EBV loads in different B-cell subsets in patients with high-level reactivation. (A) Cellular EBV loads determined by Q-PCR in bulk populations of unsorted total CD19⁺ B cells and CD19⁻ non-B cells sorted by FACS, CD27⁻IgD⁺ naive B cells and CD27⁺ memory B cells from 4 patients with high-level EBV reactivation. (B) Proportion of EBV-positive CD27⁻IgD⁺ naive and CD27⁺ memory B cells in the same 4 patients as above, determined by single-cell Q-PCR for EBV DNA. Gray shading represents the fraction of EBV-positive cells in the sorted naive and memory B-cell populations for each patient. (C) Distribution of EBV genome copy number per infected CD27⁺ memory B cell. Values are only shown for EBV-positive cells, with the solid horizontal lines indicating the median value for each patient. As a comparator, we also determined the EBV genome copy number in EBV-positive AKBM Akata-GFP cells induced into lytic cycle (Lytic Akata). Following induction, cells that had entered virus lytic cycle were single-cell sorted on the basis of GFP expression before determining the genome load per cell by Q-PCR, as above. Although the majority of sorted GFP⁺ cells have in excess of 1000 EBV genomes per cell, note that a small proportion of GFP⁺ cells have much lower (latent) viral loads because viral replication is slightly delayed with respect to GFP expression. GFP, green fluorescent protein.