In Situ Hybridization and Immunocytochemistry for Improved Assessment of Human Immunodeficiency Virus Cultures

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Abstract
The authors characterized the early intracellular events involved in human immunodeficiency virus (HIV) replication after in vitro inoculation into cultures of susceptible human T-cell lines and phytohemagglutinin-stimulated peripheralblood mononuclear cells (PMCs). Within 24 hours of infection, in situ hybridization with HIV DNA probe detected cytoplasmic viral RNA. Viral core antigen was detected in infected cells over the subsequent two to ten days by means of an immunocytochemical assay employing monoclonal antibodies. Several days later, cell-free virus was detected by both reverse transcriptase assay and a p25gag antigen-capture assay. When these methods were applied to monitor cultures of ten seropositive persons' PMCs, a similar progression of virus replication was apparent: cytoplasmic viral RNA was detected in infected PMCs by day 3, with the subsequent appearance of intracellular viral proteins (days 6-9) and cell-free virus (days 12-21). In situ hybridization and immunocytochemistry offer complementary, sensitive, and specific approaches for monitoring the early stages of acquired immune deficiency syndrome virus replication in vitro.