Abstract The outbreak of SARS-CoV-2 in late 2019 has resulted in the loss of over 6 million lives worldwide. Since then, there has been an intense focus on the development of vaccines and clinical treatments to increase the survivability of the disease caused by this virus. However, in nature other diverse sarbecoviruses circulate which may present future spillover potential and for which these treatments may be ineffective. Clade 3 sarbecoviruses originate in Africa and Europe, outside of the regions considered to be sarbecovirus hotspots in Southeast Asia. The geographical location and the absence of native human ACE2 utilization from the earliest viruses discovered in this clade resulted in this clade being discounted. However, we have recently shown that one member of this clade, BtKY72 from Kenya, has the capacity to gain human ACE2 binding within one amino acid mutation and cellular entry within two mutations. Furthermore, we demonstrated for the first time that another member of this clade, Khosta-2 from Russia, can natively bind human ACE2 as a wildtype sequence. Together, our recent observations indicate the need to develop tools to study and inhibit potential human infection by this overlooked clade of viruses. Clade 3 may be the origin of a future sarbecovirus spillover, but current tools might have limited protective capacity due to the genetic divergence between Clade 3 and the prior human sarbecoviruses in the spike protein, the viral surface glycoprotein responsible for receptor binding and fusion of the viral envelope and the host cell membrane. I hypothesize that all members of this clade can gain human ACE2 utilization within a couple of mutations in the receptor binding domain of the spike glycoprotein but that current vaccines and antibody treatments will have reduced efficacy against clade 3 sarbecoviruses. In Aim 1 of this proposal, I will uncover receptor usage of all current members of clade 3 in Rhinolophus bat species with ranges in Africa, Europe, and Asia identify mutations that enable human ACE2 binding and cellular entry of these viruses making use of safe non-replicating pseudovirus systems. In Aim 2, I will establish what clinical tools in terms of vaccines and monoclonal antibody treatments would be effective at preventing cellular entry of clade 3 sarbecoviruses. Understanding current native receptor usage combined with a sequence assessment of sarbecoviruses that may be able to coinfect a specific species of Rhinolophus will give insight into the evolutionary possibilities available to these viruses. In addition, identification of mutations that enable human ACE2 binding and cellular entry in human cell lines will provide context on how close these viruses are to achieving this first step necessary for human spillover. Finally, the assessment of current tools for their effectiveness against clade 3 sarbecoviruses and the structural characterization of clade 3 spike ectodomains, will give us a head start should t...