28 29 30 Keywords: Yellow fever virus, Brazilian outbreak, lineages, amino acid changes, phylogeography. 31 32 33 34 35 Delatorre et al., 2019 -Distinct YFV lineages co-circulated in Brazil 2 Abstract 36The current outbreak of yellow fever virus (YFV) that is afflicting Brazil since the end of 2016 probably 37 originated from a re-introduction of YFV from endemic areas into the non-endemic Southeastern 38Brazil. However, the lack of genomic sequences from endemic regions hinders the tracking of YFV's 39 dissemination routes. We assessed the origin and spread of the ongoing YFV Brazilian outbreak 40 analyzing a new set of YFV strains infecting humans, non-human primates (NHP) and mosquitoes 41 sampled across five Brazilian states from endemic and non-endemic regions between 2015 and 2018. 42We found two YFV sub-clade 1E lineages circulating in NHP from Goiás state (GO), resulting from 43 independent viral introductions into the Araguaia tributary river basin: while the strain from 2017 44 clustered intermingled with Venezuelan YFV strains from 2000, the YFV strain sampled in 2015 45 clustered with sequences of the current YFV outbreak in the Brazilian Southeastern region (named 46 YFV2015-2018 lineage), displaying the same molecular signature associated to the current YFV outbreak. 47After its introduction in GO at around mid-2014, the YFV2015-2018 lineage followed two paths of 48 dissemination outside GO, originating two major YFV sub-lineages: 1) the YFVMG/ES/RJ sub-lineage 49 spread sequentially from the eastern area of Minas Gerais state to Espírito Santo and then to Rio de 50 Janeiro states, following the Southeast Atlantic basin; 2) the YFVMG/SP sub-lineage spread from the 51 southwestern area of Minas Gerais to the metropolitan region of São Paulo state, following the Paraná 52 basin. These results indicate the ongoing YFV outbreak in Southeastern Brazil originated from a 53 dissemination event from GO almost two years before its recognition at the end of 2016. From GO this 54 lineage was introduced in Minas Gerais state at least two times, originating two sub-lineages that 55 followed different routes towards densely populated areas. The spread of YFV outside endemic regions 56 for at least four years stresses the imperative importance of the continuous monitoring of YFV to aid 57 decision-making for effective control policies aiming the increase of vaccination coverage to avoid the 58 YFV transmission in densely populated urban centers. 59 60 Delatorre et al., 2019 -Distinct YFV lineages co-circulated in Brazil 3