A total of 105 fecal samples from dogs in municipal dog shelters, veterinary clinics, and pet shops from 3 districts (Porto, Viseu, and Guarda) in Portugal were collected during December 2007–November 2008. Veterinarians evaluated the dogs for diarrhea at the time of the visit. The fecal panel consisted of 63 samples from dogs with diarrhea and 42 samples from dogs with formed, normal brown feces (i.e., controls). All samples were kept at –20°C until processed. Fecal suspensions (10%) were made in phosphate-buffered saline pH 7.2, and solids were removed by centrifugation at 8,000 × g
for 5 min. Nucleic acid was extracted by using the QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions and tested for the presence of NoV RNA by 2 broadly reactive NoV conventional reverse transcription–PCR (RT-PCR) assays (8
), selective for a partial RdRp region of the genome, by using One-Step RT-PCR kit (QIAGEN) and 37°C as annealing temperature.
Two (2%) of the 105 fecal samples tested positive for NoV with primer pair JV12y/13i, whereas no RT-PCR products were found using the p289-p290 primer pair. The 2 JV12y/13i sequences were identical and contained the GLPSG amino acid motif characteristic of viral RNA polymerases.
We designed specific canine norovirus oligonucleotide primers JV102 (5′-TGG GAT TCA ACA CAG CAG AG-3′) and JV103 (5′-TGC GCA ATA GAG TTG ACC TG-3′) and retested all 105 samples. Fecal samples from 25 (40%) of the 63 dogs with diarrhea and 4 (9%) of the 42 controls tested positive for a new canine NoV (Viseu strain) (). An ≈3.3-kb fragment from the RdRp region in ORF1 to the poly-A tail, including the complete ORF2 and ORF3 genes, was generated by long-template RT-PCR by using previously described methods (10
). Gel-purified PCR products were cloned and sequenced by primer walking, and a 3,357-nt sequence including partial ORF1, full-length ORF2 and ORF3, and the 3′ end noncoding region from the Viseu strain was obtained. The consensus sequence from 5 different clones was submitted to GenBank and assigned accession no. GQ443611. All 105 nucleic acid extracts also were tested by RT-PCR for the recently reported canine NoV strain (5
), canine coronavirus (CCV) (11
), and by PCR for canine parvovirus (CPV-2) combining oligonucleotide primers for the detection of types 2a and 2b (12
). None of the samples tested positive for the reported GIV.2 canine NoV strain (GIV.2/170/07/ITA). A total of 32 (51%) samples from dogs with diarrhea tested positive for CCV, whereas 2 (5%) of the control samples were positive. In addition, 58 (55%) samples tested positive for CPV-2, of which 18 also were positive for the novel canine NoV (). Univariate analysis (SPSS version 17.0, SPSS Inc., Chicago, IL, USA) demonstrated that the novel canine NoV and CCV were significantly associated with diarrhea ().
Detection of canine norovirus, parvovirus, and coronavirus in 105 fecal samples from dogs with and without diarrhea, Porto, Viseu, and Guarda, Portugal, December 2007–November 2008*