Search for virus nucleic acid sequences in postmortem human brain tissue using in situ hybridization technology with cloned probes: some solutions and results on progressive multifocal leukoencephalopathy and subacute sclerosing panencephalitis tissue
Abstract
In order to obtain a useful and readily applicable in situ hybridization (ISH) protocol for progressive central nervous system (CNS) diseases of unknown etiology that are possibly due to persistent viral infection, known and well described diseases were studied, namely, progressive multifocal leukoencephalopathy (PML) and subacute sclerosing panencephalitis (SSPE). The procedures described were validated by confirming results obtained by other investigators using histology, immunocytochemistry, electron microscopy, and ISH. A number of frequently encountered problems of tissue preparation are addressed as well as techniques to reduce autoradiography exposure times. A multi-staged specific, sensitive, reliable, and valid procedure for detection of viral genomes, mRNA and proteins is approached. Formalin-fixed and paraffin-embedded (FFPE) brain material from six patients who died with PML and one patient who died from SSPE were studied using ISH with a tritium-labeled cloned JC virus DNA probe and a measles-cloned nucleocapsid (NC) gene cDNA probe, respectively. This report constitutes a methodological framework as well as a detailed neuropathological analysis of identified brain cell populations within which in situ hybridizatio...Continue Reading
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