Comparative analysis of systemic and CNS responses to HSV-1 infection in vivo and in vitro. (A–C) Analysis of viral replication on day 4 after HSV-1 ocular infection (2 × 10⁶ PFU/eye) of WT (n = 12) and Irf3^WT/R278Q heterozygous (n = 12) mice. UI controls were included (WT UI, n = 3; Irf3^WT/R278Q UI, n = 3). Viral titer in (A) eyes, (B) TG, and (C) brain stem homogenates quantified by plaque assay. (D–I) Systemic cytokine levels measured in serum from WT and Irf3^R278Q/R278Q mice on day 5 after HSV-1 infection. Data points represent cytokine level (pg/ml) in serum from individual mice measured by mesoscale. (J–N) CNS response in WT and Irf3^R278Q/R278Q mice to systemic LPS stimulation in vivo. Gene expression of Ifnb, Tnfa, Il1b, Il6, and Ccl2 in whole-brain homogenates from mice treated with 5 mg/kg LPS or saline by i.p. injection. Gene expression was measured by RT-qPCR, and data were normalized to β-actin (Actb). (O) Validation of microglia depletion by qPCR of Iba1 expression in MBCs treated with 0.5 μM PLX5622 (PLX) or untreated (UT). (P and Q) Expression of Ccl2 in WT mixed brain cultures treated with (P) 0.5 μM PLX5622 (PLX) or mock treated for microglia depletion, or (Q) NF-κB activation inhibitors BMS-345541 2 µM, PDTC 25 µM, or saline, and infected with HSV-1 at MOI 1.0 for 24 h. (R and S)Isg15 and Cxcl10 infection-dose response analysis in murine microglia analyzed by RT-qPCR 24 h after infection with HSV-1 at increasing MOI. For all panels where statistical analyses were performed, the analysis was two-tailed two-way ANOVA for difference of means, followed by two-tailed unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.001.