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Review
. 2022 Jan 5:11:792584.
doi: 10.3389/fcimb.2021.792584. eCollection 2021.

Experimental Models of COVID-19

Luis A Caldera-Crespo  1   2 , Michael J Paidas  1 , Sabita Roy  3 , Carl I Schulman  3 , Norma Sue Kenyon  4   5   6 , Sylvia Daunert  7   8   9 , Arumugam R Jayakumar  1
Affiliations
Review

Experimental Models of COVID-19

Luis A Caldera-Crespo et al. Front Cell Infect Microbiol. .

Abstract

COVID-19 is the most consequential pandemic of the 21st century. Since the earliest stage of the 2019-2020 epidemic, animal models have been useful in understanding the etiopathogenesis of SARS-CoV-2 infection and rapid development of vaccines/drugs to prevent, treat or eradicate SARS-CoV-2 infection. Early SARS-CoV-1 research using immortalized in-vitro cell lines have aided in understanding different cells and receptors needed for SARS-CoV-2 infection and, due to their ability to be easily manipulated, continue to broaden our understanding of COVID-19 disease in in-vivo models. The scientific community determined animal models as the most useful models which could demonstrate viral infection, replication, transmission, and spectrum of illness as seen in human populations. Until now, there have not been well-described animal models of SARS-CoV-2 infection although transgenic mouse models (i.e. mice with humanized ACE2 receptors with humanized receptors) have been proposed. Additionally, there are only limited facilities (Biosafety level 3 laboratories) available to contribute research to aid in eventually exterminating SARS-CoV-2 infection around the world. This review summarizes the most successful animal models of SARS-CoV-2 infection including studies in Non-Human Primates (NHPs) which were found to be susceptible to infection and transmitted the virus similarly to humans (e.g., Rhesus macaques, Cynomolgus, and African Green Monkeys), and animal models that do not require Biosafety level 3 laboratories (e.g., Mouse Hepatitis Virus models of COVID-19, Ferret model, Syrian Hamster model). Balancing safety, mimicking human COVID-19 and robustness of the animal model, the Murine Hepatitis Virus-1 Murine model currently represents the most optimal model for SARS-CoV-2/COVID19 research. Exploring future animal models will aid researchers/scientists in discovering the mechanisms of SARS-CoV-2 infection and in identifying therapies to prevent or treat COVID-19.

Keywords: COVID-19; MHV-1; SARS-CoV-2; experimental models of COVID-19; in vitro model; pathology; pneumonia; variants of concern.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Major SARS-CoV-2 variants along with their dates and location of primary determination and their characteristic spike protein mutation sites. SARS-CoV-2 variants share a common mutation in D614G of the spike protein. Notable spike mutations: Alpha variant mutations include N501Y, an asparagine to tyrosine amino acid substitution at position 501, and P681H, a proline to histidine substitution at position 681have been found to be preserved in subsequent variants. The Beta variant E484K mutation, glutamate to lysine substitution at position 484 of the RBD arising from South Africa may lead to possible gain-of-function ability to confer advantage against current convalescent plasma treatments. Beta & Gamma (Brazil/Japan) lineage mutations also include K417N and N501Y and may together lead to possible gain-of-function escape from neutralizing antibodies. Delta variant, first discovered in India 2020, mutations include L452R substitution correlates with higher affinity to the ACE2 host receptor, & P681R may be responsible for increased infectivity of host cells.
Figure 2
Figure 2
The structural and functional aspects of SARS-CoV-2. Point mutations in Spike protein have been shown to increase survival of SARS-CoV-2 virion from host immune response. D614G is related to increased infectivity of cells, E484Q has been shown to increase neutralization resistance, and N501 Y has been shown to cause enhanced viral transmission.
Figure 3
Figure 3
Illustrates various animal models of COVID-19 and the mode of infection. Models were useful in displaying different levels of clinical disease severity once sensitized to SARS-CoV-2 infection. Top left: Mice engineered to express hACE2 gene using k18 promoter. SARS-CoV-2 intranasal inoculation & ACE-2 transfection was shown to cause clinical symptoms (severe lung infection, anosmia) with mild extrapulmonary symptoms (GI, neurological). Top right: Aged, wild-type mice were serially passaged with intranasal SARS-CoV-2. Over time, viral generations eventually were able to bind to mACE-2 leading to clinical interstitial pneumonia and positive titers of neutralizing antibodies. Bottom: Mouse ACE-2 promoter with Human ACE-2 coding sequence were injected into pronuclei leading to mACE-2/hACE-2 susceptible to SARS-CoV-2 infection. hACE-2 receptors were expressed in lungs, kidneys, intestines & heart with mild clinical symptoms (mild weight loss, bristled fur).
Figure 4
Figure 4
Illustrates the process of Adenovirus hACE2 mouse model transgenesis as well as clinical symptoms of model after SARS-CoV-2 inoculation. Adv-hACE-2 mice showed greater weight loss, clinical symptoms (interstitial pneumonia, weight loss), and pathology in lung sections in addition to the absence of IFN signaling. Elevated viral RNA levels are seen in lung tissue, but low levels are shown in other tissues (heart, brain, liver spleen) while other tissues are not tropic (kidney, GI, serum). Benefits of this model include the ability to quickly sensitize different species of mice due to the rapid deployability of the adenovirus delivery system for expressing hACE2.
Figure 5
Figure 5
Mice infected by MHV-1 inoculation. (A) 40% of mice were symptomatic by Day 2 post-infection and exhibition of clinical signs progressively increased to 75%. (B) Mice inoculated by MHV-1 exhibited clinical signs starting at 2 days post-infection. Mild to moderate stage (I-III) of clinical symptoms were seen on days 2-4. Severe sickness (IV-VI) was seen in mice after 6 days. Reproduced with permission from Paidas et al, Viruses; Published by MDPI, 2021.
Figure 6
Figure 6
Organs histology in infected and uninfected mice: Representative histological images of haematoxylin and eosin (H&E) stained lung tissue sections of normal mouse (A) and infected mouse lung (B). MHV-1-infected mice lung shows arterial endothelial swelling (hypertrophy, long arrow), inflammation and granular degeneration of cells (short arrow) and migration of leukocytes (arrowhead) into lung. Peribronchiolar interstitial infiltration, bronchiole epithelial cell necrosis and necrotic cell debris within alveolar lumens, alveolar exudation, infiltration, hyaline membrane formation and alveolar hemorrhage with red blood cells within the alveolar space and interstitial edema were observed in MHV-1-infected mice. Liver, kidney, heart, and brain tissue degenerative changes were observed in MHV-1-infected mice. MHV-1-infected mice at day 6 showed liver hepatocytes degeneration, severe cells necrosis [(D), long arrows] and hemorrhagic changes (short arrows) when compared to uninfected mice (C); Uninfected kidney (E) and tubular epithelial cells degenerative changes and vacuolation, and peritubular vessels congestion (F, G); Severe interstitial edema (arrows), vascular congestion and dilation (arrow heads) and red blood cells infiltrating between degenerative myocardial fibers were seen in the MHV-1 infected mice heart (I, J), as compared to uninfected mice (H). Vacuolation of cerebral matrix [arrows, (L, M)] and cytotoxic edema [arrow heads, (L, M)], congested blood vessels with perivascular edema [long arrows, (L, M)], as well as cytotoxic edema (short arrows) were seen in MHV-1infected mice brain cortex, and in hippocampus (O&P). Reproduced with permission from Paidas et al, Viruses; Published by MDPI, 2021.
Figure 7
Figure 7
Illustrates the process of Syrian hamster model SARS-CoV-2 inoculation as well as clinical symptoms. Top: Wild-type Hamster ACE-2 are readily susceptible to SARS-CoV-2 infection, particularly nasal olfactory neurons, leading to anosmia. Innate and cell-mediated infiltration were seen at lung with consolidation noted, however no histopathology was seen in extrapulmonary tissues despite viral antigen noted at duodenal and colon epithelia. Bottom: Graph illustrates viral course of SARS-CoV-2 infection in Syrian hamster model. High titers of viral RNA are noted 2 days post-inoculation (DPI), with steady decline of viral load 5 DPI and sharp decline and viral clearance at approximately 7 DPI.
Figure 8
Figure 8
Illustrates the process of Ferret model SARS-CoV-2 inoculation and clinical symptoms post-infection. Illustration of SARS-CoV-2 Ferret Direct vs. Indirect transmission experiment. Ferret ACE-2 receptors are readily susceptible to SARS-CoV-2 infection with mild clinical signs seen in in infected ferrets (hyperthermia, mild cough). High titers of viral loads in upper airways provide evidence for direct & indirect contact leading to viral infection though respiratory droplet transmission.
Figure 9
Figure 9
Illustrates the process of Non-Human Primate model SARS-CoV-2 inoculation and clinical symptoms post-infection. Multiple routes of SARS-CoV-2 inoculation allow researchers to understand which area of the upper airways are most susceptible to SARS-CoV-2 infection, with the highest levels found in the pharynx and nasal cavities. A common NHP model, Rhesus macaques, have been shown to have high levels of viremia & viral loads in feces, with seroconversion comparable to humans and mild clinical symptoms (mild-moderate interstitial pneumonia). No systemic symptoms or ARDS were seen in infected macaques making this model less useful in studying severe COVID-19.
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