DETECTION OF FELINE IMMUNODEFICIENCY PROVIRAL SEQUENCES IN LYMPHOID-TISSUES AND THE CENTRAL-NERVOUS-SYSTEM BY IN-SITU GENE AMPLIFICATION

The availability of sensitive methods for detecting and localising the feline immunodeficiency virus (FIV) may help shed light on its role i n generating tissue damage observed during infection. As immunohistoch emical and in situ hybridisation techniques might not be sufficiently sensitive for this type of study, we adapted to FIV PCR-in situ hybrid isation (PCR-ISH) that combine the extreme sensitivity of PCR with the precise localisation provided by ISH. The steps important for the suc cess of PCR-ISH, such as sample preparation, permeabilisation, amplifi cation profile, type of labels, and hybridisation conditions were opti mised using paraformaldehyde-fixed and formalin-fixed paraffin-embedde d sections of cells infected in vitro with FIV. As controls for amplif ication, the feline tumor necrosis factor-alpha gene (TNF-alpha) and t he non-related EBNA-1 gene of the human Epstein-Barr virus were used. Once the method proved sufficiently sensitive and specific with these cells, the PCR-ISH assay was applied to paraffin sections of the lymph nodes, spleen and central nervous system of a 2-year FIV infected cat that, at the time of challenge, harboured low copy numbers of provira l genomes. Comparison of the results of PCR-ISH, competitive PCR and i mmunohistochemical analysis are described. (C) 1998 Elsevier Science B .V. All rights reserved.