The unknown identity of the entry receptors utilized by many coronaviruses has significantly impeded our comprehensive understanding of these important pathogens. We recently reported an unexpected usage of angiotensin-converting enzyme 2 (ACE2), instead of Dipeptidyl peptidase-4 (DPP4), for cellular entry by NeoCoV and PDF-2180, close relatives of MERS-CoV that infect African bats. However, the presence and distribution of other ACE2-using merbecoviruses remain enigmatic. In this study, through sequence and structural analyses, we predicted that two newly discovered merbecoviruses infecting European Pipistrellus bats (Pipistrellus nathusii), namely MOW-15-22 and PnNL2018B, may also utilize ACE2 as their receptors. Functional profiling of 103 ACE2 orthologues from a variety of mammals confirmed that several ACE2 from bats efficiently facilitate the entry of MOW-15-22 and PnNL2018B. Conversely, no binding or entry signals for both viruses were detected when assessing seven DPP4 orthologues from humans, hedgehogs, and bats. Characterization of Pteronotus davyi (P.dav) ACE2 mediated entry of MOW-15-22 reveals a significant exogenous protease dependence, which can be dose-dependently neutralized by soluble P.dav ACE2 recombinant protein and a broadly neutralizing S2-targeting antibody. Verification of the previously reported critical ACE2 determinants for NeoCoV recognition reveals that MOW-15-22 and PnNL2018B displayed a glycan-independent binding mode with significantly altered interaction details. This study sheds light on two additional ACE2-using merbecoviruses circulating among European bats and underscores the potential zoonotic risk associated with these viruses.

Identification of ACE2 as the Entry Receptor for Two Novel European Bat Merbecoviruses