Releasing Intracellular NS1 from Mosquito Cells for the Detection of Dengue Virus-Infected Mosquitoes

Dengue virus (DENV), the pathogen that causes dengue fever, is mainly transmitted by Aedes aegypti. Surveillance of infected mosquitoes is a major component of integrated mosquito control methods for reducing the risk of vector-born disease outbreaks. However, a specialized rapid test for DENV detection in mosquitoes is not currently available. Utilizing immunoblotting, we found that the secretion of NS1 from both a DENV-infected mosquito cell line and mosquito bodies was below the detection threshold. However, when Triton X-100 was used to lyse infected mosquitoes, intracellular NS1 was released, and could then be effectively detected by the NS1 rapid test. The distribution of DENV NS1 in intrathoracically infected mosquitoes was different from that of orally infected mosquitoes. Next, we performed sensitivity tests by bisecting mosquitoes longitudinally; one half of each mosquito was subjected to the NS1 rapid test while the other half was used for qPCR confirmation. This modified test had a sensitivity of nearly 90% from five days post-infection onwards, while DENV had escaped from the midgut barrier. This adapted test offers a valuable, easy-to-use tool for mosquito surveillance, which is a crucial component of DENV disease control.