DIRECT EVIDENCE USING IN-SITU POLYMERASE CHAIN-REACTION THAT THE ENDOTHELIAL-CELL AND T-LYMPHOCYTE HARBOR LATENT MURINE CYTOMEGALOVIRUS

The latent viral genome, harbored indefinitely, threatens reactivation from its remote location. Although polymerase chain reaction (PCR) ha s detected the organs responsible for latency, it is not known whether latent cytomegalovirus (CMV) infection is maintained within organ-spe cific cells or ubiquitous elements such as macrophages, endothelial ce lls, or perhaps others. PCR lacks correlation with tissue structure. H owever, PCR-based in situ hybridization maintains cellular architectur e while allowing the identification of the latently infected cells. Mu rine CMV (MCMV) nucleic acid sequences in organs of latently infected Balb/C mice were amplified by PCR incorporating digoxigenin-11-dUTP, h olding the product DNA in situ (appropriate controls analyzed in paral lel). Product DNA was then hybridized in situ with a biotinylated olig onucleotide probe for detection via streptavidin-alkaline phosphatase and light microscopy. Immunohistochemistry verified the positive cell types. Using this technique, we have shown directly in multiple organs of latently infected Balb/C mice including kidney (5/5), liver (5/5), and spleen (5/5) that the endothelial cell and/or T-lymphocyte harbor latent MCMV, whereas in uninfected animals, MCMV DNA was not detected . PCR-based in situ hybridization allows detection of the specific cel l(s) harboring latent MCMV DNA while allowing conservation of cellular architecture.