Is Culex modestus a New Usutu virus vector?

Usutu virus is an emerging pathogen transmitted by mosquitoes. Culex modestus mosquitoes are widespread in Europe, but their role in disease transmission is poorly understood. Recent data from a single infectious mosquito suggested that Culex modestus could be an unrecognized vector for Usutu virus. In this study, our aim was to corroborate this finding using a larger sample size. We collected immature Culex modestus from a reedbed pond in Flemish Brabant, Belgium, and reared them in the laboratory until the third generation. Adult females were then experimentally infected with Usutu virus in a blood meal and incubated at 25 °C for 14 days. The presence of Usutu virus in the saliva, head and body of each female was determined by plaque assay and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). The transmission efficiency was 54% (n = 15/28), confirming that Belgian Culex modestus can experimentally transmit Usutu virus.

Overwintering West Nile virus in active Culex pipiens mosquito populations in Greece.

The flavivirus West Nile Virus (WNV), which is transmitted by mosquitoes, poses a significant threat to both humans and animals, and its outbreaks often challenge public health in Europe and other continents. In recent years, there is an increasing trend of WNV incidence rates across several European countries. However, whether there is a year-round circulation or seasonal introduction has yet to be elucidated. Real-time polymerase chain reaction (PCR) identified WNV-positive Culex pipiens mosquitos in 6 out of 146 pools examined in winter 2022 that correspond to three out of the 24 study areas, located in two coastal regions units in Attica, Greece. Spatial dispersion of the six positive pools in the same region suggests a clustered circulation of WNV during the winter of 2022. This is the first study that documents the identification of WNV in Cx. pipiens populations, captured in adult traps during winter period. Our findings underscore the need to extend entomological surveillance programs to include the winter period, specifically in temperate climates and historically affected areas by WNV.

Aedes aegypti Vector Competence Assay for Rift Valley Fever Virus Using Artificial Blood Meal.

Vector competence assays allow to measure, in the laboratory, the ability of a mosquito to get infected and then retransmit an arbovirus while mimicking natural vector infection route. Aedes aegypti is a major vector of arboviruses worldwide and thus a reference species used in vector competence assays. Rift Valley fever virus (RVFV) is a major public health threat, mostly in Africa, that infects humans and animals through the bite of mosquito vectors. Here, we describe vector competence assay of Aedes aegypti mosquitoes for RVFV, from mosquito exposure to the virus through an infectious artificial blood meal to the measurement of virus prevalence in the mosquito's body, head, and saliva.

Cytoskeleton-associated gelsolin responds to the midgut distention process in saline meal-fed Aedes aegypti and affects arbovirus dissemination from the midgut.

The mosquito, Aedes aegypti, is the principal vector for several arboviruses. The mosquito midgut is the initial tissue that gets infected with an arbovirus acquired along with a blood meal from a vertebrate host. Blood meal ingestion leads to midgut tissue distention thereby increasing the pore size of the surrounding basal lamina. This allows newly synthesized virions to exit the midgut by traversing the distended basal lamina to infect secondary tissues of the mosquito. We conducted a quantitative label-free proteomic time course analysis with saline meal-fed Ae. aegypti females to identify host factors involved in midgut tissue distention. Around 2000 proteins were detected during each of the seven sampling time points and 164 of those were uniquely expressed. Forty-five of 97 differentially expressed proteins were upregulated during the 96-h time course and most of those were involved in cytoskeleton modulation, metabolic activity, and vesicle/vacuole formation. The F-actin-modulating Ae. aegypti (Aa)-gelsolin was selected for further functional studies. Stable knockout of Aa-gelsolin resulted in a mosquito line, which showed distorted actin filaments in midgut-associated tissues likely due to diminished F-actin processing by gelsolin. Zika virus dissemination from the midgut of these mosquitoes was diminished and delayed. The loss of Aa-gelsolin function was associated with an increased induction of apoptosis in midgut tissue indicating an involvement of Aa-gelsolin in apoptotic signaling in mosquitoes. Here, we used proteomics to discover a novel host factor, Aa-gelsolin, which affects the midgut escape barrier for arboviruses in mosquitoes and apoptotic signaling in the midgut.

Population genetic structure of Culex tritaeniorhynchus in different types of climatic zones in China.

BACKGROUND: Culex tritaeniorhynchus is widely distributed in China, from Hainan Island in the south to Heilongjiang in the north, covering tropical, subtropical, and temperate climate zones. Culex tritaeniorhynchus carries 19 types of arboviruses. It is the main vector of the Japanese encephalitis virus (JEV), posing a serious threat to human health. Understanding the effects of environmental factors on Culex tritaeniorhynchus can provide important insights into its population structure or isolation patterns, which is currently unclear. RESULTS: In total, 138 COI haplotypes were detected in the 552 amplified sequences, and the haplotype diversity (Hd) value increased from temperate (0.534) to tropical (0.979) regions. The haplotype phylogeny analysis revealed that the haplotypes were divided into two high-support evolutionary branches. Temperate populations were predominantly distributed in evolutionary branch II, showing some genetic isolation from tropical/subtropical populations and less gene flow between groups. The neutral test results of HNQH (Qionghai) and HNHK(Haikou) populations were negative (P < 0.05), indicating many low-frequency mutations in the populations and that the populations might be in the process of expansion. Moreover, Wolbachia infection was detected only in SDJN (Jining) (2.24%), and all Wolbachia genotypes belonged to supergroup B. To understand the influence of environmental factors on mosquito-borne viruses, we examined the prevalence of Culex tritaeniorhynchus infection in three ecological environments in Shandong Province. We discovered that the incidence of JEV infection was notably greater in Culex tritaeniorhynchus from lotus ponds compared to those from irrigation canal regions. In this study, the overall JEV infection rate was 15.27 per 1000, suggesting the current risk of Japanese encephalitis outbreaks in Shandong Province. CONCLUSIONS: Tropical and subtropical populations of Culex tritaeniorhynchus showed higher genetic diversity and those climatic conditions provide great advantages for the establishment and expansion of Culex tritaeniorhynchus. There are differences in JEV infection rates in wild populations of Culex tritaeniorhynchus under different ecological conditions. Our results suggest a complex interplay of genetic differentiation, population structure, and environmental factors in shaping the dynamics of Culex tritaeniorhynchus. The low prevalence of Wolbachia in wild populations may reflect the recent presence of Wolbachia invasion in Culex tritaeniorhynchus.

The effect of repeat feeding on dengue virus transmission potential in Wolbachia-infected Aedes aegypti following extended egg quiescence.

As Wolbachia pipientis is more widely being released into field populations of Aedes aegypti for disease control, the ability to select the appropriate strain for differing environments is increasingly important. A previous study revealed that longer-term quiescence in the egg phase reduced the fertility of mosquitoes, especially those harboring the wAlbB Wolbachia strain. This infertility was also associated with a greater biting rate. Here, we attempt to quantify the effect of this heightened biting behavior on the transmission potential of the dengue virus using a combination of assays for fitness, probing behavior, and vector competence, allowing repeat feeding, and incorporate these effects in a model of R0. We show that Wolbachia-infected infertile mosquitoes are more interested in feeding almost immediately after an initial blood meal relative to wild type and Wolbachia-infected fertile mosquitoes and that these differences continue for up to 8 days over the period we measured. As a result, the infertile Wolbachia mosquitoes have higher virus prevalence and loads than Wolbachia-fertile mosquitoes. We saw limited evidence of Wolbachia-mediated blocking in the disseminated tissue (legs) in terms of prevalence but did see reduced viral loads. Using a previously published estimate of the extrinsic incubation period, we demonstrate that the effect of repeat feeding/infertility is insufficient to overcome the effects of Wolbachia-mediated blocking on R0. These estimates are very conservative, however, and we posit that future studies should empirically measure EIP under a repeat feeding model. Our findings echo previous work where periods of extensive egg quiescence affected the reproductive success of Wolbachia-infected Ae. aegypti. Additionally, we show that increased biting behavior in association with this infertility in Wolbachia-infected mosquitoes may drive greater vector competence. These relationships require further exploration, given their ability to affect the success of field releases of Wolbachia for human disease reduction in drier climates where longer egg quiescence periods are expected.

Study on the temporal and spatial distribution of Culex mosquitoes in Hanoi, Vietnam.

Arboviruses transmitted by mosquitoes, including Japanese encephalitis virus (JEV), present a substantial global health threat. JEV is transmitted by mosquitoes in the genus Culex, which are common in both urban and rural areas in Vietnam. In 2020, we conducted a 1-year survey of Culex mosquito abundance in urban, suburban, and peri-urban areas of Hanoi using CDC-light traps. Mosquitoes were identified to species and sorted into pools based on species, sex, and trap location. The mosquito pools were also investigated by RT-qPCR for detection of JEV. In total, 4829 mosquitoes were collected over a total of 455 trap-nights, across 13 months. Collected mosquitoes included Culex, Aedes, Anopheles, and Mansonia species. Culex mosquitoes, primarily Cx. quinquefasciatus, predominated, especially in peri-urban areas. Most Culex mosquitoes were caught in the early months of the year. The distribution and abundance of mosquitoes exhibited variations across urban, suburban, and peri-urban sites, emphasizing the influence of environmental factors such as degree of urbanization, temperature and humidity on Culex abundance. No JEV was detected in the mosquito pools. This study establishes baseline knowledge of Culex abundance and temporal variation, which is crucial for understanding the potential for JEV transmission in Hanoi.

The potential role of the Asian bush mosquito Aedes japonicus as spillover vector for West Nile virus in the Netherlands.

BACKGROUND: In recent years the Asian bush mosquito Aedes japonicus has invaded Europe, including the Netherlands. This species is a known vector for a range of arboviruses, possibly including West Nile virus (WNV). As WNV emerged in the Netherlands in 2020, it is important to investigate the vectorial capacity of mosquito species present in the Netherlands to estimate the risk of future outbreaks and further spread of the virus. Therefore, this study evaluates the potential role of Ae. japonicus in WNV transmission and spillover from birds to dead-end hosts in the Netherlands. METHODS: We conducted human landing collections in allotment gardens (Lelystad, the Netherlands) in June, August and September 2021 to study the diurnal and seasonal host-seeking behaviour of Ae. japonicus. Furthermore, their host preference in relation to birds using live chicken-baited traps was investigated. Vector competence of field-collected Ae. japonicus mosquitoes for two isolates of WNV at two different temperatures was determined. Based on the data generated from these studies, we developed a Susceptible-Exposed-Infectious-Recovered (SEIR) model to calculate the risk of WNV spillover from birds to humans via Ae. japonicus, under the condition that the virus is introduced and circulates in an enzootic cycle in a given area. RESULTS: Our results show that Ae. japonicus mosquitoes are actively host seeking throughout the day, with peaks in activity in the morning and evening. Their abundance in August was higher than in June and September. For the host-preference experiment, we documented a small number of mosquitoes feeding on birds: only six blood-fed females were caught over 4 full days of sampling. Finally, our vector competence experiments with Ae. japonicus compared to its natural vector Culex pipiens showed a higher infection and transmission rate when infected with a local, Dutch, WNV isolate compared to a Greek isolate of the virus. Interestingly, we also found a small number of infected Cx. pipiens males with virus-positive leg and saliva samples. CONCLUSIONS: Combining the field and laboratory derived data, our model predicts that Ae. japonicus could act as a spillover vector for WNV and could be responsible for a high initial invasion risk of WNV when present in large numbers.

Exploring the role of temperature and other environmental factors in West Nile virus incidence and prediction in California counties from 2017-2022 using a zero-inflated model.

West Nile virus (WNV) is the most common mosquito-borne disease in the United States, resulting in hundreds of reported cases yearly in California alone. The transmission cycle occurs mostly in birds and mosquitoes, making meteorological conditions, such as temperature, especially important to transmission characteristics. Given that future increases in temperature are all but inevitable due to worldwide climate change, determining associations between temperature and WNV incidence in humans, as well as making predictions on future cases, are important to public health agencies in California. Using surveillance data from the California Department of Public Health (CDPH), meteorological data from the National Oceanic and Atmospheric Administration (NOAA), and vector and host data from VectorSurv, we created GEE autoregressive and zero-inflated regression models to determine the role of temperature and other environmental factors in WNV incidence and predictions. An increase in temperature was found to be associated with an increase in incidence in 11 high-burden Californian counties between 2017-2022 (IRR = 1.06), holding location, time of year, and rainfall constant. A hypothetical increase of two degrees Fahrenheit-predicted for California by 2040-would have resulted in upwards of 20 excess cases per year during our study period. Using 2017-2021 as a training set, meteorological and host/vector data were able to closely predict 2022 incidence, though the models did overestimate the peak number of cases. The zero-inflated model closely predicted the low number of cases in winter months but performed worse than the GEE model during high-transmission periods. These findings suggests that climate change will, and may be already, altering transmission dynamics and incidence of WNV in California, and provides tools to help predict incidence into the future.

Aedes albopictus saliva contains a richer microbial community than the midgut.

BACKGROUND: Past findings demonstrate that arthropods can egest midgut microbiota into the host skin leading to dual colonization of the vertebrate host with pathogens and saliva microbiome. A knowledge gap exists on how the saliva microbiome interacts with the pathogen in the saliva. To fill this gap, we need to first define the microbial composition of mosquito saliva. METHODS: The current study aimed at analyzing and comparing the microbial profile of Aedes albopictus saliva and midgut as well as assessing the impact of Zika virus (ZIKV) infection on the midgut and saliva microbial composition. Colony-reared Ae. albopictus strains were either exposed to ZIKV infectious or noninfectious bloodmeal. At 14 ays postinfection, the 16S V3-V4 hypervariable rRNA region was amplified from midgut and saliva samples and sequenced on an Illumina MiSeq platform. The relative abundance and diversity of midgut and saliva microbial taxa were assessed. RESULTS: We observed a richer microbial community in the saliva compared with the midgut, yet some of the microbial taxa were common in the midgut and saliva. ZIKV infection did not impact the microbial diversity of midgut or saliva. Further, we identified Elizabethkingia spp. in the Ae. albopictus saliva. CONCLUSIONS: This study provides insights into the microbial community of the Ae. albopictus saliva as well as the influence of ZIKV infection on the microbial composition of its midgut and saliva. The identification of Elizabethkingia spp., an emerging pathogen of global health significance, in Ae. albopictus saliva is of medical importance. Future studies to assess the interactions between Ae. albopictus saliva microbiome and ZIKV could lead to novel strategies for developing transmission barrier tools.

Wolbachia mediates crosstalk between miRNA and Toll pathways to enhance resistance to dengue virus in Aedes aegypti.

The obligate endosymbiont Wolbachia induces pathogen interference in the primary disease vector Aedes aegypti, facilitating the utilization of Wolbachia-based mosquito control for arbovirus prevention, particularly against dengue virus (DENV). However, the mechanisms underlying Wolbachia-mediated virus blockade have not been fully elucidated. Here, we report that Wolbachia activates the host cytoplasmic miRNA biogenesis pathway to suppress DENV infection. Through the suppression of the long noncoding RNA aae-lnc-2268 by Wolbachia wAlbB, aae-miR-34-3p, a miRNA upregulated by the Wolbachia strains wAlbB and wMelPop, promoted the expression of the antiviral effector defensin and cecropin genes through the Toll pathway regulator MyD88. Notably, anti-DENV resistance induced by Wolbachia can be further enhanced, with the potential to achieve complete virus blockade by increasing the expression of aae-miR-34-3p in Ae. aegypti. Furthermore, the downregulation of aae-miR-34-3p compromised Wolbachia-mediated virus blockade. These findings reveal a novel mechanism by which Wolbachia establishes crosstalk between the cytoplasmic miRNA pathway and the Toll pathway via aae-miR-34-3p to strengthen antiviral immune responses against DENV. Our results will aid in the advancement of Wolbachia for arbovirus control by enhancing its virus-blocking efficiency.

Ecology and geography of Cache Valley virus assessed using ecological niche modeling.

BACKGROUND: Cache Valley virus (CVV) is an understudied Orthobunyavirus with a high spillover transmission potential due to its wide geographical distribution and large number of associated hosts and vectors. Although CVV is known to be widely distributed throughout North America, no studies have explored its geography or employed computational methods to explore the mammal and mosquito species likely participating in the CVV sylvatic cycle. METHODS: We used a literature review and online databases to compile locality data for CVV and its potential vectors and hosts. We linked location data points with climatic data via ecological niche modeling to estimate the geographical range of CVV and hotspots of transmission risk. We used background similarity tests to identify likely CVV mosquito vectors and mammal hosts to detect ecological signals from CVV sylvatic transmission. RESULTS: CVV distribution maps revealed a widespread potential viral occurrence throughout North America. Ecological niche models identified areas with climate, vectors, and hosts suitable to maintain CVV transmission. Our background similarity tests identified Aedes vexans, Culiseta inornata, and Culex tarsalis as the most likely vectors and Odocoileus virginianus (white-tailed deer) as the most likely host sustaining sylvatic transmission. CONCLUSIONS: CVV has a continental-level, widespread transmission potential. Large areas of North America have suitable climate, vectors, and hosts for CVV emergence, establishment, and spread. We identified geographical hotspots that have no confirmed CVV reports to date and, in view of CVV misdiagnosis or underreporting, can guide future surveillance to specific localities and species.

Update to: Assessing the efficacy of male Wolbachia-infected mosquito deployments to reduce dengue incidence in Singapore.

BACKGROUND: This trial is a parallel, two-arm, non-blinded cluster randomised controlled trial that is under way in Singapore, with the aim of measuring the efficacy of male Wolbachia-infected Aedes aegypti deployments in reducing dengue incidence in an endemic setting with all four dengue serotypes in circulation. The trial commenced in July 2022 and is expected to conclude in September 2024. The original study protocol was published in December 2022. Here, we describe amendments that have been made to the study protocol since commencement of the trial. METHODS: The key protocol amendments are (1) addition of an explicit definition of Wolbachia exposure for residents residing in intervention sites based on the duration of Wolbachia exposure at point of testing, (2) incorporation of a high-dimensional set of anthropogenic and environmental characteristics in the analysis plan to adjust for baseline risk factors of dengue transmission, and (3) addition of alternative statistical analyses for endpoints to control for post hoc imbalance in cluster-based environmental and anthropogenic characteristics. DISCUSSION: The findings from this study will provide the first experimental evidence for the efficacy of releasing male-Wolbachia infected mosquitoes to reduce dengue incidence in a cluster-randomised controlled trial. The trial will conclude in 2024 and results will be reported shortly thereafter. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT05505682. Registered on 16 August 2022. Retrospectively registered. Last updated 11 November 2023.

Ingestion of amoxicillin-clavulanic acid at therapeutic concentration during blood meal impacts Aedes aegypti microbiota and dengue virus transmission.

Dengue virus (DENV), mainly transmitted by Aedes aegypti mosquitoes, is the most prevalent arbovirus worldwide, representing a public health problem in tropical and subtropical countries. In these areas, antibiotic consumption rises which may impact both mosquito microbiota and dengue transmission. Here, we assessed how the ingestion by Ae. aegypti of therapeutic concentrations of amoxicillin-clavulanic Acid association (Amox/Clav), a broad-spectrum antibiotic used to treat febrile symptoms worldwide, impacted its microbiota. We also evaluated whether simultaneous ingestion of antibiotic and DENV impacted Ae. aegypti ability to transmit this virus. We found that Amox/Clav ingestion impacted microbiota composition in Ae. aegypti and we confirmed such impact in field-collected mosquitoes. Furthermore, we observed that Amox/Clav ingestion enhanced DENV dissemination and transmission by this mosquito at 21 days post-DENV exposure. These findings increase our understanding of factors linked to human hosts that may influence dengue transmission dynamics in regions with mass-drug administration programs.

Assessing dengue risk globally using non-Markovian models.

Dengue is a vector-borne disease transmitted by Aedes mosquitoes. The worldwide spread of these mosquitoes and the increasing disease burden have emphasized the need for a spatio-temporal risk map capable of assessing dengue outbreak conditions and quantifying the outbreak risk. Given that the life cycle of Aedes mosquitoes is strongly influenced by habitat temperature, numerous studies have utilized temperature-dependent development rates of these mosquitoes to construct virus transmission and outbreak risk models. In this study, we contribute to existing research by developing a mechanistic model for the mosquito life cycle that accurately captures its non-Markovian nature. Beginning with integral equations to track the mosquito population across different life cycle stages, we demonstrate how to derive the corresponding differential equations using phase-type distributions. This approach can be further applied to similar non-Markovian processes that are currently described with less accurate Markovian models. By fitting the model to data on human dengue cases, we estimate several model parameters, allowing the development of a global spatiotemporal dengue risk map. This risk model employs temperature and precipitation data to assess the environmental suitability for dengue outbreaks in a given area.

Virulence and transmission vary between Usutu virus lineages in Culex pipiens.

Usutu virus (USUV) is a zoonotic arbovirus infecting mainly wild birds. It is transmitted by ornithophilic mosquitoes, mainly of the genus Culex from birds to birds and to several vertebrate dead-end hosts. Several USUV lineages, differing in their virulence have emerged in the last decades and now co-circulate in Europe, impacting human populations. However, their relative transmission and effects on their mosquito vectors is still not known. We thus compared the vector competence and survival of Culex pipiens mosquitoes experimentally infected with two distinct USUV lineages, EU2 and EU3, that are known to differ in their virulence and replication in vertebrate hosts. Infection rate was variable among blood feeding assays but variations between EU2 and EU3 lineages were consistent suggesting that Culex pipiens was equally susceptible to infection by both lineages. However, EU3 viral load increased with viral titer in the blood meal while EU2 viral load was high at all titers which suggest a greater replication of EU2 than EU3 in mosquito. While their relative transmission efficiencies are similar, at least at low blood meal titer, positive correlation between transmission and blood meal titer was observed for EU3 only. Contrary to published results in vertebrates, EU3 induced a higher mortality to mosquitoes (i.e. virulence) than EU2 whatever the blood meal titer. Therefore, we found evidence of lineage-specific differences in vectorial capacity and virulence to both the vector and vertebrate host which lead to balanced propagation of both viral lineages. These results highlight the need to decipher the interactions between vectors, vertebrate hosts, and the diversity of arbovirus lineages to fully understand transmission dynamics.

Entomological surveys and insecticide susceptibility profile of Aedes aegypti during the dengue outbreak in Sao Tome and Principe in 2022.

BACKGROUND: The first dengue outbreak in Sao Tome and Principe was reported in 2022. Entomological investigations were undertaken to establish the typology of Aedes larval habitats, the distribution of Ae. aegypti and Ae. albopictus, the related entomological risk and the susceptibility profile of Ae. aegypti to insecticides, to provide evidence to inform the outbreak response. METHODOLOGY/PRINCIPAL FINDINGS: Entomological surveys were performed in all seven health districts of Sao Tome and Principe during the dry and rainy seasons in 2022. WHO tube and synergist assays using piperonyl butoxide (PBO) and diethyl maleate (DEM) were carried out, together with genotyping of F1534C/V1016I/V410L mutations in Ae. aegypti. Aedes aegypti and Ae. albopictus were found in all seven health districts of the country with high abundance of Ae. aegypti in the most urbanised district, Agua Grande. Both Aedes species bred mainly in used tyres, discarded tanks and water storage containers. In both survey periods, the Breteau (BI > 50), house (HI > 35%) and container (CI > 20%) indices were higher than the thresholds established by WHO to indicate high potential risk of dengue transmission. The Ae. aegypti sampled were susceptible to all insecticides tested except dichlorodiphenyltrichloroethane (DDT) (9.2% mortality, resistant), bendiocarb (61.4% mortality, resistant) and alpha-cypermethrin (97% mortality, probable resistant). A full recovery was observed in Ae. aegypti resistant to bendiocarb after pre-exposure to synergist PBO. Only one Ae. aegypti specimen was found carrying F1534C mutation. CONCLUSIONS/SIGNIFICANCE: These findings revealed a high potential risk for dengue transmission throughout the year, with the bulk of larval breeding occurring in used tyres, water storage and discarded containers. Most of the insecticides tested remain effective to control Aedes vectors in Sao Tome, except DDT and bendiocarb. These data underline the importance of raising community awareness and implementing routine dengue vector control strategies to prevent further outbreaks in Sao Tome and Principe, and elsewhere in the subregion.

Bloodmeals fuel dengue virus replication in the female mosquito Aedes aegypti.

Vector competence defines the ability of a vector to acquire, host, and transmit a pathogen. Understanding the molecular determinants of the mosquitos' competence to host dengue virus (DENV) holds promise to prevent its transmission. To this end, we employed RNA-seq to profile mRNA transcripts of the female Aedes aegypti mosquitos feeding on naïve vs viremic mouse. While most transcripts (12,634) did not change their abundances, 360 transcripts showed decreases. Biological pathway analysis revealed representatives of the decreased transcripts involved in the wnt signaling pathway and hippo signaling pathway. One thousand three hundred fourteen transcripts showed increases in abundance and participate in 21 biological pathways including amino acid metabolism, carbon metabolism, fatty acid metabolism, and oxidative phosphorylation. Inhibition of oxidative phosphorylation with antimycin A reduced oxidative phosphorylation activity and ATP concentration associated with reduced DENV replication in the Aedes aegypti cells. Antimycin A did not affect the amounts of the non-structural proteins 3 and 5, two major components of the replication complex. Ribavirin, an agent that reduces GTP concentration, recapitulated the effects of reduced ATP concentration on DENV replication. Knocking down one of the oxidative phosphorylation components, ATP synthase subunit ß, reduced DENV replication in the mosquitos. In summary, our results suggest that DENV enhances metabolic pathways in the female Aedes aegypti mosquitos to supply nutrients and energy for virus replication. ATP synthase subunit ß knockdown might be exploited to reduce the mosquitos' competence to host and transmit DENV. IMPORTANCE: Through evolution, the mosquito-borne viruses have adapted to the blood-feeding behaviors of their opportunist hosts to fulfill a complete lifecycle in humans and mosquitos. Disruption in the mosquitos' ability to host these viruses offers strategies to prevent diseases caused by them. With the advent of genomic tools, we discovered that dengue virus (DENV) benefited from the female mosquitos' bloodmeals for metabolic and energetic supplies for replication. Chemical or genetic disruption in these supplies reduced DENV replication in the female mosquitos. Our discovery can be exploited to produce genetically modified mosquitos, in which DENV infection leads to disruption in the supplies and thereby reduces replication and transmission. Our discovery might be extrapolated to prevent mosquito-borne virus transmission and the diseases they cause.

Mechanisms of Flavivirus Cross-Protection against Yellow Fever in a Mouse Model.

The complete lack of yellow fever virus (YFV) in Asia, and the lack of urban YFV transmission in South America, despite the abundance of the peridomestic mosquito vector Aedes (Stegomyia.) aegypti is an enigma. An immunologically naïve population of over 2 billion resides in Asia, with most regions infested with the urban YF vector. One hypothesis for the lack of Asian YF, and absence of urban YF in the Americas for over 80 years, is that prior immunity to related flaviviruses like dengue (DENV) or Zika virus (ZIKV) modulates YFV infection and transmission dynamics. Here we utilized an interferon α/ß receptor knock-out mouse model to determine the role of pre-existing dengue-2 (DENV-2) and Zika virus (ZIKV) immunity in YF virus infection, and to determine mechanisms of cross-protection. We utilized African and Brazilian YF strains and found that DENV-2 and ZIKV immunity significantly suppresses YFV viremia in mice, but may or may not protect relative to disease outcomes. Cross-protection appears to be mediated mainly by humoral immune responses. These studies underscore the importance of re-assessing the risks associated with YF outbreak while accounting for prior immunity from flaviviruses that are endemic.

Evaluation of Multiple RNA Extraction Protocols for Chikungunya Virus Screening in Aedes aegypti Mosquitoes.

Chikungunya virus (Togaviridae, Alphavirus; CHIKV) is a mosquito-borne global health threat. The main urban vector of CHIKV is the Aedes aegypti mosquito, which is found throughout Brazil. Therefore, it is important to carry out laboratory tests to assist in the virus's diagnosis and surveillance. Most molecular biology methodologies use nucleic acid extraction as the first step and require quality RNA for their execution. In this context, four RNA extraction protocols were evaluated in Ae. aegypti experimentally infected with CHIKV. Six pools were tested in triplicates (n = 18), each containing 1, 5, 10, 20, 30, or 40 mosquitoes per pool (72 tests). Four commercial kits were compared: QIAamp®, Maxwell®, PureLink®, and PureLink® with TRIzol®. The QIAamp® and PureLink® with TRIzol® kits had greater sensitivity. Two negative correlations were observed: as the number of mosquitoes per pool increases, the Ct value decreases, with a higher viral load. Significant differences were found when comparing the purity and concentration of RNA. The QIAamp® protocol performed better when it came to lower Ct values and higher RNA purity and concentration. These results may provide help in CHIKV entomovirological surveillance planning.