This has been the first year of operation for the Emerging Viral Pathogens Section. Our work this year has focused on setting the stage to initiate studies with monkeypox virus in vitro and in vivo. Studies are currently on hold until the facility receives Select Agent registration with the Centers for Disease Control and Prevention. Our work may be divided into three distinct areas: 1. readying the laboratory, 2. establishing supporting assays for in vivo and in vitro investigations, and 3. developing surrogate systems for monkeypox virus as interim investigations until monkeypox virus work is allowed. ? ? 1. The section and the laboratory are new to DIR/NIAID/NIH. Therefore substantial work was needed to purchase instrumentation not provided through the building, install and train on the instrumentation, develop standard operating procedures, train new staff on BSL 2 and 3 operations, develop standard operating procedures, and ready the facility for the Select Agent inspection by the Centers for Disease Control and Prevention.? ? 2. As part of the preparation for nonhuman primate investigations, supporting assays needed to be established in the laboratory. Specifically we established real time PCR assays to determine viral load for vaccinia virus, monkeypox virus, panorthopox virus, and an assay to differentiate the different orthopox viruses in a mixed solution. Additionally we have established a fluorescent activated cell sorting (FACS) based assay for quantitative antibody neutralization. This assay is also amenable to quantitation on fluorescent plate readers and high content readers. We have additionally modified the assay for use in drug discovery applications. FACS systems have also been established to simultaneously differentiate types of immune cell types and whether these cells are infected from nonhuman primates using surrogate systems.? ? 3. In place of monkeypox virus investigations (on hold until CDC approval is obtained) we have developed three lines of inquiry. The first is to evaluate the antiviral efficacy of Novantrone. This drug has shown antiviral efficacy against vaccinia virus and monkeypox virus. The drug will be evaluated in mice using a cowpox model. If efficacious, the drug will be further evaluated in nonhuman primates. Second, it was reported that nonhuman primates are susceptible to naturally occuring cowpox virus. Animals that became infected developed severe fatal disease. Using established parameters for our other orthopox virus nonhuman primate models we will assay whether cowpox virus could provide a safer model for orthopox virus infections. ? ? 4. Finally we we have performed a critical experiment in support of a novel filovirus vaccine, another categoty A biodefense pathogen. The vaccine is vectored by an attenuated vesicular stomatitis virus. Using SIV infected nonhuman primates, we tested whether the vaccine could be safely using in immunocompromised individuals. The vaccine produced a sunclinical, but immunogenic reaction in the immunocompromised animals. Challenge studies using authentic Ebola virus under an IAA with USAMRIID confirmed that the VSV vaccine was not only safe but also protective in monkeys whose CD4+ couints were >200. Most important, it was safe in monkeys whose CD4+ counts were vanishingly low. This study is an important contribution toward the evaluation in human subjects, and addresses one of the principal objections to the further development of VSV as a viable alternate vector for filovirus vaccines.
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