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.