Short Report
Common laboratory mice are susceptible to infection with SARS-CoV2 beta variant
https://doi.org/10.21203/rs.3.rs-762916/v1
This work is licensed under a CC BY 4.0 License
published 10 Nov, 2021
posted
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Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes consistent with COVID-19.
The COVID-19 pandemic has devastated the world since the emergence of SARS-CoV-2 in late 2019. The pathogenesis of COVID-19 includes an acute pneumonia that can develop into an acquired respiratory distress syndrome (ARDS) (1-3). The effective spread of SARS-CoV-2 among the human population is potentiated by the emergence of highly transmittable variants of concern (VOCs) some of which can evade previously acquired immunity and cause more severe disease than the “wild-type” (wt) strains originating from Wuhan, the D614G mutant of which was dominating the pandemic until the end of 2020. As declared by the World Health Organization (WHO), the nomenclature of currently circulating VOCs are alpha (B.1.1.7 by Pango nomenclature), beta (B.1.351), gamma (P.1) and delta (B.1.617.2) (4).
The rapid development of vaccines and studies of antiviral drugs against SARS-CoV-2 are of prime importance and have been made possible by animal models that recapitulate human SARS-CoV-2 infection and the associated disease. Several animal species, such as Syrian hamsters and non-human primates, have been documented as suitable platforms to experiment in vivo with various COVID-19 countermeasures (5). In contrast, common laboratory mice have proven refractory to infection due to mouse-specific differences in the SARS-CoV-2 entry receptor, the Angiotensin Converting Enzyme (ACE)-2 (1). However, using mice as the model species would provide unrivaled benefits over any other animal species by facilitating the use of an army of tools to study host genetics and immune responses developed for decades by the scientific community. Introduction of human ACE-2 (hACE-2) into mice partially overcomes this drawback but this model is less widely available and requires laborious and expensive breeding to make use of the numerous existing gene modified mouse strains. Also, hACE-2 “knock-in” mouse models are not adequate for all questions, as they do not faithfully mimic the natural expression pattern of the ACE-2 receptor (5).
Instead of mutating the host, SARS-CoV-2 infection models in mice have also been achieved by mutating the virus either by reverse genetics or by serial passaging through mouse lungs (6-9). By these means a few key residues that improve the affinity of the SARS-CoV-2 spike S1 protein to mouse ACE-2 (mACE-2) and increased infectivity of SARS-CoV-2 in standard BALB/c mice have been observed (7, 8). Interestingly, some of these mutations appear also in circulating VOCs, such as the N501Y mutation present in alpha, beta and gamma. This suggests that clinical VOC isolates possess improved infectivity over wt SARS-CoV-2 in mice. In order to test this hypothesis, we compared the infectivity and pathological effects of a clinically isolated beta variant to wt SARS-CoV-2 in the lungs of BALB/c mice after intranasal inoculation.
We intranasally inoculated 9 to 12 week old female BALB/c mice with 2 x 105 plaque-forming units (PFU) of wt and beta SARS-CoV-2 (9), and assessed virus replication, viral antigen expression and associated pathological changes in the lungs at 2 and 3 days post infection (dpi) by analyzing the viral RNA loads by RT-qPCR and antigen expression by immunohistochemistry (IHC). We detected high amounts of SARS-CoV-2 RNA in mice infected with the beta variant as indicated by low Ct values in RT-qPCR (Fig. 1) for the RNA dependent RNA polymerase (RdRp) and envelope (E) genes as well as the subgenomic E gene (subE; replication-intermediate (10)). None of the viral RNA targets were detectable in the lungs of mice infected with wt/D614G SARS-CoV-2, as shown previously (11).
Successful replication of beta SARS-CoV-2 in BALB/c mice was evident also by IHC which consistently detected virus antigen in intact and partly degenerate bronchial/bronchiolar epithelial cells and a few tracheal epithelial cells at 2 dpi (Fig. 2A-B). Scattered alveoli exhibited viral antigen in type I and II pneumocytes (Fig. 2C-D); some activated type II pneumocytes and occasional degenerate of type I pneumocytes were observed. Abundant viral antigen was often covering the luminal surface of bronchiolar epithelial cells. Bronchioles with more extensive viral antigen expression and degeneration of bronchial epithelial cells exhibited leukocyte infiltration of the wall and viral antigen within infiltrating macrophages. Adjacent arteries showed endothelial cell activation. At 3 dpi, alveolar viral antigen expression was more widespread and associated with clear evidence of alveolar damage. Focally, alveoli contained vacuolated macrophages, erythrocytes and degenerate/necrotic epithelial cells (Fig. 2F-H), consistent with focal necrotic pneumonia. In these areas, the vascular response was more intense, with endothelial cell activation and occasional rolling and emigration of leukocytes. The observed changes are morphologically similar to those observed in K18-hACE2 mice at 3 dpi with a wt strain, though less extensive (12). Bronchial lymph nodes generally exhibited SARS-CoV-2 antigen in several macrophages and/or dendritic cells (Fig.2I). In contrast, mice infected with wt SARS-CoV-2 did not show any histological changes and no viral antigen expression at 2 and 3 dpi (data not shown).
We also attempted intranasal inoculations with lower doses (6 x 104 and 6 x 103 PFU) and examined the animals at 2 dpi. In all mice the infection established in the nose, with viral antigen expression in patches of respiratory and olfactory epithelial cells (Supplemental Fig. S1A). This was occasionally associated with leukocyte recruitment (rolling) in vessels beneath the infected epithelium, consistent with an early inflammatory response. In most animals the virus had apparently not reached the lung, as IHC and PCR gave inconsistent and partly discordant results, probably because each one half of the lungs was processed for either methodological approach. However, the detection of viral antigen in tracheal, bronchiolar and, rarely, alveolar epithelial cells confirmed that infection could be achieved. Even at 4 dpi, the lungs were not consistently infected. When we co-housed 3 naïve mice with one intranasally (6 x 104 PFU) infected mouse (n =3), no evidence of infection in the co-housed animals were observed after 4 dpi as confirmed by IHC on nose and lungs, suggesting that viral shedding from the nose was insufficient for transmission to the naïve mice. These results suggest that optimal dosing of beta variant is in the 105 PFU range to consistently achieve infection of the lungs, at least when applied through the intranasal route. No new viral mutations were detected in the lungs of infected mice as compared to the inoculated SARS-CoV-2 beta variant (data not shown).
The wt SARS-CoV-2 is unable to infect mice due to a receptor mismatch, but accumulating evidence suggests that naturally occurring VOCs might be able to acquire this function (5, 13). We probed this hypothesis and show that BALB/c mice are susceptible to infection with the beta variant producing a mild pneumonia reminiscent of what is seen in the K18-hACE2 mice and other species susceptible to SARS-CoV-2 (12). Further studies are needed to determine which other variants are similarly able to infect laboratory mice, the genetic determinants required and whether the acquired ability to infect mice is related to an increased affinity towards mACE-2 specifically or to ACE-2 across species (including hACE-2); the latter is likely given the evidently increased transmissibility of VOCs in humans (4, 13).
Our findings indicate options to experimentally approach various COVID-19 countermeasures in an unprecedented scale, since the standard laboratory mouse has several advantages over other animal models used in infectious disease experimentation, including cost-effectiveness, ease of handling, and suitability for large-scale studies.
Acknowledgements
We thank Sanna Mäki and Johanna Martikainen and the laboratory technicians of the Histology Laboratory, Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, for expert technical assistance. This research is funded by the Academy of Finland iCOIN (grant number 336490).
The authors declare no competing interests.
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Supplemental Figure S1. BALB-C mouse, male. Two days post intranasal infection with 6 x 103 PFU of beta variant SARS-CoV-2. A. Nose with focal extensive viral antigen expression in ciliated epithelial cells. Bar = 20 µm. B. Trachea with small patches of positive epithelial cells (arrows). Bar = 20 µm. Bar = 20 µm. C. Lung with viral antigen deposition along the luminal surface of the bronchiolar epithelial cells. B: bronchiole. D. Lung. Small focal area with viral antigen expression in type I (arrowhead) and type II (arrow) alveolar epithelial cells. Bar = 10 µm. Immunohistochemistry, hematoxylin counterstain.
published 10 Nov, 2021
posted
You are reading this latest preprint version
