Detection of caprine arthritis--encephalitis virus RNA in macrophages by in situ hybridization using fluorescein-labelled single-stranded RNA probes.
The use of in situ hybridization (ISH) for the detection of caprine arthritis-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 PCR products were cloned into the plasmid pAM-18, and labelled single-stranded RNA probes were synthesized by the use of RNA polymerase. The LTR probe was able to detect viral RNA in CAEV-infected, cultured caprine macrophages, while probes based on the genes for the matrix and transmembrane proteins failed to do so. A few macrophages were positive for 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.
Department of Pharmacology, Microbiology and Food Hygiene, Norwegian College of Veterinary Medicine, Oslo, Norway. anne.storset@veths.no
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Storset
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Teig
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Rimstad
D000818:Animals; D016088:Arthritis-Encephalitis Virus, Caprine / Q000302:isolation & purification* / Q000502:physiology; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004279:DNA, Viral / Q000096:biosynthesis; D005814:Genes, Viral; D006041:Goats; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008264:Macrophages / Q000821:virology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000302:isolation & purification / Q000502:physiology; D015347:RNA Probes; D012367:RNA, Viral / Q000032:analysis*; D012091:Repetitive Sequences, Nucleic Acid; D015678:Viral Structural Proteins / Q000235:genetics
D017931:DNA Primers; D004279:DNA, Viral; D015347:RNA Probes; D012367:RNA, Viral; D015678:Viral Structural Proteins