Two isolates of canine parvovirus (CPV) were obtained from dogs affected with severe haemorrhagic diarrhoea. Type 2b antigenic specificity was predicted by both antigenic analysis with monoclonal antibodies and PCR characterization with type-specific primers. Nevertheless, sequence analysis of the capsid protein-encoding gene revealed two amino acid changes. One of the changes affected position 426 (Asp to Glu), in a major antigenic site of the viral capsid, determining the replacement of a residue unique to CPV type 2b. The failure of established typing methods to distinguish this antigenic variant was overcome by the development of an RFLP assay. During the early 1970s, a new infectious disease of pups, characterized by either gastroenteritis or myocarditis, was observed worldwide. A small, round, non-enveloped virus was observed by electron microscopy in stool specimens and in tissues of affected animals. Subsequently, a novel parvovirus was isolated both in canine and feline cell cultures (Kelly, 1978 ; Appel et al., 1979 ; Burtonboy et al., 1979 ; Johnson & Spradbrow, 1979). The virus was named canine parvovirus type 2 (CPV-2), to distinguish it from the previously described parvovirus canine minute virus (CMV or CPV-1), which is antigenically unrelated to CPV-2 (Carmichael & Binn, 1981 ; Carmichael et al., 1994). CPV possesses a single-stranded DNA genome of negative polarity, about 5200 nt in length. The CPV capsid is a 26 nm diameter icosahedron made up of a combination of two proteins, VP1 and VP2, formed by alternative splicing from the same RNA (Reed et al., 1988). The mutation rate of the CPV genome has not been determined ; however, since parvovirus DNA is replicated by host cell DNA polymerases (Cotmore &

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Canine coronavirus (CCoV) is usually responsible for mild, self-limiting infections restricted to the enteric tract. We report an outbreak of fatal disease in puppies caused by a pathogenic variant of CCoV that was isolated from organs with severe lesions.

The sequence of the S gene of a field canine coronavirus (CCoV), strain Elmo/02, revealed low nucleotide (61%) and amino acid (54%) identity to reference CCoV strains. The highest correlation (77% nt and 81.7% aa) was found with feline coronavirus type I. A PCR assay for the S gene of strain Elmo/02 detected analogous CCoVs of different geographic origin, all which exhibited at least 92-96% nucleotide identity to each other and to strain Elmo/02. The evident genetic divergence between the reference CCoV strains and the newly identified Elmo/02-like CCoVs strongly suggests that a novel genotype of CCoV is widespread in the dog population.

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of bovine coronavirus (BCoV) RNA in clinical samples is described. The assay is based on TaqMan technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the transmembrane-protein gene of BCoV. The BCoV real-time RT-PCR assay was able to detect the tested BCoV and BCoV-like viruses (canine respiratory coronavirus and bubaline coronavirus), whereas other common viral pathogens of cattle were not recognised by the established oligonucleotide set, thus showing that the test was specific for bovine-like CoVs. The detection limit of the assay was 20 BCoV RNA copies (1-log higher with respect to traditional gel-based RT-PCR) and the reproducibility was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. Two hundred and twenty clinical specimens (92 rectal, 82 nasal and 46 ocular swabs) were subjected to gel-based and real-time RT-PCR. By conventional amplification, 43 rectal, 54 nasal and 34 ocular samples tested positive, whereas the TaqMan assay was able to detect the BCoV nucleic acid in 49 rectal, 60 nasal and 37 ocular swabs. The rapidity and high throughput of the BCoV TaqMan assay makes this method a powerful tool for a sensitive and specific diagnosis of BCoV infection in cattle.