The SARS-CoV-2 (SARS-CoV-2) virus has caused a worldwide pandemic because of the virus's ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SARS-CoV-2, there is no practical way to assess the susceptibility of every species to SARS-CoV-2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SARS-CoV-2, we expressed the ACE2 and/or transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that expression of both human ACE2 and TMPRSS2 genes is necessary to support SARS-CoV-2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SARS-CoV-2 in these cell lines were comparable to those observed in control Vero cells. To further test the model, we developed seven additional transgenic cell lines expressing the ACE2 and TMPRSS2 derived from Felis catus (cat), Equus caballus (horse), Sus domesticus (pig), Capra hircus (goat), Mesocricetus auratus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SARS-CoV-2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. Cells expressing genes from either bat species tested demonstrated temporal replication of SARS-CoV-2 that peaked early and was not sustained. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SARS-CoV-2 and emerging variant viruses.

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Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) is believed to have a zoonotic origin. Bats are a suspected natural host of SARS-CoV-2 because of sequence homology with other bat coronaviruses. Understanding the origin of the virus and determining species susceptibility is essential for managing the transmission potential during a pandemic. In a previous study, we established an in vitro animal model of SARS-CoV-2 susceptibility and replication in a non-permissive avian fibroblast cell line (DF1) based on expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) from different animal species. In this work, we express the ACE2 of seven bat species in DF1 cells and determine their ability to support attachment and replication of the original SARS-CoV-2 Wuhan lineage virus, as well as two variants, Delta and Lambda. We demonstrate that the ACE2 receptor of all seven species: little brown bat (Myotis lucifugus), great roundleaf bat (Hipposideros armiger), Pearson's horseshoe bat (Rhinolophus pearsonii), greater horseshoe bat (Rhinolophus ferrumequinum), Brazilian free-tailed bat (Tadarida brasiliensis), Egyptian rousette (Rousettus aegyptiacus), and Chinese rufous horseshoe bat (Rhinolophus sinicus), made the DF1 cells permissible to the three isolates of SARS-CoV-2. However, the level of virus replication differed between bat species and variant tested. In addition, the Wuhan lineage SARS-CoV-2 virus replicated to higher titers (104.5 -105.5 TCID50) than either variant virus (103.5-104.5 TCID50) on pass 1. Interestingly, all viruses tested grew to higher titers (approximately 106 TCID50) when cells expressed the human ACE2 gene compared to bat ACE2. This study provides a practical in vitro method for further testing of animal species for potential susceptibility to current and emerging SARS-CoV-2 viruses.