Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. We investigated the ability of primary cells from Rhinolophus and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis, and Nyctalus noctula, to support SARS-CoV-2 replication. None of these cells were permissive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that the restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or the absence of bACE2 binding in these cells. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication, which correlated with a potent interferon response. Our data highlight the existence of species-specific and cell-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses. IMPORTANCE Bats are host ancestors of several viruses that cause serious disease in humans, as illustrated by the ongoing SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions has been hampered by a limited number of available bat cellular models. We have generated primary cells and cell lines from several bat species that are relevant for coronavirus research. The various permissivities of the cells to SARS-CoV-2 infection offered the opportunity to uncover some species-specific molecular restrictions to viral replication. All bat cells exhibited a potent entry-dependent restriction. Once this block was overcome by overexpression of human ACE2, which serves at the viral receptor, two bat cell lines controlled well viral replication, which correlated with the inability of the virus to counteract antiviral responses. Other cells potently inhibited viral release. Our novel bat cellular models contribute to a better understanding of the molecular interplays between bat cells and viruses.

Departament de Biologia Evolutiva Ecologia i Ciències Ambientals Facultat de Biologia Universitat de Barcelona Barcelona Spain

Department of Ecology and Diseases of Zoo Animals Game Fish and Bees University of Veterinary Sciences Brno Brno Czech Republic

Department of Infectious Diseases and Pathobiology Vetsuisse Faculty University of Bern Bern Switzerland

INSERM U1259 MAVIVH and Plateforme IBiSA de Microscopie Electronique Faculté de Médecine Université de Tours Tours France

Institut de Recerca de la Biodiversitat Faculty of Biology Universitat de Barcelona Barcelona Spain

Institut Pasteurgrid 428999 7 Université de Paris Cité CNRS UMR 3569 Virus and Immunity Unit Paris France

Institut Pasteurgrid 428999 7 Université de Paris Cité CNRS UMR 3569 Virus Sensing and Signaling Unit Paris France

Institut Pasteurgrid 428999 7 Université de Paris Cité Lyssavirus Epidemiology and Neuropathology Unit Paris France

Institute of Vertebrate Biology of the Czech Academy of Sciences Brno Brno Czech Republic

Institute of Virology and Immunology Bern and Mittelhäusern Switzerland

Université de Lyon LabEx Ecofect Lyon France

Université Lyon 1 CNRS Laboratoire de Biométrie et Biologie Evolutive UMR5558 Villeurbanne France

University of Lyon Université Lyon 1 INSERM INRAE Stem Cell and Brain Research Institute Bron France

Vaccine Research Institute Créteil France

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