3.3 Ferret Model:
Ferrets have been used mainly in respiratory disease related studies because their lungs share a lot of similarity to that of humans (Vinegar, Sinnett, Kosch, & Miller, 1985). Ferrets are also a popular animal model because they can mimic clinical symptoms of the SARS-CoVs such as coughing and fever. They have been previously used in Influenza (Lee et al., 2020) and Swine Influenza virus studies (van den Brand et al., 2010). Ferrets are permissive to SARS-CoV infection (Martina et al., 2003; Weingartl et al., 2004) and may be a potential model for the study of the SARS-CoV-2. Like mice models, ferrets were shown to support SARS-CoV replication with varying degrees of clinical signs but without significant mortality. When ferrets were infected with SARS-CoV at a high dose, they showed productive infection in the lungs, trachea, and nasal turbinates (Martina et al., 2003). Viral replication peaked in the lungs on days 5 or 6 as seen in humans, but another study using the same conditions failed to produce infection and mortality (Weingartl et al., 2004).
Ferrets may be a suitable model to study SARS-CoV-2 pathogenesis and human-human transmission (Y. I. Kim et al., 2020; J. Shi et al., 2020).Shi et.al tested the susceptibility of ferrets to SARS-CoV-2 by infecting pairs of ferrets by two viral strains; SARS-CoV-2/F13/environment/2020/Wuhan (F13-E) or SARS-CoV-2/CTan/human/2020/Wuhan (CTan-H) intranasally with 105 plaque-forming units. After four days of inoculation, the animals were euthanized and tissues from the nasal turbinate, soft palate, tonsils, trachea, lung, heart, liver, spleen, kidneys, pancreas, small intestine, and brain were collected. Viral RNA was detected in areas of the upper respiratory tract- nasal turbinate, soft palate and tonsils (J. Shi et al., 2020). Another study from Korea showed that when infected intranasally with NMC-nCoV02, a strain isolated from a COVID-19 patient in South Korea, showed clinical signs two days after infection (Y. I. Kim et al., 2020). Although they showed an increment in body temperature, lethargy and occasional coughs all animals recovered day 8 post-infection (Table 1) . In follow-up studies, naïve ferrets were exposed to the infected ones to study the transmission dynamics. Interestingly, nasal swabs and saliva samples of the exposed ferrets showed high viral loads. In support of this, another study from the Netherlands demonstrated aerosol mediated transmission of SARS-CoV-2 from infected to naive exposed ferrets (Mathilde Richard, 2020) (Table 1) . These studies also showed that the pathogenicity of the virus was the same in the inoculated and later exposed ferrets. These studies showed the rapidity of virus transmission and the requirement of the appropriate distancing to avoid getting infected.
Ferrets are permissive for SARS-CoV-2 infection, but they are unable to produce clinical signs and symptoms with the same degree of severity as seen in humans. Other difficulties in working with ferrets are their larger size compared to mice and hamsters, difficulty in handling, expensive and require reagents that are unique to them. Nonetheless, ferrets can reproduce the virus dynamics of infection and transmission pattern (Table 2). The respiratory illness seen in ferrets resemble humans because their lungs are proportionately larger compared to other organs in their body. Moreover, their lungs abundantly express ACE2 protein similar to human lungs, which make them excellent models to study the early events in SARS-CoV-2 attachment and entry into host cells (PETERS). Further, ferrets can cough and sneeze and in doing so transmit the disease to nearby ferrets via aerosol /droplets, which makes them useful in transmission studies. Furthermore, their immune systems share a lot of similarities with the human, thereby making them potential animal models for testing vaccines, therapeutics and antivirals against SARS-CoV-2. Finally, the fact that ferrets are long-lived animals as makes them well suited to study the impact of aging on COVID-19 pathogenesis.
3.4. Non-Human Primate (NHP) Models
Several species of NHP’s have been used in SARS-CoV and MERS-CoV studies. This includes old world monkeys such as rhesus macaques, cynomolgus macaques, and African Green monkeys, as well as new world monkeys that include the common marmosets, squirrel monkeys, and mustached tamarins (Lawler et al., 2006; McAuliffe et al., 2004; Qin et al., 2005). Squirrel monkeys and mustached tamarins are not permissive to SARS-CoV (Roberts & Subbarao, 2006). MERS-CoV can replicate in only rhesus macaques and the common marmosets (de Wit et al., 2013; Falzarano et al., 2014; Yao et al., 2014). In a study that utilized cynomolgus macaques for SARS-CoV studies, virus was retrieved from nasal secretions and lungs samples via RT-PCR including the detection of pulmonary pneumonia, which which resembled the human disease (Lawler et al., 2006). African Green monkeys, cynomolgus macaques, and rhesus macaques infected with SARS-CoV Urbani strain did not develop clinical signs but viral replication was detected in nasal swabs and tracheal lavage samples (McAuliffe et al., 2004). The virus replicated to the highest titer in African Green macaques followed by cynomolgus macaques and then rhesus macaques. Infection of the common marmoset with SARS-CoV Urbani resulted in mild clinical disease with the development of fever and diarrhea (Greenough et al., 2005). High levels of viral RNA were detected in lung samples on Days 4 and 7 after infection together with the presence of both pulmonary (interstitial pneumonia) and hepatic pathology. These macaques developed significant hepatic lesions on Days 4 and 7-post infection. The marmoset was the only NHP that showed liver pathology resembling that described in humans (Greenough et al., 2005).
The rhesus macaque and common marmoset are susceptible to MERS-CoV infection and show a wide spectrum of disease manifestations (de Wit et al., 2013; Falzarano et al., 2014). When infected intratracheally with a high dose of the EMC-2012 virus strain, rhesus macaques developed mild clinical signs such as anorexia, respiratory distress, and elevated WBCs on day 1–2 post-infection. Viral RNA could not be detected in the kidney or bladder (Yao et al., 2014). Another study by Falzarano et.al. used marmosets where the infection was done via intranasal, oral and ocular routes resulting in high virus titers (Falzarano et al., 2014). The animals showed respiratory distress, fever, nausea and lethargy at 4-6 days of infection. Viral RNA was detected from the throat and nasal swabs. Out of 9 marmosets used in the study, two of them developed multiple organ failure (kidney, liver and heart). Thus, from these studies, it was suggested that NHP models could provide deeper insights into the virus-induced pathology associated with severe Human Coronavirus infections. In summary, African Green Monkeys and Rhesus macaques were identified as good models for SARS-CoV replication studies, Among the two, Rhesus macaques showed similar viral replication kinetics that were consistent with the MERS-CoV replication in humans. However, the common marmoset remains the best model to study disease severity and multiple organ failure in both SARS-CoV and MERS-CoVs.
A recent study by Bao et. al., used the nonhuman primate models with SARS-CoV-2 infection followed by a repeat challenge with the same virus to ascertain the possibility of reinfection (Linlin Bao, 2020). In this study four adult Chinese rhesus macaques were intratracheally challenged with SARS-CoV-2/WH-09/human/2020/CHN at 1×106 TCID50 via intratracheal route. Weight loss, reduced appetite, increased respiration rate, and hunched posture was observed and the viral loads from nasal and anal swabs revealed peak viremia (RNA) at 3 days post-infection (Table 1) . After 28 days, two infected monkeys were intratracheally challenged with the same dose (1×106 TCID50) of SARS-CoV-2 to verify the possibility of reinfection. Viral loads in nasopharyngeal and anal swabs tested negative after re-exposure a SARS-CoV-2. The presence of the high levels of neutralizing antibodies in infected animals revealed that they had protective antibodies. PiCoVacc, a purified inactivated SARS-CoV-2 vaccine candidate, was tested in Rhesus macaques and showed good immunogenic response (Q. Gao et al., 2020) (Table 1) . The vaccine candidate neutralized ten representative SARS-CoV-2 strains and offered complete protection in rhesus macaques. A second set of studies was performed in the rhesus macaque model to test the efficacy of a DNA vaccine candidate against SARS-CoV-2 (J. Yu et al., 2020). A total of 35 adult rhesus macaques were injected with the DNA vaccine candidates, with various constructs of SARS-CoV-2 proteins such as S (n = 4), S.dCT (n= 4), S.dTM (n = 4), S1 (n= 4), RBD (n= 4), S.dTM.PP (n = 5) and Control (n=10), intramuscularly, and after 6 weeks they were challenged with 1.1 × 104 PFU SARS-CoV-2 intratracheally and intranasally. Nasal swabs and bronchoalveolar lavage (BAL) taken from both control and vaccinated animals found lower viral RNA in the vaccinated groups compared to the control animals. Furthermore, using the same control animals Chandrashekar et.al, showed development of protective immunity in SARS-CoV-2 infected macaques when the same animals were challenged a second time with the same virus (Chandrashekar et al., 2020). In this study 9 adult rhesus macaques, divided into three groups, were inoculated with 1.1 × 106 PFU (n=3), 1.1 × 105 PFU (n = 3) PFU or 1.1 × 104 PFU (n= 3) SARS-CoV-2 USA-WA1/2020 intranasally and intratracheally. Two days post infection; viral RNA was detected in bronchoalveolar lavage (BAL) and nasal swabs with animals experiencing loss of appetite and transient lymphopenia and neutropenia. Around day 35-post infection, these animals were re-inoculated with the same dose of SARS-CoV-2 as initial challenge. Interestingly, 2 days after the second challenge, the viral RNA in BAL was more than five logs lower than that detected after the primary challenge suggesting that recovery from the primary exposure helps the development of protective immunity against secondary exposure(Table 1) . Another, research group from Montana, USA had shown infection in Rhesus Macaque in the upper and lower respiratory tract (Vincent J. Munster, 2020). In their study, Rhesus macaques were infected with SARS-CoV-2 isolate nCoV-WA1-2020 through various routes (intranasal, oral, ocular, and intratracheal routes), showed reduced appetite, hunched posture, pale appearance and dehydration(Table 1) . The viral loads were highest in the nasal swabs followed by throat and rectal swabs. Histopathology revealed multifocal interstitial pneumonia, edematous alveoli and type II pneumocyte hyperplasia. Chest radiographs revealed the presence of pulmonary infiltrates and consolidation, which is the main clinical sign of COVID-19 in infected rhesus macaques. Near identical clinical signs and histopathology were observed when Rhesus macaques were challenged with a similar virus strain to show the therapeutic efficacy of Remdesivir (Brandi N. Williamson, 2020). The other two studies showed age-related correlations in Rhesus (P. Yu et al., 2020) and Cynomolgus macaques (Rockx et al., 2020) and have demonstrated the impact of comorbidities on SARS-CoV-2 disease severity (Table 1) . More recently, African Green monkeys (AGMs)(Woolsey et al., 2020) and baboons (Dhiraj Kumar Singh, 2020) were demonstrated to show more pronounced respiratory disease than rhesus macaques suggesting that both AGMs and baboons may be the models of choice to test novel immunomodulatory therapies to reduce disease severity. Interestingly, African green monkeys were successfully infected with a lower SARS-CoV-2 inoculum compared to rhesus macaques. Unlike rodents, macaques, baboons and AGMs are larger in size, require a larger experimental space, and costly as compared to the other animal models. However, macaques develop respiratory disease that is comparable to the human disease and therefore represent better translational animal models to study pathogenesis, vaccine efficacy, therapeutics, co-infections, including the impact of age and other preexisting co-morbidities on SARS-CoV-2 disease course.