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Studies on animal virome have mainly concentrated on chordates and medically significant invertebrates, often overlooking sylvatic mosquitoes, constituting a major part of mosquito species diversity. Despite their potential role in arbovirus transmission, the viromes of sylvatic mosquitoes remain largely unexplored. These mosquitoes may also harbor insect-specific viruses (ISVs), affecting arboviral transmission dynamics. The Cerrado biome, known for rapid deforestation and its status as a biodiversity hotspot, offers an ideal setting for investigating mosquito viromes due to potential zoonotic spillover risks from land use changes. This study aimed to characterize the viromes of sylvatic mosquitoes collected from various locations within Minas Gerais state, Brazil. The total RNA was extracted from mosquito pools of Psorophora albipes, Sabethes albiprivus, Sa. chloropterus, Psorophora ferox, and Coquillettidia venezuelensis species, followed by high-throughput sequencing (HTS). Bioinformatic analysis included quality control, contig assembly, and viral detection. Sequencing data analysis revealed 11 near-complete viral genomes (new viruses are indicated with asterisks) across seven viral families and one unassigned genus. These included: Xinmoviridae (Ferox mosquito mononega-like virus* and Albipes mosquito Gordis-like virus*), Phasmaviridae (Sabethes albiprivus phasmavirus*), Lispiviridae (Pedras lispivirus variant MG), Iflaviridae (Sabethes albiprivus iflavivirus*), Virgaviridae (Buriti virga-like virus variant MG and Sabethes albiprivus virgavirus 1*), Flaviviridae (Psorophora ferox flavivirus*), Mesoniviridae (Alphamesonivirus cavallyense variant MG), and the genus Negevirus (Biggie virus variant MG virus and Coquillettidia venezuelensis negevirus*). Moreover, the presence of ISVs and potential novel arboviruses underscores the need for ongoing surveillance and control strategies to mitigate the risk of emerging infectious diseases.

Haemagogus leucocelaenus, which is considered a major vector of wild yellow fever, exhibits acrodendrophilic habits and mainly deposits its eggs in treeholes and bamboo internodes. The selection of nursery sites is essential in the life history and reproductive success of mosquitoes. The present work investigated the preferred oviposition height and period of Hg. leucocelaenus in an Atlantic forest area in Rio de Janeiro. Sampling was performed using oviposition traps that were placed on plant material at 0, 2, 4, 6, and 8 m above the ground, from August 2015 to July 2016. Eggs were more abundant during October and May, and the height of traps placement had no significant effect on the eggs number indicating that Hg. leucocelaenus explores different levels of forest habitats, a behavior that may favor the transmission of pathogens among arboreal animals including primates and humans. The findings of the present study are discussed from an ecological and epidemiological point of view.

Background Yellow fever virus (YFV) is an arbovirus that, despite the existence of a safe and effective vaccine, continues to cause outbreaks of varying dimensions in the Americas and Africa. Between 2017 and 2019, Brazil registered un unprecedented sylvatic YFV outbreak whose severity was the result of its spread into zones of the Atlantic Forest with no signals of viral circulation for nearly 80 years. Methods To investigate the influence of climatic, environmental, and ecological factors governing the dispersion and force of infection of YFV in a naïve area such as the landscape mosaic of Rio de Janeiro (RJ), we combined the analyses of a large set of data including entomological sampling performed before and during the 2017–2019 outbreak, with the geolocation of human and nonhuman primates (NHP) and mosquito infections. Results A greater abundance of Haemagogus mosquitoes combined with lower richness and diversity of mosquito fauna increased the probability of finding a YFV-infected mosquito. Furthermore, the analysis of functional traits showed that certain functional groups, composed mainly of Aedini mosquitoes which includes Aedes and Haemagogus mosquitoes, are also more representative in areas where infected mosquitoes were found. Human and NHP infections were more common in two types of landscapes: large and continuous forest, capable of harboring many YFV hosts, and patches of small forest fragments, where environmental imbalance can lead to a greater density of the primary vectors and high human exposure. In both, we show that most human infections (~ 62%) occurred within an 11-km radius of the finding of an infected NHP, which is in line with the flight range of the primary vectors. Conclusions Together, our data suggest that entomological data and landscape composition analyses may help to predict areas permissive to yellow fever outbreaks, allowing protective measures to be taken to avoid human cases. Graphical Abstract

Background Yellow fever is a major public health concern in Brazil, transmitted in sylvatic cycles by Haemagogus and Sabethes mosquitoes. Among them, Haemagogus janthinomys and Hg. capricornii occur in sympatry in the Atlantic Forest and females are morphologically indistinguishable, complicating vector identification during outbreaks. Here, we aimed to investigate their taxonomic status and evolutionary history using an integrative approach including morphological and phylogenetic analyses. Methods Mosquitoes were collected in 17 municipalities across nine Brazilian states, including simultaneous captures of both species in sympatric areas. Males were identified by genitalia morphology and molecular analyses were performed using three mitochondrial and two nuclear genes. Diversity analyses and neutrality tests were performed, and phylogenies were reconstructed with Maximum Likelihood and Bayesian inferences. Divergence times were estimated using strict molecular clock, and population history was assessed through mismatch distribution analysis and Bayesian Skyline Plots. Results A total of 79 specimens were morphologically identified, with Hg. janthinomys showing a broader geographic and altitudinal distribution than Hg. capricornii , which was usually restricted to higher elevations. Phylogenetic analyses based on mitochondrial markers revealed two clades, but did not recover clear reciprocal monophyly, thus evidencing that these markers alone cannot separate the two species. The inclusion of nuclear markers evidenced introgression events of Hg. janthinomys mitochondria in Hg. capricornii specimens in the Paraíba River Valley and Espírito Santo State, and successive breeding of Hg. capricornii on few samples morphologically identified as Hg. janthinomys in São Paulo State. Molecular clock and population history analyses evidenced that these species have probably speciated in peripatry or parapatry during the Pleistocene era at approximately 1.2 million years ago, and a recent sudden expansion of Hg. capricornii in the last 10 thousand years ago has tripled its population and likely led to a secondary contact between the two species. Conclusions Haemagogus janthinomys and Hg. capricornii are valid and closely related species with evolutionary histories shaped by divergence during the Pleistocene era and subsequent introgression events. The use of cytochrome c oxidase subunit I gene (COI) DNA barcoding alone could not reliably distinguish them, and integrating morphology with multiple molecular markers is essential for accurate identification. Future work is needed for a finer resolution of hybridization patterns to help clarify if the observed mito-nuclear discordance reflects historical introgression or active genetic exchange between species. Graphical abstract

In Brazil, a yellow fever (YF) outbreak was reported in areas considered YF-free for decades. The low vaccination coverage and the increasing forest fragmentation, with the wide distribution of vector mosquitoes, have been related to yellow fever virus (YFV) transmission beyond endemic areas since 2016. Aiming to elucidate the molecular and phylogenetic aspects of YFV spread on a local scale, we generated 43 new YFV genomes sampled from humans, non-human primates (NHP), and primarily, mosquitoes from highly heterogenic areas in 15 localities from Rio de Janeiro (RJ) state during the YFV 2016–2019 outbreak in southeast Brazil. Our analysis revealed that the genetic diversity and spatial distribution of the sylvatic transmission of YFV in RJ originated from at least two introductions and followed two chains of dissemination, here named the YFV RJ-I and YFV RJ-II clades. They moved with similar dispersal speeds from the north to the south of the RJ state in parallel directions, separated by the Serra do Mar Mountain chain, with YFV RJ-I invading the north coast of São Paulo state. The YFV RJ-I clade showed a more significant heterogeneity across the entire polyprotein. The YFV RJ-II clade, with only two amino acid polymorphisms, mapped at NS1 (I1086V), present only in mosquitoes at the same locality and NS4A (I2176V), shared by all YFV clade RJ-II, suggests a recent clustering of YFV isolates collected from different hosts. Our analyses strengthen the role of surveillance, genomic analyses of YVF isolated from other hosts, and environmental studies into the strategies to forecast, control, and prevent yellow fever outbreaks.

Yellow fever (YF) disease is a viral infection caused by Orthoflavivirus flavi (YFV). YFV is transmitted by hematophagous daytime-biting mosquitoes, predominantly Haemagogus spp. and Sabethes spp. in the sylvatic cycles, and Aedes spp. in urban cycles. In this work, we correlated vaccination coverage with the occurrence and spread of the disease throughout Brazil during the years 2016–2018. The Vale do Mucuri and Vale do Rio Doce regions in Minas Gerais state had the highest number of reported cases. Despite being considered areas with vaccine recommendation since 2008, these regions had less than 60% and 70% vaccination coverage in 2016. The outbreak of YF in Brazil has shown that surveillance for emerging diseases should be constant, especially in relation to the national immunization program. In this study, we observed that low vaccination coverage and the lack of public policies aimed at this region with low population development may have an impact on the reemergence of YF.

The Chikungunya virus (CHIKV) presents global health challenges, with Brazil experiencing outbreaks since its introduction in 2014. In 2023, following a CHIKV outbreak in Minas Gerais (MG), social media was used to optimize an entomological survey aimed at identifying vectors and viral lineages and assessing insecticide resistance. Following Instagram posts, residents with suspected CHIKV infection were able to schedule mosquito aspirations. In total, 421 mosquitoes (165 Aedes aegypti and 256 Culex quinquefasciatus) were captured from 40 households in Salinas city (MG) and tested for the Dengue, Zika, and Chikungunya viruses through RT-qPCR. Twelve of 57 pools (10 Ae. aegypti and two Cx. quinquefasciatus) tested positive for CHIKV RNA. Viral RNA was also detected in the heads of nine Ae. aegypti, indicating viral dissemination but not in Cx. quinquefasciatus. Genome sequencing yielded the first near-complete genome from the 2023 outbreak, unveiling that the CHIKV strain belonged to the East/Central/South African (ECSA) genotype. Additionally, genetic analyses revealed high frequencies of kdr alleles, including in CHIKV-infected mosquitoes, suggesting resistance to pyrethroid insecticides in this Ae. aegypti population. Social media was important for guiding mosquito-capture efforts in CHIKV transmission hotspots, thus optimizing the opportunity for viral detection. These findings emphasize the urgent need for innovative vector studies and control strategies, as well as interdisciplinary approaches in public health interventions.

Background Zoonotic infections with epidemic potential, as non-human primate malaria and yellow fever (YF), can overlap geographically. Optimizing a small blood sample for diagnosis and surveillance is of great importance. Blood are routinely collected for YF diagnosis and blood clots usually discarded after serum obtention. Aiming to take sample advantage, the sensitivity of a PCR using extracted DNA from long-term frozen clots from human and non-human primates for detection of Plasmodium spp. in low parasitaemia conditions was assayed. Results Malaria diagnosis with DNA extracted from blood clots generated results in agreement with samples obtained with whole blood, including mixed Plasmodium vivax/simium and Plasmodium malariae/brasilianum infections. Conclusion Blood clots from human and non-human primates may be an important and low cost source of DNA for malaria surveillance in the Atlantic Forest.

Despite worldwide efforts to understand the transmission dynamics of Zika virus (ZIKV), scanty evaluation has been made on the vector competence of Aedes aegypti fed directly on viremic human and non-human primates (NHPs). We blood-fed Ae. aegypti from two districts in Rio de Janeiro on six ZIKV infected pregnant rhesus macaques at several time points, half of which were treated with Sofosbuvir (SOF). Mosquitoes were analyzed for vector competence after 3, 7 and 14 days of incubation. Although viremia extended up to eight days post monkey inoculation, only mosquitoes fed on the day of the peak of viremia, recorded on day two, became infected. The influence of SOF treatment could not be assessed because the drug was administered just after mosquito feeding on day two. The global infection, dissemination and transmission rates were quite low (4.09%, 1.91% and 0.54%, respectively); no mosquito was infected when viremia was below 1.26 × 105 RNA copies/mL. In conclusion, Ae. aegypti vector competence for ZIKV from macaques is low, likely to be due to low viral load and the short duration of ZIKV viremia in primates suitable for infecting susceptible mosquitoes. If ZIKV infection in human and macaques behaves similarly, transmission of the Zika virus in nature is most strongly affected by vector density.

Malaria remains a pressing health challenge in Brazil, primarily due to Plasmodium vivax . Most cases occur in the Amazon; however, outbreaks in the Atlantic Forest involving P. simium , a non-human primate parasite closely related to P. vivax , have posed challenges for control efforts. This study aimed to differentiate P. vivax infections from those of P. simium acquired within the Atlantic Forest. Ninety-nine samples initially identified as P. vivax -positive, comprising 95 from humans and four from non-human primates, were analyzed. The results using a refined molecular tool revealed that 93% of human samples from the Atlantic Forest were P. simium , corroborating the substantial rate of zoonotic transmission in this region. The remaining samples from the Amazon and nearby countries were confirmed as P. vivax , including five Atlantic Forest cases linked to travel to endemic areas, suggesting imported or relapsed cases. The study emphasizes the importance of molecular tools in accurately distinguishing malaria parasite species, especially in identifying the origins of the parasites infecting humans. This understanding is crucial for grasping malaria transmission dynamics, ensuring accurate epidemiological surveillance, and preventing zoonotic transmission. It also supports targeted control strategies and effective public health interventions.