The K18-Human ACE2 Transgenic Mouse Model Recapitulates Non-severe and Severe COVID-19 in Response to an Infectious Dose of the SARS-CoV-2 Virus

Abstract

A comprehensive analysis and characterization of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection model that mimics non-severe and severe coronavirus disease 2019 (COVID-19) in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in the epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2 × 10³ and 2 × 10⁴ PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lung, liver, and kidney, while lower doses (2 × 10¹ and 2 × 10² PFU) led to less severe tissue damage and some mice recovered from the infection. In this hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in postmortem samples from COVID-19 patients. Finally, the mice that recovered from infection with a low dose of virus survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human postmortem samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans being dose-dependent and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19. IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19) has reached nearly 240 million cases, caused nearly 5 million deaths worldwide as of October 2021, and has raised an urgent need for the development of novel drugs and therapeutics to prevent the spread and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed. In this study, we have comprehensively characterized a mouse model of SARS-CoV-2 infection using K18-hACE2 transgenic mice. We infected the mice with low and high doses of SARS-CoV-2 to study the pathogenesis and survival in response to different infection patterns. Moreover, we compared the pathogenesis of the K18-hACE2 transgenic mice with that of the COVID-19 patients to show that this model could be a useful tool for the development of antiviral drugs and therapeutics.

Keywords: COVID-19; K18-hACE2; SARS-CoV-2; infectious disease; lung infection; mouse model.

FIG 1

K18-hACE2 mouse infection model with high and low doses of SARS-CoV-2. (A) Experimental scheme of the K18-hACE2 mouse infection model. Mice were intranasally infected with 2 × 10¹, 2 × 10², 2 × 10³, or 2 × 10⁴ PFU virus per mouse. Blood samples were collected at 6 days postinfection. Tissue samples were collected from day 1 to day 6 postinfection. Mouse body weights (B) and survival (C) were monitored daily for 13 days. Each dot represents one mouse at the indicated time point.

FIG 2

Viral quantification in mice after SARS-CoV-2 infection. (A) Viral RNA levels are shown for the brain, trachea, lung, heart, liver, spleen, small intestine (SI), stomach, large intestine (LI), kidney, and testis. (B) Viral nucleocapsid protein (NP) was detected in the brain and lung of the mice infected with high and low doses of SARS-CoV-2 (scale bar, 40 μm). (C) Infectious virus loads in the lung at different time points. Mice were infected with 2 × 10⁴ PFU at day 0. Lungs were collected and homogenized at indicated time points. The infectious virus loads were measured by a plaque assay. Data are presented as mean ± standard deviations. Statistical analyses were performed by one-way ANOVA with P values corrected for multiple comparisons by the Bonferroni test (n = 3 or 4 mice). *, P < 0.05; **, P < 0.01.

FIG 3

Viral distribution in mice after SARS-CoV-2 infection. (A) Representative images show double staining of NP (purple) with lung club (Clara) cells and alveolar type 2 cells using the markers CCL10 (yellow) and SPC (yellow), respectively, macrophages using the CD68 marker (yellow) (20× magnification for each), and neurons using the NeuN marker (yellow) in mice infected with high-dose SARS-CoV-2 (10× magnification, with 100× magnification for the boxed area in the 2 × 10⁴ PFU group). Black arrows indicate double staining cells. (B) Representative images show double staining of the spike protein (teal) with alveolar type 2 cells using SPC (yellow) as a marker and macrophages using CD68 (yellow) as a marker in COVID-19 patient postmortem samples (20× magnification). Black arrows indicate double staining cells.

FIG 4

Pathological changes in multiple tissues of K18-hACE2 mice infected with the indicated dose of SARS-CoV-2 or postmortem tissue from COVID-19 patients. (A) Tissue damage in the brain, trachea, lung, heart, liver, spleen, small intestine, stomach, large intestine, kidney, and testis of K18-ACE2 mice after SARS-CoV-2 infection (scale bar, 40 μm). (B) Tissue damage in trachea, bowel, spleen, kidney, heart, and lung in COVID-19 patient postmortem samples (scale bar, 40 μm).

FIG 5

Tissue distribution of hACE2 in K18-hACE2 mice and human samples. (A) Detection of hACE2 RNA in multiple tissues of K18-hACE2 mice by RT-qPCR. (B) hACE2 expression in the brain, trachea, lung, and kidney of K18-hACE2 mice by IHC. (C) Human tissue array IHC staining for the hACE2 protein.

FIG 6

Protective role of sera from previously infected mice and resistance of low dose-infected surviving mice to high-dose rechallenge in the K18-hACE2 model. Body weights (A) and survival (B) of mice treated with sera from previously infected mice and prior low dose-infected mice being challenged with a high dose of SARS-CoV-2. Mice with serum protection were infected with 2 × 10⁴ PFU per mouse SASR-CoV-2 24 h after being infused with sera from mice infected with 2 × 10⁴ PFU virus per mouse for 6 days. For the rechallenge, mice were infected with the low dose of 2 × 10¹ PFU or 2 × 10² PFU per mouse SASR-CoV-2 for 2 weeks and then were rechallenged with the high dose of 2 × 10⁴ PFU per mouse SASR-CoV-2. Survival of mice was analyzed by the Kaplan–Meier method and the log-rank test. (C) Mice were infected with 1 × 10³ PFU at day 0. Sera in the peripheral blood were collected via tail vein bleeding 6 days postinfection (dpi) or right before mice were sacrificed at 13 dpi. Relative anti-spike antibody levels were measured by ELISA. Data are presented as mean ± standard deviations. Statistical analyses were performed by one-way ANOVA with P values corrected for multiple comparisons by the Bonferroni test (n = 4 or 5 mice). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.