NEW TECHNOLOGY cell-tropic HIV-1. The cervical tissues were exposed to ‘pre-titered’1× 104 median tissue culture infectious doses (TCID50) CEM-IIIB cells or cell-free HIV-1 IIIB for 24 hours, after which the virus inoculum was removed and incubation was continued for a total of 5–6 days. Viral transmission was determined during this period by measuring the infectivity of the transmitted virus in supernatants from the bottom chamber in cMAGI cells. We determined viral transmission from CEM-IIIB cells in three independent experiments (Fig. 4a). Viral transmission increased with culture time, with maximum transmission on day 6. In contrast, there was no viral transmission in the agarose control wells. Each experiment included at least triplicate tissue wells. Using fluorescein-labeled CEM-IIIB cells, we also determined that infected cells did not migrate into the epithelium layer, indicating that viral transmission could not be due to migration of infected cells into the tissue during the cultivation period (data not shown). We assessed the transmission of HIV-1 from cell-free HIV-1 IIIB in three independent experiments (Fig. 4b). The kinetics of viral transmission from cell-free IIIB were different from those of cell-associated HIV-1 IIIB: Cell-free IIIB virus showed the highest amount of transmission on day 1; CEM-IIIB cells, on day 6. There was no transmission in the agarose control wells in the cell-free experiments.

As macrophage-tropic virus is more prevalent in the general population than T-cell tropic virus, and sexual transmission is more likely to occur through cell-free virus, we assessed transmission from cell-free macrophage-tropic HIV-1 BAL. As with T-cell-tropic virus, cervical tissue was exposed to 1× 104 TCID50 of HIV-1 BAL for 24 hours, after which virus inoculum was removed and the transmission was measured.