Annamaria Pratelli

Evidence for evolution of canine parvovirus type 2 in Italy.

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.

A Canine Parvovirus Mutant Is Spreading in Italy

ABSTRACT By antigenic and genetic characterization of canine parvovirus type 2 (CPV-2) strains collected in 2001 and 2002 in Italy, it was possible to observe the spread of viruses with an unusual mutation, Glu-426, affecting a major antigenic epitope of CPV-2. Out of 67 strains analyzed, 49 (73.13%) were characterized as CPV-2a, 6 (8.95%) were characterized as CPV-2b, and 12 (17.91%) were characterized as the Glu-426 mutant.

Molecular Characterization of the VP4, VP6, VP7 and NSP4 genes of lapine rotaviruses identified in Italy: emergence of a novel VP4 genotype

The genes encoding the glycoprotein VP7, the VP8* trypsin-cleavage product of the protein VP4, a fragment of the protein VP6 associated with subgroup (SG) specificity, and the enterotoxin NSP4 of rotavirus strains identified in diarrheic fecal samples of rabbits in Italy were sequenced. The Italian lapine rotavirus (LRV) strains possessed a G3 VP7, SG I VP6, and KUN-like NSP4, a gene constellation typical of LRVs. One LRV strain (30/96), isolated in 1996, shared the closest amino acid (aa) identity (87-96%) with the P[14] genotype, composed of human and LRV strains. Conversely, three LRV strains (160/01, 229/01, and 308/01), identified in 2001, were highly identical (90-95%) among each other, but showed low aa identity (34-77%) to the VP8* genotype-specific sequences of representative rotavirus strains of all remaining P genotypes. This report confirms the worldwide genetic constellations of LRVs and identifies a novel VP4 genotype in rabbits, tentatively proposed as genotype P[22].

Genetic diversity of a canine coronavirus detected in pups with diarrhoea in Italy

Abstract 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.

Development of a nested PCR assay for the detection of canine coronavirus

Abstract A diagnostic test for canine coronavirus (CCV) infection based on a nested polymerase chain reaction (n-PCR) assay was developed and tested using the following coronavirus strains: CCV (USDA strain), CCV (45/93, field strain), feline infectious peritonitis virus (FIPV, field strain), trasmissible gastroenteritis virus (TGEV, Purdue strain), bovine coronavirus (BCV, 9WBL-77 strain), infectious bronchitis virus (IBV, M-41 strain) and fecal samples of dogs with CCV enteritis. A 230-bp segment of the gene encoding for transmembrane protein M of CCV is the target sequence of the primer. The test described in the present study was able to amplify both CCV and TGEV strains and also gave positive results on fecal samples from CCV infected dogs. n-PCR has a sensitivity as high as isolation on cell cultures, and can therefore be used for the diagnosis of CCV infection in dogs.

Quantitation of canine coronavirus RNA in the faeces of dogs by TaqMan RT-PCR.

Abstract A TaqMan® fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies of CCoV standard RNA, and was linear from 10 to 108 copies, allowing quantitation of samples with a wide range of CCoV RNA loads. A total of 78 faecal samples of diarrhoeic dogs were tested simultaneously by conventional and fluorogenic RT-PCR: 29 were negative by both techniques, whereas 27 tested positive by conventional RT-PCR and 48 by the established CCoV fluorogenic assay. One sample, which was positive by conventional RT-PCR, gave no signal in the fluorogenic assay. In addition, by the fluorogenic assay CCoV shedding in the faecal samples of an experimentally infected dog was monitored for 28 days. The high sensitivity, simplicity and reproducibility of the CCoV fluorogenic RT-PCR assay, combined with its wide dynamic range and high throughput, make this method especially suitable for efficacy trials on CCoV vaccines.

Severe enteric disease in an animal shelter associated with dual infections by canine adenovirus type 1 and canine coronavirus.

An outbreak of dual infection in dogs with canine adenovirus type 1 (CAV‐1) and canine coronavirus (CCV) infection is reported in an animal shelter that comprised approximately 200 adults stray dogs and 30 puppies. Twenty puppies died 7–8 days after the onset of the clinical signs (severe enteritis, leucopoenia, respiratory distress and dehydration). Both CAV‐1 and CCV were isolated from tissue or swab samples. Antibodies to CCV and, at high levels, to CAV‐1 also were detected in several puppies. The principal histological findings were atrophy of small intestinal villi, lymphoid depletion, hepatitis and bronchopneumonia. The persistence of CCV in the faeces, observed by the polymerase chain reaction assay, was longer than previously reported. Results demonstrated the serious consequences which may occur with dual infections by CAV‐1 and CCV in assembled groups of dogs that are housed in poorly managed kennels with inadequate vaccination programmes.

Canine Parvovirus (CPV) Vaccination: Comparison of Neutralizing Antibody Responses in Pups after Inoculation with CPV2 or CPV2b Modified Live Virus Vaccine

ABSTRACT Canine parvovirus type 2 (CPV2) emerged in 1978 as causative agent of a new disease of dogs. New antigenic variants (biotypes), designated CPV2a and CPV2b, became widespread during 1979 to 1980 and 1984, respectively. At the present time the original CPV2 has disappeared in the dog population and has been replaced by the two new viruses. In the present study the comparison of neutralizing antibody titers in two groups of pups (18 pups in each group) inoculated with CPV2 and CPV2b modified live virus vaccines is reported. Using the hemagglutination inhibition (HI) test, relevant differences between antibody titers, against either the homologous or the heterologous virus, were not constantly observed. Using the neutralization (Nt) test, however, the pups inoculated with CPV2 had antibody titers which were approximately 30 times higher to the homologous virus (mean, 4,732) than to the heterologous virus (CPV2b) (mean, 162). The results of these experiments support two conclusions: (i) the HI test may not always accurately evaluate the true immune status of dogs with respect to CPV, and (ii) dogs inoculated with CPV2 vaccine develop relatively low Nt antibody titers against the heterologous virus (CPV2b). These data may suggest an advantage for new vaccines, considering that most presently licensed vaccines are produced with CPV2, which no longer exists in the dog population.

Fatal Coronavirus Infection in Puppies following Canine Parvovirus 2b Infection

Canine coronavirus (CCV) belongs to the Coronaviridae family, which includes enveloped and pleomorphic viruses 60–220 nm in diameter, helical nucleocapsids, and petal (club-shaped) peplomeres, which are widely spaced on the envelope, resembling a solar corona.1,12 CCV was first isolated in 1971 from military dogs in Germany suffering from gastroenteritis.3 Subsequently, additional cases have been reported that were usually mild and selflimiting, unless complicated by canine parvovirus (CPV) infection.2,7,8,10,14 Coronaviral disease in dogs is extremely variable. Although adult dogs generally have no or mild clinical signs and usually recover after a brief period of illness, pups with secondary bacterial infections, parasites, or other viruses may suffer severe, even fatal, disease.1,8,10 Infected dogs shed CCV in the feces for 6–9 days, but shedding can be prolonged in some animals.1,9 The most likely mode of transmission is by the fecal–oral route. The main target of CCV is the small intestinal epithelium, where a lytic infection results in desquamation and shortening of duodenal and jejunal villi. Pathologic changes have been observed mainly in experimentally infected dogs and are characterized by dilated intestinal loops filled with watery ingesta and feces, congested or edematous mucosa, and edematous mesenteric lymph nodes.1,8,9,13,14 Microscopic changes are characterized by atrophy and fusion of intestinal villi, a deepening of crypts, and increased cellularity of the lamina propria.8,9 In this report, we describe the clinical, virologic, and histopathologic findings observed in littermate pups that died with signs of severe hemorrhagic enteritis 15 days following recovery from a CPV-2b infection. The presence of parvovirus-like intranuclear inclusions in intestinal epithelial cells .2 weeks following recovery from CPV-2b infection and the presence of pulmonary lesions were unexpected, and the CCV infection may have been complicated by infection with another agent such as the minute virus of canines (MVC, canine parvovirus 1). In the initial disease episode, 4/4 Corso (Italian breed) pups, 45 days old, developed hemorrhagic gastroenteritis. CPV. 2b was the only virus detected by laboratory examination, which included hemagglutination (HA) tests on fecal suspensions, viral isolation attempts in A-72 cell cultures,

Two Genotypes of Canine Coronavirus Simultaneously Detected in the Fecal Samples of Dogs with Diarrhea

ABSTRACT Sixty-nine fecal samples from diarrheic puppies were examined by reverse transcription-PCR assays for the M and the S genes of canine coronaviruses (CCoVs). The isolates in 10 samples were recognized as CCoV type I, and the isolates in 6 samples were recognized as CCoV type II, while isolates of both genotypes were simultaneously detected in 53 samples.