Antiviral Reponses of Culex Mosquitoes – Generating Capacity for Research on Mosquito Vectors in Temperate Regions

The long-term goal of this project will be to improve the currently available research capacity to study antiviral responses of endemic and emerging mosquito vectors of arboviruses. We also aim to increase our understanding of mosquito-arbovirus interactions. Our research will focus on currently neglected mosquito species that serve as vectors in more temperate regions such as Culex quinquefasciatus, Culex pipiens, and Culex tarsalis. These mosquitoes pose a threat to large parts of the U.S., including the state of Nevada, where Culex spp. mosquitoes are endemic and regularly test positive for West Nile virus. Less frequently, other encephalitic arboviruses such as Saint Louis encephalitis virus are detected as well.We propose to study antiviral mechanisms of Culex spp. mosquitoes in vitro and where possible in vivo. For this, we will use selected viruses representing three major arbovirus groups, namely West Nile virus (flaviviruses), Sindbis virus (alphaviruses), and California encephalitis virus (orthobunyaviruses), all of which can infect Culex mosquitoes. This research project will have two objectives as described below. Both objectives may have a long-term positive impact on Nevada mosquito control by providing useful insight into mosquito-arbovirus interactions that can be exploited for mosquito or arbovirus control.Objective 1: Determining the cellular interactions of PIWI proteins and arboviruses in Culex cells and mosquitoesThis objective will help elucidate some of the in-depth molecular interactions between a set of arboviruses and Culex cells and mosquitoes. We will use gene silencing via transient knockdown and CRISPR/Cas9 knockout to study the role of individual PIWI proteins in the control of alphaviruses, flaviviruses and orthobunyaviruses. We will also generate tagged overexpression plasmids to study the effect of PIWI gene overexpression on virus replication. These plasmids will then serve as tools for microscopy-based studies to visualize which of the PIWI proteins (if any) colocalize with viral RNA and where within the cell these proteins are expressed. These experiments will allow us to narrow down if, which, and how PIWI proteins may interfere with viral replication in Culex cells.Objective 2: To identify the role of viral DNA forms in controlling arbovirus replication in Culex cells and mosquitoes.This objective will further elucidate the role of viral DNA forms in Culex antiviral responses. We will screen various Culex mosquito species and tissues for viral DNA forms generated after infection with alphaviruses, flaviviruses and orthobunyaviruses. We will further inhibit the generation of viral DNA after infection using the RT inhibitor AZT and study the impact of RT inhibition on small RNA generation and mosquito survival. This objective will allow us to determine whether generation of viral DNA forms is a mechanism of Culex mosquitoes to control replication of RNA viruses.