Supplementary Figure 2: Kinetics of immune cell infiltration to the hippocampus.

Cx3CR1^GFPCCR2^RFP reporter animals were infected with WNV or ZIKV at 8 weeks old. Cells were isolated from the hippocampus at indicated dpi. (a) Gating strategy to identify putative microglia (P1: CD45^midCD11b⁺), macrophage (P2: CD45^hiCD11b⁺), and lymphocyte (P3: CD45^hiCD11b^-) populations. (b, c) Quantification of indicated populations at 0, 7, and 25 dpi revealed infiltration of putative macrophage and lymphocyte populations by 7 dpi, the latter of which persisted in the hippocampus at 25 dpi. Data are representative of two independent experiments (n=3 mice per group, mean ± SD, b: p<0.0001, =0.0006, <0.0001 c: p<0.0001, =0.0020, 0.0140, <0.0001). (d) Additional gates were applied to P1 and P3 populations to assess MHC-II expression in CD45^midCD11b⁺Cx3CR1⁺CCR2^- microglia populations and CD103 expression in CD45^hiCD11b^- CD8⁺ T cells (quantification in main Figure 2). (e, f) CD45^hiCD11b^- CD8⁺ T cells (identified by gating strategy in a,d) enter the hippocampus by 7 dpi and persist to 25 dpi (n=3 mice per group, mean ± SD, e: p=0.0018, <0.0001, =0.0001, 0.0017 f: p=0.0012, 0.0043). Data was analyzed by two-way (b,c) or one-way (e,f) ANOVA, and corrected for multiple comparisons. *,P<0.05, **, P<0.005, ***, P<0.001, ****, P<0.0001.