Activation of the autophagy pathway decreases dengue virus infection in Aedes aegypti cells.

BACKGROUND: Mosquito-borne dengue virus (DENV) causes major disease worldwide, impacting 50-100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology, including antiviral mechanisms, and developing new control strategies have become an important step towards the elimination of DENV disease. In the study reported here, we focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential antiviral target in the mosquito. METHODS: To understand the role played by autophagy in Ae. aegypti, we examined the activation of this pathway in Aag-2 cells, an Ae. aegypti-derived cell line, infected with DENV. Rapamycin and 3-methyladenine, two small molecules that have been shown to affect the function of the autophagy pathway, were used to activate or suppress, respectively, the autophagy pathway. RESULTS: At 1-day post-DENV infection in Aag-2 cells, transcript levels of both the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) and autophagy-related protein 1 (ATG1) increased. Rapamycin treatment activated the autophagy pathway as early as 1-h post-treatment, and the virus titer had decreased in the Aag-2 cells at 2 days post-infection; in contrast, the 3-methyladenine treatment did not significantly affect the DENV titer. Treatment with these small molecules also impacted the ATG12 transcript levels in DENV-infected cells. CONCLUSIONS: Our studies revealed that activation of the autophagy pathway through rapamycin treatment altered DENV infection in the mosquito cells, suggesting that this pathway could be a possible antiviral mechanism in the mosquito system. Here we provide fundamental information needed to proceed with future experiments and to improve our understanding of the mosquito's immune response against DENV.

Mammals Preferred: Reassortment of Batai and Bunyamwera orthobunyavirus Occurs in Mammalian but Not Insect Cells.

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the Bunyamwera orthobunyavirus and the Batai orthobunyavirus, can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of Bunyamwera orthobunyavirus and the Batai orthobunyavirus during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (Mesocricetus auratus). In contrast, no reassortment was observed in the examined insect cells from Aedes aegypti (Aag2) and Aedes albopictus (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.

Human blood microRNA hsa-miR-21-5p induces vitellogenin in the mosquito Aedes aegypti.

Mosquito vectors transmit various diseases through blood feeding, required for their egg development. Hence, blood feeding is a major physiological event in their life cycle, during which hundreds of genes are tightly regulated. Blood is a rich source of proteins for mosquitoes, but also contains many other molecules including microRNAs (miRNAs). Here, we found that human blood miRNAs are transported abundantly into the fat body tissue of Aedes aegypti, a key metabolic center in post-blood feeding reproductive events, where they target and regulate mosquito genes. Using an artificial diet spiked with the mimic of an abundant and stable human blood miRNA, hsa-miR-21-5p, and proteomics analysis, we found over 40 proteins showing differential expression in female Ae. aegypti mosquitoes after feeding. Of interest, we found that the miRNA positively regulates the vitellogenin gene, coding for a yolk protein produced in the mosquito fat body and then transported to the ovaries as a protein source for egg production. Inhibition of hsa-miR-21-5p followed by human blood feeding led to a statistically insignificant reduction in progeny production. The results provide another example of the involvement of small regulatory molecules in the interaction of taxonomically vastly different taxa.

Distinct Roles of Hemocytes at Different Stages of Infection by Dengue and Zika Viruses in Aedes aegypti Mosquitoes.

Aedes aegypti mosquitoes are vectors for arboviruses of medical importance such as dengue (DENV) and Zika (ZIKV) viruses. Different innate immune pathways contribute to the control of arboviruses in the mosquito vector including RNA interference, Toll and Jak-STAT pathways. However, the role of cellular responses mediated by circulating macrophage-like cells known as hemocytes remains unclear. Here we show that hemocytes are recruited to the midgut of Ae. aegypti mosquitoes in response to DENV or ZIKV. Blockade of the phagocytic function of hemocytes using latex beads induced increased accumulation of hemocytes in the midgut and a reduction in virus infection levels in this organ. In contrast, inhibition of phagocytosis by hemocytes led to increased systemic dissemination and replication of DENV and ZIKV. Hence, our work reveals a dual role for hemocytes in Ae. aegypti mosquitoes, whereby phagocytosis is not required to control viral infection in the midgut but is essential to restrict systemic dissemination. Further understanding of the mechanism behind this duality could help the design of vector-based strategies to prevent transmission of arboviruses.

The number of neurons in Drosophila and mosquito brains.

Various insect species serve as valuable model systems for investigating the cellular and molecular mechanisms by which a brain controls sophisticated behaviors. In particular, the nervous system of Drosophila melanogaster has been extensively studied, yet experiments aimed at determining the number of neurons in the Drosophila brain are surprisingly lacking. Using isotropic fractionator coupled with immunohistochemistry, we counted the total number of neuronal and non-neuronal cells in the whole brain, central brain, and optic lobe of Drosophila melanogaster. For comparison, we also counted neuronal populations in three divergent mosquito species: Aedes aegypti, Anopheles coluzzii and Culex quinquefasciatus. The average number of neurons in a whole adult brain was determined to be 199,380 ±3,400 cells in D. melanogaster, 217,910 ±6,180 cells in Ae. aegypti, 223,020 ± 4,650 cells in An. coluzzii and 225,911±7,220 cells in C. quinquefasciatus. The mean neuronal cell count in the central brain vs. optic lobes for D. melanogaster (101,140 ±3,650 vs. 107,270 ± 2,720), Ae. aegypti (109,140 ± 3,550 vs. 112,000 ± 4,280), An. coluzzii (105,130 ± 3,670 vs. 107,140 ± 3,090), and C. quinquefasciatus (108,530 ±7,990 vs. 110,670 ± 3,950) was also estimated. Each insect brain was comprised of 89% ± 2% neurons out of its total cell population. Isotropic fractionation analyses did not identify obvious sexual dimorphism in the neuronal and non-neuronal cell population of these insects. Our study provides experimental evidence for the total number of neurons in Drosophila and mosquito brains.

Insect-Specific Flavivirus Replication in Mammalian Cells Is Inhibited by Physiological Temperature and the Zinc-Finger Antiviral Protein.

The genus Flavivirus contains pathogenic vertebrate-infecting flaviviruses (VIFs) and insect-specific flaviviruses (ISF). ISF transmission to vertebrates is inhibited at multiple stages of the cellular infection cycle, via yet to be elucidated specific antiviral responses. The zinc-finger antiviral protein (ZAP) in vertebrate cells can bind CpG dinucleotides in viral RNA, limiting virus replication. Interestingly, the genomes of ISFs contain more CpG dinucleotides compared to VIFs. In this study, we investigated whether ZAP prevents two recently discovered lineage II ISFs, Binjari (BinJV) and Hidden Valley viruses (HVV) from replicating in vertebrate cells. BinJV protein and dsRNA replication intermediates were readily observed in human ZAP knockout cells when cultured at 34 °C. In ZAP-expressing cells, inhibition of the interferon response via interferon response factors 3/7 did not improve BinJV protein expression, whereas treatment with kinase inhibitor C16, known to reduce ZAP's antiviral function, did. Importantly, at 34 °C, both BinJV and HVV successfully completed the infection cycle in human ZAP knockout cells evident from infectious progeny virus in the cell culture supernatant. Therefore, we identify vertebrate ZAP as an important barrier that protects vertebrate cells from ISF infection. This provides new insights into flavivirus evolution and the mechanisms associated with host switching.

A Survey of Chemoreceptive Responses on Different Mosquito Appendages.

Research on the functions of insect chemoreceptors have primarily focused on antennae (olfactory receptors) and mouthparts (gustatory receptors). However, chemoreceptive sensilla are also present on other appendages, such as the leg tarsi and the anterior wing margin, and their specific roles in chemoreception and mosquito behavior remain largely unknown. In this study, electrophysiological analyses in an electroantennogram recording format were performed on Aedes aegypti (L., Diptera: Culicidae) antennae, mouthparts, tarsi, and wings during exposure to a variety of insect repellent and attractant compounds. The results provide evidence that the tarsi and wings can sense chemicals in a gaseous form, and that the odors produce differing responses on different appendages. The most consistent and strongest response occurred when exposed to triethylamine (TEA). Antennae and mouthparts showed nearly identical responses pattern to all tested compounds, and their rank orders of effectiveness were similar to those of fore- and mid-leg tarsi. Hindleg tarsi only responded to TEA, indicating that the hind legs are not as chemoreceptive. Wings responded to a range of odorants, but with a different rank order and voltage amplitude. Insights gleaned into the function of these appendages in insect chemoreception are discussed.

Establishment and characterization of a cell line from Ghanaian Aedes aegypti (Diptera: Culicidae) focusing on Aedes-borne flavivirus susceptibility.

Mosquitoes are generally considered one of the most important vectors of arboviruses, with Aedes aegypti regarded as the most important in transmission of yellow fever and dengue viruses. To investigate why there are differences in the incidence of dengue fever and Zika in different geographical areas and an absence of outbreaks in Ghana in spite of an abundance of A. aegypti mosquitoes, we established a continuous cell line from embryonic cells of A. aegypti collected in Ghana and assessed its susceptibility to dengue, yellow fever, and Zika viruses. The new cell line (designated AeAe-GH98), having an adhesive spindle-shaped web-like morphology, was serially subcultured in both VP-12 and Schneider's medium supplemented with 10% heat-inactivated fetal bovine serum. AeAe-GH98 cells were found to have a population doubling time of 1.3 d during exponential growth. The mosquito colony used to establish the cell line was confirmed to have originated from Africa using microsatellite assay. In terms of susceptibility to Aedes-borne flaviviruses, AeAe-GH98 cells were found to have different degrees of susceptibility to yellow fever, Zika, and dengue virus infection and propagation. While susceptibility of AeAe-GH98 cells to yellow fever and Zika viruses was comparable with that of C6/36 cells, susceptibility to dengue virus was significantly lower. This cell line will serve as a useful tool for determining molecular factors influencing virus-vector susceptibility in vitro.

Biodistribution of degradable polyanhydride particles in Aedes aegypti tissues.

Insecticide resistance poses a significant threat to the control of arthropods that transmit disease agents. Nanoparticle carriers offer exciting opportunities to expand the armamentarium of insecticides available for public health and other pests. Most chemical insecticides are delivered by contact or feeding, and from there must penetrate various biological membranes to reach target organs and kill the pest organism. Nanoparticles have been shown to improve bioactive compound navigation of such barriers in vertebrates, but have not been well-explored in arthropods. In this study, we explored the potential of polyanhydride micro- and nanoparticles (250 nm- 3 µm), labeled with rhodamine B to associate with and/or transit across insect biological barriers, including the cuticle, epithelium, midgut and ovaries, in female Ae. aeygpti mosquitoes. Mosquitoes were exposed using conditions to mimic surface contact with a residual spray or paint, topical exposure to mimic contact with aerosolized insecticide, or per os in a sugar meal. In surface contact experiments, microparticles were sometimes observed in association with the exterior of the insect cuticle. Nanoparticles were more uniformly distributed across exterior tissues and present at higher concentrations. Furthermore, by surface contact, topical exposure, or per os, particles were detected in internal organs. In every experiment, amphiphilic polyanhydride nanoparticles associated with internal tissues to a higher degree than hydrophobic nanoparticles. In vitro, nanoparticles associated with Aedes aegypti Aag2 cells within two hours of exposure, and particles were evident in the cytoplasm. Further studies demonstrated that particle uptake is dependent on caveolae-mediated endocytosis. The propensity of these nanoparticles to cross biological barriers including the cuticle, to localize in target tissue sites of interest, and to reach the cytoplasm of cells, provides great promise for targeted delivery of insecticidal candidates that cannot otherwise reach these cellular and subcellular locations.

Cell-to-Cell Spread of Dengue Viral RNA in Mosquito Cells.

Dengue virus (DENV) is an important mosquito-borne arbovirus that is particularly prevalent in tropical and subtropical areas of the world. The virus is generally ingested with a blood meal, replicates in host tissues, and disseminates into salivary glands for transmission to the next host. Membrane-bound vacuoles carrying DENV particles have been documented in mosquito cells and play a role in the cell-to-cell transmission of DENV2. C189 is one member of the tetraspanin family and generally increases its expression as one component of the vacuoles (C189-VCs) within C6/36 cells infected with DENV2. In the present study, we have further demonstrated via sucrose gradient centrifugation as well as magnetic immune isolation (MI) that the RNA of DENV2 was eventually carried by C189-VCs. In addition, viral RNA was shown to spread from donor to recipient cells in a coculture assay even when 20 mM NH4Cl was added to inhibit virus replication in the culture. In an alternate assay using the transwell system, viral RNA was only detected in recipient cells in the absence of 40 mM NH4Cl, suggesting that cell-cell contact is required for the intercellular spread of DENV2. In turn, the formation of viral synapse (VS) derived from aggregates of viral particles was frequently observed at sites of cell contact. Taken together, the formation of C189-VCs in C6/36 cells is induced by DENV2 infection, which may serve as a vehicle for transferring virions and also viral RNA to neighboring cells by cell-to-cell transmission after cell-cell contact. This finding provides insight into the understanding of viral spread between mosquito cells. It may also elucidate the benign persistent infection in mosquito cells and efficient dissemination of DENV infection within a mosquito vector.

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus.

BACKGROUND: Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. METHODS: After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. RESULTS: After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. CONCLUSIONS: To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation.

Zika virus noncoding RNA suppresses apoptosis and is required for virus transmission by mosquitoes.

Flaviviruses, including Zika virus (ZIKV), utilise host mRNA degradation machinery to produce subgenomic flaviviral RNA (sfRNA). In mammalian hosts, this noncoding RNA facilitates replication and pathogenesis of flaviviruses by inhibiting IFN-signalling, whereas the function of sfRNA in mosquitoes remains largely elusive. Herein, we conduct a series of in vitro and in vivo experiments to define the role of ZIKV sfRNA in infected Aedes aegypti employing viruses deficient in production of sfRNA. We show that sfRNA-deficient viruses have reduced ability to disseminate and reach saliva, thus implicating the role for sfRNA in productive infection and transmission. We also demonstrate that production of sfRNA alters the expression of mosquito genes related to cell death pathways, and prevents apoptosis in mosquito tissues. Inhibition of apoptosis restored replication and transmission of sfRNA-deficient mutants. Hence, we propose anti-apoptotic activity of sfRNA as the mechanism defining its role in ZIKV transmission.

Glycogen synthase kinase 3ß participates in late stages of Dengue virus-2 infection.

BACKGROUND: Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3ß, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3ß during Dengue virus-2 (DENV-2) infection was investigated. METHODS: GSK3ß participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS: Phosphorylation of GSK3ß-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3ß reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3ß. CONCLUSIONS: The results suggest that GSK3ß participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.

Identification and genetic analysis of a totivirus isolated from the Culex tritaeniorhynchus in northern China.

Totiviridae, a viral family of double-stranded RNA (dsRNA) viruses, contain a single dsRNA genome 4.6-7.0 kb in length. Totiviridae were initially only known to infect fungi and other eukaryotes as well as plants, but an increase in totiviruses has been detected in insects, mosquitoes, and bats. Here, we describe the isolation and characterization of a strain belonging to the family Totiviridae isolated from Culex tritaeniorhynchus in Kenli, China, in 2016. We isolated a totivirus from field-collected mosquitoes in China by cell culture in Aedes albopictus C6/36 cells, identified the virus by morphological observation and complete genome sequencing, and characterized it by phylogenetic analysis. Transmission electron microscopy identified icosahedral, non-enveloped virus particles with a mean diameter of 35-40 nm. The genome was 7612 bp in length, including two open reading frames (ORFs). ORF1 (5058 nt) encodes the capsid protein, while ORF2 (2216 nt) encodes the viral RNA-dependent RNA polymerase (RdRp). Nucleotide and amino acid homology analysis of isolate showed higher levels of sequence identity with isolate CTV_NJ2 (China, 2010) with 94.87% nucleic acid identity and 97.32% amino acid identity. The isolate was designated C. tritaeniorhynchus totivirus KL (CTV-KL). This is the first identification of a totivirus in a C. tritaeniorhynchus in northern China. Analysis of the virus's morphology, characteristic and genome organization will further enrich our understanding of the molecular and biological characteristics of dsRNA Totiviridae viruses.

Glycogen synthase kinase 3ß participates in late stages of Dengue virus-2 infection

BACKGROUND Viruses can modulate intracellular signalling pathways to complete their infectious cycle. Among these, the PI3K/Akt pathway allows prolonged survival of infected cells that favours viral replication. GSK3β, a protein kinase downstream of PI3K/Akt, gets inactivated upon activation of the PI3K/Akt pathway, and its association with viral infections has been recently established. In this study, the role of GSK3β during Dengue virus-2 (DENV-2) infection was investigated. METHODS GSK3β participation in the DENV-2 replication process was evaluated with pharmacological and genetic inhibition during early [0-12 h post-infection (hpi)], late (12-24 hpi), and 24 hpi in Huh7 and Vero cells. We assessed the viral and cellular processes by calculating the viral titre in the supernatants, In-Cell Western, western blotting and fluorescence microscopy. RESULTS Phosphorylation of GSK3β-Ser9 was observed at the early stages of infection; neither did treatment with small molecule inhibitors nor pre-treatment prior to viral infection of GSK3β reduce viral titres of the supernatant at these time points. However, a decrease in viral titres was observed in cells infected and treated with the inhibitors much later during viral infection. Consistently, the infected cells at this stage displayed plasma membrane damage. Nonetheless, these effects were not elicited with the use of genetic inhibitors of GSK3β. CONCLUSIONS The results suggest that GSK3β participates at the late stages of the DENV replication cycle, where viral activation may promote apoptosis and release of viral particles.

Cryo-Electron Microscopy Reveals That Sperm Modification Coincides with Female Fertility in the Mosquito Aedes aegypti.

Manipulating mosquito reproduction is a promising approach to reducing mosquito populations and the burden of diseases they carry. A thorough understanding of reproductive processes is necessary to develop such strategies, but little is known about how sperm are processed and prepared for fertilization within female mosquitoes. By employing cryo-electron microscopy for the first time to study sperm of the mosquito Aedes aegypti, we reveal that sperm shed their entire outer coat, the glycocalyx, within 24 hours of being stored in the female. Motility assays demonstrate that as their glycocalyx is shed in the female's sperm storage organs, sperm transition from a period of dormancy to rapid motility-a critical prerequisite for sperm to reach the egg. We also show that females gradually become fertile as sperm become motile, and that oviposition behavior increases sharply after females reach peak fertility. Together, these experiments demonstrate a striking coincidence of the timelines of several reproductive events in Ae. aegypti, suggesting a direct relationship between sperm modification and female reproductive capacity.

Transfection of Culicoides sonorensis biting midge cell lines with Wolbachia pipientis.

BACKGROUND: Biting midges of the genus Culicoides vector multiple veterinary pathogens and are difficult to control. Endosymbionts particularly Wolbachia pipientis may offer an alternative to control populations of Culicoides and/or impact disease transmission in the form of population suppression or replacement strategies. METHODS: Culicoides sonorensis cell lines were transfected with a Wolbachia infection using a modified shell vial technique. Infections were confirmed using PCR and cell localization using fluorescent in situ hybridization (FISH). The stability of Wolbachia infections and density was determined by qPCR. qPCR was also used to examine immune genes in the IMD, Toll and JACK/STAT pathways to determine if Wolbachia were associated with an immune response in infected cells. RESULTS: Here we have transfected two Culicoides sonorensis cell lines (W3 and W8) with a Wolbachia infection (walbB) from donor Aedes albopictus Aa23 cells. PCR and FISH showed the presence of Wolbachia infections in both C. sonorensis cell lines. Infection densities were higher in the W8 cell lines when compared to W3. In stably infected cells, genes in the immune Toll, IMD and JAK/STAT pathways were upregulated, along with Attacin and an Attacin-like anti-microbial peptides. CONCLUSIONS: The successful introduction of Wolbachia infections in C. sonorensis cell lines and the upregulation of immune genes, suggest the utility of using Wolbachia for a population replacement and/or population suppression approach to limit the transmission of C. sonorensis vectored diseases. Results support the further investigation of Wolbachia induced pathogen inhibitory effects in Wolbachia-infected C. sonorensis cell lines and the introduction of Wolbachia into C. sonorensis adults via embryonic microinjection to examine for reproductive phenotypes and host fitness effects of a novel Wolbachia infection.

Field-captured Aedes vexans (Meigen, 1830) is a competent vector for Rift Valley fever phlebovirus in Europe.

BACKGROUND: Aedes vexans (Meigen) is considered a nuisance species in central Europe and the Mediterranean region. It is an anthropophilic and mammalophilic floodwater mosquito involved in the transmission of several arboviruses. Rift Valley fever (RVF) is a relevant mosquito-borne zoonosis, affecting mainly humans and ruminants, that causes severe impact in public health and economic loses. Due to globalization and climate change, the European continent is threatened by its introduction. The main purpose of the present study was to evaluate the vector competence of a European field-collected Ae. vexans population. METHODS: Aedes vexans field-collected larvae were reared in the laboratory under field-simulated conditions. To assess the vector competence for Rift Valley fever phlebovirus (RVFV) transmission, adult F0 females were exposed to infectious blood meals containing the 56/74 RVFV strain. Additionally, intrathoracic inoculations with the same virus strain were performed to evaluate the relevance of the salivary gland barriers. Natural circulation of alphavirus, flavivirus and phlebovirus was also tested. RESULTS: To our knowledge, an autochthonous Ae. vexans population was experimentally confirmed as a competent vector for RVFV for the first time. This virus was capable of infecting and disseminating within the studied Ae. vexans mosquitoes. Moreover, infectious virus was isolated from the saliva of disseminated specimens, showing their capacity to transmit the virus. Additionally, a natural infection with a circulating Mosquito flavivirus was detected. The co-infection with the Mosquito flavivirus seemed to modulate RVFV infection susceptibility in field-collected Ae. vexans, but further studies are needed to confirm its potential interference in RVFV transmission. CONCLUSIONS: Our results show that field-collected European Ae. vexans would be able to transmit RVFV in case of introduction into the continent. This should be taken into consideration in the design of surveillance and control programmes.

Differential Metabolic Reprogramming by Zika Virus Promotes Cell Death in Human versus Mosquito Cells.

Zika virus is a pathogen that poses serious consequences, including congenital microcephaly. Although many viruses reprogram host cell metabolism, whether Zika virus alters cellular metabolism and the functional consequences of Zika-induced metabolic changes remain unknown. Here, we show that Zika virus infection differentially reprograms glucose metabolism in human versus C6/36 mosquito cells by increasing glucose use in the tricarboxylic acid cycle in human cells versus increasing glucose use in the pentose phosphate pathway in mosquito cells. Infection of human cells selectively depletes nucleotide triphosphate levels, leading to elevated AMP/ATP ratios, AMP-activated protein kinase (AMPK) phosphorylation, and caspase-mediated cell death. AMPK is also phosphorylated in Zika virus-infected mouse brain. Inhibiting AMPK in human cells decreases Zika virus-mediated cell death, whereas activating AMPK in mosquito cells promotes Zika virus-mediated cell death. These findings suggest that the differential metabolic reprogramming during Zika virus infection of human versus mosquito cells determines whether cell death occurs.

Conditions facilitating infection of mosquito cell lines with Wolbachia, an obligate intracellular bacterium.

Factors that influence establishment of Wolbachia, an obligate intracellular bacterium, in novel insect hosts or uninfected insect cell lines are poorly understood. Infectivity of Wolbachia strain wStr was correlated with flow cytometric profiles to define optimal conditions for harvesting an infectious inoculum. Wolbachia recovered from the cell culture supernatant after gentle pipetting of infected cells represented about 1% of the total bacterial population and were more infectious than Wolbachia that remained associated with intact cells and/or membranes after low-speed centrifugation. Optimal establishment of a robust infection in naïve cells required 6 d, at a ratio of 80 to 160 bacteria per cell. Among Aedes albopictus mosquito cell lines, an aneuploid line with a 4n + 1 karyotype was more susceptible to infection than diploid lines. These findings contribute to the in vitro manipulation of Wolbachia, illustrate some of the many factors that influence infectivity, and identify areas for future investigation.