Mila Willemsen

Introduction
The increasing prevalence of mosquito-borne diseases in Europe, particularly West Nile Virus (WNV) and Usutu Virus (USUV), emphasises the need for rapid, reliable, and fieldable diagnostic methods. Conventional molecular detection methods such as RT-qPCR offer high sensitivity and specificity but are impractical for in-field application due to their equipment demands and environmental requirements. This study, conducted within the One Health PACT framework, aimed to evaluate and establish reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays suitable for in-field virus detection while maintaining performance comparable to that of RT-qPCR.

Methods
Diluted virusstocks of WNV and USUV were used for comparison between RT-LAMP (New England Biolabs, E1708) in combination with visual SYBR Green I detection, and RT-qPCR (Thermo Fisher, 4444434). Using the same diluted virusstocks, the limit of detection (LOD) was established and cross reactivity between viruses was ruled out for both assays. Finally, 65 RNA isolates supplied by Erasmus MC originating from USUV-positive birds of the genera Turdus, Pica, Strix, and Cyanistes were used to determine the sensitivity and specificity of the USUV RT-LAMP assay.

Results
RT-qPCR reliably detected WNV and USUV up to Ct-values of 36.38 and 34.79 respectively. RT-LAMP demonstrated a remarkably lower LOD, detecting WNV up to an approximated Ct-value of 44.49, and USUV up to an approximated Ct-value of 38.99 in virusstocks. This was confirmed both visually via SYBR Green colorimetric change and by gel electrophoresis. For USUV, a sensitivity of 82% and a specificity of 75% were found.

Conclusion
RT-LAMP demonstrated adequate sensitivity and specificity for the detection of WNV and USUV and showed a lower LOD compared to RT-qPCR in virusstocks. Its minimal equipment requirements and visual readout make it a promising tool for in-field arbovirus surveillance.