Infectious Clones Produce SARS-CoV-2 That Causes Severe Pulmonary Disease in Infected K18-Human ACE2 Mice

Abstract

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has caused extensive mortality and morbidity and wreaked havoc on socioeconomic structures. The urgent need to better understand SARS-CoV-2 biology and enable continued development of effective countermeasures is aided by the production of laboratory tools that facilitate SARS-CoV-2 research. We previously created a directly accessible SARS-CoV-2 toolkit containing user-friendly reverse genetic (RG) infectious clones of SARS-CoV-2. Here, using K18-human ACE2 (hACE2) mice, we confirmed the validity of RG-rescued SARS-CoV-2 viruses to reproduce the infection profile, clinical disease, and pathogenesis already established in mice infected with natural SARS-CoV-2 isolates, often patient derived. RG-rescued SARS-CoV-2-infected K18-hACE2 mice developed substantial clinical disease and weight loss by day 6 postinfection. RG-rescued SARS-CoV-2 was recovered from the lungs and brains of infected K18-hACE2 mice, and infection resulted in viral pneumonia with considerable changes in lung pathology, as seen previously with natural SARS-CoV-2 infection. In mice infected with RG-rescued SARS-CoV-2-mCherry, mCherry was detected in areas of lung consolidation and colocalized with clinically relevant SARS-CoV-2-assocated immunopathology. RG-rescued SARS-CoV-2 viruses successfully recapitulated many of the features of severe COVID-19 associated with the K18-hACE2 model of SARS-CoV-2 infection. With utility in vivo, the RG-rescued SARS-CoV-2 viruses will be valuable resources to advance numerous areas of SARS-CoV-2 basic research and COVID-19 vaccine development.IMPORTANCE To develop COVID-19 countermeasures, powerful research tools are essential. We produced a SARS-COV-2 reverse genetic (RG) infectious clone toolkit that will benefit a variety of investigations. In this study, we further prove the toolkit's value by demonstrating the in vivo utility of RG-rescued SARS-CoV-2 isolates. RG-rescued SARS-CoV-2 isolates reproduce disease signs and pathology characteristic of the K18-hACE2 mouse model of severe COVID-19 in infected mice. Having been validated as a model of severe COVID-19 previously using only natural SARS-CoV-2 isolated from patients, this is the first investigation of RG-rescued SARS-CoV-2 viruses in K18-hACE2 mice. The RG-rescued SARS-CoV-2 viruses will facilitate basic understanding of SARS-CoV-2 and the preclinical development of COVID-19 therapeutics.

Keywords: coronavirus; cytokines; infectious clones; lung infection; respiratory viruses.

FIG 1

Weight loss and clinical disease score of K18-hACE2 mice infected with SARS-CoV-2 or SARS-CoV-2-mCherry. Twenty-one-week-old male and female K18-hACE2 mice were intranasally inoculated with 4 × 10⁴ PFU of SARS-CoV-2 (n = 7) or SARS-CoV-2-mCherry (n = 3) (20 μl per mouse). Mock-infected mice (n = 5) received 20 μl sterile DMEM with 2% FBS intranasally. Individual mice were monitored daily until reaching a clinical score of >3, when twice daily monitoring was performed. (A) Weight change was monitored and compared with the initial weight on day 0. *, P < 0.05 (two-way ANOVA with Bonferroni posttest). (B) Mice were given a clinical score according to general health (eating habit, locomotion, behavior), appearance, and weight loss. All values represent the means ± standard errors of the mean from one experiment.

FIG 2

Viral burden in tissues. Twenty-one-week-old male and female K18-hACE2-transgenic mice were inoculated intranasally with 4 × 10⁴ PFU SARS-CoV-2 or SARS-CoV-2-mCherry. (A and B) At day 7 postinfection, we determined the titer of infectious virus in the lung, nasal cavity, trachea, and brain by plaque assay (A), and the viral genome copy number in tissues (lung, nasal cavity, trachea, brain, heart, liver, spleen, and kidney) was measured by RT-qPCR (B). Each symbol represents the mean titer for one mouse. Bars represent means ± standard errors, and a horizontal dotted line indicates the limit of detection.

FIG 3

Histopathological analysis of lung tissue from SARS-CoV-2- and SARS-CoV-2-mCherry-infected K18-hACE2 mice. Twenty-one-week-old male and female K18-hACE2-transgenic mice were inoculated intranasally with 4 × 10⁴ PFU SARS-CoV-2 or SARS-CoV-2-mCherry. (A) At day 7 postinfection, lung tissue was harvested and stored in 4% PFA for 24 h prior to macroimaging. White arrows show the dark-red lesions throughout the dorsal region of the lung in infected mice. Hematoxylin and eosin (H&E)-stained lung sections demonstrate thickened interalveolar septa (black arrow) and a hemorrhage (black arrowhead) in SARS-CoV-2- and SARS-CoV-2-mCherry-infected mice. Scale bars, 6 mm (black), 500 μm (red), 100 μm (green). Black dashed boxes indicate the area of magnification. Each image is representative of ≥3 mice. (B) Cell density was quantified using ImageScope software. Statistical analyses were performed using an unpaired t test. Data represent means ± standard errors.

FIG 4

Cytokine and chemokine expression in the lung tissue of SARS-CoV-2-infected K18-hACE2 mice. Twenty-one-week-old male and female K18-hACE2-transgenic mice were inoculated intranasally with 4 × 10⁴ PFU SARS-CoV-2 or SARS-CoV-2-mCherry. Transcriptional profiles of immune mediators, namely, TNF-α, CXCL10, IL-1β, CCL5, IFN-γ, IL-6, and IFN-β, were determined by qRT-PCR in the lung at day 7 postinfection. Data were normalized to GAPDH levels and are shown as fold change from the level in mock-infected mice. Data are presented as means ± standard errors. No statistical difference in the levels of expression of inflammatory mediators was observed between SARS-CoV-2- and SARS-CoV-2-mCherry-infected lungs by the Student unpaired t test.

FIG 5

Immunofluorescence analysis of lung sections from SARS-CoV-2-mCherry-infected K18-hACE2 mice. Lung cryosections were labeled with Hoechst 33258 dye (nuclei), CD169 (alveolar macrophages), Ly6G (neutrophils), and cytokeratin 14 (parenchymal tissue), and images were acquired by confocal microscopy. (A) Localization of SARS-CoV-2-mCherry in the lungs of infected mice at 7 days postinfection. The image was acquired as a z-stack using a 10× (0.75-NA) objective. (B) Colocalization of CD169⁺ alveolar macrophages and mCherry⁺ signal in the lung parenchyma. Arrows show cytoplasmic colocalization (insets). The image was acquired as a z-stack using a 20× (0.95-NA) objective.

FIG 6

Immunofluorescence analysis of heart, kidney, and trachea sections from SARS-CoV-2-mCherry-infected K18-hACE2 mice. Tissue cryosections were labeled with Hoechst 33258 dye (nuclei), collagen type IV (basement membrane), and F4/80 (macrophage), and images were acquired by confocal microscopy. Localization of SARS-CoV-2-mCherry in the heart (cross-section) (A), kidney (cross-section) (B), and trachea (longitudinal section) (C) of infected mice at 7 days postinfection. Images were acquired as a z-stack using a 20× (0.95-NA) objective. Arrows show mCherry-positive cells (insets). Scale bars (50 μm) are shown in individual panels.