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Journal of Clinical Microbiology, September 2003, p. 4343-4352, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4343-4352.2003

Use of Discriminatory Probes for Strain Typing of Formalin-Fixed, Rabies Virus-Infected Tissues by In Situ Hybridization

Susan A. Nadin-Davis,* Mary Sheen, and Alexander I. Wandeler

Rabies Centre of Expertise, Ottawa Laboratory—Fallowfield, Canadian Food Inspection Agency, Nepean, Ontario K2H 8P9, Canada

Received 9 December 2002/ Returned for modification 20 February 2003/ Accepted 18 June 2003

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.

* Corresponding author. Mailing address: Rabies Centre of Expertise, Ottawa Laboratory—Fallowfield, Canadian Food Inspection Agency, 3851 Fallowfield Rd., Nepean, Ontario K2H 8P9, Canada. Phone: (613) 228-6698. Fax: (613) 228-6669. E-mail: nadindaviss{at}inspection.gc.ca.

Journal of Clinical Microbiology, September 2003, p. 4343-4352, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4343-4352.2003






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