Animal models of mechanisms of SARS‐CoV‐2 infection and COVID‐19 pathology

The coronavirus disease 2019 (COVID‐19) pandemic caused by SARS‐CoV‐2 infections has led to a substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS‐CoV‐2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID‐19. At the time of writing, in vivo studies of SARS‐CoV‐2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partly model the mechanisms involved in human severe COVID‐19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID‐19. These models need to reproduce the key mechanisms of COVID‐19 severe acute respiratory distress syndrome and the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS‐CoV‐2 infection and COVID‐19‐related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID‐19 pathogenesis and for assessing potential treatments. Linked Articles This article is part of a themed issue on The Pharmacology of COVID‐19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc

The coronavirus disease 2019  pandemic caused by SARS-CoV-2 infections has led to a substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS-CoV-2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID-19. At the time of writing, in vivo studies of SARS-CoV-2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partly model the mechanisms involved in human severe COVID-19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID-19. These models need to reproduce the key mechanisms of COVID-19 severe acute respiratory distress syndrome and the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS-CoV-2 infection and COVID-19-related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID-19 pathogenesis and for assessing potential treatments. essential information on route of administration, pharmacokinetics, and pharmacodynamics and to identify key mechanisms driving pathology in vivo. The pandemic of coronavirus disease 2019 , the disease manifestation of SARS-CoV-2 infection, has led to the rapid development of animal models of SARS-CoV-2 infection which have already provided insights into the natural history of the disease and, together with data from previous studies, have permitted identification of potential antiviral approaches identified in studies of other viral infections (Alexander et al., 2020).
Notably, at the time of writing (late April 2020), studies using animal models have already provided some evidence that immunity might confer protection from reinfection (Bao et al., 2020) and that convalescent plasma might decrease viral burden .
Animal studies have also confirmed the susceptibility of domestic cats to infection  and have provided evidence of the importance of ACE2 in enabling infection (Bao et al., 2020).
These animal models are well suited for proof of concept studies into the efficacy of potential vaccines or antivirals. However, each model system has its drawbacks, and at the time of writing (April 2020), no reported animal model of SARS-CoV-2 infection fully reproduces every key feature of severe COVID-19 (Table 1 and Figure 1). This deficit in preclinical modelling is important as, until vaccines are widely available, there is a particularly urgent need to identify potential treatments for patients who already have established SARS-CoV-2 infection and who are at risk of progressing to severe COVID-19 requiring hospitalisation and the need for respiratory support in a high dependency or intensive care unit. The ideal evidence from preclinical efficacy studies of potential therapeutic interventions to prevent or promote resolution of severe COVID-19 in patients who are already symptomatic will be meaningful improvements in clinically relevant endpoints in models which deterministically progress to severe disease following infection with SARS-CoV-2. Interventions should also be applied at realistic time points in these models ( Figure 2). This ideal standard of evidence will of course have to be balanced with practical and ethical considerations (Table 2). SARS-CoV-2 has zoonotic origins (Andersen, Rambaut, Lipkin, Holmes, & Garry, 2020), but several major factors complicate the study of this virus in model organisms. These include lack of infectivity of clinical isolates of SARS-CoV-2 in some model species including mice and dogs (Bao, Deng, Huang, et al., 2020;Shi et al., 2020) and an absence of the persistent infection, immunopathology, severe acute respiratory distress syndrome, and systemic complications which characterise COVID-19 clinically. Furthermore, SARS-CoV-2 predominately causes severe COVID-19 in older people with co-morbidities , presenting a construct validity problem with attempted use of young and immunologically naïve laboratory animals in COVID-19 testing.
Scientists developing animal models therefore face a challenging set of trade-offs as well as a sense of urgency. Despite the pressing needs for treatments, design and implementation of preclinical studies for COVID-19 should maintain quality to produce meaningful results, avoid needless duplication, and avoid undue reduction in consideration of animal welfare issues (London & Kimmelman, 2020).
Here, we review the mechanisms involved in COVID-19 development and reported approaches to model SARS-CoV-2 infection responses.
We then identify key areas in which studies using animal models might improve the reproduction of important characteristics of human COVID-19 to better help in the identification and assessment of new therapeutic interventions.
2 | BACKGROUND ON SARS-COV-2 AND COVID-19 New modelling approaches are needed to improve our understanding of SARS-CoV-2 and COVID-19 because the virus has only recently infected humans and because aspects of the disease are different to previously described syndromes developing as a result of viral infections including SARS. Fortunately, clinical and preclinical data related to the COVID-19 pandemic have been disseminated at a neverbefore-seen rate.
SARS-CoV-2 is a positive-sense single-stranded RNA coronavirus thought to be the descendant from a bat coronavirus which spilled over to infecting humans after infecting an intermediate host, potentially a pangolin (Andersen et al., 2020;. The genome of SARS-CoV-2 is 79% homologous with that of SARS-CoV which caused severe acute respiratory syndrome (SARS) outbreaks in 2003 (Lu et al., 2020). Studies indicate that, as with SARS-CoV, the Spike glycoprotein expressed by SARS-CoV-2 undergoes activating cleavage by host proteases on epithelial surfaces which permits high affinity interaction of Spike with host epithelial ACE2 (Bao, Deng, Huang, et al., 2020;Hoffmann et al., 2020). When it is bound by Spike, cleavage of ACE2 by further host proteases such as TMPRSS2 allows viral entry into host epithelial cells for viral replication (Hoffmann et al., 2020). Damas et al. (2020) assessed ACE2 polymorphism and similarity to human ACE2 in 410 vertebrates, including 252 mammals. Based on conservation of 25 amino acids that are essential for interaction between ACE2 and SARS-CoV-2, a scoring system that predicts the likelihood of infectivity was devised.
Although experimental data would be required to substantiate the merit of this system, this study does provide a rationale for choice of animal species to study the infectivity, pathogenesis, and treatment of COVID-19 and suggested that species such as Old World monkeys would be highly susceptible to infection, while most rodents are less likely to be infected. SARS-CoV-2 and SARS-CoV exhibit similar stability outside of the body (van Doremalen et al., 2020) but differ in their incubation and transmission kinetics. Compared with SARS-CoV, SARS-CoV-2 appears to replicate more rapidly in the upper respiratory tract (Zou et al., 2020), where it achieves transmission in asymptomatic carriers , a factor which is likely to contribute to the rapid global spread of COVID-19.
The reported clinical symptoms of COVID-19 range from mild to critical . Mild disease varies from no symptoms to mild pneumonia. Severe disease is characterised by moderate to severe pneumonia. Critical COVID-19 involves diagnosis of acute respiratory distress syndrome (ARDS), septic shock, and/or multi-organ failure ( McGoogan, 2020). Risk factors including advanced age, male sex, obesity, diabetes, and immunodeficiency predispose towards development of severe or critical COVID-19. SARS-CoV-2 appears to be less likely to cause severe disease than SARS-CoV (Ruan, 2020), a factor which is likely also to allow a greater spread of SARS-CoV-2 and may also present an additional challenge for development of animal models.
Pathology of COVID-19 is centred around immunopathology with persistent lung infection leading to ARDS, and clinical progression follows a time course characteristic of a dysregulated viral immune response (Gattinoni, Coppola, Cressoni, Busana, & Chiumello, 2020;Tian et al., 2020; (Figure 1). It is clear, however, that critical cases of COVID-19 involve damage to other systems in the body which may both be a result of ARDS and may also be causative of greater lung injury. Notably, COVID-19 has been associated with a coagulopathy driven by inflammation and characterised by elevated fibrinogen and Ddimer levels indicating increased thrombin generation and fibrinolysis . Patients suffer from increased thrombotic risk against which standard prophylactic anticoagulants appear only partly effective (Llitjos et al., 2020), and there is evidence that platelets, autoantibodies and neutrophil extracellular traps (NETs) may be involved in the pathogenesis (Barnes et al., 2020;Bikdeli et al., 2020;Zuo et al., 2020).
The ARDS presentation within COVID-19 is heterogenous and may involve both gas exchange and perfusion abnormalities  (Bao, Deng, Gao, et al., 2020;Munster et al., 2020;Yu et al., 2020) and cynomolgus macaques (Rockx et al., 2020). For studies of SARS-CoV, African green monkeys, common marmosets, squirrel monkeys, and moustached tamarins have also been used (Gong & Bao, 2018). Although nonhuman primate models can resemble human systems more closely than models which use more phylogenetically distant animals, it is important to note that these studies frequently use limited numbers of animals in their experiments (as low as one or two animals per group), and so their results should be interpreted with due caution (Curtis et al., 2018). Investigators using these intelligent animals do however have limited numbers of non-human primates available for terminal proof of concept pathology studies, especially as many of these animals will soon be needed for vaccine tests, and so experimental designers are forced to make trade-offs between group sizes in early pathology studies and those in later preclinical trials of therapeutics.
The most convincing demonstration of an animal model of COVID-19 has been reported by Munster et al. (2020). Rhesus macaques were inoculated intranasally, intratracheally, by mouth, and onto both eyes.
Pulmonary infiltrates were observed radiologically, and oedema was measured gravimetrically, although superficial inspection found lesions to be focal and sporadic. Alveolar flooding with the presence of hyaline membranes, which are rarely seen in other animal models of ARDS (Matute-Bello et al., 2011), were observed in lung histology. This was accompanied by an irregular breathing pattern and increased respiratory rate in some animals suggestive of hypoxaemia, although this readout may also be a marker of pain or distress. Together these results suggest some degree of ARDS development. However, gas exchange impairment was not measured, and serum cytokine analysis detected no consistent evidence of systemic inflammation.
As age is a major risk factor for severe COVID-19, the response of aged rhesus macaques (15 years old) to intratracheal inoculation with SARS-CoV-2 has also been compared with that of younger controls (3-5 years old) . This report suggests that there are age-related increases in viral load 7 days after inoculation. Radiology and histology were indicative of mild interstitial infiltrates in younger animals with signs of more severe oedema including alveolar flooding in aged macaques. Although preliminary and largely qualitative, this study suggests that aged macaque models may be useful for modelling more severe disease.
Rhesus macaques have also been used to test whether seroconversion provides protective immunity against SARS-CoV-2. In one reported study, two animals were inoculated intratracheally with SARS-CoV-2 and then challenged again 28 days later (Bao, Deng, Gao, et al., 2020). Lack of viral shedding after re-challenge in both macaques suggested development of protective immunity. This study should not be overinterpreted, however, as other studies suggests low, or even undetectable, titres of neutralising antibodies in previously infected patients ,

| Ferrets
Ferrets are useful in both studies of viral transmission and the pharmacology of the most frequently reported symptom of SARS-CoV-2 infection as, unlike mice and rats, they exhibit a cough reflex. There are also ferret models used to study cystic fibrosis (Sun et al., 2010).
Ferret studies also have potential veterinary and zoonotic relevance as an outbreak of SARS-CoV-2 infections with respiratory symptoms has been reported in minks, related to ferrets within the Mustilidae family, in two farms in the Netherlands (Dutch Parliament report, 2020).
Following inoculation with SARS-CoV-2, ferrets have been shown to develop symptoms similar to those described in human COVID-19, namely, elevated body temperature suggestive of pyresis, reduced activity and appetite, and coughing between 2 and 12 days post infection (Kim et al., 2020;Shi et al., 2020). Histologically, SARS-CoV-2 infected ferret lungs have exhibited severe pulmonary lymphoplasmacytic perivasculitis and vasculitis at 13 days post infection .
Transmission of SARS-CoV-2 was studied by placing naïve ferrets in direct or indirect contact with inoculated ferrets (Kim et al., 2020).
It was observed that all naïve ferrets placed in direct contact with

| Cats
Although cats are not widely used to study respiratory diseases, the close association of humans and domestic cats means that investiga-

| Mice
Mice are widely used in studies of immunology and lung injury and have a highly characterised immune system, rapid breeding cycle, and can be used with a range of readily available research tools. Importantly, the size and rapid breeding and growth of mice is useful for accelerating the completion of studies with group numbers high enough for statistical testing of efficacy of potential interventions.
However, the Spike proteins of SARS-CoV-2, and of its relative, SARS-CoV, are thought to have insufficient affinity for the murine ACE2 entry receptor for infection of mice (Wan, Shang, Graham, Baric, & Li, 2020). Clinical isolates of SARS-CoV have therefore been adapted by serial passage in the respiratory tract of mice to produce related viruses (MA15 and v2163) that can cause lung injury and mortality in wild-type mice (Day et al., 2009;Roberts et al., 2007) Initial studies preprints show that SARS-CoV-2 can also infect another strain of mice expressing hACE2 driven by the mouse Ace2 promoter in a transgene-dependent manner (Bao, Deng, Huang, et al., 2020;Yang, Deng, et al. 2007). In these mice, inoculation with SARS-CoV-2 at 10 5 TCID 50 caused weight loss, antibody responses, and both superficial and histological evidence of lung inflammation in a hACE2 transgene-dependent manner, although lung injury was limited and not quantified (Bao, Deng, Huang, et al., 2020). In contrast to previous SARS-CoV experiments, however, no mortality was reported with SARS-CoV-2 infection in hACE2 mice, perhaps related to the lower virulence of SARS-CoV-2 observed in humans, although only one dose of virus was studied. Further adaptations of the experimental approach to infecting transgenic mice may be required to study lethal lung injury resulting from SARS-CoV-2 infection.
Another issue for further studies is whether the tissue distribution and surface expression levels of hACE2 in hACE2 mice fully replicate those in humans, as murine ACE2 expression appears to be highly localised to bronchial epithelium in mice (Sodhi et al., 2019;Sun, Gu, Ma, & Duan, 2020), with ACE2 perhaps more generally distributed in human lungs (Hamming et al., 2004) and when transgenic hACE2 expression is driven by the mouse ACE2 promoter in mouse lungs (Bao, Deng, Huang, et al., 2020). There are also species differences in distribution of ACE2 expression outside the lungs which may have implications for systemic responses to SARS-CoV-2 infection.
Additionally, ACE2 down-regulation may play a role in disease progression (Kuba et al., 2005), and hACE2 have both human and non-Spike reactive murine ACE2 and so may be resistant to ACE2 down-regulation related complications. The targeted insertion of human ACE2 into the endogenous mouse locus may therefore be useful in generating a better model of severe COVID-19.

| Other species
SARS-CoV-2 probably originated from a coronavirus infecting bats (Andersen et al., 2020;. The route or method used for viral inoculation can affect the severity of viral disease models, and a range of inoculation routes have been used in reported animal studies (Table 1). Intranasal delivery has been widely used as this delivery method is simple, some inoculum can be aspirated into airways, and because the intranasal route is potentially reflective of the real-world route of entry for viral droplets. However, this method can lead to limited and focal delivery of inoculum to lungs (Su, Looney, Robriquet, Fang, & Matthay, 2004), and viral diseases affecting the lower respiratory tract such as COVID-19 may result from inhalation of aerosols which can enter deeper into the lungs or from widespread deposition of virus in lungs due to high upper respiratory tract viral load and mucociliary escalator disruption.
Aerosolised delivery of viral inoculum may therefore be useful in the induction of widespread lung injury which might be sufficient to provoke severe disease, as well as in the standardisation of the dose of inoculum that reaches the lower airways and respiratory lungs. Evidence that aerosolised delivery can provoke injury resembling severe ARDS with elevated cytokine release has been produced in cynomolgus macaque models of H5N1 influenza (Wonderlich et al., 2017). There have also been recent developments in the quantitative delivery of aerosols and powders to lungs of laboratory animals (Lexmond, Keir, Terakosolphan, Page, & Forbes, 2018). Aerosolised or intratracheal administration approaches for controlled delivery of inoculum may be worthy of consideration in animal models for more severe COVID-19.

| Demonstration of ARDS-like lung pathology in animals
Histological analysis, superficial visual inspection, and radiological imaging have been the predominant approaches used to assess the development of lung inflammation and injury following experimental SARS-CoV-2 infection. However, these pathological observations have so far often been reported in only some animals under study and only some regions of the lungs of those animals (Table 1). This is likely to be due to probabi-  (Cascella, Rajnik, Cuomo, Dulebohn, & Di Napoli, 2020). These points are reminders that some degree of hypoxaemia and pneumonia is indicative of some disease but not conclusive evidence of progression to severe COVID-19-like ARDS.
Where possible, serial non-invasive imaging approaches as reported in some macaque studies (Table 1) are also useful for tracking the time course of pulmonary oedema development. It is notable that some patients with COVID-19 ARDS have more limited oedema with severe hypoxaemia (Gattinoni et al., 2020), and so additional measurements of lung perfusion (pulmonary dead space fraction or ventilation perfusion scans) in animals may also be useful for better understanding of COVID-19 as disease sub-phenotypes may be also be present within animal models (Carla et al., 2020).

Planning of terminal measurements should involve consideration
of how the maximum number of high-value measurements can be made. If inoculations can affect the lungs widely and relatively evenly, this can involve the use of different lobes for different potentially confounding measurements. For example, bronchoalveolar lavage is useful for simultaneous measurements of viruses, inflammatory cells, inflammatory mediators, and protein deposits in the bronchoalveolar spaces but causes artefacts in histological analysis and prevents later measurement of pulmonary oedema. Likewise, formalin inflation is useful for histological analysis which can be performed outside BSL3 conditions but prevents the most direct and quantitative methods of measurement of viral load in lungs and pulmonary oedema. Lobes of lungs can therefore be sequentially tied off and sampled to permit functional and histological measurements related to ARDS as well as viral load in lungs. Experimental designs will of course require appropriate tradeoffs between study complexity and welfare and safety of animals and laboratory workers.

| Non-BSL3 approaches for modelling mechanisms relevant to COVID-19
The serious and potentially lethal nature of COVID-19 means that BSL3 laboratories are required for experiments using SARS-CoV-2.
However, insights into mechanisms driving COVID-19 may still be derived from non-infectious models.
One example directly related to SARS-CoV-2 is the effects of absence of ACE2 function on enhancement of the pulmonary oedema response, which were established using a mouse acid inhalation model which to provoke rapid and severe pulmonary oedema (Kuba et al., 2005). This finding has led to ongoing trials to assess whether administration of recombinant ACE2 may address infection-related deficiencies in lung barrier protective ACE2-derived Ang 1-7 generation from angiotensin II in addition to its potential action as a decoy preventing viral entry into cells (Sriram & Insel, 2020).
Some urgently needed studies of putative therapeutic agents for COVID-19, particularly those targeting host responses, may therefore be possible or supportive using other lung injury models with higher throughput. Influenza viruses have been adapted to infect mouse although it is likely that in many cases efficacy studies with SARS-CoV-2 infection will also be needed.

| Incorporating known COVID-19 risk factors into models
Known risk factors for severe COVID-19 offer opportunities to drive models towards pathological states (Figure 1). Advanced age, obesity, and diabetes are additional risk factors associated with high casefatality rates COVID-19 (Onder, Rezza, & Brusaferro, 2020;Petrilli et al., 2020). These features may predispose towards severe disease through complicating medical treatment, by hypoventilation as a result of low cardiorespiratory fitness, and through dysregulated immunity with imbalance of inflammation and repair mechanisms, as well as a propensity for underlying cardiovascular disease and changes to haemostasis that will influence progression of respiratory infection towards ARDS (Sattar, McInnes, & McMurray, 2020;Tzoran, Hoffman, & Monreal, 2018).
The use of aged animals might allow for modelling of the age risk factor. It is encouraging in this light that early development of preclinical models of COVID-19 includes the influence of age in rhesus macaques. Infection with SARS-CoV-2 reportedly led to heightened lung pathology and viral replication in older macaques (15 years old) compared to younger animals (5 years old) . Additionally, aged mice have previously demonstrated exacerbated inflammation and lung injury following SARS-CoV infection (Rockx et al., 2009).
Animal models which require ageing over years may not permit the rapid testing of treatments which is required for the COVID-19 pandemic. Models incorporating other risk factors such as induced obesity (e.g., high fat diet-induced), diabetes (e.g., leptin receptor deficiency; Paul, Queen, Page, & Ferro, 2007), or impaired immune responses (e.g., STAT1 knockout/inhibition; Frieman et al., 2010) might be made available more quickly. Additionally, COVID-19 may progress to severe disease status as a result of immune priming, secondary infections, or intravascular sequelae, and so combining SARS-CoV-2 infection models with a first or second hit such as immune priming with LPS or additional challenges such as the non-BSL3 approaches to cause lung injury described above (i.e., inoculation with pathogenic bacteria or injections of injurious antibodies) might be capable of driving host responses towards a pathological state.

| Platelet responses, coagulopathy, and hyperinflammation
As discussed above, severe COVID-19 is also associated with low blood platelet counts and thrombosis, a consumptive coagulopathy, and a hyperinflammatory state involving platelet activation, release of NETs, and prolonged systemic elevations of cytokines such as IL-6 and CXCL10. These pathological features associated with severe disease have not yet been demonstrated in animal models (Table 1).
The degree of thrombocytopaenia has been reported as a potential biomarker for severe COVID-19 (Lippi, Plebani, & Henry, 2020). A large retrospective study revealed the degree of thrombocytopaenia to be dynamic after presentation to clinic, with decreasing platelet numbers being predictive of mortality later on . Furthermore, a shift in platelet-lymphocyte ratio may indicate the occurrence of an acute inflammatory or thrombotic event and therefore have prognostic value (Qu et al., 2020). Thus, thrombocytopaenia may reflect (a) alteration in thrombopoiesis due to the bone marrow or lungs (and potentially spleen) being inflamed or receiving inflammatory and trauma-related thrombopoietic cues; (b) localised lung recruitment of platelets as a facet of their role in the immune response or alveolar coagulation; (c) disseminated intravascular coagulation (DIC) throughout the body (Xu, Zhou, & Xu, 2020); or (d) platelet-viral interaction, although this remains hypothetical as an engagement of platelets with SARS-CoV-2 has not been described (Amgalan & Othman, 2020 Results of studies in mice modelling influenza agree on the necessity for platelets in the immune response and inflammation but conflict as to whether this is beneficial (Campbell et al., 2019;Guo et al., 2017), or detrimental (Boilard et al., 2014;Lê et al., 2015).  (Amison et al., 2018) suggest that the induction of severe experimental thrombocytopenia (>95% depletion) in animal models of SARS-CoV-2 may allow these infection models to demonstrate the progression from a mild to a severe disease phenotype. Additionally, pulmonary platelet The use of these antibodies combined with infection models might be useful in modelling the contributions of platelets and NETs to severe COVID-19 associated with lung hypoperfusion, release of NETs, and autoantibody production (Zhang, Xiao, et al., 2020). The involvement of FcγRIIA in viral platelet responses suggests that transgenic expression of human FcγRIIA on platelets may be useful in driving greater platelet responses in murine SARS-CoV-2 infection models, as mice lack activating Fcγ receptors on platelets (Boilard et al., 2014).
Although heparin is classically used as an anticoagulant, it has also been demonstrated to bind and reduce the activity of a range of cytokines implicated in the COVID-19-associated cytokine storm (Mulloy, Hogwood, Gray, Lever, & Page, 2015) and In addition to platelet number, the severity of COVID-19 disease progression also correlates closely with levels of the pro-inflammatory mediators such as CXCL10 (IFN-inducible protein 10) and TNFα, both associated with the pathology and progression associated with hyperinflammatory condition associated with ARDS (Bautista et al., 2013;Tang et al., 2020;Yang et al., 2020). The amelioration of LPS induced lung inflammation following CXCL10 neutralisation in rats, and the proposition of anti-TNFα therapy in COVID-19 patients, therefore highlights the potential for combining SARS-CoV-2 infections with TNFα and CXCL10 challenge as a further method in the development of models with a more severe phenotype. Lang et al., 2017). Dysregulated immune responses by myeloid leukocytes may also be important in pathogenesis of severe disease (Barnes et al., 2020).

Persistence of infection and inflammation due to ineffective
Although the impact of COVID-19 on cystic fibrosis patients is still unclear (Colombo et al., 2020), mouse models of cystic fibrosis mutations involve heightened platelet activation and a hyperinflammatory state with increased neutrophil responses, so these models might be useful in driving increased severity of pathology (Ortiz-Muñoz et al., 2020).

| Time course of interventions relative to disease progression
Efficacy studies using animal models of many disease processes have been criticised for over-use of the prophylactic application of interventions (i.e., pretreatment) when such approaches would not be realistic for clinical treatments (Denayer, Stöhrn, & Van Roy, 2014).
Severe COVID-19 has a characteristic time course which suggests that there are several windows where different treatment approaches might be useful   (Figure 2).
The users of animal models for COVID-19 research should wherever possible assess the time course of readouts in their models and consider the type of therapy being assessed, the potential of risk to patients from any anticipated adverse effects, and whether the intervention is likely to need to take place in a hospital setting. For example, as viral replication increases, likelihood of transmission and persistent infection mediates severe disease, thus antivirals with low toxicity might be useful for all diagnosed cases and perhaps even prophylactically in high-risk populations. Some interventions such as nebulised heparin may be of benefit as antiviral agents (Mycroft-West et al., 2020) and in reducing the inflammatory response (Dixon et al., 2010;Mulloy et al., 2015), and they may need to be dosed at different time points across the development of COVID-19. Once severe disease develops, interventions which are aimed at resolving ARDS and systemic complications would be useful, and robust preclinical testing might require models which include aspects of intensive care support such as invasive ventilation (Alexander et al., 2020). Careful model characterisation will be required to identify analogous phases of pathology development in animal models for appropriate timing of interventions under study.

| CONCLUSION
Animal models have been rapidly mobilised to address the need for greater understanding of COVID-19 and for testing of new therapeutic approaches to this pandemic. While many useful observations have already been shared, it is important that animal studies are adapted to measure and report key readouts that are relevant to clinical COVID-19 such as evidence of lung injury resembling ARDS, physiological gas exchange impairment, hyperinflammation, platelet responses, and coagulopathy. Study designs may need to incorporate additional risk factors or disease triggers to shift from modelling protective and selfresolving infection and inflammation to replicating severe disease with ARDS and multi-organ failure ( Table 3). Development of models that reproduce these hallmarks of disease will permit better understanding of COVID-19 pathogenesis and facilitate improved efficacy testing of desperately needed drugs and other therapeutic actions.
T A B L E 3 Limitations of animal models of COVID-19 and potential opportunities for model development