Intranasal HSV‑1 infection drives region‑specific interferon‑dominant microglial remodeling

Herpes simplex virus type 1 (HSV-1) is a neurotropic pathogen capable of invading the central nervous system (CNS) and is associated with chronic neuroinflammation, cognitive impairment, and neurodegenerative disease. While microglia orchestrate the initial immune response to HSV‑1, the molecular mechanisms that regulate their sustained neuroinflammatory activity in vivo remain poorly understood. To define the transcriptional and epigenetic mechanisms that shape microglial responses during acute HSV-1 infection in vivo, we have, for the first time, integrated single‑nucleus RNA sequencing, chromatin accessibility profiling, and spatial proteomics and transcriptomics in an intranasal HSV‑1 infection model. Single-cell multiome analysis of CD11b⁺ nuclei identified transcriptionally and epigenetically distinct microglial and macrophage populations. HSV-1 infection redistributed monocyte-lineage states, with a marked overrepresentation of interferon (IFN)‑responsive microglia and macrophage-associated populations characterized by amplification of Stat1/2, Irf1, and Cebpb rather than reflecting uniform inflammatory activation. In parallel, homeostatic microglial gene signatures ( e.g., ApoE, Cst3 ) were reduced following HSV-1 infection. Spatial transcriptomics localized HSV‑1 antigen to discrete brainstem regions, which were enriched for predicted STAT1/2, IRF1, and CEBPB‑regulated targets identified through single‑nuclei analysis, demonstrating that these inferred transcriptional networks are activated within anatomically defined regions in vivo . Complementary spatial proteomics further confirmed increased microglial activation and astrocytic responses specifically within HSV-1 antigen-positive regions. Together, these integrated multiomic analyses demonstrate that HSV-1 induces localized transcriptional and epigenetic remodeling of microglia dominated by IFN-driven regulatory programs and a loss of homeostatic signatures. By linking single-cell regulatory architecture to spatially restricted tissue responses, our findings provide mechanistic insight into how localized viral infection sustains neuroinflammation, contributing to long-term neurological vulnerability and neurodegenerative disease risk.