Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes.

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects 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 which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen 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 both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-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 of Wolbachia-based strategies to control vector-borne pathogens.

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.

Culex tarsalis Is a Competent Host of the Insect-Specific Alphavirus Eilat Virus (EILV).

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.

Anopheles gambiae densovirus (AgDNV) negatively affects Mayaro virus infection in Anopheles gambiae cells and mosquitoes.

BACKGROUND: Recent studies demonstrate that insect-specific viruses can influence the ability of their mosquito hosts to become infected with and transmit arboviruses of medical and veterinary importance. The aim of this study was to evaluate the interactions between Anopheles gambiae densovirus (AgDNV) (Parvoviridae) (a benign insect-specific virus that infects An. gambiae mosquitoes) and Mayaro virus (MAYV) (Togaviridae) (an emerging human pathogen that can be transmitted by An. gambiae) in both insect cell culture and mosquitoes. METHODS: For in vitro studies, An. gambiae Mos55 cells infected or uninfected with AgDNV were infected with MAYV. For in vivo studies, An. gambiae mosquitoes were injected intrathoracically with AgDNV and 4 days later orally infected with MAYV. Mosquitoes were dissected 10 days after MAYV infection, and MAYV titers in the body, legs and saliva samples quantified using focus-forming assay. RESULTS: MAYV virus replication was reduced 10-100-fold in An. gambiae Mos55 cells infected with AgDNV. In mosquitoes, there was a significant negative correlation between AgDNV and MAYV body titers 10 days post-blood meal. CONCLUSIONS: AgDNV infection was associated with reduced production of MAYV in cell culture, and reduced body titers of MAYV in An. gambiae mosquitoes. As densovirus infections are common in natural mosquito populations, these data suggest that they may affect the epidemiology of viruses of medical importance.