Quest for a reliable, valid, and sensitive in situ hybridization procedure to detect viral nucleic acids in the central nervous system

摘要

In situ hybridization (ISH) to detect and to quantitate viral nucleic acid sequences in cryopreserved central nervous system (CNS) tissue is a reliable, valid and sensitive molecular technique. On the other hand, utilization of formaldehyde fixed paraffin embedded (FFPE) tissue to improve cytomorphology requires fundamental changes in the procedure since it is necessary to cleave the elaborate protein network cross-linked by formaldehyde using elevated concentration of proteinases in order to permit diffusion of complementary DNA probes to the targets (genomic viral nucleic acid sequences and/or viral mRNA). Adversely, this procedure hydrolized the proteinaceous glues generally used to fix tissue to glass slides resulting in loss of tissue sections during the ISH protocol. This report describes the application of a novel procedure utilizing a silano-organic compound to covalently bond to glass slides FFPE sections as well as cryopreserved tissue sections and cultured cells with and without virus infections. This covalent bonding procedure has permitted optimization of the ISH procedure for virus detection and quantification, especially for exploratory studies of specificity and wash stringency in relation to the Tm of the hybridized product. Progressive multifocal leucoencephalopathy (PML) caused by an opportunistic papovavirus (JC) was chosen because of the ready availability of tissue, stability of papovavirus nucleic acids, and specificity of3H-and35S-radiolabeled JC cloned DNA probes. Further, this laboratory is utilizing the optimized sensitive procedure to search for several virus etiologies in human diseases such as multiple sclerosis, temporal lobe epilepsy, Alzheimer's disease, schizophrenia, and Parkinson's disease, as well as normal aging. Fanally, the procedure permits study of 100 of thin serial sections; hence, alternate sections can be hybridized with sense and antisense riboprobes to detect viral genome and its mRNA or stained, immunocytochemically, to detect viral proteins. Accordingly, it is anticipated that the mechanism of persistent CNS viral infections will be deciphered, at least in part by advances in cytological molecular hybridization.