key: cord-0875233-3ftmv5zf
authors: Moreau, G. Brett; Burgess, Stacey L.; Sturek, Jeffrey M.; Donlan, Alexandra N.; Petri, William A.; Mann, Barbara J.
title: Evaluation of K18-hACE2 mice as a model of SARS-CoV-2 infection
date: 2020-06-26
journal: bioRxiv
DOI: 10.1101/2020.06.26.171033
sha: 86dbfcebdd70fbed44757d05a89cd04393a7cd22
doc_id: 875233
cord_uid: 3ftmv5zf

Murine models of SARS-CoV-2 infection are critical for elucidating the biological pathways underlying COVID-19 disease. Because human ACE2 is the receptor for SARS-CoV-2, mice expressing the human ACE2 gene have shown promise as a potential model for COVID-19. Five mice from the transgenic mouse strain K18-hACE2 were intranasally inoculated with SARS-CoV-2 Hong Kong/VM20001061/2020. Mice were followed twice daily for five days and scored for weight loss and clinical symptoms. Infected mice did not exhibit any signs of infection until day four, when weight loss, but no other obvious clinical symptoms were observed. By day five all infected mice had lost around 10% of their original body weight, but exhibited variable clinical symptoms. All infected mice showed high viral titers in the lungs as well as altered lung histology associated with proteinaceous debris in the alveolar space, interstitial inflammatory cell infiltration and alveolar septal thickening. Overall, these results show that symptomatic SARS-CoV-2 infection can be established in the K18-hACE2 transgenic background and should be a useful mouse model for COVID-19 disease.

An invaluable step in identifying effective vaccines and therapies to combat COVID-19 is the availability of a mouse model of infection. The host receptor for SARS-CoV-2 is the human angiotensin-converting enzyme 2 (hACE2) (Wan et al., 2020) , which was previously identified as the receptor for the SARS-CoV-1 virus (Li et al., 2003) that causes Severe Acute Respiratory Syndrome (SARS), a disease that emerged from China in 2002 (WHO, Tsang). The mouse ACE2 ortholog, which has significant amino acid sequence variation in the viral receptor binding domain, cannot serve as an efficient receptor for either SARS-CoV-2 or CoV-1 (Shang et al., 2020) . To develop a mouse model to study SARS-CoV-1 infection, McCray et al. developed a transgenic mouse line, K18-hACE2, which expresses the hACE2 gene under the control of the human cytokeratin 18 promoter. Infection of these mice with SARS-CoV-1 results in a rapidly lethal infection that spreads from the lungs to the brain, and induces proinflammatory cytokines and chemokines (McCray et al., 2007) . Four other hACE2-expressing mouse lines have been created to date and tested for the ability to support SARS-CoV-2 infection (Lutz, 2020) . Two lines express the hACE2 gene under the control of the mouse ACE2 promotor (Bao et al., 2020) ; one was made using CRISPR/Cas9 technology (Sun et al., 2020) . The third strain uses the lung ciliated epithelial cell HFH4 promoter (Menachery et al., 2016) (Dinnon et al., 2020 ). An additional approach was to transfect wild type mice with an adenovirus carrying the hACE2 gene (Hassan et al., 2020) . Overall, with the exception of the HFH4 mice, in which there was some lethality, infection of these three mouse strains with SARS-CoV-2 results in mild clinical symptoms, and no lethality. Here we report the infection of K18-hACE2 with SARS-CoV-2. While this infection resembled that of other strains, we observed variable clinical presentation, with some mice exhibiting more severe symptoms than reported using other models. Overall, this work supports the usefulness of K18-hACE2 transgenic mice as a model for human COVID-19 infections.

To investigate the potential of this transgenic mouse strain as a model for COVID-19 infection, five K18-hACE2 mice were intranasally inoculated with 8 x 10 4 TCID50 of SARS-CoV-2, while five mice were mock-infected with sterile DMEM. Mice were followed twice daily for five days, and scored for clinical symptoms (weight loss, eye closure, appearance of fur and posture, and respiration). The mock-infected mice did not exhibit any clinical symptoms or experience any weight loss throughout the experiment. Infected mice did not exhibit any measurable clinical symptoms until day four, and these were limited to weight loss. On day five all of the infected mice had lost around 10% of their original weight ( Figure 1A ) and exhibited variability in other clinical signs of infection, with clinical scores ranging from 3-9 (maximal score 14) ( Figure 1B ). While two of the infected K18-hACE2 mice showed only mild symptoms at day five (weight loss, and reduced activity), two mice exhibited piloerection. The most severe mouse had increased respiration, lethargy, and slight eye closure, and met our criteria for euthanasia. Because the study was ended on day five, it is unclear whether the remaining four mice would have recovered if the study was carried past day five.

While the clinical severity was variable between infected K18-hACE2 mice, our results suggest that these mice present with more symptomatic disease than other hACE2 mouse models of SARS-CoV-2 infection. In the mouse model expressing hACE2 under the mouse ACE2 promoter, infected mice did not exhibit any clinical symptoms other than maximal weight loss on day three post-infection, and those mice recovered (Sun et al, 2020) . Only mild ruffling of fur and up to 8% weight loss on day five were observed in the other model using the mouse ACE2 promoter, and once again all mice recovered (Bao et al, 2020) . In mice transfected with an adenovirus carrying the hACE2 gene mice exhibited about a 10% weight loss on day four postinfection, but no lethality (Hassan et al., 2020) . In contrast to these models, in which mice exhibited mild symptoms and recovered, only 60% of the mice survived past day five in the mouse strain expressing hACE2 under the lung ciliated epithelial cell HFH4 promoter (Dinnon et al., 2020) . While this model had higher lethality, weight loss was only about 5% and these mice had no respiratory symptoms. The authors hypothesize that mortality may be due to neuroinvasion, as virus was detected in the brain. In K18-hACE2 mice infected with SARS-CoV-1 the course of infection is clearly different; the infection is uniformly fatal, beginning on day four post-infection, and mice were symptomatic with labored breathing and lethargy. (McCray et al., 2007) . While the numbers of mice used in this study are small and we were not able to measure survival, our data support a difference in the disease progression between these two viruses.

All mice were euthanized on day five and tissue was collected for dissection and enumeration of viral loads. No significant differences in histology of the spleen, small intestine, or liver were observed between infected and mock-infected mice, and these tissues were normal in size and appearance. Dissection of the lungs of infected mice revealed a mottled or marbled appearance that was not observed in mock-infected mice (data not shown). Lung sections were analyzed after staining with hematoxylin and eosin (H&E) and scored based on tissue pathology (Matute-Bello et al., 2011) . SARS-CoV-2-infected mice exhibited significantly higher histopathology scores than mock-infected mice (Figure. 2). The major histopathology findings in infected mice were proteinaceous debris in the alveolar space, neutrophils in the interstitial space, and alveolar septal thickening ( Figure 2) ; these observations were consistent with other hACE2 mouse models, which also detected signs of lung injury including interstitial pneumonia, inflammatory cell infiltrates, and alveolar septal thickening (Sun et al, 2020; Bao et al, 2020 ) (Dinnon et al., 2020) (Hassan et al., 2020) . Consistent with the observed infiltrating neutrophils, granulocytes and inflammatory monocytes were also elevated in the bronchoalveolar lavage (BAL) fluid from infected mice (Figure 3 ).

Other hACE2 mouse models of COVID-19 infection have observed high viral titers in the lungs with limited viral load in organs such as the liver and spleen during intranasal infection (Sun et al, 2020; Bao et al, 2020) . While we did not investigate viral load in the liver or spleen, these organs appeared normal by histology, suggesting that there was limited viral titer in these tissues. Virus was detected in the lungs of all infected mice, with titers generally in the range of 1 x 10 5 PFU/ml (Table 1 ). Viral titers in the lungs appeared somewhat associated with disease severity: mouse 1390, which had the highest lung titer, had the highest clinical score, histopathology score, percent weight loss at day five (Table 1) , and the highest numbers of neutrophils, monocytes and eosinophils in the BAL (Figure 3 ). In addition, mouse 1413, which had the lowest titer, had the lowest clinical score, second lowest percent weight loss at day five (Table 1) , and lowest number of eosinophils and monocytes in BAL (Figure 3 ). Of note, mouse 1413 did not have the lowest histopathology score (Table 1) . While there were trends towards higher viral titers in the lungs being associated with higher clinical and histopathology scores, these trends were not significant, and viral titer was not a strong predicter of percent weigh loss. The power of this analysis is limited by the small sample size, but these results suggest that factors in addition to viral load, such as inflammatory responses, are driving the severity of disease. This would also potentially explain the sudden onset of clinical symptoms at five days post-inoculation.

In this report we have described the course of SARS-CoV-2 infection in K18-hACE2 transgenic mice. Our findings are consistent with other studies utilizing hACE2 mice, which observed successful infection with SARS-CoV-2 and a milder disease severity compared to SARS-CoV-1 (Bao et al., 2020) (Sun et al., 2020) . The onset of symptoms was abrupt, manifesting on day five. Mice exhibited similar degree of weight loss, but a varying degree of symptoms and clinical/histopathological scores. The number of mice used in this study was too small to determine whether this was a result of experimental variability or natural variability in outcomes. The variance in clinical and histopathological scores may be partially explained by viral titer, but there are likely other factors, such as the host immune response, that contribute to the variance observed. The observation of more severe disease in a subset of the K18-hACE2 mice is distinct from other hACE2-expressing COVID-19 models, which typically observed only mild clinical symptoms (Bao et al., 2020) (Sun et al., 2020) . This could be due to differences in the level of hACE2 receptor expression or tissue distribution. Based on these findings, the K18-hACE2 transgenic mice may be a particularly useful for studying the biological processes underlying the clinical symptoms of COVID-19 disease.

Challenge: Ten week-old Tg(K18-hACE2)2Prlmn (Jackson Laboratories) ((McCray et al., 2007) were challenged with 8 x 10 4 TCID50 in 50 μl of Hong Kong/VM20001061/2020 (NR-52282, obtained from BEI Resources, NIAID, NIH) by intranasal route under ketamine/xylazine. Mockinfected animals received 50 μl DMEM. Mice were followed twice daily for clinical symptoms, which included weight loss, activity, fur appearance and posture, eye closure, and respiration. On day five all mice were euthanized. All mouse work was approved by the University's Institutional Animal Care and Use, and all procedures were performed in the University's certified animal Biosafety Level Three laboratory.

Histology: Tissues were fixed in formaldehyde. Slides were scanned at 20X magnification. Histopathological scoring for lung tissue was done according to the guidelines of the American Thoracic Society (Matute-Bello et al., 2011) . Viral titers: The left lobe of the lung was removed and placed in a disposable tissue grinder with 1 ml of serum free DMEM. Plaque assays were performed based on the protocol described in Baer and Hall, 2014. Briefly, Vero C1008, Clone E6 (ATCC CRL-1586) cells grown in DMEM (GIBCO 11995-040) supplemented with fetal bovine serum (FBS) were seeded into 12 well tissue culture plates at a concentration of 2 x 10 5 cells/well the night before the assay. Lung homogenates were diluted in cold serum-free DMEM and serial dilutions added to the wells. The plate was incubated at 37°C, 5% CO2 for two hours to allow viral infection of the cells, shaking the plates every 15 minutes. After two hours, the media was replaced with a liquid overlay of DMEM, 2.5% FBS containing 1.2 % Avicel PH-101 (Sigma Aldrich) and incubated at 37°C, 5% CO2. After three days the overlay was removed, wells were fixed with 10% formaldehyde and stained with 0.1% crystal violet, and plaques enumerated to calculate viral PFU/ml. 

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Acknowledgements: The authors would like to gratefully acknowledge the advice and assistance of Angelina Angelucci and Young Hahn at the University of Virginia as well as Caitlin Woodson and Kylene Kehn-Hall at George Mason University. This work was supported by NIH grants R01 AI124214 to WAP, R01 AI146257 to SLB, and 5T32AI007496-23 to AND, and the University of Virginia's Global Infectious Diseases Institute. JMS is a an iTHRIV Scholar, a program is supported in part by the National Center For Advancing Translational Sciences of the National Institutes of Health under Award Numbers UL1TR003015 and KL2TR003016. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors have no competing interests.