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

## TL;DR

Researchers used small RNA sequencing to identify seven insect-specific viruses in Culex tarsalis mosquitoes collected under poor conditions, showing the method's reliability even with suboptimal samples.

## Contribution

Demonstrates the effectiveness of small RNA-seq for virome profiling in real-world, non-ideal sample conditions.

## Key findings

- Seven insect-specific viruses were identified in Culex tarsalis mosquitoes across five U.S. states.
- Two viruses, Marma virus and Culex narnavirus 1, were found in all sampled states.
- Small RNA-seq proved reliable for virome analysis despite suboptimal sample integrity.

## Abstract

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.

Mosquitoes are capable of transmitting pathogenic viruses to humans and other vertebrate hosts during feeding. However, they can also carry viruses that infect only mosquitoes, which are known as insect specific viruses (ISVs). Some ISVs are have the potential to affect how mosquitoes interact with viruses that can cause diseases in vertebrates. When a virus infects a mosquito, the insect’s immune system responds by producing small RNA fragments complementary to the virus’s genetic code. In this study, we sequenced these fragments and assembled them to identify seven ISVs present in Culex tarsalis mosquitoes collected under non-ideal storage conditions from five USA states. It is important to note that although the samples were not collected under ideal conditions for RNA studies, valuable information was still recovered by studying the small RNAs. Our work highlights the diversity of ISVs that Cx. tarsalis mosquitoes carry, and demonstrates how even suboptimal samples can provide insights into mosquito viral communities and their relationships.