While the success of combination antiretroviral therapy (ART) has extended the lifespan of people with human immunodeficiency virus (HIV)(PWH), approximately half of PWH on suppressive ART will experience HIV-associated neurological dysfunction. While ART has decreased the incidence of severe neurological disease and dementia in PWH, the incidence of milder neurological and cognitive complications remains stable. Despite the frequency of HIV neurological disease, contributing factors and inflammatory pathogenesis are difficult to observe in PWH over time. Extracellular vesicles (EVs) constitute an understudied method of intercellular communication and molecule delivery in viral infections. EVs carry inflammatory mediators to areas of the periphery during ART suppression but are understudied in the brain. In this dissertation, we use a biologically relevant simian-human immunodeficiency virus (SHIV)-infected non-human primate (NHP) model of HIV persistence in the central nervous system (CNS) to investigate the formation of a myeloid viral reservoir, inflammation during ART-mediated viral suppression, and the roles of myeloid EVs in persistent SHIV neuropathogenesis.In Chapter 2, we characterized viral and immune persistence in the CNS using SHIV.D, a novel model of HIV-1 in rhesus macaques (RM). Here, we demonstrate viral replication in the brain and neuropathogenesis after ART in RM using novel macrophage-tropic transmitted/founder (TF) SHIV.D.191859. Using quantitative immunohistochemistry (IHC) and DNA/RNAscope, we demonstrated myeloid-mediated neuroinflammation, viral replication, and proviral DNA in the brain in all animals. These findings were replicated in a second cohort of RM necropsied after 6 months of suppressive ART. We concluded that TF SHIV.D models HIV-1 CNS replication, pathogenesis, and persistence on ART in rhesus macaques, and is a biologically relevant model to study HIV neuropathogenesis. In Chapter 3, we investigated EVs in a SHIV.D/RM model of HIV. To determine the potential roles of different cell-derived EV populations in SHIV/HIV neuropathogenesis, we developed a method to investigate changes in the cellular origin of EVs in vivo in RM. EVs that are released by neural and glial cells into the blood circulatory system can serve as biomarkers for injury and illness as well as give insight into CNS dysfunction and other disease processes in a non-invasive manner. Here, we present a bead-free multiparameter conventional flow cytometry method to phenotype, characterize, and determine cellular origin of plasma extracellular vesicles. Using RM plasma and two four-parameter panels, we identified the following subsets of plasma EVs: tetraspanin CD81+, CD11b+ macrophage-derived, CD14+ monocyte-derived, TMEM119+ microglia-derived, CD171+ neuron-derived, CD3+ T cell-derived, and CD31+ endothelium-derived EVs. EVs were isolated from RM plasma before infection with SHIV.D, during acute viremia, and after ART suppression. EV flow cytometry on these samples revealed a significant increase in TMEM119+ microglial EVs and CD171+ neuronal EVs in RM plasma during viremia and ART suppression. In Chapter 4, we investigate myeloid-specific EVs in an in vitro SHIV.D/RM model. Using primary RM monocyte-derived macrophages (MDM), we determined that MDMs increased EV production after SHIV.D infection. Whole proteomic analysis was conducted on EVs from SHIV-infected and uninfected MDM. Gene ontology pathway analysis and gene set enrichment analysis reveal pathways associated with overrepresented proteins in myeloid EVs. Finally, differential abundance analysis demonstrated that myeloid EVs isolated from SHIV.D-infected MDMs carried significantly increased levels of neuropathogenic and inflammatory proteins. Altogether, these studies improve our understanding of SHIV.D viral persistence and persistent neuropathogenesis in the RM brain as a model for HIV-1 chronic neuropathogenesis and describe the contribution of myeloid EVs to neurological disease during SHIV/HIV infection. / Biomedical Sciences