Automated molecular detection of West Nile Virus in mosquito pools using the Panther Fusion system

West Nile Virus (WNV) is an arthropod-borne virus that is spread through mosquito vectors. WNV emerged in the US in 1999 and has since become endemic in the US, causing the most domestically acquired arboviral disease in the country. Mosquito surveillance for WNV is useful to monitor arboviral disease burden over time and across different locations. RT-qPCR is the preferred method for WNV surveillance, but these methods are labor-intensive. The Panther Fusion System has an Open Access feature that allows for laboratory-developed tests (LDTs) to run on a fully automated system for nucleic acid extraction, RT-qPCR, and result generation. This study demonstrates the successful optimization of a WNV multiplex LDT (assay targets: ENV and NS1 genes) for high-throughput environmental surveillance testing of mosquito pool homogenates on the Panther Fusion System. Analytical sensitivity of the assay was 186 and 744 copies/PCR reaction for the ENV and NS1 targets, respectively. To assess the performance of this assay, a total of 80 mosquito pools were tested, including 60 previously tested pools and 20 spiked negative mosquito pools. Among the 60 previously tested specimens, the Panther Fusion WNV LDT demonstrated 100% positive and negative agreement with the CDC West Nile RT-qPCR assay. The Panther Fusion WNV LDT also detected all 20 spiked specimens. The Panther Fusion WNV LDT assay was successfully developed and optimized for high throughput testing with similar performance to the previously used CDC West Nile RT-qPCR assay.