São Paulo, 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 São Paulo, 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 São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs 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 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 São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species. 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 (NHPs), and human cases represent isolated spill over events from this predominantly sylvatic cycle. Understanding the epidemiological dynamics and spread of YFV in NHPs 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 São Paulo. We analyse the geographic and temporal distribution of observed cases of YFV in NHPs in São Paulo state, and identify three distinct phases of the epizootic. We generate virus genome sequences 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 NHPs primates and sylvatic vectors across forested patches.

Background: Yellow fever (YF) is an arboviral disease which is endemic to Brazil due to a sylvatic transmission cycle maintained by infected mosquito vectors, non-human primate (NHP) hosts, and humans. Despite the existence of an effective vaccine, recent sporadic YF epidemics have underscored concerns about sylvatic vector surveillance, as very little is known about their spatial distribution. Here, we model and map the environmental suitability of YF’s main vectors in Brazil, Haemagogus spp. and Sabethes spp., and use human population and NHP data to identify locations prone to transmission and spillover risk. Methodology/Principal findings: We compiled a comprehensive set of occurrence records on Hg. janthinomys, Hg. leucocelaenus, and Sabethes spp. from 1991–2019 using primary and secondary data sources. Linking these data with selected environmental and land-cover variables, we adopted a stacked regression ensemble modelling approach (elastic-net regularized GLM, extreme gradient boosted regression trees, and random forest) to predict the environmental suitability of these species across Brazil at a 1 km x 1 km resolution. We show that while suitability for each species varies spatially, high suitability for all species was predicted in the Southeastern region where recent outbreaks have occurred. By integrating data on NHP host reservoirs and human populations, our risk maps further highlight municipalities within the region that are prone to transmission and spillover. Conclusions/Significance: Our maps of sylvatic vector suitability can help elucidate potential locations of sylvatic reservoirs and be used as a tool to help mitigate risk of future YF outbreaks and assist in vector surveillance. Furthermore, at-risk regions identified from our work could help disease control and elucidate gaps in vaccination coverage and NHP host surveillance.

The risk to human health from mosquito-borne viruses such as dengue, chikungunya and yellow fever is increasing due to increased human expansion, deforestation and climate change. To anticipate and predict the spread and transmission of mosquito-borne viruses, a better understanding of the transmission cycle in mosquito populations is needed. We present a pathogen-agnostic combined sequencing protocol for identifying vectors, viral pathogens and their hosts or reservoirs using portable Oxford Nanopore sequencing. Using mosquitoes collected in São Paulo, Brazil, we extracted RNA for virus identification and DNA for blood meal and mosquito identification. Mosquitoes and blood meals were identified by comparing cytochrome c oxidase I (COI) sequences against a curated Barcode of Life Data System (BOLD). Viruses were identified using the SMART-9N protocol, which allows amplified DNA to be prepared with native barcoding for nanopore sequencing. Kraken 2 was employed to detect viral pathogens and Minimap2 and BOLD identified the contents of the blood meal. Due to the high similarity of some species, mosquito identification was conducted using blast after generation of consensus COI sequences using RACON polishing. This protocol can simultaneously uncover viral diversity, mosquito species and mosquito feeding habits. It also has the potential to increase understanding of mosquito genetic diversity and transmission dynamics of zoonotic mosquito-borne viruses.

Metagenomic studies of mosquito viromes demonstrated a more diverse composition than just an exclusive composition of pathogenic arboviruses transmitted to humans. In our study, the virome of 866 female mosquitoes collected throughout 2020 at the São Paulo Zoo, located in the city of São Paulo/SP—Brazil, was obtained. Specifically, in this paper, we describe a new virus found by viral RNA extraction and next-generation MiSeq sequencing of a group of 23 specimens of Anopheles (Nys.) strodei. The complete genome with a length of 9709 nucleotides was characterized by a positive orientation and a single strand, with a single large ORF, which encodes a polyprotein of 2987 amino acids. The phylogenetic analysis showed an association with the viral family Iflaviridae and the Riboviria realm. We carried out comparisons with translated sequences of the capsid regions of other iflavirus, and the identities in relation to our sequence were below the minimum limit of 90%, indicating that possibly it is a new species of iflavirus. Our findings contribute to expanding knowledge of virome composition among mosquito species in Brazil and globally. Moreover, we provide a viral genome reference specific to this geographic region and Culicidae family of mosquitoes. This resource facilitates future in silico recognition and assembly of viral genomes within metagenomic datasets.

Hand-held insect nets are the standard method for capturing vector mosquitoes of sylvatic arboviruses; however, occupational risks and biases due to individual skill and attractiveness are important limitations. The use of chemical attractants and automatic traps could be an alternative to resolve these limitations. This study compares the yields achieved using nets with those employing electrical traps with CO₂ and BG-Lure^®, near the ground and in the canopy strata (6.0 and 8.0 m high). The study was conducted at the Cantareira State Park, which is in the Brazilian Atlantic Forest biome. In the 18 collections performed, 3570 specimens of 52 taxa were obtained. The most frequent species captured near the ground were Wyeomyia confusa and Limatus durhamii, whereas Sabethes albiprivus, Sabethes purpureus, and Haemagogus leucocelaenus were the most frequent in the canopy. The nets resulted in greater species richness and abundance, followed by the trap employing CO₂. The combination of CO₂ traps with BG-Lure^® did not improve performance. The use of BG-Lure^® alone resulted in low abundance and a low number of species. Our results demonstrate that the use of traps with CO₂ can be complementary to collections with nets; however, for species of epidemiological interest such as those of the genera Haemagogus and Sabethes, especially in the canopy, the net remains the method of choice.