First evaluation of antibody responses to Culex quinquefasciatus salivary antigens as a serological biomarker of human exposure to Culex bites: A pilot study in Côte d'Ivoire.

BACKGROUND: Culex mosquitoes are vectors for a variety of pathogens of public health concern. New indicators of exposure to Culex bites are needed to evaluate the risk of transmission of associated pathogens and to assess the efficacy of vector control strategies. An alternative to entomological indices is the serological measure of antibodies specific to mosquito salivary antigens. This study investigated whether the human IgG response to both the salivary gland extract and the 30 kDa salivary protein of Culex quinquefasciatus may represent a proxy of human exposure to Culex bites. METHODOLOGY/PRINCIPAL FINDINGS: A multidisciplinary survey was conducted with children aged 1 to 14 years living in neighborhoods with varying exposure to Culex quinquefasciatus in the city of Bouaké, Côte d'Ivoire. Children living in sites with high exposure to Cx quinquefasciatus had a significantly higher IgG response to both salivary antigens compared with children living in the control site where only very few Culex were recorded. Moreover, children from any Culex-high exposed sites had significantly higher IgG responses only to the salivary gland extract compared with children from the control village, whereas no difference was noted in the anti-30 kDa IgG response. No significant differences were noted in the specific IgG responses between age and gender. Sites and the use of a bed net were associated with the level of IgG response to the salivary gland extract and to the 30 kDa antigen, respectively. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the IgG response to Culex salivary gland extracts is suitable as proxy of exposure; however, the specificity to the Culex genus needs further investigation. The lower antigenicity of the 30 kDa recombinant protein represents a limitation to its use. The high specificity of this protein to the Culex genus makes it an attractive candidate and other specific antibody responses might be more relevant as a biomarker of exposure. These epidemiological observations may form a starting point for additional work on developing serological biomarkers of Culex exposure.

Impacts of fungal entomopathogens on survival and immune responses of Aedes albopictus and Culex pipiens mosquitoes in the context of native Wolbachia infections.

Microbial control of mosquitoes via the use of symbiotic or pathogenic microbes, such as Wolbachia and entomopathogenic fungi, are promising alternatives to synthetic insecticides to tackle the rapid increase in insecticide resistance and vector-borne disease outbreaks. This study evaluated the susceptibility and host responses of two important mosquito vectors, Ae. albopictus and Cx. pipiens, that naturally carry Wolbachia, to infections by entomopathogenic fungi. Our study indicated that while Wolbachia presence did not provide a protective advantage against entomopathogenic fungal infection, it nevertheless influenced the bacterial / fungal load and the expression of select anti-microbial effectors and phenoloxidase cascade genes in mosquitoes. Furthermore, although host responses from Ae. albopictus and Cx. pipiens were mostly similar, we observed contrasting phenotypes with regards to susceptibility and immune responses to fungal entomopathogenic infection in these two mosquitoes. This study provides new insights into the intricate multipartite interaction between the mosquito host, its native symbiont and pathogenic microbes that might be employed to control mosquito populations.

Adaptations to haematophagy: Investigations on how male and female Culex quinquefasciatus (Diptera: Culicidae) deal with human complement activation after a blood meal.

Culex quinquefasciatus is a mosquito species with an anthropophilic habit, often associated with areas with poor sanitation in tropical and urban regions. Adult males and females feed on sugars but only females feed on blood in natural conditions for egg maturation. During haematophagy, female C. quinquefasciatus transmit pathogens such as the West Nile virus, Oropouche virus, various encephalitis viruses, and Wuchereria bancrofti to human hosts. It has been observed in laboratory conditions that male C. quinquefasciatus may feed on blood during an artificial feed. Experiments were carried out to understand how males and females of this species deal with human complement activation. Our results showed that female C. quinquefasciatus, but not males, withstand the stress caused by the ingestion of normal human serum. It was observed that the salivary gland extracts from female mosquitoes were able to inhibit the classical and lectin pathways, whereas male salivary gland extracts only inhibited the lectin pathway. The male and female intestinal contents inhibited the classical and lectin pathways. Neither the salivary glands nor the intestinal contents from males and females showed inhibitory activity towards the alternative pathway. However, the guts of male and female C. quinquefasciatus captured factor H from the human serum, permitting C3b inactivation to its inactive form iC3b, and preventing the formation of the C3 convertase. The activity of the antioxidant enzyme catalase is similar in C. quinquefasciatus females and males. This article shows for the first time that males from a haematophagous arthropod species present human anti-complement activity in their salivary gland extracts and gut contents. The finding of an activity that helps to protect the damage caused by blood ingestion in sugar-feeding male mosquitoes suggests that this may be a pre-adaptation to blood-feeding.

Alteration in the Culex pipiens transcriptome reveals diverse mechanisms of the mosquito immune system implicated upon Rift Valley fever phlebovirus exposure.

Rift Valley fever phlebovirus (RVFV) causes an emerging zoonotic disease and is mainly transmitted by Culex and Aedes mosquitoes. While Aedes aegypti-dengue virus (DENV) is the most studied model, less is known about the genes involved in infection-responses in other mosquito-arboviruses pairing. The main objective was to investigate the molecular responses of Cx. pipiens to RVFV exposure focusing mainly on genes implicated in innate immune responses. Mosquitoes were fed with blood spiked with RVFV. The fully-engorged females were pooled at 3 different time points: 2 hours post-exposure (hpe), 3- and 14-days post-exposure (dpe). Pools of mosquitoes fed with non-infected blood were also collected for comparisons. Total RNA from each mosquito pool was subjected to RNA-seq analysis and a de novo transcriptome was constructed. A total of 451 differentially expressed genes (DEG) were identified. Most of the transcriptomic alterations were found at an early infection stage after RVFV exposure. Forty-eight DEG related to immune infection-response were characterized. Most of them were related with the RNAi system, Toll and IMD pathways, ubiquitination pathway and apoptosis. Our findings provide for the first time a comprehensive view on Cx. pipiens-RVFV interactions at the molecular level. The early depletion of RNAi pathway genes at the onset of the RVFV infection would allow viral replication in mosquitoes. While genes from the Toll and IMD immune pathways were altered in response to RVFV none of the DEG were related to the JAK/STAT pathway. The fact that most of the DEG involved in the Ubiquitin-proteasome pathway (UPP) or apoptosis were found at an early stage of infection would suggest that apoptosis plays a regulatory role in infected Cx. pipiens midguts. This study provides a number of target genes that could be used to identify new molecular targets for vector control.

"Submergence" of Western equine encephalitis virus: Evidence of positive selection argues against genetic drift and fitness reductions.

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.

Wolbachia limits pathogen infections through induction of host innate immune responses.

BACKGROUND: Wolbachia has been reported to suppress a variety of pathogen infections in mosquitoes, but the mechanism is undefined. Two possibilities have been proposed. One is that Wolbachia activates host immune responses, and the other one is that Wolbachia competes with pathogens for limited nutrients. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we compared host immune responses and the densities of two different strains of Wolbachia in naturally occurring parental and artificially created hybrid host genetic backgrounds. No significant difference in Wolbachia density was found between these hosts. We found that Wolbachia could activate host innate immune responses when the host genetic profile was different from that of its natural host. When these hosts were challenged with pathogenic bacteria, mosquitoes in new host-Wolbachia symbioses had a higher survival rate than in old host-Wolbachia symbioses. CONCLUSIONS/SIGNIFICANCE: The presence of Wolbachia per se does not necessarily affect pathogen infections, suggesting that a competition for limited nutrients is not the main reason for Wolbachia-mediated pathogen suppression. Instead, host immune responses are responsible for it. The elucidation of an immunity nature of PI is important to guide future practice: Wolbachia may be genetically engineered to be more immunogenic, it is desired to search and isolate more strains of Wolbachia, and test more host-Wolbachia symbioses for future applications. Our results also suggest Wolbachia-based PI may be applied to naturally Wolbachia-infected mosquito populations, and extend to the control of a broader range of mosquito-borne diseases.

Insights into the impact of Ivermectin on some protein aspects linked to Culex pipiens digestion and immunity.

In developing countries, low-cost control and treatment programs that offer combined approaches against diseases and their vectors are certainly needed. Ivermectin (IVM) has been well known for its role in the treatment of parasitic diseases, due to its effect on glutamate-gated chloride channels. These same channels are also present in the mosquito vector, and thus, research has focused on the insecticidal effects of this drug. Possible alternative mechanisms of IVM on the physiology of mosquitoes, however, have not been sufficiently elaborated. We assessed the protease activity, lipid peroxidation, and local expression of STAT, p53, caspase-3, and Bax markers to study the effect of this antibiotic on digestion and immunity in Culex pipiens. Sugar- and blood-feeding assays were employed to investigate the potential influence of blood feeding on the dynamics of these parameters. IVM was found to have an effect on protease activity, lipid peroxidation as well as the expression of different markers investigated in this work. The focus on the detailed effect of this drug certainly opens the gate to broadening the spectrum of IVM and expanding its health and economic benefit, especially that it is relatively more affordable than other antibiotics on the market.

Insulin Potentiates JAK/STAT Signaling to Broadly Inhibit Flavivirus Replication in Insect Vectors.

The World Health Organization estimates that more than half of the world's population is at risk for vector-borne diseases, including arboviruses. Because many arboviruses are mosquito borne, investigation of the insect immune response will help identify targets to reduce the spread of arboviruses. Here, we use a genetic screening approach to identify an insulin-like receptor as a component of the immune response to arboviral infection. We determine that vertebrate insulin reduces West Nile virus (WNV) replication in Drosophila melanogaster as well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, we show that insulin signaling activates the JAK/STAT, but not RNAi, pathway via ERK to control infection in Drosophila cells and Culex mosquitoes through an integrated immune response. Finally, we validate that insulin priming of adult female Culex mosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to human pathogens.

Evaluation of environment safety of a Japanese encephalitis live attenuated vaccine.

JE vaccination is the most effective and economical method of preventing JE. A live attenuated JE vaccine has been widely used in many countries since 1989, playing an important role in controlling JE outbreaks. However, whether the large-scale use of the live attenuated JE vaccine will lead to the dissemination of the vaccine virus in the environment and whether reversion of the neuroattenuation of the virus will occur during the transmission process remain major concerns for some researchers. To evaluate the transmission of a live attenuated JEV vaccine in mosquitoes and hosts, JE SA14-14-2 attenuated vaccine virus was intrathoracically (i.t.) inoculated into Culex tritaeniorhynchus, a native vector. Subsequently, virus harvested from inoculated mosquitoes was inoculated into pigs, a mammalian reservoir. The virus was isolated from the pigs and passaged once again in Culex tritaeniorhynchus. The genome sequences and virulence of the passaged viruses were then investigated. While a few nucleotide substitutions occurred during passaging, there was no change in the encoded amino acids. After intracerebral (i.c.) inoculation of mice with the vaccine, no pathological effects were observed. In addition, virus virulence remained low after inoculation of suckling mouse brains. These results indicate that vaccination of individuals with the live vaccine will not result in transmission of the live SA14-14-2 vaccine virus through mosquito biting and virus amplified in pigs.

Curious entanglements: interactions between mosquitoes, their microbiota, and arboviruses.

Mosquitoes naturally harbor a diverse community of microorganisms that play a crucial role in their biology. Mosquito-microbiota interactions are abundant and complex. They can dramatically alter the mosquito immune response, and impede or enhance a mosquito's ability to transmit medically important arboviral pathogens. Yet critically, given the massive public health impact of arboviral disease, few such interactions have been well characterized. In this review, we describe the current state of knowledge of the role of microorganisms in mosquito biology, how microbial-induced changes to mosquito immunity moderate infection with arboviruses, cases of mosquito-microbial-virus interactions with a defined mechanism, and the molecular interactions that underlie the endosymbiotic bacterium Wolbachia's ability to block virus infection in mosquitoes.

Small RNA responses of Culex mosquitoes and cell lines during acute and persistent virus infection.

RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication of different viruses.

Presencia larval de Culex (Culex) interrogator (Dyar and Knab) (Diptera: Culicidae) en Cuba / Larval presence of Culex (Culex) interrogator (Dyar and Knab) (Diptera: Culicidae) in Cuba

Actualmente se conocen más de 3 500 especies de mosquitos y cada día se registran otras de importancia en la transmisión de enfermedades al hombre y los animales. El objetivo de este trabajo es registrar por primera vez la presencia larval de Cx interrogator en la provincia de Santiago de Cuba y en Cuba. Para ello se realizaron los muestreos larvarios en sitios de cría naturales y artificiales con frecuencia semanal durante el 2013-2017, según lo establecido por el Programa de Vigilancia y Control de otros culícidos para la provincia de Santiago de Cuba y adjunto al Programa de Control de Aedes aegypti y Aedes albopictus en Cuba. Las larvas se enviaron al Laboratorio Provincial de Entomología de Santiago de Cuba para su clasificación utilizando claves morfológicas. Posteriormente el material biológico fue enviado al Laboratorio de Referencia Nacional del Instituto de Medicina Tropical Pedro Kourí donde se verificó la identificación taxonómica. Culex interrogator fue colectado en los municipios Palma Soriano y San Luis en el 2013 en Santiago de Cuba, y Songo la Maya y II Frente en 2015 y 2017, respectivamente, todos pertenecientes a la provincia Santiago de Cuba. Los sitios de cría correspondieron a fosas sépticas, sótanos inundados debajo de edificios, lagunas de oxidación, pomos plásticos, zanjas, charcos y cañadas. Se encontró asociado en sitios de cría naturales con Culex nigripalpus, Culex quinquefasciatus y Anopheles albimanus. En conclusión, Cx interrogator constituye un nuevo registro para la entomofauna cubana, de ahí la importancia de ampliar los conocimientos sobre su distribución, asociación con otras especies de culícidos y posible rol en la transmisión de enfermedades al hombre(AU)

Nowadays there are more than 3 500 known species of mosquitoes and everyday are registered some more of importance in disease transmission to men and animals every day. The aim of this study is to register for the first time larval presence of Cx. interrogator in Santiago de Cuba province and in Cuba. In this regard, larval sampling was carried out by the established Surveillance and Control Program of other mosquitoes in Santiago de Cuba province and attached to the Control Program of Aedes aegypti and Aedes albopictus in Cuba; its was carried out in natural and artificial breeding sites with weekly frequency during 2013-2017. The larvae were sent to the Provincial Laboratory of Enthomology of Santiago de Cuba for classification using morphological keys. Subsequently, the biological material was sent to the National Reference Laboratory of Pedro Kourí Institute where the taxonomic identification was verified. Culex interrogator was collected in Palma Soriano and San Luis municipalities in 2013, in Santiago de Cuba, and Songo la Maya and II Frente in 2015 and 2017, respectively, all belonging to Santiago de Cuba province. The breeding sites were septic tanks, flooded basements under buildings, oxidation ponds, plastic bottles, ditches, puddles and streams. This mosquito was found in natural breeding sites associated to Culex nigripalpus, Culex quinquefasciatus and Anopheles albimanus. As a conclusion, it can be said that Cx. interrogator constitutes a new registry for Cuban entomofauna, hence the importance of expanding knowledge about its distribution, association with other culicid´s species and its possible role in the transmission of diseases to man(AU)

Highly specific host-pathogen interactions influence Metarhizium brunneum blastospore virulence against Culex quinquefasciatus larvae.

Entomopathogenic fungi are potential biological control agents of mosquitoes. Our group observed that not all mosquitoes were equally susceptible to fungal infection and observed significant differences in virulence of different spore types. Conidiospores and blastospores were tested against Culex quinquefasciatus larvae. Blastospores are normally considered more virulent than conidia as they form germ tubes and penetrate the host integument more rapidly than conidia. However, when tested against Cx. quinquefasciatus, blastospores were less virulent than conidia. This host-fungus interaction was studied by optical, electron and atomic force microscopy (AFM). Furthermore, host immune responses and specific gene expression were investigated. Metarhizium brunneum (formerly M. anisopliae) ARSEF 4556 blastospores did not readily adhere to Culex larval integument and the main route of infection was through the gut. Adhesion forces between blastospores and Culex cuticle were significantly lower than for other insects. Larvae challenged with blastospores showed enhanced immune responses, with increased levels of phenoloxidase, glutathione-S-transferase, esterase, superoxide dismutase and lipid peroxidase activity. Interestingly, M. brunneum pathogenicity/stress-related genes were all down-regulated in blastospores exposed to Culex. Conversely, when conidia were exposed to Culex, the pathogenicity genes involved in adhesion or cuticle degradation were up-regulated. Delayed host mortality following blastospore infection of Culex was probably due to lower adhesion rates of blastospores to the cuticle and enhanced host immune responses deployed to counter infection. The results here show that subtle differences in host-pathogen interactions can be responsible for significant changes in virulence when comparing mosquito species, having important consequences for biological control strategies and the understanding of pathogenicity processes.

Conglomeration of novel Culex quinquefasciatus salivary proteins to contrive multi-epitope subunit vaccine against infections caused by blood imbibing transmitter.

The southern house vector, Culex quinquefasciatus is the paramount cause of Japanese encephalitis, West Nile fever and Lymphatic Filariasis, which is globally affecting the worldwide population. Many attempts were made by researchers with different perceptions to discover regimen against these aforementioned ailments but the output was not that effectual. Consequently, there is an imminent need to develop very effective and potential treatment against these perilous diseases. Employing immunoinformatic approaches, we have designed the multi-epitope subunit vaccine by exploring salivary proteins of Culex quinquefasciatus, which possess both antigenic and potent immunogenic behaviour. The immunogenic epitopes from the immune cells (B-cell, CTL, and HTL) were predicted and linked together with the help of linkers. Apart from this, at the N-terminal of the construct, an adjuvant was added in order to enhance the immunogenicity of the vaccine. The physiological parameters, antigenicity and allergenicity were also evaluated for the designed vaccine construct. Molecular docking between ligand (vaccine construct) and TLR-4 receptor was performed. Molecular dynamics simulation of the docked complex was performed to identify the stability, patterns, macromolecules interactions and their behaviour. Finally, to ensure the translation and gene expression efficiency of designed construct, insilico restriction cloning was executed into suitable expression vector pET28a.

Are tree squirrels involved in the circulation of flaviviruses in Italy?

West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) are emerging zoonotic flaviviruses (family Flaviviridae), which have circulated in Europe in the past decade. A cross-sectional study was conducted to assess exposure to these antigenically related flaviviruses in eastern grey squirrels (Sciurus carolinensis) in Italy. Seventeen out of 158 (10.8%; CI95% : 5.9-15.6) squirrels' sera tested through bELISA had antibodies against flaviviruses. Specific neutralizing antibodies to WNV, USUV and TBEV were detected by virus neutralization tests. Our results indicate that tree squirrels are exposed to Culex and tick-borne zoonotic flaviviruses in Italy. Moreover, this study shows for the first time USUV and TBEV exposure in grey squirrels, broadening the host range reported for these viruses. Even though further studies are needed to define the real role of tree squirrels in the epidemiology of flaviviruses in Europe, this study highlights that serology could be an effective approach for future investigations aimed at broadening our knowledge about the species exposed to these zoonotic infections.

Assessment of the ability of V920 recombinant vesicular stomatitis-Zaire ebolavirus vaccine to replicate in relevant arthropod cell cultures and vector species.

V920, rVSVΔG-ZEBOV-GP, is a recombinant vesicular stomatitis-Zaire ebolavirus vaccine which has shown an acceptable safety profile and provides a protective immune response against Ebola virus disease (EVD) induced by Zaire ebolavirus in humans. The purpose of this study was to determine whether the V920 vaccine is capable of replicating in arthropod cell cultures of relevant vector species and of replicating in live mosquitoes. While the V920 vaccine replicated well in Vero cells, no replication was observed in Anopheles or Aedes mosquito, Culicoides biting midge, or Lutzomyia sand fly cells, nor in live Culex or Aedes mosquitoes following exposure through intrathoracic inoculation or feeding on a high-titer infectious blood meal. The insect taxa selected for use in this study represent actual and potential epidemic vectors of VSV. V920 vaccine inoculated into Cx. quinquefasciatus and Ae. aegypti mosquitoes demonstrated persistence of replication-competent virus following inoculation, consistent with the recognized biological stability of the vaccine, but no evidence for active virus replication in live mosquitoes was observed. Following administration of an infectious blood meal to Ae. aegypti and Cx. quinquefasciatus mosquitoes at a titer several log10 PFU more concentrated than would be observed in vaccinated individuals, no infection or dissemination of V920 was observed in either mosquito species. In vitro and in vivo data gathered during this study support minimal risk of the vector-borne potential of the V920 vaccine.

Flaviviruses Produce a Subgenomic Flaviviral RNA That Enhances Mosquito Transmission.

Mosquito-borne flaviviruses (MBFVs) are a global public health burden. MBFVs have several unique 3'UTR structures that inhibit the host RNA decay machinery to produce subgenomic flaviviral RNAs (sfRNAs). Number of sfRNA species and their relative quantities are dependent on the 3'UTR tertiary structures and can vary between tissues. Two recent in vivo studies demonstrated that sfRNA enhances mosquito transmission, resulting in increased infection rate of saliva. Transmission efficiency is determined by the immune response. First evidence points to sfRNA interference with the Toll and RNAi immune pathways. However, a more complex picture that includes flexibility in sfRNA production and interaction with immune-related proteins remains to be explored.

Blood meal acquisition enhances arbovirus replication in mosquitoes through activation of the GABAergic system.

Mosquitoes are hematophagous insects that carry-on and transmit many human viruses. However, little information is available regarding the common mechanisms underlying the infection of mosquitoes by these viruses. In this study, we reveal that the hematophagous nature of mosquitoes contributes to arboviral infection after a blood meal, which suppresses antiviral innate immunity by activating the GABAergic pathway. dsRNA-mediated interruption of the GABA signaling and blockage of the GABAA receptor by the specific inhibitors both significantly impaired arbovirus replication. Consistently, inoculation of GABA enhanced arboviral infection, indicating that GABA signaling facilitates the arboviral infection of mosquitoes. The ingestion of blood by mosquitoes resulted in robust GABA production from glutamic acid derived from blood protein digestion. The oral introduction of glutamic acid increased virus acquisition by mosquitoes via activation of the GABAergic system. Our study reveals that blood meals enhance arbovirus replication in mosquitoes through activation of the GABAergic system.

Iron availability affects West Nile virus infection in its mosquito vector.

BACKGROUND: Mosquitoes are responsible for transmission of viruses, including dengue, West Nile and chikungunya viruses. Female mosquitoes are infected when they blood-feed on vertebrates, a required step for oogenesis. During this process, mosquitoes encounter high iron loads. Since iron is an essential nutrient for most organisms, including pathogens, one of the defense mechanisms for the host includes sequestration of iron away from the invading pathogen. Here, we determine whether iron availability affects viral replication in mosquitoes. METHODS: To elucidate effect of iron availability on mosquito cells during infection, Culex cells were treated with either ferric ammonium citrate (FAC) or the iron chelator, deferoxamine (DFX). Real time RT-PCR was performed using ferritin (heavy chain) and NRAMP as a measure of iron homeostasis in cells. To determine iron requirement for viral replication, Culex cells were knocked down for NRAMP using dsRNA. Finally, the results were validated in Culex mosquito-infection model, by treating infected mosquitoes with DFX to reduce iron levels. RESULTS: Our results show that infection of Culex cells led to induction in levels of ferritin (heavy chain) and NRAMP mRNAs in time-dependent manner. Results also showed that treatment of cells with FAC, reduced expression of NRAMP (iron transporter) and increase levels of ferritin (heavy chain). Interestingly, increasing iron levels increased viral titers; while reducing intracellular iron levels, either by NRAMP knock-down or using DFX, reduced viral titers. The results from Culex mosquito infection showed that mosquitoes treated with DFX had reduced viral titers compared with untreated controls in midgut as well as carcass 8 days pi. Saliva from mosquitoes treated with DFX also showed reduced viral titers compared with untreated controls, indicating low viral transmission capacity. CONCLUSIONS: Our results indicate that iron is required for viral replication in mosquito cells. Mosquitoes respond to viral infection, by inducing expression of heavy chain ferritin, which sequesters available iron, reducing its availability to virus infected cells. The data indicates that heavy chain ferritin may be part of an immune mechanism of mosquitoes in response to viral infections.

Transmission Incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for Zika Virus.

AbstractIn late 2014, Zika virus (ZIKV; Flaviviridae, Flavivirus) emerged as a significant arboviral disease threat in the Western hemisphere. Aedes aegypti and Aedes albopictus have been considered the principal vectors of ZIKV in the New World due to viral isolation frequency and vector competence assessments. Limited reports of Culex transmission potential have highlighted the need for additional vector competence assessments of North American Culex species. Accordingly, North American Culex pipiens and Culex quinquefasciatus were orally exposed and intrathoracically inoculated with the African prototype ZIKV strain and currently circulating Asian lineage ZIKV strains to assess infection, dissemination, and transmission potential. Results indicated that these two North American Culex mosquito species were highly refractory to oral infection with no dissemination or transmission observed with any ZIKV strains assessed. Furthermore, both Culex mosquito species intrathoracically inoculated with either Asian or African lineage ZIKVs failed to expectorate virus in saliva. These in vivo results were further supported by the observation that multiple mosquito cell lines of Culex species origin demonstrated significant growth restriction of ZIKV strains compared with Aedes-derived cell lines. In summation, no evidence for the potential of Cx. pipiens or Cx. quinquefasciatus to serve as a competent vector for ZIKV transmission in North America was observed.