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We detected yellow fever virus by using quantitative PCR in Aedes albopictus mosquitoes and isolated the virus in C6/36 cells in 4 of 18 pools, including 118 specimens collected in an urban green area in São Paulo State, Brazil. Additional monitoring to detect shifts in transmission of this species is warranted.

The hand-net is the standard method for capturing mosquitoes with sylvatic diurnal activity in disease outbreaks in Brazil. However, occupational risks and biases related to the collectors' abilities and attractiveness are important limitations. In this study, we compared hand-nets with automatic traps (CDC) associated to CO2 and BG-Lure in the Vassununga State Park, a Brazilian Savanna protection area. The collections carried out over 27 days on the ground and the forest canopy. A total of 1555 mosquitoes were obtained in 20 taxa. The diversity index ranged between 1.12 and 1.79 and the dominance index from 0.22 to 0.40. The dominant species on the ground was 'Aedes scapularis' (46.0%), and in the canopy, Hg. 'janthinomys/capricornii' (31.9%). 'Haemagogus leucocelaenus' was rare ('n' = 2). The hand-net resulted in the greatest diversity and abundance of species in both strata, followed by the traps associated with CO2. A low degree of similarity was observed between the hand-net on the ground compared to the other capture methods. The use of BG-Lure alone resulted in a low number of specimens. In conclusion, the hand-net is still the method of choice for collecting arbovirus vectors in the diurnal period, especially yellow fever vectors.

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.

Yellow Fever (YF) is a severe disease caused by Yellow Fever Virus (YFV), endemic in some parts of Africa and America. In Brazil, YFV is maintained by a sylvatic transmission cycle involving non-human primates (NHP) and forest canopy-dwelling mosquitoes, mainly Haemagogus -spp and Sabethes- spp. Beginning in 2016, Brazil faced one of the largest Yellow Fever (YF) outbreaks in recent decades, mainly in the southeastern region. In São Paulo city, YFV was detected in October 2017 in Aloutta monkeys in an Atlantic Forest area. From 542 NHP, a total of 162 NHP were YFV positive by RT-qPCR and/or immunohistochemistry, being 22 Callithrix- spp. most from urban areas. Entomological collections executed did not detect the presence of strictly sylvatic mosquitoes. Three mosquito pools were positive for YFV, 2 Haemagogus leucocelaenus , and 1 Aedes scapularis . In summary, YFV in the São Paulo urban area was detected mainly in resident marmosets, and synanthropic mosquitoes were likely involved in viral transmission.

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. 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. 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.

Mosquito-borne diseases have a significant public health threat worldwide, with arboviruses accounting for a high proportion of infectious diseases and mortality annually. Brazil, in particular, has been suffering outbreaks of diseases transmitted by mosquito viruses, notably those of the genus, such as dengue, Zika, and chikungunya. Against this background, the São Paulo Zoo is an intriguing ecological niche to explore the virome of mosquitoes, potentially shedding light on the dynamics of arbovirus transmission within a confined setting. In this study, we conducted a comprehensive metagenomic analysis of mosquitoes collected from diverse habitats within the zoo, focusing on the , , and genera. From 1,039 contigs of viral origin, we identified 229 viral species infecting mosquitoes, with the orders , and being the most prevalent and abundant. The difference in virome composition was primarily driven by mosquito host species rather than specific collection sites or trap height. Despite environmental disparities, the virome remained remarkably uniform across different areas of the zoo, emphasizing the strong association between mosquito species and their viral communities. Furthermore, we identified a core virome shared among mosquito species, highlighting potential cross-species transmission events and underscoring the need for targeted surveillance and control measures. These results contribute to our understanding of the interplay between mosquitoes, the environment, and viruses, providing valuable insights for disease intervention strategies in mosquito-borne diseases.

Yellow fever virus (YFV) is an endemic arbovirus in parts of Africa and the Americas. In Brazil, following the eradication of the urban transmission cycle, YFV is maintained in a sylvatic cycle involving several species of neotropical primates and mosquitoes of the genera Haemagogus and Sabethes, which serve as primary and secondary vectors, respectively. During the 2016–2019 outbreak in São Paulo State, a total of 3731 mosquito pools were collected from sites with ongoing epizootic events in 192 municipalities. The RT-qPCR analysis detected YFV in 46 pools (1.4%) across nine mosquito species, including both primary and secondary vectors, as well as species from the genera Aedes and Psorophora. Differences in viral loads were observed among species. While Aedes aegypti was not found to be positive, the detection of natural YFV infection in other Aedes species raises concerns about potential virus reurbanization. Further studies are needed to clarify the role of additional mosquito species in YFV transmission in Brazil.

Brazil constitutes a hotspot for arbovirus emergence, posing persistent public health challenges. In São José do Rio Preto (SJdRP), dengue has historically predominated, whereas only 62 chikungunya virus (CHIKV) cases were reported between 2015 and 2023, suggesting cryptic circulation. However, from 2024 onwards, CHIKV notifications began to increase across São Paulo State. Because arboviral febrile illnesses frequently overlap clinically, early detection necessitates robust surveillance systems. To address this, mosquito collections were conducted over 15 months in SJdRP and screened for CHIKV RNA. Positive specimens were further analyzed through virus isolation and whole-genome sequencing. Overall, 5.6% of specimens tested positive, with prevalence rising from December 2023 and peaking in May 2024, coinciding with human cases. Phylogenetic reconstruction indicated multiple introductions, primarily from southeastern Brazil, driving viral establishment. These events likely facilitated transmission within a dense, susceptible vector population, contributing to extensive spread and culminating in a major CHIKV outbreak in northwestern São Paulo.

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.

The identification of mosquito species is necessary for determining the entomological components of disease transmission. However, identification can be difficult in species that are morphologically similar. The cytochrome c oxidase subunit I (COI) DNA barcode region is considered a valuable and reliable diagnostic tool for mosquito species recognition, including those that belong to species complexes. Mansonia mosquitoes are found in forests near swampy areas. They are nocturnal and are highly attracted to light. Hematophagous adult females exhibit aggressive biting behavior and can become infected with and transmit pathogens during their feeding, including some epizootic viruses and avian malaria. In Brazil, twelve Mansonia species have been reported. In a recent study from the São Paulo Zoo in Brazil, three morphologically distinct species were collected and identified, namely: Mansonia (Mansonia) indubitans, Ma. (Man.) pseudotitillans and Ma. (Man.) titillans. However, confirmation of these species by molecular identification was unsuccessful due to a lack of COI sequences in the GenBank database. Thus, this research aimed to describe the COI DNA barcode sequences of some morphologically characterized Mansonia (Man.) species from Brazil and to determine their utility in delimiting species collected from the Atlantic Forest and Brazilian Savanna. Accordingly, we provide tools for the genetic identification of species that play a significant role in pathogen transmission in wildlife and potentially humans. We show that the delimitation of Mansonia species via five different approaches based on COI DNA sequences (BI, NJ, ASAP, bPTP and GMYC) yield basically the same groups identified by traditional taxonomy, and we provide the identification of specimens that were previously identified only up to the subgenus level. We also provide COI sequences from two Mansonia species that were not previously available in sequence databases, Ma. wilsoni and Ma. pseudotitillans, and thus contribute to the ongoing global effort to standardize DNA barcoding as a molecular means of species identification.