Significance: SARS-CoV-2 emerged because of viral spillover from animals to humans, and spillback to other animal species has been observed with accelerating frequency. Cross-species transmission generally results in the rapid adaptation of the virus to the new host, and repeated transmissions may hasten viral evolution and novel strain emergence. We report the surprisingly rapid selection of numerous SARS-CoV-2 variants in cell culture and following infection of nonhuman mammalian hosts, including dogs and cats. These molecular changes in SARS-CoV-2 provide insight into mechanisms of viral host adaptation, lay the groundwork for additional studies assessing dominant variant fitness and phenotype, and highlight the potential for human reinfection with new viral variants arising in species in close and frequent contact with humans.

Significance: Pseudomonas aeruginosa is one of the leading causes of hospital-acquired infections worldwide primarily due to its ability to develop antibiotic-resistant biofilms. This report describes a mechanism whereby human airway epithelial cells (AECs) increase the ability of antibiotics to kill P. aeruginosa and reduce the formation of drug-resistant biofilms. We demonstrate that human AECs secrete extracellular vesicles containing the microRNA let-7b-5p, that the vesicles deliver let-7b-5p to P. aeruginosa , and that let-7b-5p decreases biofilm formation and increases the ability of beta-lactam antibiotics to reduce biofilm formation by down-regulating key antibiotic-resistance and biofilm genes. A combination of let-7b, which is in clinical trials as an anti-inflammatory agent, and beta-lactam antibiotics may help combat antibiotic-resistant P. aeruginosa infections.

SARS-CoV-2 (SARS2) infection of a novel permissive host species can result in rapid viral evolution. Data suggest that felids are highly susceptible to SARS2 infection, and species-specific adaptation following human-to-felid transmission may occur. We employed experimental infection and analysis of publicly available SARS2 sequences to observe variant emergence and selection in domestic cats. Three cohorts of cats (N = 23) were inoculated with SARS-CoV-2 USA-WA1/2020 or infected via cat-to-cat contact transmission. Full viral genomes were recovered from RNA obtained from nasal washes 1–3 days post-infection and analyzed for within-host viral variants. We detected 118 unique variants at ≥3 per cent allele frequency in two technical replicates. Seventy of these (59 per cent) were nonsynonymous single nucleotide variants (SNVs); the remainder were synonymous SNVs or structural variants. On average, we observed twelve variants per cat, nearly 10-fold higher than what is commonly reported in human patients. We observed signatures of positive selection in the spike protein and the emergence of eleven within-host variants located at the same genomic positions as mutations in SARS2 variant lineages that have emerged during the pandemic. Fewer variants were noted in cats infected from contact with other cats and in cats exposed to lower doses of cultured inoculum. An analysis of ninety-three publicly available SARS2 consensus genomes recovered from naturally infected domestic cats reflected variant lineages circulating in the local human population at the time of sampling, illustrating that cats are susceptible to SARS2 variants that have emerged in humans, and suggesting human-to-felid transmission occurring in domestic settings is typically unidirectional. These experimental results underscore the rapidity of SARS2 adaptation in felid hosts, representing a theoretical potential origin for variant lineages in human populations. Further, cats should be considered susceptible hosts capable of shedding virus during infections occurring within households.

SARS-CoV-2 spillback from humans into domestic and wild animals has been well-documented. We compared variants of cell culture-expanded SARS-CoV-2 inoculum and virus recovered from four species following experimental exposure. Five nonsynonymous changes in nsp12, S, N and M genes were near fixation in the inoculum, but reverted to wild-type sequences in RNA recovered from dogs, cats and hamsters within 1-3 days post-exposure. Fourteen emergent variants were detected in viruses recovered from animals, including substitutions at spike positions H69, N501, and D614, which also vary in human lineages of concern. The rapidity of in vitro and in vivo SARS-CoV-2 selection reveals residues with functional significance during host-switching, illustrating the potential for spillback reservoir hosts to accelerate evolution, and demonstrating plasticity of viral adaptation in animal models. One-Sentence Summary SARS-CoV-2 variants rapidly arise in non-human hosts, revealing viral evolution and potential risk for human reinfection.

In late 2019, SARS-CoV-2 spilled over from an animal host into humans, where it efficiently spread, resulting in the COVID-19 pandemic. Through both natural and experimental infections, we learned that many animal species are susceptible to SARS-CoV-2. Importantly, animals in close proximity to humans, including companion, farmed, and those at zoos and aquariums, became infected, and many studies demonstrated transmission to/from humans in these settings. In this study, we first review the literature of SARS-CoV-2 infections in tigers and lions and compare species, sex, age, virus and antibody detection assay, and types, frequency, and length of clinical signs, demonstrating broad heterogeneity among infections. We then describe a SARS-CoV-2 outbreak in lions, tigers, and hyenas at Denver Zoo in late 2021. Animals were tested for viral RNA (vRNA) for 4 months. Lions had significantly more vRNA in nasal swabs than both tigers and hyenas, and many individual lions experienced viral recrudescence after weeks of undetectable vRNA. Infectious virus was correlated with high levels of vRNA and was more likely to be detected earlier during infection. Four months post-infection, all tested animals generated robust neutralizing antibody titers. Animals were infected with Delta lineage AY.20 identical to a variant circulating at less than 1% in Colorado humans at that time, suggesting a single spillover event from an infected human spread within and between species housed at the zoo. Better understanding of epidemiology and susceptibility of SARS-CoV-2 infections in animals is critical to limit the current and future spread and protect animal and human health. IMPORTANCE: Surveillance and experimental testing have shown many animal species, including companion, wildlife, and conservatory, are susceptible to SARS-CoV-2. Early in the COVID-19 pandemic, big cats at zoological institutions were among the first documented cases of naturally infected animals; however, challenges in the ability to collect longitudinal samples in zoo animals have limited our understanding of SARS-CoV-2 kinetics and clearance in these settings. We measured SARS-CoV-2 infections over 4 months in lions, tigers, and hyenas at Denver Zoo and detected viral RNA, infectious virus, neutralizing antibodies, and recrudescence after initial clearance. We found lions had longer and higher levels of virus compared to the other species. All animals were infected by a rare viral lineage circulating in the human population, suggesting a single spillover followed by interspecies transmission. These data are important in better understanding natural SARS-CoV-2 spillover, spread, and infection kinetics within multiple species of zoo animals.

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.