title: Ultrastructural localization of viral nucleic acid by in situ hybridization.
abstract: In situ hybridization has become a standard technique in the localization of viral nucleic acids in tissue sections and cytologic preparations at the light microscopic level. We have extended this technique to the electron microscopic level using human cytomegalovirus (CMV) infection in cultured human foreskin fibroblasts, and have shown for the first time that colloidal gold can be used to study intranuclear localization of viral replication. CMV-infected fibroblasts exhibiting early (4-day) and late (18-day) cytopathic effect were fixed in formalin, gently permeabilized with detergent and protease, and hybridized with a biotinylated CMV DNA probe. Hybridized sequences were localized by a pre-embedding technique using streptavidin-conjugated 15 to 20 nm colloidal gold particles. Ultrastructural nuclear and cytoplasmic architecture were well preserved through permeabilization and hybridization steps. Viral DNA was clearly detected in fibroblast nuclei containing nascent and well-formed electron-dense viral inclusions. Gold particles were localized to the periphery of electron-dense nuclear inclusions, occasionally in association with 70 nm nuclear dense bodies, but not with complete viral nucleocapsids. DNA hybridization was abolished by pretreatment of infected cells with DNase. Cross-hybridization of CMV DNA sequences with human DNA or with herpes simplex virus genome was not observed. The ultrastructural findings suggest that CMV DNA replication may occur at the margins of electron-dense regions in maturing viral inclusions, and that viral DNA associated with core dense bodies is available for hybridization with complementary nucleic acid sequences. This technique can be useful in studies of viral pathogenesis.
authors.affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor.
authors.forename: R A
authors.initials: RA
authors.lastname: Wolber
authors.forename: T F
authors.initials: TF
authors.lastname: Beals
authors.forename: R V
authors.initials: RV
authors.lastname: Lloyd
authors.forename: H F
authors.initials: HF
authors.lastname: Maassab
mesh_terms: D002478:Cells, Cultured; D003102:Colloids; D003429:Cross Reactions; D003587:Cytomegalovirus / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D003588:Cytopathogenic Effect, Viral; D004261:DNA Replication; D004279:DNA, Viral / Q000032:analysis* / Q000648:ultrastructure; D005347:Fibroblasts; D006046:Gold; D006801:Humans; D008854:Microscopy, Electron; D009693:Nucleic Acid Hybridization; D014779:Virus Replication
chemical_list: D003102:Colloids; D004279:DNA, Viral; D006046:Gold