Identification of vertebrate hosts of mosquito blood meals following the 2017–2018 yellow fever outbreak in São Paulo State

Background The 2016–2019 sylvatic yellow fever epidemic in Brazil spread into the peri-urban areas of São Paulo State, raising concerns about the potential resurgence of urban yellow fever transmission in the Americas. In Alberto Löfgren State Park (PEAL), Serra da Cantareira, one of the world’s largest urban forests, southern brown howler monkeys became locally extinct in early January 2018 following a short-lived outbreak. Methods To investigate mosquito feeding patterns during and after the yellow fever outbreak, we collected 5,001 female mosquitoes between December 2017 and March 2020, and screened 143 blood-engorged specimens using Nanopore sequencing. Results Vertebrate hosts were identified as the source of blood meals in 74.8% (107/143) of samples, revealing that mosquitoes fed predominantly on mammals (83.2%, 89/107), but also on birds (10.3%, 11/107), and on multiple hosts (6.5%, 7/107). Humans accounted for 76.6% (82/107) of identified blood meals. Other hosts included non-human primate, dog, opossum, mouse and passerine birds. Among Haemagogus leucocelaenus , the primary yellow fever virus (YFV) vector at PEAL, 94.6% (35/37) of blood meals were human-derived, while 5.4% (2/37) contained non-human primate blood. Additionally, two engorged Haemagogus capricornii (100%, 2/2), another YFV vector, fed on humans. Untargeted sequencing recovered a complete YFV genome from an Hg. leucocelaenus specimen with human blood, providing direct evidence of active viral circulation within PEAL. Conclusions Sylvatic YFV vectors captured in urban forest fragments of the Serra da Cantareira exhibited marked anthropophilic feeding behaviour. Our findings underscore the risk of mosquito-mediated spillover into densely populated São Paulo, emphasising the urgent need for sustained vaccination together with entomological and virological surveillance to track and respond to yellow fever outbreaks in peri-urban settings.