Increased frequencies of an inflammatory monocyte population in the HSV-1–infected Irf3 R278Q/R278Q brain. (A) Schematic illustration of single-cell omics pipeline. Mice were infected via the ocular route using 2 × 106 PFU/eye in 5 μl. On day 5 after infection, mice were PBS perfused and brain stems harvested for either CyTOF (WT, n = 6; Irf3R278Q/R278Q, n = 6; UI, n = 6 mice); analysis of CD45+ cells or scRNAseq (WT, n = 3, each sample contains pools of two to four brain stems; Irf3R278Q/R278Q, n = 3, each sample contains pools of two to four brain stems; UI, n = 3, each sample contains pools of two brain stems). Image was generated in BioRender. (B) UMAP and cell cluster analysis of live CD45+ cells acquired by CyTOF from UI or HSV-1–infected brain stems. (C and D) Frequency of (C) MHC-IIhi MoMØ and (D) MoDC gated as % of live CD45+ cells. (E) Table of phenotypic markers characterizing the MHC-IIhi MoMØ and MoDC cell populations, respectively. The frequencies (C and D) were analyzed with two-tailed Student’s t test. Error bars; SD. (F) UMAP and cell type annotation of brain stem scRNAseq of WT and Irf3R278Q/R278Q mice. (G) Average cell number of annotated cell types per mouse brain stem of infected WT and Irf3R278Q/R278Q mice. (H) Average cell number of identified monocyte subpopulations per mouse brain stem of infected WT and Irf3R278Q/R278Q mice. Cell numbers within each subpopulation were first compared across the WT UI compartment using ANOVA test. This was followed by pairwise comparisons between WT and Irf3R278Q/R278Q mice using Tukey’s honestly significant difference (HSD) test. (I) Average number of monocyte #1 population in the brain of mice infected with Japanese encephalitis virus grouped according to severity of disease. P values <0.05 were considered statistically significant, *P < 0.05, **P < 0.01, and ***P < 0.001. MoMø, monocyte-derived macrophage-like cell; MoDC, monocytic-derived dendritic-like cell; VLMC, vascular and leptomeningeal cell; vSMC, vascular smooth muscle cell.