Novel African Rhinolophus bat ACE2 sequences reveal the determinants of Afro-Eurasian sarbecovirus entry

Sarbecoviruses, including SARS-CoV and SARS-CoV-2, are frequently linked to Rhinolophus bats as their putative natural reservoirs. Angiotensin-converting enzyme 2 (ACE2), a host carboxypeptidase widely expressed in mammalian tissues, plays a critical role in sarbecovirus infection by serving as the cellular receptor for the viral spike (S) protein. Given recent human outbreaks and pandemics caused by members of sarbecoviruses, and the wide distribution of Rhinolophus bats, it is essential to maintain surveillance of these viruses while improving our understanding of their interactions with bat hosts, particularly the ACE2 receptor. However, while Rhinolophus bats from Asia have been relatively well studied, African Rhinolophus bats remain underrepresented and require further investigation. In this study, five Rhinolophus bat lung samples were obtained from Zambia, and ACE2 genes from these individuals were cloned and sequenced. We further evaluated the susceptibility of ACE2 variants to a panel of sarbecoviruses, revealing key residues that influence viral infectivity. ACE2 polymorphism was observed among Rhinolophus simulator individuals, revealing multiple ACE2 genotypes within the sampled population. However, R. simulator ACE2s did not permit infection by the clade 3 Afro-Eurasian sarbecoviruses tested in this study. Notably, RhGB01 and BM48-31 virus utilized only Rhinolophus blasii ACE2. Mutational analyses further suggested that ACE2 residues 31 and 41 play important roles in modulating spike-ACE2 interactions. This study reports 4 unique ACE2 sequences of R. simulator and R. blasii, and provides new insights into the molecular interactions between African Rhinolophus species ACE2s and the S protein of sarbecoviruses circulating in Africa and Europe. ImportanceAs putative natural reservoirs of sarbecoviruses, including SARS-CoV and SARS-CoV-2, Rhinolophus bats play a critical role in the emergence of zoonotic coronaviruses, making it essential to understand their interactions with these viruses for future pandemic preparedness. While Asian Rhinolophus bats have been relatively well studied, African species remain underrepresented, highlighting the need for further investigation. In this study, we cloned and sequenced ACE2 genes of five Rhinolophus bats collected in Zambia, Africa. We identified ACE2 polymorphism among Rhinolophus simulator individuals, although this variation was not associated with susceptibility to the clade 3 Afro-Eurasian sarbecoviruses examined. In addition, we identified key ACE2 residues that govern SARS-CoV-2 spike-ACE2 interactions and contribute to distinct infectivity patterns across species. These findings expand our understanding of the molecular determinants of sarbecovirus host range and support improved surveillance and risk assessment of emerging coronaviruses.