Selection of animal models for COVID-19 research

Abstract

The researcher community across the globe is on a search for a promising animal model that closely mimics the clinical manifestation of SARS-CoV-2. Though some developments were seen such as serial adaptation in various animal species or the creation of genetically engineered models, a suitable animal model remains elusive. A model that could display the severity of human illness and can be used for the fast-track evaluation of potential drugs as well as for the clinical trials of vaccines is an urgent need of the hour. In the light of huge information generated on SARS-CoV-2 and daily updates received from the research community, we have chosen to review the current status of animal models of SARS-CoV-2 in encompassing the areas of viral replication, transmission, active/passive immunization, clinical disease, and pathology. The review is intended to help the researchers in the selection of appropriate animal models for SARS CoV-2 research in the fight against the current global pandemic.

Introduction

Emerging and re-emerging coronavirus outbreaks and nonavailability of vaccines and antiviral therapy for the recent worldwide pandemic of SARS-CoV-2/COVID-19, across the world, has encouraged the interdisciplinary collaborative efforts based on the concept of a “One Health approach” between human health, veterinary medicine, and environmental sciences [27]. The unpredictable nature of these viruses in terms of host transmission [2, 35], interspecies shift [8], evasion from host responses [11], high adaptation and relapsing nature [1] have severely hampered the evaluation of therapeutic modalities against these agents directly in humans. To recognize the epidemiology of various factors that contribute to the diseases, the use of various animal models of disease is of paramount importance for vaccine or antiviral therapy. In this article, we explored the animal models that can be used to study SARS-CoV-2 pathogenesis with the primary aim of the evaluation of antiviral therapies and vaccines.

Requirements of an animal model against highly pathogenic viruses

Direct clinical trials on humans for highly pathogenic viruses are not feasible and also not ethically permitted without prior preclinical studies. Therefore, animal studies play an essential role in characterizing the viral pathogenesis and evaluation of antiviral agents and vaccines for these viruses. The ideal animal models should be permissive to infection and must reproduce the clinical course and pathology observed in humans [30]. A disease animal model can truly replicate the type of human disease as closely as possible in immunocompetent animals with a challenge dose and with a suitable exposure route that can be administered in humans. The animal model should be customized to the goals of the study. The model should replicate the key aspects of the disease. The availability of immunological reagents and the demographic background of animals should be taken into consideration. In addition to assessing vaccine/antiviral efficacy, the studies must demonstrate meaningful differences between vaccinated and unvaccinated control groups followed by detectable immunological responses to animals from different demographic backgrounds to the vaccine/antiviral under investigation [16].

Different animal models used

Mouse models

One of the best mouse models used for COVID-19 is the K18-hACE2 transgenic mouse. A transgene of human ACE2 (hACE2) expression is driven into the mouse epithelial cells under the control of the human cytokeratin 18 (K18) promoter [24]. K18-hACE2 mice when treated with doses of SARS-CoV (2.3 × 104 PFU) that induced severe lung damage and neuronal damage of CNS in transgenic mice. But it did not induce CNS pathology in nontransgenic mice. The transgenic mice showed replication of the virus in the lungs, had weight loss, and developed pathological lung inflammation, and died at 4 days post-infection. However, a recent study showed that SARS-CoV-2 can infect K18- hACE2 mice in a transgene-dependent manner [4]. SARS-CoV-2 at 105 TCID50 caused weight loss, evoked antibody responses, and developed histological evidence of lung inflammation in a K18-hACE2 transgene-dependent manner with interstitial congestion, inflammatory exudate, epithelial damage, etc. [4, 24]. Additional transgenic mouse strains have been developed to express hACE 2 using various promotors. HFH4-ACE2 transgenic mice developed using the human HFH4 promoter [19], AC70 transgenic mice developed using CAG promoter [36], and mouse ACE2 promoter-driven hACE2 Tg mice using mouse ACE2 promoter [34]. The above ACE2 transgenic mice models will be useful to study on SARS-CoV-2 replication in the lungs and its pathogenesis.

The limitation in these ACE2 transgenic mouse model is its lethal effects caused by neuroinvasion affecting CNS. The other limitations are its limited availability and the comparatively long breeding time.

An adeno-associated virus (AAV) delivery based mouse model that expresses the SARS-CoV-2 receptor in the mouse lungs [14, 15, 29] was developed to study SARS-CoV-2 pathogenesis. The model was a more efficient and rapid, and reproducible murine model for SARS-CoV-2. The limitations are that this model artificially expresses ACE2 in non-relevant cell types in the mouse respiratory system making pathology and immune response data hard to interpret in the situation of human SARS-CoV-2 infection. However, this model can be suitable for drug therapy and antibody testing.

SARS-CoV-2 cannot infect wild type and laboratory inbred mouse strains due to incompatible interactions between the viral spike (S) protein and the murine ortholog of the human receptor, ACE2. Standard inbred mouse strains such as BALB/c, C57BL/6 cannot be directly used for the studies. Nevertheless, these mouse strains by adaptation can be used to study the development of neutralizing antibodies against the spike protein, pseudo viral vaccine candidate, and antiviral drugs [32]. However, a mouse model was developed using a clinical isolate of SARS-CoV-2 by serial passaging in the respiratory tract of aged BALB/c mice [14]. The resulting mouse-adapted strain showed marked infection in mouse lung leading interstitial pneumonia and inflammatory responses after intranasal inoculation. In another study, reverse genetics was used to develop a mouse model using inbred BALB/c mice where the interaction between S and mACE2 was remodeled resulting in a recombinant virus (SARS-CoV-2 MA) that utilized mACE2 for entry in BALB/c mice. The SARS-CoV-2 MA replicated well in adult and aged BALB/c mice causing more severe infections in aged mice. It was concluded that this model is more clinically relevant phenotypes than HFH4-hACE2 transgenic mice [10].

Besides the above transgenics mouse, a few knock out mouse model has been developed to understand the pathology of SARS-CoV-2. Among them, ACE−/− knockout mice can be used to study the effects of Angiotensin conversion enzyme during acute lung injury study [17]. Using TMPRSS2−/−knockout mice the role of TMPRSS2 during SARS-CoV entry into cells can be studied for new drugs against SARS-CoV-2 infections [18]. Humanized DPP4 mice and STAT-1−/−knockout mice having susceptibility coronavirus infection and used as a model for MERS can help in SARS-CoV-2 also [12, 22].

Hamsters

Sia et al., 2020 opined that the Goldern syrian hamster is a good model for the study of COVID- 19. Golden Syrian hamsters after infection with the SARS-CoV-2 virus showed weight loss, efficient viral replication in the nasal mucosa, and epithelial cells of the lower respiratory system. The virus was shown to be transmitted from the naive to co-housed animals either by aerosols or by fomites. The infected hamsters also generated neutralizing antibody responses in response to SARS-CoV-2 infection [28]. A significant drawback with hamster was that 14 days post-infection, the lung pathology had resolved to normalcy. Hamsters can be used to study mild SARS-CoV-2 infections in humans and host defense response to the virus [5].

Ferrets

Ferrets have previously been used as a good model for the study of viral diseases especially for respiratory pathogens like influenza. For studying SARS-CoV-2 the animals were inoculated with the virus and found to develop similar symptoms as in humans within 2 and 12 days post-infection [20]. Histologically, SARS-CoV-2 infected ferret lungs have exhibited severe pulmonary lymphoplasmacytic perivasculitis and vasculitis at 13 days post-infection [20]. However, in another study using ferrets it was found that SARS-CoV-2 was transmitted efficiently via direct contact and via the air (via respiratory droplets and/or aerosols) between ferrets [26]. Considering the above, it can be seen that ferret can be a suitable model for disease transmission studies.

Non-human primates

Nonhuman primates (NHP) in particular M. mulatta (Rhesus macaques) can be a good model to study COVID-19 pathogenesis. NHP models have been developed for SARS-CoV-2 to resemble the condition seen in human pathogenesis.

Three Non-human primates species, rhesus macaques (Macaca mulatta), crab-eating macaques (Macaca fascicularis), and common marmosets (Callithrix jacchus) were compared for SARS-CoV-2 infections and found that rhesus macaques and c rab-eating macaques had developed typical lung pathology lesions seen in humans. Additionally, the viral antigens were detected in alveolar epithelial cells and macrophages in these macaques [23]. Yu et al., 2020, compared the severity of interstitial pneumonia between young and aged rhesus macaques and concluded that severity is more pronounced in aged rhesus monkeys [38]. In another study by Munster et al., adult rhesus monkeys were challenged using a live virus and were found to have viral replication and shedding along with COVID-19 symptoms with pathological lesions [25].

Passive immunity studies on [6, 9] rhesus monkeys were done to evaluate whether re-exposure to the virus led to the reoccurrence of the virus and it was found that there was no recurrence of SARS-CoV-2 infection, suggestive of protective immunity [3]. Cynomolgus macaques were also evaluated for susceptibility to SARS-CoV-2, and pathological lesions including alveolar and bronchiolar epithelial necrosis, alveolar edema, hyaline membrane formation, and accumulation of immune cells could be demonstrated which can make them an additional model for COVID-19 research for the development of COVID-19 therapeutics and vaccines [21].

In addition to infection studies, the therapeutic efficacy of drugs such as Remdesivir has been studied in rhesus monkeys. Rhesus macaques were primarily used to evaluate many vaccine candidates before undergoing testing in humans in clinical trials [7, 31, 33, 37].

From the above studies, one could say that NHP models may be an appropriate model for SARS-CoV-2 for virus replication, mimicking mild human COVID-19. Besides, NHP models can be applied in drug evaluation and vaccine candidate efficacy testing before the initiation of various phases in human clinical trials.

Conclusion

Several models have been attempted thus far. However, there is no clear model that is preferred for studying SARS-CoV-2 infection as the clinical signs, recovery, and transmission vary between and within species. Each animal model seems to have its own merits and demerits, and careful consideration is required before the selection of animal models. In transgenic animal model selection, various transgenic mouse models developed for SARS-CoV are in use for COVID-19 research. However, before their application in SARS-CoV-2, they must be aptly validated and ascertained that these mouse models could replicate the same pathogenesis as seen in humans. The researchers using these mouse strains should be careful in interpreting the data obtained from these mouse models. Possibly the study on the transgenic mouse can provide a proof of concept for understanding pathogenesis.

Hamsters can be best used to study replicate mild SARS-CoV-2 infections seen in humans and host defense response to the virus and will also help to understand SARS-CoV-2 pathogenesis and for testing vaccines and antiviral drugs. Ferrets on the other hand can be appropriate for disease transmission and lung infections. Considering that the SARS-CoV-2 infection in Non-human primates, rhesus macaque most closely resembles that observed in humans, it could be a valuable model to evaluate vaccines and drug efficacy.

Given the above, it is understood that a comprehensive model for SARS- CoV-2 infection that would exactly replicate human disease remains elusive. Yet an appropriate animal model susceptible to SARS-CoV-2 virus with comorbidity conditions as seen in humans needs to be developed for complete understanding and treatment of the COVID-19 pandemic. Nevertheless, the models available so far suggest that it is possible to choose models depending on the scientific goals of the researchers (Table 1).

Table 1 Animal models used for study clinical manifestation/pathogenesis, drug and vaccine efficacy

Availability of data and material

Not applicable.

References

  1. 1.

    Angeletti S, Benvenuto D, Bianchi M, Giovanetti M, Pascarella S, Ciccozzi M. COVID-2019: the role of the nsp2 and nsp3 in its pathogenesis. J Med Virol. 2020;92(6):584–8.

    CAS  Article  Google Scholar 

  2. 2.

    Banerjee A, Kulcsar K, Misra V, Frieman M, Mossman K. Bats and coronaviruses. Viruses. 2019;11(1):41.

    Article  Google Scholar 

  3. 3.

    Bao L, Deng W, Gao H, Xiao C, Liu J, Xue J, Lv Q, Liu J, Yu P, Xu Y, Qi F. Reinfection could not occur in SARS-CoV-2 infected rhesus macaques. BioRxiv. 2020.

  4. 4.

    Bao L, Deng W, Huang B, Gao H, Liu J, Ren L, Wei Q, Yu P, Xu Y, Qi F, Qu Y. The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice. Nature. 2020;583:830–3.

    CAS  Article  Google Scholar 

  5. 5.

    Chan JF, Zhang AJ, Yuan S, Poon VK, Chan CC, Lee AC, Chan WM, Fan Z, Tsoi HW, Wen L, Liang R. Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin Infect Dis. 2020an Official Publication of the Infectious Diseases Society of America. 2020. https://doi.org/10.1093/cid/ciaa325

  6. 6.

    Chandrashekar A, Liu J, Martinot AJ, McMahan K, Mercado NB, Peter L, Tostanoski LH, Yu J, Maliga Z, Nekorchuk M, Busman-Sahay K. SARS-CoV-2 infection protects against rechallenge in rhesus macaques. Science. 2020;369(6505):812–7.

    CAS  Article  Google Scholar 

  7. 7.

    Corbett KS, Flynn B, Foulds KE, Francica JR, Boyoglu-Barnum S, Werner AP, Flach B, O’Connell S, Bock KW, Minai M, Nagata BM. Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates. New England J Med. 2020;. https://doi.org/10.1056/nejmoa2024671.

    Article  Google Scholar 

  8. 8.

    Cui J, Li F, Shi ZL. Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol. 2019;17(3):181–92.

    CAS  Article  Google Scholar 

  9. 9.

    Deng W, Bao L, Liu J, Xiao C, Liu J, Xue J, Lv Q, Qi F, Gao H, Yu P, Xu Y. Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques. Science. 2020;369(6505):818–23.

    CAS  Article  Google Scholar 

  10. 10.

    Dinnon KH, Leist SR, Schäfer A, Edwards CE, Martinez DR, Montgomery SA, West A, Yount BL, Hou YJ, Adams LE, Gully KL. A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures. Nature. 2020;27:1–9.

    Google Scholar 

  11. 11.

    Duffy S. Why are RNA virus mutation rates so damn high? PLoS Biol. 2018;16(8):e3000003.

    Article  Google Scholar 

  12. 12.

    Frieman MB, Chen J, Morrison TE, Whitmore A, Funkhouser W, Ward JM, Lamirande EW, Roberts A, Heise M, Subbarao K, Baric RS. SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism. PLoSPathog. 2010;6(4):e1000849.

    Google Scholar 

  13. 13.

    Gao Q, Bao L, Mao H, Wang L, Xu K, Yang M, Li Y, Zhu L, Wang N, Lv Z, Gao H. Development of an inactivated vaccine candidate for SARS-CoV-2. Science. 2020;369(6499):77–81.

    CAS  Article  Google Scholar 

  14. 14.

    Gu H, Chen Q, Yang G, He L, Fan H, Deng YQ, Wang Y, Teng Y, Zhao Z, Cui Y, Li Y. Adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy. Science. 2020;369(6511):1603–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Hassan AO, Case JB, Winkler ES, Thackray LB, Kafai NM, Bailey AL, McCune BT, Fox JM, Chen RE, Alsoussi WB, Turner JS. A SARS-CoV-2 infection model in mice demonstrates protection by neutralizing antibodies. Cell. 2020;182(3):744–53.

    CAS  Article  Google Scholar 

  16. 16.

    Herati RS, Wherry EJ. What is the predictive value of animal models for vaccine efficacy in humans? Consideration of strategies to improve the value of animal models. Cold Spring Harb Perspect Biol. 2018;10(4):a031583. https://doi.org/10.1101/cshperspect.a031583.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, Yang P, Sarao R, Wada T, Leong-Poi H, Crackower MA. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature. 2005;436(7047):112–6.

    CAS  Article  Google Scholar 

  18. 18.

    Iwata-Yoshikawa N, Okamura T, Shimizu Y, Hasegawa H, Takeda M, Nagata N. TMPRSS2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection. J Virol. 2019;93(6):e01815–8. https://doi.org/10.1128/JVI.01815-18.PMID:30626688;PMCID:PMC6401451.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Jiang RD, Liu MQ, Chen Y, Shan C, Zhou YW, Shen XR, Li Q, Zhang L, Zhu Y, Si HR, Wang Q. Pathogenesis of SARS-CoV-2 in transgenic mice expressing human angiotensin-converting enzyme 2. Cell. 2020;182(1):50–8.

    CAS  Article  Google Scholar 

  20. 20.

    Kim YI, Kim SG, Kim SM, Kim EH, Park SJ, Yu KM, Chang JH, Kim EJ, Lee S, Casel MA, Um J. Infection and rapid transmission of SARS-CoV-2 in ferrets. Cell Host Microb. 2020;27(5):704–709.e2.

    CAS  Article  Google Scholar 

  21. 21.

    Le Bras A. SARS-CoV-2 causes COVID-19-like disease in cynomolgus macaques. Lab Anim. 2020;49(6):174. https://doi.org/10.1038/s41684-020-0571-8.

    Article  Google Scholar 

  22. 22.

    Li K, Mc Cray PB Jr. Development of a Mouse-Adapted MERS Coronavirus. Methods Mol Biol. 2020;2099:161–71. https://doi.org/10.1007/978-1-0716-0211-9_13.

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Lu S, Zhao Y, Yu W, Yang Y, Gao J, Wang J, Kuang D, Yang M, Yang J, Ma C, Xu J. Comparison of nonhuman primates identified the suitable model for COVID-19. Signal Trans Target Ther. 2020;5(1):1–9.

    Article  Google Scholar 

  24. 24.

    McCray PB, Pewe L, Wohlford-Lenane C, Hickey M, Manzel L, Shi L, Netland J, Jia HP, Halabi C, Sigmund CD, Meyerholz DK. Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus. J Virol. 2007;81(2):813–21.

    CAS  Article  Google Scholar 

  25. 25.

    Munster VJ, Feldmann F, Williamson BN, Van Doremalen N, Pérez-Pérez L, Schulz J, Meade-White K, Okumura A, Callison J, Brumbaugh B, Avanzato VA. Respiratory disease and virus shedding in rhesus macaques inoculated with SARS-CoV-2. BioRxiv. 2020.

  26. 26.

    Richard M, Kok A, de Meulder D, Bestebroer TM, Lamers MM, Okba NMA, Fentener van Vlissingen M, Rockx B, Haagmans BL, Koopmans MPG. SARS-CoV-2 is transmitted via contact and via the air between ferrets. Nat Commun. 2020;11:3496.

    CAS  Article  Google Scholar 

  27. 27.

    Rubin C, Myers T, Stokes W, Dunham B, Harris S, Lautner B, Annelli J. Review of institute of medicine and national research council recommendations for one health initiative. Emerg Infect Dis. 2013;19(12):1913.

    Article  Google Scholar 

  28. 28.

    Sia SF, Yan L, Chin AWH, et al. Pathogenesis and transmission of SARS-CoV-2 in golden hamsters. Nature. 2020;583:834–8. https://doi.org/10.1038/s41586-020-2342-5.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Sun J, Zhuang Z, Zheng J, Li K, Wong RL, Liu D, Huang J, He J, Zhu A, Zhao J, Li X. Generation of a broadly useful model for COVID-19 pathogenesis, vaccination, and treatment. Cell. 2020;182(3):734–43.

    CAS  Article  Google Scholar 

  30. 30.

    Sutton TC, Subbarao K. Development of animal models against emerging coronaviruses: from SARS to MERS coronavirus. Virology. 2015;1(479):247–58.

    Article  Google Scholar 

  31. 31.

    van Doremalen N, Lambe T, Spencer A, Belij-Rammerstorfer S, Purushotham JN, Port JR, Avanzato VA, Bushmaker T, Flaxman A, Ulaszewska M, Feldmann F. ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. Nature. 2020;30:1–8.

    Google Scholar 

  32. 32.

    Wang J, Shuai L, Wang C, Liu R, He X, Zhang X, Sun Z, Shan D, Ge J, Wang X, Hua R. Mouse-adapted SARS-CoV-2 replicates efficiently in the upper and lower respiratory tract of BALB/c and C57BL/6 J mice. Protein Cell. 2020;4:1–7.

    Article  Google Scholar 

  33. 33.

    Williamson BN, Feldmann F, Schwarz B, Meade-White K, Porter DP, Schulz J, Van Doremalen N, Leighton I, Yinda CK, Pérez-Pérez L, Okumura A. Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2. BioRxiv. 2020.

  34. 34.

    Yang XH, Deng W, Tong Z, Liu YX, Zhang LF, Zhu H, Gao H, Huang L, Liu YL, Ma CM, Xu YF. Mice transgenic for human angiotensin-converting enzyme 2 provide a model for SARS coronavirus infection. Comp Med. 2007;57(5):450–9.

    CAS  PubMed  Google Scholar 

  35. 35.

    Ye ZW, Yuan S, Yuen KS, Fung SY, Chan CP, Jin DY. Zoonotic origins of human coronaviruses. Int J Biol Sci. 2020;16(10):1686.

    CAS  Article  Google Scholar 

  36. 36.

    Yoshikawa N, Yoshikawa T, Hill T, Huang C, Watts DM, Makino S, Milligan G, Chan T, Peters CJ, Tseng CT. Differential virological and immunological outcome of severe acute respiratory syndrome coronavirus infection in susceptible and resistant transgenic mice expressing human angiotensin-converting enzyme 2. J Virol. 2009;83(11):5451–65.

    CAS  Article  Google Scholar 

  37. 37.

    Yu J, Tostanoski LH, Peter L, Mercado NB, McMahan K, Mahrokhian SH, Nkolola JP, Liu J, Li Z, Chandrashekar A, Martinez DR. DNA vaccine protection against SARS-CoV-2 in rhesus macaques. Science. 2020;369(6505):806–11.

    CAS  Article  Google Scholar 

  38. 38.

    Yu P, Qi F, Xu Y, Li F, Liu P, Liu J, Bao L, Deng W, Gao H, Xiang Z, Xiao C. Age-related rhesus macaque models of COVID-19. Anim Models Exp Med. 2020;3(1):93–7.

    Article  Google Scholar 

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Kumar, S., Yadav, P.K., Srinivasan, R. et al. Selection of animal models for COVID-19 research. VirusDis. 31, 453–458 (2020). https://doi.org/10.1007/s13337-020-00637-4

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Keywords

  • Animal models
  • SARS-CoV2
  • Macaques
  • Transgenic mice
  • Immunogenicity
  • Antiviral
  • Vaccines