Intracellular localization of parvovirus B19 nucleic acid at the ultrastructural level by in situ hybridization with digoxigenin-labelled probes

Abstract

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.

Combined immunocytochemistry and non-isotopic in situ hybridization for the ultrastructural investigation of human parvovirus B19 infection

“…Proteolytic digestion had likewise been found to be unnecessary. Hybridization was performed by floating grids face-down on 15 l, tl drops of denatured hybridization mix for 4 h at 37~ Subsequent post-hybridization washes and blocking were performed as previously described (Morey et al, 1993a). Detection of bound probe was achieved using either (i) a one-step procedure employing 60 min incubation at 37~ with a direct anti-digoxigenin-5 nm gold conjugate (Biocell) diluted 1:50 in TBS-BSA with 0.5% Triton, or (it) a three-step amplified protocol employing 60 rain incubation in sheep polyclonal anti-digoxigenin antibody (Boerhinger Mannheim; diluted to 2 lag ml ~ in TBS-BSA-Triton), followed by 30 rain incubations in polyclonal rabbit anti-sheep antibody (Dako; diluted to 25 lag ml * in TBS-BSA-Triton), and goat anti-rabbit 10 nm gold conjugate (Biocell: 1:50 in TBS-BSA-Triton) with 5 x 2 min rinses in TBS between steps.…”

“…Signal was also found at the periphery of the nucleus, apparently in association with nuclear pores. Cytoplasmic signal was either sparsely distributed or over crystalline arrays (for a detailed description see Morey et al, 1993a).…”

“…Recent studies have demonstrated that B19 nucleic acid can be localized at the ultrastructural level in infected cells embedded in hydrophilic acrylic resins by non-isotopic in situ hybridization with a digoxigenin-labelled probe (Morey et al, 1993a). We have previously reported the successful detection of B19 capsid protein at the light microscope level in formalin-fixed paraffin-embedded material (Morey et al, 1992) using monoclonal antibody R92F6 (O'Neill & Coyle, 1992), demonstrating that the Fig.…”

Non-isotopic electron microscope in situ hybridization for studying the functional sub-compartmentalization of the cell nucleus

“…1993), cytomegalovirus (Cenacchi etal. 1993), papilloma virus (Hagari et al 1993;Multhaupt et al 1992;Yun and Sherwood 1992) and parvovirus (Morey et al 1993.…”

Detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp by in situ hybridization at the electron microscope level

“…For example, long exposure times, limited resolution and low sensitivity associated with isotopic probes (Binder 1987) have been overcome by the use of highly sensitive non-radioactive probes when combined with improved embedding resins (Binder 1987, Beals 1992, Morey 1995, Le Guellec 1998. Intracellular replication of at least 7 different viruses has now been studied using this approach (Geuskens & May 1974, Puvion-Dutilleul & Puvion 1989, Escaig-Haye et al 1992, Multhaupt et al 1992, Morey et al 1993.…”