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 Western 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 a total of seven ISVs, all of which have been previously associated with Cx. tarsalis , along with their respective sRNA (siRNA and piRNA) profiles. The ISVs found here did not show a clear distribution pattern, but two of them (Marma virus and Culex narnavirus 1) were found in all sampled states. 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.