The Ferret as a Model for Filovirus Pathogenesis and Countermeasure Evaluation.

The domestic ferret (Mustela putorius furo) has long been a popular animal model for evaluating viral pathogenesis and transmission as well as the efficacy of candidate countermeasures. Without question, the ferret has been most widely implemented for modeling respiratory viruses, particularly influenza viruses; however, in recent years, it has gained attention as a novel animal model for characterizing filovirus infections. Although ferrets appear resistant to infection and disease caused by Marburg and Ravn viruses, they are highly susceptible to lethal disease caused by Ebola, Sudan, Bundibugyo, and Reston viruses. Notably, unlike the immunocompetent rodent models of filovirus infection, ferrets are susceptible to lethal disease caused by wild-type viruses, and they recapitulate many aspects of human filovirus disease, including systemic virus replication, coagulation abnormalities, and a dysregulated immune response. Along with the stringency with which they reproduce Ebola disease, their relatively small size and availability make ferrets an attractive choice for countermeasure evaluation and pathogenesis modeling. Indeed, they are so far the only small animal model available for Bundibugyo virus. Nevertheless, ferrets do have their limitations, including the lack of commercially available reagents to dissect host responses and their unproven predictive value in therapeutic evaluation. Although the use of the ferret model in ebolavirus research has been consistent over the last few years, its widespread use and utility remains to be fully proven. This review provides a comprehensive overview of the ferret models of filovirus infection and perspective on their ongoing use in pathogenesis modeling and countermeasure evaluation.

Characterization of Reston virus infection in ferrets.

Among the five currently recognized type viruses within the genus Ebolavirus, Reston virus (RESTV) is not known to cause disease in humans, although asymptomatic infections have been confirmed in the past. Intriguingly, despite the absence of pathogenicity in humans, RESTV is highly lethal to nonhuman primates and has been isolated from domestic pigs co-infected with other viruses in the Philippines and China. Whether infection in these animals can support the eventual emergence of a human-pathogenic RESTV remains unclear and requires further investigation. Unfortunately, there is currently no lethal small animal model available to investigate RESTV pathogenicity or pan-ebolavirus therapeutics. Here we show that wild type RESTV is uniformly lethal in ferrets. In this study, ferrets were challenged with 1260 TCID50 of wild type RESTV either intramuscularly or intranasally and monitored for clinical signs, survival, virus replication, alteration in serum biochemistry and blood cell counts. Irrespective of the route of challenge, viremia occurred in all ferrets on day 5 post-infection, and all animals succumbed to infection between days 9 and 11. Additionally, several similarities were observed between this model and the other ferret models of filovirus infection, including substantial decreases in lymphocyte and platelet counts and abnormalities in serum biochemistry indicating hepatic injury. The ferret model represents the first uniformly lethal model for RESTV infection, and it will undoubtedly prove useful for evaluating virus pathogenicity as well as pan-ebolavirus countermeasures.

Assays to Measure Suppression of Type I Interferon Responses by Filovirus VP35 Proteins.

Innate immunity is the first line of defense against virus infections and is marked by production of type I interferons (IFN), a family of cytokines that includes IFN-ß and several IFN-αs. For the filoviruses and many other RNA viruses that replicate in the cytoplasm, the RIG-I-like pattern recognition receptors (RLRs) are potential triggers of IFN production. To counteract such innate antiviral responses, many viruses encode proteins that antagonize RLR signaling. Ebola virus (EBOV) and other filoviruses produce VP35 proteins that block IFN induction via RLR signaling. We describe here cell-based reporter gene assays that quantify the IFN-antagonist function of filovirus VP35 proteins by assessing activation of the IFN-ß promoter.

Rodent-Adapted Filoviruses and the Molecular Basis of Pathogenesis.

Ebola, Marburg, and Ravn viruses, all filoviruses, are the causative agents of severe hemorrhagic fever. Much of what we understand about the pathogenesis of filovirus disease is derived from work with animal models, including nonhuman primates, which are considered the "gold standard" filovirus model since they faithfully recapitulate the clinical hallmarks of filovirus disease. However, rodent models, including the mouse, guinea pig, and hamster, also exist for Ebola, Marburg, and Ravn viruses, and although they may not reproduce all the clinical signs of filovirus disease, thanks to their relative ease of use and low cost, they are often the first choice for initial descriptions of virus pathogenesis and evaluation of antiviral prophylactics and therapeutics. Since filoviruses do not cause significant disease in adult, immunocompetent rodents, these models rely on "rodent-adapted" viruses that have been passaged several times through their host until virulence and lethality are achieved. In the process of adaptation, the viruses acquire numerous nucleotide/amino acid mutations that contribute to virulence in their rodent host. Interestingly, virus protein 24 (VP24) and nucleoprotein (NP) appear to be major virulence factors for ebolaviruses in rodents, whereas VP40 appears to be the major virulence factor for marburgviruses. By characterizing these mutations and understanding the molecular mechanisms that lead to the acquisition of virulence, we can gain better insight into the pathogenic processes that underlie filovirus disease in humans. These processes, and the viral and/or cellular proteins that contribute to them, will make attractive targets for the development of novel therapeutics and counter-measures.

The Role of Cytokines and Chemokines in Filovirus Infection.

Ebola- and marburgviruses are highly pathogenic filoviruses and causative agents of viral hemorrhagic fever. Filovirus disease is characterized by a dysregulated immune response, severe organ damage, and coagulation abnormalities. This includes modulation of cytokines, signaling mediators that regulate various components of the immune system as well as other biological processes. Here we examine the role of cytokines in filovirus infection, with an emphasis on understanding how these molecules affect development of the antiviral immune response and influence pathology. These proteins may present targets for immune modulation by therapeutic agents and vaccines in an effort to boost the natural immune response to infection and/or reduce immunopathology.

Filoviruses: One of These Things is (not) Like the Other.

The family Filoviridae contains several of the most deadly pathogens known to date and the current Ebola virus disease (EVD) outbreak in Western Africa, due to Ebola virus (EBOV) infection, highlights the need for active and broad research into filovirus pathogenesis. However, in comparison, the seven other known filovirus family members are significantly understudied. Many of these, including Marburgviruses and Ebolaviruses other than EBOV, are also highly virulent and fully capable of causing widespread epidemics. This review places the focus on these non-EBOV filoviruses, including known immunological and pathological data. The available animal models, research tools and currently available therapeutics will also be discussed along with an emphasis in the large number of current gaps in knowledge of these less highlighted filoviruses. It is evident that much research is yet to be done in order to bring the non-EBOV filovirus field to the forefront of current research and, importantly, to the development of more effective vaccines and therapeutics to combat potential future outbreaks.

[Haemostasis dysregulation in filovirus infections]. / Dérégulation de l'hémostase dans les infections à filovirus.

Filoviruses are responsible for highly lethal infections. Those viruses are found in intertropical areas of Africa and Asia where they circulate in their supposed natural reservoir, fruit bats. During filovirus outbreaks and depending on the strains, various modifications in hemostasis have been observed in patients. The disseminated intravascular coagulation identified in these infections is multicausal and involves both viral factors and abnormal physiological responses. In this review we will describe the mechanisms responsible for these disturbances and we will highlight some aspects of the basis of filovirus high pathogenicity.

Immunohistochemistry in the diagnosis of cutaneous viral infections- part II: cutaneous viral infections by parvoviruses, poxviruses, paramyxoviridae, picornaviridae, retroviruses and filoviruses.

BACKGROUND: Cutaneous viral infections are increasing in recent years, particularly in immunocompromised patients. OBJECTIVE: Immunohistochemistry (IHC) provides a rapid and helpful tool that can be applied to confirm the diagnosis of specific viral infections that may be difficult to diagnose with certainty using routine microscopy alone. METHODS: Several immunostains that are useful in histopathology have been reviewed and tested in cutaneous samples of viral infections. Emphasis is placed on new stains and novel uses of existing stains. RESULTS: This article is an up-to-date overview of the potential uses of IHC in the histopathologic diagnosis of cutaneous viral infections by parvoviruses, polyomaviruses, poxviruses, paramyxoviridae, picornaviridae, retroviruses, and filoviruses. LIMITATIONS: Specific monoclonal antibodies are commercially available only for some members of these virus families. CONCLUSIONS: IHC may assist dermatopathologists to appropriately diagnose viral infections by parvoviruses, polyomaviruses, poxviruses, paramyxoviridae, picornaviridae, retroviruses, and filoviruses.

Establishment and characterization of a lethal mouse model for the Angola strain of Marburg virus.

UNLABELLED: Infections with Marburg virus (MARV) and Ebola virus (EBOV) cause severe hemorrhagic fever in humans and nonhuman primates (NHPs) with fatality rates up to 90%. A number of experimental vaccine and treatment platforms have previously been shown to be protective against EBOV infection. However, the rate of development for prophylactics and therapeutics against MARV has been lower in comparison, possibly because a small-animal model is not widely available. Here we report the development of a mouse model for studying the pathogenesis of MARV Angola (MARV/Ang), the most virulent strain of MARV. Infection with the wild-type virus does not cause disease in mice, but the adapted virus (MARV/Ang-MA) recovered from liver homogenates after 24 serial passages in severe combined immunodeficient (SCID) mice caused severe disease when administered intranasally (i.n.) or intraperitoneally (i.p.). The median lethal dose (LD50) was determined to be 0.015 50% TCID50 (tissue culture infective dose) of MARV/Ang-MA in SCID mice, and i.p. infection at a dose of 1,000× LD50 resulted in death between 6 and 8 days postinfection in SCID mice. Similar results were obtained with immunocompetent BALB/c and C57BL/6 mice challenged i.p. with 2,000× LD50 of MARV/Ang-MA. Virological and pathological analyses of MARV/Ang-MA-infected BALB/c mice revealed that the associated pathology was reminiscent of observations made in NHPs with MARV/Ang. MARV/Ang-MA-infected mice showed most of the clinical hallmarks observed with Marburg hemorrhagic fever, including lymphopenia, thrombocytopenia, marked liver damage, and uncontrolled viremia. Virus titers reached 10(8) TCID50/ml in the blood and between 10(6) and 10(10) TCID50/g tissue in the intestines, kidney, lungs, brain, spleen, and liver. This model provides an important tool to screen candidate vaccines and therapeutics against MARV infections. IMPORTANCE: The Angola strain of Marburg virus (MARV/Ang) was responsible for the largest outbreak ever documented for Marburg viruses. With a 90% fatality rate, it is similar to Ebola virus, which makes it one of the most lethal viruses known to humans. There are currently no approved interventions for Marburg virus, in part because a small-animal model that is vulnerable to MARV/Ang infection is not available to screen and test potential vaccines and therapeutics in a quick and economical manner. To address this need, we have adapted MARV/Ang so that it causes illness in mice resulting in death. The signs of disease in these mice are reminiscent of wild-type MARV/Ang infections in humans and nonhuman primates. We believe that this will be of help in accelerating the development of life-saving measures against Marburg virus infections.

A characterization of aerosolized Sudan virus infection in African green monkeys, cynomolgus macaques, and rhesus macaques.

Filoviruses are members of the genera Ebolavirus, Marburgvirus, and "Cuevavirus". Because they cause human disease with high lethality and could potentially be used as a bioweapon, these viruses are classified as CDC Category A Bioterrorism Agents. Filoviruses are relatively stable in aerosols, retain virulence after lyophilization, and can be present on contaminated surfaces for extended periods of time. This study explores the characteristics of aerosolized Sudan virus (SUDV) Boniface in non-human primates (NHP) belonging to three different species. Groups of cynomolgus macaques (cyno), rhesus macaques (rhesus), and African green monkeys (AGM) were challenged with target doses of 50 or 500 plaque-forming units (pfu) of aerosolized SUDV. Exposure to either viral dose resulted in increased body temperatures in all three NHP species beginning on days 4-5 post-exposure. Other clinical findings for all three NHP species included leukocytosis, thrombocytopenia, anorexia, dehydration, and lymphadenopathy. Disease in all of the NHPs was severe beginning on day 6 post-exposure, and all animals except one surviving rhesus macaque were euthanized by day 14. Serum alanine transaminase (ALT) and aspartate transaminase (AST) concentrations were elevated during the course of disease in all three species; however, AGMs had significantly higher ALT and AST concentrations than cynos and rhesus. While all three species had detectable viral load by days 3-4 post exposure, Rhesus had lower average peak viral load than cynos or AGMs. Overall, the results indicate that the disease course after exposure to aerosolized SUDV is similar for all three species of NHP.

Clinical management of filovirus-infected patients.

Filovirus infection presents many unique challenges to patient management. Currently no approved treatments are available, and the recommendations for supportive care are not evidence based. The austere clinical settings in which patients often present and the sporadic and at times explosive nature of filovirus outbreaks have effectively limited the information available to evaluate potential management strategies. This review will summarize the management approaches used in filovirus outbreaks and provide recommendations for collecting the information necessary for evaluating and potentially improving patient outcomes in the future.

Antiviral activity of a small-molecule inhibitor of filovirus infection.

There exists an urgent need to develop licensed drugs and vaccines for the treatment or prevention of filovirus infections. FGI-103 is a low-molecular-weight compound that was discovered through an in vitro screening assay utilizing a variant of Zaire ebolavirus (ZEBOV) that expresses green fluorescent protein. In vitro analyses demonstrated that FGI-103 also exhibits antiviral activity against wild-type ZEBOV and Sudan ebolavirus, as well as Marburgvirus (MARV) strains Ci67 and Ravn. In vivo administration of FGI-103 as a single intraperitoneal dose of 10 mg/kg delivered 24 h after infection is sufficient to completely protect mice against a lethal challenge with a mouse-adapted strain of either ZEBOV or MARV-Ravn. In a murine model of ZEBOV infection, delivery of FGI-103 reduces viremia and the viral burden in kidney, liver, and spleen tissues and is associated with subdued and delayed proinflammatory cytokine responses and tissue pathology. Taken together, these results identify a promising antiviral therapeutic candidate for the treatment of filovirus infections.

Fibroblastic reticular cells and their role in viral hemorrhagic fevers.

Viral hemorrhagic fevers (VHFs) caused by Ebola, Marburg and Lassa viruses often manifest as multiple organ dysfunction and hemorrhagic shock with high mortality. These viruses target numerous cell types, including monocytes and dendritic cells, which are primary early targets that mediate critical pathogenetic processes. This review focuses on fibroblastic reticular cells (FRCs), another prevalent infected cell type that is known as a key regulator of circulatory and immune functions. Viral infection of FRCs could have debilitating effects in secondary lymphoid organs and various other tissues. FRCs may also contribute to the spread of these deadly viruses throughout the body. Here, we review the salient features of these VHFs and the biology of FRCs, emphasizing the potential role of these cells in VHFs and the rapid deterioration of immune and hemovascular sytems that are characteristic of such acute infections.

Ebolavirus and Marburgvirus: insight the Filoviridae family.

Ebolavirus and Marburgvirus (belonging to the Filoviridae family) emerged four decades ago and cause epidemics of haemorrhagic fever with high case-fatality rates. The genome of filoviruses encodes seven proteins. No significant homology is observed between filovirus proteins and any known macromolecule. Moreover, Marburgvirus and Ebolavirus show significant differences in protein homology. The natural maintenance cycle of filoviruses is unknown, the natural reservoir, the mode of transmission, the epidemic disease generation, and temporal dynamics are unclear. Lastly, Ebolavirus and Marburgvirus are considered as potential biological weapons. Vaccine appears the unique therapeutic frontier. Here, molecular and clinical aspects of filoviral haemorrhagic fevers are summarized.

Dendritic cells in viral pathogenesis: protective or defective?

Dendritic cells (DC) are potent antigen-presenting cells that are critical in the initiation of immune responses to control and/or eliminate viral infections. Recent studies have investigated the effects of virus infection on the biology of DC. This review summarizes these changes, focusing on both the DC parameters affected and the viral factors involved. In addition, the central role of DC biology in the pathogenesis of several viral families, including herpesviruses, paramyxoviruses and retroviruses, is explored. The field of pathogen recognition by DC is addressed, focusing on its role in protecting the host from viral infection, as well as the ability of viruses to exploit such host receptor ligation and signalling to their replicative advantage. The hypothesis is proposed that virus and host have evolved a symbiotic relationship to ensure both viral transmission and host survival.