The purpose of this research project is to identify markers and develop rapid quantitative methods for 1) detecting and quantifying low levels of infectious HIV in blood and blood products, 2) estimating viral burden in clinical material, 3) characterizing virus target cells and 4) distinguishing among various virus strains. Rapid assays for infectious virus have been developed in the laboratory based on the indicator T-cell line (H938) containing multiple integrated copies of HIV-1-LTR-chloramphenicol acetyl transferase (CAT). The indicator T-cells signal infection with quantitative increase in enzyme activity. The T-cell assay has been used to determine the half-life of HIV-1 in packed red blood cells (see Z01 BG 0699-02(LR), and has formed the basis for a neutralization assay to assess potency and efficacy of monoclonal antibodies (see Z01 BG 0631-03(LR). A rapid reverse transcriptase assay developed under this project has been used to titer the representative strains of HIV-1 virus (IIIB, RF, Z, MN) used as inocula for the neutralization test system. Currently a monocyte indicator cell line containing stably integrated copies of the HIV-1-LTR-CAT construct is being characterized with representative strains of HIV-1 (RF, IIIB, Z, MN) and adapted for use as a neutralization target cell. The CAT signal produced in the indicator assays presently use 3H- coenzyme A as substrate. Alternative substrates which produce non- radioactive products, but which are equally sensitive, are under active investigation.
