Transgenic Expression of Bcl-2 in B Cells Does Not Rescue the Defect in Splenic Latency Establishment by γHV68-IκBαM after Intranasal Infection

<div><p>Bulk unsorted splenocytes or magnetic bead–enriched CD19⁺ splenocytes from infected C57BL/6-Tg(BCL2) were analyzed by limiting-dilution viral genome PCR (A, C, and D) and limiting-dilution ex vivo reactivation assays (B) as described in Materials and Methods.</p><p>(A) Frequency of unsorted, bulk splenocytes and CD19⁺ B cells from mice infected with 1,000 PFU of IκBαM.MR or IκBαM.1 harboring viral genomes at 16 dpi. 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 by magnetic activated cell sorting. Postsort FACS analysis indicated that the mean purities for CD19⁺ cells were 97.4 for WT, and 96.5 ± 1.5% for IκBαM.1. The frequency of splenocytes that harbored viral genome in mice infected with IκBαM was significantly different (<i>p</i> = 0.05) from that of mice infected with IκBαM.MR virus.</p><p>(B) Frequency of unsorted, bulk splenocytes and CD19⁺ B cells reactivating virus from mice infected with 1,000 PFU of IκBαM.MR or IκBαM.1 at 16 dpi. The frequency of splenocytes and CD19⁺ B cells that harbored viral genome in mice infected with IκBαM was significantly different from that of mice infected with IκBαM.MR in a <i>t-</i>test as follows: <i>p</i> = 0.0460 (unsorted splenocytes) and <i>p</i> = 0.0286 (CD19⁺).</p><p>(C) Frequency of unsorted, bulk splenocytes reactivating virus from mice infected with 1,000 PFU of IκBαM.MR or IκBαM.1 at 41 to 43 dpi. CD19⁺ B cells comprise greater than 67% of total splenocytes as determined by FACS analysis.</p><p>(D) Frequency of unsorted, bulk splenocytes reactivating virus from mice infected with 1,000 PFU of IκBαM.MR or IκBαM.1 at 235 dpi. FACS analysis indicated that the unsorted splenocytes were composed of 66.3 ± 2.0% (IκBαM.MR) and 60.3 ± 7.7% (IκBαM.1) CD19⁺ B cells. Symbols represent the mean percentage of wells positive for virus (viral DNA or cytopathic effect) ±SEM. The data shown represent two independent experiments with cells pooled from three to five mice per experimental group (16 and 41 to 43 dpi) or one experiment with four individual mice at 235 dpi.</p>