DETECTION OF BLUETONGUE VIRUS-RNA IN CELL-CULTURES AND IN THE CENTRAL-NERVOUS-SYSTEM OF EXPERIMENTALLY INFECTED MICE USING IN-SITU HYBRIDIZATION

Two radiolabelled complementary DNA (cDNA) probes (1663 bp and 200 bp respectively) were prepared from the genome segment that encodes the n on-structural protein 1 (NS1) of bluetongue virus serotype 4 (BTV4). T he probes were used to optimize the in situ hybridization (ISH) method on baby hamster kidney-21 (BHK-21) cells and to investigate the use o f the technique as a diagnostic procedure. Cells were infected with BT V4 at a multiplicity of infection of 0,5 PFU/cell. An intense cytoplas mic hybridization signal could be detected from 3 hours post-infection onwards, reaching a peak at 17 hours. The ISH procedure has potential use as a diagnostic technique, but will probably find a wider applica tion in pathogenesis studies. An in situ hybridization method was also developed for the detection of BTV RNA in the central nervous system of newborn mice after intracranial inoculation with BTV10. Viral RNA-p ositive cells were detected from Day 3 onwards, predominantly in areas where the virus caused necrotic encephalitis.