The coronavirus disease 2019 (COVID-19) pandemic caused by the betacoronavirus “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2) represents an unprecedented public health emergency. Most patients with COVID-19 clear the virus upon resolution of the acute infection but ongoing, persistent, SARS-CoV-2 replication has been documented in immunocompromised individuals. In these chronically infected patients, recovery of replication-competent virus over several weeks to months is linked to stepwise acquisition of mutations within and outside of spike. Our preliminary data show that such prolonged intra-host viral evolution plays a role in the emergence of new, antigenically distinct, SARS-CoV-2 variants. We propose to systematically elucidate the determinants of persistent SARS-CoV-2 infections using an integrated translational research approach combining real-world clinical metadata with bioinformatics, genomics and molecular virology. We will leverage an existing large surveillance dataset covering two and a half years of SARS-CoV-2 spread in New York City going back to the beginning of the pandemic in the spring of 2020 when the NY metropolitan area emerged as one of the early epicenters of the pandemic. Specific Aim 1 will dissect the clinical features and therapeutic interventions associated with persistent SARS-CoV-2 replication using existing longitudinal data from electronic medical records. These studies will be complemented by the analysis of the B and T cell populations of persistently infected patients. Specific Aim 2 will dissect the viral genotypes representative of intra-host evolution of prolonged periods with a special emphasis on co-circulating viral variants. Specific Aim 3 will examine the phenotypic properties of persistent SARS-CoV-2 variants with an emphasis on convergent evolution within and outside of the spike region (susceptibility to neutralization, fusogenicity, spike processing and interferon antagonism). Altogether, the proposed studies address a critical knowledge gap regarding the biological drivers and viral dynamics fueling the selection of SARS-CoV-2 viral variants during persistent SARS-CoV-2 infection. This knowledge will provide the scientific basis needed to treat and prevent such chronic infections thereby limiting the emergence and spread of increasingly neutralization-resistant yet transmissible SARS-CoV-2 variants.