Use of the Syrian Hamster as a New Model of Ebola Virus Disease and Other Viral Hemorrhagic Fevers

Historically, mice and guinea pigs have been the rodent models of choice for therapeutic and prophylactic countermeasure testing against Ebola virus disease (EVD). Recently, hamsters have emerged as a novel animal model for the in vivo study of EVD. In this review, we discuss the history of the hamster as a research laboratory animal, as well as current benefits and challenges of this model. Availability of immunological reagents is addressed. Salient features of EVD in hamsters, including relevant pathology and coagulation parameters, are compared directly with the mouse, guinea pig and nonhuman primate models.


Introduction
Ebola virus (EBOV), a member of the family Filoviridae, is the etiologic agent of Ebola virus disease (EVD), a severe hemorrhagic fever syndrome with unusually high case-fatality rates, ranging between 65-90%. Filoviruses are emerging/reemerging zoonotic agents that are highly virulent in primates, and the frequency of outbreaks in Africa and Asia and impact on ape populations have been increasing in recent years. Introduction of filoviruses into human populations leads to serious, albeit limited, epidemics. Interhuman transmission occurs by direct person-to-person contact and possibly by fomites and droplets. Filoviruses infect, among others, monocytes, macrophages, dendritic cells, hepatocytes, and endothelial cells. In the infected primate, these viral infections lead to severe cytokine imbalances that impair the innate and adaptive immune responses, disseminated intravascular coagulation (e.g., hemorrhages, thrombi), and organ necroses that result in multi-organ failure and shock. No approved vaccines or effective therapeutics are currently available. Because of the high case-fatality rates of EVD and the lack of an approved vaccine or therapy, EBOV is classified as a category A pathogen requiring biosafety level-4 (BSL-4) biocontainment.

Existing Animal Models of EVD
The development of animal models that accurately reflect human disease is critical to our understanding of the pathogenesis of EVD and evaluation of countermeasures against filoviruses. Because of the sporadic and geographically isolated nature of EVD outbreaks, clinical efficacy studies may not be feasible. Clinical data and access to human tissues from fatal cases are limited.
Another option for licensing new drugs and vaccines for EVD is extrapolation of data derived from accurate, validated animal models supported by human safety evaluation data and pharmacokinetic information. The "Animal Rule" from the U.S. Food and Drug Administration [19] requires that a countermeasure be evaluated in animal models in which the route and dose of virus administration, time to onset of disease, and time course/progression of disease optimally mimic the pathophysiology of human disease. One of the challenges with this regulatory pathway is the development of animal models that recapitulate human disease, as data on the clinical presentation of EVD disease in humans are quite limited.
The nonhuman primate (NHP) model of EVD is the gold-standard for the study of EVD pathogenesis that most closely resembles what we currently know regarding human disease. Guinea pigs and mice are regarded as models for preliminary evaluation of therapeutic interventions against EVD. As wild-type EBOV does not produce appreciable disease in these rodent models, EBOV was adapted by serial passage to produce fatal infection following intraperitoneal (IP) inoculation [20,21]. The pathogenesis of EVD from adapted rodent viruses differs in a number of aspects from EVD in humans and NHPs. Important clinical signs of EVD in humans and NHPs such as fever and maculopapular rash are not present in mice infected with mouse-adapted Ebola virus (MA-EBOV) [20,22]. Fever is present in guinea pigs infected with guinea pig-adapted Ebola virus (GPA-EBOV), but maculopapular rash does not develop in these animals [21]. Mice infected with MA-EBOV do not consistently display coagulation abnormalities (Table 1) [20,23,24]. Compared to mice, guinea pigs infected with GPA-EBOV develop coagulation defects, including a drop in platelet Viruses 2012, 4 3757 counts and an increase in coagulation time, but fibrin deposition and coagulopathy (i.e., disseminated intravascular coagulation) are not as marked as that observed in NHPs [21,25]. Further, bystander lymphocyte apoptosis, an important feature in primates and mice, has not been determined in guinea pigs infected with GPA-EBOV. Mice infected with MA-EBOV differ from guinea pigs infected with GPA-EBOV and monkeys infected with wild-type EBOV in that they display a decrease in blood urea nitrogen (BUN), rather than an increase [20]. Because of these differences in EVD in rodent models, a number of therapeutic interventions that are effective in rodents challenged with adapted EBOV fail to protect NHPs challenged with wild-type EBOV from EVD (Tables 2-3, see Supplemental Tables 1-2 for unabridged versions). Of the five equivalent vaccines tested in rodents and NHPs with a comparable degree of immunocompetence, two vaccines had equivalent protection in all animal models tested, and three vaccines that provided some protection in rodents were not protective in NHPs. In evaluation of peri-exposure treatment of EVD, vesicular stomatitis virus (VSV) viral vectors provided roughly the same percentage of protection in guinea pigs and NHPs. Transfer of immune serum or equivalent polyclonal or monoclonal antibodies to naïve infected animals provided no protection to NHPs and some protection to rodents. Administration of equivalent antisense phosphorodiamidate morpholino oligomers to NHPs provided less protection against EVD than rodents.

History of Outbred Strains of Syrian Hamster in U.S.
Syrian hamsters (Mesocricetus auratus) are used in research studies of infectious diseases and cancer. In particular, Syrian hamsters are recognized as valuable animal models for studying emerging, acute human viral diseases caused by highly pathogenic RNA viruses. Outbred strains of Syrian hamsters in the U.S. are currently available from 3 sources: Simonsen Laboratories, Charles River Laboratories, and Harlan Laboratories. Most or all of these sources obtained Syrian hamster stock from Jerusalem. In 1930, lengthy experiments on leishmaniasis at the Hebrew University of Jerusalem were hampered by limitations of the only animal model known for the disease, the Chinese hamster (Cricetus griseus) [61]. Continuous replenishment of Chinese hamster stocks from China was needed as conditions for successful breeding of captive Chinese hamsters were not known, and such hamsters succumbed to a Pasturella epidemic in the region. Instead, an endemic species of hamster, the Syrian or golden hamster was substituted for the Chinese hamster. Syrian hamsters are easily bred in captivity, relatively free from natural hamster diseases, but are susceptible to experimental pathogens and have a short life cycle [61,62] [70]. These strains are used as disease models for carcinogenicity, dental caries, cardiomyopathy, muscular dystrophy, diabetes, atherosclerosis, and hypertension.
Among the Syrian hamster models of viral hemorrhagic fevers, liver and lungs are the commonly affected organs. Signs of tachycardia and tachypnea and results of hematological, blood chemistry, and coagulation tests indicative of vascular leakage or shock are noted in Syrian hamsters infected with Andes virus, [77] Pichindé or Pirital viruses, [80,84,86] or yellow fever virus [80]. Upregulation of cytokines in one study of hamsters challenged with Pichindé virus preceded vascular leakage [80]. The search for an animal model that more closely resembles human EVD than other rodent models to date led to the development of the Syrian hamster model.

Syrian Hamsters as an Ebola Virus Disease Model
Data from a study of Syrian hamsters challenged IP or SC with MA-EBOV or wild-type EBOV indicate that only MA-EBOV given IP causes EVD reminiscent of human disease including, severe coagulopathy, lymphocyte apoptosis, cytokine dysregulation (e.g., suppression of early type I IFN responses), target organ necrosis and/or apoptosis (i.e., lymph nodes, spleen, liver), and lethal outcome (Tables 5-6) ( Figure 2) [27]. Such suppression/non-induction of type-I IFN response and aberrant pro-inflammatory responses are suggested as some of the key pathogenic processes that lead to lethal outcome [20,27]. In contrast to MA-EBOV challenge, wild-type EBOV given IP in hamsters causes activation of early type-I IFN responses, mild inflammatory responses, induction of less-prominent apoptosis, and minimal pathological changes [27].  The severity of coagulopathy observed in Syrian hamsters infected with MA-EBOV is similar to that observed in rhesus macaques following challenge with wild-type EBOV (Table 1). Hepatic changes in Syrian hamsters closely resemble those of rhesus macaques, including disseminated hepatocellular degeneration and necrosis with infiltration of moderate numbers of neutrophils and fewer macrophages than neutrophils (Table 6). In contrast to macaques, little fibrin deposition occurs within hepatic sinusoids of hamsters ( Figure 3). Likewise, splenic lesions in hamsters are also similar to those observed in macaques and are characterized by necrosis of lymphocytes and marked loss of white pulp. Additionally, multifocal acute splenitis is characterized by moderate numbers of viable and degenerate neutrophils and fewer macrophages than neutrophils mixed with necrotic debris within the red pulp. Lymph nodes also display diffuse lymphoid necrosis and loss along with acute lymphadenitis and draining hemorrhage (Figure 3). In terminal Syrian hamsters, all cytokines tested (IL-1β, IL-2, IL-4, IL-6, and IL-12p35; tumor growth factor [TGF]-β; IFN-γ induced protein [IP]-10 and IFN-γ; TNF-α) are upregulated in the spleen, liver, and blood, indicating potentially uncontrolled immune responses.

Reagent and Assay Development
Until recently, lack of available reagents and specific assays to monitor host responses in hamsters (including early innate immune responses) limited investigators to studies on disease progression, humoral immune responses, and pathology. The lack of a complete genome sequence of the Syrian hamster has retarded the development of molecular, genetic, and antibody-based assays. In lieu of a complete genome, a number of studies evaluated the cross reactivity of antibody-based (ELISA, Luminex ® ) assays developed for other rodents against Syrian hamster cytokines, chemokines, adherins, growth factors, and other serum factors (Table 7) [76,80,94]. Data from most of these studies indicate limited cross reactivity of Syrian hamster proteins to other rodent antibodies. However, monoclonal antibodies from rats, mice, goats, and rabbits specific for hamsters successfully identified hamster surface markers of immune cells (T cells, B cells, dendritic cells, macrophages) via flow cytometry [74,[95][96][97]. Microarray proteome expression studies have quantified hamster responses to disease through cross species hybridization of Syrian hamster RNA to cDNA from other species (e.g., rat, mouse, human) [98][99][100][101][102]. The complete transcriptome of the Syrian hamster has been determined but is not yet publicly available, and a microarray chip is currently under development. As hamster-specific antibodies have not been made against cytokines/chemokines, gene expression of these factors during infection is tracked through quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) [103][104][105][106][107][108][109]. Recently, use of qRT-PCR has been extended to include 51 registered hamster gene sequences targeting apoptosis, cell junction integrity, cell proliferation, and coagulation in addition to immunological responses [94]. qRT-PCR assays were utilized to profile host responses in hamsters infected with yellow fever virus, Andes virus, and EBOV [27, 85,94,109]. Use of qRT-PCR assays will also contribute to identification of host response factors needed for survival in animals treated with antiviral drugs and of protective immune response in vaccinated animals. Such assays will be used until the full genome sequence is available for the development of large scale microarrays. Table 7. Cross-reactive or hamster-specific reagents.

Future Perspectives
Although the predictive value of the hamster model for efficacy testing of vaccines and therapeutics remains to be shown, numerous research tools are now available that will facilitate the use of this animal model in future research on Ebola virus pathogenesis. The newly developed hamster EVD model will certainly augment and perhaps may one day replace mice and guinea models as an alternative model for pathogenesis studies and efficacy testing. Hamsters infected with MA-EBOV currently exhibit EVD manifestations most similar to primates, particularly with respect to coagulation abnormalities. Of particular interest is employment of this hamster model to confirm the efficacy of drugs used in NHPs to control coagulopathy (e.g., recombinant activated protein C, recombinant nematode anticoagulant protein c2).