Recent news continues to show that the COVID-19 pandemic situation globally is far from being over. Across the entire world, the pneumonia-like COVID-19 caused by the novel coronavirus (SARS-CoV-2) remains a huge medical challenge. To provide a wholistic guide for developing clinical treatments, vaccines, and antiviral drugs, it is of great significance to study the interaction mechanisms between severe acute respiratory syndrome (SARS) coronaviruses and host molecules, while also exploring the pathogenic process of coronaviruses. Accurate animal models are necessary for verifying the pathogenesis and immune mechanisms of the illness to accelerate research across vaccine development, new drug development, gene therapy, and more. 

 

What are the advantages of using mouse models to study SARS coronavirus diseases?

In studying human-infecting coronavirus diseases, the advantages of the mouse model compared to other in vivo models are readily apparent - lower cost, easy breeding, faster reproduction, and more litters. More importantly, the genetic background of inbred mice is consistent, permitting their genes to be edited to reliably construct the required models. These custom models are undoubtedly helpful for the studying the role of specific host genes in pathogenic processes, as well as in understanding host immunity response and mechanisms of protection initiated by viral challenge. 

 

However, mouse models have their limitations: species differences between mice and humans influence viral pathology, resulting in some human-infecting viruses being unable to directly infect mice, or that mice exhibit no obvious symptoms even after in vivo virus replication. The mechanism of viral infection requires binding to specific host cell surface receptors, and influences the associated innate immune response of the host. In the case of SARS-CoV-2, the virus’ receptor binding motif (RBM) binds to human ACE2 receptors, but not endogenous mouse ACE2. Given that the genetic background of inbred mice is consistent, editing the mouse genes can overcome the shortcomings of wild-type mice to a certain extent. One noteworthy example is the development of humanized mouse models, which integrate human genes that are relevant to the pathology of interest. Such genetic engineering approaches help to elucidate the role of the host’s specific genes in viral pathogenesis, providing a mechanism for research across a range of applications - from investigating preliminary genetic targets, through evaluating the safety and efficacy of vaccines and drugs.

 

What are the strategies for mouse models used to evaluate the pathogenicity of SARS-CoV infection?

Mouse models of SARS-CoV infection fall into three main classifications based on construction strategy and methods:

  1. Direct infection of inbred mice with human-susceptible SARS-CoVs.
  2. Application of gene-editing technology: knockout related genes in mice or transfer human host cell virus-binding receptors (such as ACE2) to develop humanized mouse models.
  3. Adapt wild-type SARS-CoVs to mice by repeated exposure to obtain a more mouse-adapted viral pathogenesis, thereby establishing a virus-infected mouse model that can present obvious clinical phenotypes.
  4. Application of Collaborative Cross (CC) technology to establish genetically diverse mouse strains to research how genetic diversity within host species may contribute to susceptibility and clinical outcome of viral pathogenesis.

 

Regarding the mouse model for SARS-CoV infection, the main research progress focuses on the following aspects: 

 

a) Direct application of inbred strain mouse model to study the pathogenicity of SARS virus

The higher SARS-CoV infection fatality rate has correlated with the increasing number of older patients (> 60 years old) diagnosed with acute respiratory distress syndrome (ARDS). In order to explore a mouse model that is more suitable for simulating the infection of SARS-CoV in the elderly, there are studies adopting BALB/c, C57BL/6, and 129S6 inbred wild-type (WT) mice in 12-14 month age range for experiments separately. The results show that these three mice infected by SARS-CoV all developed with short-term (an average of about 7 days) clinical phenotypes, such as weight loss, disordered hair, arched back, dehydration, etc. In addition, histopathology results showed infiltration of inflammatory cells around the blood vessels and bronchioles, necrosis of bronchial cells, and the occurrence of interstitial pneumonia. Moreover, the extensive alveolar destruction in SARS-CoV infected BALB/c mice can last 9 days, which is remarkably close to the clinicopathological symptoms of human SARS-CoV infection. These studies clarified for the first time that the genetic and age characteristics of different hosts can significantly affect the pathogenicity of SARS-CoV infection. It also demonstrated that the occurrence of severe disease resulting from viral infection is not only related to the characteristics of the virus, but also depend on differences among potential hosts.

 

b) Application of gene-editing technology to construct SARS-CoV infection mouse model

In regards to the construction strategy of a humanized ACE2 (hACE2) mouse model, researchers are currently using different (tissue-specific or widely expressed) promoters and expression vectors constructed by the hACE2 gene, to obtain a randomly inserted transgenic mouse model by prokaryotic injection. Examples of the promoters used in hACE2 models include cytokeratin (epithelial cell specific expression-K18), CAG (widely expressed), or the endogenous mouse ACE2 gene promoter. The results of studies using such humanized ACE2 mouse models indicate that the level of hACE2 expression is directly related to the severity of the disease. Although these mouse models all showed respiratory epithelial cell infections from exposure to SARS-CoV, the high expression of the hACE2 gene also seen in the mouse brain may have led to additional affects. In the SARS-CoV infection model of humanized mice, the virus in the brain increased and spread extensively - eventually mice were found to die with encephalitis.

 

c) Apply adaptive experimental evolution to obtain SARS-CoV mutant virulent strains

Repeatedly introducing the virus in mouse-specific tissues may be performed to encourage viral evolution to infect mice. This approach forces the virus to adapt to its specific living environment and mutate under specific selective pressure, to persist in reproduction and achieve more effective replication. In order to adapt SARS-CoV to replicate in the lungs of wild-type mice and cause severe acute respiratory syndrome (SARS) phenotypes, researchers repeatedly inoculated SARS-CoV clinical strains in the nasal cavity of BALB/c mice (6-weeks-old) for 15 times until obvious clinical symptoms (such as weight loss, etc.) arose in wild-type mice. Through comparing the adapted lethal virus strains obtained from different passages, it is helpful to analyze the correlation between the variation of severe respiratory infections caused by the virus and the specific mutations of its proteins. This approach provides insights for further research on the role of adaptive mutations in specific SARS-CoV protein interactions between the virus and the hosts.

 

d) Application of Collaborative Cross (CC) technology to establish genetically diverse mouse strains

Using the collaborative cross (CC) panel of inbred mouse strains to establish the SARS-CoV infection model helps to identify genetically related factors influencing the process of viral pathogenicity that lead to different responses towards viral infectious diseases among host species. To quickly identify genes related to particular phenotypes, it is suggested to analyze and compare CC mice infected with SARS-CoV across different phenotypes, such as variability of weight loss and virus titers, etc. Some researchers have found CC mouse strains (CC003 and CC053/Unc) with the opposite susceptibility to the pathogenesis of SARS-CoV infection. In addition, gene mapping analysis found that Ticam2, an adapter protein in the toll-like receptor (TLR) signaling pathway, is a potential contributor to severe respiratory disease phenotypes. According to the results of such studies, the CC platform can be used to identify additional mouse models that recapitulate the human clinical outcomes caused by viral infectious diseases.

 

Advances in the application of mouse models in the study of the novel coronavirus (SARS-CoV-2)

Comparisons of both nucleic and amino acid sequences have confirmed a high similarity between SARS-CoV-2 and SARS-CoV - approximately 80% and 76%, respectively. Additionally, SARS-CoV-2 initiates infection through interactions with the human ACE2 receptor on the cell surface, causing clinical manifestations similar to SARS-CoV infection (though not exactly the same). Therefore, strategies and methods for constructing mouse models for SARS-CoV infection described above are also suitable for establishing SARS-CoV-2 infection mouse models. Scientists worldwide have the latest applications of the mouse model for studying the novel coronavirus (SARS-CoV-2) and the resulting infection, known as COVID-19.

 

i. Transgenic hACE2 Mouse Model

The development of the hACE2 mouse model for studying SARS-CoV-2 infection was first reported by the research team led by Qin Chuan of the Institute of Laboratory Animals Science, CAMS & PUMC. This transgenic hACE2 mouse model applies mouse mACE2 promoter and hACE2 gene in expression vector construction, which is injected through prokaryotic cells to obtain a dynamic hACE2 mouse model. After SARS-CoV-2 infection, hACE2 transgenic mice were detected to have elevated levels of viral load in lungs, and develops pathological features, such as weight loss, moderate interstitial pneumonia, significant interstitial infiltration of lymphocytes and monocytes, and aggregates of alveolar macrophages.

 

In addition, researchers also applied the hACE2 transgenic mice for the first time to evaluate the safety and effectiveness of an inactivated SARS-CoV-2 vaccine. According to the experiment, inactivated vaccines of different doses and times were administered to hACE2 mice - no inflammation or adverse reactions were observed in the mice. Immunogenicity analysis of the inactivated vaccine showed that it can induce mice to produce immunoglobulin G (IgG) antibodies against the SARS-CoV-2 spike (S) protein, specifically the S1 subunit of the receptor-binding domain (RBD). Additionally, colocalization of the SARS-CoV-2 S protein and the human ACE2 receptor was confirmed through a combination of immunohistochemistry (IHC), gross pathology, and confocal microscopy.

 

ii. Targeted Insertion of hACE2 Mouse Model

The research team of Sun et al. reported the successful construction of a hACE2-KI/NIFDC mouse model through fixed insertion. The construction strategy of this model targets the first coding exon of the mouse ACE2 gene for insertion of the hACE2 and tdTomato genes - to realize co-expression of hACE2 and tdTomato under the control of the endogenous mAce2 gene. After intranasal inoculation of SARS-CoV-2, both young and old hACE2 mice were found with higher viral loads in the lungs, trachea, and brain, while no viral RNA was detected in their spleen, kidney, liver, intestine, and serum. Interstitial pneumonia occurred in infected hACE2 mice, as indicated by inflammatory cell infiltration, thickened alveolar septa, and obvious vascular system damage - but no death of infected mice was observed.

 

Analyzing the colocalization of SARS-CoV-2 with cells of infected mice revealed that Clara cell 10kDa protein positive (CC10+) cells are the main target cells of SARS-CoV-2 in the airway. The study also provided evidence that intragastric exposure to SARS-CoV-2 can also result in infection and lead to pathological changes in the lungs of hACE2 mice.

 

iii. Develop Mouse Model with Adenovirus Vector

Professor Zhao Jincun's team at the State Key Laboratory of Respiratory Diseases in Guangzhou successfully established the world's first non-transgenic SARS-CoV-2 mouse model by exogenously delivering human ACE2 with replication-deficient adenovirus (Ad5-hACE2) vector to express hACE2 in the lungs of mice. Transnasal transduction of the Ad5-CMV-hACE2 vector, expressing hACE2 under control of the CMV promoter, successfully established a mouse model of hACE2 gene expression in the lungs of mice as compared to wild mice and related gene knockout mice (e.g. IFN-I receptor-deficient, STAT1 [key interferon pathway gene] knockout).

 

After the Ad5-hACE2-transduced mice are infected with SARS-CoV-2, not only are high titers of virus detected in the lungs, but the Ad5-hACE2 mice also exhibit weight loss and clinicopathological manifestations of patients with COVID-19. The mouse model experiment also demonstrated that both type I interferon (IFN-I) and STAT1 genes offer potential protection against SARS-CoV-2 infection.

 

Conclusions

As a leading provider of professional genetically modified animal model services, Cyagen’s research and development (R&D) team has prioritized the development of mouse models catered to studying SARS-CoV-2 infections since the early stages of the outbreak. With our proprietary TurboKnockout® Gene Targeting services and optimized CRISPR technology, we have simultaneously prepared hACE2 mice across multiple background strains, including BALB/c and C57BL/6J. Additionally, Cyagen continues to develop comprehensive gene editing methods and design construction strategies catered to different research purposes and applications.

 

It has been half a year since the outbreak of COVID-19 and the development of vaccines and antiviral drugs has made great progress. Although most studies cannot be used immediately for the treatment of patients, it is obvious that the incremental research on SARS-CoV-2 is of vital importance in the process of identifying treatments and preventatives for COVID-19. In this process, mouse models have able to recapitulate aspects of COVID-19 pathology in humans, making them valuable tools for COVID-19 immunology research.

 

References:

  1. Bao, L., Deng, W., Chuan, Q.,Huang, B. et al. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature (2020). https://doi.org/10.1038/s41586-020-2312-y
  2. Sun SH, Chen Q, Gu HJ, et al. A Mouse Model of SARS-CoV-2 Infection and Pathogenesis. Cell Host Microbe. 2020;S1931-3128(20)30302-4.DOI:10.1016/j.chom.2020.05.020
  3. Sun J, Zhuang Z, Zheng J, et al. Generation of a Broadly Useful Model for COVID-19 Pathogenesis, Vaccination, and Treatment. Cell. 2020;DOI:10.1016/j.cell.2020.06.010