Quantitation of KSHV and rKSHV.219 infection of CD34⁺cells cultured in vitro. (A) CD34⁺ HPCs were infected with wild-type KSHV or UV-irradiated KSHV (UV, 10 μW/cm²  for 1 hour) (100 viral genome equivalents per cell) or were mock-infected. Infected CD34⁺ HPCs (5 × 10⁶  cells) were cultivated in IMDM supplemented with 10% FBS at 37°C, 5% CO₂ for up to 25 days. Cell aliquots were collected daily, and DNA was extracted and analyzed by RQ-PCR using KSHV KS330₂₃₃ (ORF 26 minor capsid protein, 233 bp) and human β-globin (240 bp) primers. KSHV genomic copy number was normalized to 0.5 × β-globin copy number, representing one human cell. Bars represent mean KSHV genomic copies per human cell at indicated periods after infection, and error bars represent standard error of the mean (SEM). (B) CD34⁺ HPCs were infected with rKSHV.219, UV-irradiated rKSHV.219 (UV, 10 μW/cm²  for 1 hour), heat-inactivated rKSHV.219 (HEAT, 65°C for 1 hour) (600 viral genome equivalents per cell), or mock-infected. Infected CD34⁺ HPCs were cultivated, cell aliquots collected, and DNA was extracted and analyzed by RQ-PCR as in panel A. Infected CD34⁺ HPCs were cultivated for up to 16 days. Bars represent mean rKSHV.219 genomic copies per human cell at indicated time periods after infection, and error bars represent SEM.