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. The following markers of HIV infection and replication in vitro have been shown to be equally sensitive to acute HIV infection of susceptible cell lines: reverse transcriptase (RT, determined by rapid microassay), HIV-p24 antigen, gag DNA-PCR and CAT enzyme of transfected indicator cells (3 day assay). Results indicate that cell-associated RT activity and p24 antigen levels used to monitor HIV replication in vitro precede and tend to be greater than levels measured in culture supernatants. Marker signal strength was greatest using the HIV-TAT-sensitive indicator T-cell line which contains stably integrated copies of HIV-LTR-CAT gene. The quantitative relationship of various markers (cell-associated and cell- free) to virus titer, particle count and virus burden is currently under investigation. Studies are ongoing to develop methods and reagents capable of distinguishing among various isolates of HIV. Inoculum has been prepared of representative strains of HIV. Following infection, the sensitivity and signal strength of viral markers are under study as a means of characterizing isolates. Rapid detection of HIV infection of several monocyte/macrophage cell lines have been more difficult. Changes in markers appear to be less sensitive, less consistent and require longer culture times than T-cells. Other monocyte/macrophage cell lines are being evaluated as possible target cells.
