Single-cell RNA sequencing of mouse brain immune cells following peripheral SARS-CoV-2 infection

Neurological and neuropsychiatric symptoms are among the most prevalent Post-Acute Sequelae of COVID-19 (PASC), collectively referred to as neuroPASC. Despite growing recognition, the mechanisms driving neuroPASC remain elusive, hindering therapeutic development. Brain immune cells - particularly microglia - have been implicated, yet current knowledge largely derives from post-mortem tissues with variable disease courses and comorbidities and limited spatial coverage. To address this, we established a PASC animal model by infecting C57BL/6 mice with a sublethal dose of mouse-adapted SARS-CoV-2. Infected mice exhibited persistent behavioural alterations and prolonged neuroinflammation in the absence of direct viral neuroinvasion. Single-cell RNA sequencing of brain immune cells collected at 0, 6, 30, and 100 days post-infection (dpi) revealed underlying dynamic, longitudinal immune responses. Microglia, the predominant brain-resident sentinel cells, displayed sustained expansion of subclusters characterized by inflammatory, stress response, and metabolic reprogramming signatures across all time points. Border-associated macrophages upregulated monocyte attractants during acute infection and were partially replaced by infiltrating monocytes. Concurrently, inflammatory monocytes and neutrophils showed maximal brain infiltration and antiviral activity at 6 dpi, potentially triggering long-term microglial activation. Together, these findings provide a high-resolution atlas of brain myeloid immune dynamics during neuroPASC and highlight a central role of microglia in sustaining chronic neuroinflammation, offering insight into potential therapeutic targets. Overall design: C57BL/6 mice were intranasally infected with 1000 PFU of SARS-CoV-2 N501YMA30. Brain immune cells were isolated from mock-infected mice or at 6, 30, 100 days post-infection using CD45+ magnetic beads. 4-5 mice per group were pooled together for scRNA-seq library preparation.