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Given the recent human outbreaks and pandemics caused by members of sarbecoviruses and the wide distribution of\n Rhinolophus<\/jats:italic>\n 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\n Rhinolophus<\/jats:italic>\n bats from Asia have been relatively well studied, African\n Rhinolophus<\/jats:italic>\n bats remain underrepresented and require further investigation. In this study, five\n Rhinolophus<\/jats:italic>\n 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\n Rhinolophus simulator<\/jats:italic>\n individuals, revealing multiple ACE2 genotypes within the sampled population. However,\n R. simulator<\/jats:italic>\n ACE2s did not permit infection by the clade 3 Afro-Eurasian sarbecoviruses tested in this study. Notably, the RhGB01 and BM48-31 viruses utilized only\n Rhinolophus blasii<\/jats:italic>\n ACE2. Mutational analyses further suggested that ACE2 residues 31 and 41 play important roles in modulating spike-ACE2 interactions. This study reports four unique ACE2 sequences of\n R. simulator<\/jats:italic>\n and\n R. blasii<\/jats:italic>\n , and provides new insights into the molecular interactions between African\n Rhinolophus<\/jats:italic>\n species ACE2s and the S protein of sarbecoviruses circulating in Africa and Europe.\n <\/jats:p>\n <\/jats:sec>\n \n IMPORTANCE<\/jats:title>\n \n As 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\n Rhinolophus<\/jats:italic>\n 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\n Rhinolophus<\/jats:italic>\n bats collected in Zambia, Africa. We identified ACE2 polymorphism among\n Rhinolophus simulator<\/jats:italic>\n 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.\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1128\/jvi.00540-26","type":"journal-article","created":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T14:08:48Z","timestamp":1787321328000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Novel African\n Rhinolophus<\/i>\n bat ACE2 sequences reveal the determinants of Afro-Eurasian sarbecovirus entry"],"prefix":"10.1128","volume":"100","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-2246-8172","authenticated-orcid":true,"given":"Yueying","family":"Zhang","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/057zh3y96","id-type":"ROR","asserted-by":"publisher"}],"name":"Division of Systems Virology, Department of Microbiology and Immunology, The Institute of Medical 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