Splenic Latency Is Markedly Reduced in Mice Infected with 1,000 PFU of γHV68-IκBαM by the Intranasal Route of Inoculation

<div><p>Bulk splenocytes or flow cytometry-sorted CD19⁺ splenic cell populations were harvested from infected C57BL/6 mice at 15 or 16 dpi and analyzed by limiting-dilution viral genome PCR (A and C) and limiting-dilution ex vivo reactivation assays (B and D) as described in Materials and Methods.</p><p>(A) Frequency of unsorted, bulk splenocytes harboring viral genomes.</p><p>(B) Frequency of unsorted, bulk splenocytes reactivating virus. Significant levels of preformed virus were not detected.</p><p>(C) Frequency of sorted CD19⁺ B cells harboring viral genome. Using a PE-conjugated antibody to the pan–B-cell marker CD19 (CD19-PE), splenic B cells were separated into B-cell (CD19⁺) and non–B-cell (CD19⁻) populations isolated by FACS. Postsort FACS analysis indicated that the mean purities for CD19⁺ cells were 97.7 ± 1.1% for WT and 96.7 ± 3.4% for IκBαM.1.</p><p>(D) Frequency of sorted CD19⁺ B cells reactivating virus. For both limiting-dilution assays, curve fit lines were derived from nonlinear regression analysis, and symbols represent the mean percentage of wells positive for virus (viral DNA or cytopathic effect) ±SEM. The dashed line represents 63.2%, from which the frequency of viral genome–positive cells or the frequency of cells reactivating virus was calculated based on Poisson distribution. The data shown represent three to five independent experiments with spleen cells pooled from five mice per experimental group. Where examined, the frequency of splenocytes and CD19⁺ B cells that harbored viral genome in mice infected with IκBαM.1 or IκBαM.2 was significantly different (<i>p</i> < 0.02) from that of mice infected with WT or IκBαM.MR control viruses.</p>