(A) Clinical scoring (left) and the associated area under the curve (right) of SPF (n = 14) or GF C57BL/6 mice (n = 11) following intracranial (i.c.) infection with 150–300 PFU JHMV. Clinical disease was measured as described in the Materials and methods. Results shown from two independent experiments. (B) Supernatant from homogenized brains of JHMV infected SPF (n = 6–8) or GF mice (n = 6–9) at the indicated time points were used for determining viral titers. (C) Representative image of luxol fast blue/H&E stained spinal cords isolated from JHMV infected SPF or GF 21 days post-infection (p.i.), dashed lines highlight loss of myelin staining indicating demyelination. (D) Quantification of demyelination of SPF (n = 3) or GF (n = 3) mice at 21 days p.i. as represented in 1 c. (E–J) Brains from GF and SPF mice were analyzed via flow cytometry 7 and 21 days p.i. with 150 PFU of JHMV (n = 3–12). Results from one to two independent experiments. (E) Total number of brain cells isolated after homogenization of brain tissue and percoll gradient centrifugation. (F) CD3+, CD4+ frequency of recovered cells (G) CD3+, CD8+ frequency of recovered cells. Numbers of virus-specific CD4+ (H) and CD8+ (I) T cells as determined by intracellular IFN-γ staining in response to defined viral epitopes at indicated time points. (J) Foxp3+ frequency of CD4+ T cells recovered. All data displayed as mean with SEM. Means are of biological replicates (i.e. each data point is from a different mouse). Clinical score significance determined using two-way ANOVA statistical test with multiple comparisons. Viral titer significance determined by Mann-Whitney test. All other significance determined using Student’s t-test. *p<0.05, **p<0.01, ***p<0.005.