Single-cell and spatiotemporal transcriptomic profiling of brain immune infiltration following Venezuelan equine encephalitis virus infection

Neurotropic alphaviruses such as Venezuelan equine encephalitis virus (VEEV) are critical human pathogens that continually expand to naive populations and for which there are no licensed vaccines or therapeutics. VEEV is highly infectious via the aerosol route and is a recognized weaponizable biothreat that causes neurological disease in humans. The neuropathology of VEEV has been attributed to an inflammatory immune response in the brain yet the underlying mechanisms and specific immune cell populations involved are not fully elucidated. This study uses single-cell RNA sequencing to produce a comprehensive transcriptional profile of immune cells isolated from the brain over a time course of infection in a mouse model of VEEV. Analyses reveal differentially activated subpopulations of microglia, including a distinct type I interferon-expressing subpopulation. This is followed by the sequential infiltration of myeloid cells and cytotoxic lymphocytes, also comprising subpopulations with unique transcriptional signatures. We identify a subpopulation of myeloid cells that form a distinct localization pattern in the hippocampal region whereas lymphocytes are widely distributed, indicating differential modes of recruitment, including that to specific regions of the brain. Altogether, this study provides a high-resolution analysis of the immune response to VEEV in the brain and highlights potential avenues of investigation for therapeutics that target neuroinflammation in the brain. Author SummaryVenezuelan equine encephalitis virus (VEEV) causes brain inflammation in both animals and humans when transmitted by mosquito bite or infectious aerosols. The mechanisms underlying disease caused by VEEV, including the role of the immune response in brain pathology, are not well understood. Here we performed a comprehensive assessment of the immune response to VEEV in the brain over time using two advanced sequencing techniques. Following infection, immune cells infiltrate the brain in a sequential fashion and display different activation profiles. Different types of immune cells also display strikingly different spatial patterns throughout the brain. This study provides the most comprehensive description of the immune response to VEEV in the brain performed to date and advances our understanding of immune-driven neuropathology and identification of therapeutic targets.