PROJECT SUMMARY An overarching goal of the Mutant Mouse Resource and Research Center at the University of Missouri (MU- MMRRC) is to optimize and refine mouse models so that biomedical research using these models can proceed rapidly and effectively. The study of COVID-19 has relied on mouse models such as the B6.Cg-Tg(K18- ACE2)2Prlmn/J mouse (K18-hACE2). While great advances have already been made, some studies, notably those assessing Post-Acute Sequela of COVID-19 (PASC) are hampered by the fact that infected mice are currently maintained at animal biosafety level 3. These facilities often have limited capacity, resulting in delays in research and the elevated costs associated with this level of biocontainment. The study of disease pathogenesis associated with reinfection of SARS-CoV-2 and cross infection with variants is also hampered as such studies are by nature longer in duration and also require the need for PASC assessment. Thus, there is a pressing need to establish strategies by which studies of PASC can be accomplished more quickly and economically. Herein we propose that such studies can be performed in modified biosafety level 2 conditions, provided that viral clearance is established to a point acceptable to investigators as well as experts in institution biosafety and animal care and use. The objective of this proposal is to assess the kinetics / natural history of SARS-CoV-2 in the K18-hACE2 mouse model to determine when clearance is reliably observed and to establish standard operating procedures for the safe transfer of mice from ABSL-3 facilities to more accessible and less expensive ABSL-2 facilities. To broaden the applicability of these strategies to future research, studies will be performed using two different routes of infection and three different variants of the virus. Moreover, we will also perform these studies using two gut microbiomes that differ in species richness and that have been associated with differing disease susceptibility in preliminary studies. A second objective will be to similarly assess the kinetics of reinfection by SARS-CoV-2 and cross infection with two different variants of virus. The resulting expansion of the utility of models of COVID-19 for PASC studies will be critical to research on this devastating disease.