Simple SummaryEpstein-Barr virus (EBV) infects a high proportion of the human population. After initial infection, EBV is maintained in healthy individuals in an inactive latent state in memory B-cells of the immune system. Reactivation of EBV from latency can drive B-cell tumours. EBV mimics immune signals to drive B-cells into specific activation states, expanding the virally infected B-cell population. Here we use a laboratory model of the human memory B-cell immune response to study the frequency and patterns of B-cell activation that occur upon spontaneous EBV reactivation. To do this we use single cell analysis of RNA expression. Consistent with the high prevalence of latent EBV infection we find evidence of EBV in cells from all eight healthy adult donors tested. While the extent varies between donors, we identify four recurrent cell states that EBV-associated B-cells adopt upon viral reactivation. Our results establish a model for studying polymorphic EBV-driven B-cell lymphoproliferation.AbstractEpstein-Barr virus (EBV)-driven B cell neoplasms arise from the reactivation of latently infected B cells. In a subset of patients, EBV was seen to drive a polymorphous lymphoproliferative disorder (LPD) in which B cell differentiation was retained. In this work, spontaneous EBV reactivation following B cell mitogen stimulation was shown to provide a potential model of polymorphic EBV-driven LPD. Here, we developed an in vitro model of plasma cell (PC) differentiation from peripheral blood memory B cells. To assess the frequency and phenotypes of EBV-associated populations derived during differentiation, we analysed eight differentiations during the PC stage with a targeted single-cell gene expression panel. We identified subpopulations of EBV-gene expressing cells with PC and/or B cell expression features in differentiations from all tested donors. EBV-associated cells varied in frequency, ranging from 3–28% of cells. Most EBV-associated cells expressed PC genes such asXBP1orMZB1,and in all samples these included a quiescent PC fraction that lacked cell a cycle gene expression. With increasing EBV-associated cells, populations with B cell features became prominent, co-expressing a germinal centre (GC) and activating B cell gene patterns. The presence of highly proliferative EBV-associated cells was linked to retainedMS4A1/CD20expression andIGHMandIGHDco-expression, whileIGHMclass-switched cells were enriched in quiescent PC fractions. Thus, patterns of gene expression in primary EBV reactivation were shown to include features related to GC B cells, which was also observed in EBV-transformed lymphoblastoid cell lines. This suggests a particular association between spontaneously developing EBV-expansions and IgM+ IgD+ non-switched B cells.