Animal Models of COVID-19: Transgenic Mouse Model

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged in December 2019 in Wuhan, China, and rapidly spread throughout the world, threatening global public health. An animal model is a valuable and a crucial tool that allows understanding of nature in the pathogenesis of SARS-CoV-2 and its associated COVID-19 disease. Here we introduce detailed protocols of SARS-CoV-2 infection and COVID-19 disease using C57BL/6 (B6) transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) from the human cytokeratin 18 promoter (K18 hACE2). To mimic natural SARS-CoV-2 infection, K18 hACE2 transgenic mice are infected intranasally under anesthesia. Upon infection, viral pathogenesis is determined by monitoring changes in body weight (morbidity) and monitoring survival (mortality), cytokine/chemokine responses, gross-lung pathology, histopathology, and viral replication in tissues. The presence of the virus and viral replication is evaluated by immunohistochemistry (IHC) and viral titrations, respectively, from the upper (nasal turbinate) and the lower (lungs) respiratory tracts, and nervous system (brain). Also, the immune response to SARS-CoV-2 infection is measured by cytokine/chemokine enzyme-linked immunosorbent assay (ELISA) from lung, spleen and brain homogenates to characterize the cytokine storm that hallmarks as one of the major causes of death caused by SARS-CoV-2 infection. This small rodent animal model based on the use of K18 hACE2 transgenic mice represents an excellent option to understand the pathogenicity of natural SARS-CoV-2 strains and its recently described Variants of Concern (VoC), and will be applicable to the identification and characterization of prophylactic (vaccine) and therapeutic (antiviral and/or neutralizing monoclonal antibodies) strategies for the prevention or treatment of SARS-CoV-2 infection or its associated COVID-19 disease.

Keywords: COVID-19; K18 hACE2 transgenic mice; Mouse model; SARS-CoV-2; hACE2.

Fig. 1

Schematic experimental procedures for SARS-CoV-2 infection in K18 hACE2 transgenic mice and parameters to measure. (a) Viral infection: K18 hACE2 transgenic mice are weighed and anesthetized using isoflurane before intranasal (i.n.) infection with 10⁵ PFU/ mouse of SARS-CoV-2, WA-1/US strain. (b) Morbidity and mortality: From 0 to 14 dpi, mice are weighed daily to evaluate changes in body weight loss (morbidity) and mortality (survival percentage). (c) Organ lesions, viral titration, histology and cytokine changes: At 2 and 4 dpi, SARS-CoV-2 infected K18 hACE2 transgenic mice are sacrificed and internal organs such as nasal turbinate, lung, spleen, and brain are collected for gross pathology changes, viral titers, histopathology and IHC, and cytokine/chemokine analysis. All experiments to characterize SARS-CoV-2 in the K18 hACE2 transgenic mouse model are performed in ABSL3 laboratories. All experiments with SARS-CoV-2 in K18 hACE2 transgenic mice discussed are approved by the IBC and IACUC committees at Texas Biomedical Research Institute. dpi: days postinfection

Fig. 2

Morbidity and mortality of SARS-CoV-2 infected K18 hACE2 transgenic mice: K18 hACE2 transgenic mice are mock-infected or infected (10⁵ PFU/mouse) with SARS-CoV-2 intranasally (n = 14). Changes in body weight (a) and survival (b) are evaluated daily for 14 days. Mice that loss more than 20% of their initial body weight are humanely euthanized. Error bars represent standard deviations (SD) of the mean for each group of mice. dpi: days postinfection

Fig. 3

Gross lung pathology and viral titers. (a, b) Macroscopic lung pathology lesions: K18 hACE2 transgenic mice are mock-infected or infected with 10⁵ PFU/mouse of SARS-CoV-2 and sacrificed at 2 and 4 dpi. Lungs are collected from all sacrificed mice to observe gross pathological changes (a). Scale bars = 1 cm. Macroscopic pathology scoring is determined by measuring the distributions of pathological lesions, including consolidation, congestion, and pneumonic lesions using ImageJ software (NIH) (b). (c) Viral titers: Presence of SARS-CoV-2 in the nasal turbinate (left panel), lung (middle panel) and brain (right panel) is determined by plaque assay (PFU/ml) from homogenized tissues at the indicated dpi. Dotted line indicates limit of detection (100 PFU/ml). Each symbol represents an individual animal and bars the geometric means of viral titers. dpi: days postinfection

Fig. 4

Histopathologic and IHC examination from SARS-CoV-2 infected K18 hACE2 transgenic mice: Nasal turbinate (left), lung (middle), and brain (right) tissues from K18 hACE2 transgenic mice infected with SARS-CoV-2 are fixed in 10% NBF, embedded in paraffin blocks, and sectioned for hematoxylin and eosin (H&E) (a) and confocal microscopy (b). For H&E staining, nasal turbinate, lung, and brain. Nasal turbinate at 2 dpi are microscopically unremarkable. At 4 dpi there is minimal neutrophilic inflammation (arrowheads) within the submucosa (bracket). Scale bars = 200 μm (upper row) and 50 μm (lower row). Lung at 2 dpi has mild to moderate multifocal interstitial pneumonia associated with inflammatory cell infiltrations (asterisks), mild type II pneumocyte hyperplasia (arrowhead), endothelial cells hyperplasia and vasculitis (bracket). At 4 dpi, it progressed to severe diffuse interstitial pneumonia with inflammatory cell infiltrations in alveolar spaces (asterisks) and interstitium (bracket). Brain at 2 dpi presented multifocal necrotic cellular debris within the brain parenchyma (arrowheads) and vasculitis (inflammation of blood vessels, asterisks). This phenotype extends on the brain over time (4 dpi) causing encephalitis. Scale bars = 1 mm (upper row) and 50 μm (lower row). For IHC staining, nasal turbinate, lung and brain tissues from SARS-CoV-2 infected K18 hACE2 transgenic mice at 2 and 4 dpi are immune-stained with hACE2 (red), and SARS-CoV N (green) antibodies. Cell nucleus is stained by DAPI (blue). Note of the tropism of SARS-CoV-2 for pyramidal neurons in the brain. Scale bars = 50 μm. Dpi: days postinfection

Fig. 5

Cytokine and chemokine responses in SARS-CoV-2 infected K18 hACE2 transgenic mice: Cytokine (TNF, IFN-γ, IL-6, IL-10, IL-17/IL-17A, and IFN-α) (a) and chemokine (CCL-5 and CXCL-10) responses in SARS-CoV-2 infected K18 hACE2 transgenic mice at 2 and 4 dpi are measured in lung (a), spleen (b), and brain (c). A 2-WAY ANOVA K18 hACE2 transgenic mice over time. n = 8 (per time-point studied, except mock n =3). dpi: days postinfection