Abstract
SARS-CoV-2 rapidly adapts to new hosts following cross-species transmission; this is highly relevant as unique within-host variants have emerged following infection of susceptible wild and domestic animal species. Furthermore, SARS-CoV-2 transmission from animals (e.g., white-tailed deer, mink, domestic cats, and others) back to humans has been observed, documenting the potential of animal-derived variants to infect humans. Here, we investigate SARS-CoV-2 evolution and host-specific adaptation during an outbreak in Amur tigers (Panthera tigris altaica), African lions (Panthera leo), and spotted hyenas (Crocuta crocuta) at Denver Zoo in 2021. SARS-CoV-2 genomes from longitudinal samples from 16 individuals are evaluated for within-host variation and genomic signatures of selection, and we determine that the outbreak was likely initiated by a single spillover of a rare Delta sublineage. Within-host virus populations rapidly expand and diversify, and we detect signatures of purifying and positive selection, including strong positive selection in hyenas and in the nucleocapsid (N) gene in all animals. Four candidate species-specific adaptive mutations are identified: N A254V in lions and hyenas, and ORF1a E1724D, spike T274I, and N P326L in hyenas. These results reveal accelerated SARS-CoV-2 adaptation following host shifts in three non-domestic species in daily contact with humans.