Histochemical Journal 25, 421-429 (1993) Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes A. L. MOREY 1, D. J. P. FERGUSON 1, K. O. LESLIE e, D. J. TAATJES z and K. A. FLEMING ~ ~University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital Oxford OX3 9DU, UK and 2University of Vermont, Department of Pathology, Burlington, VT 05405-0068, USA Received 2 September 1992 and in revised form 14 December 1992 Summary Conditions suitable for immunogold detection of digoxigenin-labelled DNA probes hybridized to parvovirus B19-infectederythroid 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, BI9 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 c0~posed of mainly 'empty' capsids. BI9 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. Introduction Infection with human parvovirus B19 can cause aplastic crises in patients with underlying haemolytic anaemias, persistent infection leading to chronic bone marrow failure in the immunosuppressed and fetal death associ- ated with hydrops (see review by Harris, 1992). This single-stranded DNA virus has a specific tropism for human erythroid precursor cells and cannot be cultured in standard cell lines or animal models. Erythroid precur- sors infected in vitro demonstrate characteristic cytopathic effects at the ultrastructural level, including margination of the cellular heterochromatin and cytoplasmic vacuo- lation (Young et aI., 1984; Morey et at., 1993). Crystalline arrays of viral capsids are occasionally found in such cells, but the small size of this non-enveloped virus (22 nm) and its unremarkable ribosome-like appearance have made detailed investigations of the intracellular lifecycle of this agent difficult. The ability to localize B19 nucleic acids at the ultrastructural level would be of considerable benefit in such studies. 0018-2214 9 1993 Chapman & Hall Although in situ hybridization has become a standard technique for the localization of nucleic acid sequences in cellular preparations at the light microscope level, it is only very recently, with the development of non-isotopic probe labelling strategies and immunogold detection methods, that the technique has been successfully trans- posed to the electron microscope level (see review by Beals, 1992). Just as the localization of viral nucleic acid sequences has been one of the most fruitful applications of in situ hybridization technology at the light microscope level (Fleming, 1992), the analysis of viral life cycles within cells is potentially one of the most promising applications of the technique at the ultrastructural level. Biotinylated probes and immunogold (or strep- tavidin-gold) detection have been used to localize viral nucleic acid in material embedded in hydrophilic acrylic resins (Puvion-Dutilleul & Puvion, 1989, 1990, 1991; Troxler et al., 1990; Fournier et al., 1991) and in perme- abilized cells prior to embedding (Wolber et al., 1988, 1989). A recurring problem with the use of biotinylated probes for in situ hybridization at the light-microscope HIST 25/(~-B