Syrian Hamster as an Animal Model for the Study on Infectious Diseases

Infectious diseases still remain one of the biggest challenges for human health. In order to gain a better understanding of the pathogenesis of infectious diseases and develop effective diagnostic tools, therapeutic agents, and preventive vaccines, a suitable animal model which can represent the characteristics of infectious is required. The Syrian hamster immune responses to infectious pathogens are similar to humans and as such, this model is advantageous for studying pathogenesis of infection including post-bacterial, viral and parasitic pathogens, along with assessing the efficacy and interactions of medications and vaccines for those pathogens. This review summarizes the current status of Syrian hamster models and their use for understanding the underlying mechanisms of pathogen infection, in addition to their use as a drug discovery platform and provides a strong rationale for the selection of Syrian hamster as animal models in biomedical research. The challenges of using Syrian hamster as an alternative animal model for the research of infectious diseases are also addressed.


INTRODUCTION
According to data released by the World Health Organization (WHO), infectious agents causing lower respiratory infections, diarrheal diseases, and tuberculosis were ranked in the top ten causes of death worldwide, resulting in 5.7 million deaths in 2016 (1). It is clear that we need to improve our understanding of these diseases and pathogenic agents in order to develop more effective drugs and vaccines. To this end, we need a suitable animal model that can most accurately mimic the pathogenesis of infection as infection usually induces a complex process of host immune responses that in vitro experiments are unable to simulate. Only in vivo models can accurately assess the complexity of host responses and allow the efficacy and adverse effects of drugs or vaccine to be evaluated.
The Syrian hamster (Mesocricetus auratus) has been used as an animal model to study humanassociated diseases for over 60 years. A number of studies have documented that Syrian hamsters represent better models for analysis of viral infections compared to murine models as the similarity to humans with regard to disease symptoms, pathognesis and immune responses is greater (2)(3)(4). It has been demonstrated by us and others that human cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-12 , are fully functional in hamster models, but not in mouse models (5,6). Together with other advantages, such as fast reproductive rate and ease of handling, Syrian hamsters are a superior choice compared with other small animals.
Although Syrian hamsters have historically been used in diseases research, their value as an animal model in the study of infectious diseases has only recently been realized. With advancements in gene editing technologies, their popularity has increased significantly (Figure 1). The use of genetically engineered Syrian hamster (GESH) models is critical for understanding disease progression and for developing prophylactic and therapeutic treatment regimens. The first STAT2 gene knockout (KO) Syrian hamster was developed in 2014, using the CRISPR/Cas9 system to target the hamster germline (7). STAT2 is a crucial element of the type I interferon (IFN) signal transduction pathway and the hamster model has emerged as the only small animal model permissive for Adenovirus (AdV) infection, thus, the STAT2 KO model has been critical for the characterization of Adenovirus pathogenesis (8).

SYRIAN HAMSTER USED FOR RESEARCH IN VIRAL INFECTIONS
The Syrian hamster is an ideal small animal model to study the disease caused by virus infection. Previous studies have shown that some human-specific viruses can also infect, replicate and cause similar pathological alterations in Syrian hamsters (9). In particular, Syrian hamsters are recognized as valuable model for studying emerging and acute human viral diseases caused by highly pathogenic RNA viruses (10). Thus, these animals are of great value for testing potential vaccines and new therapeutic drugs for human use. At present, over 70 different viruses have been investigated using Syrian hamster, and more viral infection studies will be explored in the future ( Table 1). In this review, we focus on six viruses for which the use of the Syrian hamster has provided valuable insight into disease pathogenesis. The search was performed with the keywords "Syrian hamster" or "golden hamster" AND "model" AND (1) "viral" or "virus," (2) "bacteria," (3) "infection" or "disease". transmitted via bites on infected arthropods (mosquitos). In humans, the majority of WNV infections are asymptomatic, with only 20% of infected individuals developing symptomatic West Nile fever (WNF) (39). However, WNV is an important emerging neurotropic virus causing severe encephalitis in human posing a significant threat to global health (40). Syrian hamsters can be readily infected by mosquito bite, ingestion (oral) or needle inoculation and infected hamsters develop viremia and illness, with symptoms similar to those experienced during human infection (41,42). Using this hamster model, Xiao et al. observed both histologic abnormalities and appearance of viral antigen in the brain first followed by the spinal cord, with infection eventually leading to acute central nervous system (CNS) injury (13). Infected hamsters developed neurological disease (43)(44)(45)(46) and association of suppressed diaphragmatic electromyographs (EMGs) with infection of the medulla oblongata (47

Yellow Fever Virus
YFV is an arthropod-borne virus of the genus Flavivirus (family Flaviviridae) and has high morbidity and mortality rates in regions of sub-Saharan Africa and South America (53). It was one of the first viruses of humans to be identified, isolated, propagated in vitro and studied by genomic sequencing (54).
The study of infection mechanism of YFV has historically been hindered by the lack of appropriate small animal model and non-human primate (NHP) models have typically been used. More recently, several research groups have generated animal models using Syrian hamsters that can be successfully infected with YFV (55-58). McArthur et al. reported adapted viral strains (Asibi/hamster p7) allow the reproduction of yellow fever disease in hamsters with features similar to the human disease (59). Further, studies have also shown that infection of Syrian hamster results in immune responses that correspond to those observed in infected humans, with marked increases in IFN-γ, IL-2, TNF-α in the spleen, kidney, and heart, but reduced levels of these seen in the liver. In addition, these studies found increased levels of IL-10 and reduced levels of TGF-β in the liver, spleen, and heart in early and mid-stages of infection (60). Syrian hamster can be used both to study the pathogenesis of the YFV infection, and to validate antiviral drugs and antiviral therapies. Recent findings have shown that treatment with the anti-viral compounds 2 ′ -C-methyl cytidine (61)

Nipah Virus
Nipah is paramyxovirus of the genus Henipavirus (family Paramyxoviridae) with a high fatality rate (69). Infection in humans usually causes severe encephalitic and respiratory disease (70). After inoculation with Nipah virus (NiV), Syrian hamsters also develop characterisitic neurological disease (12). Similar to symptoms after human infection, pathological lesions are the most severe and extensive in the hamster brain and viral antigen and RNA can be detected in neurons (11), lung (71), kidney, and spleen (11). The Syrian hamsters in the majority of NiV infection studies are treated by intraperitoneal (IP) injection or intranasal (IN.) delivery and these models have revealed that different inoculation method can cause diverse pathological responses (11). In Wong's work, IP injection of NiV in Syrian hamsters caused primarily neurological disease, while IN delivery developed neurological symptoms as well as labored breathing due to lung infection in the final stages of disease (11). Disease progression is usually much rapid and the time to death post-infection is shorter following intraperitoneal rather than intranasal inoculation (72). Since the Syrian hamster has shown suitability for studying NiV infection, it was further used to study the viral transmission (73)(74)(75), demonstrating that Nipah virus is transmitted efficiently via direct contact and inefficiently via fomites, but not via aerosols. Regarding the use of these models for development of disease treatment and prophylaxis, recent studies have shown that pretreatment with Poly(I)-poly(C 12 U) can significantly decrease the mortality caused by NiV infection of Syrian hamster (76). In addition, the model was used as a platform for evaluation of vaccines for NiV (77)(78)(79)(80). Walpita et al. discovered purified NiV-like particles (VLP) can protect the Syrian hamster using either multiple-dose or single-dose vaccination regimens followed by NiV challenge (81).

Ebola Virus
Ebola virus (EBOV) is one of five known viruses within the genus Ebolavirus (family Filoviridae) (10). It's classified as biosafety level 4 (BSL-4) pathogen by the WHO. Not only can Syrian hamsters be effectively infected with mouse adapted (MA)-EBOV, they additionally display major hallmarks infection and pathogenesis seen in humans and non-human primates (NHPs). Syrian hamsters could be inoculated via intraperitoneal injection with mouse adapted Zaire Ebola virus (MA-ZEBOV). The pathology caused by this infection is similar to that of humans, which includes significant spleen and liver damage, cytokine dysregulation, severe coagulopathy, lymphocyte apoptosis, and infected organ necrosis or apoptosis (21,82). The immune responses of infected Syrian hamsters include activation of T cell and antibody production. In a recent study, the results of Ebola virus infection in hamsters demonstrate that CD4 + T cells are required for natural immunity and CD4-dependent antibody responses are required for immunity against the virus in this model (83). Syrian hamsters can be used to evaluate a bivalent vaccine comprising recombinant Vesicular stomatitis virus (VSV) expressing two different immunogens derived from ZEBOV envelope glycoprotein (84) and Andes Virus (ANDV) (32). The results showed that a single immunization with this vaccine provides hamsters complete and sterile protection against lethal challenge with MA-ZEBOV or ANDV (85).

Marburg Virus
Marburg virus (MARV) is also a negative sense RNA virus belonging to the family Filoviridae that causes hemorrhagic fever (86

SYRIAN HAMSTER USED FOR RESEARCH IN BACTERIAL AND PARASITIC INFECTIONS
Syrian hamster is also an ideal animal model for the study of a series of human bacterial and parasite infections, and its application has been well-reported in the literature ( Table 2). Among the pathogens studied, some, such as Babesiosis, Leptospirosis, and Leishmaniasis can cause fatal infection. As for studies on virus pathogenesis, the value of the Syrian hamster model is not only reflected in the study of pathological and immune response to these infections, but also in the discovery of potential drugs and treatments.

Leptospira interrogan
Pathogenic Leptospira interrogans is spirochete bacteria responsible for leptospirosis, a widespread and emerging neglected zoonotic. Syrian hamster is the preferred model to study the infection of serovars of Leptospira interrogans, with bacteria traveling rapidly to the bloodstream via the lymphatics, Frontiers in Immunology | www.frontiersin.org then invading tissues and infecting all organs prior to the death of the hamsters (108). Similar to human, the presence of Leptospira interrogans can be detected in liver and kidney, with the destruction of hepatocyte junctions that leads to jaundice, thrombotic glomerulopathy, and interstitial nephritis (109, 110). Infected animals develop the enhanced expression of pro-inflammatory cytokines by peripheral blood cells, such as IL-1α, IL-10, TNF-α (111). Dramatic imbalance in the cytokine production upon Leptospira infection might play an important role in the development of severe leptospirosis (112). Since the Syrian hamster has been shown to be a suitable model, it has been used to test the efficacy of vaccines against this disease. Palaniappan et al. demonstrate that the immunization of Syrian hamster with recombinant LigA (rLigA) prevents fatalities, with decreased histopathological lesions in kidney and inhibited the growth of the organisms (113). In another study, a vaccine using a conserved region of the leptospiral immunoglobulin-like B protein (LigB, 131-645) and aluminum hydroxide (AH) can significantly increase IgG and IgM levels in the hamster, protecting the animal from mortality after challenge (114).

Clostridium difficile
Clostridium difficile disease caused by Clostridium difficile infection (CDI) is one of the most common infectious diseases worldwide (115). The increasing threat of morbidity and mortality caused by the infection is mostly due to the emergence of hypervirulent strains, increased use and misuse of antibiotics (116). The use of mouse animal model has been unable to provide CDI drug discoveries, so it is necessary to find new animal models (117,118). Several groups have generated Syrian hamster models for CDI, which developed many of clinical symptoms observed in infected humans (119)(120)(121). In these studies, Syrian hamsters were conditioned with a single subcutaneous injection of clindamycin to induce Clostridium difficile colitis model. Using this infection model, the efficacy of LFF571 antibiotic against Clostridium difficile was assessed (122) and the oral mixture of kefir-isolated bacteria and yeasts to prevent diarrhea and enterocolitis triggered by Clostridium difficile was tested (123).

Leishmania donovani
Visceral leishmaniasis (VL; also known as kala-azar) is the most severe form of leishmaniasis caused by Leishmania donovani and Leishmania infantum (Leishmania chagasi in the Americas) (124). The Syrian hamster is highly susceptible to infection with visceralizing Leishmania species and is considered the best experimental model to study VL as it reproduces the clinicopathological features of human disease and quite distinct from those noted in murine models of infection (125). In the majority of studies, animals were infected by the intracardial route. Infected animals demonstrate up-regulated expression of Th1-associated cytokine mRNA, such as IFN-γ, IL-2, and TNF-α in the spleen, but limited induction of IL-4 mRNA (126). In murine models, Leishmania is controlled through nitric oxide (NO) generation, and however in hamsters, as in humans, NO does not have a role in macrophage function. Inducible NO synthetase (iNOS) mRNA was not detected in livers or spleen of hamsters, which may explain the uncontrolled parasite replication occurring in hamsters and humans, despite the induction of a strong Th1 cytokine response (126

Leishmania infantum
Besides Leishmania donovani, Leishmania infantum has also been studied using the Syrian hamster model. Moreira et al. generated a model using Syrian hamsters featuring a similar human clinical picture on Leishmania infantum infection (129). The animals developed hepatosplenomegaly, severe weight loss, anemia, and leucopenia. A study found increased levels of IgG in hamsters infected with Leishmania infantum (130). Similar to humans, Syrian hamsters can develop the progressive fatal disease, with major sites of parasites replication being the liver, spleen, and bone marrow, eventually causing death of the host (131). Infection of the hamsters showed a strong humoral response against Leishmania antigens, and high antibody levels (131). Study have tested the LJM19 (Immunization with 16 DNA plasmids coding for salivary proteins of Lu. longipalpis) protein protected hamsters against the fatal outcome of VL (132).

Entamoeba histolytica
Entamoeba histolytica is a popular protozoan parasite causing amebiasis in humans that is a major source of morbidity and mortality in the developing countries (133). Parasitic Entamoeba histolytica produces amebic colitis and an amebic liver abscess (ALA). Syrian hamster can be successfully infected with Entamoeba histolytica (104). Similar to symptoms after human infection, the main extraintestinal complication, ALA, is also found in the hamster. In the hamster, liver recruitment of neutrophils is the initial host response to Entamoeba histolytica infection (134). A study indicated that leukocytes can induce Entamoeba histolytica trophozoites to undergo cell death (135). Although an anti-parasitic drug (Metronidazole) exists, side effects of toxicity exist in patients; thus this model has been used to develop alternative therapeutic agents. One research group showed that bovine lactoferrin protected against hepatic amoebiasis in Syrian hamster model (136). Hamsters were also used to show that intraperitoneal injection of Entamoeba histolytica surface metalloprotease (EhMSP-1), an antigen vaccine, protected against the amebic liver abscesses (137). In this study, EhMSP-1 immunization stimulated a robust IgG antibody response, IgG bound to the surface of Entamoeba histolytica trophozoites and accelerated amebic lysis via activation of the classical complement cascade. The same animal model used for Entamoeba histolytica infection was used to show that baculovirus driving the expression of the Gal-Lectin LC3 fragment, when administeredvia intramuscular injection, increased IFN γ and IL-4 levels in the liver to protect against ALA (138).

Schistosoma haematobium
Schistosoma haematobium (urinary blood fluke) is the etiologic agent for urogenital schistosomiasis, a source of morbidity and mortality for over 112 million people worldwide (139).

CONCLUDING REMARKS
In this review, we described the use of the Syrian hamster model as an extraordinarily effective and relevant platform for evaluation of the molecular mechanisms of immune responses to infectious diseases. These studies focus on several infectious pathogens including those of viral, parasitic, and bacterial origins. The results indicate that the Syrian hamster immune response is more physiological similar to the human immune response when compared to other animals, thus offering unique advantages when studying the disease pathogenesis and for novel drug and treatment discovery. Future studies should consider determining additional similarities between the Syrian hamster and human immune response activation through pathogen manipulation of host metabolism. Increased research efforts will ultimately allow for the development of new technologies and tools to study the Syrian hamster, such as more accurate sequencing technology along with specific antibodies against hamster proteins that are currently limited in comparison to similar tools for studying murine responses to infection. We believe that the recent advances that the Syrian hamster model has contributed enormously to our understanding of infectious diseases and disease management and demonstrates the strong potential for future research and development of anti-viral drug discovery. However, as discussed, the lack of research tools represents a major barrier to effective use of Syrian hamster models. Immunologic reagents for examing host immune response and particular gene expression, and transgenic disease models will all be required for a more complete evaluation of the value of this model. To overcome this, research groups are developing or identifying a considerable number of antibodies against Syrian hamster ( Table 3) and hamster specific quantitative real-time PCR (RT-qPCR), transcriptome analysis and microarrays have also been developed (169). Most strikingly, CRISPR/Cas9 technology has rapidly sped up the creation of transgenic Syrian hamster disease models (170). These tools will overcome the limitations to research using Syrian hamsters, opening up a powerful platform for recapitulation of human disease pathogensis.