SMALL ANIMAL MODELS FOR EVALUATING ... - ACS Publications

Subscriber access provided by the Henry Madden Library | California State University, Fresno

Perspective

Small Animal Models for Evaluating Filovirus Countermeasures Logan Banadyga, Gary Wong, and Xiangguo Qiu ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00266 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Infectious Diseases is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Infectious Diseases

SMALL ANIMAL MODELS FOR EVALUATING FILOVIRUS COUNTERMEASURES

AUTHOR INFORMATION Logan Banadyga1,2, Gary Wong1,2,3, Xiangguo Qiu1,2, * 1

Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of

Canada, 1015 Arlington St., Winnipeg, MB R3E 3R2, Canada 2

Department of Medical Microbiology and Infectious Diseases, University of Manitoba, 745

Bannatyne St., Winnipeg, MB R3E 0J9, Canada 3

Guangdong Key Laboratory for Diagnosis and Treatment of Emerging Infectious Diseases,

Shenzhen Key Laboratory of Pathogen and Immunity, Shenzhen Third People’s Hospital, 29 Bulan Rd., Longgang District, Shenzhen, China, 518000

*Corresponding Author: Xiangguo Qiu, M.D. Special Pathogens Program, National Microbiology Laboratory Public Health Agency of Canada 1015 Arlington St. Winnipeg, MB R3E 3R2 Canada E-mail: [email protected]

ACS Paragon Plus Environment

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The development of novel therapeutics and vaccines to treat or prevent disease caused by filoviruses, such as Ebola and Marburg viruses, depends on the availability of animal models that faithfully recapitulate clinical hallmarks of disease as it is observed in humans. In particular, small animal models (like mice and guinea pigs) are historically and frequently used for the primary evaluation of antiviral countermeasures, prior to testing in nonhuman primates, which represent the gold-standard filovirus animal model. In the last several years, however, the filovirus field has witnessed the continued refinement of the mouse and guinea pig models of disease, as well as the introduction of the hamster and ferret models. We now have small animal models for most human-pathogenic filoviruses, many of which are susceptible to wild type virus and demonstrate key features of disease, including robust virus replication, coagulopathy, and immune system dysfunction. Although none of these small animal model systems perfectly recapitulates Ebola virus disease or Marburg virus disease on its own, collectively they offer a nearly complete set of tools in which to carry out the pre-clinical development of novel antiviral drugs.

Keywords: filovirus; Ebola virus; Marburg virus; countermeasure development; vaccine; therapeutic; animal models; rodent models; ferrets

2


Page 2 of 36

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Filoviruses (family: Filoviridae) comprise a group of single-stranded, negative-sense RNA viruses that are among the most renowned and feared viruses in the world (Table 1)1-2. Ebola virus (EBOV), the prototypical member of the Ebolavirus genus, is by far the most infamous member of this family and has been responsible for causing sporadic outbreaks of Ebola virus disease (EVD; also referred to as Ebola hemorrhagic fever) throughout central Africa, at least since its discovery in 1976. Historically, EBOV has caused small, self-limiting outbreaks, with case totals numbering in the hundreds, albeit with relatively high case fatality rates that occasionally approach 90%. Our conception of this virus and its capabilities changed in 2013, however, when EBOV appeared in Western Africa and instigated an epidemic that lasted until 2016, infecting nearly 30,000 people, killing over 10,000, and devastating the countries of Liberia, Guinea, and Sierra Leone3-4. Although the overall case fatality rate in the West African EBOV epidemic was lower than typically expected for a filovirus, the outbreak still took more lives than all previous filovirus outbreaks combined and served as a stark reminder of the global public health threat posed by these viruses. Most of the other filoviruses also pose a potential risk to public health (Table 1)1-2. All but one of the remaining ebolaviruses—Sudan virus (SUDV), Bundibugyo virus (BDBV), and Taï Forest virus (TAFV)—cause severe human disease (also called EVD), with the former two viruses responsible for multiple outbreaks that have left hundreds dead. The only ebolavirus that appears to be apathogenic in humans is Reston virus (RESTV), which nevertheless causes severe EVD in some nonhuman primates (NHPs). Marburg virus (MARV) and Ravn virus (RAVV), both of which belong to the related Marburgvirus genus, have caused multiple outbreaks of Marburg virus disease (MVD; also referred to as Marburg hemorrhagic fever), which can be at least as deadly as EVD. Conversely, Lloviu virus (LLOV), the only member of the Cuevavirus genus, is not known to have caused any human infections and has yet to be isolated.

3



Both EVD and MVD are life-threatening diseases characterized by systemic virus replication leading to a dysregulated immune response, multi-organ damage, coagulation disorders, and, in severe cases, death1-2. Within a few days following infection, non-specific signs of illness, including fever, malaise, and headache, are typically reported. As the infection progresses, virus- and immune-mediated damage to various organs throughout the body, including the liver, spleen, kidneys, and lymphoid organs, results in a variety of vascular, gastrointestinal, respiratory, and neurological abnormalities5. Hemorrhagic manifestations are occasionally evident and are thought to be derived primarily from defects in the coagulation cascade. Death usually occurs within 10 to 16 days following onset of symptoms from multiple organ failure and a syndrome that resembles septic shock. Despite the severe disease caused by filoviruses—not to mention the public health and bioterrorism threat that these viruses present—no clinically approved therapeutic or prophylactic exists to treat EVD or MVD, although many candidates did move through Phase I, II, and III clinical trials during and after the West African EBOV outbreak6. The continued successful development of filoviral countermeasures depends on animal models that accurately recapitulate the disease as it is observed in humans. Ideally, this animal model should be abundant, inexpensive, and small in size, thus permitting high-throughput experiments within the logistically challenging environment of a maximum containment laboratory. Moreover, the animal should be of limited sentience, thereby reducing the ethical considerations of experimentation. The animal should be well characterized, with a variety of assays and reagents readily available to dissect the host response—particularly the immune response, which should preferably resemble its human counterpart. And, finally, the animal should not only be susceptible to all wild type (WT) variants of human-pathogenic filoviruses, but it should also possess an anatomy and physiology sufficiently similar to humans so that the manifestation of disease in both humans and the animal model is indistinguishable. Obviously, no such model exists, and researchers are forced instead to use multiple different model

4


Page 4 of 36

Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


systems—each with their own advantages and disadvantages—to establish the efficacy of novel countermeasures. Because NHPs (typically rhesus and cynomolgus macaques) offer the most faithful recapitulation of EVD and MVD, they are considered the “gold-standard” filovirus model and are used for all confirmatory testing7. Prior to their use, however, potential countermeasures are typically first evaluated in one or more small animal models, traditionally mice and/or guinea pigs. Small animal models of filovirus infection are critical to the development of novel drugs and vaccines because they permit rigorous pre-clinical evaluation and they conserve precious NHP resources. Here we will briefly discuss the most recently developed and popular small animal models for filovirus infection, followed by a detailed discussion of their potential use in countermeasure evaluation.

MICE Laboratory mice (typically of the species Mus musculus; usually the BALB/c or C57BL/6 strains) are, at least by absolute numbers, the most commonly used animal models in the filovirus research field, and it is easy to see why. In general, mice offer several advantages: they are cheap and abundant, they are easy to house and manipulate in high-containment laboratories, they are extremely well characterized, and they come with numerous reagents and tools. However, since adult, immunocompetent mice are resistant to filovirus infection, the mouse model must rely on one of two significant compromises: either the use of an immunodeficient mouse strain or the use of a serially passaged, host-adapted virus. Immunodeficient mice—such as STAT1 knockout (STAT1 KO), interferon α/β-receptor KO (IFNAR KO), and severe combined immunodeficiency (scid) mice—are sensitive to infection with most WT filoviruses (typically via the intraperitoneal route of infection) and develop partial to uniformly lethal disease that recapitulates some major clinical features observed in NHPs and humans (Table 2)8-12. The recently reported IFNAR KO mouse models for WT-EBOV and SUDV may be especially useful, particularly when partial (as opposed to complete) lethality is

5



Page 6 of 36

desired10. Nevertheless, due to the absence of a complete immune system, immunodeficient mice are often overlooked when it comes to modeling filovirus disease and, in particular, evaluating countermeasures such as vaccines. Conversely, mouse-adapted filoviruses have been created by serially passaging WT-EBOV, -MARV, and -RAVV in young or immunocompromised mice until uniform lethality is achieved in adult, immunocompetent mice (Table 2)13-16. The process of adaptation introduces multiple point mutations into the virus genome that are responsible for the increase in virulence17, yet whether this artificially introduced genetic divergence compromises the relevance of the adapted viruses in the first place remains to be fully elucidated. This issue notwithstanding, following intraperitoneal inoculation, mouse-adapted viruses recapitulate many hallmark features of filovirus disease— with the notable exception of severe and consistent coagulopathy—and are often used for preliminary

evaluations

of

novel

therapeutics

and

vaccines.

Indeed,

the

adult,

immunocompetent mouse model has proved vital in the development of most of the current filovirus countermeasure candidates18, even if the model is not always completely predictive of therapeutic success in NHPs19. The details of the various filovirus mouse models have been reviewed extensively elsewhere17-18, 20-23, and they will not be repeated here. Instead, we will focus on two recent advancements that attempt to address the deficiencies in the filovirus mouse model through a more refined manipulation of the mouse itself.

CC-RIX Mice The Collaborative Cross (CC) is a large panel of recombinant inbred (CC-RI) mouse strains bred specifically to have randomly distributed, uniformly high genetic variation. Collectively, these mice model the complexity of the human genome and permit systems analyses of diseases with complex genetic interactions24. CC-RIX mice represent the progeny of intercrossed CC-RI mice and further increase the genetic diversity in a completely reproducible manner. In an effort to replicate the coagulation abnormalities and hemorrhagic manifestations

6


Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


that characterize EVD but are not otherwise observed in the immunocompetent mouse model of filovirus infections, mouse adapted EBOV (MA-EBOV) was used to infect 47 different CC-RIX strains via the intraperitoneal inoculation route (Table 2)25. Remarkably, 16 of these strains produced a lethal disease with severe coagulopathy, mimicking what is observed in NHPs and humans (Table 3). Nine of the strains proved completely resistant to MA-EBOV infection, while the remaining 22 strains developed disease with mortality rates ranging from 100% to 20% but an absence of hemorrhagic manifestations. Notably, WT-EBOV did not produce disease in any of the eight CC-RI founding strains, but whether any one of the other CC-RI or CC-RIX strains is susceptible to WT infection remains to be determined. Although the CC-RIX model of EBOV infection requires further development, it offers the promise of identifying a strain of immunocompetent mouse that more accurately recapitulates EVD.

Human Immune System Mice Human immune system (HIS) mice are immunodeficient mice that, following sublethal irradiation, have been engrafted with human cells in order to reconstitute aspects of the human immune system26-27. Ideally, HIS mice combine many of the advantages of a typical mouse model with the opportunity to evaluate human-specific immune parameters, and they are used in a variety of research fields, including infectious diseases28. Three different HIS mouse models for EBOV infection have recently been published, and all three models recapitulate key aspects of EVD using—remarkably—WT-EBOV (Table 2)29-31. The severely immunodeficient “NSG” mouse (nonobese diabetic (NOD)/scid/interleukin2 (IL-2) receptor-γ chain knockout) forms the basic platform from which all three EBOV HIS mouse models were engineered. NSG mice lack T and B cells, macrophages, dendritic cells, natural killer cells, and several cytokine receptors, and although they are susceptible to WTEBOV infection, disease can take longer than six weeks to develop29-31. The EBOV HIS mouse models improve upon the NSG mouse model by effectively “humanizing” it through the

7



engraftment of human CD34+ hematopoietic cells (HSC) along with either the expression of transgenic human immune genes or the transplantation of human immune organs. In every case, humanized NSG (hu-NSG) mice recapitulate at least some features of EVD better than the traditional mouse model (Table 3). The first EBOV HIS mouse model to be reported relied on NSG mice expressing human HLA-A2 and engrafted with HSCs purified from umbilical cord blood of HLA-A2+ donors (huNSG-A2)31. hu-NSG-A2 mice showed good engraftment, with the presence of fully differentiated lymphocyte and myeloid cell populations in both lymphoid and peripheral tissues. Following intraperitoneal inoculation with WT-EBOV, all of the animals with high levels of engraftment died, albeit by day 20 post-infection, while only three of the four animals with lower levels of engraftment died, after an even more protracted disease course. In animals with both low and high levels of engraftment, moderate viremia was observed throughout illness, and infectious virus was detected in the typical EBOV target organs (spleen, liver, and kidney) as well as the lung and brain. Accordingly, liver damage was evident, and one out of six necropsied mice showed signs of hemorrhage, although no evidence of coagulopathy was reported. The second reported EBOV HIS mouse model made use of hu-NSG-BLT (bone marrow/liver/thymus) mice, which are NSG mice engrafted with human HSCs and cotransplanted with autologous fetal liver and thymus fragments30. These mice reconstitute a complete human immune system with high levels of engraftment and HLA-restricted T cells, and they have been used extensively to model numerous other virus infections, particularly HIV-132. Intraperitoneal inoculation with WT-EBOV produced severe disease that was uniformly lethal at higher doses, with animals succumbing to disease as soon as 6 days post-infection. Viremia was relatively high, and virus was disseminated to the spleen, liver, kidney, and lung. Liver and adrenal gland damage was extensive and reminiscent of human EBOV infections, although no signs of hemorrhage were observed and coagulation parameters were not investigated. Notably,

8


Page 8 of 36

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the levels of several human cytokines, such as TNF-α, IP-10, and MCP-1, were significantly elevated, indicating a functional response to the infection by human immune cells. The latest EBOV HIS mouse model to be reported used NSG mice engrafted with HSCs and expressing three human cytokines: stem cell factor (SCF), granulocyte/monocyte colonystimulating factor (GM-CSF), and IL-329. These mice, known as hu-NSG-SGM3 mice, exhibited the highest levels of engraftment, with correspondingly high levels of myeloid, T, and B cells. Although hu-NSG-SGM3 mice were susceptible to WT-EBOV infection by both intraperitoneal and intramuscular routes of inoculation, disease was not uniformly lethal, with a maximum of ~67% of infected mice succumbing. Interestingly, relatively high levels of viral RNA were detected in the blood, and high levels of viral RNA and antigen were detected in the spleen and liver; however, the liver and spleen pathology normally associated with EBOV infection was not present. The reasons for this remain unclear, but further investigation (including analysis of blood chemistry, hematology, and coagulopathy) may ultimately provide insight into the mechanisms behind EBOV immunopathology.

GUINEA PIGS Guinea pigs (Cavia porcellus) have been widely used in filovirus research, dating back to the discovery of MARV in 1967. Initial work demonstrated that, upon inoculation with WT filoviruses, outbred guinea pigs developed a mild febrile illness but did not succumb to disease33-36. This early work also demonstrated that serial passaging of MARV increased lethality34-36, and since then several guinea pig-adapted, uniformly lethal variants of EBOV, MARV, RAVV, and SUDV have been established and thoroughly characterized (Table 2)17. All recent reports of guinea pig-adapted (GPA) filovirus pathogenesis in outbred guinea pigs reveal a similar course of disease that mirrors what is observed in NHPs and humans (Table 3)37-40. GPA-MARV resulted in death within 8 to 10 days post-infection, while GPA-RAVV resulted in death at day 1038, 40. Cross et al. did not specifically publish time-to-death data for

9



GPA-EBOV37, although animals appeared to reach the terminal stages of disease around day 8 post-infection, which is within an 8-12 day range consistent with other reports34, 37, 41-42. GPASUDV resulted in death within 9 to 14 days post-infection39. Fever was also reported for GPAMARV-, -RAVV-, and -EBOV-infected animals beginning on day 3 and peaking around day 737-38. GPA-EBOV-infected animals reached peak viremia by day 3 post-infection, while GPA-MARV, SUDV, and -RAVV reached peak viremia at days 7, 9, and 10, respectively37-40. In all infected animals, virus replication occurred systemically and to high levels. Inflammatory markers were elevated in animals infected with GPA-EBOV, -MARV, and -RAVV; however, similar data does not exist for GPA-SUDV-infected animals37-39. Histopathologic lesions in the liver and spleen were typical for filovirus disease, and evidence of apoptosis was observed in GPA-EBOV-, MARV-, and -RAVV-infected animals but not reported for GPA-SUDV-infected animals37-40. Dramatic changes in serum biochemistry late during the disease course provided further evidence of severe organ damage in all infected animals37-40. Likewise, severe coagulopathy in all animals was indicated by thrombocytopenia, along with increases in prothrombin and activated partial thromboplastin times, as well as dysregulated fibrinogen levels37-40. Accordingly, evidence of hemorrhage was reported for GPA-EBOV-, -MARV-, and -RAVV-infected animals; however, no animals appeared to exhibit a petechial rash, which is consistently observed in NHPs and occasionally observed in humans37-40. All animals exhibited lymphopenia, and neutrophilia was reported for GPA-EBOV-, -MARV-, and -RAVV-infected animals37-38. Overall, similar pathogenic processes were observed in inbred (strain 2 and 13) guinea pigs infected with MA-EBOV and an independently derived GPA-EBOV41, 43.

HAMSTERS Compared to the other rodent models of filovirus disease, the Syrian golden hamster (Mesocricetus auratus) model is perhaps the least well-characterized and the most under used, despite the remarkable degree to which it recapitulates both EVD and MVD. Preliminary work

10


Page 10 of 36

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with WT-MARV-infected hamsters was performed following the initial outbreak in 196744-46; however, more detailed characterizations of EBOV and MARV hamster infections were not published until 2013 and 2016, respectively (Table 2)47-48. As is the case with the other immunocompetent rodent models of filovirus infection, the hamster model requires the prior adaptation of the virus to a rodent host, although, interestingly, the EBOV hamster model uses MA-EBOV47, and a hamster-adapted (HA) EBOV has so far not been reported. Conversely, the MARV hamster model relies on a bespoke HA-MARV48. Following intraperitoneal inoculation, both MA-EBOV and HA-MARV cause disease in hamsters that bears remarkable similarity to what is observed in humans and NHPs, displaying almost all the typical clinical hallmarks of infection (Table 3)47-48. Animals infected with MAEBOV underwent a relatively short course of disease, succumbing within 4 to 5 days postinfection, whereas HA-MARV-infected animals succumbed by days 8 to 9. Virus replication was robust and systemic for both viruses, with exceptionally high viremia peaking around days 4 and 5, and high titers of virus detectable in all major organs, including the liver and spleen. Viral replication was reflected in the typically severe lesions detected by histopathology, and significant bystander lymphocyte apoptosis was evident in both models. Moreover, both MAEBOV and HA-MARV infection resulted in a highly dysregulated immune response, characterized in part by the upregulation of several pro-inflammatory cytokines and chemokines in multiple organs. Lymphopenia and neutrophilia were reported for hamsters infected with HAMARV, and suggested by histological examination of MA-EBOV-infected animals. The most striking feature of the hamster model, however, was the severe coagulopathy, substantiated in both MA-EBOV- and HA-MARV-infected hamsters by significantly prolonged clotting times. Interestingly, thrombocytopenia was observed at the terminal stage of disease in MA-EBOVinfected hamsters, but it was not obvious during HA-MARV infection. It is also worth noting that while signs of hemorrhage were evident in both model systems, hamsters infected with HAMARV are the only rodents so far shown to develop a petechial rash.

11



FERRETS The domestic ferret (Mustela putorius furo) has long been used as a model system for human respiratory diseases, in particular influenza49. Thanks to certain anatomic, physiologic, and metabolic features shared between ferrets and humans, the ferret is naturally susceptible to influenza A virus and recapitulates many of the key disease features observed in humans. Indeed, the last 80 years of influenza research are a testament to the value of this animal model and the key contributions its use has made to the field. Ferrets have also been used with varying degrees of success to model infections caused by paramyxoviruses, coronaviruses, rhabdoviruses, togaviruses, and bunyaviruses49. Thus, given the historically widespread use of the ferret model with a variety of different viruses, including numerous mononegaviruses, it was likely only a matter of time before the ferret was finally conscripted for use in ebolavirus research. Recently, three papers were published describing the use of the ferret as an animal model for EBOV, SUDV, and BDBV infection (Table 2)50-52. Disease in these animals is uniformly lethal and remarkably similar to what is observed in NHPs and humans, with robust and systemic virus replication, evidence of multi-organ failure and a dysregulated inflammatory response, and clear signs of coagulopathy. Most notably, the ferret model, like the NHP models, uses outbred and immunocompetent animals that are susceptible to WT—rather than hostadapted—ebolaviruses, thereby overcoming a shortcoming of the rodent models. In general, the disease described for EBOV, SUDV, and BDBV in ferrets, whether using an intramuscular or intranasal inoculation route, followed a similar course and reflected the species-specific differences in virulence typically associated with these three viruses (Table 3)5052

. EBOV appeared to be the most virulent, resulting in death within 5 to 6 days post-infection,

followed by SUDV, which resulted in death within 6 to 9 days, and finally BDBV, which resulted in death within 8 to 9 days. All animals developed a fever around 3 to 4 days post-infection,

12


Page 12 of 36

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


followed by significant weight loss beginning a day or two later. EBOV-infected animals reached peak viremia the earliest, around day 6, while SUDV-infected animals reached peak viremia around day 7-8 and BDBV-infected animals reached peak viremia on day 8. All viruses were detected systemically, with the highest titers generally observed in the liver, spleen, and kidney, regardless of inoculation route. Increased levels of circulating TNF-α and nitric oxide, beginning around 4 days post-infection, suggested a pro-inflammatory response50. Histopathologic lesions typically associated with ebolavirus disease were detected, along with viral antigen, in the livers and spleens of all models, and evidence of lymphocyte apoptosis was reported by one group in the spleens of EBOV-, SUDV-, and BDBV-infected ferrets50. Accordingly, serum biochemistry indicated liver damage, kidney damage, and vascular leakage in all animals. Thrombocytopenia was observed in all animals infected with each virus, as were increases in activated partial thromboplastin time, thrombin time, prothrombin time, and fibrinogen levels, indicating severe consumptive coagulopathy. Indeed, all three viruses caused a petechial rash on most infected animals (with the exception of Kroeker et al.’s SUDV model, which did not exhibit a rash), and evidence of gross internal hemorrhaging caused by EBOV, SUDV, and BDBV was reported by Cross et al.50, 52. The same group reported the typically observed lymphopenia and neutrophilia in all infected ferrets50; whereas, Kroeker et al. reported no significant changes in the hematological parameters of SUDV-infected ferrets52 and Kozak et al. reported lymphopenia in BDBV- but not EBOV-infected ferrets51. Although further work is necessary, the ferret model seems to be a promising intermediate model system of ebolavirus infection, for use prior to NHPs but after rodents.

APPLICATIONS IN COUNTERMEASURE DEVELOPMENT To date, countermeasure development in the filovirus field has followed a common trajectory: novel vaccines or therapeutics are usually first tested in mice, followed by secondary testing in the guinea pig model and eventually confirmatory evaluation in the gold-standard NHP

13



model. The traditional mouse models, whether immunocompetent or immunodeficient, provide a practical and cost-effective first-choice model of evaluation, particularly for evaluating large numbers of candidate therapeutics simultaneously; however, mice do not fully recapitulate human disease and their predictive value can be low, with therapies that work in mice not necessarily equally effective in NHPs19. While guinea pigs may not be completely accurate in predicting therapeutic success in NHPs either18-19, 53, they are generally considered to be a more stringent model since they mimic more hallmark features of EVD and MVD and are therefore the standard choice for follow-up testing after mice. Following this experimental triaging through rodents, promising countermeasures are ultimately evaluated in NHPs, which provide the bestavailable approximation of human filovirus disease. It is important to note, however, that there exist some critical distinctions in filovirus pathophysiology between humans and NHPs— including the occasionally milder presentation of disease in humans—underscoring the unfortunate reality that no model system is perfect7. Nevertheless, testing in these various animal models—particularly NHPs—potentially helps satisfy the FDA’s “animal rule” for developing novel therapeutics: that is, when human clinical trials are either impractical or unethical, as is the case for filovirus infections, efficacy testing can be performed in well characterized, immunocompetent animal models that faithfully recapitulate disease, ideally using WT virus and a realistic challenge dose/route54. Recent developments over the last several years have resulted in the characterization of multiple new—and, in many ways, improved—models of filovirus infection that have the potential to modify the standard pre-clinical assessment of viral countermeasures and help further advance their development (Table 4). In spite of its limitations, the immunocompetent mouse model has been a staple in filovirus research and countermeasure development for nearly twenty years, and the recent development of the CC-RIX and HIS mouse models has the potential to further enhance the utility of these animals. Although more characterization is required, the CC-RIX mouse model of EBOV infection appears to at least theoretically advance the mouse model in two key areas25.

14


Page 14 of 36

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


First, the fact that certain CC-RIX strains exhibit coagulopathy and hemorrhaging brings the immunocompetent mouse model in line with other filovirus models and, presumably, offers a better recapitulation of human disease. This feature also makes the CC-RIX mouse model potentially more suitable for the evaluation of countermeasures that seek to specifically target coagulopathy. Second, the vast, random, and reproducible genetic diversity of CC-RIX strains will not only facilitate the identification of genetic factors that contribute to filovirus disease susceptibility (as has already been demonstrated by Rasmussen et al.), but it will also facilitate the identification of factors that contribute to disease resistance25. Presumably, a greater understanding of such factors will assist in the targeted development of therapeutics; however, in order for the potential of this model system to be realized, additional work is required. Although EBOV infection has been partially characterized in a number of CC-RIX lines, detailed characterization has so far been limited to just two lines: one that exhibits complete resistance to MA-EBOV (strain ID: 15156x1566) and one that exhibits complete susceptibility with coagulopathy (strain ID: 13140x3015)25. Besides demonstrating severe coagulopathy and hemorrhaging, the susceptible strain also displayed typical EBOV cell/tissue tropism and lymphocyte apoptosis; however, hematological, biochemical, and inflammatory parameters remain to be investigated. It must also be kept in mind that CC-RIX mice are not, as yet, commercially available, and the breeding performance of the parental CC-RI strains may be poor, limiting the availability of CC-RIX mice55. Thus, whether any of the CC-RIX mice truly has the capacity to replace the traditional mouse model remains to be determined. Unlike CC-RIX mice, it is improbable that HIS mice (hu-NSG-A2, hu-NSG-BLT, and huNSG-SGM3) will ever serve as the primary initial animal model for filovirus therapeutic evaluation. Nonetheless, these mice do offer several potential advantages to the study of filovirus immunopathogenesis and the development of countermeasures, especially those that target the immune system. Unlike any other filovirus animal model, HIS mice permit interrogation of the interactions that occur specifically at the interface between EBOV infection

15



and human immunity. Indeed, these mice offer the only opportunity to experiment with aspects of the human immune system in an in vivo model. The fact that WT-EBOV can be used to infect these mice via multiple routes of infection further increases the robustness of the models. Whether other filoviruses also cause disease in HIS mice remains to be evaluated, and it will be interesting to see if these models broadly reflect species- or variant-specific differences in virulence, as appears to be the case for EBOV variants Mayinga and Makona in hu-NSG-BLT mice30. HIS mice will also presumably make excellent systems for evaluating vaccines and antiviral therapeutics that aim to specifically modulate the immune system. Although anti-filoviral countermeasures have yet to be evaluated in these models, limited testing of vaccines and therapeutics for other infectious organisms in HIS mouse models is promising and demonstrates proof of principle56-59. Ultimately, however, the usefulness of the HIS mouse models will depend on our understanding of the complex immune processes that they reconstitute, and, to this end, much more work is required. Improving the degree to which the entire human immune system is effectively reconstituted in these animals remains a critical—albeit technical—hurdle, as does improving our understanding of the composition of the reconstituted immune system itself. Interspecies interactions also remain a disadvantage to this model, with the hu-NSG-BLT mice, in particular, susceptible to graft-versus-host disease that limits their experimental longevity. Additionally, the generation of HIS mice requires human donors, which not only increases the practical and ethical issues surrounding their use, but also introduces the possibility of donor-todonor variation, particularly with hu-NSG-BLT mice. Whether such variation represents an advantage or a disadvantage of this model is largely a matter of perspective, but the increase in variability must nevertheless be accounted for in experimental design. Finally, it is also worth noting that the strict housing requirements for immunodeficient mice, along with the subsequent manipulations required to humanize these mice, increase their cost well beyond that of a traditional mouse model, which may impact the use of these animals in efficiently testing large

16


Page 16 of 36

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


numbers of candidate countermeasures simultaneously. As such, HIS mice are unlikely to be first-choice models for countermeasure evaluation, but they will likely serve important roles for downstream testing of specific parameters. In comparison to the other rodent models of filovirus infection, the hamster model offers the closest recapitulation of disease as it is observed in humans (with the notable proviso that time-to-death in MA-EBOV-infected animals is remarkably short). Combined with the typical advantages associated with any other rodent model (i.e., their low cost and ease of handling), and in light of their most glaring disadvantage (i.e., the genetic changes associated with host adaptation), hamsters, in principle, seem to make the best available rodent model for filovirus infections. Indeed, they have been widely used to study a variety of other viral hemorrhagic fevers, all caused by RNA viruses19, and they appear well poised to subsume the role traditionally played by mice in the preliminary evaluation of antiviral drugs and vaccines. Yet, the filovirus field continues to await the widespread adoption of this animal model. Historically, one of the major factors limiting the use of hamsters has been the lack of hamster-specific reagents, although recently developed tools, including transcriptome, kinome, and microarray platforms, in addition to validated RT-qPCR assays for a variety of host response genes, have begun to alleviate this issue60-63. The hamster model is also currently limited to EBOV and MARV, and its adoption may be further hindered by the absence of data demonstrating its predictive value in testing novel countermeasures. However, we expect that the continued adoption of the Syrian golden hamster as a model system, in general, along with the concomitant development of species-specific tools and reagents, will undoubtedly, although gradually, aid in mitigating its shortcomings as a model for filovirus infections. Like hamsters and mice, guinea pigs offer many of the same practical research advantages associated with small-animal models—namely that they are relatively cheap and easy to house and handle—however, guinea pigs also offer additional advantages. The larger size of guinea pigs means a proportionally greater blood volume, which permits an increased

17



frequency of sampling and easier dosing, thereby easing countermeasure evaluation and disease characterization37. Furthermore, the development of a catheterized guinea pig model for EBOV infection simplifies sequential sampling and provides an opportunity to limit the use of sharps in a BSL-4 environment64. Unlike mice, outbred guinea pigs, in particular, seem to more fully recapitulate filovirus disease as it is observed in humans, with the notable manifestation of severe coagulopathy, lymphocyte apoptosis, and an elevated pro-inflammatory response. Moreover, these hallmark features of disease are either less striking or less well characterized in the inbred strain 13 guinea pigs, which are also difficult to acquire and appear to have defective immune responses that may make them less suitable for therapeutic development65-66. Outbred guinea pigs have therefore become a model of choice in which to conduct preliminary evaluations of vaccines and therapeutics, particularly after initial evaluation in mice. Guinea pigs have been successfully used to model EBOV transmission67-68, opening up additional avenues of investigation typically limited to NHPs. Finally, it is noteworthy that the guinea pig is the only immunocompetent rodent model currently available for SUDV, an often-overlooked ebolavirus that nevertheless poses a significant public health threat69. Indeed, a recent publication demonstrated the first successful use of the SUDV guinea pig model in evaluating a panebolavirus neutralizing monoclonal antibody70, highlighting the important role that guinea pigs continue to play in filovirus countermeasure development. As is the case with hamsters, the continued and increased use of guinea pigs as a filovirus model system should eventually help alleviate one of its major disadvantages: the lack of reagents and molecular tools for dissecting, in particular, the host response to infection. Although recent work has offered a glimpse at the guinea pig inflammatory response37-38, a more detailed characterization awaits the development of guinea pig-specific, or at least crossreactive, molecular tools—a goal that depends, in part, on the as yet uncompleted guinea pig genome sequence. Moreover, like the traditional mouse and hamster models, the guinea pig model relies on adapted viruses, which may not truly reflect the WT virus. It must also be kept in

18


Page 18 of 36

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mind that multiple guinea pig-adapted filoviruses have been generated independently, each with a largely unique set of mutations that contribute to virulence in an incompletely understood manner17. As such, a small amount of caution may be necessary when drawing comparisons between different guinea pig-adapted viruses, even within the same study. While rodents of some description will likely always be the first-choice model for evaluation of filovirus countermeasures, the introduction of the ferret as a model for ebolavirus infection provides an important intermediate model for more stringent testing prior to the involvement of NHPs. Indeed, the advantages of the ferret model are numerous. The degree to which ferrets recapitulate ebolavirus disease is truly remarkable, and the importance of being able to use WT instead of host-adapted viruses should not be underestimated, particularly when it comes to quickly and efficiently evaluating novel clinical isolates. Not only does the ferret model allow investigators to sidestep the laborious process of virus adaptation, but it also avoids any questions of relevance regarding the genome mutations acquired during virus adaptation. Compared to NHPs, ferrets are easier to house and handle, cheaper, and less ethically problematic. Larger numbers of animals can therefore be used in experiments, and, unlike rodents, ferrets offer the theoretical advantage of serial sampling. Although ferrets are practically more challenging (and expensive) to work with than rodents, their greater fidelity as an ebolavirus model system may outweigh these inconveniences and make them a first-choice intermediate model system for ebolavirus infections. Moreover, the fact that ferrets are susceptible to both intranasal and intramuscular inoculation permits the modeling of physiologically relevant routes of exposure. This, in combination with evidence of viral shedding from nasal, oral, and rectal mucosa, may also make ferrets a suitable alternative to NHPs in virus transmission modeling51-52. Finally, it must be noted that the ferret is currently the only animal model system other than NHPs that can be used for BDBV infection, and the BDBV ferret model has already been used to demonstrate the protective efficacy of pan-ebolavirus neutralizing monoclonal antibodies70-71. It is encouraging that extensive data from the influenza

19



research field suggests ferrets possess high predictive value for vaccine and therapeutic evaluation49, and we expect the continued use of ferrets for ebolavirus research will reveal the same utility. Ferrets are not without their disadvantages, however. Aside from the logistical challenges that make these animals slightly more difficult to work with than rodents, the biggest disadvantage for the ferret model is the relative paucity of ferret-specific molecular or genetic tools that have been developed and validated. In particular, the immune systems of these animals are poorly characterized, and combined with the lack of reagents, this makes dissecting the host response to ebolavirus infection difficult. Nevertheless, researchers from other fields have been addressing this problem by evaluating the cross-reactivity of existing reagents and developing novel, species-specific reagents72-75. The publication of the ferret transcriptome76 and annotated ferret genome77 should at least permit the quantification of specific gene transcripts over the course of infection—similar to what has been developed for hamsters60— and this should allow for a more detailed characterization of the ferret immune response. Similarly, the development of a CFTR-knockout ferret proves that it is technically feasible to generate transgenic ferrets78, although whether genetic manipulation of ferrets is practical enough to become widespread remains to be seen.

CONCLUSIONS AND FUTURE PERSPECTIVES Over the last several years, the filovirus field has witnessed the introduction of a number of new small animal models, and it has also ushered in numerous advancements to existing models. While none of these new (or improved) small animal models will replace the goldstandard NHP model, they each offer certain key advantages that should better facilitate drug development and promote a keener understanding of filovirus pathogenesis. Nevertheless, more work remains to be done. Notably, with the exception of the outbred guinea pig, the majority of the animal models discussed in detail in this review remain under used—or even

20


Page 20 of 36

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


unused—in filovirus countermeasure development (Table 5). Undoubtedly, this is at least partially due to the relative novelty of most of these model systems; however, without a substantial track record, the true value of these animal models to the filovirus research field is uncertain. In addition to the models discussed in this review, it would also be advantageous to possess a single small animal model in which novel therapeutics and vaccines against all human-pathogenic filoviruses—including the so-called “neglected filoviruses”69—could be evaluated. Unfortunately, the field still lacks a model for TAFV, and the models for BDBV and RAVV remain relatively under-characterized. Moreover, as our understanding of EVD and MVD continues to evolve, especially in light of the West African EBOV outbreak and its repercussions, we must continue to develop animal models in line with the features of disease observed in humans. In particular, it is now becoming clear that viral persistence is a significant aspect of disease79, yet we have few animal models in which we can accurately interrogate this feature80. Indeed, uniformly lethal animal models have been traditionally sought for their stringency in therapeutic evaluation; however, models of viral persistence may be just as valuable in developing countermeasures that can eliminate persistent virus and prevent disease recrudescence. Undoubtedly, this should be a major focus of filovirus animal model development going forward.

21



Page 22 of 36

ACKNOWLEDGEMENTS The authors are grateful to Amber Sturgis for her help in preparing figures.

FUNDING SOURCES This work was supported by the Public Health Agency of Canada (LB, GW, and XQ), the National Key Research and Development Program of China (GW; 2016YFE0205800), and the National Key Program for Infectious Disease of China (GW; 2016ZX10004222). This work was partially supported by grants from the Canadian Institutes of Health Research (XQ; IER143487), the Sanming Project of Medicine in Shenzhen (GW; ZDSYS201504301534057), the Shenzhen

Science

and

Technology

Research

and

Development

Project

(GW;

JCYJ20160427151920801), and the National Natural Science Foundation of China International Cooperation and Exchange Program (GW; 816110193).

ABBREVIATIONS

22


Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


EBOV, Ebola virus; SUDV, Sudan virus; BDBV, Bundibugyo virus; TAFV, Taï Forest Virus; RESTV, Reston virus; EVD, Ebola virus disease; MARV, Marburg virus; RAVV, Ravn virus; MVD, Marburg virus disease; WT, wild type; MA, mouse-adapted; GPA, guinea pig-adapted; HA, hamster-adapted; NHP, nonhuman primate; CC-RIX, collaborative cross recombinant interbred intercrossed; HIS, human immune system; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

CONFLICT OF INTEREST The authors declare no conflict of interest

REFERENCES 1. Feldmann, H.; Sanchez, A.; Geisbert, T. W., Filoviridae: Marburg and Ebola Viruses. In Fields Virology, 6th ed.; Fields, B. N.; Knipe, D. M.; Howley, P. M., Eds. Wolters Kluwer/Lippincott Williams & Wilkins Health: Philadelphia, 2013. 2. Banadyga, L.; Ebihara, H., Epidemiology and Pathogenesis of Filovirus Infections. In Biology and Pathogenesis of Rhabdo- and Filoviruses, Pattnaik, A.; Whitt, M. A., Eds. World Scientific Publishing: Singapore, 2015; pp 453-486. 3. Ebola Situation Report - 30 March 2016. http://apps.who.int/ebola/currentsituation/ebola-situation-report-30-march-2016. 4. Coltart, C. E.; Lindsey, B.; Ghinai, I.; Johnson, A. M.; Heymann, D. L., The Ebola outbreak, 2013-2016: old lessons for new epidemics. Philos Trans R Soc Lond B Biol Sci 2017, 372 (1721), DOI 10.1098/rstb.2016.0297. 5. Baseler, L.; Chertow, D. S.; Johnson, K. M.; Feldmann, H.; Morens, D. M., The Pathogenesis of Ebola Virus Disease. Annu Rev Pathol 2017, 12, 387-418, DOI 10.1146/annurev-pathol-052016-100506. 6. Liu, G.; Wong, G.; Su, S.; Bi, Y.; Plummer, F.; Gao, G. F.; Kobinger, G.; Qiu, X., Clinical Evaluation of Ebola Virus Disease Therapeutics. Trends Mol Med 2017, 23 (9), 820-830, DOI 10.1016/j.molmed.2017.07.002. 7. Shurtleff, A. C.; Warren, T. K.; Bavari, S., Nonhuman primates as models for the discovery and development of ebolavirus therapeutics. Expert Opin Drug Discov 2011, 6 (3), 233-250, DOI 10.1517/17460441.2011.554815. 8. Warfield, K. L.; Alves, D. A.; Bradfute, S. B.; Reed, D. K.; VanTongeren, S.; Kalina, W. V.; Olinger, G. G.; Bavari, S., Development of a model for marburgvirus based on severecombined immunodeficiency mice. Virol J 2007, 4, 108, DOI 10.1186/1743-422X-4-108. 9. Raymond, J.; Bradfute, S.; Bray, M., Filovirus infection of STAT-1 knockout mice. J Infect Dis 2011, 204 Suppl 3, S986-990, DOI 10.1093/infdis/jir335.

23



10. Brannan, J. M.; Froude, J. W.; Prugar, L. I.; Bakken, R. R.; Zak, S. E.; Daye, S. P.; Wilhelmsen, C. E.; Dye, J. M., Interferon alpha/beta Receptor-Deficient Mice as a Model for Ebola Virus Disease. J Infect Dis 2015, 212 Suppl 2, S282-294, DOI 10.1093/infdis/jiv215. 11. Bray, M., The role of the Type I interferon response in the resistance of mice to filovirus infection. J Gen Virol 2001, 82 (Pt 6), 1365-1373, DOI 10.1099/0022-1317-82-6-1365. 12. de Wit, E.; Munster, V. J.; Metwally, S. A.; Feldmann, H., Assessment of rodents as animal models for Reston ebolavirus. J Infect Dis 2011, 204 Suppl 3, S968-972, DOI 10.1093/infdis/jir330. 13. Qiu, X.; Wong, G.; Audet, J.; Cutts, T.; Niu, Y.; Booth, S.; Kobinger, G. P., Establishment and characterization of a lethal mouse model for the Angola strain of Marburg virus. J Virol 2014, 88 (21), 12703-12714, DOI 10.1128/JVI.01643-14. 14. Warfield, K. L.; Bradfute, S. B.; Wells, J.; Lofts, L.; Cooper, M. T.; Alves, D. A.; Reed, D. K.; VanTongeren, S. A.; Mech, C. A.; Bavari, S., Development and characterization of a mouse model for Marburg hemorrhagic fever. J Virol 2009, 83 (13), 6404-6415, DOI 10.1128/JVI.00126-09. 15. Bray, M.; Davis, K.; Geisbert, T.; Schmaljohn, C.; Huggins, J., A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J Infect Dis 1998, 178 (3), 651-661, DOI. 16. Lofts, L. L.; Wells, J. B.; Bavari, S.; Warfield, K. L., Key genomic changes necessary for an in vivo lethal mouse marburgvirus variant selection process. J Virol 2011, 85 (8), 3905-3917, DOI 10.1128/JVI.02372-10. 17. Banadyga, L.; Dolan, M. A.; Ebihara, H., Rodent-Adapted Filoviruses and the Molecular Basis of Pathogenesis. J Mol Biol 2016, 428 (17), 3449-3466, DOI 10.1016/j.jmb.2016.05.008. 18. Bradfute, S. B.; Warfield, K. L.; Bray, M., Mouse models for filovirus infections. Viruses 2012, 4 (9), 1477-1508, DOI 10.3390/v4091477. 19. Wahl-Jensen, V.; Bollinger, L.; Safronetz, D.; de Kok-Mercado, F.; Scott, D. P.; Ebihara, H., Use of the Syrian hamster as a new model of ebola virus disease and other viral hemorrhagic fevers. Viruses 2012, 4 (12), 3754-3784, DOI 10.3390/v4123754. 20. St Claire, M. C.; Ragland, D. R.; Bollinger, L.; Jahrling, P. B., Animal Models of Ebolavirus Infection. Comp Med 2017, 67 (3), 253-262, DOI. 21. Shurtleff, A. C.; Bavari, S., Animal models for ebolavirus countermeasures discovery: what defines a useful model? Expert Opin Drug Discov 2015, 10 (7), 685-702, DOI 10.1517/17460441.2015.1035252. 22. Nakayama, E.; Saijo, M., Animal models for Ebola and Marburg virus infections. Front Microbiol 2013, 4, 267, DOI 10.3389/fmicb.2013.00267. 23. Yamaoka, S.; Banadyga, L.; Bray, M.; Ebihara, H., Small Animal Models for Studying Filovirus Pathogenesis. Curr Top Microbiol Immunol 2017, DOI 10.1007/82_2017_9. 24. Threadgill, D. W.; Miller, D. R.; Churchill, G. A.; de Villena, F. P., The collaborative cross: a recombinant inbred mouse population for the systems genetic era. ILAR J 2011, 52 (1), 24-31, DOI. 25. Rasmussen, A. L.; Okumura, A.; Ferris, M. T.; Green, R.; Feldmann, F.; Kelly, S. M.; Scott, D. P.; Safronetz, D.; Haddock, E.; LaCasse, R.; Thomas, M. J.; Sova, P.; Carter, V. S.; Weiss, J. M.; Miller, D. R.; Shaw, G. D.; Korth, M. J.; Heise, M. T.; Baric, R. S.; de Villena, F. P.; Feldmann, H.; Katze, M. G., Host genetic diversity enables Ebola hemorrhagic fever pathogenesis and resistance. Science 2014, 346 (6212), 987-991, DOI 10.1126/science.1259595. 26. Spengler, J. R.; Prescott, J.; Feldmann, H.; Spiropoulou, C. F., Human immune system mouse models of Ebola virus infection. Curr Opin Virol 2017, 25, 90-96, DOI 10.1016/j.coviro.2017.07.028.

24


Page 24 of 36

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


27. Shultz, L. D.; Brehm, M. A.; Garcia-Martinez, J. V.; Greiner, D. L., Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol 2012, 12 (11), 786-798, DOI 10.1038/nri3311. 28. Ernst, W., Humanized mice in infectious diseases. Comp Immunol Microbiol Infect Dis 2016, 49, 29-38, DOI 10.1016/j.cimid.2016.08.006. 29. Spengler, J. R.; Lavender, K. J.; Martellaro, C.; Carmody, A.; Kurth, A.; Keck, J. G.; Saturday, G.; Scott, D. P.; Nichol, S. T.; Hasenkrug, K. J.; Spiropoulou, C. F.; Feldmann, H.; Prescott, J., Ebola Virus Replication and Disease Without Immunopathology in Mice Expressing Transgenes to Support Human Myeloid and Lymphoid Cell Engraftment. J Infect Dis 2016, 214 (suppl 3), S308-S318, DOI 10.1093/infdis/jiw248. 30. Bird, B. H.; Spengler, J. R.; Chakrabarti, A. K.; Khristova, M. L.; Sealy, T. K.; ColemanMcCray, J. D.; Martin, B. E.; Dodd, K. A.; Goldsmith, C. S.; Sanders, J.; Zaki, S. R.; Nichol, S. T.; Spiropoulou, C. F., Humanized Mouse Model of Ebola Virus Disease Mimics the Immune Responses in Human Disease. J Infect Dis 2016, 213 (5), 703-711, DOI 10.1093/infdis/jiv538. 31. Ludtke, A.; Oestereich, L.; Ruibal, P.; Wurr, S.; Pallasch, E.; Bockholt, S.; Ip, W. H.; Rieger, T.; Gomez-Medina, S.; Stocking, C.; Rodriguez, E.; Gunther, S.; Munoz-Fontela, C., Ebola virus disease in mice with transplanted human hematopoietic stem cells. J Virol 2015, 89 (8), 4700-4704, DOI 10.1128/JVI.03546-14. 32. Karpel, M. E.; Boutwell, C. L.; Allen, T. M., BLT humanized mice as a small animal model of HIV infection. Curr Opin Virol 2015, 13, 75-80, DOI 10.1016/j.coviro.2015.05.002. 33. Bowen, E. T.; Lloyd, G.; Harris, W. J.; Platt, G. S.; Baskerville, A.; Vella, E. E., Viral haemorrhagic fever in southern Sudan and northern Zaire. Preliminary studies on the aetiological agent. Lancet 1977, 1 (8011), 571-573, DOI. 34. Simpson, D. I.; Zlotnik, I.; Rutter, D. A., Vervet monkey disease. Experiment infection of guinea pigs and monkeys with the causative agent. Br J Exp Pathol 1968, 49 (5), 458-464, DOI. 35. Smith, C. E.; Simpson, D. I.; Bowen, E. T.; Zlotnik, I., Fatal human disease from vervet monkeys. Lancet 1967, 2 (7526), 1119-1121, DOI. 36. Siegert, R.; Shu, H. L.; Slenczka, H. L.; Peters, D.; Muller, G., The aetiology of an unknown human infection transmitted by monkeys (preliminary communication). Ger Med Mon 1968, 13 (1), 1-2, DOI. 37. Cross, R. W.; Fenton, K. A.; Geisbert, J. B.; Mire, C. E.; Geisbert, T. W., Modeling the Disease Course of Zaire ebolavirus Infection in the Outbred Guinea Pig. J Infect Dis 2015, 212 Suppl 2, S305-315, DOI 10.1093/infdis/jiv237. 38. Cross, R. W.; Fenton, K. A.; Geisbert, J. B.; Ebihara, H.; Mire, C. E.; Geisbert, T. W., Comparison of the Pathogenesis of the Angola and Ravn Strains of Marburg Virus in the Outbred Guinea Pig Model. J Infect Dis 2015, 212 Suppl 2, S258-270, DOI 10.1093/infdis/jiv182. 39. Wong, G.; He, S.; Wei, H.; Kroeker, A.; Audet, J.; Leung, A.; Cutts, T.; Graham, J.; Kobasa, D.; Embury-Hyatt, C.; Kobinger, G. P.; Qiu, X., Development and Characterization of a Guinea Pig-Adapted Sudan Virus. J Virol 2015, 90 (1), 392-399, DOI 10.1128/JVI.02331-15. 40. Wong, G.; Cao, W.; He, S.; Zhang, Z.; Zhu, W.; Moffat, E.; Ebihara, H.; Embury-Hyatt, C.; Qiu, X., Development and characterization of a guinea pig model for Marburg virus. Zoological Research 2017, 38 (5), 1-11, DOI. 41. Connolly, B. M.; Steele, K. E.; Davis, K. J.; Geisbert, T. W.; Kell, W. M.; Jaax, N. K.; Jahrling, P. B., Pathogenesis of experimental Ebola virus infection in guinea pigs. J Infect Dis 1999, 179 Suppl 1, S203-217, DOI 10.1086/514305. 42. Subbotina, E.; Dadaeva, A.; Kachko, A.; Chepurnov, A., Genetic factors of Ebola virus virulence in guinea pigs. Virus Res 2010, 153 (1), 121-133, DOI 10.1016/j.virusres.2010.07.015. 43. Bray, M.; Hatfill, S.; Hensley, L.; Huggins, J. W., Haematological, biochemical and coagulation changes in mice, guinea-pigs and monkeys infected with a mouse-adapted variant of Ebola Zaire virus. J Comp Pathol 2001, 125 (4), 243-253, DOI 10.1053/jcpa.2001.0503.

25



44. Simpson, D. I., Vervent monkey disease. Transmission to the hamster. Br J Exp Pathol 1969, 50 (4), 389-392, DOI. 45. Zlotnik, I.; Simpson, D. I., The pathology of experimental vervet monkey disease in hamsters. Br J Exp Pathol 1969, 50 (4), 393-399, DOI. 46. Zlotnik, I., “Marburg Disease” The Pathology of Experimentally Infected Hamsters. In Marburg Virus Disease, Martini, G. A.; Siegert, R., Eds. Springer: New York, 1971; pp 129-135. 47. Ebihara, H.; Zivcec, M.; Gardner, D.; Falzarano, D.; LaCasse, R.; Rosenke, R.; Long, D.; Haddock, E.; Fischer, E.; Kawaoka, Y.; Feldmann, H., A Syrian golden hamster model recapitulating ebola hemorrhagic fever. J Infect Dis 2013, 207 (2), 306-318, DOI 10.1093/infdis/jis626. 48. Marzi, A.; Banadyga, L.; Haddock, E.; Thomas, T.; Shen, K.; Horne, E. J.; Scott, D. P.; Feldmann, H.; Ebihara, H., A hamster model for Marburg virus infection accurately recapitulates Marburg hemorrhagic fever. Sci Rep 2016, 6, 39214, DOI 10.1038/srep39214. 49. Enkirch, T.; von Messling, V., Ferret models of viral pathogenesis. Virology 2015, 479480, 259-270, DOI 10.1016/j.virol.2015.03.017. 50. Cross, R. W.; Mire, C. E.; Borisevich, V.; Geisbert, J. B.; Fenton, K. A.; Geisbert, T. W., The Domestic Ferret (Mustela putorius furo) as a Lethal Infection Model for 3 Species of Ebolavirus. J Infect Dis 2016, 214 (4), 565-569, DOI 10.1093/infdis/jiw209. 51. Kozak, R.; He, S.; Kroeker, A.; de La Vega, M. A.; Audet, J.; Wong, G.; Urfano, C.; Antonation, K.; Embury-Hyatt, C.; Kobinger, G. P.; Qiu, X., Ferrets Infected with Bundibugyo Virus or Ebola Virus Recapitulate Important Aspects of Human Filovirus Disease. J Virol 2016, 90 (20), 9209-9223, DOI 10.1128/JVI.01033-16. 52. Kroeker, A.; He, S.; de La Vega, M. A.; Wong, G.; Embury-Hyatt, C.; Qiu, X., Characterization of Sudan Ebolavirus infection in ferrets. Oncotarget 2017, 8 (28), 46262-46272, DOI 10.18632/oncotarget.17694. 53. Feldmann, H.; Jones, S.; Klenk, H. D.; Schnittler, H. J., Ebola virus: from discovery to vaccine. Nat Rev Immunol 2003, 3 (8), 677-685, DOI 10.1038/nri1154. 54. Snoy, P. J., Establishing efficacy of human products using animals: the US food and drug administration's "animal rule". Vet Pathol 2010, 47 (5), 774-778, DOI 10.1177/0300985810372506. 55. UNC Systems Genetics: Available Strains. https://csbio.unc.edu/CCstatus/index.py?run=availableLines (accessed January 25, 2018). 56. Cheng, L.; Zhang, Z.; Li, G.; Li, F.; Wang, L.; Zhang, L.; Zurawski, S. M.; Zurawski, G.; Levy, Y.; Su, L., Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system. Vaccine 2017, 35 (45), 6143-6153, DOI 10.1016/j.vaccine.2017.09.052. 57. Yao, Y.; Lai, R.; Afkhami, S.; Haddadi, S.; Zganiacz, A.; Vahedi, F.; Ashkar, A. A.; Kaushic, C.; Jeyanathan, M.; Xing, Z., Enhancement of Antituberculosis Immunity in a Humanized Model System by a Novel Virus-Vectored Respiratory Mucosal Vaccine. J Infect Dis 2017, 216 (1), 135-145, DOI 10.1093/infdis/jix252. 58. Mishra, S.; Lavelle, B. J.; Desrosiers, J.; Ardito, M. T.; Terry, F.; Martin, W. D.; De Groot, A. S.; Gregory, S. H., Dendritic cell-mediated, DNA-based vaccination against hepatitis C induces the multi-epitope-specific response of humanized, HLA transgenic mice. PLoS One 2014, 9 (8), e104606, DOI 10.1371/journal.pone.0104606. 59. Matsuda, G.; Imadome, K.; Kawano, F.; Mochizuki, M.; Ochiai, N.; Morio, T.; Shimizu, N.; Fujiwara, S., Cellular immunotherapy with ex vivo expanded cord blood T cells in a humanized mouse model of EBV-associated lymphoproliferative disease. Immunotherapy 2015, 7 (4), 335-341, DOI 10.2217/imt.15.2. 60. Zivcec, M.; Safronetz, D.; Haddock, E.; Feldmann, H.; Ebihara, H., Validation of assays to monitor immune responses in the Syrian golden hamster (Mesocricetus auratus). J Immunol Methods 2011, 368 (1-2), 24-35, DOI 10.1016/j.jim.2011.02.004.

26


Page 26 of 36

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


61. Tchitchek, N.; Safronetz, D.; Rasmussen, A. L.; Martens, C.; Virtaneva, K.; Porcella, S. F.; Feldmann, H.; Ebihara, H.; Katze, M. G., Sequencing, annotation and analysis of the Syrian hamster (Mesocricetus auratus) transcriptome. PLoS One 2014, 9 (11), e112617, DOI 10.1371/journal.pone.0112617. 62. Falcinelli, S.; Gowen, B. B.; Trost, B.; Napper, S.; Kusalik, A.; Johnson, R. F.; Safronetz, D.; Prescott, J.; Wahl-Jensen, V.; Jahrling, P. B.; Kindrachuk, J., Characterization of the host response to pichinde virus infection in the Syrian golden hamster by species-specific kinome analysis. Mol Cell Proteomics 2015, 14 (3), 646-657, DOI 10.1074/mcp.M114.045443. 63. Ying, B.; Toth, K.; Spencer, J. F.; Aurora, R.; Wold, W. S., Transcriptome sequencing and development of an expression microarray platform for liver infection in adenovirus type 5infected Syrian golden hamsters. Virology 2015, 485, 305-312, DOI 10.1016/j.virol.2015.07.024. 64. Dowall, S.; Taylor, I.; Yeates, P.; Smith, L.; Rule, A.; Easterbrook, L.; Bruce, C.; Cook, N.; Corbin-Lickfett, K.; Empig, C.; Schlunegger, K.; Graham, V.; Dennis, M.; Hewson, R., Catheterized guinea pigs infected with Ebola Zaire virus allows safer sequential sampling to determine the pharmacokinetic profile of a phosphatidylserine-targeting monoclonal antibody. Antiviral Res 2013, 97 (2), 108-111, DOI 10.1016/j.antiviral.2012.11.003. 65. Munoz, J., Comparison between Hartley and strain 13 guinea pigs--antibody production and delayed hypersensitivity. J Immunol 1967, 99 (1), 31-39, DOI. 66. Stone, S. H., Differences in reactivity associated with sex or strain of inbred or randombred guinea pigs in the massive hemorrhagic reaction and other manifestations of delayed hypersensitivity. Int Arch Allergy Appl Immunol 1962, 20, 193-202, DOI. 67. Wong, G.; Richardson, J. S.; Cutts, T.; Qiu, X.; Kobinger, G. P., Intranasal immunization with an adenovirus vaccine protects guinea pigs from Ebola virus transmission by infected animals. Antiviral Res 2015, 116, 17-19, DOI 10.1016/j.antiviral.2015.01.001. 68. Wong, G.; Qiu, X.; Richardson, J. S.; Cutts, T.; Collignon, B.; Gren, J.; Aviles, J.; Embury-Hyatt, C.; Kobinger, G. P., Ebola virus transmission in guinea pigs. J Virol 2015, 89 (2), 1314-1323, DOI 10.1128/JVI.02836-14. 69. Burk, R.; Bollinger, L.; Johnson, J. C.; Wada, J.; Radoshitzky, S. R.; Palacios, G.; Bavari, S.; Jahrling, P. B.; Kuhn, J. H., Neglected filoviruses. FEMS Microbiol Rev 2016, 40 (4), 494519, DOI 10.1093/femsre/fuw010. 70. Zhao, X.; Howell, K. A.; He, S.; Brannan, J. M.; Wec, A. Z.; Davidson, E.; Turner, H. L.; Chiang, C. I.; Lei, L.; Fels, J. M.; Vu, H.; Shulenin, S.; Turonis, A. N.; Kuehne, A. I.; Liu, G.; Ta, M.; Wang, Y.; Sundling, C.; Xiao, Y.; Spence, J. S.; Doranz, B. J.; Holtsberg, F. W.; Ward, A. B.; Chandran, K.; Dye, J. M.; Qiu, X.; Li, Y.; Aman, M. J., Immunization-Elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability. Cell 2017, 169 (5), 891-904 e815, DOI 10.1016/j.cell.2017.04.038. 71. Wec, A. Z.; Herbert, A. S.; Murin, C. D.; Nyakatura, E. K.; Abelson, D. M.; Fels, J. M.; He, S.; James, R. M.; de La Vega, M. A.; Zhu, W.; Bakken, R. R.; Goodwin, E.; Turner, H. L.; Jangra, R. K.; Zeitlin, L.; Qiu, X.; Lai, J. R.; Walker, L. M.; Ward, A. B.; Dye, J. M.; Chandran, K.; Bornholdt, Z. A., Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses. Cell 2017, 169 (5), 878-890 e815, DOI 10.1016/j.cell.2017.04.037. 72. Layton, D. S.; Xiao, X.; Bentley, J. D.; Lu, L.; Stewart, C. R.; Bean, A. G.; Adams, T. E., Development of an anti-ferret CD4 monoclonal antibody for the characterisation of ferret T lymphocytes. J Immunol Methods 2017, 444, 29-35, DOI 10.1016/j.jim.2017.02.009. 73. Pillet, S.; Kobasa, D.; Meunier, I.; Gray, M.; Laddy, D.; Weiner, D. B.; von Messling, V.; Kobinger, G. P., Cellular immune response in the presence of protective antibody levels correlates with protection against 1918 influenza in ferrets. Vaccine 2011, 29 (39), 6793-6801, DOI 10.1016/j.vaccine.2010.12.059. 74. Fang, Y.; Rowe, T.; Leon, A. J.; Banner, D.; Danesh, A.; Xu, L.; Ran, L.; Bosinger, S. E.; Guan, Y.; Chen, H.; Cameron, C. C.; Cameron, M. J.; Kelvin, D. J., Molecular characterization

27



of in vivo adjuvant activity in ferrets vaccinated against influenza virus. J Virol 2010, 84 (17), 8369-8388, DOI 10.1128/JVI.02305-09. 75. Rutigliano, J. A.; Doherty, P. C.; Franks, J.; Morris, M. Y.; Reynolds, C.; Thomas, P. G., Screening monoclonal antibodies for cross-reactivity in the ferret model of influenza infection. J Immunol Methods 2008, 336 (1), 71-77, DOI 10.1016/j.jim.2008.04.003. 76. Bruder, C. E.; Yao, S.; Larson, F.; Camp, J. V.; Tapp, R.; McBrayer, A.; Powers, N.; Granda, W. V.; Jonsson, C. B., Transcriptome sequencing and development of an expression microarray platform for the domestic ferret. BMC Genomics 2010, 11, 251, DOI 10.1186/14712164-11-251. 77. Peng, X.; Alfoldi, J.; Gori, K.; Eisfeld, A. J.; Tyler, S. R.; Tisoncik-Go, J.; Brawand, D.; Law, G. L.; Skunca, N.; Hatta, M.; Gasper, D. J.; Kelly, S. M.; Chang, J.; Thomas, M. J.; Johnson, J.; Berlin, A. M.; Lara, M.; Russell, P.; Swofford, R.; Turner-Maier, J.; Young, S.; Hourlier, T.; Aken, B.; Searle, S.; Sun, X.; Yi, Y.; Suresh, M.; Tumpey, T. M.; Siepel, A.; Wisely, S. M.; Dessimoz, C.; Kawaoka, Y.; Birren, B. W.; Lindblad-Toh, K.; Di Palma, F.; Engelhardt, J. F.; Palermo, R. E.; Katze, M. G., The draft genome sequence of the ferret (Mustela putorius furo) facilitates study of human respiratory disease. Nat Biotechnol 2014, 32 (12), 1250-1255, DOI 10.1038/nbt.3079. 78. Sun, X.; Yan, Z.; Yi, Y.; Li, Z.; Lei, D.; Rogers, C. S.; Chen, J.; Zhang, Y.; Welsh, M. J.; Leno, G. H.; Engelhardt, J. F., Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets. J Clin Invest 2008, 118 (4), 1578-1583, DOI 10.1172/JCI34599. 79. Caviness, K.; Kuhn, J. H.; Palacios, G., Ebola virus persistence as a new focus in clinical research. Curr Opin Virol 2017, 23, 43-48, DOI 10.1016/j.coviro.2017.02.006. 80. Gupta, M.; Mahanty, S.; Greer, P.; Towner, J. S.; Shieh, W. J.; Zaki, S. R.; Ahmed, R.; Rollin, P. E., Persistent infection with ebola virus under conditions of partial immunity. J Virol 2004, 78 (2), 958-967, DOI. 81. Dowall, S. D.; Bosworth, A.; Watson, R.; Bewley, K.; Taylor, I.; Rayner, E.; Hunter, L.; Pearson, G.; Easterbrook, L.; Pitman, J.; Hewson, R.; Carroll, M. W., Chloroquine inhibited Ebola virus replication in vitro but failed to protect against infection and disease in the in vivo guinea pig model. J Gen Virol 2015, 96 (12), 3484-3492, DOI 10.1099/jgv.0.000309. 82. Madrid, P. B.; Panchal, R. G.; Warren, T. K.; Shurtleff, A. C.; Endsley, A. N.; Green, C. E.; Kolokoltsov, A.; Davey, R.; Manger, I. D.; Gilfillan, L.; Bavari, S.; Tanga, M. J., Evaluation of Ebola Virus Inhibitors for Drug Repurposing. ACS Infect Dis 2015, 1 (7), 317-326, DOI 10.1021/acsinfecdis.5b00030. 83. Richardson, J. S.; Pillet, S.; Bello, A. J.; Kobinger, G. P., Airway delivery of an adenovirus-based Ebola virus vaccine bypasses existing immunity to homologous adenovirus in nonhuman primates. J Virol 2013, 87 (7), 3668-3677, DOI 10.1128/JVI.02864-12. 84. Martins, K.; Carra, J. H.; Cooper, C. L.; Kwilas, S. A.; Robinson, C. G.; Shurtleff, A. C.; Schokman, R. D.; Kuehl, K. A.; Wells, J. B.; Steffens, J. T.; van Tongeren, S. A.; Hooper, J. W.; Bavari, S., Cross-protection conferred by filovirus virus-like particles containing trimeric hybrid glycoprotein. Viral Immunol 2015, 28 (1), 62-70, DOI 10.1089/vim.2014.0071. 85. Mire, C. E.; Geisbert, J. B.; Versteeg, K. M.; Mamaeva, N.; Agans, K. N.; Geisbert, T. W.; Connor, J. H., A Single-Vector, Single-Injection Trivalent Filovirus Vaccine: Proof of Concept Study in Outbred Guinea Pigs. J Infect Dis 2015, 212 Suppl 2, S384-388, DOI 10.1093/infdis/jiv126. 86. Matassov, D.; Marzi, A.; Latham, T.; Xu, R.; Ota-Setlik, A.; Feldmann, F.; Geisbert, J. B.; Mire, C. E.; Hamm, S.; Nowak, B.; Egan, M. A.; Geisbert, T. W.; Eldridge, J. H.; Feldmann, H.; Clarke, D. K., Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus. J Infect Dis 2015, 212 Suppl 2, S443-451, DOI 10.1093/infdis/jiv316. 87. Cong, Y.; Dyall, J.; Hart, B. J.; DeWald, L. E.; Johnson, J. C.; Postnikova, E.; Zhou, H.; Gross, R.; Rojas, O.; Alexander, I.; Josleyn, N.; Zhang, T.; Michelotti, J.; Janosko, K.; Glass, P.

28


Page 28 of 36

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


J.; Flint, M.; McMullan, L. K.; Spiropoulou, C. F.; Mierzwa, T.; Guha, R.; Shinn, P.; Michael, S.; Klumpp-Thomas, C.; McKnight, C.; Thomas, C.; Eakin, A. E.; O'Loughlin, K. G.; Green, C. E.; Catz, P.; Mirsalis, J. C.; Honko, A. N.; Olinger, G. G., Jr.; Bennett, R. S.; Holbrook, M. R.; Hensley, L. E.; Jahrling, P. B., Evaluation of the Activity of Lamivudine and Zidovudine against Ebola Virus. PLoS One 2016, 11 (11), e0166318, DOI 10.1371/journal.pone.0166318. 88. Miller, J. L.; Spiro, S. G.; Dowall, S. D.; Taylor, I.; Rule, A.; Alonzi, D. S.; Sayce, A. C.; Wright, E.; Bentley, E. M.; Thom, R.; Hall, G.; Dwek, R. A.; Hewson, R.; Zitzmann, N., Minimal In Vivo Efficacy of Iminosugars in a Lethal Ebola Virus Guinea Pig Model. PLoS One 2016, 11 (11), e0167018, DOI 10.1371/journal.pone.0167018. 89. Dowall, S. D.; Bewley, K.; Watson, R. J.; Vasan, S. S.; Ghosh, C.; Konai, M. M.; Gausdal, G.; Lorens, J. B.; Long, J.; Barclay, W.; Garcia-Dorival, I.; Hiscox, J.; Bosworth, A.; Taylor, I.; Easterbrook, L.; Pitman, J.; Summers, S.; Chan-Pensley, J.; Funnell, S.; Vipond, J.; Charlton, S.; Haldar, J.; Hewson, R.; Carroll, M. W., Antiviral Screening of Multiple Compounds against Ebola Virus. Viruses 2016, 8 (11), DOI 10.3390/v8110277. 90. Dowall, S. D.; Bosworth, A.; Rayner, E.; Taylor, I.; Landon, J.; Cameron, I.; Coxon, R.; Al Abdulla, I.; Graham, V. A.; Hall, G.; Kobinger, G.; Hewson, R.; Carroll, M. W., Post-exposure treatment of Ebola virus disease in guinea pigs using EBOTAb, an ovine antibody-based therapeutic. Sci Rep 2016, 6, 30497, DOI 10.1038/srep30497. 91. Dowall, S. D.; Callan, J.; Zeltina, A.; Al-Abdulla, I.; Strecker, T.; Fehling, S. K.; Krahling, V.; Bosworth, A.; Rayner, E.; Taylor, I.; Charlton, S.; Landon, J.; Cameron, I.; Hewson, R.; Nasidi, A.; Bowden, T. A.; Carroll, M. W., Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection. J Infect Dis 2016, 213 (7), 1124-1133, DOI 10.1093/infdis/jiv565. 92. Dowall, S. D.; Jacquot, F.; Landon, J.; Rayner, E.; Hall, G.; Carbonnelle, C.; Raoul, H.; Pannetier, D.; Cameron, I.; Coxon, R.; Al Abdulla, I.; Hewson, R.; Carroll, M. W., Post-exposure treatment of non-human primates lethally infected with Ebola virus with EBOTAb, a purified ovine IgG product. Sci Rep 2017, 7 (1), 4099, DOI 10.1038/s41598-017-03910-7. 93. Reynard, O.; Jacquot, F.; Evanno, G.; Mai, H. L.; Salama, A.; Martinet, B.; Duvaux, O.; Bach, J. M.; Conchon, S.; Judor, J. P.; Perota, A.; Lagutina, I.; Duchi, R.; Lazzari, G.; Le Berre, L.; Perreault, H.; Lheriteau, E.; Raoul, H.; Volchkov, V.; Galli, C.; Soulillou, J. P., Anti-EBOV GP IgGs Lacking alpha1-3-Galactose and Neu5Gc Prolong Survival and Decrease Blood Viral Load in EBOV-Infected Guinea Pigs. PLoS One 2016, 11 (6), e0156775, DOI 10.1371/journal.pone.0156775. 94. Zheng, X.; Wong, G.; Zhao, Y.; Wang, H.; He, S.; Bi, Y.; Chen, W.; Jin, H.; Gai, W.; Chu, D.; Cao, Z.; Wang, C.; Fan, Q.; Chi, H.; Gao, Y.; Wang, T.; Feng, N.; Yan, F.; Huang, G.; Zheng, Y.; Li, N.; Li, Y.; Qian, J.; Zou, Y.; Kobinger, G.; Gao, G. F.; Qiu, X.; Yang, S.; Xia, X., Treatment with hyperimmune equine immunoglobulin or immunoglobulin fragments completely protects rodents from Ebola virus infection. Sci Rep 2016, 6, 24179, DOI 10.1038/srep24179. 95. Howell, K. A.; Qiu, X.; Brannan, J. M.; Bryan, C.; Davidson, E.; Holtsberg, F. W.; Wec, A. Z.; Shulenin, S.; Biggins, J. E.; Douglas, R.; Enterlein, S. G.; Turner, H. L.; Pallesen, J.; Murin, C. D.; He, S.; Kroeker, A.; Vu, H.; Herbert, A. S.; Fusco, M. L.; Nyakatura, E. K.; Lai, J. R.; Keck, Z. Y.; Foung, S. K. H.; Saphire, E. O.; Zeitlin, L.; Ward, A. B.; Chandran, K.; Doranz, B. J.; Kobinger, G. P.; Dye, J. M.; Aman, M. J., Antibody Treatment of Ebola and Sudan Virus Infection via a Uniquely Exposed Epitope within the Glycoprotein Receptor-Binding Site. Cell Rep 2016, 15 (7), 1514-1526, DOI 10.1016/j.celrep.2016.04.026. 96. Wu, S.; Kroeker, A.; Wong, G.; He, S.; Hou, L.; Audet, J.; Wei, H.; Zhang, Z.; Fernando, L.; Soule, G.; Tran, K.; Bi, S.; Zhu, T.; Yu, X.; Chen, W.; Qiu, X., An Adenovirus Vaccine Expressing Ebola Virus Variant Makona Glycoprotein Is Efficacious in Guinea Pigs and Nonhuman Primates. J Infect Dis 2016, 214 (suppl 3), S326-S332, DOI 10.1093/infdis/jiw250.

29



97. Meyer, M.; Huang, E.; Yuzhakov, O.; Ramanathan, P.; Ciaramella, G.; Bukreyev, A., Modified mRNA-Based Vaccines Elicit Robust Immune Responses and Protect Guinea Pigs From Ebola Virus Disease. J Infect Dis 2018, 217 (3), 451-455, DOI 10.1093/infdis/jix592. 98. Domi, A.; Feldmann, F.; Basu, R.; McCurley, N.; Shifflett, K.; Emanuel, J.; Hellerstein, M. S.; Guirakhoo, F.; Orlandi, C.; Flinko, R.; Lewis, G. K.; Hanley, P. W.; Feldmann, H.; Robinson, H. L.; Marzi, A., A Single Dose of Modified Vaccinia Ankara expressing Ebola Virus Like Particles Protects Nonhuman Primates from Lethal Ebola Virus Challenge. Sci Rep 2018, 8 (1), 864, DOI 10.1038/s41598-017-19041-y. 99. Mire, C. E.; Geisbert, J. B.; Borisevich, V.; Fenton, K. A.; Agans, K. N.; Flyak, A. I.; Deer, D. J.; Steinkellner, H.; Bohorov, O.; Bohorova, N.; Goodman, C.; Hiatt, A.; Kim, D. H.; Pauly, M. H.; Velasco, J.; Whaley, K. J.; Crowe, J. E., Jr.; Zeitlin, L.; Geisbert, T. W., Therapeutic treatment of Marburg and Ravn virus infection in nonhuman primates with a human monoclonal antibody. Sci Transl Med 2017, 9 (384), DOI 10.1126/scitranslmed.aai8711. 100. Falzarano, D.; Safronetz, D.; Prescott, J.; Marzi, A.; Feldmann, F.; Feldmann, H., Lack of protection against ebola virus from chloroquine in mice and hamsters. Emerg Infect Dis 2015, 21 (6), 1065-1067, DOI 10.3201/eid2106.150176.

30


Page 30 of 36

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Table 1: Filoviruses ORDER

FAMILY

GENUS

Ebolavirus Mononegavirales

Filoviridae

a

SPECIES

VIRUS

DISEASE

Zaire ebolavirus

Ebola virus (EBOV)

EVD

Sudan ebolavirus

Sudan virus (SUDV)

EVD

Bundibugyo ebolavirus

Bundibugyo virus (BDBV)

EVD

Taï Forest ebolavirus

Taï Forest virus (TAFV)

EVD

Reston ebolavirus

Reston virus (RESTV)

-

Marburg virus (MARV)

MVD

Ravn virus (RAVV)

MVD

Lloviu virus (LLOV)

?

Marburgvirus

Marburg marburgvirus

Cuevavirus

Lloviu cuevavirus

a

In humans EVD, Ebola virus disease; MVD, Marburg virus disease


ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 32 of 36

Table 2: Filoviruses Used in Various Small Animal Models MOUSE CC-RIX

Immunocompetent Immunodeficient

EBOV

MA

WT

9-11

SUDV

-

WT

9, 10

BDBV

-

TAFV RESTV

15

HIS

25

29-31

50, 51

-

WT

50, 52

-

-

WT

50, 51

-

-

-

-

-

-

-

-

-

-

-

GPA

-

-

-

-

-

-

-

-

-

-

-

WT

13, 16

MARV

MA

RAVV

MA

14, 16

WT

9, 11

9

9,11

, GPA 8 MA 11

, 8

WT , GPA , MA

WT, wild type; MA, mouse-adapted; GPA, guinea pig-adapted; HA, hamster-adapted

32


GPA

39

GPA

38, 40

38

GPA

47

FERRET WT

WT

37, 41, 43

HAMSTER MA

MA

12

GUINEA PIG

48

HA

-

-

-

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Table 3: Features of Select Small Animal Models of Filovirus Disease CC-RIXa Virus

MOUSE hu-NSGhu-NSGb A2 BLT

GUINEA PIG

hu-NSGSGM3

HAMSTER

FERRET

MA-EBOV WT-EBOV WT-EBOV WT-EBOV GPA-EBOV GPA-SUDV GPA-MARV GPA-RAVV MA-EBOV HA-MARV 1000 FFU 105 TCID50 1000 FFU 5000 PFU 53 TCID50 5000 PFU 5000 PFU 1000 FFU (IP) (IP/IM)c (IP) (IP) (IP) (IP) (IP) (IP)

Inoculation (Route)

100 FFU (IP)

Peak Fever

ND

ND

ND

>4 log10 FFU/ml (~8)

Peak Liver Titer (DPI)

~5 log10 FFU/ml (4)

ND

Peak Spleen Titer (DPI)

~4 log10 FFU/ml (4)

ND

Time of Death/ Euthanasia (Lethality)

5-6 DPI (100%)

Lymphopenia

ND

WT-EBOV

WT-SUDV

WT-BDBV

1 PFU (IP)

1000 PFU (IN)

200 TCID50 (IM/IN)c

1000 PFU (IN)

1000 TCID50 (IM/IN)c

1000 PFU (IN)

159 TCID50 (IM)

5 DPI

4 DPI

6 DPI

4 DPI

7 DPI

6 DPI

ND

7 DPI

ND

7 DPI

7 DPI

ND

6 DPI

>107 TCID50 eq/ml (6) >109 TCID50 eq/ml (6) ~108 TCID50 eq/ml (6)

~6 log10 FFUeq/ml (8-terminal)

~5 log10 PFU/ml (3)

>106 GEQ/ml (9)

~8 log10 PFU/ml (7)

>6 log10 PFU/ml (10)

~8 log10 FFU/ml (4)

~8 log10 TCID50 (5, 6, 8)

~8 log10 ~109 >107 ~107 ~8 log10 ~7 log10 PFU/ml TCID50/ml PFU/ml TCID50/ml PFU/ml TCID50/ml (6) (7) (8) (5-6) (8) (8)

~8-10 log10 FFUeq/ml (8-terminal)

~4 log10 PFU/ml (8)

~106 GEQ/ml (5)

~8 log10 PFU/ml (7)

~8 log10 PFU/ml (10)

~7 log10 FFU/ml (5)

~7 log10 TCID50 (6)

~6 log10 ~10 >10 ~10 -10 >5 log10 >10 PFU/ml TCID50/ml PFU/ml TCID50/ml PFU/ml TCID50/ml (terminal) (terminal) (terminal) (terminal) (terminal) (terminal)

~7-9 log10 FFUeq/ml (8-terminal)

~4 log10 PFU/ml (3)

>106 GEQ/ml (5)

>7 log10 PFU/ml (10)

~8 log10 PFU/ml (7)

~7 log10 FFU/ml (5)

>6 log10 TCID50 (5, 6)

>6 log10 ~109 ~106 ~108-109 >5 log10 ~104 PFU/ml TCID50/ml PFU/ml TCID50/ml PFU/ml TCID50/ml (terminal) (terminal) (terminal) (terminal) (terminal) (terminal)

~8-20 DPI (100%)

6 DPI (100%)

9-13 DPI (50-67%)

8? DPI (100%)

9-14 DPI (100%)

8-10 DPI (100%)

10 DPI (100%)

4-5 DPI (100%)

8-9 DPI (100%)

6 DPI 5-6 DPI 7-8 DPI 6-9 DPI 8-9 DPI 8-9 DPI (100%) (100%) (100%) (100%) (100%) (100%)

ND

ND

-

ND

+

+

+

+

+

+

+

-

+

-

+

+

Neutrophilia

ND

ND

ND

ND

+

ND

+

+

+

+

+

+

+

-

+

+

Coagulopathy

+

ND

ND

ND

+

+

+

+

+

+

+

+

+

+

+

+

Thrombocytopenia

ND

ND

-

ND

+

+

+

+

+

-

+

+

+

+

+

+

Hemorrhage

+

+

-

-

+

-

+

+

+

+

+

+

+

+

+

+

Petechial Rash

-

-

-

-

-

-

-

-

-

+

+

+

+

-

+

+

Dysregulated Immune Response

+

ND

+

ND

+

ND

+

+

+

+

+

+

+

+

+

+

Reference

25

31

30

29

37

39

38

38

47

48

50

51

50

52

50

51

Peak Viremia (DPI)

9

5

7

8

6

a Specifically, strain ID 13140x3015; bSpecifically, mice with high levels of stem cell engraftment; cTwo routes of inoculation were tested in independent experiments, and this column includes data representing a range of values from all infected animals ND, not determined; FFU, focus-forming units; TCID, tissue culture infective dose; PFU, plaque-forming units; IP, intraperitoneal; IM, intramuscular; IN, intranasal; DPI, days post-infection; GEQ, genome equivalents; “-“ denotes phenomenon not observed; “+” denotes phenomenon observed.



Page 34 of 36

Table 4: Advantages and Disadvantages of Filovirus Small Animal Models MOUSE Immunocompetent Immunodeficient

CC-RIX

HIS

GUINEA PIG

HAMSTER

FERRET

NHP

++++

+++

++

+

++++

++++

++++

+

+

++

++

++++

+

+

+++

++++

Ease of Handling

++++

+++

+++

+

+++

++++

++

+

Availability of Tools and Reagents

++++

++++

++++

++++

++

++

++

+++

+

+

+

+++

++

+

+++

++++

++++

+++

++

+

+++

++++

++

+

+

+

+++

+++

+++

+++

++++

++++

Adapted

WT/Adapted

WT

WT

Adapted

Adapted

WT

WT

Availability of Animal Cost

Ethical Concerns High Throughput Use Stringency Filovirus Genome

“+” symbols denote the degree of the model feature listed on the left, with “++++” indicating a very high degree, “+++” indicating a high to moderate degree, “++” indicating a moderate to low degree, and “+” indicating a low degree.

34


Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


Table 5: Filovirus Countermeasures Evaluated in Small Animal Models MODEL SYSTEM CC-RIX Mice HIS Mice

VIRUS WT-EBOV WT-EBOV

GPA-EBOVa

Outbred Guinea Pig

GPA-SUDV

GPA-MARVa

GPA-RAVVa

COUNTERMEASURES TESTED None None Chloroquine Azithromycin/chloroquine Adjuvanted Adenovirus-based vaccine (intranasal) VLP-based trimeric hybrid GP vaccine rVSV-based vaccine (trivalent, containing EBOV, MARV, SUDV GP) Attenuated rVSV-based vaccine Lamivudine Iminosugars 17-DMAG, BGB324, NCK-8 EBOTab (ovine IgG)

OUTCOME 0% survival 0 – 10% survival

CONFIRMATION IN NHPs ND ND

REFERENCE -

100% survival

~67% survival

67,83

0 – 75% survival

ND

84

100% survival

ND

85

100% survival ND ND ND 25 – 100% survival ND ND ND 100% survival ND ND 100% survival

86

100% survival 0% survival 0 – 25% survival 0%, ~16%, and ~16% survival, respectively ~33 – 100% survival 0% survival Swine IgG lacking α1-3 Galactose and Neu5Gc Equine IgG or IgG fragments 100% survival Monoclonal antibody (FVM04) ~33% survival Adenovirus-based vaccine 100% survival Monoclonal antibody (CA45) 100% survival mRNA-based vaccine 100% survival MVA-based vaccine 100% survival rVSV-based vaccine (trivalent, containing 100% survival (prevented disease in nonEBOV, MARV, SUDV GP) lethal GPA-SUDV model) Monoclonal antibody (FVM04) 100% survival Monoclonal antibody (CA45) 100% survival VLP-based trimeric hybrid GP vaccine 75 – 100% survival rVSV-based vaccine (trivalent, containing 100% survival EBOV, MARV, SUDV GP) Monoclonal Antibodies (MR78-N, MR191-N)

60% and 100% survival, respectively

Monoclonal Antibodies (MR78-N, MR191-N)

100% survival

MA-EBOV HA-MARV WT-EBOV WT-SUDV

35


82

87 88 89 90-92 93 94 95 96 70 97 98

ND

85

ND ND ND

95

ND

85

100% survival with MR191-N treatment on 4, 7DPI; 80% survival with MR191-N with treatment on 5, 8DPI 100% survival with MR191-N treatment on 5, 8DPI ND ND ND

Chloroquine 0% survival None None None Ferret Monoclonal antibody (CA45) 100% survival WT-BDBV Monoclonal Antibodies (ADI-15742, ADI-15878) 50% and 75% survival, respectively a Only reports of countermeasure evaluation published from 2015 to present are listed VLP, virus-like particles; rVSV, recombinant vesicular stomatitis virus; MVA, modified vaccinia virus Ankara; siRNA, small interfering RNA; ND, not determined; DPI, days post-infection Hamster

81

70 84

99

99

100

70 71


For table of contents use only:

36


Page 36 of 36

SMALL ANIMAL MODELS FOR EVALUATING ... - ACS Publications

Recommend Documents