In vitro viral rescue assay in CD4+ T cells isolated by magnetic column at 126 weeks PI. FIV gag PCR products were present in samples from FIV-infected cat (165) vDNA (lane 3) and vRNA (lane 4) (a). Amplicons were not identified in RT- samples (lane 5, cat 165 cDNA), or in samples from the uninfected control cat (cat 183, DNA and cDNA, lanes 6 and 7 respectively). Positive [pDNA (plasmid), lane 2] and negative controls (water template, lane 1) were appropriate. Appropriate-sized amplicons were generated with the feline GAPDH primer set (b) from the same samples as in (a). CD4+ T cells derived from cat 165 were cultured ex vivo for 14 days and subsequently co-cultured for 6 days with SPF CD4+ T cells (lane 3, negative) or PBMCs (lane 4, positive) and evaluated for FIV circle junctions (primer set B) (c). Positive (pDNA, lane 1) and negative controls (water template, lane 2) were appropriate. Appropriate-sized amplicons (arrowhead) were generated for 18s rRNA gene (d) from the same samples as in (c). Co-culture of latently infected CD4+ T cells with uninfected feline PBMCs results in detectable 2 LTR circle junction amplicons (e). Freshly isolated CD4+ T cells derived from FIV-infected cats 165, 184, 187 and 186 (day 0, lanes 3-6, respectively) were cultured ex vivo for 14 days (165) or 3 days (184, 187 and 186) and subsequently co-cultured for 6 (165), 5 (187 and 184) or 10 (186) additional days with FIV-negative PBMCs (lanes 7-10, respectively). Samples derived from cat 186 PBMCs cultured ex vivo for 13 days served as the positive control (lane 1) while water template served as the negative control (lane 2). Expected PCR amplicons of 250 bp were generated with 2 LTR circle junction primer set C in lanes 1 (positive control) and 7-10 (arrowhead). Appropriate-sized amplicons (arrowhead) were generated for 18s rRNA gene (f) from the same samples as in (e).