Single-nucleus detection of rare HIV-infected cells defines the cellular landscape of HIV persistence in the human brain

Background HIV-1 enters the central nervous system early after infection and establishes a long-lived reservoir that persists despite antiretroviral therapy. Single-cell and single-nucleus RNA sequencing provide powerful approaches to study HIV infection in the human brain, yet standardized and sensitive methods for identifying rare HIV-infected cells in these datasets remain limited. Here, we present a scalable multi-reference framework for detecting HIV RNA–positive cells in human CNS single-nucleus RNA-seq data. The pipeline integrates a modified HIV reference genome, subject-specific variant-updated HIV references, and a comprehensive HIV strain collection to improve viral read recovery and specificity. Results We applied this framework to 250 post-mortem brain samples from the SCORCH (Single Cell Opioid Responses in the Context of HIV) consortium spanning 12 brain regions and 102 donors, including people with and without HIV (PWH and PWoH). After screening, 48 samples from 35 donors comprising 559,207 high-quality nuclei were analyzed in depth. We identified 1,939 HIV RNA–positive cells exclusively in samples from PWH. Using conservative thresholds, 908 high-confidence infected cells were retained for downstream analyses. HIV RNA-positive cells were rare overall and strongly enriched in cases with HIV encephalitis. Microglia constituted the predominant infected population (79% of HIV RNA-positive cells), with substantially smaller contributions from oligodendrocytes, astrocytes, and neurons. In non-encephalitic brains, detectable infection was largely restricted to microglia, whereas in encephalitic tissue HIV RNA–positive cells were distributed across multiple CNS (Central Nervous System) lineages. Viral RNA burden followed a long-tailed distribution, with microglia retaining higher HIV transcript counts than other cell types. Recovered HIV reads were concentrated in the U3 region of the 5′ LTR and in the env gene, implicating regulatory and entry-associated regions as focal points of viral diversity in the brain. Conclusions Together, these data establish a harmonized framework for identifying rare HIV-infected cells in CNS single-cell datasets and provide large-scale quantitative evidence that microglia represent the dominant and most persistent HIV-infected population in the human brain. This work offers a reference strategy and resource for future NeuroHIV studies aimed at defining, monitoring, and ultimately targeting CNS viral reservoirs.