PREFACE

Coronavirus disease-19 (COVID-19) is a complex disease that causes illnesses ranging from mild to severe respiratory problems. It is caused by a novel coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), an enveloped, positive-sense, and single-stranded RNA (+ssRNA) virus that belongs to the coronavirus family. It has a fast-spreading potential worldwide, which leads to high death cases regardless of lows death rates. Yet, there are no animal models or specific drugs for disease prevention and/or treatment. Therefore, it is highly demanded to identify the known drugs and test them as a possible therapeutic approach. In this critical situation, one or more of these drugs may represent the only option to treat or reduce the severity of the disease until a specific drug or vaccine is developed and approved. A wide variety of therapeutics have been explored to treat COVID-19, initially suggested for other diseases and already established safety profiles, and approved by the Food and Drug Administration (FDA). Such treatments are referred to by the World Health Organization (WHO) as repurpose medications. Still, there are many ongoing clinical trials regarding the safety and effectiveness of repurposing immune-therapeutics to mitigate the symptoms of COVID-19.

In this book volume-2 proposal, we consolidate the various animal models and treatment strategies widely used for the global emergency of COVID-19. Since SARS-CoV-2 is the closest to SARS-CoV and MERS-CoV, the approaches brought here will be similar and/or varying with a slight degree. It is cleared that in the last 17-18 years, this is the third outbreak of the same coronavirus with a small mutation that shock the whole world. The chapters in this book should be prioritized as up-to-date literature of techniques used in the study SARS-CoV-2 and will act as a suitable reference if any such wary appear soon.

The 2^nd volume of the proposed book proposal has been classified into Part IV: Models for SARS-CoV-2 and Part V: Treatment Strategies for SARS-CoV-2. With the emergence of new coronavirus variants, epidemiology, different host tropism permits a thorough analysis of their evolution and acquired adaptability to their host. The 1^st volume already discussed the entry, epidemiology, genetic alteration, and diagnostic approaches. In the 2^nd volume, part IV, we have planned to describe chapter-wise models used in COVID-19. No studies are complete without animal models closely related to human physiology to replicate the disease and observe the pathology conditions as in human cases. Such animal models play a vital role in virus pathogenesis and prepare a therapeutic immune response. Here describe bio-engineered transgenic mouse model inserting with specific genes, or CRISPR-Case9 gene-editing tool has been used previously for SARS-CoV and MERS-CoV. The chapter will deal with culture techniques or cell lines for COVID-19-also histopathology of COVID-19, essential proteins that up or down-regulate SARS-CoV-2. The last chapter of this part will describe other diseases having similar signs and symptoms and their differentiation. In the last part of the book proposal, part V, chapters will deal with therapeutic approaches to attenuate SARS-CoV-2 as there is no specific treatment available to date, just symptomatic therapy. However, scientists will elucidate effective antiviral drugs in clinical trials, phytochemicals, photomedicine such as ultraviolet A & B, homemade remedies, blood plasma transfusion, stem cell therapy, and computational approaches in vivo and in vitro trials.

This book will appear as a baseline for academicians, scientists, and health professionals as still, research is going to overcome this outbreak of COVID-19, the novelty of best animal models, and find an effective treatment. However, just a single book proposal like this wouldn't have flourished without enthusiasm and determined publishers' and investigators' strength to take time from their busy schedule and subsidize on time.

We thank the whole investigators who contributed, directly and indirectly, to bring it to reality.

Kamal Niaz

Department of Pharmacology and Toxicology

Faculty of Bio-Sciences, Cholistan University of Veterinary and Animal Sciences

Bahawalpur-63100

Pakistan

&

Muhammad Farrukh Nisar

Department of Physiology and Biochemistry

Faculty of Bio-Sciences, Cholistan University of Veterinary and Animal Sciences (CUVAS)

Bahawalpur-63100

Pakistan

List of Contributors

Part I: Models for COVID-19

Genetically Engineered Mouse Models for COVID-19

Aadil Javed^{1, *}, Kamal Niaz², Ovais Sideeq³

¹ Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University, Bornova, Izmir, Turkey

² Department of Pharmacology & Toxicology, Faculty of Bio-Sciences, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100, Pakistan

³ School of Medicine, Tehran University of Medical Sciences (TUMS), Tehran, Iran

Abstract

Previous severe acute respiratory syndrome-coronavirus (SARS-CoV) outbreaks resulted in a cohort of preclinical studies that utilized various mice models for determining the pathogenesis of the infection, including the viral replication, spread, and mortality of the disease. Such studies have provided a framework upon which new investigations have been launched for understanding the outbreak of new coronavirus disease-19 (COVID-19) causing viral agents and their interaction with the host and its body. Recent investigations showed that the previous SARS-CoV and the recently discovered severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) both require the spike S protein to enter the host cell upon infection the binding with the receptors on the surface of the cells. The viral entry also requires proteases from the host cells. Since there are key similarities between the structure of the viruses and the construct of the viral transmission along with the spread inside the host's body in animal models. They were developed for the previous viral agent. The disease can be emulated or manipulated to bring forth novel investigations leading to key data that can broaden the sphere of COVID-19 studies being conducted. There are several different options to choose the right animal model for the question being raised in the experimental design with the pathogenesis of COVID-19. This chapter focused on the already established animal models for other coronavirus outbreaks and some of the strategies that can be exploited to develop new animal models. For COVID-19, research aimed at targeting the therapy or basic investigations for understanding cellular or organ level mechanisms involved in the disease.

Keywords: COVID-19, Knockout, Mouse models, SARS-CoV-2, Transgenic.

^* Corresponding author Aadil Javed: Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University, Bornova, Izmir, Turkey. E-mail: adiljaved313@hotmail.com

INTRODUCTION

In December 2019, a novel coronavirus outbreak was reported in Wuhan, China which caused a pandemic and is an ongoing public health concern worldwide [1]. The disease known as coronavirus disease-19 (COVID-19) is caused by the novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which leads to deadly pneumonia and severe health issues related to lung damage [2]. The genomic properties of the viral agent responsible for the local epidemic in China were timely shared across the scientific community. Since then, a valiant effort to combat the disease and understanding the agent responsible for the pandemic has been ongoing. Across the world, millions of people have been affected by the pandemic leading to a change in lifestyle globally [3^-5]. The diverse scientific community took upon understanding the pandemic and the viral agent responsible for the COVID-19 from the get-go during the earlier days of the pandemic in 2020 [6]. Millions of people have been infected, and hundreds of thousands of people have lost their lives due to the COVID-19 pandemic outbreak [7]. Various attempts have been reported to classify the epidemiological features of the pandemic, including the case fatality rate, reproduction number, and recovery rate. To date, it is being assumed that various factors that affect the rigorous testing and other socio-economic factors hamper any progress in determining the true features of the pandemic [8^-13].

The SARS-CoV-2 virus belonging to the Coronaviridae family of the virus has been studied extensively due to the urgency of pandemics. The vaccine development approaches have been challenging so far, and various mouse models have been utilized for understanding different aspects of the physiology of this novel coronavirus. Previously, the Middle East Respiratory Syndrome (MERS) and SARS candidates for vaccines were evaluated in the different mouse models [14, 15]. The clinical features, including the pathogenesis of the coronavirus, can be studied in great detail upon the development of animal models that can efficiently mimic the properties of the disease. For this purpose, numerous models have been developed in animals such as hamsters, guinea pigs, mice, cats, and rabbits, etc., [16^-20]. The major receptor of SAR-CoV is angiotensin-converting enzyme 2 (ACE2) and is utilized to develop transgenic mouse models containing the human ACE2 gene [21]. Rhesus macaques were the first animal model for vaccine development against MERS-CoV, showing the symptoms of the infections as seen in clinical patients [22]. Golden Syrian hamsters were also used as animal models for establishing the vaccine candidate's safety and viral pathogenesis for different strains of SARS-CoV [19]. NSP16 CoV attenuated vaccine development approaches utilized mice as well [23]. Developing animal models, MERS-CoV had several drawbacks due to the inefficiency of the virus being replicated in the respiratory system. Therefore, new approaches like gene targeting paved the way for demonstrating modified models containing genes of interest or lack thereof for more naturalized infectivity of the virus, e.g., human DPP4 transgenic mice [24]. Clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) the system has been efficiently employed for developing mouse models that are engineered to get infected by the virus and show high replication [25, 26]. Small animals like rabbits and mice are used to develop mouse models specific for a particular infection such as SAR-CoV-2 for various reasons, including efficacy, cost issues, and manipulation approaches. There is still much work that is needed to better understand the behavior of COVID-19 and transgenic animal models can efficiently smoothen the process of understanding due to ease in receptor identification, protection, pathogenesis models and immune response studies [15, 27]. Various applications and interference points for different types of mouse models in understanding or combating SARS have been depicted in Fig. (1). The aim of this chapter is to summarize the well-known animal models that can be used for coronavirus along with some of the strategies that can be exploited to develop new animal models for COVID-19 as a model to target the therapy or basic investigations the underline molecular mechanisms involved in the disease.

Emerging Role of CRISPR/Cas9

In molecular diagnostics, CRISPR, and the Cas protein-containing CRISPR-Cas systems, have advanced the process of functional research. Since its discovery almost 30 years ago, the field of genome editing using CRISPR systems has been revolutionized [28]. In clinical sciences and modern molecular biology approaches, CRISPR-Cas9 systems are routinely applied for targeting the cells of mammalian origin involving gene editing methodology [29]. Such approaches have been utilized to detect Zika virus and methicillin-resistant Staphylococcus aureus along with nucleic acid detection-based diagnosis procedures that use RNA-guided or RNA-targeting CRISPR-Cas systems [30^-32]. In the wake of the COVID-19 pandemic of 2020, CRISPR-Cas13 approached for diagnostic applications are being considered [33]. The potential for this technology is deemed as tremendous, as described by Xiang et al. [34]. A fast and accurate method for detecting SARS-CoV-2 is the need for the hour, and SHERLOCK protocols can provide the scientific community an approach that has huge potential in this area. A recently reported DETECTR assay for the detection of SARS-CoV-2 via employing CRISPR leads to a 95% positive prediction along with a 100% negative prediction agreement in a real-time RT-PCR [35]. Genome-wide CRISPR screens were employed to determine the therapeutic targets for COVID-19. Apart from already established protease cathepsin and receptor ACE2, SWI/SNF chromatin complex proteins that comprise the transforming growth factor-beta (TGF-β) signaling network were also identified using the technology [36]. The protocol for designing a transgenic mouse model for SARS-CoV-2 that conforms to the CRISPR-Cas9 technology has also been reported recently [37]. Such models can successfully provide representativeness of the infection with tissue specificity of the human genes as well [37].

Fig. (1))

Experimental animals of SARS-CoV, MERS-CoV, and SARS-CoV-2. The coronaviruses with high infectivity and pathogenicity break the species barrier and infect humans in the past two decades. Besides NHP, mice, hamsters, ferrets, and rabbits, the other possible natural hosts might be able to support the studies of coronavirus infection, pathogenesis, and drug discovery.

Human ACE2 Expression

A carboxypeptidase named ACE2, which is encoded by the gene ACE2 resides on chromosome Xp22. It gives rise to a type I type transmembrane protein containing an extracellular N-terminal domain with carboxypeptidase which is heavy, and a short C-terminal domain having a cytoplasmic tail intracellularly [38, 39]. The SARS-CoV binding site in ACE2 is present on the N-terminal peptidase domain [40]. Cellular ACE2 manifests in either soluble or membrane-bound form and is usually expressed in pneumocytes and enterocytes located in the small intestine [41]. Vascular endothelial cells located in the heart, brain, and kidneys also exhibit ACE2 expression [42, 43]. The major site of infection for the coronavirus, especially SARS-CoV, is the respiratory tract. In human airway epithelia and lung parenchyma, ACE2 is expressed and is considered the receptor for these coronaviruses. SARS-CoV also seemed to infect the differentiated epithelial cells more than undifferentiated cells as the differentiated epithelia are reported to have higher expression of ACE2 receptor via SARS S protein [44].

ACE2 gene in humans produces the protein receptor that is identified by SARS coronaviruses, including SARS-CoV and NL63, along with the novel SARS-CoV-2 virus that caused the pandemic of 2020 [45]. In vitro studies suggest that SARS-CoV infection correlates with the expression of the ACE2 receptor. The variations or mutations in the ACE2 sequence lead to a reduced association or linkage between the spike protein on the SARS-CoV or NL63 with the ACE2 receptor [40, 46]. It has been concluded that the severity, susceptibility, or even the symptoms of SARS-CoV-2 infection is dependent on the expression of the receptor ACE2. Recently, ACE2 expression levels from single-cell RNA-sequencing experiments concluded that Asian males show higher expression of the receptor [47]. However, more studies need to be conducted to profile a linkage with ethnicities based on ACE2 expression and COVID-19 epidemiological features. The overall function of ACE2 in different populations and the genetic background correlations, are yet to be understood in detail [48]. A systematic analysis of variants of ACE2 (coding region variants) that can influence the expression pattern has been performed. The genetic analysis from microarray data showed that the East Asian populations have a higher allelic frequency of different eQTL variants associated with higher expression of ACE2. More studies on the pilot data need to be performed in other populations to report concrete evidence related to receptor and virus spike protein [49].

ACE2 Knockout Mouse for Acute respiratory Distress Syndrome (ARDS) Investigation

Major causes of death resulting from SARS include acute lung injury along with ARDS. ACE2 knockout mice are reported to be protected from infections by SARS [50]. Moreover, studies showing data from knockout ACE2 receptors have depicted that the spike protein of the virus can cause the downregulation of the ACE2 expression and ACE2 being involved in the protection of lungs from acute injury of the lungs [50, 51]. The introduction of recombinant ACE2 protein can reduce ARDS in mice due to the angiotensin and renin pathway [51]. Moreover, knockout ACE2 mice have been reported to be more prone to impaired lung functionality and exacerbated fibrosis of the lung along with reduced exercise capacity when compared with wild-type mice [52]. In another investigation, knockout ACE2 mice depicted an increased level of lung edema, increased vascular permeability and demonstrated severe lung disease [51].

On the one hand, knockout ACE2 shows severe lung disease, the introduction of recombinant ACE leads to elevated symptoms related to ARDS [53]. It can be deduced from the studies on knockout ACE2 models that ACE2 is a key factor involved in the pathogenesis of SARS viruses and could be the most promising target for therapy that does not include the immunization procedures that can target ARDS. Based on the data obtained through knockout studies, recombinant human ACE2 studies showed promising results in improving the pulmonary circulatory hemodynamics and arterial hypoxemia associated with ARDS [54]. ACE2 knockout mice are known to be resistant to SARS infection as the viral titers determined from the knockout model were a hundred thousand folds lower than the wild-type mice lungs [50].

K18-hACE2 Transgenic Mouse

Coronaviruses, especially those that cause SARS, infect many animal species, including ferrets, mice, cynomolgus, hamsters, and rhesus macaques, among others [55]. Apart from different types of coronaviruses available for studies that demonstrate the infectivity comparable to human coronaviruses. The clinical disease that arises from infection by the SARS-CoV is difficult to replicate in animals in terms of clinical signs and severity as observed in the patients. To determine the vaccine efficacy along with the overall pathogenesis of SARS-CoV, a reproducible and equivalent animal model is the need of the hour that can mimic the immune response in the host as well. Transgenic models are preferred to overcome this drawback, e.g., hACE2 transgenic mice, which show overexpression of the receptor ACE against the SARS-CoV [56]. Since mACE2 does not bind as efficiently with the virus as hACE2. Therefore hACE2 is the specific target for generating transgenic mice. Moreover, the expression of ACE2 being higher in transgenic hACE2 mice also result in a severe disease upon infection with the SARS-CoV in the epithelia of mice [57]. pK18-hACE2 transgenic mice were generated by cloning into pCR2.1-TOPO vector the amplified hACE2 coding sequence from IMAGE consortium clone ID 5243048. The final construct for generating transgenic mice contained a promoter and intron for human cytokeratin 18 (K18 gene) and the enhancer sequence obtained from the alfalfa mosaic virus upstream to the hACE2 coding sequence. Besides, 2 exons and 1 intron of K18 along with polyA signal tail were included for bringing higher specificity towards the epithelia in terms of selective translation in the downstream sequence [57, 58]. For the final generation of the mice, the transgene was injected into pronuclei of the fertilized mice eggs (C57BL/6J) for embryo generation and validated by PCR specific for transgene primers. In one of the recent reports, the SARS-CoV-2 viral agent responsible for the COVID-19 pandemic (one of the earlier isolates) was infected in transgenic hACE2 mice for determining the pathogenicity of the novel SARS-CoV-2 in hACE2 mice [59]. As a result of this experimental infection, the mice lost weight and demonstrated interstitial pneumonia, known as the clinical signs of COVID-19 [59, 60]. Specific immunoglobulins (IgG) targeting SARS-CoV-2 were also observed in the lungs of transgenic hACE2 mice along with virus replication. More studies are needed to determine the immune response against the infection in these animals.

TMPRSS2 Knockout Mouse for Viral Entry and Pathogenesis Studies

Coronavirus fusion glycoproteins which are also known as spike proteins, are cleaved by transmembrane protease serine-type 2 (TMPSS2) belongs to a family of transmembrane serine protease type II. Thus activates the spike protein resulting in the fusion of the cell-virus membrane to facilitate the next step of infection further; the entry of the virus in the host cell [61^-63]. There are reports which suggest that TMPRSS2 knockout mice are resistant to severe outcomes from infection by the influenza virus. At the same time, higher expression of this gene leads to severe outcomes after infection with the influenza virus [64, 65]. Mice deficient in TMPRSS2 are reported to have shown a reduced loss of weight and replication of viral agents in the lungs. It spreads the virus in the airways of the animals when infected with SARS-CoV and MERS-CoV [61]. The viral entry of coronaviruses and influenza viruses depends critically on TMPRSS2 as the viral protein interacts and binds with ACE2 localized in the epithelia. Viral protein is cleaved by special proteases such as TMPRSS2 to activate the process of internalization of the virus, leading to the next phases of the infection [66]. SARS-CoV-2 and other coronaviruses and influenza viruses require TMPRSS2 to activate the virus and its entry into the host cell for carrying the infection [62, 67, 68]. Therefore, TMPRSS2 can be placed at the center of the pathogenesis of COVID-19 and other pandemics of the past [69].

Since it has been established that TMPRSS2 is vital for entry of SARS-CoV-2 and involved in the dysregulation of ACE2. Therefore, it has been hypothesized by various accounts that it can play a vital role in preventing the critical circumstances of COVID-19 disease if used in a well-defined elaborated therapeutic strategy [70]. TMPRSS2 mediated entry of the SARS-CoV-2 can be inhibited by the serine protease inhibitor known as camostat mesylate [71, 72]. The envelope and plasma membrane fusion between SARS-CoV-2 mediated by TMPRSS2 and ACE2 can also be inhibited by nafamostat mesylate showing 10-times more efficacy than the camostat mesylate [73]. TMPRSS2 is now considered one of the most promising candidates as a therapeutic target for COVID-19. TMPRSS2 knockout mice model study indicated that the influenza virus entry into the cell is inhibited compared to wild-type mice, further implying the role of TMPRSS2 in regulating the pathogenesis of viruses as the crucial step of viral entry inhibition can lead to reduced mortality [74]. TMPRSS2 knockout mouse model can be generated in C57BL/6 Embryonic Stem Cells (ESCs) usually obtained from KOMP (knockout Mouse Project) repository. It is followed by injection of ESCs into blastocyst for generating chimeric mice, followed by male and female mice for creating homologous genotype [61, 75]. The viral kinetics observed in the lungs of TMPRSS2 knockout mice upon infection by SARS-CoV were reduced along with a reduced weight loss upon infection. Moreover, the inflammatory cytokine and chemokine response usually associated with such infections were also observed to be weakened in TMPRSS2 knockout mice [61]. For SARS-CoV-2 experimental infections in mice models, more studies need to be performed to understand the underlying mechanisms of the inflammation-mediated responses and immune responses generated via infections that lead to severe clinical circumstances of COVID-19 disease.

STAT1 Knockout Mouse for Studying Pneumonia and Antiviral Strategies

Signal transducer and activator of transcription 1 (STAT1) is considered one of the STAT family members of proteins involved in the signaling responses of the innate immune system. The signaling works by cytokines or growth factors that elicit response inside the cells, leading to phosphorylation of these proteins by the kinases associated with certain receptors, further causing the translocation of these proteins to the nucleus. One of the most important cytokine-mediated signalings in immune responses caused by viruses is interferon receptor signaling, which requires STAT1 responses [76]. The severe cases of COVID-19 show a cytokine storm phenotype, which is considered to be caused by SARS-CoV-2 nsp3 due to the de-mono-ADP-ribosylation of STAT1. It further highlights the importance of STAT1 as a therapeutic target in the precarious times of the COVID-19 pandemic [77]. A group of cytokines known as type 1 interferons (IFN-1) having further subtypes is known to be secreted by different cells like dendritic cells when pattern recognition receptors (PRRs) recognize the viral antigens or components [78]. IFN-1 is considered the first type of cytokine produced and released upon infection by a viral agent.

Interferon gets fixated upon interferon-α/β receptor (IFNAR), transcription factors including STAT1 are phosphorylated, leading to their translocation to the nucleus, further causing activation interferon-stimulated genes (ISGs). ISGs are known for the modulation of immune responses and signaling related to inflammatory pathways. After activation, ISGs lead to the further secretion of cytokines promoting adaptive immunity and interference with viral replication or spread and slowing down the cell's metabolism [79, 80]. Previously available IFN-1 treatment data on SARS-CoV and MERS-CoV can be valuable in determining the SARS-CoV-2 related mechanisms of pathogenesis and immune responses, as both of these viruses led to the disruption of the interferon signaling pathway. SARS-CoV Orf6 protein has been reported to inhibit the STAT1 transport to the nucleus upon interferon response by disrupting karyopherin transport [81, 82]. Therefore, STAT1 knockout models are important in understanding the role of viral proteins and their subsequent action in determining the immune response resulting from the infection [83]. The innate immune response that leads to clearance of the SARS-CoV also requires STAT1 activity, which modulates the IFN signaling [84]. IFN deficient mice showed that these components of innate immune responses are required STAT1 deficiency to prolong the viral shedding or susceptibility, allowing for lethal infection outcomes. STAT1 deficient mice data revealed the mechanistic role of these proteins in regulating SARS-CoV pathogenesis in the lungs.

Moreover, the pathogenesis of SARS-CoV seemed to be dependent on STAT1 and not on IFN subtypes, highlighting the role of STAT1 in controlling SARS especially viral replication, wound repairing, or cell proliferation in the lethal type disease [85]. Profibrotic phenotype resulting from the lethal outcome of SARS-CoV in the lungs of STAT1 knockout mice was also reported [86]. Therefore, the role of STAT1 in clearing the SARS-CoV-2 infection cannot be avoided and can be used in determining the therapeutic strategies for lethal conditions of COVID-19 [87]. Moreover, the role of STAT1 knockout, as highlighted in studies, demonstrated the role of an innate immune response about interferon signaling, can be utilized for potential therapeutic approaches for COVID-19.

Standard Mouse Strains (BALB/c mice, C57BL/6, and 129S6)

The development of COVID-19 therapy approaches along with vaccine studies, and preclinical investigations can be performed in these mice as they are known to support the replication of viral agents (SARS-CoV and MERS). The standard mice used in laboratory research, like BALB/c mice, C57BL/6, and 129S6, demonstrate the SARS-CoV replication along with other mice models in conjunction with the experimental designs [55]. Mouse-adapted SARS-CoV-2 infection in BALB/c mice showed severe symptoms compared to young mice, showing the clinical similarities observed in human patients of COVID-19 [88, 89]. Moreover, acute lung injury models have also been developed using C57BL/6 mice, which may further understand the therapeutic agent selection against ARDS associated with the severity of COVID-19 [90]. Similarly, 126S6 mice have been used in viral studies investigating the immune responses elicited upon viral infection [91].

Inbred Mice for Pathogenesis and Therapeutic Response Studies

Mouse strains of inbred nature that are available widely can also be adapted in the experimental designs to understand various parameters associated with the pathogenesis and pathophysiology of COVID-19. For example, age-related mortality can be studied in more detail in mouse models that are infected with SARS-CoV-2 [19]. The pneumonitis and clinical symptoms of SARS have been mimicked in several inbred mouse species, including BALB/c, C57BL/6, and 129S [84, 92^-94]. The main reason for inbred mice being animals of choice for COVID-19 disease is their cost-effectiveness, smaller size, larger numbers to include a valid statistical output, and ease of manipulation at the genetic level [95]. 129S6 strain of mice is observed to report SARS susceptibility higher than BALB/c or B6 mice because the infection led to mild interstitial pneumonitis 2 days post-infection [84].

Similarly, TNF-alpha, a known pro-inflammatory cytokine, was reported at higher levels in BALB/c mice when recorded 2-9 days post-infection [94]. Age-related severity of SARS has already been demonstrated in aged BALB/c mice, which is observed in previous SARS outbreaks as well [93]. As seen in COVID-19 patients, viral replication is observed in the respiratory tract, and BALB/c also shows the same pattern of viral replication upon infection with viral agents in experimental conditions, leading to respiratory distress. Other clinical signs of weight loss, edema, dehydration, and pneumonitis were also observed in such mice strains when infected with SARS-CoV [96].

Humanized Immune System Mice for Vaccine Approaches

COVID-19 has overwhelmed many healthcare systems and has impacted economies negatively since its spread globally. If a COVID-19 vaccine becomes available for public immunization, life globally can go back to normalcy. Many vaccine candidates for COVID-19 are under investigation, and some are in clinical trials [74, 97]. Preclinical studies to develop an approach against SARS-CoV-2 require animal models corresponding to the immune responses as elicited in humans against the viral agent [98]. The antigens presented to the Major Histocompatibility Complex (MHC), however, vary between different species. Therefore, the mice models that can carry human leukocyte antigen genes can mimic the human response to the epitope-based vaccines and assist in developing or discovering the vaccines for COVID-19 in animal models [99].

Moreover, the vaccine discovery process can become rapid if the humanized immune system containing mice model is used for this purpose [100]. They can enhance the discovery process by speedily eliciting the response for efficacy studies and the immune response or memory generated upon vaccinated and un-immunized mice [37, 101^-103]. Human immune cell engrafted mice have also been instrumental in determining the immune response for a vaccine candidate and include a promising approach in combating COVID-19 [104, 105]. Immuno-deficient mice, when engrafted with peripheral blood mononuclear cells, exhibit another vaccine modeling approach [106].

CONCLUSION AND FUTURE PERSPECTIVES

Although there are many mice strains available that have assisted in understanding the pathogenesis of SARS and SARS-CoV previously, COVID-19 caused by SARS-CoV-2 presents a new challenge to the scientific community. Although mice and humans have shared genes, mice as in vivo replicas of COVID-19 or disease models require overcoming the challenges that are unique to the virus. The challenges include the model not possessing the receptor for the virus as human cells have mouse protein expression not being similar to human proteins. The binding affinity is different among viral and animal proteins. It also has a slightly distinct immune system among different species. Due to the aforementioned challenges, certain approaches have been utilized to overcome certain challenges, including transgenic mice representing the human version of certain aspects of the pathogenesis, knockout mice revealing the functions of intracellular programs required for the disease patterns, and inbred mice as age-related mortality models for disease, etc. In the end, mice models can also provide a great tool in discovering the therapeutic targets for the disease, which are not obvious at first but can be unearthed upon functional studies. Moreover, the information obtained through studies conducted in various types of mouse models consequently leads to the development phases of the vaccine or a drug target. Therefore, recently used immunized or immuno-deficient mice for investigating vaccine candidates or drug targets will ultimately lead to our understanding of rapidly concluding the different phases of discovery, therefore providing a gateway to quick response in times like COVID-19.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The author confirms that this chapter contents have no conflict of interest.

ACKNOWLEDGEMENT

Declared none.

REFERENCES