2008 Annual Report 1a.Objectives (from AD-416) To develop diagnostic reagents and vaccines for RVF that can be safely produced and distributed in North America. 1b.Approach (from AD-416) Neither ARS nor the Canadian Food Inspection Agency (CFIA) has modern validated diagnostic tests or vaccines for Rift Valley Fever. ARS and CFIA will collaborate to generate reagents to develop modern diagnostic tests and evaluate candidate vaccines. CFIA will conduct experimental infection studies necessary to evaluate the diagnostic tests. ABADRL will develop an immunohistochemical protocol using fixed (virus-inactivated) tissue samples generated. ABADRL and CFIA will collaborate on immunologic dose response studies to determine the optimal vaccination protocol. CFIA will then test the optimized vaccine protocol for efficacy by virulent challenge studies. 3.Progress Report Bacterial expression and purification of the recombinant N protein, and plasmids containing the S segment, the Ns and N genes, were obtained. However, following sequencing the two genes were cloned de novo at NCFAD. The recombinant N protein was semi-purified and shipped to the University of Calgary for antibody development. Three sets of primers and probes were designed for detecting each of the gene-specific sequences of RVFV N protein or Ns proteins, based on the genomic sequence of the RVFV strain ZH501. Using cell culture virus, the N3 set appears to be the most sensitive, however the assay did not work well for detection of RVFV in sheep sera. We switched to the protocol by Bird et al. (J Clin Microbiol 2007, 45: 3506 - 3513), using the L gene as a target. The assay is working for calf sera, although analytical sensitivity must be improved. After comparing sensitivity of detection of virus both by virus isolation and real time RT-PCR, it was determined that whole blood will not be used in sampling, as serum appears to provide a better quality sample, and could additionally be used for antibody detection. Stocks of the N and Ns plasmids were also prepared as controls for the real time RT-PCR, and transferred to ABADRL. In addition, cDNA of the entire S segment of the RVFV strain ZH501 was produced using RT-PCR, and cloned into pQEM-T easy vector. The sequence of the cDNA has 100% homology with the RVFV strain ZH501. The polymerase L protein gene was cloned into pGEM-T easy to be used as the positive control for the real-time RT-PCR assay. Tissues for pathology were processed and embedded, and shipped to ARS, ABADRL for development of the IHC. Sera obtained from sheep on final bleed were transferred to ARS, ABADRL. The sheep sera (28 dpi) had high neutralizing titers, between 1280 and 2560. The sera also worked well in Western blots. This research supports NP104 Action Plan Component 1: Ecology and Epidemiology. The ADODR monitors activities to evaluate research progress thru conference calls and meetings with the cooperator's personnel.