Detection of caprine arthritis--encephalitis virus RNA in macrophages by in situ hybridization using fluorescein-labelled single-stranded RNA probes

Vet Microbiol. 1996 Sep;52(1-2):25-35. doi: 10.1016/0378-1135(96)00063-6.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arthritis-Encephalitis Virus, Caprine / isolation & purification*
  • Arthritis-Encephalitis Virus, Caprine / physiology
  • Base Sequence
  • Cells, Cultured
  • DNA Primers
  • DNA, Viral / biosynthesis
  • Genes, Viral
  • Goats
  • In Situ Hybridization, Fluorescence / methods
  • Macrophages / virology*
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Proviruses / isolation & purification
  • Proviruses / physiology
  • RNA Probes
  • RNA, Viral / analysis*
  • Repetitive Sequences, Nucleic Acid
  • Viral Structural Proteins / genetics

Substances

  • DNA Primers
  • DNA, Viral
  • RNA Probes
  • RNA, Viral
  • Viral Structural Proteins