Project Summary We request funds via a competitive revision to our existing NIA award (R21 AG065776) to support research on COVID-19. These supplemental funds will enable us to build and validate a multimodal contrast agent that reports the presence of Mpro—a protease intricately linked to the life cycle of the SARS-CoV-2. Existing tools to detect and monitor SARS-type viruses are based on PCR. While quantitative, these in vitro tools are limited in their ability to map the spatiotemporal distribution of the virus in living subjects. The missing element is a contrast agent for specific imaging of Mpro to map and measure this specific byproduct of SARS-CoV-2 infection. This work will accomplish this and report the presence of Mpro with conventional fluorescence as well as novel photoacoustic imaging. The fluorescence will allow for rapid and routine in vitro assays while the photoacoustic modality will be used for deep tissue in vivo imaging via rodent models. Aim 1 will build the probe based on a peptide sequence that is selectively cleaved by Mpro with cell penetration based on charge. The probe will be decorated with sonophores that are in a deactivated state until the peptide is cleaved. Once cleaved, these molecules produce both fluorescence and photoacoustic signal. Aim 2 will validate this contrast agent with a less infectious analogue of SARS-CovV-2 (Sindbis virus). Sindbis virus can easily be handled in BSL-2 facilities and will allow work to commence immediately. We will validate the probe with infected cells and infected animals. These aims are feasible because of Dr. Jokerst’s prior work in optical imaging and contrast agent construction and Dr. Siqueira-Neto’s work in infectious disease including image-based screening tools for therapies and pathogens. The innovation of this work is the first contrast agent to image COVID-19 infection. The significance is that, once completed, the community will have a powerful chemical tool to quantify and locate SARS-Cov-2 infection to answer key questions about this disease: What is the time course of infection and biodistribution?; How does biodistribution change by route of infection? Are there latent disease reservoirs?; How do protease levels change in response to therapy? Unfortunately, none of these questions can be answered because there are no in vivo imaging methods specific for viruses much less SARS- CoV-2.