Use of Discriminatory Probes for Strain Typing of Formalin-Fixed, Rabies Virus-Infected Tissues by In Situ Hybridization
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 o...
Published in: | Journal of Clinical Microbiology |
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Main Authors: | , , |
Format: | Text |
Language: | English |
Published: |
American Society for Microbiology
2003
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Subjects: | |
Online Access: | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC193863 http://www.ncbi.nlm.nih.gov/pubmed/12958267 https://doi.org/10.1128/JCM.41.9.4343-4352.2003 |
Summary: | 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. |
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