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Aedes aegypti and A. albopictus mosquitoes are the main vectors for dengue virus (DENV) and other arboviruses, including Zika virus (ZIKV). Understanding the factors that affect transmission of arboviruses from mosquitoes to humans is a priority because it could inform public health and targeted interventions. Reasoning that interactions among viruses in the vector insect might affect transmission, we analysed the viromes of 815 urban Aedes mosquitoes collected from 12 countries worldwide. Two mosquito-specific viruses, Phasi Charoen-like virus (PCLV) and Humaita Tubiacanga virus (HTV), were the most abundant in A. aegypti worldwide. Spatiotemporal analyses of virus circulation in an endemic urban area revealed a 200% increase in chances of having DENV in wild A. aegypti mosquitoes when both HTV and PCLV were present. Using a mouse model in the laboratory, we showed that the presence of HTV and PCLV increased the ability of mosquitoes to transmit DENV and ZIKV to a vertebrate host. By transcriptomic analysis, we found that in DENV-infected mosquitoes, HTV and PCLV block the downregulation of histone H4, which we identify as an important proviral host factor in vivo.

Collecting blood-fed mosquitoes to monitor pathogen presence or to gather information on the host blood meal is often challenging. Fermenting molasses can be used to produce carbon dioxide to attract host-seeking mosquitoes, however, earlier work indicated that it may also attract blood-fed mosquitoes in the field. In the current study, these field results were validated in an experimental setting using a large cage setup with Anopheles coluzzii (Diptera, Culicidae). Blood-fed mosquitoes were indeed attracted to fermenting molasses with the highest attraction at 72 hours post feeding, which was used for subsequent experiments. Next, it was tested if fermentation of molasses is required for attraction, and whether it acts as an oviposition attractant, increases egg laying, or increases mosquito survival. The compounds that could be responsible for attraction were identified by combined electrophysiology and chemical analyses and formulated into a synthetic blend. Fermenting molasses attracted blood-fed mosquitoes in the large cage study, while fermenting sugar and non-fermenting molasses did not. The fecundity of blood-fed mosquitoes increased after feeding on fermenting molasses, however, compounds emanating from molasses did not trigger oviposition. The synthetic blend attracted blood-fed mosquitoes and may be used to determine mosquito host selection and for xenomonitoring, as ‘flying syringes’ to detect non-vector borne pathogens.

Usutu virus (USUV) is a mosquito-borne flavivirus of African origin. Over the past decades, USUV has spread through Europe causing mass die-offs among multiple bird species. The natural transmission cycle of USUV involves Culex spp. mosquitoes as vectors and birds as amplifying hosts. Next to birds and mosquitoes, USUV has also been isolated from multiple mammalian species, including humans, which are considered dead-end hosts. USUV isolates are phylogenetically classified into an African and European branch, subdivided into eight genetic lineages (Africa 1, 2, and 3 and Europe 1, 2, 3, 4, and 5 lineages). Currently, multiple African and European lineages are co-circulating in Europe. Despite increased knowledge of the epidemiology and pathogenicity of the different lineages, the effects of co-infection and transmission efficacy of the co-circulating USUV strains remain unclear. In this study, we report a comparative study between two USUV isolates as follows: a Dutch isolate (USUV-NL, Africa lineage 3) and an Italian isolate (USUV-IT, Europe lineage 2). Upon co-infection, USUV-NL was consistently outcompeted by USUV-IT in mosquito, mammalian, and avian cell lines. In mosquito cells, the fitness advantage of USUV-IT was most prominently observed in comparison to the mammalian or avian cell lines. When Culex pipiens mosquitoes were orally infected with the different isolates, no overall differences in vector competence for USUV-IT and USUV-NL were observed. However, during the in vivo co-infection assay, it was observed that USUV-NL infectivity and transmission were negatively affected by USUV-IT but not vice versa.

Background Tick-borne encephalitis virus (TBEV) can cause severe neurological disease in humans. Its geographical distribution is expanding in Western Europe with unresolved causes and spatial patterns, necessitating enhanced surveillance. Monitoring the virus in the environment is complicated, as it usually relies on destructive sampling of small rodents to test organs for TBEV, which in addition to ethical considerations also raises issues for long-term monitoring or longitudinal studies. Moreover, even when the virus is not detected in the blood or organs of the rodent, TBEV can still be transmitted from an infected tick to uninfected ticks feeding nearby. This is due to the ability of TBEV to replicate and migrate locally within the epidermis of small mammals, including those that do not appear to have systemic infection. This suggests that the virus may be detectable in skin biopsies, which has been confirmed in experimentally infected laboratory rodents, but it remains unknown if this sample type may be a viable alternative to destructively obtained samples in the monitoring of natural TBEV infection. Here we test ear tissue and dried blood spot (DBS) samples from rodents to determine whether TBEV-RNA can be detected in biological samples obtained non-destructively. Methods Rodents were live-trapped and sampled at three woodland areas in The Netherlands where presence of TBEV has previously been recorded. Ear tissue (n = 79) and DBSs (n = 112) were collected from a total of 117 individuals and were tested for TBEV-RNA by real-time RT-PCR. Results TBEV-RNA was detected in five rodents (4.3% of tested individuals), all of which had a TBEV-positive ear sample, while only two out of four of these individuals (for which a DBS was available) had a positive DBS. This equated to 6.3% of ear samples and 1.8% of DBSs testing positive for TBEV-RNA. Conclusions We provide the first evidence to our knowledge that TBEV-RNA can be detected in samples obtained non-destructively from naturally infected wild rodents, providing a viable sampling alternative suitable for longitudinal surveillance of the virus. Graphical Abstract

Background Evidence suggests a vicious cycle between rice cultivation and malaria control in Rwanda. Rice fields offer an attractive breeding ground for malaria vectors, which increases the disease burden in rice farming communities, and, consequently, reduces productivity in the rice sector. Community-based larval source management in rice fields is propagated as a sustainable solution to break this cycle. A sense of agency and ownership of malaria control interventions, as well as the mobilization of resources at the local level, are often considered preconditions for success. However, an evidence gap exists regarding the interaction between the agentive and financial dimension of local sustainability. Methods We conduct a larviciding pilot involving three groups; one group where rice farmers sprayed their fields under expert supervision, one group where rice farmers organised the larviciding campaign themselves, and a (non-sprayed) control group. We test whether the difference in agency between the intervention groups affects farmers’ willingness-to-pay for a larviciding campaign. Willingness-to-pay is elicited in a contingent valuation exercise, more specifically a bidding game, and is assessed both before and after the pilot (n = 288). Difference-in-difference estimates are computed, using a propensity score matching technique. Supplementary data were collected in a survey and two focus group discussions for triangulation. Results The high-agency (self-organised) group significantly outperforms the low-agency (expert-supervised) group in terms of maintaining its willingness to contribute financially. However, higher willingness-to-pay in the high-agency group does not appear to be driven by a stronger sense of ownership per se. The supplementary data indicate high levels of ownership in both treatment groups compared to the control group. A tentative explanation lies in diverging perceptions concerning the effectiveness of the pilot. Conclusions The study supports the idea that community-led organization of larval source management can prove instrumental in mobilizing finance for malaria control in low-income settings where rice production interferes with the fight against malaria. However, the causality is complex. Feelings of ownership do not appear the main driver of willingness-to-pay, at least not directly, which opens up the possibility of initiating community-driven malaria control interventions that promote the agentive and financial dimension of local sustainability simultaneously.

BackgroundThe Asian bush mosquitoAedes japonicusis rapidly invading North America and Europe. Due to its potential to transmit multiple pathogenic arthropod-borne (arbo)viruses including Zika virus, West Nile virus and chikungunya virus, it is important to understand the biology of this vector mosquito in more detail. In addition to arboviruses, mosquitoes can also carry insect-specific viruses that receive increasing attention due to their potential effects on host physiology and arbovirus transmission. In this study, we characterized the collection of viruses, referred to as the virome, circulating inAe. japonicuspopulations in the Netherlands and France.ResultsApplying a small RNA-based metagenomic approach toAe. japonicus, we uncovered a distinct group of viruses present in samples from both the Netherlands and France. These included one known virus,Ae. japonicusnarnavirus 1 (AejapNV1), and three new virus species that we namedAe. japonicustotivirus 1 (AejapTV1),Ae. japonicusanphevirus 1 (AejapAV1) andAe. japonicusbunyavirus 1 (AejapBV1). We also discovered sequences that were presumably derived from two additional novel viruses:Ae. japonicusbunyavirus 2 (AejapBV2) andAe. japonicusrhabdovirus 1 (AejapRV1). All six viruses induced strong RNA interference responses, including the production of 21 nucleotide sized small interfering RNAs, a signature of active replication in the host. Notably, AejapBV1 and AejapBV2 belong to different viral families, however, no RNA-dependent RNA polymerase sequence has been found for AejapBV2. Intriguingly, our small RNA-based approach identified a ∼1 kb long ambigrammatic RNA that is associated with AejapNV1 as a secondary segment but showed no similarity to any sequence in public databases. We confirmed the presence of AejapNV1 primary and secondary segments, AejapTV1, AejapAV1 and AejapBV1 by reverse-transcriptase PCR in wild-caughtAe. japonicusmosquitoes. AejapNV1 and AejapTV1 were found at high prevalence (87-100%) in adult females, adult males and larvae.ConclusionsUsing a small RNA-based, sequence-independent metagenomic strategy, we uncovered a conserved and prevalent virome amongAe. japonicusmosquito populations. The high prevalence of AejapNV1 and AejapTV1 across all tested mosquito life stages suggests that these viruses are intimately associated withAe. japonicusand may affect different aspects of the physiology of this vector mosquito.

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika. Zika virus (ZIKV), the causative agent of virus-induced brain damage in newborns, is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus. Here, Obadia et al. characterize ZIKV vector competence of 50 mosquito populations from six species collected in 12 different countries to inform about epidemic risk. They find that African ZIKV strain shows higher transmission efficiency compared to American and Asian ZIKV strains and that Ae. aegypti mosquitoes have highest susceptibility to infections, while Culex mosquitoes are largely non-susceptible.

Contains fulltext : 250889.pdf (Publisher’s version ) (Open Access) Larval source management (LSM) programs for control of malaria vectors are often vertically organized, while there is much potential for involving local communities in program implementation. To address this, we evaluated the entomological impact of community-based application of Bacillus thuringiensis var. israelensis (Bti) in a rice irrigation scheme in Ruhuha, Rwanda. A non-randomized trial with control compared a Bti implementation program that was supervised by the project team (ES) with a program that was led and carried out by local rice farming communities (CB). One other area served as a control to assess mosquito populations without Bti application. Entomological surveys were carried out every two weeks and assessed the presence and abundance of the larval, pupal, and adult stages of Anopheles mosquitoes. In ES, the per round reduction in Anopheles larval habitats was estimated at 49%. This reduction was less in CB (28%) and control (22%) although the per round reduction in CB was still significantly higher than in control. Pupal production was almost completely prevented from round 5 (out of 10) onwards in both CB (average habitat occupancy 0.43%) and ES intervention arms (average habitat occupancy 0.27%), whereas pupal occupancy rates were on average 12.8% from round 5 onwards in the control. Emergence of adult mosquitoes from rice fields was thus prevented although this was not directly noticeable in adult An. gambiae populations in houses nearby the rice fields. Together with our earlier work on the willingness to financially contribute to the LSM program and the high perceived safety and acceptance of the Bti product, the current study demonstrates that, in an environment with limited resources, communities could become more engaged in LSM program implementation and contribute directly to malaria vector control in their environment. 14 p.

Bluetongue virus (BTV) serotype 8 has been circulating in Europe since a major outbreak occurred in 2006, causing economic losses to livestock farms. The unpredictability of the biting activity of midges that transmit BTV implies difficulty in computing accurate transmission models. This study uniquely integrates field collections of midges at a range of European latitudes (in Sweden, The Netherlands, and Italy), with a multi-scale modelling approach. We inferred the environmental factors that influence the dynamics of midge catching, and then directly linked predicted midge catches to BTV transmission dynamics. Catch predictions were linked to the observed prevalence amongst sentinel cattle during the 2007 BTV outbreak in The Netherlands using a dynamic transmission model. We were able to directly infer a scaling parameter between daily midge catch predictions and the true biting rate per cow per day. Compared to biting rate per cow per day the scaling parameter was around 50% of 24 h midge catches with traps. Extending the estimated biting rate across Europe, for different seasons and years, indicated that whilst intensity of transmission is expected to vary widely from herd to herd, around 95% of naive herds in western Europe have been at risk of sustained transmission over the last 15 years. Funding Agencies|Ministry of Economic Affairs, The NetherlandsMinistry of Economic Affairs, Netherlands [1300018161]; DEFRADepartment for Environment, Food & Rural Affairs (DEFRA); EPSRCUK Research & Innovation (UKRI)Engineering & Physical Sciences Research Council (EPSRC) [EP/S022244/1]; BBSRC, United KingdomUK Research & Innovation (UKRI)Biotechnology and Biological Sciences Research Council (BBSRC) [BB/S01750X/1]; Swedish Research Council (FORMAS), Sweden; Italian Ministry of Education, University and Research (MIUR) (Prin 2012)Ministry of Education, Universities and Research (MIUR) [2012T85B3R]

Background Application of the larvicide Bacillus thuringiensis var. israelensis (Bti) is a viable complementary strategy for malaria control. Efficacy of Bti is dose-dependent. There is a knowledge gap on the effects of larval exposure to sublethal Bti doses on emerging adult mosquitoes. The present study examined the effect of larval exposure to sublethal doses of Bti on the survival, body size and oviposition rate in adult Anopheles coluzzii. Methods Third-instar An. coluzzii larvae were exposed to control and sublethal Bti concentrations at LC20, LC50 and LC70 for 48 h. Surviving larvae were reared to adults under standard colony conditions. Thirty randomly selected females from each treatment were placed in separate cages and allowed to blood feed. Twenty-five gravid females from the blood-feeding cages were randomly selected and transferred into new cages where they were provided with oviposition cups. Numbers of eggs laid in each cage and mortality of all adult mosquitoes were recorded daily. Wing lengths were measured of 570 mosquitoes as a proxy for body size. Results Exposure to LC70Bti doses for 48 h as third-instar larvae reduced longevity of adult An. coluzzii mosquitoes. Time to death was 2.58 times shorter in females exposed to LC70Bti when compared to the control females. Estimated mortality hazard rates were also higher in females exposed to the LC50 and LC20 treatments, but these differences were not statistically significant. The females exposed to LC70 concentrations had 12% longer wings than the control group (P < 0.01). No differences in oviposition rate of the gravid females were observed between the treatments. Conclusions Exposure of An. coluzzii larvae to sublethal Bti doses reduces longevity of resultant adults and is associated with larger adult size and unclear effect on oviposition. These findings suggest that anopheline larval exposure to sublethal Bti doses, though not recommended, could reduce vectorial capacity for malaria vector populations by increasing mortality of resultant adults.

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations that are unable to spread autonomously, are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that incomplete virus particles are capable of co-infecting mosquitoes, resulting in the rescue of infectious virus that is able to disseminate to the mosquito salivary glands. Our findings reveal a significant role of incomplete particles in within-host spread and between-host transmission, reminiscent of the life cycle of multipartite viruses.

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented arthropod-borne Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations unable to spread autonomously are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that complementing incomplete virus particles can co-infect mosquitoes, resulting in the reconstitution of infectious virus that is able to disseminate to the mosquito salivary glands. Computational models of infection dynamics predict that incomplete virus particles can positively impact virus spread over a wide range of conditions, with the strongest effect at intermediate multiplicities of infection. Our findings suggest that incomplete particles may play a significant role in within-host spread and between-host transmission, reminiscent of the infection cycle of multipartite viruses.

Mosquito species belonging to the genus Aedes have attracted the interest of scientists and public health officers for their invasive species traits and efficient capacity of transmitting viruses affecting humans. Some of these species were brought outside their native range by human activities such as trade and tourism, and colonised new regions thanks to a unique combination of eco-physiological traits.Considering mosquito physiological and behavioural traits to understand and predict the spatial and temporal population dynamics is thus a crucial step to develop strategies to mitigate the local densities of invasive Aedes populations.Here, we synthesised the life cycle of four invasive Aedes species (Ae. aegypti, Ae. albopictus, Ae. japonicus and Ae. koreicus) in a single multi-scale stochastic modelling framework which we coded in the R package dynamAedes. We designed a stage-based and time-discrete stochastic model driven by temperature, photo-period and inter-specific larval competition that can be applied to three different spatial scales: punctual, local and regional. These spatial scales consider different degrees of spatial complexity and data availability, by accounting for both active and passive dispersal of mosquito species as well as for the heterogeneity of the input temperature data.Our overarching aim was to provide a flexible, open-source and user-friendly tool rooted in the most updated knowledge on species biology which could be applied to the management of invasive Aedes populations as well as for more theoretical ecological inquiries.

Bacteria are part of the insect gut system and influence many physiological traits of their host. Gut bacteria may even reduce or block the transmission of arboviruses in several species of arthropod vectors. Culicoides biting midges are important arboviral vectors of several livestock and wildlife diseases, yet limited information is available on their gut bacterial communities. Addressing this gap will help inform how these communities can be manipulated and ultimately used as novel tools to control pathogens. To assess how bacterial communities change during the life stages of lab-reared C. nubeculosus and C. sonorensis, endosymbiotic bacteria were identified using Illumina sequencing of 16S rRNA and taxonomically characterised. Analyses were conducted to determine how gut bacterial communities in adults are influenced by species identity and geographic distance among biting midge populations. Communities of the two lab-reared Culicoides species significantly changed after pupation and with maturation into 6-day-old adults. Pseudomonas, Burkholderiaceae and Leucobacter bacteria were part of a core community that was trans-stadially transmitted and found throughout their life cycle. Among field-collected biting midges, the bacterial communities were unique for almost each species. Cardinium, Rickettsia and Wolbachia were some of the most abundant bacteria in midges collected from wetlands. Only Pseudomonas was present in high relative abundance in all field-collected species. In this study, species identity, as well as geographic distance, influenced the gut bacterial communities and may partly explain known inter- and intra-species variability in vector competence. Additionally, stably associated bacterial species could be candidates for paratransgenic strategies to control vector-borne pathogens.

Despite their past importance as vectors of indigenous malaria, the species composition and spatial distribution of the members of the Anopheles maculipennis complex have been studied to a limited extent in the Netherlands. Therefore, this investigation focuses on the distribution of the members of this complex in the Netherlands, including Anopheles daciae, which has recently been found in countries bordering the Netherlands. In the framework of a national mosquito surveillance between 2010 and 2021, a total of 541 specimens of An. maculipennis s.l. were analyzed from 161 locations covering the entire territory. In addition, 89 specimens were analyzed from overwintering sites during the winter of 2020/2021. All individual mosquitoes were identified to species-level using Sanger sequencing of the ribosomal internal transcribed spacer 2. To characterize the habitat of An. maculipennis s.l. in the Netherlands, land cover use data was extracted in a 1 km buffer area around each finding location. For populations collected in summers between 2010 and 2021, the most frequent species was An. messeae, present in 88.19% of the locations, followed by An. maculipennis s.s. (11.80%), An. atroparvus (3.72%) and An. daciae (3.72%). Anopheles daciae was found in the southern inland areas of the country. Furthermore, An. messeae and An. daciae occurred in sympatry at overwintering sites. This study provides relevant information on the occurrence of species of the Anopheles maculipennis complex in the Netherlands, contributing to a better estimation of the risk of mosquito-borne disease in the country.

Contains fulltext : 240210.pdf (Publisher’s version ) (Open Access) There is broad consensus that successful and sustained larval source management (LSM) interventions, including bio-larviciding campaigns, require embeddedness in local community institutions. Ideally, these community structures should also be capable of mobilizing local resources to (co-)finance interventions. To date, farmer cooperatives, especially cooperatives of rice growers whose economic activity facilitates mosquito breeding, have remained under the radar in designing community-based bio-larviciding campaigns. This study explores the potential of rice farmer cooperatives in Bugesera district, Rwanda, to take up the aforementioned roles. To this purpose, we surveyed 320 randomly selected rice farmers who belonged to one of four rice cooperatives in the area and elicited their willingness-to-pay (WTP) for application of Bti, a popular bio-larvicide, in their rice paddies. Results from a (non-incentivized) bidding game procedure, which tested two alternative contribution schemes showed that financial contributions would be significantly different from zero and sufficient to carry a co-financing share of 15–25 per cent. A strong heterogeneity in mean WTP is revealed across cooperatives, in addition to variation among individual farmers, which needs to be anticipated when engaging farmer cooperatives in LSM. 20 p.

Usutu virus (USUV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that are mainly transmitted between bird hosts by vector mosquitoes. Infections in humans are incidental but can cause severe disease. USUV is endemic in large parts of Europe, while WNV mainly circulates in Southern Europe. In recent years, WNV is also frequently detected in Northern Europe, thereby expanding the area where both viruses co-circulate. However, it remains unclear how USUV may affect the future spread of WNV and the likelihood of human co-infection. Here we investigated whether co-infections with both viruses in cell lines and their primary mosquito vector, Culex pipiens, affect virus replication and transmission dynamics. We show that USUV is outcompeted by WNV in mammalian, avian and mosquito cells during co-infection. Mosquitoes that were exposed to both viruses simultaneously via infectious blood meal displayed significantly reduced USUV transmission compared to mosquitoes that were only exposed to USUV (from 15% to 3%), while the infection and transmission of WNV was unaffected. In contrast, when mosquitoes were pre-infected with USUV via infectious blood meal, WNV transmission was significantly reduced (from 44% to 17%). Injection experiments established the involvement of the midgut in the observed USUV-mediated WNV inhibition. The competition between USUV and WNV during co-infection clearly indicates that the chance of concurrent USUV and WNV transmission via a single mosquito bite is low. The competitive relation between USUV and WNV may impact virus transmission dynamics in the field and affect the epidemiology of WNV in Europe.

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

Background Infectious blood meal experiments have been frequently performed with different virus-vector combinations to assess the transmission potential of arthropod-borne (arbo)viruses. A wide variety of host blood sources have been used to deliver arboviruses to their arthropod vectors in laboratory studies. The type of blood used during vector competence experiments does not always reflect the blood from the viremic vertebrate hosts in the field, but little is known about the effect of blood source on the experimental outcome of vector competence studies. Here we investigated the effect of avian versus human blood on the infection and transmission rates of the zoonotic Usutu virus (USUV) in its primary mosquito vector Culex pipiens. Methods Cx. pipiens biotypes (pipiens and molestus) were orally infected with USUV through infectious blood meals containing either chicken or human whole blood. The USUV infection and transmission rates were determined by checking mosquito bodies and saliva for USUV presence after 14 days of incubation at 28 °C. In addition, viral titers were determined for USUV-positive mosquito bodies and saliva. Results Human and chicken blood lead to similar USUV transmission rates for Cx. pipiens biotype pipiens (18% and 15%, respectively), while human blood moderately but not significantly increased the transmission rate (30%) compared to chicken blood (17%) for biotype molestus. USUV infection rates with human blood were consistently higher in both Cx. pipiens biotypes compared to chicken blood. In virus-positive mosquitoes, USUV body and saliva titers did not differ between mosquitoes taking either human or chicken blood. Importantly, biotype molestus had much lower USUV saliva titers compared to biotype pipiens, regardless of which blood was offered. Conclusions Infection of mosquitoes with human blood led to higher USUV infection rates as compared to chicken blood. However, the blood source had no effect on the vector competence for USUV. Interestingly, biotype molestus is less likely to transmit USUV compared to biotype pipiens due to very low virus titers in the saliva. Graphical Abstract

Current tools for vector control are insufficient to curb vector-borne disease transmission. Recent outbreaks of 'new' vector-borne diseases, such as Zika and chikungunya, and the ongoing fight against malaria underscore this. Scientists and public health authorities collaborate on a continued search for innovative strategies to address this challenge. To guide the integration of currently available and new tools in vector control programs, the World Health Organization (WHO) developed the Global Vector Control Response (GVCR).This initiative was unanimously endorsed by the World Health Assemb

The detection of Aedes albopictus in Lucky bamboo (Dracaena sanderiana) greenhouses and Ae. atropalpus at used tire importers illustrates that the Netherlands is exposed to the risk of introductions of invasive mosquito species (IMS). In this study we implemented a risk-based and adaptive surveillance (2010–16) in order to detect introductions and prevent potential proliferation of IMS at these locations. Results at Lucky bamboo greenhouses show that interceptions of Ae. albopictus occurred every year, with 2010 and 2012 being the years with most locations found positive for this species (n = 6), and 2015 the year with the highest percentage of positive samples (4.1%). Furthermore, our results demonstrate that Ae. japonicus can also be associated with the import of Lucky bamboo. At used tire companies, IMS were found at 12 locations. Invasive mosquito species identified were Ae. albopictus, Ae. atropalpus, Ae. aegypti, and Ae. japonicus, of which Ae. albopictus has been found every year since 2010. The proportion of samples containing IMS was significantly higher before application of a covenant between the used tire importers and the Dutch government in 2013 (12.96%) than in the successive 3 years (2014 [6.93%], 2015 [4.24%], 2016 [5.09%], 1-sided binomial test, P < 0.01). It is concluded that risk-based and adaptive surveillance is an effective methodology for detection of IMS, and that application of governmental management measures in combination with mosquito control has stabilized the situation.

Additional file 3. Members of the Anopheles gambiae complex found among samples of Anopheles gambiae s.l. tested from Ruhuha and Busoro sector, Ruhuha

Background As part of malaria prevention and control efforts, the distribution and density of malaria mosquitoes requires continuous monitoring. Resources for long-term surveillance of malaria vectors, however, are often limited. The aim of the research was to evaluate the value of citizen science in providing insight into potential malaria vector hotspots and other malaria relevant information, and to determine predictors of malaria vector abundance in a region where routine mosquito monitoring has not been established to support vector surveillance. Methods A 1-year citizen science programme for malaria mosquito surveillance was implemented in five villages of the Ruhuha sector in Bugesera district, Rwanda. In total, 112 volunteer citizens were enrolled and reported monthly data on mosquitoes collected in their peridomestic environment using handmade carbon-dioxide baited traps. Additionally, they reported mosquito nuisance experienced as well as the number of confirmed malaria cases in their household. Results In total, 3793 female mosquitoes were collected, of which 10.8% were anophelines. For the entire period, 16% of the volunteers reported having at least one confirmed malaria case per month, but this varied by village and month. During the study year 66% of the households reported at least one malaria case. From a sector perspective, a higher mosquito and malaria vector abundance was observed in the two villages in the south of the study area. The findings revealed significant positive correlations among nuisance reported and confirmed malaria cases, and also between total number of Culicidae and confirmed malaria cases, but not between the numbers of the malaria vector Anopheles gambiae and malaria cases. At the sector level, of thirteen geographical risk factors considered for inclusion in multiple regression, distance to the river network and elevation played a role in explaining mosquito and malaria mosquito abundance. Conclusions The study demonstrates that a citizen science approach can contribute to mosquito monitoring, and can help to identify areas that, in view of limited resources for control, are at higher risk of malaria.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that is pathogenic to ruminants and humans. The virus is endemic to Africa and the Arabian Peninsula where outbreaks are characterized by abortion storms and mortality of newborns, particularly in sheep herds. Vector competence experiments in laboratory settings have suggested that over 50 mosquito species are capable of transmitting RVFV. Transmission of mosquito-borne viruses in the field is however influenced by numerous factors, including population densities, blood feeding behavior, extrinsic incubation period, longevity of vectors, and viremia levels in vertebrate hosts. Animal models to study these important aspects of RVFV transmission are currently lacking. In the present work, RVFV was transmitted to European (Texel-swifter cross-breed) lambs by laboratory-reared Aedes aegypti mosquitoes that were infected either by membrane feeding on a virus-spiked blood meal or by feeding on lambs that developed viremia after intravenous inoculation of RVFV. Feeding of mosquitoes on viremic lambs resulted in strikingly higher infection rates as compared to membrane feeding. Subsequent transmission of RVFV from lamb to lamb by infected mosquitoes was highly efficient in both models. The animal models described here can be used to study mosquito-mediated transmission of RVFV among the major natural target species and to evaluate the efficacy of vaccines against mosquito-mediated RVFV infection.

Additional file 1. Mosquito species collected using CDC light traps in selected villages in Busoro and Ruhuha sector, Rwanda (2017)

Additional file 2. Mosquito species collected using CDC light traps in selected villages in Busoro and Ruhuha sector, Rwanda (2018)

BackgroundMalaria remains a major public health concern in the Democratic Republic of the Congo (DRC) and its control is affected by recurrent conflicts. Médecins Sans Frontières (MSF) initiated several studies to better understand the unprecedented incidence of malaria to effectively target and implement interventions in emergency settings. The current study evaluated the main vector species involved in malaria transmission and their resistance to insecticides, with the aim to propose the most effective tools and strategies for control of local malaria vectors.MethodsThis study was performed in 52 households in Shamwana (Katanga, 2014), 168 households in Baraka (South Kivu, 2015) and 269 households in Kashuga (North Kivu, 2017).Anophelesvectors were collected and subjected to standardized Word Health Organization (WHO) and Center for Disease Control (CDC) insecticide susceptibility bioassays. Mosquito species determination was done using PCR andPlasmodium falciparuminfection in mosquitoes was assessed by ELISA targeting circumsporozoite protein.ResultsOf 3517Anophelesspp. mosquitoes collected,Anopheles gambiaesensu lato (s.l.) (29.6%) andAnopheles funestus(69.1%) were the main malaria vectors. Plasmodium falciparuminfection rates forAn. gambiaes.l. were 1.0, 2.1 and 13.9% for Shamwana, Baraka and Kashuga, respectively.Anopheles funestusshowed positivity rates of 1.6% in Shamwana and 4.4% in Baraka. NoAn. funestuswere collected in Kashuga. Insecticide susceptibility tests showed resistance development towards pyrethroids in all locations. Exposure to bendiocarb, malathion and pirimiphos-methyl still resulted in high mosquito mortality.ConclusionsThis is one of only few studies from these conflict areas in DRC to report insecticide resistance in local malaria vectors. The data suggest that current malaria prevention methods in these populations are only partially effective, and require additional tools and strategies. Importantly, the results triggered MSF to consider the selection of a new insecticide for indoor residual spraying (IRS) and a new long-lasting insecticide-treated net (LLIN). The reinforcement of correct usage of LLINs and the introduction of targeted larviciding were also included as additional vector control tools as a result of the studies.

Background Many countries, including Rwanda, have mosquito monitoring programmes in place to support decision making in the fight against malaria. However, these programmes can be costly, and require technical (entomological) expertise. Involving citizens in data collection can greatly support such activities, but this has not yet been thoroughly investigated in a rural African context. Methods Prior to the implementation of such a citizen-science approach, a household entomological survey was conducted in October–November 2017 and repeated one year later in Busoro and Ruhuha sectors, in southern and eastern province of Rwanda, respectively. The goal was to evaluate the perception of mosquito nuisance reported by citizens as a potential indicator for malaria vector hotspots. Firstly, mosquito abundance and species composition were determined using Centers for Disease Control and Prevention (CDC) light traps inside the houses. Secondly, household members were interviewed about malaria risk factors and their perceived level of mosquito nuisance. Results Tiled roofs, walls made of mud and wood, as well as the number of occupants in the house were predictors for the number of mosquitoes (Culicidae) in the houses, while the presence of eaves plus walls made of mud and wood were predictors for malaria vector abundance. Perception of mosquito nuisance reported indoors tended to be significantly correlated with the number of Anopheles gambiae sensu lato (s.l.) and Culicidae collected indoors, but this varied across years and sectors. At the village level, nuisance also significantly correlated with An. gambiae s.l. and total mosquito density, but only in 2018 while not in 2017. Conclusions Perception of mosquito nuisance denoted in a questionnaire survey could be used as a global indicator of malaria vector hotspots. Hence, involving citizens in such activities can complement malaria vector surveillance and control.

BackgroundMalaria control remains a challenge globally and in malaria-endemic countries in particular. In Rwanda, a citizen science programme has been set up to improve malaria control. Citizens are involved in collecting mosquito species and reporting mosquito nuisance. This study assessed what people benefit from such a citizen science programme. The analysis was conducted on how the citizen science programme influenced perceptions and behaviour related to malaria control.MethodsThis study employed a mixed-methods approach using dissemination workshops, a survey, and village meetings as the main data collection methods. Dissemination workshops and village meetings involved 112 volunteers of the citizen science programme and were conducted to explore: (1) the benefits of being involved in the programme and (2) different ways used to share malaria-related information to non-volunteers. The survey involved 328 people (110 volunteers and 218 non-volunteers) and was used to compare differences in malaria-related perceptions and behaviour over time (between 2017 and 2019), as well as between volunteers and non-volunteers.ResultsMalaria-related perceptions and behaviour changed significantly over time (between 2017 and 2019) and became favourable to malaria control. When the findings were compared between volunteers and non-volunteers, for perceptions, only perceived self-efficacy showed a significant difference between these two groups. However, volunteers showed significantly more social interaction, participation in malaria-related activities at the community level, and indoor residual spraying (IRS) acceptance. In addition, both volunteers and non-volunteers reported to have gained knowledge and skills about the use of malaria control measures in general, and mosquito species in particular among volunteers.ConclusionThe reported knowledge and skills gained among non-volunteers indicate a diffusion of the citizen science programme-related information in the community. Thus, the citizen science programme has the potential to provide individual and collective benefits to volunteers and society at large.

Zika virus (ZIKV) is a mosquito-borne pathogen that caused a large outbreak in the Americas in 2015 and 2016. The virus is currently present in tropical areas around the globe and can cause severe disease in humans, including Guillain-Barr&eacute, syndrome and congenital microcephaly. The tropical yellow fever mosquito, Aedes aegypti, is the main vector in the urban transmission cycles of ZIKV. The discovery of ZIKV in wild-caught Culex mosquitoes and the ability of Culex quinquefasciatus mosquitoes to transmit ZIKV in the laboratory raised the question of whether the common house mosquito Culex pipiens, which is abundantly present in temperate regions in North America, Asia and Europe, could also be involved in ZIKV transmission. In this study, we investigated the vector competence of Cx. pipiens (biotypes molestus and pipiens) from the Netherlands for ZIKV, using Usutu virus as a control. After an infectious blood meal containing ZIKV, none of the tested mosquitoes accumulated ZIKV in the saliva, although 2% of the Cx. pipiens pipiens mosquitoes showed ZIKV&ndash, positive bodies. To test the barrier function of the mosquito midgut on virus transmission, ZIKV was forced into Cx. pipiens mosquitoes by intrathoracic injection, resulting in 74% (molestus) and 78% (pipiens) ZIKV&ndash, positive bodies. Strikingly, 14% (molestus) and 7% (pipiens) of the tested mosquitoes accumulated ZIKV in the saliva after injection. This is the first demonstration of ZIKV accumulation in the saliva of Cx. pipiens upon forced infection. Nevertheless, a strong midgut barrier restricted virus dissemination in the mosquito after oral exposure and we, therefore, consider Cx. pipiens as a highly inefficient vector for ZIKV.

Background To further reduce malaria, larval source management (LSM) is proposed as a complementary strategy to the existing strategies. LSM has potential to control insecticide resistant, outdoor biting and outdoor resting vectors. Concerns about costs and operational feasibility of implementation of LSM at large scale are among the reasons the strategy is not utilized in many African countries. Involving communities in LSM could increase intervention coverage, reduce costs of implementation and improve sustainability of operations. Community acceptance and participation in community-led LSM depends on a number of factors. These factors were explored under the Majete Malaria Project in Chikwawa district, southern Malawi. Methods Separate focus group discussions (FGDs) were conducted with members from the general community (n = 3); health animators (HAs) (n = 3); and LSM committee members (n = 3). In-depth interviews (IDIs) were conducted with community members. Framework analysis was employed to determine the factors contributing to community acceptance and participation in the locally-driven intervention. Results Nine FGDs and 24 IDIs were held, involving 87 members of the community. Widespread knowledge of malaria as a health problem, its mode of transmission, mosquito larval habitats and mosquito control was recorded. High awareness of an association between creation of larval habitats and malaria transmission was reported. Perception of LSM as a tool for malaria control was high. The use of a microbial larvicide as a form of LSM was perceived as both safe and effective. However, actual participation in LSM by the different interviewee groups varied. Labour-intensiveness and time requirements of the LSM activities, lack of financial incentives, and concern about health risks when wading in water bodies contributed to lower participation. Conclusion Community involvement in LSM increased local awareness of malaria as a health problem, its risk factors and control strategies. However, community participation varied among the respondent groups, with labour and time demands of the activities, and lack of incentives, contributing to reduced participation. Innovative tools that can reduce the labour and time demands could improve community participation in the activities. Further studies are required to investigate the forms and modes of delivery of incentives in operational community-driven LSM interventions.