A nested PCR approach for unambiguous typing of pestiviruses infecting cattle.

An atypical pestivirus ('Hobi'-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that 'Hobi'-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5' untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay (n=2) or reacted as 'Hobi'-like pestivirus BVDV-3 (n=3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true 'Hobi'-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of 'Hobi'-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle.

Genetic heterogeneity and recombination in canine noroviruses.

Alphatronlike (genogroup IV [GIV]) noroviruses (NoVs) have been recently identified in carnivores. By screening a collection of 183 fecal samples collected during 2007 from dogs with enteric signs, the overall NoV prevalence was found to be 2.2% (4/183). A unique strain, Bari/91/07/ITA, resembled GIV.2 NoVs in its ORF1 (polymerase complex), while it was genetically unrelated in its full-length ORF2 (capsid gene) to GIV animal and human NoVs (54.0 to 54.4% amino acid identity) and to any other NoV genogroup (<54.7% amino acid identity). It displayed the highest identity (58.1% amino acid identity) to unclassified human strain Chiba/040502/04/Jp. Interestingly, the very 5' end of ORF2 of the canine virus matched short noroviral sequences (88.9% nucleotide identity and 98.9% amino acid identity) identified from oysters in Japan, indicating that similar viruses may be common environmental contaminants.

Specific identification of feline panleukopenia virus and its rapid differentiation from canine parvoviruses using minor groove binder probes.

Taking into account reports of the isolation of canine parvoviruses (CPVs) from faecal samples of cats, we developed a real-time PCR assay, based on minor groove binder (MGB) probe technology, for rapid discrimination between true feline panleukopenia viruses (FPLVs) from CPVs. The assay takes advantage of a single nucleotide polymorphism at position 3753 of the viral genome (corresponding to residue 323 of the capsid VP2 protein) and of the ability of MGB probes to bind specifically only to perfectly complementary sequences. The FPV/CPV assay was proven to be highly specific, sensitive and reproducible and correlated well with a TaqMan assay able to recognise canine as well as feline parvoviruses. Using this assay for extensive molecular surveys will provide precise information on the real circulation of the CPV antigenic variants, including the new variant 2c, in cat population worldwide.

Detection of a canine parvovirus type 2c with a non-coding mutation and its implications for molecular characterisation.

An epidemiological survey for canine parvovirus (CPV) was conducted by collecting 615 faecal samples from dogs with diarrhoea in different European countries. Molecular methods showed that CPV-2a was predominant in most countries, followed by CPV-2c and CPV-2b, whereas 30 strains were not characterised. By sequence analysis of the full-length VP2 gene, 20 of these viruses were characterised as CPV-2c mutants having the synonymous mutation A4061G in the probe-binding region that prevented correct strain characterisation. A real-time polymerase chain reaction (PCR) assay using a minor groove binder probe was able to recognise both mutant and classical CPV-2c strains. These results indicate that the emergence of CPVs with mutations affecting the oligonucleotide-binding region needs a continuous update of molecular diagnostic tools in order to detect efficiently those emerging strains.

Molecular characterization of Canine minute virus associated with neonatal mortality in a litter of Jack Russell terrier dogs.

The molecular characterization of a strain of Canine minute virus (CnMV) associated with neonatal death is reported. Three newborn puppies of a litter of Jack Russell terrier dogs died after displaying systemic disease, whereas 2 surviving puppies showed no clinical signs with the exception of transient cardiac abnormalities that were evident by electrocardiography. Necropsy of 1 dead puppy revealed severe lesions in the internal organs. A strain of Canine minute virus was detected in tissue samples collected from the puppy, and virus circulation was demonstrated by molecular or serological testing in the dam, puppies of the same litter, and other puppies in the same kennel. By sequence and phylogenetic analysis of the gene encoding for the VP2 capsid protein, the strain circulating in the kennel was found to be related to recent Asian CnMV isolates. Continuous molecular surveillance for CnMV in kennels, shelters, and rescue centers would expand the knowledge base on the epidemiological and pathogenetic features of CnMV, which has been known for several decades but still poorly understood.

Canine parvovirus epidemiology in Bulgaria.

Canine parvovirus 2 (CPV-2) emerged in 1978 as one of the most pathogenic etiologic agents in dogs. Under the influence of evolution, the original CPV-2 was replaced, a few years later, by 2 variants, CPV-2a and CPV-2b. In 2000, a new variant, CPV-2c, was detected first in Italy and later in other countries. The current study was conducted to provide data about the CPV types circulating in Bulgaria. Forty-two fecal samples from dogs with clinical signs of parvovirosis, collected between June 2009 and February 2010, were tested for CPV using a rapid test based on detection of CPV antigens and a real-time polymerase chain reaction (PCR) for detection of viral DNA. Positive samples were characterized by means of minor groove binder probe PCR assays. Forty samples were positive, of which 30 were identified as CPV-2a, 9 as CPV-2b, and 1 as CPV-2c. The results from this molecular investigation of CPV show the prevalence of type 2a and occurrence of type 2c for the first time in Bulgaria.

Canine parvovirus type 2c infection in a kitten associated with intracranial abscess and convulsions.

A case of canine parvovirus type 2c (CPV-2c) infection in a 3-month-old feral kitten with a cerebral abscess and neurological disease is reported. The cat displayed ataxia and convulsions together with signs of gastroenteritis and profound alteration of the total and differential white blood cell counts. A parvovirus strain was detected by a TaqMan assay in the blood and faeces of the affected kitten, which was characterised as CPV by means of molecular assays but did not react with any of the CPV type-specific probes. By sequence and phylogenetic analyses of the VP2-protein gene, the CPV-2c strain displayed a non-coding mutation in the probe-binding region. Although the role of CPV-2c in this particular case is unclear, it is possible that it predisposed the kitten to the clinical signs seen. Continuous surveillance is needed to monitor future spreading of this CPV-2c mutant, and any associated clinical signs, in the dog and cat population.

Characterisation of canine parvovirus strains isolated from cats with feline panleukopenia.

Unlike the original canine parvovirus type 2 (CPV-2), CPV-2 variants have gained the ability to replicate in vivo in cats but there is limited information on the disease patterns induced by these variants in the feline host. During 2008, two distinct cases of parvoviral infection were diagnosed in our laboratories. A CPV-2a variant was identified in a 3-month-old Persian kitten displaying clinical sign of feline panleukopenia (FPL) (acute gastroenteritis and marked leukopenia) and oral ulcerations, that died eight days after the onset of the disease. Two pups living in the same pet shop as the cat were found to shed a CPV-2a strain genetically identical to the feline virus and were likely the source of infection. Also, non-fatal infection by a CPV-2c strain occurred in a 2.5-month-old European shorthair kitten displaying non-haemorrhagic diarrhoea and normal white blood cell counts. By sequence analysis of the major capsid protein (VP2) gene, the feline CPV-2c strain showed 100% identity to a recent canine type-2c isolate. Both kittens had been administered multivalent vaccines against common feline pathogens including FPL virus. Whether and to which extent the FPL vaccines can protect cats adequately from the antigenic variants of CPV-2 should be assessed.

Detection of canine parvovirus type 2c by a commercially available in-house rapid test.

Diagnosis of canine parvovirus (CPV) infection is usually carried out by means of rapid immunochromatographic assays, but the ability of these tests to detect all CPV variants, including the recently identified CPV-2c, is still debated. To determine if the assays detect the different CPV variants, 201 CPV PCR-positive faecal samples or rectal swabs were tested using a commercially available in-house test. Specimens (CPV-2a, n=51; CPV-2b, n=50; CPV-2c, n=100), containing CPV DNA loads >10(5) DNA copies/mg faeces, as determined by real-time PCR, were selected from previous studies. The percentage of positive in-house tests was 80.4%, 78.0% and 77.0% for CPV types 2a, 2b and 2c, respectively, confirming the ability of the test to detect the new variant CPV-2c. However, considering the sensitivity limits of the in-house tests that have been observed previously, negative results from the in-house test kit should be confirmed by PCR-based methods.

Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1.

A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 10(1) and 1.20 x 10(1) DNA copies per 10 microl(-1) of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs.