Virome profiling of Culex tarsalis through small RNA-seq: A challenge of suboptimal samples

Viral infections in mosquitoes trigger the RNA interference (RNAi) pathway, a key antiviral defense mechanism that generates virus-derived small RNAs (vsRNAs). Given the natural enrichment of vsRNAs during infection and their stability, small RNA sequencing (sRNA-seq) has emerged as a powerful tool for virome characterization. Culex tarsalis is a widely distributed mosquito species in North America and is an important vector of West Nile virus (WNV). Previous studies have shown that co-infection with insect-specific viruses (ISVs) can modulate WNV replication in Cx. tarsalis , highlighting the importance of characterizing the virome of this species. Here, we investigated the virome of Cx. tarsalis populations across 5 states of the Midwestern United States using sRNA-seq. We analyzed samples from 17 geographic locations which were collected under suboptimal field conditions during the COVID-19 pandemic, presenting challenges related to sample integrity. Despite these challenges, sRNA-seq proved to be a reliable method for virome analysis. We identified seven viruses associated with Cx. tarsalis , along with their respective sRNA (siRNA and piRNA) profiles. These findings not only deepen our understanding of ISVs, but also demonstrate the utility of sRNA-seq in non-ideal situations, enabling the collection and analysis of samples under real-world surveillance scenarios.