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. 2022 Apr 28;96(10):e00027-22. doi: 10.1128/jvi.00027-22

FIG 3.

FIG 3

Latency establishment in the spleen is severely ablated at 16 dpi by the expression of IKKα-SA. C57BL/6 mice were infected with 1,000 PFU of the indicated viruses via the intranasal route, and spleens were analyzed at 16 dpi. (A) Spleen weights from individual uninfected naive or infected mice are represented by symbols. Lines indicate mean weights; open and closed symbols indicate matched replicate experiments. Data are from two independent experiments with 3 to 5 infected mice per group. (B) Latency was measured in bulk splenocytes by determining the frequency of viral genome-positive splenocytes at 16 dpi by limiting dilution PCR. Briefly, intact splenocytes were serially diluted and subjected to nested PCRs to detect viral ORF50. (C) Reactivation was measured by coculturing serial dilutions of intact splenocytes from infected mice onto a monolayer of MEFs and scoring for CPE 2 to 3 weeks after coculture. For the limiting dilution analyses, curve fit lines were determined by nonlinear regression analysis. Using Poisson analysis, the intersection of the nonlinear regression curves with the dashed line at 63.2% was used to determine the frequency of cells that were positive for either the viral genome (B) or reactivation (C). Data were generated from three independent experiments with 3 to 5 mice per group for IKKα-SA and IKKα-SA.STOP viruses and two independent experiments with WT BAC-derived MHV68. Error bars indicate SEM. (D) Flow cytometric analysis of splenocytes to enumerate percentages of CD19+ B cells, CD21− CD23− CD19+ newly formed (NF) cells, CD21int CD23− CD19+ marginal zone (MZ) cells, CD21+ CD23+ CD19+ follicular (FO) cells, CD95+ GL7+ CD19+ germinal center (GC) cells, and MHV68-YFP-infected CD19+ cells. Symbols represent individual mice; closed symbols are IKKα-SA1 and -SA.STOP1 viruses, and open symbols are IKKα-SA2 and -SA.STOP2 viruses. Significance was determined by an unpaired, two-tailed t test (B and C) (***, P < 0.001) or one-way ANOVA with Tukey’s multiple comparisons (A and D) (*, P < 0.05; **, P < 0.010; ***, P < 0.0005).