Natural or experimental infection of domestic cats and virus transmission from humans to captive predatory cats suggest that felids are highly susceptible to SARS-CoV-2 infection. However, it is unclear which cells and compartments of the respiratory tract are infected. To address this question, primary cell cultures derived from the nose, trachea, and lungs of cat and lion were inoculated with SARS-CoV-2. Strong viral replication was observed for nasal mucosa explants and tracheal air–liquid interface cultures, whereas replication in lung slices was less efficient. Infection was mainly restricted to epithelial cells and did not cause major pathological changes. Detection of high ACE2 levels in the nose and trachea but not lung further suggests that susceptibility of feline tissues to SARS-CoV-2 correlates with ACE2 expression. Collectively, this study demonstrates that SARS-CoV-2 can efficiently replicate in the feline upper respiratory tract ex vivo and thus highlights the risk of SARS-CoV-2 spillover from humans to felids.

The emergence of the coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inspired rapid research efforts targeting the host range, pathogenesis and transmission mechanisms, and the development of antiviral strategies. Genetically modified mice, rhesus macaques, ferrets, and Syrian golden hamsters have been frequently used in studies of pathogenesis and efficacy of antiviral compounds and vaccines. However, alternatives to in vivo experiments, such as immortalized cell lines, primary respiratory epithelial cells cultured at an air–liquid interface, stem/progenitor cell-derived organoids, or tissue explants, have also been used for isolation of SARS-CoV-2, investigation of cytopathic effects, and pathogen–host interactions. Moreover, initial proof-of-concept studies for testing therapeutic agents can be performed with these tools, showing that animal-sparing cell culture methods could significantly reduce the need for animal models in the future, following the 3R principles of replace, reduce, and refine. So far, only few studies using animal-derived primary cells or tissues have been conducted in SARS-CoV-2 research, although natural infection has been shown to occur in several animal species. Therefore, the need for in-depth investigations on possible interspecies transmission routes and differences in susceptibility to SARS-CoV-2 is urgent. This review gives an overview of studies employing alternative culture systems like primary cell cultures, tissue explants, or organoids for investigations of the pathophysiology and reverse zoonotic potential of SARS-CoV-2 in animals. In addition, future possibilities of SARS-CoV-2 research in animals, including previously neglected methods like the use of precision-cut lung slices, will be outlined.

Several animal species are susceptible to SARS-CoV-2 infection, as documented by case reports and serological and in vivo infection studies. However, the susceptibility of many animal species remains unknown. Furthermore, the expression patterns of SARS-CoV-2 entry factors, such as the receptor angiotensin-converting enzyme 2 (ACE2), as well as transmembrane protease serine subtype 2 (TMPRSS2) and cathepsin L (CTSL), cellular proteases involved in SARS-CoV-2 spike protein activation, are largely unexplored in most species. Here, we generated primary cell cultures from the respiratory tract of domestic and wildlife animals to assess their susceptibility to SARS-CoV-2 infection. Additionally, the presence of ACE2, TMPRSS2 and CTSL within respiratory tract compartments was investigated in a range of animals, some with unknown susceptibility to SARS-CoV-2. Productive viral replication was observed in the nasal mucosa explants and precision-cut lung slices from dogs and hamsters, whereas culture models from ferrets and multiple ungulate species were non-permissive to infection. Overall, whereas TMPRSS2 and CTSL were equally expressed in the respiratory tract, the expression levels of ACE2 were more variable, suggesting that a restricted availability of ACE2 may contribute to reduced susceptibility. Summarized, the experimental infection of primary respiratory tract cell cultures, as well as an analysis of entry-factor distribution, enable screening for SARS-CoV-2 animal reservoirs.

Formation of neutrophil extracellular traps (NETs) is a two-faced innate host defense mechanism, which, on the one hand, can counteract microbial infections, but on the other hand, can contribute to massive detrimental effects on the host. Cholesterol depletion from the cellular membrane by Methyl-β-cyclodextrin (MβCD) is known as one of the processes initiating NET formation. Since neutrophils mainly act in an inflammatory environment with decreased, so-called hypoxic, oxygen conditions, we aimed to study the effect of oxygen and the oxygen stress regulator hypoxia-inducible factor (HIF)-1α on cholesterol-dependent NET formation. Thus, murine bone marrow-derived neutrophils from wild-type and HIF-knockout mice or human neutrophils were stimulated with MβCD under normoxic (21% O₂) compared to hypoxic (1% O₂) conditions, and the formation of NETs were studied by immunofluorescence microscopy. We found significantly induced NET formation after treatment with MβCD in murine neutrophils derived from wild-type as well as HIF-1α KO mice at both hypoxic (1% O₂) as well as normoxic (21% O₂) conditions. Similar observations were made in freshly isolated human neutrophils after stimulation with MβCD or statins, which block the HMG-CoA reductase as the key enzyme in the cholesterol metabolism. HPLC was used to confirm the reduction of cholesterol in treated neutrophils. In summary, we were able to show that NET formation via MβCD or statin-treatment is oxygen and HIF-1α independent.

Culturing respiratory epithelial cells at an air–liquid interface (ALI) represents an established method for studies on infection or toxicology by the generation of an in vivo-like respiratory tract epithelial cellular layer. Although primary respiratory cells from a variety of animals have been cultured, an in-depth characterization of canine tracheal ALI cultures is lacking despite the fact that canines are a highly relevant animal species susceptible to various respiratory agents, including zoonotic pathogens such as severe acute respiratory coronavirus 2 (SARS-CoV-2). In this study, canine primary tracheal epithelial cells were cultured under ALI conditions for four weeks, and their development was characterized during the entire culture period. Light and electron microscopy were performed to evaluate cell morphology in correlation with the immunohistological expression profile. The formation of tight junctions was confirmed using transepithelial electrical resistance (TEER) measurements and immunofluorescence staining for the junctional protein ZO-1. After 21 days of culture at the ALI, a columnar epithelium containing basal, ciliated and goblet cells was seen, resembling native canine tracheal samples. However, cilia formation, goblet cell distribution and epithelial thickness differed significantly from the native tissue. Despite this limitation, tracheal ALI cultures could be used to investigate the pathomorphological interactions of canine respiratory diseases and zoonotic agents.