Abstract As of April 30, 2021, the COVID-19 pandemic has resulted in more that 148 million cases and with 3.1 million deaths worldwide. Typically, people recover from COVID-19 after 2 to 6 weeks; however, in a significant fraction of patients symptoms may linger or recur for weeks or months following initial recovery. These “long COVID-19” symptoms or Post-Acute Sequelae of SARS-CoV-2 infection (PASC) include fatigue or muscle weakness, sleep disorders, loss of taste or smell, confusion, anxiety, and depression. While the clinical characteristics and pathogenesis of acute COVID-19 disease are being intensively studied, the long-term consequences of disease remain largely unknown. Furthermore, the small number of PASC research studies published to date are limited by a relatively short follow-up after patients are discharged from the hospital; a lack of pre-infection data; and limited sampling of tissues. Here, taking advantage of a recently established nonhuman primate (NHP) model of SARS-CoV-2 infection, we aim to establish biosafety guidelines to validate and standardize PASC NHP studies in rhesus macaques- up to 18 weeks post-infection – in which animals repeatedly testing negative for SARS-CoV-2 will be transferred from ABSL-3 to ABSL-2+ facilities (Aim 1). This ability to transfer animals from ABSL-3 facilities will be critical to allow the study of PASC in NHP with acceptable costs and labor, while preserving the safety of personnel and the NHP colonies. Transfer of animals to BSL-2+ will allow us to characterize neurological and behavioral manifestations observed in human PASC. Additionally, we are proposing extensive and state-of-the-art immunologic and virologic analyses in a long-term study of SARS-CoV- 2-infected rhesus macaques (RMs) to define the pathogenesis and identify mechanisms underlying PASC (Aim 2). Importantly, our study will contribute to the establishment of NHP models of SARS-CoV-2 infection and could prove essential for understanding long-term effects of SARS-CoV-2 pathogenesis. This model provides longitudinal assessment of disease findings, pathogenesis, immune responses, and viral persistence. Furthermore, collection of multiple tissues virtually impossible to obtain in humans, such as gut, brain, heart and lung, will allow us to identify pulmonary and extra-pulmonary long-term and/or permanent damage. Finally, specimens collected longitudinally and at necropsy will be cryo-banked and will be key in bridging our discoveries with data compiled in SARS-CoV-2 recovery human cohorts. These studies will allow us to dissect biological causes underlying PASC, thus providing key insights for preventing and treating the effects of long-term COVID- 19 disease. The proposed studies are within the scope of the parent award and are highly likely to foster additional research funding leading to the progress of the overall goals of the parent award. However, there is no overlap with work already funded in the parent award.