Fig 3. MHV68 ORF75A is essential for efficient seeding of intermediate reservoirs and the establishment of latency in the spleen at early, but not late times of chronic infection after intranasal inoculation.

C57BL/6 mice were infected at 1000 PFU by the intranasal route with the indicated viruses. (A) Frequency of cells in the mediastinal lymph node harboring viral genomes 9 dpi. Data is a compilation of 75A.stop1.1 (n = 1) with 75A.stop1.2 (n = 4) and of H2BYFP (n = 1) with 75A.stop1MR (n = 4). (B) Frequency of peripheral blood mononuclear cells harboring genomes 9 dpi. Data is generated from 3 independent experiments, 5 mice per group. (C) Splenomegaly 16 dpi with indicated viruses. Each symbol represents an individual mouse. Line indicates geometric mean titer. ORF75A mutants are blue and WT control viruses are black. (D) Frequency of splenocytes harboring genomes 16 dpi. (E) Frequency of splenocytes undergoing reactivation from latency upon explant 16 dpi. (F) Frequency of splenocytes harboring genomes at six weeks post-infection. 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 either positive for the viral genome or reactivating virus. (C-F) Data is generated from two independent experiments for 75A.stop2 and 75a.dbl.stop viruses, and three independent experiments for 75A.stop1.2 and 75A.stop1MR viruses. Each experiment contained three to six mice per group for 16 dpi. Data for 42–49 dpi is generated from two experiments with four to five mice per group. Error bars indicate SEM. * p ≤ 0.05, *** p ≤ 0.0005, and **** p ≤ 0.00005; for A, B, D-F, significance determined by two-way paired t-test; for C, significance determined by two-way unpaired t-test.