SummaryOropouche virus (OROV), initially detected in Trinidad and Tobago in 1955, has been historically confined to the Amazon Basin. However, since late 2022, OROV has been reported in northern Brazil as well as urban centers in Bolivia, Colombia, Cuba, and Peru. Herein, we describe the generation of 133 new publicly available full genomes. We show how the virus evolved via genome component reassortment and how it rapidly spread across multiple states in Brazil, causing the largest outbreak ever recorded outside the Amazon basin, including the first detected deaths. This work highlights the need for heightened epidemiological and genomic surveillance and the implementation of adequate measures in order to mitigate transmission and the impacts on the population.BackgroundOropouche virus was first identified in 1955 in Trinidad and Tobago and later found in Brazil in 1960. Historically, it has been reported to have caused around 30 outbreaks, mostly within the Amazon Basin, where it circulates among forest animals, but also in urban areas where it is known to be transmitted by the midgeCulicoides paraensis. Recently, Brazil has seen a surge in cases, with more than 7000 reported by mid-2024 alone.MethodsIn a collaboration with Central Public Health Laboratories across Brazilian regions, we integrated epidemiological metadata with genomic analyses of recently sampled cases. This initiative resulted in the generation of 133 whole genome sequences from the three genomic segments (L, M, and S) of the virus, including the first genomes obtained from regions outside the Amazon and from the first ever recorded fatal cases.FindingsAll of the 2024 genomes form a monophyletic group in the phylogenetic tree with sequences from the Amazon Basin sampled since 2022. Our analyses revealed a rapid north-to-south viral movement from the Amazon Basin into historically non-endemic regions. We identified 21 reassortment events, although it remains unclear if genomic evolution of the virus enabled the virus to adapt to local ecological conditions and evolve new phenotypes of public health importance.InterpretationBoth the recent rapid spatial expansion and the first reported fatalities associated with Oropouche (and other outcomes under investigation) underscore the importance of enhancing surveillance for this evolving pathogen across the Region. Without any obvious changes in the human population over the past 2 years, it is possible that viral adaptation, deforestation and recent climate change, either alone or in combination, have propelled Oropouche virus beyond the Amazon Basin.Research in contextEvidence before this studyBefore this study, Oropouche virus (OROV) was known to cause periodic outbreaks primarily within the Amazon Basin. Initially identified in Trinidad and Tobago in 1955, the virus had been responsible for approximately 30 outbreaks in Latin America, mostly confined to the Amazon region. The virus typically circulates among forest animals and is transmitted to humans by the bite of the midgeCulicoides paraensis. There has been an historical dearth of available genomic data, and so far the spread beyond the Amazon Basin has not been well-documented.Added value of this studyThis study provides a timely and comprehensive analysis of epidemiological and genomic data of Oropouche virus from regions outside the Amazon Basin. By generating 133 whole genome sequences from various regions across Brazil, this study reveals the movement of OROV over the past few years across Brazil, with a north-south spatial movement from regions of historic endemicity to regions with clear epidemic potential. We identify 21 reassortment events, with the possible occurrence of virus adaptation to new environments. We also report the first fatal cases of Oropouche virus infection in patients without underlying relevant comorbidities, underscoring the public health risk of future outbreaks and the need for increased awareness and surveillance.Implications of all the available evidenceThe rapid spread of Oropouche virus beyond the Amazon Basin into regions of Brazil more than 3500 Km distant, coupled with the identification of genome reassortment events, raises the possibility that the virus is adapting to the new environments of its increasing spatial landscape. This evolution could lead to the emergence of new viral phenotypes, with potential changes at various levels, from vector efficiency to disease outcome, raising the challenge of managing future outbreaks. This underscores the critical need for enhanced surveillance systems at national and continental levels, particularly in urban centers that appear to have been hit hard during the spatial expansion, to detect and respond to Oropouche virus outbreaks promptly.