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Over 400 million people are estimated to be at risk of acquiring dengue virus (DENV). Despite efforts to mitigate the impact of DENV epidemics, the virus remains a public health problem in the Americas: more than one million DENV cases were reported in the continent between January and July 2019 DENV was first detected in Brazil in 1982, and Brazil has reported 88% (1,127,244 cases) of all DENV cases in the Americas during 2019 to date. São Paulo state in the southeast of Brazil has reported nearly half of all DENV infections in the country. Here we characterised the genetic diversity of DENV strains circulating in São Paulo state in 2019, at the epicentre of the ongoing DENV epidemic. Using portable nanopore sequencing we generated 20 new DENV genome sequences from viremic patients with suspected dengue infection residing in two of the most-affected municipalities, Araraquara and São José do Rio Preto. We conducted a comprehensive phylogenetic analysis with 1,630 global DENV strains to better understand the evolutionary history of the DENV lineages that currently circulate in the region. The new outbreak strains were classified as DENV2 genotype III (American/Asian genotype). Notably, phylogenetic analysis indicated that the 2019 outbreak is the result of a novel DENV lineage that was recently introduced to Brazil from the Caribbean region. Our genetic analysis further indicates that the introduction and onwards spread of the outbreak lineage (named here DENV2-III lineage 4) indicates a new DENV2 lineage replacement in Brazil. Molecular clock analyses suggest that DENV2-III lineage 4 was introduced to Brazil in or around early 2014. Our study describes the early detection of a newly introduced and rapidly-expanding DENV2 virus lineage in Brazil.

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