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West Nile virus (WNV) is the leading cause of mosquito-borne illness in the USA. There are currently no human vaccines or therapies available for WNV, and vector control is the primary strategy used to control WNV transmission. The WNV vectorCulex tarsalisis also a competent host for the insect-specific virus (ISV) Eilat virus (EILV). ISVs such as EILV can interact with and cause superinfection exclusion (SIE) against human pathogenic viruses in their shared mosquito host, altering vector competence for these pathogenic viruses. The ability to cause SIE and their host restriction make ISVs a potentially safe tool to target mosquito-borne pathogenic viruses. In the present study, we tested whether EILV causes SIE against WNV in mosquito C6/36 cells andC. tarsalismosquitoes. The titres of both WNV strains – WN02-1956 and NY99 – were suppressed by EILV in C6/36 cells as early as 48–72 h post-superinfection at both m.o.i. values tested in our study. The titres of WN02-1956 at both m.o.i. values remained suppressed in C6/36 cells, whereas those of NY99 showed some recovery towards the final timepoint. The mechanism of SIE remains unknown, but EILV was found to interfere with NY99 attachment in C6/36 cells, potentially contributing to the suppression of NY99 titres. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. InC. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes,EILV enhanced NY99 infection titres at 3 days post-superinfection, but this effect disappeared at 7 days post-superinfection. In contrast, WN02-1956 infection titres were suppressed by EILV at 7 days post-superinfection. The dissemination and transmission of both WNV strains were not affected by superinfection with EILV at either timepoint. Overall, EILV caused SIE against both WNV strains in C6/36 cells; however, inC. tarsalis, SIE caused by EILV was strain specific potentially owing to differences in the rate of depletion of shared resources by the individual WNV strains. 

ABSTRACT Eilat virus (EILV) is an insect-specific alphavirus that has the potential to be developed into a tool to combat mosquito-borne pathogens. However, its mosquito host range and transmission routes are not well understood. Here, we fill this gap by investigating EILV’s host competence and tissue tropism in five mosquito species: Aedes aegypti , Culex tarsalis , Anopheles gambiae , Anopheles stephensi , and Anopheles albimanus . Of the tested species, C. tarsalis was the most competent host for EILV. The virus was found in C. tarsalis ovaries, but no vertical or venereal transmission was observed. Culex tarsalis also transmitted EILV via saliva, suggesting the potential for horizontal transmission between an unknown vertebrate or invertebrate host. We found that reptile (turtle and snake) cell lines were not competent for EILV infection. We tested a potential invertebrate host ( Manduca sexta caterpillars) but found they were not susceptible to EILV infection. Together, our results suggest that EILV could be developed as a tool to target pathogenic viruses that use Culex tarsalis as a vector. Our work sheds light on the infection and transmission dynamics of a poorly understood insect-specific virus and reveals it may infect a broader range of mosquito species than previously recognized. IMPORTANCE The recent discovery of insect-specific alphaviruses presents opportunities both to study the biology of virus host range and to develop them into tools against pathogenic arboviruses. Here, we characterize the host range and transmission of Eilat virus in five mosquito species. We find that Culex tarsalis —a vector of harmful human pathogens, including West Nile virus—is a competent host of Eilat virus. However, how this virus is transmitted between mosquitoes remains unclear. We find that Eilat virus infects the tissues necessary for both vertical and horizontal transmission—a crucial step in discerning how Eilat virus maintains itself in nature. 

Abstract Insect epithelial cells contain cellular extensions such as bristles, hairs, and scales. These cellular extensions are homologous structures that differ in morphology and function. They contain actin bundles that dictate their cellular morphology. While the organization, function, and identity of the major actin-bundling proteins in bristles and hairs are known, this information on scales is unknown. In this study, we characterized the development of scales and the role of actin bundles in the mosquito, Aedes aegypti . We show that scales undergo drastic morphological changes during development, from a cylindrical to flat shape with longer membrane invagination. Scale actin-bundle distribution changes from the symmetrical organization of actin bundles located throughout the bristle membrane to an asymmetrical organization. By chemically inhibiting actin polymerization and by knocking out the forked gene in the mosquito ( Ae-Forked ; a known actin-bundling protein) by CRISPR-Cas9 gene editing, we showed that actin bundles are required for shaping bristle, hair, and scale morphology. We demonstrated that actin bundles and Ae-Forked are required for bristle elongation, but not for that of scales. In scales, actin bundles are required for width formation. In summary, our results reveal, for the first time, the developmental process of mosquito scale formation and also the role of actin bundles and actin-bundle proteins in scale morphogenesis. Moreover, our results reveal that although scale and bristle are thought to be homologous structures, actin bundles have a differential requirement in shaping mosquito scales compared to bristles. 

Abstract MultipleWolbachiastrains can block pathogen infection, replication and/or transmission inAedes aegyptimosquitoes under both laboratory and field conditions. However,Wolbachiaeffects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in whichWolbachiagoverns pathogen transmission phenotypes; rather, the genetics of the host can significantly modulateWolbachia‐mediated pathogen blocking. Specifically, previous work linked variation inWolbachiapathogen blocking to polymorphisms in the mosquito alpha‐mannosidase‐2 (αMan2) gene. Here we use CRISPR‐Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on bothWolbachiaand virus levels, using dengue virus (DENV;Flaviviridae) and Mayaro virus (MAYV;Togaviridae).Wolbachiatitres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. InWolbachia‐uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in aWolbachia‐infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication inWolbachia‐uninfected mosquitoes and did not affectWolbachia‐mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection inA. aegyptimosquitoes in a pathogen‐ andWolbachia‐specific manner, and thatWolbachia‐mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use ofWolbachia‐based strategies to control vector‐borne pathogens.