Human angiotensin-converting enzyme 2 (hACE2) protein is the functional receptor used by severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) to gain entry to cells. Recently, hACE2 has also been described as the receptor for acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent responsible for coronavirus disease 2019 (COVID-19). SARS-CoV-2 emerged in Wuhan, China, in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. Importantly, hACE2 is widely expressed in the lung, central nervous system, cardiovascular system, kidneys, gut, and adipose tissues where it negatively regulates the renin-angiotensin system and facilitates amino-acid transport. K18 hACE2 transgenic mice [B6.Cg-Tg(K18-ACE2)2Prlmn/J] are susceptible to SARS-CoV-1 infection and recent reports suggest that K18 hACE2 transgenic mice can also be infected with SARS-CoV-2. hACE2 expression in K18 hACE2 transgenic mice is driven by the human cytokeratin 18 (K18) promoter. Importantly, hACE2 expression in K18 hACE2 transgenic mice is observed in airway epithelial cells where SARS-CoV-1 and SARS-CoV-2 infections are typically initiated. Recent research indicates that hACE2-expressing mice are useful for studies related to SARS-CoV-2 pathogenesis and COVID-19. A validated rodent model of SARS-CoV-2 infection could help accelerate testing of vaccines (prophylactic) and antivirals (therapeutic) for the prevention and treatment, respectively, of SARS-CoV-2 infection and associated severe COVID-19 disease. Compared with large animals, a murine model would have desirable features of tractability, ease of use and availability, be cost-efficient and permit mechanistic studies to identify attributes of severe COVID-19 outcomes in some but not all people who are infected. Transgenic mice expressing hACE2 have been developed using various promoters that produce mild-to-moderate SARS-CoV-2 infection in a variety of organs, in addition to physiological (weight loss, interstitial pneumonia) or immunological (anti-spike IgG) changes. No transgenic mice models to date have led to SARS-CoV-2 infection-induced mortality. Adenovirus-based delivery of hACE2 (Ad4-hACE2) to wild-type (WT) C57BL/6 mice resulted in susceptibility to SARS-CoV-2 infection but not mortality. K18 hACE2 transgenic mice have previously been shown to represent a good animal model for SARS-CoV-1 infection and associated disease. However, SARS-CoV-2 lethality in K18 hACE2 transgenic mice has not yet been fully determined. In this study, we infected K18 hACE2 transgenic mice with SARS-CoV-2 to assess the feasibility of its use as an animal model of SARS-CoV-2 infection and associated COVID-19 disease.

The study references numerous publications on SARS-CoV-1 and SARS-CoV-2, including research on the hACE2 receptor, the virus's entry into cells, the genetic characterization and epidemiology of the virus, the clinical course and risk factors for mortality in COVID-19 patients, and the cytokine storm associated with severe COVID-19. Previous work on K18-hACE2 transgenic mice as models for SARS-CoV-1 infection is also reviewed. The authors highlight the limitations of existing mouse models, noting that none have previously demonstrated SARS-CoV-2 infection-induced mortality. Several studies utilizing transgenic mice with varying hACE2 expression levels are discussed, emphasizing the need for a robust model to facilitate vaccine and antiviral development.

The study used specific-pathogen-free, 4-5-week-old, female and male B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18 hACE2) hemizygotes and wild-type (WT) C57BL/6 control mice. Mice were either mock-infected (PBS) or infected intranasally (i.n.) with 1 x 105 PFU of SARS-CoV-2 (USA-WA1/2020 strain). Mice were monitored daily for morbidity (body weight loss) and mortality. At 2, 4, and 6 days post-infection (DPI), mice were euthanized, and various tissues (nasal turbinate, trachea, lung, heart, kidney, liver, spleen, small intestine, large intestine, and brain) were collected. Viral titers were determined using plaque assays in Vero E6 cells. A custom 18-multiplex panel mouse magnetic bead Luminex assay was used to measure cytokines and pro-inflammatory markers. Enzyme-linked immunosorbent assays (ELISAs) measured interferon (IFN)-α and IFN-λ. Histopathology analyses (H&E staining) were performed, and immunohistochemistry (IHC) was used to label SARS-CoV-2 nucleocapsid protein (NP) antigen and hACE2 receptor. Confocal microscopy was employed for double staining to visualize co-localization of hACE2 and SARS-CoV-2 NP. Statistical analyses included unpaired two-tailed Student's t-tests, one-way ANOVA with Tukey's post-test, and hierarchical clustered Pearson correlation tests.

K18 hACE2 transgenic mice showed high susceptibility to SARS-CoV-2 infection, with all mice succumbing to the infection by 6 DPI. Significant weight loss was observed in infected mice, correlating with viral replication in the nasal turbinates, lungs, and brains. SARS-CoV-2 was detected in the upper and lower respiratory tracts by 2 DPI and in the brain by 4 DPI. A marked chemokine storm was observed in the lungs and spleen at 2 DPI, with some chemokines (e.g., RANTES/CCL5) sustaining high levels at 4 DPI. A chemokine storm was also detected in the brain at 4 and 6 DPI. A mixed cytokine storm (pro-inflammatory, TH1, TH2, and TH17 cytokines) was observed in the lungs at 2 DPI, resolving by 4 DPI except for TNF and type I and III IFNs. The cytokine response was largely localized to the lungs, with less pronounced systemic effects. However, some TH1 and TH17 cytokines and chemokines increased in the brain at 4 DPI, suggesting delayed viral spread to the brain. Sex differences in brain cytokine responses were observed, with males exhibiting a stronger TH1/TH17 response at 4 DPI compared to females. Histopathological examination revealed progressive pneumonia in K18 hACE2 transgenic mice, with evidence of alveolar histiocytosis, vasculitis, and edema. IHC showed heterogeneous distribution of viral NP in the lungs, more focalized in the nasal turbinates and brain. hACE2 receptor expression was observed in the lung and nasal turbinate epithelium and in the choroid plexus. Double staining confirmed co-localization of hACE2 and SARS-CoV-2 NP primarily in the lungs, nasal turbinates, and brain. In the brain, SARS-CoV-2 NP was found in the cell bodies of cortical neurons.

The study demonstrates that K18 hACE2 transgenic mice represent a valuable model for studying SARS-CoV-2 pathogenesis and evaluating potential therapeutics. The lethal phenotype observed in these mice, unlike other hACE2 mouse models, is attributed to the high hACE2 expression in airway epithelial cells driven by the K18 promoter. The early and robust cytokine and chemokine storms observed mirror human COVID-19, suggesting that this model can be useful for studying the pathogenesis of severe disease. The presence of SARS-CoV-2 in the brain and the observed cytokine profiles suggest a potential mechanism for neurological manifestations in COVID-19. The authors discuss potential regulatory mechanisms that might explain the observed reduction in cytokine production by 4 DPI, including the role of TH2 cytokines and IL-10. The observed sex differences in brain cytokine responses highlight the potential for differences in disease susceptibility between sexes. The study also provides insights into the correlations between different cytokines and chemokines with the progression of the disease, which could be crucial for the development of effective treatments.

The K18 hACE2 transgenic mouse model provides a valuable tool for studying SARS-CoV-2 pathogenesis and evaluating the efficacy of vaccines and antivirals. The model recapitulates key features of severe COVID-19, including lethality, cytokine/chemokine storms, and lung pathology. Future research using this model could focus on elucidating the roles of specific cytokines and chemokines in disease progression and on investigating potential therapeutic targets.

The limited availability of K18 hACE2 mice from the vendor constrained the sample size, limiting the ability to perform studies with different viral doses or more detailed time-course analysis. The high viral dose used (1 x 10⁵ PFU) might not fully represent the range of infection severity seen in humans. The detection limit of the viral titer assay might have missed low levels of virus in some organs, potentially affecting the interpretation of some results. The study focuses on a specific strain of SARS-CoV-2 (USA-WA1/2020), and the findings might not be directly generalizable to other strains.