(A) Study pathogenesis and pathophysiology of high biocontainment viral pathogens utilizing molecular technologies including reverse genetics systems: Bunyaviruses: We are currently in the process of establishing the first nonhuman primate model for Crimean-Congo hemorrhagic fever virus (CCHF) utilizing Cynomolgus macaques. The model will be extremely valuable for pathogenesis studies and countermeasure development. (studies ongoing) Filoviruses: The Syrian hamster model for Ebola virus infection displays almost all hallmark features of human disease. Using this model, we have investigated the mechanisms in which hamsters are protected from wild-type Ebola virus infection and determined that a CD4+ T cell response, facilitating a neutralizing antibody response, is responsible for natural immunity. (studies ongoing) We continued to utilize the Collaborative Cross (CC), a panel of reproducible, recombinant inbred strains spanning the genetic breadth of three murine subspecies, to identify host factors important for Ebola virus disease progression. Transcriptomics revealed that the host responses dependent on genetic background determine susceptibility to infection independent of virus replication. (studies ongoing) We have studied the virulence of several Ebola-Makona isolates from the West African epidemic in macaque models and could show that these isolates are slightly attenuated compared to the Ebola-Mayinga prototype strain. More interesting, these new isolates did not cause lethal infection in the IFNR-/- mouse model, an established rodent model for non-rodent-adapted Ebola isolates. We have started to utilize humanized mice to study Ebola virus pathogenesis and immune responses. (studies ongoing) (B) Study immune responses to infection and vaccination of high containment viral pathogens and develop new vaccine candidates: Arenaviruses: We continued a project focused on the elimination of Lassa virus from the reservoir species, Mastomys natalensis. For this, we used two vaccine platforms, a recombinant attenuated strain of Salmonella and a recombinant cytomegalovirus vector, to immunize the peri-domestic reservoir with the hope to block Lassa virus transmission among rodents and to humans. (studies ongoing) Bunyaviruses: We have continued to characterize and optimize the adenovirus based vaccine for CCHF. Ongoing transfer and depletion studies using the adenovirus-based vaccines expressing the CCHF nucleoprotein or glycoproteins indicated that antibodies are the mechanism of protection. We also developed VSV-based vaccine vectors. Efficacy testing in the mouse model has shown that the glycoprotein itself is insufficient to protect animals. Filoviruses: The VSV vaccine efforts are reported under the 'Trivalent Filovirus Vaccine' project. In the meantime, we have proceeded with the development of alternative vaccine candidates for filoviruses. One approach is targeted towards wildlife vaccination, in particular the great apes, using the concept of a disseminating vaccine vector. The idea is the introduction of a recombinant CMV expressing the Ebola virus glycoprotein. Proof-of-concept studies have been successful in the mouse and rhesus macaque model using a murine and rhesus CMV vector, respectively. WE have also continued to investigate a vaccine based on Modified Vaccinia Ankara (MVA) expressing filovirus-like particles; initial rodent and nonhuman primate studies have revealed very promising results. (C) Study vector/reservoir transmission of high containment viral pathogens using appropriate animal models: Arenaviruses: We have studied infection kinetics of different Lassa virus in the Mastomys reservoir utilizing a unique colony established here at RML. The animals support virus replication and shedding for several weeks before Lassa virus gets cleared. The model will allow for important transmission studies. (studies ongoing) (D) Utilize in vitro and in vivo systems to study the interactions between viral pathogen or viral components and host cells and develop new antiviral strategies: Arenaviruses: Previously we had tested the efficacy of two antivirals, T-705 and ribavirin, in the guinea pig model of Lassa virus infection. In particular, T-705 showed promise as a treatment for Lassa virus with protection even when treatment was started post-disease onset. More recently, we were able to demonstrate efficacy of T-705 against Lassa infection in the nonhuman primate model. (studies ongoing) Filoviruses: Ribavirin can effectively extend the time-to-death of hamsters infected with Ebola virus, but resistance will rapidly develop. Surprisingly, T-705 protected hamsters against Ebola virus infection when animals were treated for two weeks beginning the day after infection. This promising treatment option is currently being followed up. We have also identified a few promising broad spectrum antivirals against Ebola virus, which are in development as treatment options for other RNA viruses. Further confirmation is underway. In contrast, treatment with anti-malaria drugs, including chloroquine, failed in Ebola rodent models and should not be further considered. (studies ongoing) (E) Study the epidemiology and ecology of high biocontainment pathogens utilizing newly developed rapid, sensitive and specific diagnostic test systems including those that can be applied under field conditions: Filoviruses: From August 2014 through May 2015 we have operated a diagnostic laboratory in Monrovia, Liberia to support the international response to the West African Ebola epidemic. We have started to analyze data for scientific purposes under approved protocols. So far we have demonstrated the importance of performing on-line clinical chemistry and malaria differential diagnostics. Most importantly, we have identified the interesting observation that plasmodium co-infection increases the survival rate of Ebola-infected patients. Future studies will be designed to test this observation in animal models and to understand the underlying mechanism.
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