Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes.
Conditions suitable for immunogold detection of digoxigenin-labelled DNA probes hybridized to parvovirus B19-infected erythroid cells embedded in Lowicryl K4M and LR White acrylic resins were established at the electron microscope level. The protocol was initially optimized using a positive control probe for whole human DNA which produced signal over the heterochromatin of all nucleated cells. In cultures harvested 2 days postinfection, B19 nucleic acid was detected mainly within the centrinuclear region of erythroid cells exhibiting characteristic margination of the chromatin. The B19 hybridization signal was largely unaffected by denaturation and was resistant to RNase digestion but sensitive to DNase digestion, indicating that it was mainly single-stranded B19 DNA. Relatively few gold particles were found over crystalline arrays of viral capsids, consistent with the observation that they are composed of mainly 'empty' capsids. B19 nucleic acid was detected in apparent transit from nucleus to cytoplasm through pores in the nuclear membrane. While the sensitivity of this system is limited by the fact that hybridization occurs only at the surface of the section, it is a rapid and specific means of localizing viral nucleic acids with a high degree of resolution.
Morey
A L
AL
University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, UK.
Ferguson
D J
DJ
Leslie
K O
KO
Taatjes
D J
DJ
Fleming
K A
KA
D002478:Cells, Cultured; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D004076:Digoxigenin / Q000737:chemistry; D015672:Erythroid Precursor Cells / Q000382:microbiology* / Q000648:ultrastructure; D006801:Humans; D017403:In Situ Hybridization; D008854:Microscopy, Electron; D016732:Parvovirus B19, Human / Q000235:genetics / Q000302:isolation & purification*
D015342:DNA Probes; D004279:DNA, Viral; D004076:Digoxigenin