A pigtailed macaque model of Kyasanur Forest disease virus and Alkhurma hemorrhagic disease virus pathogenesis.

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.

Guinea Pig Transferrin Receptor 1 Mediates Cellular Entry of Junín Virus and Other Pathogenic New World Arenaviruses.

Several clade B New World arenaviruses (NWAs) can cause severe and often fatal hemorrhagic fever, for which preventive and therapeutic measures are severely limited. These NWAs use human transferrin receptor 1 (hTfR1) as a host cell receptor for virus entry. The most prevalent of the pathogenic NWAs is Junín virus (JUNV), the etiological agent of Argentine hemorrhagic fever. Small animal models of JUNV infection are limited because most laboratory rodent species are refractory to disease. Only guinea pigs are known to develop disease following JUNV infection, but the underlying mechanisms are not well characterized. In the present study, we demonstrate marked susceptibility of Hartley guinea pigs to uniformly lethal disease when challenged with as few as 4 PFU of the Romero strain of JUNV. In vitro, we show that infection of primary guinea pig macrophages results in greater JUNV replication compared to infection of hamster or mouse macrophages. We provide evidence that the guinea pig TfR1 (gpTfR1) is the principal receptor for JUNV, while hamster and mouse orthologs fail to support viral entry/infection of pseudotyped murine leukemia viruses expressing pathogenic NWA glycoproteins or JUNV. Together, our results indicate that gpTfR1 serves as the primary receptor for pathogenic NWAs, enhancing viral infection in guinea pigs.IMPORTANCE JUNV is one of five known NWAs that cause viral hemorrhagic fever in humans. Countermeasures against JUNV infection are limited to immunization with the Candid#1 vaccine and immune plasma, which are available only in Argentina. The gold standard small animal model for JUNV infection is the guinea pig. Here, we demonstrate high sensitivity of this species to severe JUNV infection and identify gpTfR1 as the primary receptor. Use of hTfR1 for host cell entry is a feature shared by pathogenic NWAs. Our results show that expression of gpTfR1 or hTfR1 comparably enhances JUNV virus entry/infectivity. Our findings shed light on JUNV infection in guinea pigs as a model for human disease and suggest that similar pathophysiological mechanisms related to iron sequestration during infection and regulation of TfR1 expression may be shared between humans and guinea pigs. A better understanding of the underlying disease process will guide development of new therapeutic interventions.

Correlation of CD38 expression with the progression of hemorrhagic fever with renal syndrome.

To assess the relationship between the expression of CD38 and the progression of hemorrhagic fever with renal syndrome (HFRS), we determined the levels of CD38 during different phases of HFRS and evaluated the relationship between changes in CD38 expression and the progression of HFRS. The expression of CD38 in 68 patients with HFRS was analyzed by flow cytometry, and this method was also used to determine the levels of CD4+T, CD8+T, and B lymphocytes and NK cells. Furthermore, creatinine (Cr), uric acid (UA), and urea in serum at each stage of HFRS were measured using commercial kits. The basic clinical reference values for leukocytes, platelets (PLT), and red blood cells were determined by conventional methods. The colloidal gold method was used to measure HFRS antibody levels in the patients. A significant change in CD38 expression was observed from the fever phase to the recovery phase in patients with HFRS. Moreover, the expression of CD38 was proportionally correlated with the levels of Cr, UA, and urea in serum. In contrast, there was an inverse correlation between CD38 and PLT. Interestingly, an increase in CD38 expression correlated with an increase in CD8+T lymphocytes, B cells, and NK cells, but with a decrease in CD4+T lymphocytes. The expression of CD38 is associated with the progression of HFRS, suggesting that it may be a potent indicator of the stages of this disorder.

A Vaccine Platform against Arenaviruses Based on a Recombinant Hyperattenuated Mopeia Virus Expressing Heterologous Glycoproteins.

Several Old World and New World arenaviruses are responsible for severe endemic and epidemic hemorrhagic fevers, whereas other members of the Arenaviridae family are nonpathogenic. To date, no approved vaccines, antivirals, or specific treatments are available, except for Junín virus. However, protection of nonhuman primates against Lassa fever virus (LASV) is possible through the inoculation of the closely related but nonpathogenic Mopeia virus (MOPV) before challenge with LASV. We reasoned that this virus, modified by using reverse genetics, would represent the basis for the generation of a vaccine platform against LASV and other pathogenic arenaviruses. After showing evidence of exoribonuclease (ExoN) activity in NP of MOPV, we found that this activity was essential for multiplication in antigen-presenting cells. The introduction of multiple mutations in the ExoN site of MOPV NP generated a hyperattenuated strain (MOPVExoN6b) that is (i) genetically stable over passages, (ii) has increased immunogenic properties compared to those of MOPV, and (iii) still promotes a strong type I interferon (IFN) response. MOPVExoN6b was further modified to harbor the envelope glycoproteins of heterologous pathogenic arenaviruses, such as LASV or Lujo, Machupo, Guanarito, Chapare, or Sabia virus in order to broaden specific antigenicity while preserving the hyperattenuated characteristics of the parental strain. Our MOPV-based vaccine candidate for LASV, MOPEVACLASV, was used in a one-shot immunization assay in nonhuman primates and fully protected them from a lethal challenge with LASV. Thus, our hyperattenuated strain of MOPV constitutes a promising new live-attenuated vaccine platform to immunize against several, if not all, pathogenic arenaviruses.IMPORTANCE Arenaviruses are emerging pathogens transmitted to humans by rodents and responsible for endemic and epidemic hemorrhagic fevers of global concern. Nonspecific symptoms associated with the onset of infection make these viruses difficult to distinguish from other endemic pathogens. Moreover, the unavailability of rapid diagnosis in the field delays the identification of the virus and early care for treatment and favors spreading. The vaccination of exposed populations would be of great help to decrease morbidity and human-to-human transmission. Using reverse genetics, we generated a vaccine platform for pathogenic arenaviruses based on a modified and hyperattenuated strain of the nonpathogenic Mopeia virus and showed that the Lassa virus candidate fully protected nonhuman primates from a lethal challenge. These results showed that a rationally designed recombinant MOPV-based vaccine is safe, immunogenic, and efficacious in nonhuman primates.

Inhibition of Innate Immune Responses Is Key to Pathogenesis by Arenaviruses.

Mammalian arenaviruses are zoonotic viruses that cause asymptomatic, persistent infections in their rodent hosts but can lead to severe and lethal hemorrhagic fever with bleeding and multiorgan failure in human patients. Lassa virus (LASV), for example, is endemic in several West African countries, where it is responsible for an estimated 500,000 infections and 5,000 deaths annually. There are currently no FDA-licensed therapeutics or vaccines available to combat arenavirus infection. A hallmark of arenavirus infection (e.g., LASV) is general immunosuppression that contributes to high viremia. Here, we discuss the early host immune responses to arenavirus infection and the recently discovered molecular mechanisms that enable pathogenic viruses to suppress host immune recognition and to contribute to the high degree of virulence. We also directly compare the innate immune evasion mechanisms between arenaviruses and other hemorrhagic fever-causing viruses, such as Ebola, Marburg, Dengue, and hantaviruses. A better understanding of the immunosuppression and immune evasion strategies of these deadly viruses may guide the development of novel preventative and therapeutic options.

Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy.

Delayed inflammatory and cell death responses are associated with reduced pathogenicity in Lujo virus-infected cynomolgus macaques.

UNLABELLED: To identify host factors associated with arenavirus virulence, we used a cynomolgus macaque model to evaluate the pathogenesis of Lujo virus (LUJV), a recently emerged arenavirus that caused an outbreak of severe viral hemorrhagic fever in southern Africa. In contrast to human cases, LUJV caused mild, nonlethal illness in macaques. We then compared this to contrasting clinical outcomes during arenavirus infection, specifically to samples obtained from macaques infected with three highly pathogenic lines of Lassa virus (LASV), the causative agent of Lassa fever (LF). We assessed gene expression in peripheral blood mononuclear cells (PBMC) and determined genes that significantly changed expression relative to that in uninfected animals over the course of infection. We detected a 72-h delay in the induction of host responses to infection during LUJV infection compared to that of the animals infected with LASV. This included genes associated with inflammatory and antiviral responses and was particularly apparent among groups of genes promoting cell death. We also observed early differential expression of a subset of genes specific to LUJV infection that accounts for the delayed inflammatory response. Cell type enrichment analysis suggested that host response induction delay and an LUJV-specific profile are due to a different proportion of natural killer cells responding in LUJV infection than that in the LASV-infected animals. Together, these data indicate that delayed proinflammatory and proapoptotic host responses to arenavirus infection could ameliorate disease severity. This conclusion provides insight into the cellular and molecular mechanisms of arenaviral hemorrhagic fever and suggests potential strategies for therapeutic development. IMPORTANCE: Old World arenaviruses are significant human pathogens that often are associated with high mortality. However, mechanisms underlying disease severity and virulence in arenavirus hemorrhagic fever are largely unknown, particularly regarding host responses that contribute to pathogenicity. This study describes a comparison between Lujo and Lassa virus infection in cynomolgus macaques. Lujo virus-infected macaques developed only mild illness, while Lassa virus-infected macaques developed severe illness consistent with Lassa fever. We determined that mild disease is associated with a delay in host expression of genes linked to virulence, such as those causing inflammation and cell death, and with distinct cell types that may mediate this delay. This is the first study to associate the timing and directionality of gene expression with arenaviral pathogenicity and disease outcome and evokes new potential approaches for developing effective therapeutics for treating these deadly emerging pathogens.

Vaccination strategies against highly pathogenic arenaviruses: the next steps toward clinical trials.

Vaccination is one of the most valuable weapons against infectious diseases and has led to a significant reduction in mortality and morbidity. However, for most viral hemorrhagic fevers caused by arenaviruses, no prophylactic vaccine is available. This is particularly problematic as these diseases are notoriously difficult to diagnose and treat. Lassa fever is globally the most important of the fevers caused by arenaviruses, potentially affecting millions of people living in endemic areas, particularly in Nigeria. Annually, an estimated 300,000 humans are infected and several thousands succumb to the disease. The successful development of the vaccine "Candid#1" against Junin virus, the causative agent of Argentine hemorrhagic fever, proved that an effective arenavirus vaccine can be developed. Although several promising studies toward the development of a Lassa fever vaccine have been published, no vaccine candidate has been tested in human volunteers or patients. This review summarizes the immunology and other aspects of existing experimental arenavirus vaccine studies, discusses the reasons for the lack of a vaccine, and proposes a plan for overcoming the final hurdles toward clinical trials.

[The vaccines based on the replicon of the venezuelan equine encephalomyelitis virus against viral hemorrhagic fevers].

The status of the various recombinant DNA and RNA-derived candidate vaccines, as well as the Venezuelan equine encephalomyelitis virus (VEEV) replicon vaccine system against extremely hazardous viral hemorrhagic fevers, were reviewed. The VEEV-based replication-incompetent vectors offer attractive features in terms of safety, high expression levels of the heterologous viral antigen, tropism to dendritic cells, robust immune responses, protection efficacy, low potential for pre-existing anti-vector immunity and possibility of engineering multivalent vaccines were tested. These features of the VEEV replicon system hold much promise for the development of new generation vaccine candidates against viral hemorrhagic fevers.

Undiagnosed acute viral febrile illnesses, Sierra Leone.

Sierra Leone in West Africa is in a Lassa fever-hyperendemic region that also includes Guinea and Liberia. Each year, suspected Lassa fever cases result in submission of ≈500-700 samples to the Kenema Government Hospital Lassa Diagnostic Laboratory in eastern Sierra Leone. Generally only 30%-40% of samples tested are positive for Lassa virus (LASV) antigen and/or LASV-specific IgM; thus, 60%-70% of these patients have acute diseases of unknown origin. To investigate what other arthropod-borne and hemorrhagic fever viral diseases might cause serious illness in this region and mimic Lassa fever, we tested patient serum samples that were negative for malaria parasites and LASV. Using IgM-capture ELISAs, we evaluated samples for antibodies to arthropod-borne and other hemorrhagic fever viruses. Approximately 25% of LASV-negative patients had IgM to dengue, West Nile, yellow fever, Rift Valley fever, chikungunya, Ebola, and Marburg viruses but not to Crimean-Congo hemorrhagic fever virus.

Comparative analysis of disease pathogenesis and molecular mechanisms of New World and Old World arenavirus infections.

Arenaviruses can cause fatal human haemorrhagic fever (HF) diseases for which vaccines and therapies are extremely limited. Both the New World (NW) and Old World (OW) groups of arenaviruses contain HF-causing pathogens. Although these two groups share many similarities, important differences with regard to pathogenicity and molecular mechanisms of virus infection exist. These closely related pathogens share many characteristics, including genome structure, viral assembly, natural host selection and the ability to interfere with innate immune signalling. However, members of the NW and OW viruses appear to use different receptors for cellular entry, as well as different mechanisms of virus internalization. General differences in disease signs and symptoms and pathological lesions in patients infected with either NW or OW arenaviruses are also noted and discussed herein. Whilst both the OW Lassa virus (LASV) and the NW Junin virus (JUNV) can cause disruption of the vascular endothelium, which is an important pathological feature of HF, the immune responses to these related pathogens seem to be quite distinct. Whereas LASV infection results in an overall generalized immune suppression, patients infected with JUNV seem to develop a cytokine storm. Additionally, the type of immune response required for recovery and clearance of the virus is different between NW and OW infections. These differences may be important to allow the viruses to evade host immune detection. Understanding these differences will aid the development of new vaccines and treatment strategies against deadly HF viral infections.

An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses.

Five New World (NW) arenaviruses cause human hemorrhagic fevers. Four of these arenaviruses are known to enter cells by binding human transferrin receptor 1 (hTfR1). Here we show that the fifth arenavirus, Chapare virus, similarly uses hTfR1. We also identify an anti-hTfR1 antibody, ch128.1, which efficiently inhibits entry mediated by the glycoproteins of all five viruses, as well as replication of infectious Junín virus. Our data indicate that all NW hemorrhagic fever arenaviruses utilize a common hTfR1 apical-domain epitope and suggest that therapeutic agents targeting this epitope, including ch128.1 itself, can be broadly effective in treating South American hemorrhagic fevers.

Infection and pathogenesis of Huaiyangshan virus (a novel tick-borne bunyavirus) in laboratory rodents.

A novel tick-borne bunyavirus (Huaiyangshan virus, HYSV), which causes haemorrhagic fever-like disease, has recently been reported in China. So far no animal experiments have been performed to study its pathogenesis. Towards developing an animal model for HYSV fever, newborn and adult mice and rats and golden hamsters were inoculated intracerebrally or intraperitoneally with HYSV. Newborn rats and newborn mice, especially Kunming (KM) mice, appeared highly susceptible. Remarkably, the KM mice that died of the HYSV infection developed large necrotic areas in the liver, while no obvious pathological changes were observed within the other organs. PCR and immunohistochemical analyses of the post-mortem material detected both HYSV antigen and RNA in almost all organs, indicating a systemic infection. Our data demonstrate that HYSV can cause a lethal infection of both newborn mice and newborn rats with apparent pathological damage of the liver. This animal model may help to understand the pathogenesis of the HYSV infection in humans.

Viral hemorrhagic fevers: advancing the level of treatment.

The management of viral hemorrhagic fevers (VHFs) has mainly focused on strict infection control measures, while standard clinical interventions that are provided to patients with other life-threatening conditions are rarely offered to patients with VHFs. Despite its complexity, a proper clinical case management of VHFs is neither futile nor is it lacking in scientific rationale. Given that patient outcomes improve when treatment is started as soon as possible, development and implementation of protocols to promptly identify and treat patients in the earliest phases of diseases are urgently needed. Different pharmacological options have been proposed to manage patients and, as for other life-threatening conditions, advanced life support has been proved effective to address multiorgan failure. In addition, high throughput screening of small molecular libraries has emerged as a novel promising way to find new candidates drugs for VHFs therapy and a relevant number of new molecules are currently under investigation. Here we discuss the current knowledge about VHF clinical management to propose a way to step up the approach to VHFs beyond the mere application of infection control measures.

Seroprevalence of Alkhurma and other hemorrhagic fever viruses, Saudi Arabia.

A 2009 deployment of military units from several Saudi Arabian provinces to Jazan Province, Saudi Arabia, enabled us to evaluate exposure to Alkhurma, Crimean-Congo, dengue, and Rift Valley hemorrhagic fever viruses. Seroprevalence to all viruses was low; however, Alkhurma virus seroprevalence was higher (1.3%) and less geographically restricted than previously thought.

Subarachnoid hemorrhage associated with epidemic hemorrhagic fever: a rare case report.

Nervous system injuries associated with epidemic hemorrhagic fever (EHF) are not rarely seen. However, cerebrovascular disease arising from EHF is rarely reported in the literature. A 50-year-old male patient suffered from subarachnoid hemorrhage (SAH). No abnormal condition was found in intracranial vascular digital subtraction angiography (DSA). But, this patient presented with positive hantavirus-IgM and IgG, with typical clinical process, which lead to the diagnosis of EHF followed by SAH. To our knowledge, SAH associated with EHF has not been previously reported. A meticulous assessment of EHF patients with a serious condition had one or more central nervous system (CNS) abnormalities, such as sudden headache, vomiting, confusion, meningismus, and convulsions, which is necessary for diagnosing and giving timely treatment to improve the prognosis.

Vaccines against viral hemorrhagic fevers: non-human primate models.

Viral hemorrhagic fevers are a group of disease syndromes caused by infection with certain RNA viruses. The disease is marked by a febrile response, malaise, coagulopathy and vascular permeability culminating in death. Case fatality rates can reach 90% depending on the etiologic agent. Currently, there is no approved antiviral treatment. Because of the high case fatality, risk of importation and the potential to use these agents as biological weapons, development of countermeasures to these agents is a high priority. The sporadic nature of disease outbreaks and the ethical issues associated with conducting a human trial for such diseases make human studies impractical; therefore, development of countermeasures must occur in relevant animal models. Non-human primates are superior models to study infectious disease because their immune system is similar to humans and they are good predictors of efficacy in vaccine development and other intervention strategies. This review article summarizes viral hemorrhagic fever non-human primate models.

The use of mice lacking type I or both type I and type II interferon responses in research on hemorrhagic fever viruses. Part 1: Potential effects on adaptive immunity and response to vaccination.

For more than 20 years, researchers have used laboratory mice lacking type I or both type I and type II interferon (IFN) responses to study viruses that cause hemorrhagic fever (HF) in humans. Whereas immunocompetent mice do not become ill when infected with Ebola, Lassa, dengue and other HF viruses, IFN-deficient mice typically develop severe or fatal disease when inoculated with these pathogens. The ease of employment of these "mouse models" has led to their extensive use in biocontainment laboratories to assess the efficacy of novel vaccines, often without consideration of whether adaptive immune responses in IFN-deficient mice accurately mirror those in humans. Failure to consider these questions may lead to inappropriate expectations of the predictive value of mouse experiments. In two invited articles, we investigate this question. This paper examines how the lack of type I or both type I and type II IFN signaling may affect the development of adaptive immune responses in mice and the outcome of vaccine studies. A second article reviews the published literature on the use of IFN-deficient mice for the assessment of novel vaccines against HF viruses.

The use of mice lacking type I or both type I and type II interferon responses in research on hemorrhagic fever viruses. Part 2: Vaccine efficacy studies.

For more than 20 years, researchers have used laboratory mice lacking type I or both type I and II interferon (IFN) responses to study high-containment viruses that cause hemorrhagic fevers (HF) in humans. With the exception of Rift Valley fever virus, agents that cause viral HF in humans, such as Ebola and Lassa virus, do not cause disease in mature immunocompetent mice. In contrast, IFN-deficient mice typically develop severe or fatal disease when inoculated with these agents. The sensitivity of IFN-deficient mice to disease has led to their widespread use in biocontainment laboratories to assess the efficacy of novel vaccines against HF viruses, often without considering whether adaptive immune responses in IFN-deficient mice accurately mirror those in immunocompetent humans. Failure to recognize these questions may lead to inappropriate expectations of the predictive value of mouse experiments. In two invited articles, we investigate these questions. The present article reviews the use of IFN-deficient mice for assessing novel vaccines against HF viruses, including Ebola, Lassa, Crimean-Congo hemorrhagic fever and Rift Valley fever viruses. A companion paper examines the general question of how the lack of IFN signaling may affect adaptive immune responses and the outcome of vaccine studies in mice.

Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis.

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.