# Genomic characterization of a severe West Nile Virus transmission season using a single reaction amplicon sequencing approach

## TL;DR

Researchers used a new sequencing method to study West Nile Virus in Nebraska, finding high genetic diversity and early transmission in the west part of the state.

## Contribution

Validated a single-reaction amplicon sequencing method for generating high-quality WNV genomes from mosquito pools.

## Key findings

- The IDT xGen WNV panel produced coding-complete and accurate WNV genomes from mosquito pools.
- The 2023 Nebraska outbreak showed significant genetic diversity with minimal clustering of WNV genomes.
- Early and intense WNV transmission occurred in western Nebraska without seeding transmission elsewhere.

## Abstract

West Nile virus (WNV) is an endemic arthropod-borne virus that has routinely caused seasonal outbreaks in the United States since it was first detected in 1999. While phylogenetic studies have shown how WNV has diversified and undergone genotype replacement since introduction, more geographically focused studies are needed to understand intricate transmission dynamics at local and regional scales. In this study, we validate the IDT xGen WNV panel, a novel single reaction amplicon-based Next-Generation Sequencing approach, to generate high-quality WNV genomes and compare it to the “Primal Scheme” assay for WNV, a common amplicon sequencing strategy. By generating >250 genomes from mosquito pools, we show that the IDT xGen WNV panel generated coding-complete and accurate WNV genomes when compared to the current sequencing approaches. Additionally, we used this approach to generate 100 coding-complete WNV genomes from surveillance pools of mosquitoes collected in Nebraska during the 2023 outbreak. Our discrete phylogeographic analysis revealed substantial genetic diversity in WNV genomes from 2023 with minimal clustering across the state. This study demonstrated the utility of a single reaction amplicon-based sequencing approach to generate quality WNV genomes from routine surveillance samples and characterize WNV transmission dynamics in a high-incidence setting.

West Nile virus (WNV) remains the most impactful arthropod-borne virus in the United States. Genomic epidemiology of WNV can inform our understanding of virus emergence and transmission, ultimately informing mosquito surveillance and control programs. In this study, we validated a simple, single-reaction amplicon sequencing approach by generating >250 coding-complete WNV genomes from WNV positive pools of mosquitoes. We then characterized the 2023 transmission in Nebraska, a high-incidence state, and showed that the western portion of the state saw early and intense WNV transmission, as evidenced by high infection rates in both mosquitoes and humans. We showed there was significant genetic diversity in WNV genotypes with minimal clustering of WNV genomes across the state, indicating the early transmission initiation in the western portion did not seed transmission in other areas of the state. Our study demonstrated the utility of a single-reaction sequencing approach to generate high-quality WNV genomes directly from pools of mosquitoes collected for surveillance purposes while also highlighting the substantial WNV genetic diversity present during a severe WNV transmission season.