Rapid detection of West Nile and Dengue viruses from mosquito saliva by loop-mediated isothermal amplification and displaced probes.

Arthropod-borne viruses are major causes of human and animal disease, especially in endemic low- and middle-income countries. Mosquito-borne pathogen surveillance is essential for risk assessment and vector control responses. Sentinel chicken serosurveillance (antibody testing) and mosquito pool screening (by RT-qPCR or virus isolation) are currently used to monitor arbovirus transmission, however substantial time lags of seroconversion and/or laborious mosquito identification and RNA extraction steps sacrifice their early warning value. As a consequence, timely vector control responses are compromised. Here, we report on development of a rapid arbovirus detection system whereby adding sucrose to reagents of loop-mediated isothermal amplification with displaced probes (DP-LAMP) elicits infectious mosquitoes to feed directly upon the reagent mix and expectorate viruses into the reagents during feeding. We demonstrate that RNA from pathogenic arboviruses (West Nile and Dengue viruses) transmitted in the infectious mosquito saliva was detectable rapidly (within 45 minutes) without RNA extraction. Sucrose stabilized viral RNA at field temperatures for at least 48 hours, important for transition of this system to practical use. After thermal treatment, the DP-LAMP could be reliably visualized by a simple optical image sensor to distinguish between positive and negative samples based on fluorescence intensity. Field application of this technology could fundamentally change conventional arbovirus surveillance methods by eliminating laborious RNA extraction steps, permitting arbovirus monitoring from additional sites, and substantially reducing time needed to detect circulating pathogens.

Infrared light sensors permit rapid recording of wingbeat frequency and bioacoustic species identification of mosquitoes.

Recognition and classification of mosquitoes is a critical component of vector-borne disease management. Vector surveillance, based on wingbeat frequency and other parameters, is becoming increasingly important in the development of automated identification systems, but inconsistent data quality and results frequently emerge from different techniques and data processing methods which have not been standardized on wingbeat collection of numerous species. We developed a simple method to detect and record mosquito wingbeat by multi-dimensional optical sensors and collected 21,825 wingbeat files from 29 North American mosquito species. In pairwise comparisons, wingbeat frequency of twenty six species overlapped with at least one other species. No significant differences were observed in wingbeat frequencies between and within individuals of Culex quinquefasciatus over time. This work demonstrates the potential utility of quantifying mosquito wingbeat frequency by infrared light sensors as a component of an automated mosquito identification system. Due to species overlap, wingbeat frequency will need to integrate with other parameters to accurately delineate species in support of efficient mosquito surveillance, an important component of disease intervention.