Eilat virus (EILV) causes superinfection exclusion against West NILE virus (WNV) in a strain specific manner in Culex tarsalis mosquitoes

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the United States. 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 vector Culex tarsalis is 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 and Culex tarsalis mosquitoes. The titers 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 multiplicity of infections (MOIs) tested in our study. The titers of WN02-1956 at both MOIs 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 titers. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. In Cx. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes, EILV enhanced NY99 infection titers at 3 days post superinfection, but this effect disappeared at 7 days post superinfection. In contrast, WN02-1956 infection titers 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, in Cx. 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. AUTHOR SUMMARYWest Nile virus (WNV) is the main cause of mosquito-borne disease in the United States. In the absence of a human vaccine or WNV-specific antivirals, vector control is the key strategy to reduce WNV prevalence and transmission. The WNV mosquito vector, Culex tarsalis, is a competent host for the insect-specific virus Eilat virus (EILV). EILV and WNV potentially interact within the mosquito host, and EILV can be used as a safe tool to target WNV in mosquitoes. Here, we characterize the ability of EILV to cause superinfection exclusion (SIE) against two strains of WNV--WN02-1956 and NY99--in C6/36 cells and Cx. tarsalis mosquitoes. EILV suppressed both superinfecting WNV strains in C6/36 cells. However, in mosquitoes, EILV enhanced NY99 whole-body titers at 3 days post superinfection and suppressed WN02-1956 whole-body titers at 7 days post superinfection. Vector competence measures, including infection, dissemination, and transmission rates and transmission efficacy, as well as leg and saliva titers of both superinfecting WNV strains, were not affected by EILV at both timepoints. Our data show the importance of not only validating SIE in mosquito vectors but also testing multiple strains of viruses to determine the safety of this strategy as a control tool.