Correction: Broad dengue neutralization in mosquitoes expressing an engineered antibody.

[This corrects the article DOI: 10.1371/journal.ppat.1008103.].

Broad dengue neutralization in mosquitoes expressing an engineered antibody.

With dengue virus (DENV) becoming endemic in tropical and subtropical regions worldwide, there is a pressing global demand for effective strategies to control the mosquitoes that spread this disease. Recent advances in genetic engineering technologies have made it possible to create mosquitoes with reduced vector competence, limiting their ability to acquire and transmit pathogens. Here we describe the development of Aedes aegypti mosquitoes synthetically engineered to impede vector competence to DENV. These mosquitoes express a gene encoding an engineered single-chain variable fragment derived from a broadly neutralizing DENV human monoclonal antibody and have significantly reduced viral infection, dissemination, and transmission rates for all four major antigenically distinct DENV serotypes. Importantly, this is the first engineered approach that targets all DENV serotypes, which is crucial for effective disease suppression. These results provide a compelling route for developing effective genetic-based DENV control strategies, which could be extended to curtail other arboviruses.

Engineered resistance to Zika virus in transgenic Aedes aegypti expressing a polycistronic cluster of synthetic small RNAs.

Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive-based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered synthetic small RNAs targeting ZIKV is expressed and fully processed in Aedes aegypti, ensuring the formation of mature synthetic small RNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.

Outbreak of Oropouche Virus in French Guiana.

Oropouche fever is a zoonotic dengue-like syndrome caused by Oropouche virus. In August-September 2020, dengue-like syndrome developed in 41 patients in a remote rainforest village in French Guiana. By PCR or microneutralization, 23 (82.1%) of 28 tested patients were positive for Oropouche virus, documenting its emergence in French Guiana.

Zika virus-induced hyper excitation precedes death of mouse primary neuron.

BACKGROUND: Zika virus infection in new born is linked to congenital syndromes, especially microcephaly. Studies have shown that these neuropathies are the result of significant death of neuronal progenitor cells in the central nervous system of the embryo, targeted by the virus. Although cell death via apoptosis is well acknowledged, little is known about possible pathogenic cellular mechanisms triggering cell death in neurons. METHODS: We used in vitro embryonic mouse primary neuron cultures to study possible upstream cellular mechanisms of cell death. Neuronal networks were grown on microelectrode array and electrical activity was recorded at different times post Zika virus infection. In addition to this method, we used confocal microscopy and Q-PCR techniques to observe morphological and molecular changes after infection. RESULTS: Zika virus infection of mouse primary neurons triggers an early spiking excitation of neuron cultures, followed by dramatic loss of this activity. Using NMDA receptor antagonist, we show that this excitotoxicity mechanism, likely via glutamate, could also contribute to the observed nervous system defects in human embryos and could open new perspective regarding the causes of adult neuropathies. CONCLUSIONS: This model of excitotoxicity, in the context of neurotropic virus infection, highlights the significance of neuronal activity recording with microelectrode array and possibility of more than one lethal mechanism after Zika virus infection in the nervous system.

Detection of Elizabethkingia spp. in Culicoides Biting Midges, Australia.

The bacterial pathogen Elizabethkingia is known to exist in certain species of mosquito but was unknown in other arthropods. We report the detection and identification of Elizabethkingia in species of Culicoides biting midge in Australia, raising the possibility of bacterial transmission via this species.

Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes.

Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.

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.

Zika vector transmission risk in temperate Australia: a vector competence study.

BACKGROUND: Zika virus is an emerging pathogen of global importance. It has been responsible for recent outbreaks in the Americas and in the Pacific region. This study assessed five different mosquito species from the temperate climatic zone in Australia and included Aedes albopictus as a potentially invasive species. METHODS: Mosquitoes were orally challenged by membrane feeding with Zika virus strain of Cambodia 2010 origin, belonging to the Asian clade. Virus infection and dissemination were assessed by quantitative PCR on midgut and carcass after dissection. Transmission was assessed by determination of cytopathogenic effect of saliva (CPE) on Vero cells, followed by determination of 50% tissue culture infectious dose (TCID50) for CPE positive samples. Additionally, the presence of Wolbachia endosymbiont infection was assessed by qPCR and standard PCR. RESULTS: Culex mosquitoes were found unable to present Zika virus in saliva, as demonstrated by molecular as well as virological methods. Aedes aegypti, was used as a positive control for Zika infection and showed a high level of virus infection, dissemination and transmission. Local Aedes species, Ae. notoscriptus and, to a lesser degree, Ae. camptorhynchus were found to expel virus in their saliva and contained viral nucleic acid within the midgut. Molecular assessment identified low or no dissemination for these species, possibly due to low virus loads. Ae. albopictus from Torres Strait islands origin was shown as an efficient vector. Cx quinquefasciatus was shown to harbour Wolbachia endosymbionts at high prevalence, whilst no Wolbachia was found in Cx annulirostris. The Australian Ae. albopictus population was shown to harbour Wolbachia at high frequency. CONCLUSIONS: The risk of local Aedes species triggering large Zika epidemics in the southern parts of Australia is low. The potentially invasive Ae. albopictus showed high prevalence of virus in the saliva and constitutes a potential threat if this mosquito species becomes established in mainland Australia. Complete risk analysis of Zika transmission in the temperate zone would require an assessment of the impact of temperature on Zika virus replication within local and invasive mosquito species.

Genotyping of whole genome amplified reduced representation libraries reveals a cryptic population of Culicoides brevitarsis in the Northern Territory, Australia.

BACKGROUND: The advent of genotyping by Next Generation Sequencing has enabled rapid discovery of thousands of single nucleotide polymorphism (SNP) markers and high throughput genotyping of large populations at an affordable cost. Genotyping by sequencing (GBS), a reduced representation library sequencing method, allows highly multiplexed sequencing of genomic subsets. This method has limitations for small organisms with low amounts of genomic DNA, such as the bluetongue virus (BTV) vectors, Culicoides midges. RESULTS: This study employed the GBS method to isolate SNP markers de novo from whole genome amplified Culicoides brevitarsis genomic DNA. The individuals were collected from regions representing two different Australian patterns of BTV strain distribution: the Northern Territory (NT) and the east coast. We isolated 8145 SNPs using GBS. Phylogenetic analysis conducted using the filtered 3263 SNPs revealed the presence of a distinct C. brevitarsis sub-population in the NT and this was confirmed by analysis of mitochondrial DNA. Two loci showed a very strong signal for selection and were unique to the NT population. Bayesian analysis with STRUCTURE indicated a possible two-population cluster. CONCLUSIONS: The results suggest that genotyping vectors with high density markers in combination with biological and environmental data is useful. However, more extensive sampling over a wider spatial and temporal range is needed. The presence of sub-structure in populations and loci under natural selection indicates the need for further investigation of the role of vectors in shaping the two Australian systems of BTV transmission. The described workflow is transferable to genotyping of small, non-model organisms, including arthropod vectors of pathogens of economic and medical importance.

Ecology of urban malaria vectors in Niamey, Republic of Niger.

BACKGROUND: Urbanization in African cities has major impact on malaria risk. Niamey, the capital of the Republic of Niger, is situated in the West African Sahel zone. The short rainy season and human activities linked with the Niger River influence mosquito abundance. This study aimed at deciphering the factors of distribution of urban malaria vectors in Niamey. METHODS: The distribution of mosquito aquatic stages was investigated monthly from December 2002 to November 2003, at up to 84 breeding sites, throughout Niamey. An exploratory analysis of association between mosquito abundance and environmental factors was performed by a Principal Component Analysis and confirmed by Kruskall-Wallis non-parametric test. To assess the relative importance of significant factors, models were built for Anopheles and Culicinae. In a second capture session, adult mosquitoes were collected weekly with pyrethrum sprays and CDC light-traps from June 2008 to June 2009 in two differentiated urban areas chosen after the study's first step. Members of the Anopheles gambiae complex were genotyped and Anopheles females were tested for the presence of Plasmodium falciparum circumsporozoite antigens using ELISA. RESULTS: In 2003, 29 % of 8420 mosquitoes collected as aquatic stages were Anopheles. They were significantly more likely to be found upstream, relatively close to the river and highly productive in ponds. These factors remained significant in regression and generalized linear models. The Culicinae were found significantly more likely close to the river, and in the main temporary affluent stream. In 2009, Anopheles specimens, including Anopheles gambiae s.l. (95 %), but also Anopheles funestus (0.6 %) accounted for 18 % of the adult mosquito fauna, with a large difference between the two sampled zones. Three members of the An. gambiae complex were found: Anopheles arabiensis, Anopheles coluzzii, and An. gambiae. Nineteen (1.3 %) out of 1467 females tested for P. falciparum antigen were found positive. CONCLUSION: The study provides valuable update knowledge on malaria vector ecology and distribution in Niamey. The identification of spatial and environmental risk factors could pave the way to larval source management strategy and allow malaria vector control to focus on key zones for the benefit of the community.

Detection of Low-Level Cardinium and Wolbachia Infections in Culicoides.

Bacterial endosymbionts have been identified as potentially useful biological control agents for a range of invertebrate vectors of disease. Previous studies of Culicoides (Diptera: Ceratopogonidae) species using conventional PCR assays have provided evidence of Wolbachia (1/33) and Cardinium (8/33) infections. Here, we screened 20 species of Culicoides for Wolbachia and Cardinium, utilizing a combination of conventional PCR and more sensitive quantitative PCR (qPCR) assays. Low levels of Cardinium DNA were detected in females of all but one of the Culicoides species screened, and low levels of Wolbachia were detected in females of 9 of the 20 Culicoides species. Sequence analysis based on partial 16S rRNA gene and gyrB sequences identified "Candidatus Cardinium hertigii" from group C, which has previously been identified in Culicoides from Japan, Israel, and the United Kingdom. Wolbachia strains detected in this study showed 98 to 99% sequence identity to Wolbachia previously detected from Culicoides based on the 16S rRNA gene, whereas a strain with a novel wsp sequence was identified in Culicoides narrabeenensis. Cardinium isolates grouped to geographical regions independent of the host Culicoides species, suggesting possible geographical barriers to Cardinium movement. Screening also identified Asaia bacteria in Culicoides. These findings point to a diversity of low-level endosymbiont infections in Culicoides, providing candidates for further characterization and highlighting the widespread occurrence of these endosymbionts in this insect group.

Assessment of population genetic structure in the arbovirus vector midge, Culicoides brevitarsis (Diptera: Ceratopogonidae), using multi-locus DNA microsatellites.

Bluetongue virus (BTV) is a major pathogen of ruminants that is transmitted by biting midges (Culicoides spp.). Australian BTV serotypes have origins in Asia and are distributed across the continent into two distinct episystems, one in the north and another in the east. Culicoides brevitarsis is the major vector of BTV in Australia and is distributed across the entire geographic range of the virus. Here, we describe the isolation and use of DNA microsatellites and gauge their ability to determine population genetic connectivity of C. brevitarsis within Australia and with countries to the north. Eleven DNA microsatellite markers were isolated using a novel genomic enrichment method and identified as useful for genetic analyses of sampled populations in Australia, northern Papua New Guinea (PNG) and Timor-Leste. Significant (P < 0.05) population genetic subdivision was observed between all paired regions, though the highest levels of genetic sub-division involved pair-wise tests with PNG (PNG vs. Australia (FST = 0.120) and PNG vs. Timor-Leste (FST = 0.095)). Analysis of multi-locus allelic distributions using STRUCTURE identified a most probable two-cluster population model, which separated PNG specimens from a cluster containing specimens from Timor-Leste and Australia. The source of incursions of this species in Australia is more likely to be Timor-Leste than PNG. Future incursions of BTV positive C. brevitarsis into Australia may be genetically identified to their source populations using these microsatellite loci. The vector's panmictic genetic structure within Australia cannot explain the differential geographic distribution of BTV serotypes.

Secreted Vago restricts West Nile virus infection in Culex mosquito cells by activating the Jak-STAT pathway.

Although West Nile virus (WNV) and other arthropod-borne viruses are a major public health problem, the mechanisms of antiviral immunity in mosquitoes are poorly understood. Dicer-2, responsible for the RNAi-mediated response through the C-terminal RNase-III domain, also contains an N-terminal DExD/H-box helicase domain similar to mammalian RIG-I/MDA5 which, in Drosophila, was found to be required for activation of an antiviral gene, Vago. Here we show that the Culex orthologue of Vago (CxVago) is up-regulated in response to WNV infection in a Dicer-2-dependent manner. Further, our data show that CxVago is a secreted peptide that restricts WNV infection by activation of the Jak-STAT pathway. Thus, Vago appears to function as an IFN-like antiviral cytokine in mosquitoes.

Epidemiology of malaria in an area of seasonal transmission in Niger and implications for the design of a seasonal malaria chemoprevention strategy.

BACKGROUND: Few data are available about malaria epidemiological situation in Niger. However, implementation of new strategies such as vaccination or seasonal treatment of a target population requires the knowledge of baseline epidemiological features of malaria. A population-based study was conducted to provide better characterization of malaria seasonal variations and population groups the most at risk in this particular area. METHODS: From July 2007 to December 2009, presumptive cases of malaria among a study population living in a typical Sahelian village of Niger were recorded, and confirmed by microscopic examination. In parallel, asymptomatic carriers were actively detected at the end of each dry season in 2007, 2008 and 2009. RESULTS: Among the 965 presumptive malaria cases recorded, 29% were confirmed by microscopic examination. The incidence of malaria was found to decrease significantly with age (p < 0.01). The mean annual incidence was 0.254. The results show that the risk of malaria was higher in children under ten years (p < 0.0001). The number of malaria episodes generally followed the temporal pattern of changes in precipitation levels, with a peak of transmission in August and September. One-thousand and ninety subjects were submitted to an active detection of asymptomatic carriage of whom 16% tested positive; asymptomatic carriage decreased with increasing age. A higher prevalence of gametocyte carriage among asymptomatic population was recorded in children aged two to ten years, though it did not reach significance. CONCLUSIONS: In Southern Niger, malaria transmission mostly occurs from July to October. Children aged two to ten years are the most at risk of malaria, and may also represent the main reservoir for gametocytes. Strategies such as intermittent preventive treatment in children (IPTc) could be of interest in this area, where malaria transmission is highly seasonal. Based on these preliminary data, a pilot study could be implemented in Zindarou using IPTc targeting children aged two to ten years, during the three months of malaria transmission, together with an accurate monitoring of drug resistance.

Mosquitoes (Diptera: Culicidae) of the Amazonian savannas of French Guiana with a description of two new species.

Amazonian savannas are among the most noteworthy landscape components of the coastal plain of French Guiana. Although they cover only 0.22% of the territory, they bring together a large part of the animal and plant diversity of this overseas region of France. This article outlines the results of the first study dedicated to mosquitoes (Diptera: Culicidae) of Amazonian savannas. Samplings were conducted in eight independent savannas evenly distributed along a transect of 170 km on the coastal plain of French Guiana. A total of 50 mosquito species were recorded, which is about 20% of the culicid fauna currently known in French Guiana. Among them, Culex (Melanoconion) organaboensis sp. nov. and Cx. (Mel.) zabanicus sp. nov. are newly described based on both morphological features of the male genitalia and a DNA barcode obtained from type specimens. Diagnostic characters to assist their identification are provided and their placement within the infrasubgeneric classification of the subgenus Melanoconion is discussed.

Ribosomal RNA (rRNA) sequences from 33 globally distributed mosquito species for improved metagenomics and species identification.

Total RNA sequencing (RNA-seq) is an important tool in the study of mosquitoes and the RNA viruses they vector as it allows assessment of both host and viral RNA in specimens. However, there are two main constraints. First, as with many other species, abundant mosquito ribosomal RNA (rRNA) serves as the predominant template from which sequences are generated, meaning that the desired host and viral templates are sequenced far less. Second, mosquito specimens captured in the field must be correctly identified, in some cases to the sub-species level. Here, we generate mosquito rRNA datasets which will substantially mitigate both of these problems. We describe a strategy to assemble novel rRNA sequences from mosquito specimens and produce an unprecedented dataset of 234 full-length 28S and 18S rRNA sequences of 33 medically important species from countries with known histories of mosquito-borne virus circulation (Cambodia, the Central African Republic, Madagascar, and French Guiana). These sequences will allow both physical and computational removal of rRNA from specimens during RNA-seq protocols. We also assess the utility of rRNA sequences for molecular taxonomy and compare phylogenies constructed using rRNA sequences versus those created using the gold standard for molecular species identification of specimens-the mitochondrial cytochrome c oxidase I (COI) gene. We find that rRNA- and COI-derived phylogenetic trees are incongruent and that 28S and concatenated 28S+18S rRNA phylogenies reflect evolutionary relationships that are more aligned with contemporary mosquito systematics. This significant expansion to the current rRNA reference library for mosquitoes will improve mosquito RNA-seq metagenomics by permitting the optimization of species-specific rRNA depletion protocols for a broader range of species and streamlining species identification by rRNA sequence and phylogenetics.

Identification of Neotropical Culex Mosquitoes by MALDI-TOF MS Profiling.

The mosquito (Diptera: Culicidae) fauna of French Guiana encompasses 242 species, of which nearly half of them belong to the genus Culex. Whereas several species of Culex are important vectors of arboviruses, only a limited number of studies focus on them due to the difficulties to morphologically identify field-caught females. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been reported as a promising method for the identification of mosquitoes. Culex females collected in French Guiana were morphologically identified and dissected. Abdomens were used for molecular identification using the COI (cytochrome oxidase 1) gene. Legs and thorax of 169 specimens belonging to 13 Culex species, (i.e., Cx. declarator, Cx. nigripalpus, Cx. quinquefasciatus, Cx. usquatus, Cx. adamesi, Cx. dunni, Cx. eastor, Cx. idottus, Cx. pedroi, Cx. phlogistus, Cx. portesi, Cx. rabanicolus and Cx. spissipes) were then submitted to MALDI-TOF MS analysis. A high intra-species reproducibility and inter-species specificity of MS spectra for each mosquito body part tested were obtained. A corroboration of the specimen identification was revealed between MALDI-TOF MS, morphological and molecular results. MALDI-TOF MS protein profiling proves to be a suitable tool for identification of neotropical Culex species and will permit the enhancement of knowledge on this highly diverse genus.

Review on Main Arboviruses Circulating on French Guiana, An Ultra-Peripheric European Region in South America.

French Guiana (FG), a French overseas territory in South America, is susceptible to tropical diseases, including arboviruses. The tropical climate supports the proliferation and establishment of vectors, making it difficult to control transmission. In the last ten years, FG has experienced large outbreaks of imported arboviruses such as Chikungunya and Zika, as well as endemic arboviruses such as dengue, Yellow fever, and Oropouche virus. Epidemiological surveillance is challenging due to the differing distributions and behaviors of vectors. This article aims to summarize the current knowledge of these arboviruses in FG and discuss the challenges of arbovirus emergence and reemergence. Effective control measures are hampered by the nonspecific clinical presentation of these diseases, as well as the Aedes aegypti mosquito's resistance to insecticides. Despite the high seroprevalence of certain viruses, the possibility of new epidemics cannot be ruled out. Therefore, active epidemiological surveillance is needed to identify potential outbreaks, and an adequate sentinel surveillance system and broad virological diagnostic panel are being developed in FG to improve disease management.

The Culex Mosquitoes (Diptera: Culicidae) of French Guiana: A Comprehensive Review With the Description of Three New Species.

The taxonomically intricate genus Culex Linnaeus includes numerous known vector species of parasites and viruses to humans. The aim of this article is to comprehensively review the Culex species which occur in French Guiana to provide a stronger taxonomic foundation for future studies on this genus in South America. The occurrence of Culex species was investigated in light of current taxonomic knowledge through an extensive examination of voucher specimens deposited in the entomological collections of four French depositories and additional specimens recently collected at various localities in French Guiana. Based on this review, 104 Culex species classified in eight subgenera are confirmed to occur in French Guiana. Compared to the most recent checklist, 18 species are added, and 10 species excluded, resulting in a total number of 242 valid mosquito species known to occur in French Guiana. Three nominal species are synonymized, three others are newly described, and a last one is transferred to another informal infrasubgeneric group. Overall, this review also highlights the limits of the use of only bibliographic data when dealing with taxonomically complex groups of insects.