Project Summary/Abstract ? Advanced Bioaerosol Technology Core The Advanced Bioaerosol Technology Core (ABTC) will develop a comprehensive suite of innovative technologies for improved sampling, fractionation, culture, and characterization of influenza virus aerosols to enable new analytical capabilities in support of the U19 research project goals. The advanced technologies developed through the ABTC will be employed within each individual research project to yield more effective studies of airborne transmission and infectivity through a combination of efficient ambient sampling to evaluate size-dependent distributions of infective virus within the clinical environment, and high-resolution individual exhaled breath sampling to elucidate the fundamental source terms needed for accurate transmission modeling. The ABTC team will leverage expertise across the fields of bioengineering, bioaerosol system engineering, and respiratory virology to develop 5 interconnected technologies for advanced bioaerosol analysis. In Aim 1, a set of compact instruments will be developed for the collection and parallel size-based fractionation of ambient environmental aerosols to enable distributed monitoring of airborne virus during the proposed clinical studies. In Aim 2, a second aerosol sampling instrument employing a unique exhaled breath sampling technology will be developed to provide high resolution aerosol fractionation and collection, allowing us to evaluate both viable and non-infective virus emission from individual donors across narrow particle size ranges. To enhance infectivity characterization for sampled virus particles, a synthetic mucus hydrogel will be advanced in Aim 3 as a novel sample target for the environmental sampler, providing improved virus capture and cell culture, greatly improving virus capture and viability for downstream infectivity assays. A digital cell culture array technology will be developed in Aim 4, enabling the direct isolation of individual aerosol particles captured by the exhaled breath sampler, and allowing us to evaluate distribution and clustering of both viable and non-infective virus units at single particle resolution. The ABTC team will also develop an optimized cell line that will be employed for the analysis of virus collected with the developed instruments to enhance evaluation of infectivity. The core will validate the developed technologies for aerosol sampling, capture, isolation, culture, and analysis through a set of ferret studies before integration with the proposed U19 research projects, and will execute the required assays using clinical samples to support the goals of RP1 and RP2. The results of the ABTC research efforts will yield the instruments and technologies needed to enhance our understanding of influenza aerobiology, from the determination of critical source terms to the evaluation of infective particle distributions within the environment, supporting significant improvements to influenza transmission modeling, risk analysis, and mitigation, while making important contributions toward advancing our understanding of other respiratory viral pandemic threats.