Virological and molecular characterization of a mammalian orthoreovirus type 3 strain isolated from a dog in Italy

Abstract

A mammalian orthoreovirus (MRV) strain was isolated from a pup with fatal diarrhea, which had a concurrent infection by canine parvovirus type 2. The reovirus isolate showed an atypical hemagglutination pattern and a retarded electrophoretic mobility of the S1 segment, which is characteristic of MRV type 3 (MRV-3). Assignment of the isolated virus to MRV-3 was confirmed by type-specific RT-PCR assays, targeting the S1 gene, and by subsequent sequence analysis of the PCR product. By phylogeny based on the S1 gene of several MRVs, the isolate fell into lineage E, along with the murine strain T3C9/61 and the bovine strains T3C18/61 and T3C31/59. Conversely, L1 sequences were found to segregate regardless of the viral type. A total of 110 fecal samples, 56 nasal and 31 ocular swabs from dogs with diarrhea or nasal/ocular discharge were tested by a nested-PCR assay specific for reoviruses, and no sample was found to contain MRV RNA, a finding that is apparently in contrast with the seroprevalence (25.77%) observed in dogs.

Fig. 1

Reovirus-like particles from CrFK cells infected with strain T3D/04. The viral preparation was negative-stained with sodium phosphotungstate and observed by EM (25,000×).

Fig. 2

Perinuclear inclusion bodies (arrows) in the cytoplasm of CrFK cells infected with strain T3D/04. HE staining 400×.

Fig. 3

Electrophoretic analysis of T3D/04 dsRNA in a silver-stained polyacrylamide gel. The arrow indicates the position of the serotype-determining S1 segment.

Fig. 4

PCR assays carried out on fecal samples of pups 214/04-A and 214/04-B and inoculated cells. Amplification of the L1 fragment by RT-PCR (416 bp) and nested-PCR (344 bp). T1L (strain T1L/53); Af (sample 214/04-A, feces); Ac (sample 214/04-A, inoculated cells); Bf (sample 214/04-B, feces); Bc (sample 214/04-B, inoculated cells); N (negative fecal sample); M (marker GeneRuler 50 bp DNA Ladder, MBI Fermentas GmbH, Germany).

Fig. 5

Strain characterization by the type 3-specific RT-PCR assay targeting the S1 segment (primer pair S1-R3F/S1-R3R, PCR product of 326 bp). M (marker GeneRuler 100 bp DNA Ladder, MBI Fermentas GmbH, Germany); T3D (strain T3D/55); T3A (strain T3A/57); Bf (sample 214/04-B, feces); T1L (strain T1L/53); T2J (strain T2J/55); N (negative fecal sample).

Fig. 6

Maximum parsimony trees based on partial L1 (a) and S1 (b) nucleotide sequences of MRV strains. Accession numbers of the strains used for phylogeny are reported in Table 2. Trees are unrooted and drawn to scale. Bootstrap values were calculated and are indicated at each node.