The recurrent Spike A222V mutation in SARS-CoV-2 enhances replication in primary deer lung cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects humans and animals and is therefore a pathogen of grave concern within a One Health framework. Identifying animal-adaptive mutations is critical to preserving One Health, as these mutations could also lead to the persistence of SARS-CoV-2 in animal reservoirs with continual spillover to humans. Therefore, we sought to pair experimental evolution and epidemiological data to identify putative human- and animal-adaptive viral residues and determine their impact on replication-competent SARS-CoV-2 in both human and animal cells. We passaged SARS-CoV-2 in cells expressing human, dog, cat, mink, and white-tailed deer ACE2 and sequenced the passaged populations. In addition, we searched SARS-CoV-2 sequences for mutations following patterns of convergent evolution that were common to both human- and animal-derived SARS-CoV-2 sequences. We identified the epidemiologically relevant Spike A222V mutation from our passaging experiment in cells expressing cat ACE2, a mutation that has also arisen independently across eight lineages of SARS-CoV-2 from human- and animal-derived sequences. To assess its impact on replication in human and animal cells, we constructed SARS-CoV-2 Spike A222V in the Wuhan-Hu-1 backbone with Spike D614G; this virus replicated similarly to the WT SARS-CoV-2 in human lung epithelial cells. In contrast, SARS-CoV-2 Spike A222V demonstrated an advantage in replication in primary deer lung cells, which was not mediated by the deer ACE2 receptor. Infection