The Ferret as a Model for Filovirus Pathogenesis and Countermeasure Evaluation
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ILAR Journal, 2021, Vol. 00, No. 00, 1–10
doi: 10.1093/ilar/ilab011
Review
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The Ferret as a Model for Filovirus Pathogenesis
and Countermeasure Evaluation
Zachary Schiffman 1,2 , Guodong Liu2 , Wenguang Cao2 , Wenjun Zhu2 ,
Karla Emeterio1,2 , Xiangguo Qiu2 and Logan Banadyga 2,*
1 Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg, Manitoba,
Canada and 2 Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada,
Winnipeg, Manitoba, Canada
*Corresponding Author: Dr Logan Banadyga, Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, 1015
Arlington Street, Winnipeg, Manitoba R3E 3R2, Canada. E-mail: logan.banadyga@canada.ca.
Abstract
The domestic ferret (Mustela putorius furo) has long been a popular animal model for evaluating viral pathogenesis and
transmission as well as the efficacy of candidate countermeasures. Without question, the ferret has been most widely
implemented for modeling respiratory viruses, particularly inf luenza viruses; however, in recent years, it has gained
attention as a novel animal model for characterizing filovirus infections. Although ferrets appear resistant to infection and
disease caused by Marburg and Ravn viruses, they are highly susceptible to lethal disease caused by Ebola, Sudan,
Bundibugyo, and Reston viruses. Notably, unlike the immunocompetent rodent models of filovirus infection, ferrets are
susceptible to lethal disease caused by wild-type viruses, and they recapitulate many aspects of human filovirus disease,
including systemic virus replication, coagulation abnormalities, and a dysregulated immune response. Along with the
stringency with which they reproduce Ebola disease, their relatively small size and availability make ferrets an attractive
choice for countermeasure evaluation and pathogenesis modeling. Indeed, they are so far the only small animal model
available for Bundibugyo virus. Nevertheless, ferrets do have their limitations, including the lack of commercially available
reagents to dissect host responses and their unproven predictive value in therapeutic evaluation. Although the use of the
ferret model in ebolavirus research has been consistent over the last few years, its widespread use and utility remains to be
fully proven. This review provides a comprehensive overview of the ferret models of filovirus infection and perspective on
their ongoing use in pathogenesis modeling and countermeasure evaluation.
INTRODUCTION ebolavirus (Sudan virus [SUDV]), Bundibugyo ebolavirus (Bundibu-
gyo virus [BDBV]), Reston ebolavirus (Reston virus [RESTV]), Taï
Filoviruses Forest ebolavirus (Taï Forest virus [TAFV]), and Bombali ebolavirus
Filoviruses (family Filoviridae; order Mononegavirales) are single- (Bombali virus [BOMV]). In contrast, the genus Marburgvirus con-
stranded, non-segmented, negative-sense RNA viruses, some of sists of a single species, Marburg marburgvirus, that contains 2
which are highly pathogenic in humans and the causative agents viruses, Marburg virus (MARV) and Ravn virus (RAVV). With the
of filovirus disease (FVD).1,2 The family Filoviridae encompasses exception of TAFV, for which there has been only 1 documented
5 distinct genera, namely Ebolavirus, Marburgvirus, Cuevavirus, non-fatal case,3 RESTV, which is non-pathogenic to humans
Striavirus, and Thamnovirus, among which only members of the (reviewed in Miranda and Miranda),4 and BOMV, which has yet
Ebolavirus and Marburgvirus genera are known to cause disease to be isolated,5 the aforementioned filoviruses are highly lethal,
in humans. The genus Ebolavirus consists of 6 species, each con- with case fatality rates ranging from approximately 34% to 90%,
taining a single virus: Zaire ebolavirus (Ebola virus [EBOV]), Sudan depending on the virus (reviewed in Kuhn et al and Jacob et al).1,6
Received: June 15, 2020. Revised: December 4, 2020. Accepted: January 14, 2021
Published by Oxford University Press on behalf of the National Academies of Sciences, Engineering, and Medicine 2021.
© Her Majesty the Queen in Right of Canada, as represented by the Minister of Health, 2021.
1
2 Schiffman et al
Since 1976, there have been 30 outbreaks linked to ebolavirus- countermeasures owing to their ease of handling, low cost, avail-
es, the largest being the 2014–2016 West African EBOV epidemic, ability of reagents, and general suitability for high-throughput
which resulted in over 28 000 cases and more than 11 000 studies. Notably, however, because immunocompetent mice are
deaths.1,6 To this day, EBOV in particular continues to pose naturally resistant to wild-type filoviruses, host adaptation via
a significant threat to public health, as exemplified by the serial passaging is required to generate filovirus strains capable
2018–2020 EBOV outbreak in the Democratic Republic of the of causing uniformly lethal disease. Aside from being a relatively
Congo, which was the second largest EBOV outbreak in history,7 laborious process, adaptation results in several mutations to
as well as the ongoing EBOV outbreak in the same country, which the viral genome, and the impact of these mutations on virus
was declared in June 2020.8 In contrast to the ebolaviruses, pathobiology remains poorly understood.18 Although some wild-
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there have been only 14 marburgvirus outbreaks since the type filoviruses can causes lethal disease in STAT1 and IFNAR
viruses were first discovered in 1967.1 Although these outbreaks knock-out mice, the absence of an intact immune system may
have been smaller in magnitude compared with the ebolavirus confound evaluation of certain countermeasures. Conversely,
outbreaks, they have often been severe, with an overall case “humanized mice” with reconstituted human immune systems
fatality rate of 80% (388 deaths among 482 reported cases).1 have been shown to be susceptible to wild-type EBOV; however,
Filovirus infections in humans are acquired by direct contact their relatively high cost and associated ethical considerations
with infectious bodily fluids, whereby virus enters through have limited the use of this model. Guinea pigs are also routinely
breaks in the skin or mucosal surfaces.6,9,10 On entry into the used as model systems for filoviruses, and, like mice, they are
body, filoviruses initially infect macrophages, monocytes, and low in cost and relatively easy to handle in high containment
dendritic cells, ultimately suppressing the immune response laboratories. Additionally, guinea pigs are also naturally resistant
and facilitating virus dissemination. Generally, the clinical to filovirus infection, and host adaptation is required to produce
course of FVD involves an incubation period of 7–10 days prior uniformly lethal disease. Unlike mice, however, guinea pigs seem
to the onset of signs and symptoms. On symptom onset, the to more closely recapitulate FVD and are therefore thought
patient enters the early phase of FVD, which is characterized by to offer a more stringent evaluation of countermeasures.
non-specific signs such as fever, fatigue, anorexia, myalgia, and Hamster models of EBOV and MARV, which also rely on host-
headache as well as gastrointestinal symptoms such as nausea, adapted viruses, may offer enhanced fidelity compared with the
diarrhea, and abdominal pain. Throughout the early phase, guinea pig models, but a lack of reagents for dissecting disease
which can last anywhere from 0 to 7 days following symptom pathways in this animal, combined with a poor understanding
onset, the clinical manifestations of FVD become more severe of their predictive value, appears to have limited their use
as the patient enters the peak phase of disease, around day 7. so far.
Characteristic of the peak phase is systemic replication of the
virus in a number of different tissues, resulting in more severe
clinical manifestations, including rash, renal failure, respiratory
The Ferret
failure, cardiac dysfunction, and hemorrhage. This phase of The domestic ferret (Mustela putorius furo) is a small carnivore
disease is also characterized by high viremia and severe immune belonging to the Mustelidae family, members of which can be
dysregulation, resulting in the so-called “cytokine storm.” In found across the globe in a number of highly diverse ecosys-
the most severe cases, organ failure and hypovolemic shock tems ranging from arctic tundra to tropical rain forests.19 These
culminate in death anywhere from 7 to 14 days to as late as 21 animals, thought to have been domesticated from the Euro-
to 28 days. Recovery from FVD is accompanied by both cellular pean polecat more than 2000 years ago,20 share many anatomi-
and humoral immune responses, although survivors can suffer cal, metabolic, and physiological features with humans, which
from a number of complications, including arthralgia as well has long made them appealing models for infectious disease
as ocular and neurological issues. Moreover, virus is known to research.
persist in immune-privileged niches such as the central nervous The utility of the ferret as an animal model in virus research
system and eyes as well as the urogenital system. Indeed, virus first became apparent in the early 1930s, following the discov-
persistence in the semen has been linked to sexual transmission ery of their susceptibility to influenza virus.21 To this day, the
of FVD.11 ferret remains one of the best animal models for evaluating
viral pathogenesis and transmission of influenza viruses as
well as efficacy testing of candidate countermeasures.22 Their
Filovirus Animal Models suitability as an animal model for influenza virus research is in
Animal models play a central role in filovirus research (Fig. 1). most part due to their susceptibility to wild-type viruses without
Models that faithfully recapitulate the hallmarks of FVD are not the need for host adaptation, their ability to efficiently transmit
only useful for understanding virus pathogenesis, but they are the virus, and their recapitulation of many aspects of human
also critical for evaluating the efficacy of novel countermeasures. disease.
Although several different filovirus animal models have been Although the ferret has proven to be an exceptional model
developed—ranging from mice to nonhuman primates (NHPs)— for influenza viruses, it has also been shown to be a valuable
each has a unique set of advantages and disadvantages, all of model for a number of other respiratory viruses, such as
which have been reviewed extensively elsewhere.12–17 NHPs human respiratory syncytial virus,23,24 severe acute respiratory
(typically rhesus or cynomolgus macaques) are considered to syndrome coronavirus,25–27 and, more recently, severe acute
be the gold-standard animal model for filoviruses because respiratory syndrome coronavirus-2,28,29 the causative agent
they are susceptible to wild-type filoviruses and most closely of coronavirus disease 2019. Furthermore, as the natural host of
reproduce FVD as it is observed in humans. For this reason, canine distemper virus, ferrets also represent a suitable model
NHPs are most often used for the confirmatory evaluation of for many morbilliviruses.30 For example, canine distemper
novel countermeasures after efficacy has already been proven virus infection in ferrets reproduces many of the clinical
in rodent models and before they are advanced into human manifestations associated with measles virus in humans,
trials. Mice, on the other hand, are used for primary evaluation of serving as a surrogate model system for evaluating pathogenesisThe Ferret as a Model for Filovirus Pathogenesis and Countermeasure Evaluation 3
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Figure 1: Comparison of filovirus animal models. General features of filovirus disease (FVD) and various elements affecting research use are compared among humans,
non-human primates (rhesus or cynomolgus macaques), domestic ferrets, guinea pigs, hamsters, and mice. #, Indicates average lethality of all filoviruses from all
outbreaks; virus-specific lethality ranges from 0% to approximately 81%. ∗ , Indicates uniform lethality is commonly observed, but may be lower depending on the virus
or virus variant. GPA = guinea pig adapted; HA = hamster adapted; MA = mouse adapted; A = not applicable.
and efficacy of candidate countermeasures. A number of FILOVIRUS PATHOGENESIS IN FERRETS
additional viruses have also been evaluated in the ferret
Ebolaviruses
model with varying degrees of success, including Middle East
respiratory syndrome coronavirus,31 rabies virus,32–34 Hendra Ferret models for ebolavirus infections have been developed
virus,35,36 Nipah virus,37 mumps virus,38– 40 simian virus 5,41 primarily by 2 different research groups: 1 at the University
and canine42 and human parainfluenza viruses (reviewed in of Texas Medical Branch (UTMB) and 1 at the Public Health
Enkirch and von Messing).43 More recently, the ferret has become Agency of Canada (PHAC). At UTMB, Cross and colleagues
increasingly popular as a model for filoviruses, particularly established models for EBOV (variant Kikwit), BDBV (variant
for evaluating pathogenesis and transmission as well as for Uganda 2007), and SUDV (variant Gulu) in 2016,44 while at PHAC,
efficacy testing of candidate countermeasures. In this review, we Kozak et al and Kroeker et al developed similar models for EBOV
describe the ferret models of filovirus infection that have been (variant Makona-C07) and BDBV (variant Uganda 2007) in 201645
developed so far, and we offer a perspective on the continued as well as SUDV (variant Boneface) in 2017.46 Additionally, in
use of this animal model for understanding viral pathogenesis 2019, Yan and colleagues, also at PHAC, established a model
and evaluating countermeasures. for RESTV (variant AZ-1435).47 Remarkably, in all cases, ferrets4 Schiffman et al
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Figure 2: Hallmarks of pathogenesis for different filoviruses in ferrets. Virulence, peak viremia, and distinctive features of filovirus disease (FVD) are compared among
different filoviruses, including Ebola virus (EBOV; blue), Sudan virus (SUDV; orange), Bundibugyo virus (BDBV; green), Reston virus (RESTV, red), Marburg virus (MARV;
purple), and Ravn virus (RAVV; brown). Virulence parameters include dosage, expressed in 50% tissue culture infective dose (TCID50 ) or plaque forming unit (PFU); time
to death (TTD); route of infection (intramuscular [IM], intranasal [IN], or intraperitoneal [IP]); and percent lethality. Time range of peak viremia days post infection (DPI)
are shown along with infectious titers, reported as TCID50 /mL and/or PFU/mL, and presence of viral RNA presented as genome equivalents (GEQ)/mL. + and − depict
the presence or absence of a feature, respectively; ND indicates a feature was not analyzed.
were susceptible to wild-type viruses without the requirement Regardless of the inoculation dose or route, disease caused
of virus adaptation. In general, all animals developed disease by all ebolaviruses followed a similar course but with species-
that recapitulated many of the major hallmarks of Ebola disease specific variances (Fig. 2). Ferrets infected either IM or IN with
(EBOD) that have been observed in NHPs and humans, including EBOV succumbed to disease at 5 to 6 days post infection (DPI),
fever, weight loss, vascular leakage, dysregulated immune with viremia first detected at 4 DPI and peaking at the terminal
response, uncontrolled systemic virus replication, multi-organ time point (5–6 DPI).44,45 Ferrets infected with SUDV succumbed
failure, and coagulopathy (Fig. 2; Supplementary Tables 1 and to infection at 7 or 8 DPI, depending on the variant used, with
2). Disease was lethal in all cases, although the time to death viremia first observed at 4 DPI.44,46 In BDBV-infected animals,
varied, possibly reflecting differences in virulence inherent to viremia was first detected at 4 DPI and peaked at 8 DPI, and
each virus. Notably, animals were uniformly susceptible to both the animals died between 8 and 9 DPI.44 Animals survived the
intranasal (IN) and intramuscular (IM) challenge, with a range of longest after infection with RESTV—between 9 to11 days—and
inoculation doses from 159 median tissue culture infectious the viremia increased slowly following detection at 3 and 5
dose (TCID50 ) to 1000 plaque-forming units (PFU). Indeed, a DPI for IM- and IN-inoculated groups, respectively.47 Fever was
separate study demonstrated that doses as low as 0.1 PFU of usually the first sign of disease observed in all the models,
reverse genetics-derived EBOV (variant Makona-C07) caused beginning at 3 to 6 DPI, depending on the virus. Marked weight
100% lethality, with a median lethal dose calculated to be 0.015 loss was also observed from 4 to 7 DPI in all animals, although the
PFU.48 decrease was not as pronounced in animals infected with EBOVThe Ferret as a Model for Filovirus Pathogenesis and Countermeasure Evaluation 5
variant Makona-C07.44 Other common clinical signs observed in the spleens of many animals included red pulp hyperplasia,
the ferrets throughout infection included progressively worsen- increased macrophages, and multifocal necrotic areas, as well
ing depression, diarrhea, dehydration, and labored breathing. In as lymphocytosis, although an increase in macrophages was
addition, a petechial rash—an important feature of EBOD seen in not found for SUDV-infected animals.46 Kidney lesions were less
humans and NHPs—was observed in all animals except for those common, but interstitial nephritis associated with necrotic cells
infected with SUDV variant Gulu.44 was found in RESTV- and SUDV-infected animals, and scattered
Systemic viral spread was identified in all cases, with high epithelial tubular degeneration was mostly seen in animals
virus titers found in the liver, spleen, kidney, and lung at terminal infected with EBOV, BDBV, or RESTV. In contrast, pathology in
time points, regardless of the virus or route of infection. In the lung was usually mild to moderate, with the most consis-
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addition, EBOV, BDBV, and SUDV were detected in the brain and tent lesion being the expansion of alveolar walls by inflam-
heart. Virus shedding from the oral, nasal, and rectal mucosae matory cells and edema. Notably, however, severe necrotizing
was observed in all animals, regardless of virus, and a separate pneumonia, bronchiolitis, and perivasculitis was observed in
study demonstrated the capacity for both direct and indirect ferrets inoculated IN with EBOV, and severe bronchointerstitial
EBOV transmission among ferrets.49 pneumonia was observed in animals inoculated IN with SUDV.
Dysregulation of the immune system, including depletion Importantly, all of these histological changes were associated
of immune cells, is another hallmark of FVD in humans, with the detection of virus antigen, suggesting the presence of
and evidence for these processes was observed in ferrets. replicating virus.
Although the magnitude varied among studies, most animals Together, these 4 studies provide a thorough characterization
infected with EBOV, BDBV, SUDV, or RESTV showed some of ferret models for EBOV, SUDV, BDBV, and RESTV, and they
degree of reduction in white blood cells and/or lymphocytes, are particularly notable for introducing the first, and so far
with disturbances in monocytes, eosinophils, and basophils only, small animal model for BDBV infection. Nonetheless, some
observed by the UTMB group.44 Increased levels of circulating outstanding issues remain unresolved. Firstly, some caution may
tumor necrosis factor alpha (TNF-α) and nitric oxide were also be required in making direct comparisons between results from
found beginning around 4 DPI in the UTMB-developed models the UTMB and PHAC studies, because both groups used dif-
for EBOV, BDBV, and SUDV, suggesting a pro-inflammatory ferent virus variants and inoculation doses. Secondly, with the
response.44 Moreover, transcriptomic analyses of the blood of exception of the RESTV study, all other studies that character-
ferrets infected with EBOV revealed a dramatic upregulation ized ebolavirus pathogenesis in ferrets used exclusively female
in a number of pro-inflammatory cytokines, chemokines, animals. It is therefore unclear whether significant differences
and interferon-stimulated genes, suggestive of a dysregulated exist in disease presentation between male and female ferrets,
immune response and similar to what is observed in humans but considering that many subsequent papers—assessing trans-
and NHPs.50 mission or evaluating countermeasures—use both males and
Disruption of the coagulation pathway was a consistent find- females, it may be important to better understand this issue.
ing in all animals infected with the ebolaviruses. A dramatic Finally, it is worth noting that to date, ferret models for TAFV
reduction in platelet levels, indicating thrombocytopenia, was and BOMV have not been published. Although public health and
observed in all animals. Moreover, serum biochemical analy- research interest in these viruses is relatively low, a ferret model
ses in animals infected with EBOV, BDBV, or SUDV indicated for either virus may provide additional insight into the biology
prolonged activated partial thromboplastin times, prothrombin and pathogenesis of ebolaviruses in general.
time, and thrombin time as well as the increased level of fib-
rinogen (although the latter was not significant in BDBV-infected
Marburgviruses
animals). Together, these data are suggestive of consumptive
coagulopathy.44,45 The same 2 research groups at UTMB and PHAC responsible
Abnormalities in the levels of certain enzymes and biomark- for developing the ferret models of ebolavirus infection also
ers in the serum of infected ferrets were indicative of multi- evaluated the pathogenesis of marburgviruses in ferrets (Fig. 2;
organ failure. Liver damage was consistently found in all ani- Supplementary Tables 1 and 2). In 2 separate studies, male and
mals, indicated by increased levels of alanine aminotransferase, female ferrets were challenged with MARV (variant Angola or
alkaline phosphatase, and total bilirubin and decreased levels Musoke) or RAVV via a number of different routes (IM, IN, or
of albumin. Renal failure was also indicated by increases in intraperitoneal [IP]) and doses (317 TCID50 –100,000 PFU) in an
blood urea nitrogen and creatinine. The organ damage sug- attempt to establish a lethal model.51,52 Surprisingly, however,
gested by the serum biochemical analyses was further con- regardless of challenge dose, route, or virus/variant, ferrets were
firmed by histopathology. The most significant lesions reported found to be not susceptible to infection with marburgviruses.
by the UTMB group for EBOV, SUDV, and BDBV infection were Among the 36 ferrets challenged across both studies, none dis-
marked lymphohistiocytic and neutrophilic necrotizing hepati- played any of the disease signs—such as weight loss, fever,
tis and necrotizing splenitis.44 In addition, increased lymphocyte or decreased food/water intake—that are observed in ferrets
apoptosis in the spleen was observed in all infected ferrets. infected with ebolaviruses. Furthermore, no significant changes
Accordingly, viral antigens were detected in hepatic sinusoidal from baseline hematology or serum biochemistry were observed
mononuclear cells, hepatocytes, and mononuclear cells within at any point, nor could any infectious virus be detected in the
the red and white pulp of the spleen. Similar histological lesions blood or tissues. Interestingly, viral RNA (8.35 × 106 genome
were also observed in the other ferret models developed by equivalents [GEQ]/mL) was detected in the blood of a single ani-
the PHAC research group.45–47 Moderate to severe pathology mal at 6 DPI; however, no additional details on this animal were
was observed in the liver, including diffuse or multifocal vac- available.51 Despite the lack of viremia, all animals seroconverted
uolar degeneration and loss of hepatocytes that led to dis- and demonstrated moderate neutralizing antibody titers at end
ruption of normal structure as well as penetration of inflam- point.
matory cells into the portal area. In addition, viral inclusion Taken together, the findings from these 2 studies demon-
bodies were observed in some animals. Lesions observed in strate that, in contrast to ebolaviruses, the domestic ferret is not6 Schiffman et al
susceptible to lethal infection with marburgviruses. While the and strongly neutralize recombinant vesicular stomatitis viruses
lack of detectable viral RNA and infectious virus in both blood pseudotyped with GPs of the 5 human-pathogenic ebolaviruses
and tissues indicates that inoculation with marburgviruses does as well as authentic EBOV, SUDV, and BDBV. Both mAbs were
not lead to a productive infection, the presence of MARV-specific highly protective against mouse-adapted (MA)-EBOV and MA-
IgG and moderate neutralizing antibody titers indicates that the SUDV in mice, and thanks to the recently developed ferret model
animals did mount a humoral immune response. Although it of BDBV infection, they were also tested for their efficacies
remains unclear as to why ferrets are refractory to infection against BDBV. Ferrets were infected with a target dose of 1000
with marburgviruses, yet susceptible to ebolaviruses, it is rea- TCID50 wild-type BDBV via IM injection and treated IP with 15 mg
sonable to speculate that differences in the immune response of ADI-15878 or ADI-15742 on 3 DPI followed by a second dose
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to infection, including differences in innate immune evasion of 10 mg at 6 DPI. Control animals, who received phosphate-
between ebolaviruses and marburgviruses may be at least partly buffered saline (PBS) post challenge, all succumbed to infec-
responsible.53 Additionally, ferret cells themselves may differ in tion within 10 days. By contrast, significant survival rates were
their permissiveness to filovirus infection, with species-specific observed among the treated animals, with ADI-15878 and ADI-
differences in viral entry and/or replication possibly affecting the 15742 resulting in 75% and 50% survival, respectively. Both mAbs
development (or not) of FVD. Further investigations are required clearly delayed and/or significantly reduced peak viral loads in
to fully elucidate the molecular determinants of filovirus viru- the blood; however, among the survivors, animals treated with
lence in ferrets, and future directions could involve the estab- ADI-15878 showed lower viral loads and earlier virus clearance
lishment of a ferret-adapted virus that could prove critical for than those treated with ADI-15742. Interestingly, this discrep-
the identification of mutations responsible for the avirulence of ancy has been linked to possible escape mutation (G528S) in the
marburgviruses in these animals. BDBV GP found in animals treated with ADI-15742.
CA45 is another broadly neutralizing antibody, this time iso-
lated from memory B cells of a cynomolgus macaque immunized
with recombinant GPs of EBOV, SUDV, and MARV, that shows
FERRETS AND COUNTERMEASURE strong binding activity against multiple ebolaviruses.57 Similar
EVALUATION to ADI-15878, CA45 also targets the GP1-GP2 interface at the GP
Candidate countermeasures against filoviruses are typically base but in a distinct region with major contacts predominantly
screened for efficacy in rodents before confirmatory evaluation in and around the internal fusion loop stem. In concordance with
in NHPs; however, the recent success in recapitulating ebolavirus its GP binding profile, CA45 is a strong neutralizer of EBOV, BDBV,
infection in ferrets has offered a potential alternative model. The and SUDV in both GP-pseudotyping and authentic systems and
apparent stringency of the ferret ebolavirus model, along with a moderate neutralizer of RESTV GP-mediated infection. CA45
the ability to use wild-type viruses, may position the ferret as an was highly protective against MA-EBOV in mice and both MA-
intermediate model system to be used after primary evaluation and guinea pig-adapted (GPA)-SUDV in mice and guinea pigs,
in rodents and prior to evaluation in NHPs. However, to date, respectively, but it was only partially protective against GPA-
there are still relatively few efficacy studies involving ferrets EBOV in guinea pigs. When combined with another NHP-derived
and therefore little understanding of the predictive value of mAb, known as FVM04, which targets the receptor binding region
this model. Moreover, the majority of efficacy studies that have of GP, the cocktail provided full protection against GPA-EBOV
been performed have been limited to evaluation of monoclonal and GPA-SUDV in guinea pigs. The authors then decided to test
antibodies (mAbs) against BDBV infection, for which the ferret the cocktail for efficacy against BDBV in ferrets. Equal numbers
is the only small animal model available. of male and female ferrets challenged via the IM route with
a lethal dose (253 TCID50 ) of BDBV followed by IP treatment
with PBS or the antibody cocktail (20 mg of each mAb) at 3
and 6 DPI. The control animals treated with PBS died at 7 DPI,
Monoclonal Antibodies
whereas the animals treated with the cocktail all survived with-
The past decade has witnessed remarkable progress in the out clinical signs of diseases or weight loss. The control animals
development of therapeutics against filoviruses.54 Among them, exhibited high viral loads in the blood, peaking at nearly 1010
mAbs targeting the surface glycoprotein (GP) of ebolaviruses GEQ/mL on the day of death, whereas, in contrast, viral RNA was
have shown exceptional efficacy in several pre-clinical eval- undetectable in the treated male animals and only transiently
uations. Indeed, a clinical trial conducted during the recent detected at low levels (approximately 104 GEQ/mL) in the females
outbreak in the Democratic Republic of the Congo demonstrated on 6 DPI. No viral RNA was detected in the oral, nasal, or rectal
that a single mAb known as Mab114, as well as a cocktail of mucosa of the treated animals, suggesting the absence of virus
3 mAbs known as REGN-EB3, conferred a significant survival shedding in the treated animals, while up to approximately 106
advantage in patients.55 Although these mAbs solely target EBOV, GEQ/mL of viral RNA was detected in the control animals. Blood
the therapeutic promise of mAbs in general has generated great chemistry features associated with organ damage, including
interest in developing broadly cross-reactive mAbs capable of dramatic elevation in alanine aminotransferase and alkaline
protecting against multiple ebolaviruses or filoviruses. Ferrets phosphatase levels, were observed in the control animals but not
have played a key role in the pre-clinical development of many in the treated animals. In addition, lymphopenia and thrombo-
of these candidate mAb therapeutics, primarily because they cytopenia were at least partially reversed in all treated animals,
offer a suitable model in which to test efficacy against BDBV. which was not the case in the controls. Thus, the rodent studies
Among the first mAbs to be evaluated in the ferret model combined with the ferret study indicate that CA45, particularly
were ADI-15878 and ADI-15742.56 These mAbs were isolated when combined with FVM04, is highly potent against the 3 most
from a human survivor and shown to bind a GP base region span- pathogenic ebolaviruses.
ning both GP1 and GP2, which includes the conserved internal In 2018, two studies from the same group were published
fusion loop that is essential for the fusion of viral and cellular describing broadly neutralizing mAbs isolated from human
membranes. The 2 mAbs each recognize multiple ebolavirus GPs survivors of ebolavirus infection.58,59 The first study, by FlyakThe Ferret as a Model for Filovirus Pathogenesis and Countermeasure Evaluation 7
et al, identified 3 cross-neutralizing mAbs (termed BDBV223, cocktail containing 1 × 107 PFU of each vaccine through IN
BDBV317, and BDBV340) from human survivors of BDBV.58 All inoculation and challenged 4 weeks later with a target dose of
3 mAbs targeted the canonical heptad repeat 2 region close 1 × 103 PFU BDBV via IM injection. The vaccine cocktail induced
to the membrane-proximal external region in GP and showed a strong antibody response against BDBV, with GP binding capac-
neutralizing activity against BDBV as well as EBOV, RESTV, ity and neutralizing activity comparable with that elicited by
and SUDV. BDBV223 completely protected mice from MA-EBOV the BDBV monovalent vaccine, and weaker responses against
infection and partially protected guinea pigs from GPA-EBOV EBOV and SUDV. The BDBV challenge (1000 PFU) was uniformly
infection. To assess the protective efficacy of BDBV223 against lethal among the control animals, which experienced contin-
BDBV, male and female ferrets were IM-inoculated with a target uous weight loss starting at 3 DPI and suffered rapid disease
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dose of 1000 PFU BDBV and treated with 20 mg of antibody progression, with signs of disease appearing on or 1 day before
on 3 and 6 DPI. While the control animals treated with an the day of death at 7 DPI. By contrast, both the vaccine cock-
irrelevant mAb died at 7 DPI, 2 of the 4 animals treated with tail and the monovalent HPIV3-BDBV provided full protection,
BDBV223 survived, having exhibited no weight loss or clinical with all vaccinated animals testing negative for viremia and no
signs of illness. In the second study, the same group reported animals exhibiting weight loss or showing signs of disease. The
the protective efficacy of EBOV-520, a mAb isolated from a successful evaluation of the efficacy of these vaccines against
human survivor of EBOV.59 EBOV-520 exhibited neutralizing BDBV in ferrets suggests that this animal model may be useful
activity against EBOV, SUDV, and BDBV in vitro as well as in vivo for testing vaccines against other pathogenic ebolaviruses.
efficacy against MA-EBOV, MA-SUDV, and GPA-SUDV. To assess
the antibody’s protective efficacy against BDBV, male and female
ferrets were IM-inoculated with a target dose of 1000 PFU virus
PERSPECTIVES ON FERRETS AS A FILOVIRUS
and treated IP with 18 mg EBOV-520 on 3 and 6 DPI. Surprisingly,
3 out of 4 animals treated with EBOV-520 succumbed by 10 DPI
ANIMAL MODEL
even though all animals had undetectable viremia in the blood Ferrets occupy a unique niche in the landscape of filovirus
at 6 DPI, just prior to the second treatment. Only 1 of the 3 animal models, somewhere between the larger NHPs and the
ferrets that succumbed to disease showed evidence of viremia, smaller rodents. Like NHPs, ferrets are susceptible to lethal
with virus levels peaking on 10 DPI, the terminal time point. infection by wild-type viruses, negating the need for the host
Unlike the animal that survived, all 3 ferrets that succumbed adaptation that is required for all immunocompetent rodent
showed disturbances in clinical chemistry parameters, including models. Not only does this mean that the ferret model hews
alkaline phosphatase and alanine aminotransferase, suggesting more closely to the NHP and human conditions, but it also
organ damage. However, the presence of virus in tissues at the circumvents any questions of relevance that come from using
time of death was not investigated. an adapted virus strain, which may possess a number of
To date, only a single study has evaluated mAb efficacy genome mutations of unknown consequence, or an immune-
against an ebolavirus other than BDBV in the ferret model.60 deficient animal model, which may confound the evaluation
The mAb cocktail known as MBP134AF consists of 2 broadly of vaccines and therapeutics. Moreover, the ferret model
neutralizing mAbs, ADI-15878 (discussed above) and ADI-23774, faithfully reproduces key hallmarks of disease as they are
which have individually been shown to neutralize GP and protect observed in humans and NHPs, including robust viral replication
from disease in small animal models.56,61 In ferrets, MBP134AF and systemic spread, disturbances in hematology and blood
successfully protected all animals (female) from EBOV, SUDV, biochemistry, multi-organ damage, coagulation abnormalities,
and BDBV (1000 PFU delivered IN) following two 15-mg doses and a pro-inflammatory immune response. Like rodents, ferrets
of antibody given IP on 2 and 5 DPI (EBOV) or 3 and 6 DPI are considered a small animal model, and they possess some
(SUDV, BDBV). None of the animals infected with EBOV or SUDV of the attendant advantages. Compared with NHPs, ferrets are
showed evidence of viremia, and only 1 animal infected with relatively cheap, easier to handle, easier to house, and of lower
BDBV exhibited low levels of viral RNA and infectious virus in the sentience. As a result, higher animal numbers can often be used
blood. Interestingly, a lower dose of 5-mg MBP134AF , given via the in experiments involving ferrets. Yet, compared with rodents,
same regimen, did not protect against SUDV infection, offered ferrets are much more expensive, and the husbandry associated
partial protection against BDBV, and was not tested against with their care is more complex inside high containment,
EBOV. Notably, efficacy of MBP134AF was also evaluated against particularly given their playful and inquisitive dispositions.
EBOV and SUDV in rhesus macaques—in which it provided Like NHPs, the larger size of ferrets permits serial sampling,
full protection—and against BDBV in cynomolgus macaques— which helps maximize the utility of each animal, in addition
in which it offered near-complete protection. Thus, this study is to providing important longitudinal data. Indeed, the unique
the only one to date that provides a direct comparison between combination of advantages and disadvantages possessed by the
the virus-specific efficacy of a therapeutic in ferrets and in NHPs, ferret suggests that it may serve as an “intermediate” animal
providing some idea of the predictive value of the ferret model. model for the efficacy testing of novel countermeasures to
be used after primary evaluation in rodent models and before
confirmatory evaluation in the gold-standard NHP model.
Vaccines Despite the promise it holds as a model for countermea-
Recently, a study evaluated the protective efficacy of a vaccine sure development, the ferret remains largely underused. Since
cocktail against BDBV infection in ferrets.62 This trivalent cock- the introduction of this animal model in 2016, only 6 stud-
tail is composed of 3 vaccines, each expressing EBOV, BDBV, or ies have been published that used ferrets to evaluate medical
SUDV GP in a human parainfluenza virus type 3 (HPIV3) vector. countermeasures, primarily mAbs. Of these 6 studies, 5 used
The cocktail demonstrated potent protective efficacy against the ferret model exclusively for BDBV infection,56–59,62 and 1
GPA-EBOV and GPA-SUDV infection in guinea pigs. To evaluate used the model for BDBV infection as well as EBOV and SUDV
its efficacy against BDBV, female ferrets were vaccinated with infection.60 Given that ferrets are currently the only available
1 × 107 PFU of the monovalent HPIV3-BDBV vaccine or the small animal model for BDBV, the choice to use these animals8 Schiffman et al
when evaluating countermeasure efficacy against this virus is The fact that neither MARV nor RAVV causes disease in ferrets,
an easy one. Indeed, the field has had tremendous success despite causing severe disease in NHPs and humans, is puzzling.
demonstrating remarkable therapeutic efficacy of cross-reactive Besides clearly excluding ferrets as a model for marburgvirus
mAbs or antibody cocktails in ferrets infected with BDBV. Why infection, it also raises questions about how well the animals
then has this animal not seen wider adoption in the filovirus reflect disease processes for the ebolaviruses. In humans, unlike
field? in ferrets, EBOD is not uniformly lethal and often involves other
Although the ferret model possesses many clear advantages, features, such as long-term complications and virus persistence
it also suffers from a few crucial disadvantages in addition to that are not reproduced by the ferret model. Moreover, although
some broader weaknesses that have yet to be resolved. The EBOV, SUDV, and BDBV disease in ferrets seems to mirror the
Downloaded from https://academic.oup.com/ilarjournal/advance-article/doi/10.1093/ilar/ilab011/6264820 by guest on 23 November 2021
greatest practical disadvantage to working with ferrets is the lack virus-specific differences in virulence observed in humans, this
of specific reagents and tools for interrogating host-response is not the case for RESTV, which was surprisingly found to cause
pathways; however, advances are being made. The publication of lethal disease in ferrets despite being apathogenic in humans.
the ferret’s draft genome63 and transcriptome64 helped facilitate Without a clearer understanding of the mechanisms underly-
transcriptional analyses of the ferret immune response,65 ing these discrepancies in virulence, interpretation of results
including to EBOV infection,50 thereby setting the stage for future obtained in ferrets should be performed with caution.
investigations. Additionally, genetically engineered knockout Given the significant threat that filoviruses pose to global
ferrets have also been developed,66,67 raising the prospect of public health, along with their potential for misuse as biological
future studies directed at understanding specific components weapons, the development of novel countermeasures to treat
of the animal’s immune system. Work with other viruses has and prevent FVD is urgently required. Although a vaccine has
also helped shed light on the poorly understood ferret immune recently been clinically approved for EBOV,69 no vaccines exist
system as well as identify ferret-reactive reagents that can be for the other filoviruses, and no effective therapeutics have
used to study it.68 Nevertheless, the current paucity of these been licensed for the treatment of infection. Animal models are
reagents—especially compared with those available for other critical to the pre-clinical development of countermeasures, and
animals—is likely to continue to handicap the ferret model for immunocompetent animal models that accurately recapitulate
many years to come. all facets of disease using wild-type viruses are especially use-
Beyond the utilitarian disadvantages of the ferret model exist ful. The domestic ferret, a stalwart of influenza virus research,
questions regarding the animal’s predictive value and stringency represents one of the newest animals to be added to the arsenal
in countermeasure evaluation. The limited number of published of filovirus models, and it possesses several qualities that have
studies reporting vaccine or therapeutic efficacy in the ferret made it amenable to pathogenesis modeling and countermea-
model, especially against EBOV and SUDV infection, makes it sure evaluation. Nonetheless, its true value in understanding
unclear whether results observed in ferrets will ultimately be filoviruses—or at least ebolaviruses—has yet to be fully realized
translatable to NHPs or humans. This creates a paradox whereby and will ultimately depend on its continued use and develop-
researchers may be reluctant to test a novel therapeutic in ferrets ment.
because of their unknown predictive value even though the
only way to determine their predictive value is by using them
to test novel therapeutics. In contrast, the well-studied guinea Supplementary Data
pig model already has a reputation for offering good predictive
Supplementary materials are available at ILAR Journal online.
value,14 and for the time being, it may remain the preferred
choice of small animal (when possible) for evaluating novel
countermeasures.
The stringency of the ferret model is also worth considering. Acknowledgments and Funding
Ferrets are extremely sensitive to ebolaviruses, especially EBOV, This work was supported by the Public Health Agency of Canada.
and they experience a relatively quick, uniformly lethal disease, All figures were created using BioRender.com.
even after inoculation with low doses of virus. It is conceivable
that the severity and rapidity of disease caused by EBOV, and
perhaps also SUDV, elevates the stringency of these models
Potential conf licts of interest
above what is practically useful or informative, setting a high
bar that otherwise effective countermeasures may struggle to All authors: No reported conflicts.
meet. Whether this high bar also exists for BDBV remains to be
determined. Compared with EBOV and SUDV, the slower progres-
sion of BDBV disease in ferrets offers a wider window for treat-
ment that may enhance the odds of therapeutic success. Indeed,
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