2009 Annual Report 1a.Objectives (from AD-416) The objective of this cooperative research is to develop a real-time RT-PCR assay that differentiates candidate attenuated Rift Valley fever (RVF) vaccine RNA from wild-type (virulent) RVF vaccine RNA. The second objective is to evaluate new diagnostic tests that would facilitate a differentiating infected form vaccinated animals (DIVA) control strategy. 1b.Approach (from AD-416) Published real-time RT-PCR assays will be evaluated and modified to a multiplex assay that differentiates RNA infected from vaccinated animals (DIVA). If the published assays cannot be multiplexed or prove unreliable then new PCR signatures will be developed. Efforts are underway to establish a reference sample collection of veterinary RVF diagnostic specimens that will be used to validate these assays. In addtion, the ARS is developing new diagnostic reagents are being generated because the availability of current reagents is limited and commercially available only from foreign countries. This includes E ELISA and immunohistochemistry assays for detection antibodies to RVF and RVF antigens. The reference sample collection will be used to evaluate these immunodiagnostic assays. 3.Progress Report A staff person to accomplish this project was hired, real-time PCR technology was transferred and initial analyses were performed. All three published real-time PCR assays were run successfully. A multiplex format utilizing all three primer sets in a single assay was also successful. During the multiplex PCR optimization it was determined that two of the three segments were not sufficiently sensitive. These primers sets have been redesigned and the new format is being evaluated. In addition, the staff person performed the flow cytometer analysis in conjunction with a related RVF vaccine trial to characterize host immune responses. This preliminary study provided interesting but inconclusive results. As a result of these findings this project will be extended and expanded to include immunological characterization of sheep responses to RVF vaccination and/or infection. ADODR is directly involved in performance of the research and also monitors activities to evaluate research progress through site visits, email and phone calls.