Background: Spatial repellents that create airborne concentrations of an active ingredient (AI) within a space offer a scalable solution to further reduce transmission of malaria, by disrupting mosquito behaviours in ways that ultimately lead to reduced human-vector contact. Passive emanator spatial repellents can protect multiple people within the treated space and can last for multiple weeks without the need for daily user touchpoints, making them less intrusive interventions. They may be particularly advantageous in certain use cases where implementation of core tools may be constrained, such as in humanitarian emergencies and among mobile at-risk populations. The purpose of this study was to assess the efficacy of Mosquito Shield™ deployed in experimental huts against wild, free-flying, pyrethroid-resistant Anopheles arabiensis mosquitoes in Tanzania over 1 month. Methods: The efficacy of Mosquito Shield™ transfluthrin spatial repellent in reducing mosquito lands and blood-feeding was evaluated using 24 huts: sixteen huts were allocated to Human Landing Catch (HLC) collections and eight huts to estimating blood-feeding. In both experiments, half of the huts received no intervention (control) while the remaining received the intervention randomly allocated to huts and remained fixed for the study duration. Outcomes measured were mosquito landings, blood-fed, resting and dead mosquitoes. Data were analysed by multilevel mixed effects regression with appropriate dispersion and link function accounting for volunteer, hut and day. Results: Landing inhibition was estimated to be 70% (57–78%) [IRR 0.30 (95% CI 0.22–0.43); p < 0.0001] and blood-feeding inhibition was estimated to be 69% (56–79%) [IRR 0.31 (95% CI 0.21–0.44; p < 0.0001] There was no difference in the protective efficacy estimates of landing and blood-feeding inhibition [IRR 0.98 (95% CI 0.53–1.82; p = 0.958]. Conclusions: This study demonstrated that Mosquito Shield™ was efficacious against a wild pyrethroid-resistant strain of An. arabiensis mosquitoes in Tanzania for up to 1 month and could be used as a complementary or stand-alone tool where gaps in protection offered by core malaria vector control tools exist. HLC is a suitable technique for estimating bite reductions conferred by spatial repellents especially where direct blood-feeding measurements are not practical or are ethically limited.

Pyriproxyfen (PPF) is an insect growth regulator used in the co-treatment of long-lasting insecticidal nets for its ability to sterilize female mosquitoes. To evaluate the efficacy of PPF-treated nets on mosquito reproductivity, most studies observe oviposition (egg-laying) rates in the laboratory. This technique has several technical disadvantages. Our study assessed if ovarial dissection could serve as an effective proxy for evaluating sterility in Anopheles gambiae mosquitoes. Blood-fed females were exposed to untreated or PPF-treated nets in cylinder assays and followed over several days to observe oviposition rates or egg development by dissection. For identifying PPF-exposed mosquitoes, both techniques demonstrated high sensitivity (oviposition: 99.1%; dissection: 100.0%), but for identifying non-exposed mosquitoes, specificity was significantly higher in the dissection group (52.5% vs. 18.9%). To assess whether dissection could be applied to nets treated with a pyrethroid or co-treated with a pyrethroid and PPF in tunnel tests, a blinded investigator performed dissections to predict the PPF exposure status across different treatment groups. The exposure status of dissected females was predicted with >90% accuracy. We report that dissection is a sensitive technique to assess sterility in female Anopheles gambiae mosquitoes and can be used as a predictor of PPF exposure.

Dengue virus (DENV) is the most widespread mosquito-borne virus worldwide, but no antiviral therapies are available yet. The pan-serotype DENV inhibitor JNJ-A07 has shown potent activity in a mouse model. It remains unknown whether an antiviral drug ingested by mosquitoes could inhibit virus replication and thus reduce transmission to other hosts. Here, we investigated the antiviral activity of JNJ-A07 when administered in the blood meal to Aedes aegypti mosquitoes. JNJ-A07 blocked DENV-2 transmission by the mosquitoes in both pre-exposure and post-exposure settings. In addition, JNJ-A07 remained in the mosquito bodies for 7 days after blood meal. Reductions of DENV systemic infection in the mosquitoes suggested a potential for decreased proportions of DENV outbreaks in a simulated environment when the mosquitoes ingested JNJ-A07 via the blood meal.

Background: West Nile virus (WNV) and Usutu virus (USUV) are emerging arboviruses in Europe transmitted by Culex mosquitoes. In Belgium, it is currently unknown which Culex species are competent vectors for WNV or USUV and if these mosquitoes carry Wolbachia , an endosymbiotic bacterium that can block arbovirus transmission. The aims of our study were to measure the vector competence of Belgian Culex mosquitoes to WNV and USUV and determine if a naturally acquired Wolbachia infection can influence virus transmission. Methodology/Principal Findings: We captured 876 non-engorged female Culex mosquitoes from urban and peri-urban sites in Leuven, Belgium. We provided females with an infectious bloodmeal containing WNV lineage 2, USUV European (EU) lineage 3, or USUV African (AF) lineage 3. Blood-fed females (n=154) were incubated for 14 days at 25°C after which the body, head, and saliva were collected to measure infection (IR), dissemination (DR), and transmission (TR) rates, respectively. Mosquito species were identified by qRT-PCR or Sanger sequencing, the presence of infectious virus in mosquitoes was confirmed by plaque assays, and viral genome copies were quantified by qRT-PCR. We found that Culex pipiens pipiens were able to transmit WNV (11% IR, 40% DR, 100% TR) but not USUV (EU lineage: 13% IR, 0% DR; AF lineage: 16% IR, 17% DR, 0% TR). In contrast, Culex modestus was able to transmit USUV (AF lineage: 60% IR, 67% DR, 50% TR), but not WNV (0% IR). We found that the presence or absence of Wolbachia was species-dependent and did not associate with virus transmission. Conclusions/Significance: This is the first report that Belgian Culex mosquitoes can transmit both WNV and USUV, forewarning the risk of human transmission. More research is needed to understand the potential influence of Wolbachia on arbovirus transmission in Culex modestus mosquitoes. Author Summary: West Nile virus and Usutu virus can cause seasonal epidemics in humans. They are maintained in a transmission cycle between wild birds and Culex mosquitoes, and humans that are bitten by infected mosquitoes can develop life-threatening neurological disease. Certain Culex species carry the symbiotic bacterium Wolbachia which can block virus transmission in mosquitoes. In Belgium, it is currently unknown which Culex species can transmit West Nile virus and/or Usutu virus, or if they carry Wolbachia bacteria. In our study, we captured wild mosquitoes from Belgium and infected them with West Nile virus or Usutu virus. We found that a common European species ( Culex pipiens pipiens , the Northern House mosquito) could transmit West Nile virus, whereas a lesser known species ( Culex modestus ) could transmit Usutu virus. Wolbachia bacteria could be found in almost all Culex pipiens pipiens , but not in Culex modestus , suggesting that Wolbachia prevalence is species-specific. More research is needed to understand if Wolbachia can block virus transmission in Culex modestus . This is the first report on the ability of Culex mosquitoes to transmit West Nile virus and Usutu virus in Belgium, forewarning the risk of transmission to humans.

The continuous re-emergence of yellow fever virus (YFV) poses a serious public health threat, with no approved specific antiviral drugs for treatment. Sofosbuvir and uprifosbuvir, developed for treatment of chronic hepatitis C, are nucleotide analogs with anti-YFV activity in vitro, and sofosbuvir has been used off-label in YFV-infected individuals. To study the barrier to resistance of these drugs, escape experiments were performed with the YFV 17D vaccine strain in human hepatoma Huh7.5 cells. Compared to the original virus, sofosbuvir and uprifosbuvir escape variants exhibited 8.6- to 10.7-fold increased 50% effective concentration (EC₅₀) values. Escape viruses developed putative resistance-associated-substitutions (RAS) M478K and A482G in the NS5 RNA-dependent RNA polymerase domain. Reverse-engineered YFV 17D mutants demonstrated that A482G was the primary determinant of drug resistance, resulting in 5.3- to 5.7-fold increased EC₅₀; however, the substitutions acted synergistically when combined, leading to 12.3- and 11.1-fold increased EC₅₀. A482G conferred slight cross-resistance to bemnifosbuvir, while no cross-resistance was observed for remdesivir, galidesivir, or NITD008. Escape and mutant viruses had slight delays in growth kinetics in Huh7.5 cells and were viable in C6/36 and Aag2-AF5 mosquito cells. Structural analysis of a predicted AlphaFold3 YFV NS5-RNA structure suggests that M478K and A482G induce conformational changes in RdRp motif F, which may reduce sofosbuvir and uprifosbuvir affinities. Sofosbuvir RAS described for other orthoflaviviruses did not substantially affect drug susceptibility in reverse-engineered YFV 17D. This study demonstrates that YFV can develop resistance to sofosbuvir and uprifosbuvir with only two NS5 substitutions that have slight impact on viral fitness.

Background: Mosquito saliva plays a key role in arbovirus transmission and pathogenesis. It was shown that saliva contains several molecules that are essential for blood feeding. Recently, bacteria were also reported to be present in the saliva of Aedes albopictus and Anopheles mosquitoes. Nevertheless, information on the bacterial communities in Aedes and Culex saliva is still scarce. Methods: This study isolated and identified culturable fungal and bacterial colonies from saliva harvested from Aedes aegypti (laboratory strain) and Culex pipiens (field-collected) mosquitoes. 16S metagenomic sequencing was performed to identify bacterial communities in saliva and mosquito organs. Furthermore, it was assessed how these microbial communities were affected upon blood feeding and upon oral treatment with antibiotics and an antifungal drug. Results: The fungal species Penicillium crustosum was identified in mosquito saliva. Culturable bacteria detected in mosquito saliva included Serratia marcescens , Serratia nematodiphila , Enterobacter spp., and Klebsiella spp., which were previously identified as mosquito or insect endosymbionts in the midgut or other organs. Analysis with 16S metagenomics showed that bacterial communities in saliva were more diverse than those in the midgut. Blood feeding did not affect the fungal or bacterial load in mosquito saliva. Oral treatment of adult mosquitoes with antibiotics or an antifungal drug resulted in a significant reduction of bacteria or fungi present in the mosquito saliva. Notably, co-incubation of the mosquito-borne Semliki Forest virus with saliva from antibiotic- or antifungal-treated mosquitoes triggered a decrease in viral infection in human skin fibroblasts compared with nontreated saliva. Conclusions: These findings indicate that bacteria and fungi can be present in mosquito saliva and provide a foundation for further exploration of the impact of salivary fungi and bacteria on both vector competence and arbovirus infection in the mammalian host. Graphical