August 2020

A mouse model of SARS-CoV-2 infection and pathogenesis

Perhaps there is not a better time to bridge two of the topics that mark this decade: CRISPR-Cas9 and “Coronavirus”. CRISPR-Cas9 is a current topic of this website (see previous issues) and “Coronavirus” or SARS-CoV-2 virus gained its highest highlight across all media after bringing a pandemic known as Covid-19 following an outbreak in China in 2019. (1)

After many diagnostic tools have been developed in an attempt to determine individuals that were infected by the virus, CRISPR-Cas9 has also been equated as one of the possibilities to such end (2). Likewise, one of the objectives ever since has been to demonstrate the mechanisms that underlie the infection caused by SARS-CoV-2 in these individuals. One of the features that is known so far, is the targeting of the receptor human angiotensin-converting enzyme 2 (hACE2) which is thought to be responsible for respiratory-associated issues often manifested by symptomatic patients (3).

This paper tries to pave a way to understand such mechanism and elucidate better on how the infectious route is driven by SARS-CoV-2. For that sake, a mouse model has been established with the mouse ACE2 (mACE2) gene being replaced by the human ACE2 (hACE2) through a knock-in mechanism induced by CRISPR-Cas9 technology usage. Briefly, 370 zygotes of C57BL/6 mice were injected with both ACE2 sgRNA and Cas9 mRNA in order to allow for integration of hACE2 at the Exon2 site on a region of chromosome X belonging to mACE2 remaining under the control of its promoter. This has been achieved in roughly one-fifth of the zygotes as confirmed by qPCR on founders’ cDNA, which were bred afterwards with wild-type C57BL/6 mice to allow for absence of random insertion of the construct in their offspring (F1+). F1+ population has been confirmed to have faithfully integrated the construct of choice by genomic sequencing and southern blotting and upon a dispersion of hACE2 expression seen across lung and kidney tissues as shown by western-blotting patterns.

After the establishment of the humanized hACE2 mouse model, two independent routes of injection of SARS-CoV-2 virus were carried in both young and aged mice envisaging to understand the mechanism of infection - intranasal and intragastric – whereas wild-type mice have been used as a control model. Respiratory route has been clearly infected upon demonstration of virus replication by qPCR and protein expression by immunofluorescence in lung and trachea tissues. Viral S protein is co-localized with hACE2 and CC10 proteins in Clara10 cells which are associated to lung airway. Interestingly, a particular high replication of this viral protein in hACE2 mice was detected in brain cells. Moreover, aged hACE2 mice had high levels of virus in their feces. Furthermore, a pathological pulmonary response has been observed in young and aged hACE2 mice with features of interstitial pneumonia being manifested. In aged hACE2 mice an elevated cytokine production, higher neutrophil and macrophage infiltration and alveolar lesions and focal hemorrhage has been observed by comparison to young ones whereas wild-type mice revealed none of these symptoms. In what concerns intragastric injection, once again high levels of viral RNA were detected in tissues associated to respiratory tract – trachea and lung – in hACE2 mice and S viral protein has also been detected in lung airway similar to what has been found in hACE2 mice following intranasal infection route injection. Associated to this were also pulmonary pathological signs common to hACE2 mice that were infected intranasally.

Several inferences can be taken from this model. Firstly, there is a clear relationship between the infection in mice that are “equipped” with hACE2 by SARS-CoV-2 and the resemblance of pathological symptoms in mice similar to what is found in patients with such infection. Secondly, despite similar levels of viral RNA particles observed across several tissues, aged and young mice have a different expression of such particles as well as a clear difference on the type of inflammatory responses that they initiate. Thirdly, both intranasal and intragastric routes seem to develop rather common features of interstitial pneumonia and presence of viral loads in the same type of tissues. Finally, in this model the mechanism of infection route seems to be driven by targeting Clara cells present in the lung airways where CC10 co-localizes with hACE2 protein.
There are, however, some questions that remain unanswered, namely: why did brain cells have been expressing such viral proteins? What could be its relationship with neurological symptoms sometimes associated to Covid-19 patients? And also, how cells not expressing hACE2 have been invaded by SARS-CoV-2 virus?
This model established with the help of CRISPR-Cas9 seems to be a candidate for the study on the mechanism driven by SARS-CoV-2 upon infection and with that, lead to advances in therapeutic studies to be applied later in humans.

Links:
https://www.who.int/csr/don/12-january-2020-novel-coronavirus-china/en/

https://www.medicaldevice-network.com/comment/crispr-biotechnology-disrupt-covid-19-testing-market/

https://theconversation.com/what-is-the-ace2-receptor-how-is-it-connected-to-coronavirus-and-why-might-it-be-key-to-treating-covid-19-the-experts-explain-136928