Field Detection of arboviruses directly from mosquito traps Firebird Biomolecular Sciences, LLC

Project Summary/Abstract: Field Detection of arboviruses directly from mosquito traps Firebird Biomolecular Sciences, LLC. Abstract Mosquitoes are the world's deadliest creatures. In the US, mosquitoes are vectors for West Nile, many kinds of encephalitis, and viruses arriving Latin America and Africa. In the developing world, they cause millions of cases of disease and death each year. Routine insect spraying is not a ""fix"", as it leads to pesticide resistance, encounters public opposition, and destroys beneficial insects. Thus, the EPA, CDC, ECDPC, and their counterparts around the globe, strongly recommend surveillance before spraying. Florida law requires it. In Phase 1, we created (a) innovative technology to generate CO2 to attract mosquitoes, (b) innovative tech- nology to collect viral RNA from the attracted mosquitoes, (c) data showing that collected RNA is stable under Florida summer conditions for at least 3 days, (d) processes where captured viral RNA is amplified directly without handling mosquitos or Qiagen RNA preps, and (e) reagents that allow robust multiplex amplification. These specs, which can transform the science and practice of arbovirus surveillance, attracted TrakITNow, an innovator in instrument design. Its award-winning trap (Moskeet) counts and speciates mosquitoes, doing everything short of collecting samples from the mosquitoes and determining if these hold arboviruses. This Phase 2 project will re-configure and metric the Phase 1 innovations to allow them, in Phase 3, to be placed on Moskeet in two operational architectures (OA1 and OA2). A series of improvement and adaptations will be evaluated using tightly crafted metrics in two Aims. Meeting these metrica will trigger a commitment by TrakITNow to support Phase 3 funding to launch a combined product, Aim 1 will develop and metric Firebird's chemistry for an OA1 architecture that will convert Moskeet from a trap-count-speciate-communicate product into a trap-count-speciate-sample-communicate product. This will exploit Firebird's Phase 1 innovation (SterileQTM) that autonomously traps and stabilizes viral RNA. Different baits, RNA-stabilizing preservatives, and pre-amplification enzymes will be metricked to create, at the end of Year 1, a Moskeet-ready OA1 chemistry that allows continuous sampling of mosquitoes. Metrics include (a) rt50% of infected mosquitoes leaving detectable sample, (b) rt200 hours half-life of RNA, and (c) capture of rt1 pg of RNA. An instrument from Insilixa (Hydra), also enhanced by Firebird's reagents, will be used for on-site virus detection. Hydra is portable, and its low complexity design allows it to be used by amateurs. Aim 2 will develop and metric Firebird's chemistry for an OA2 architecture. This will convert Moskeet into a trap-count-speciate-sample-analyze-communicate product. Here, we will develop and benchmark point-of- sampling amplification tools, specifically: (a) RT-PCR, and (b) loop amplification (LAMP). These will, as needed, incorporate reagent innovations (AEGIS, SAMRS, Biversals) to support highly multiplexed detection of arboviruses, create uniform amplification of multiple analytes, manage primer-primer products, prevent loss of PCR resources to off-target processes that create ""monsters"", and manage arbovirus sequence divergence.