An Extraction-Free Colorimetric Reverse Transcription-Loop-Mediated Isothermal Amplification Platform for Rapid and Scalable Arbovirus Surveillance Using Mosquito Pools and Saliva.

Mosquitoes transmit a wide range of arboviruses that cause significant morbidity and mortality worldwide. Surveillance approaches (sentinel animals, mosquito pool screening using reverse transcription quantitative polymerase chain reaction [RT-qPCR], human serology) are limited by cost, processing time, and assay complexity, which can delay detection of arbovirus activity and timely implementation of vector control interventions. Here, we evaluated the performance of a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay under diverse conditions simulating real-world surveillance scenarios, using West Nile virus (WNV) and Everglades virus (EVEV). Colorimetric RT-LAMP assays detected WNV and EVEV without ribonucleic acid (RNA) extraction from a range of samples after 30 mins isothermal amplification at 65°C. The WNV RT-LAMP assay enabled detection of WNV from a single infected mosquito within pooled mosquito homogenates (1:24 and 1:49) without RNA extraction. Additionally, WNV was detected in mosquito saliva collected from sugar-saturated swabs after feeding, demonstrating the potential of behavior-based surveillance for infectious mosquitoes. The EVEV RT-LAMP assay enabled detection of EVEV across high (7.25 log10 PFU/ml) and low (1.25 log10 PFU/ml) titers, as well as heat-inactivated virus, without RNA extraction. Two of the three WNV primer sets tested produced false positives. The colorimetric RT-LAMP platform enables rapid, extraction-free detection of arboviruses with simple visual readout and has strong potential as a scalable pre-screening tool for arbovirus surveillance programs. The RT-LAMP assays require in-house validation and confirmatory testing for positive field samples.