Culex Mosquito Piwi4 Is Antiviral against Two Negative-Sense RNA Viruses

Culex spp. mosquitoes transmit several pathogens concerning public health, including West Nile virus and Saint Louis encephalitis virus. Understanding the antiviral immune system of Culex spp. mosquitoes is important for reducing the transmission of these viruses. Mosquitoes rely on RNA interference (RNAi) to control viral replication. While the siRNA pathway in mosquitoes is heavily studied, less is known about the piRNA pathway. The piRNA pathway in mosquitoes has recently been connected to mosquito antiviral immunity. In Aedes aegypti, Piwi4 has been implicated in antiviral responses. The antiviral role of the piRNA pathway in Culex spp. mosquitoes is understudied compared to Ae. aegypti. Here, we aimed to identify the role of PIWI genes and piRNAs in Culex quinquefasciatus and Culex tarsalis cells during virus infection. We examined the effect of PIWI gene silencing on virus replication of two arboviruses and three insect-specific viruses in Cx. quinquefasciatus derived cells (Hsu) and Cx. tarsalis derived (CT) cells. We show that Piwi4 is antiviral against the La Crosse orthobunyavirus (LACV) in Hsu and CT cells, and the insect-specific rhabdovirus Merida virus (MERDV) in Hsu cells. None of the silenced PIWI genes impacted replication of the two flaviviruses Usutu virus (USUV) and Calbertado virus, or the phasivirus Phasi-Charoen-like virus. We further used small RNA sequencing to determine that LACV-derived piRNAs, but not USUV-derived piRNAs were generated in Hsu cells and that PIWI gene silencing resulted in a small reduction in vpiRNAs. Finally, we determined that LACV-derived DNA was produced in Hsu cells during infection, but whether this viral DNA is required for vpiRNA production remains unclear. Overall, we expanded our knowledge on the piRNA pathway and how it relates to the antiviral response in Culex spp mosquitoes.