2008 Annual Report 1a.Objectives (from AD-416) 1. Determine the vector competence of North American mosquitoes to virulent and marked Rift Valley fever virus (RVFV) vaccine strains, including amplification and vertical transmission. 2. Develop expression and delivery systems to advance the discovery of diagnostics and vaccines specifically designed for the control and eradication of RVF. 3. Develop direct and indirect diagnostic tests for the early detection of RVFV, including the differentiation of infected from vaccinated animals. 1b.Approach (from AD-416) The approach to determine the vector competence of North American mosquito species for RVFV will be to focus on key mosquito species that feed on RVFV susceptible livestock and will include genetic studies of mosquito vector competence for RVFV. Differences in vector competence among populations of the same species throughout the U.S. will be examined using virulent RVFV and candidate RVF marked vaccine virus strains. The potential for RVFV to reassort with indigenous Bunyaviruses will be assessed. This project will provide scientific information critical for assessing the risk of RVFV spreading via endemic mosquito species if it is introduced into the U.S. The approach to develop expression and delivery systems to advance diagnostic and vaccine technology will be to develop alphavirus replicon vectors expressing the RVFV glycoproteins, leading to vaccines that elicit high levels of neutralizing anti-RVFV antibody, which prevent the amplification of wild type RVFV in susceptible ruminant hosts. The development of transmission blocking vaccines that target virus development in the insect vector will be explored. In addition, the immunogenic characteristics of RVFV proteins will be evaluated to support the development of companion diagnostic tests that can support control strategies. The approach to development of early detection technology for RVFV will be the discovery and transfer of quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) technology to the National Animal Health Laboratory Network. The research will also focus on the development of antibody-based diagnostic tests using non-infectious expressed antigens that will enable early detection and differentiate infected from vaccinated animals. 3.Progress Report The following work aligns with the Bio-defense research and the control of zoonotic diseases components of the Animal Health Action Plan. The ABADRL has established formal collaborations with numerous national and international collaborators necessary to address the objectives of this project. This includes: the Canadian Food Inspection Agency (CFIA), the U.S. Army Medical Research Institute for Infectious Diseases (USAMRIID), Kenya Research Institutes, and others. Five ABADRL staff members have received RVF vaccinations and are certified to safely work with RVFV. Mosquito collection sites have been established and preliminary work has been done to identify target mosquitoes that would be important in RVF transmission. Additional sites could be added as deemed necessary by research results. Three mosquito species (Anopheles quadrimaculatus, Aedes vexans, and Aedes dorsalis) and one biting midge species (Culicoides sonorensis) have been tested for infection with and transmission of wild type RVF. ABADRL acquired Cache Valley, Rio Grande, Toscana, and Punta Toro viruses for the RVF reassortment study. Once wild-type RVF vector competence studies have been completed, a competent mosquito species will be selected for use in reassortment studies. Sufficient quantities of the Sindbis virus RVF vaccine and control replicons for a sheep immunization study have been produced and preparations for the initial trial are in progress. Virulent RVFV challenge studies of lambs and calves have been conducted in collaboration with CFIA. Sera that were collected from these animals has been heat-inactivated, safety tested and imported to ABADRL. This provides positive control sera for assay development and evaluation of the antigenicity of viral proteins. Positive control tissues have also been produced and embedded in paraffin for immunohistochemistry assay development. All required permits and agreements and the transfer of RVF MP-12 vaccine candidate to ABADRL have been completed. Plasmids containing the RVFV genes for Gn, Gc, N, NSs have been received from various collaborators. These have been sub-cloned into an expression plasmid vector. The proteins have been expressed and will soon be inoculated into mice and rabbits to generate antibody reagents, however, they have not yet been incorporated into a cELISA. Three RVFV real-time RT-PCR assays based on three separate genome segments are being evaluated using RNA transcript from various recombinant plasmids generated for this purpose. Kenyan collaborators have generated a wildlife sample collection. The RVF Loop-mediated isothermal amplification (LAMP) RVF assay is working with cell-culture samples but some modifications are needed to increase reliability with tissue samples. ABADRL sent U.S. sera to the Onderstepoort Veterinary Institute, South Africa to be run on their IgG recombinant N antigen ELISA. There were false positives with the U.S. sera using this ELISA although all of the U.S. sera were RVF virus neutralization negative. This indicates the possibility of cross-reactive virus circulating in the U.S. This ELISA, although useful, will require an additional confirmatory test. 4.Accomplishments 1. Three mosquito species (Anopheles quadrimaculatus, Aedes vexans, and Aedes dorsalis) and one biting midge species (Culicoides sonorensis) have been tested for infection with and transmission of wild type RVFV. None of the species tested was able to transmit RVFV efficiently under laboratory conditions. Although Ae. dorsalis was the most susceptible to infection and had the highest dissemination rate, this species had a salivary gland barrier and rarely transmitted RVFV by bite. In contrast, a lower percentage of Ae. vexans became infected and developed a disseminated infection, however, had a higher rate of virus transmission by bite. None of the C. sonorensis became infected indicating that this species would not be a competent vector. In addition to laboratory vector competence, factors such as seasonal density, feeding preference, longevity, and foraging behavior also need to be considered when determining the role these species could play in RVFV transmission. 6.Technology Transfer Number of the New MTAs (providing only) 1 Review Publications Linthicum, K., Anyamba, A., Britch, S.C., Chretien, J., Erickson, R.L., Small, J., Tucker, C.J., Bennett, K.E., Mayer, R.T., Schmidtmann, E.T., Andreadis, T.G., Anderson, J.F., Wilson, W.C., Freier, J., James, A., Miller, R., Drolet, B.S., Miller, S., Tedrow, C., Bailey, C., Strickman, D.A., Barnard, D.R., Clark, G.G., Zou, L. 2007. A Rift Valley fever Risk Surveillance System for Africa Using Remotely Sensed Data: Potential for use on Other Continents. Veterinaria Italiana 43(3): 663-674. Mcholland, L.E., Mecham, J.O. 2005. Bluetongue virus mammalian cell surface receptors: Role of glycosaminologycans. Journal of General Virology.