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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.

Aedes aegypti mosquitoes carrying self-spreading, virus-blocking Wolbachia bacteria are being deployed to suppress dengue transmission. However, there are challenges in applying this technology in extreme environments. We introduced two Wolbachia strains into Ae . aegypti from Saudi Arabia for a release program in the hot coastal city of Jeddah. Wolbachia reduced infection and dissemination of dengue virus (DENV2) in Saudi Arabian mosquitoes and showed complete maternal transmission and cytoplasmic incompatibility. Wolbachia reduced egg hatch under a range of environmental conditions, with the Wolbachia strains showing differential thermal stability. Wolbachia effects were similar across mosquito genetic backgrounds but we found evidence of local adaptation, with Saudi Arabian mosquitoes having lower egg viability but higher adult desiccation tolerance than Australian mosquitoes. Genetic background effects will influence Wolbachia invasion dynamics, reinforcing the need to use local genotypes for mosquito release programs, particularly in extreme environments like Jeddah. Our comprehensive characterization of Wolbachia strains provides a foundation for Wolbachia -based disease interventions in harsh climates.

Japanese encephalitis virus (JEV) is transmitted by species of mosquitoes. In 2022, JEV belonging to a previously unrecognized lineage of genotype IV (GIV) caused a major outbreak of JE in South-eastern Australia, resulting in human cases and affecting piggeries. has previously been implicated as the major vector of JEV in northern Australia where the virus has circulated since its first detection in 1995. Here, we showed that experimental infection of a laboratory colony of Australian with the isolate JEV NSW/22 resulted in a 100% mosquito infection rate, with 87% of mosquito saliva samples testing positive by RT-qPCR at 14 days post-infection. Immunohistochemistry confirmed the presence of a replicating virus in the mosquito midgut and dissemination throughout the body, including the salivary glands. Our results also showed evidence of transovarial transmission of this virus; however, transstadial transmission from the eggs to the adult stage was not found. Comparison with an Indonesian isolate of GIV JEV and previous Australian isolates belonging to genotypes I and II showed that infection with JEV NSW/22 resulted in higher viral titres in the early stage of infection and higher proportions of mosquitoes with JEV-positive saliva, indicating a greater transmission potential compared to other isolates. This study provides compelling experimental evidence that Australian is a highly efficient vector for the 2022 Australian JEV GIV outbreak strain.

Chikungunya virus (CHIKV) is an emerging pathogen around the world and causes significant morbidity in patients. A single amino acid mutation in the envelope protein of CHIKV has led to a shift in vector preference towards Aedes albopictus. While mosquitoes are known to mount an antiviral immune response post-infection, molecular interactions during the course of infection at the tissue level remain largely uncharacterised. We performed whole transcriptome analysis on dissected midguts of Aedes albopictus infected with CHIKV to identify differentially expressed genes. For this, RNA was extracted at two days post-infection (2-dpi) from pooled midguts. We initially identified 25 differentially expressed genes (p-value < 0.05) when mapped to a reference transcriptome. Further, multiple differentially expressed genes were identified from a custom de novo transcriptome, which was assembled using the reads that did not align with the reference genome. Thirteen of the identified transcripts, possibly involved in immunity, were validated by qRT-PCR. Homologues of seven of these genes were also found to be significantly upregulated in Aedes aegypti midguts 2 dpi, indicating a conserved mechanism at play. These results will help us to characterise the molecular interaction between Aedes albopictus and CHIKV and can be utilised to reduce the impact of this viral infection.

Current arbovirus surveillance strategies in Australia involve mosquito collection, species identification, and virus detection. These processes are labour-intensive, expensive, and time-consuming and can lead to delays in reporting. Mosquito excreta has been proposed as an alternative sample type to whole mosquito collection, with potential to streamline the virus surveillance pipeline. In this study, we investigated the feasibility of Aedes aegypti excreta as a sample type in the detection of Dengue virus serotype 2 (DENV2). DENV2 could be detected from as little as one DENV2-infected mosquito excreta spot, with virus levels in individual excreta spots varying within and between mosquitoes and depending highly on mosquito viral load. Detectability was improved by pooling up to 20 DENV2-infected mosquitoes and collecting excreta into liquid substrate, followed by virus concentration using magnetic nanoparticles. Virus concentration improves quantification accuracy in comparison to unconcentrated samples and increases the amount of material available for detection, expanding detection capabilities to techniques with higher limits of detection. Mosquito excreta as a sample type, coupled with magnetic virus concentration, expands the current detection toolbox for DENV2 and has the potential to improve arbovirus surveillance strategies in Australia.

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

Biting midges ( spp.) are important vectors of several insect borne arboviruses but are underrepresented in terms of availability of high-resolution genomic resources. We assembled and annotated complete mitochondrial genomes for two species, namely and which are proven vectors for Bluetongue Virus (BTV). We used both long read and short read sequencing technologies to assemble the circular genomes. The genome sizes are 17,100 bp and 17,031 bp, respectively, all comprising 37 genes, including 13 protein, 22 tRNA, two rRNA coding genes, and one non-coding AT rich control region. The gene organizations and orientations are comparable to other available mitogenomes, except for a translocation of and six tRNA genes in both and . Eleven protein-coding genes encode a full TAA stop codon, while two ( , ) are completed by mRNA polyadenylation. Phylogenetic analysis of the mitogenomes showed and form a monophyletic group. The sequences of these mitogenomes contribute to a baseline of molecular tools for diagnostics and surveillance for use by World Organisation for Animal Health (WOAH) reference laboratories for monitoring vectors of emerging diseases.

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.

Escalating vector disease burdens pose significant global health risks, so innovative tools for targeting mosquitoes are critical. We engineered an antiviral strategy termed REAPER (v NA xpression ctivates oisonous ffector ibonuclease) that leverages the programmable RNA-targeting capabilities of CRISPR Cas13 and its potent collateral activity. Akin to a stealthy Trojan Horse hiding in stealth awaiting the presence of its enemy, REAPER remains concealed within the mosquito until an infectious blood meal is up taken. Upon target viral RNA infection, REAPER activates, triggering programmed destruction of its target arbovirus such as chikungunya. Consequently, Cas13 mediated RNA targeting significantly reduces viral replication and its promiscuous collateral activity can even kill infected mosquitoes. This innovative REAPER technology adds to an arsenal of effective molecular genetic tools to combat mosquito virus transmission.

Aedes aegypti mosquitoes carrying self-spreading, virus-blocking Wolbachia bacteria are being deployed to suppress dengue transmission. However, there are challenges in applying this technology in extreme environments. We introduced two Wolbachia strains into Ae. aegypti from Saudi Arabia for a release program in the hot coastal city of Jeddah. Wolbachia reduced infection and dissemination of dengue virus (DENV2) in Saudi Arabian mosquitoes and showed complete maternal transmission and cytoplasmic incompatibility. Wolbachia reduced mosquito heat tolerance and egg viability, with the Wolbachia strains showing differential thermal stability. Wolbachia effects were similar across mosquito genetic backgrounds but we found evidence of local adaptation, with Saudi Arabian mosquitoes having lower egg viability but higher adult desiccation tolerance than Australian mosquitoes. Genetic background effects will influence Wolbachia invasion dynamics, reinforcing the need to use local genotypes for mosquito release programs, particularly in extreme environments like Jeddah. Our comprehensive characterization of Wolbachia strains provides a foundation for Wolbachia-based disease interventions in harsh climates. Competing Interest Statement The authors have declared no competing interest. Footnotes * The manuscript has been updated to correct a spelling error in the author list