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

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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 hybridization was detected. In early PML lesions, swollen nuclei or oligodendrocytes were the predominant cells labeled, whereas older lesions revealed increased numbers of reactive and bizarre hypertrophic astrocytes hybridized at the outer periphery of the demyelinated lesions. The hybridization varied greatly in intensity in different cells. Intense hybridization was noted very rarely in microglial cells, including rod cells and rarely in venular pericytes, intravascular mononuclear cells, or in vascular endothelial cells. These results, considered together with previous findings, indicate that in PML the viral infection runs different courses in the various cells: in astrocytes the viral genome persists for a long time inducing pathological changes in some cells. In oligodendrocytes the infection rapidly lyses the cells. There was a good correlation between chromatic changes observable in routinely stained sections and virus presence. In addition, in situ hybridization using a measles-NP-cloned probe in white matter from FFPE SSPE brain is presented confirming earlier results in SSPE cryopreserved brain.