Project Summary While knowledge of the transmission modes for respiratory pathogenic viruses is critical, understanding their transmission mechanisms is hampered by existing sampling methods. Commonly used bioaerosol samplers have low collection efficiencies for particles < 0.3 m (e.g., influenza virus and the recent SARS- CoV-2), and the process involved in the collection often reduces infectivity, lowering the chances to accurately assess the extent of the occurrence. To address these issues, we developed under a NIAID grant the VIable Virus Aerosol Sampler (VIVAS), which has been proven more efficient and reliable in collecting viable viruses than the industry standard samplers. This system has been tested in the laboratory and the field, and it is now commercially available as the BioSpot Sampler (sold by our licensee Aerosol Devices Inc.). Nevertheless, none of the commercial bioaerosol samplers, including the BioSpot, has the capability to collect size-fractionated airborne particles. Although previous efforts have tried to separate virus-containing particles by aerodynamic size, maintaining their infectivity during sampling remains challenging. Here, we aim to develop a novel sampling system, the BioCascade that will allow the collection into liquid of four different particle fractions: >10 m, PM4-10, PM1-4 and PM1 (fine) while maintaining infectivity. In Phase I, we will build a BioCascade prototype that can be attached to a VIVAS unit and to a gelatin filter collection device. The proposed approach will create a powerful tool, not available before, that can transform our current disease- control paradigm from a reactive approach (to an outbreak after its fact) to a proactive approach (informing us the forthcoming outbreak.) Specific aims of this proposal are: 1) Achieve the desired particle size cut-offs (>10 m, PM4-10, PM1-4 and PM1) while minimizing particle losses in the impactors and efficiently delivering each fraction simultaneously into a liquid collection medium. Preliminary tests to determine the effective cut-off of the BioCascade will be conducted at Aerosol Dynamics Inc. in combination with the Viable Virus Aerosol Sampler, using aerosol particles of known sizes. (2) Demonstrate a high viability/infectivity in the delivered samples while achieving optimal size separation. The BioCascade unit combined with the VIVAS or the gelatin filter, will be further tested at the University of Florida using aerosolized microorganisms of different sizes. The effect of the system on the viability of the samples will be determined. (3) Evaluate the exposure level to influenza viruses at a Student Health Care Center (SHCC). The BioCascade with the VIVAS will be placed at the waiting room in the SHCC for collecting respiratory viruses in the four size fractions during the “flu season” to capture the circulating strains and to determine the pathogen loading in each size fraction. This information will be of utmost importance to understan...