Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 – 2018

São Paulo (SP), a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in SP, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in SP, we generated and analysed virus genomic data and epizootic case data from NHP in SP. We report the occurrence of three spatiotemporally distinct phases of the outbreak in SP prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in SP, mostly sampled from non-human primates between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo state at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of SP state. Our results shed light on the sylvatic transmission of yellow fever in highly fragmented forested regions in SP state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species. Author’s Summary Since July 2016, the southeast region of Brazil has experienced the largest yellow fever virus (YFV) outbreak in decades. São Paulo is the most densely populated state in southeast Brazil. The outbreak has caused serious public health concern in the state, as YFV does not normally circulate widely there and most of the 21 million inhabitants were correspondingly unvaccinated against YFV when the outbreak began. In Brazil, YFV typically circulates among non-human primates, and human cases represent isolated spillover events from this predominantly sylvatic cycle. Understanding the epidemiological dynamics and spread of YFV in non-human primates is therefore critical for contextualising human cases, and guiding vaccination strategies that can better protect local human populations. Here, we aim to contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in SP. We analyse the geographic and temporal distribution of observed cases of YFV in non-human primates in São Paulo state, and identify three distinct phases of the epizootic. We generate sequence data from 51 YFV-positive cases and perform phylogenetic and phylogeographic analyses aimed at understanding the spatial spread of YFV in São Paulo state. Analyses of these data indicate that YFV spread from the north of São Paulo state into more densely populated southern regions. Although we observe substantial heterogeneity in the rate at which different sampled YFV lineages spread, the typical rate of spread was low with a mean rate of ~1 km per day. This is consistent with a scenario in which the majority of transmission events occurred between non-human primates and sylvatic vectors across forested patches. Article Summary Line Genomic surveillance of yellow fever in São Paulo during the 2016-2018 epizootic