Replication and cytopathicity of the R3 isolates in HF-TOC. In each experiment, fetal thymus fragments were infected with mock supernatant, NL4-3, R3A, or R3B. (A) Thymocytes were isolated and cell-associated p24 was measured by ELISA. The results from at least three independent experiments are summarized. (B–C) Isolated thymocytes were stained for CD4 and CD8 and gated by scatter profiles. A representative FACS plot showing pathogenesis mediated by R3A at 9 days post-infection relative to mock, NL4-3, and R3B is shown. Shown are the percentage of cells gated live or in each quadrant of CD4/ CD8 staining. (D) Pathogenesis of the R3 isolates was summarized by measuring total CD4+ thymocyte depletion. The total percent CD4+ cells was calculated by adding the % CD4+CD8+ immature thymocytes with the % CD4+CD8- thymocytes of cells gated live by forward and side scatter. The results from at least three independent experiments are summarized.