diversity. In addition to a common set of genes for replicase and structural proteins, each coronavirus may carry multiple group-specific genes apparently acquired through relatively recent heterologous recombination events. Here we describe an accessory gene, ORF3, unique to canine coronavirus type I (CCoV-I) and characterize its product, glycoprotein gp3. Whereas ORF3 is conserved in CCoV-I, only remnants remain in CCoV-II and CCoV-II-derived porcine and feline coronaviruses. Our findings provide insight into the evolutionary history of coronavirus group 1a and into the dynamics of gain and loss of accessory genes.

Molecular characterisation of the virulent canine coronavirus CB/05 strain.

This paper characterises a virulent strain (CB/05) of canine coronavirus (CCoV) isolated from the internal organs of pups that had died of a systemic disease without evidence of other common canine pathogens. High viral RNA titres were detected in the internal organs by a real-time RT-PCR assay specific for CCoV type II. Sequence analysis of the 3' end (8.7kb) of the genomic RNA of strain CB/05 revealed conserved structural as well as non-structural proteins, with the exception of a truncated form of non-structural protein 3b. The exceptional form was due to a 38-nucleotide deletion and a frame shift in ORF3b that introduced an early stop codon. By phylogenetic analysis of the structural proteins, the spike (S) protein was found to cluster with feline coronavirus type II strain 79-1683, whereas, the envelope (E), membrane (M) and nucleocapsid (N) proteins segregated together with the reference strain Purdue of transmissible gastroenteritis virus of swine.

Evaluation of antibody response to canine coronavirus infection in dogs by Western Blotting analysis.

We investigated by Western Blotting the antibody responses against the three major structural proteins of Canine coronavirus (CCoV) in dogs naturally infected. A pool of Elisa positive sera were also tested to clearly identify the binding profiles of CCoV proteins. The immune response to S protein was barely detectable in naturally infected dogs, whereas anti-M and anti-N antibodies were detected with a very strong reaction and for a long time post infection. The limited response to S protein may explain the poor protection of dogs and the possibility of persisting infection.

Feline and canine coronaviruses are released from the basolateral side of polarized epithelial LLC-PK1 cells expressing the recombinant feline aminopeptidase-N cDNA.

In this study feline (FECV and FIPV) and canine (CCoV) coronavirus entry into and release from polarized porcine epithelial LLC-PK1 cells, stably expressing the recombinant feline aminopeptidase-N cDNA, were investigated. Virus entry appeared to occur preferentially through the apical membrane, similar to the entry of the related porcine coronavirus transmissible gastroenteritis virus (TGEV) into these cells. However, whereas TGEV is released apically, feline and canine coronaviruses were found to be released from the basolateral side of the epithelial cells. These observations indicate that local infections as caused by TGEV, FECV and CCoV do not strictly correlate with apical release, as suggested by earlier work.

Feline aminopeptidase N is a receptor for all group I coronaviruses.

Human coronavirus HCV-229E and porcine transmissible gastroenteritis virus (TGEV), both members of coronavirus group I, use aminopeptidase N (APN) as their cellular receptors. These viruses show marked species specificity in receptor utilization as they can only use APN of their respective species to initiate virus infection. Feline and canine coronaviruses are also group I coronaviruses. To determine whether feline APN could serve as a receptor for feline coronaviruses (FCoVs), we cloned the cDNA encoding feline APN (fAPN) by PCR from feline cells and stably expressed it in FCoV-resistant mouse or hamster cells. These became susceptible to infection with either of several biotypes of FCoVs. The expression of recombinant fAPN also made hamster and mouse cells susceptible to infection with other group I coronaviruses, including several canine coronavirus strains, transmissible gastroenteritis virus (TGEV), and human coronavirus HCV-229E. Thus, fAPN served as a functional receptor for each of these coronaviruses in group I. As expected, fAPN could not serve as a receptor for mouse hepatitis virus (MHV), a group II coronavirus which uses murine biliary glycoproteins as receptors. Thus, fAPN acts as a common receptor for coronaviruses in group I, in marked contrast to human and porcine APN glycoproteins which serve as receptors only for human and porcine coronaviruses, respectively. These observations suggest that cats could serve as a "mixing vessel" in which simultaneous infection with several group I coronaviruses could lead to recombination of viral genomes.

Interspecies aminopeptidase-N chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus.

We report that cells refractory to canine coronavirus (CCV) and feline infectious peritonitis virus (FIPV) became susceptible when transfected with a chimeric aminopeptidase-N (APN) cDNA containing a canine domain between residues 643 and 841. This finding shows that APN recognition by these viruses is species related and associated with this C-terminal domain. The human/canine APN chimera was also able to confer susceptibility to the porcine transmissible gastroenteritis virus (TGEV), whereas its human/porcine homolog failed to confer susceptibility to CCV and FIPV. A good correlation was observed between the capacity of CCV, FIPV, and TGEV to recognize the different interspecies APN chimeras and their ability to infect cells derived from the relevant species. As an exception, TGEV was found to use a human/bovine APN chimera as a receptor although itself unable to replicate in bovine cells.

Comparison of the amino acid sequence and phylogenetic analysis of the peplomer, integral membrane and nucleocapsid proteins of feline, canine and porcine coronaviruses.

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU-2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79-1683. Only M and N genes were analyzed in strain KU-2 and strain 79-1683 which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79-1146, canine coronavirus (CCV) strain Insavc-1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein-deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein-deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein-deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.

Further characterization of aminopeptidase-N as a receptor for coronaviruses.

We recently reported that porcine aminopeptidase-N (pAPN) acts as a receptor for transmissible gastroenteritis virus (TGEV). In the present work, we addressed the question of whether TGEV tropism is determined only by the virus-receptor interaction. To this end, different non-permissive cell lines were transfected with the porcine APN cDNA and tested for their susceptibility to TGEV infection. The four transfected cell lines shown to express pAPN at their membrane became sensitive to infection. Two of these cell lines were found to be defective for the production of viral particles. This suggests that other factor(s) than pAPN expression may be involved in the production of infectious virions. The pAPN-transfected cells were also tested for their susceptibility to several viruses which have a close antigenic relationship to TGEV. So far, we failed to evidence permissivity to the feline infectious peritonitis coronavirus FIPV and canine coronavirus CCV. In contrast, we found clear evidence that porcine respiratory coronavirus PRCV, a variant of TGEV which replicates efficiently in the respiratory tract but to a very low extent in the gut, may also utilise APN to gain entry into the host cells. This suggests that the switch between TGEV and PRCV tropisms in vivo may involve other determinant(s) than receptor recognition.