Productive EBV replication and the relevance between EBV infection and immune activation in infected NOG-hCD34 mice. (A) Distribution of EBV DNA in splenic human MNCs. Splenic MNCs were isolated from EBV-infected mice (n = 7), and human leukocytes (CD4⁺, CD8⁺, and CD19⁺ cells, respectively) and murine CD45⁺ leukocytes (mCD45⁺ cells, as the background control) were separated by cell sorting. Results are presented in EBV copies per cell of each cell populations. Asterisk represents statistic significance (P < .05 by Student t test) versus the value obtained from mCD45⁺ cells. n.s. indicates no statistical significance. (B) Systemic EBV replication in multiple organs. DNA was extracted from the human MNCs that were isolated from spleen, liver, BM, lung, kidney, and ascitic lavage fluid of EBV-infected mice (n = 7), and the copy number of EBV DNA was measured. (C) Immunostaining for ZEBRA and gp110. Representatives of spleen (top) and liver (bottom) of EBV-infected mice are shown. ZEBRA and gp110 were shown in green, and nuclei were shown in blue by staining with Hoechst. Areas enclosed with squares are enlarged in bottom right of each panel. Scale bars represent 50 μm. (D) Expression of EBV genes. RNA was extracted from splenic human MNCs of 5 EBV-infected mice, a mock-infected mouse, and anti-IgG–stimulated Akata cells (“Lytic Akata”), and the expression of EBV latent genes (lmp1, lmp2a, and ebna2a) and EBV lytic genes (bzlf1, balf2, bxlf1, barf1, bclf1, and bllf1) was determined by RT-PCR. As the internal control, GAPDH expression was also determined. IE indicates immediate early gene; E, early genes; and L, late genes. (E-G) Correlation between viral load in plasma, activation frequency of CD8⁺ T cells, and the level of IFN-γ in plasma. The percentage of Ki67⁺ cells in splenic CD8⁺ T cells (x-axis) and the concentration of IFN-γ in plasma (y-axis; E), the percentage of Ki67⁺ cells in splenic CD8⁺ T cells (x-axis) and EBV DNA copies in plasma (y-axis; F), and the concentration of IFN-γ in plasma (x-axis) and EBV DNA copies in plasma (y-axis; G) are, respectively shown. Red dots represent the results from the EBV-infected mice exhibited severe weight loss and were killed before 10 wpi (n = 13), whereas gray dots represent the results from the EBV-infected mice survived until 10 wpi (n = 5). The lines present exponential approximation. Spearman rank correlation coefficient (r_s) was adopted to determine statistically significant correlation between each value.