DETECTION OF CAPRINE ARTHRITIS-ENCEPHALITIS VIRUS-RNA IN MACROPHAGES BY IN-SITU HYBRIDIZATION USING FLUORESCEIN-LABELED SINGLE-STRANDED RNAPROBES

The use of in situ hybridization (ISH) for the detection of caprine ar thritis-encephalitis virus (CAEV) RNA with fluorescein-11-UTP-labelled single-stranded RNA probes is described. Three different probes were made by PCR amplification of proviral CAEV DNA (strain 75-G63). The PC R products were cloned into the plasmid pAM-18, and labelled single-st randed RNA probes were synthesized by the use of RNA polymerase. The L TR probe was able to detect viral RNA in CAEV-infected, cultured capri ne macrophages, while probes based on the genes for the matrix and tra nsmembrane proteins failed to do so. A few macrophages were positive f or CAEV RNA 24 h post infection (p.i.) while most cells were positive 96 h p.i. The use of fluorescein-labelled RNA probes made this method feasible for kinetic in vitro studies of CAEV.