2010 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 NP-103 Animal Health Action Plan. The ABADRU has established formal collaborations with numerous national and international collaborators necessary to address the objectives of this project. Five ABADRU staff members have received the investigational RVF vaccinations and their immune status is annually certified. Unfortunately, only two of the five have chosen to relocate to KS. Efforts will be made to maintain collaboration at least until ABADRU has sufficient vaccinated staff to complete the project goals. To determine which mosquitoes should be targeted for control should RVFV be detected in North America, we evaluated eight mosquito and one biting midge U.S. species for their ability to serve as potential vectors of RVFV. Cx. tarsalis transmitted RVFV efficiently. Ae. Vexans, however, could play a role but would be expected to transmit RVFV less efficiently. Population differences were noted within species. The ability to perform the whole RVF genome amplification protocol has been demonstrated and five strains were sequenced to indentify which strains should be used for RVF genetic reassortment in mosquito studies. Several virulent RVFV challenge studies of lambs and calves have been conducted in collaboration with CFIA. Sera that were collected from these animals have been heat-inactivated, safety tested and imported to ABADRU. 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 and used for immunohistochemistry assay development. The RVFV immunohistochemical assay has been optimized and can be used for mammalian and insect tissues as well as cell culture without modification of the assay protocol. The multiplex three-gene target assay RVFV real-time RT-PCR assay has been redesigned and optimized. The initial field test has been conducted in Kenya. Additional data is needed to fully validate the protocol. Additions of external and/or internal amplification controls are being evaluated. The recombinant RVF N, NSs and truncated G2 proteins have been expressed, purified and shown to react specifically with polyclonal mouse anti-MP12 in a binding ELISA. These reagents were incorporated into cELISA for both proteins that detected specific antibody in sheep experimentally infected with RVF MP12 vaccine or RVF wild-type virus. Initial laboratory validation has been completed. Field evaluation is planned for Fall of 2010. ABADRU assisted in the validation of the IgG recombinant N antigen indirect ELISA developed by National Institute for Communicable Diseases, South Africa. There were false positives with the U.S. although all of the U.S. sera were RVF virus neutralization negative. The overall sensitivity and specificity of this assay was ~98%. The RVF multiplex qRT-PCR assay has undergone a preliminary field investigation. Additionally, RVF MP-12 vaccine candidate trials have been conducted at ABADRU. hese studies determined that feeding on vaccinated animals did not infect mosquitoes. 4.Accomplishments 1. Development and preliminary validation of a Rift Valley Fever (RVF) immunological assay. Rift Valley Fever is a deadly Sub-Saharan African disease of livestock and humans. There are limited diagnostic assays available should RVF be introduced into the United States. Diagnostic reagents that do not pose health risks to the producer and user based on recombinantly expressed target proteins were developed. These reagents were incorporated into assays by ARS scientists in Manhattan, KS, that detected specific antibodies in sheep experimentally infected with RVF MP12 or wild-type virus. Testing of sequential serum samples from experimentally infected sheep obtained from South Africa and the Canadian Food Inspection Agency showed a rise in specific antibody to RVF. These assays provide a safer assay to produce and use thus providing a tool to be used for early detection through distribution to the National Animal Health Laboratory Network.