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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 BackgroundVector competence inAedes aegyptiis influenced by various factors. Crucial new control methods can be developed by recognizing which factors affect virus and mosquito interactions. MethodsIn the present study we used three geographically distinctAe. aegyptipopulations and compared their susceptibility to infection by dengue virus serotype 2 (DENV-2). To identify any differences among the three mosquito populations, we evaluated expression levels of immune-related genes and assessed the presence of microbiota that might contribute to the uniqueness in their vector competence. ResultsBased on the results from the DENV-2 competence study, we categorized the three geographically distinctAe. aegyptipopulations into a refractory population (Vilas do Atlântico), a susceptible population (Vero) and a susceptible but low transmission population (California). The immune-related transcripts were highly expressed in the California population but not in the refractory population. However, the Rel-1 gene was upregulated in the Vilas do Atlântico population following ingestion of a non-infectious blood meal, suggesting the gene’s involvement in non-viral responses, such as response to microbiota. Screening of the bacteria, fungi and flaviviruses revealed differences between populations, and any of these could be one of the factors that interfere with the vector competence. ConclusionsThe results reveal potential factors that might impact the virus and mosquito interaction, as well as influence theAe. aegyptirefractory phenotype. Graphical Abstract