Objective (A) Arenaviruses: Lassa virus isolates from Mali are less virulent then classical isolates, and in macaques disease progression was atypical with primarily pulmonary disease manifestation (Safronetz 2013). Our results suggest that clinical presentation of Lassa fever in Mali may be atypical, which might explain the low incidence of disease in this region. Bunyaviruses: We have characterized two animal models for the study of Sin nombre virus; an infection model in Syrian hamsters (Safronetz 2013) and a disease model in macaques (Safronetz, submitted). Using the novel nonhuman primate model we performed the first temporal characterization of the pathophysiology of Hantavirus pulmonary syndrome and identified several key factors, including viremia, hematological abnormalities and tissue specific immune dysregulation in the development of this rare but frequently fatal disease. The ability of Crimean-Congo hemorrhagic fever (CCHF) virus to infect animals without causing overt disease signs has hampered research into human disease. We have developed a model using interferon alpha/beta receptor knockout (IFNAR-/-) mice, which closely mimics human CCHF. The IFNAR-/- mouse model is the first animal model for studying disease progression and countermeasures (Zivcec 2013). Filoviruses: We have developed and characterized a novel small animal model for Ebola virus infection. The Syrian hamster displays almost all hallmark features of human disease including coagulopathy. This is likely to replace current small animal models for Ebola virus research (Ebihara 2013). Paramyxoviruses: We have compared two strains of Nipah virus in the Syrian hamster model. Both strains cause similar disease and pathology with enhanced disease progression for the Malaysian strain. (DeBuysscher 2013). We performed a spatio-temporal analysis of Nipah virus entry into the CNS of Syrian hamsters and showed that Nipah virus rapidly enters the CNS via the olfactory route (Munster 2013). In collaboration we have further defined henipavirus pathogenesis in human respiratory epithelial cells (Escaffe 2013). Objective (B) Bunyaviruses: We have continued our efforts to evaluate and characterize newly designed adenovirus-based CCHF vaccines expressing the nucleoprotein or glycoproteins of CCHF virus. Neither vaccine vector alone provided complete protection, but nearly complete protection could be achieved with a blended approach and complete protection could be achieved through booster immunizations. Ongoing transfer and depletion studies indicated that antibodies are the mechanism of protection of these vaccine vectors (Zivcec, in preparation). Filoviruses: We have investigated the mechanism of protection of the recombinant Vesicular stomatitis virus (rVSV) based Ebola vaccine in nonhuman primates. Depletion studies demonstrated that neutralizing antibody responses are critical for the efficacy of the vaccine (Wong 2012; Marzi 2013). Furthermore, we have evaluated the protective efficacy of live or inactivated rabies-based Ebola vaccines expressing the Zaire ebolavirus glycoprotein in a nonhuman primate model. Protection was achieved with all vaccination approaches using a prime/boost strategy. Protection is largely dependent on the quality of the humoral immune response (Blaney 2013). All our vaccine efforts were summarized and discussed in a recent review article (Falzarano & Feldmann 2013). We have further characterized and investigated the role of antibodies to different proteins of Ebola and Marburg virus (Changula 2013). Interestingly, non-neutralizing antibodies to the glycoprotein of Marburg virus inhibited the budding of live virus and thus, present a potential treatment strategy (Kajihara 2012, 2013). Paramyxoviruses: We developed rVSV-based Nipah virus vaccines expressing either the nucleoprotein (N), fusion protein (F) or glycoprotein (G) of Nipah virus, strain Malaysia. Efficacy studies were performed in the Syrian hamster model and showed that the F and G protein expressing vaccine vectors provide full protection in this animal model. Current ongoing studies support the notion that the humoral immune responses are critical for vaccine efficacy (DeBuyscher, in preparation). Objective (C) Bunyaviruses: We are currently characterizing the immune response to hantaviruses in their natural hosts. For this we have started experiments using our in-house deer mouse colony, the reservoir for Sin nombre virus (North American hantavirus). We have established Andes virus (South American hantavirus) infection in deer mice (not the natural reservoir) and compared it to Sin nombre virus infection in these animals (deer mouse is the natural reservoir). In contrast to Sin nombre, which persistently infects deer mice, Andes virus infection is efficiently cleared by deer mice. We have found that deer mice mount an anti-inflammatory response to Sin nombre virus allowing for persistence, but mount a strong inflammatory response to Andes virus (Spengler 2013). It is thought that hantaviruses cause disease via an immunopathogenic mechanism. To examine this, we have performed T cell depletion in hamsters, a model for human disease. Depletion did not alter the pathogenicity or replication of Andes virus indicating that pro-inflammatory T cell responses do not contribute to disease (Prescott 2013; Schountz 2013). Objective (D) Bunyaviruses: We tested the effect of favipiravir against Andes and Sin nombre virus replication in the established lethal Syrian hamster model. We concluded that favipiravir treatment is beneficial for postexposure prophylaxis against HPS-causing hantaviruses (Safronetz et al., Antimicrob Agents Chemother 2013). Filoviruses: Ribavirin can effectively extend the time-to-euthanasia of hamsters infected with Ebola virus, but as observed in cell culture appears to rapidly develop resistance that is either viral or cellular based. Doxycycline and 5-fluoruracil have shown some activity against Ebola in vitro. A novel platelet activating factor inhibitor (WEB2086) has been show to partially protect hamsters when given following challenge. Following optimization of the treatment schedule, in future combinde treatment with ribavirin and/or other agents will be attempted (Chen 2013; Hoenen 2013; Falzarano, ongoing). In the past year we have further intensified our work with recombinant Ebola viruses to study virus biology (Hoenen 2012; Groseth 2013; Shabman 2013), as well as used them to develop a novel platform for rapid antiviral screening (Hoenen 2013). We will continue the work with recombinant Ebola viruses in order to gain further insights into virus biology, and are now in the process of moving this work into animal models to study pathogenesis. Cellular cysteine proteases are proposed to play an important role in Ebola virus replication. We tested Ebola virus replication in cathepsin B & L knockout mice and conclude that other proteases can substitute for cathepsins making it difficult to use proteases as antiviral targets (Marzi 2012). We have studied the mechanism of Ebola virus RNA editing. Studies have identified primary and secondary structural requirements and a potential role for VP30 as a necessary factor for RNA editing. (Mehedi, in press). Objective (E) Arenaviruses: Our continuous ecological studies have further defined endemic areas for a unique Lassa virus, Soromba R, in Mali (Safronetz, in preparation). We have started breeding Mastomys natalensis in order to generate a colony for experimental work in the natural reservoir of Lassa virus. Bunyaviruses: In collaboration we have developed a new indirect immunofluorescence assay for the detection of antibodies against clinically relevant hantaviruses (Lederer 2013), which will be helpful for our field work.