title: Use of discriminatory probes for strain typing of formalin-fixed, rabies virus-infected tissues by in situ hybridization.
abstract: An in situ hybridization (ISH) method has been developed to overcome difficulties encountered in the viral typing of formalin-fixed rabies virus-infected brain tissue. Rabies viruses representative of all strains normally encountered in diagnostic submissions throughout Canada, including 3 strains of terrestrial hosts (arctic fox, western skunk, mid-Atlantic raccoon), 10 strains circulating in several bat reservoirs (BBCAN1 to BBCAN7, LACAN, SHCAN, and MYCAN), and the Evelyn-Rokitniki-Abelseth (ERA) strain, used as an oral vaccine for fox rabies control in Ontario, were targeted. Partial phosphoprotein gene fragments generated from reverse transcription (RT)-PCR products of specimens of each viral type were molecularly cloned and used to produce negative-sense digoxigenin-labeled RNA transcripts. Conditions permitting the use of these transcripts as strain-specific probes were optimized by blotting analyses with RT-PCR amplicons generated with representative rabies viruses and by ISH applied to mouse brains inoculated with these strains. The successful application of this methodology to two rabies virus-positive specimens that were also identified by traditional methods and the retrospective typing of two archival rabies virus-positive equine specimens is described. This technique provides a typing regimen for rabies virus isolates submitted in a form that is normally recalcitrant to alternate typing strategies.
authors.affiliation: Rabies Centre of Expertise, Ottawa Laboratory-Fallowfield, Canadian Food Inspection Agency, Nepean, Ontario K2H 8P9, Canada. nadindaviss@inspection.gc.ca
authors.forename: Susan A
authors.initials: SA
authors.lastname: Nadin-Davis
authors.forename: Mary
authors.initials: M
authors.lastname: Sheen
authors.forename: Alexander I
authors.initials: AI
authors.lastname: Wandeler
mesh_terms: D000818:Animals; D014644:Genetic Variation; D017403:In Situ Hybridization / Q000379:methods*; D051379:Mice; D015347:RNA Probes / Q000235:genetics*; D011820:Rabies virus / Q000145:classification* / Q000235:genetics; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity; D016707:Tissue Fixation
chemical_list: D015347:RNA Probes