PROJECT SUMMARY/ABSTRACT Cholera continues to be a major cause of global morbidity and mortality, with continuing spread into new areas and recurrences in areas thought to be free of cholera, driven by draught, flood, war, mass population movements, and the associated breakdown of public health infrastructure. Haiti was the site of a major cholera epidemic beginning in 2010, with over 820,000 reported cases and close to 10,000 deaths. No clinical cases from this initial epidemic were reported after February 2019, and in 2022 it was announced that cholera had been “eliminated” from Haiti. However, on September 25, 2022, cholera cases were again identified, with 64,618 suspect cholera cases and 60,171 hospitalizations subsequently reported. In collaboration with GHESKIO, Haiti’s leading medical research institute, we have sequenced Vibrio cholerae strains isolated from patients infected during the “new” 2022/2023 epidemic. Our genetic analysis indicates that the epidemic strain originated from an “older” Haitian V. cholerae serogroup Ogawa strain that had been circulating at sub-epidemic levels in the aquatic environment, rather than from more recent clinical serogroup Inaba isolates or from strains introduced from outside of the country. These findings raise critically important (and controversial) questions about the impact of strain and ecologic factors on persistence of environmental foci of V. cholerae; factors influencing spread of epidemic disease within the Haitian population; and best approaches to long-term cholera prevention, in Haiti and similar settings globally. Haiti provides a unique environment in which to address questions related to cholera: it is on an island, with an initial epidemic caused by introduction of a single well characterized strain in 2010. Since 2010 we have established a strong infrastructure in Haiti for cholera research with excellent local collaborators and have built extensive environmental and clinical databases. The current study focuses on understanding factors driving re-emergence of the disease after three years of quiescence, including use of causal Artificial Intelligence (AI) to see if it is possible to model/predict the occurrence of future epidemic waves. Specific Aim 1: Identify strain characteristics and ecologic factors that influence evolution, virulence, and survival of V. cholerae O1 in environmental reservoirs, through environmental studies and evolutionary- informed laboratory microcosm model systems. Specific Aim 2: Monitor levels of exposure/immunity to V. cholerae O1 in communities and case cohorts to determine rates of infection in the population and monitor waning of immune responses. Specific Aim 3: Assess phylogeographic and phylodynamic patterns driving evolution of V. cholerae from clinical and environmental sources. Specific Aim 4: Develop a causal AI approach to predict the occurrence of future epidemic waves and estimate effectiveness of interventions.