2009 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 ABADRL has established formal collaborations with numerous national and international collaborators necessary to address the objectives of this project. Five ABADRL staff members have received the investigational RVF vaccinations and their immune status is annually certified. To determine which mosquitoes should be targeted for control should RVFV be detected in North America, we evaluated eight mosquito and one biting midge United States 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. This is a useful epidemiological tool and for studies on the potential of RVF genetic reasortment. 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 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. The original design for a multiplex three-gene target assay RVFV real-time RT-PCR assay lacked sensitivity. The assay has been redesigned and is being optimized. The initial field test will be conduct late August 2009 in Kenya. The recombinant RVF N 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 MP12. Testing of sequential serum samples from experimentally RVFV infected sheep showed a rise in specific antibody to RVFV. ABADRL 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%. Additionally, RVF MP-12 vaccine candidate trials have been conducted at ABADRL. These studies are designed to determine if feeding on vaccinated animals can infect mosquitoes. 4.Accomplishments 1. Vector competence of North American Insects for Rift Valley fever virus: To determine which mosquitoes should be targeted for control should RVFV be detected in North America, ARS scientists at Laramie, Wyoming in collaboration with USAMRID evaluated eight additional U.S. mosquito species that previously had not been evaluated for their ability to serve as potential vectors for RVFV. This included Aedes dorsalis, Ae. vexans, Culex tarsalis, and biting midge, Culicoides sonorensis, from the Midwestern United States. Cx. tarsalis transmitted RVFV efficiently. However, Ae. vexans would be expected to transmit RVFV less efficiently. Population differences were noted in that Eastern Ae. vexans were more capable of transmitting RVFV than Western populations. None of the C. sonorensis became infected, even after intrathoracic inoculation, and none of the Anopheles quadrimaculatus tested transmitted RVFV by bite, even after intrathoracic inoculation, indicating that these species would not be competent vectors of RVFV. 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. 2. Development of a Rift Valley Fever virus detection assay. The recombinant RVF N 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 MP12. Experimental animals showed variation in their immune response to different viral proteins. This information is critical in determining the configuration of a cELISA to use in a field situation. Hybridoma cultures to MP12 are being tested for production of monoclonal antibodies that can be incorporated into the cELISA. This will provide a consistent, reliable source of well-characterized antibody for incorporation into the test. The cELISA tests detected specific antibody in the serum from sheep experimentally infected with virulent RVFV. Testing of sequential serum samples from experimentally infected sheep obtained from South Africa showed a rise in specific antibody to RVFV. 3. Validation of a commercial recombinant RVFV ELISA. ARS scientists in Laramie, Wyoming participated in the validation of the IgG recombinant N antigen indirect ELISA developed by National Institute for Communicable Diseases, South Africa. The overall sensitivity and specificity of this assay was ~98%. The assay did have strong false positives with the U.S. sera, although all of the U.S. sera were RVF virus neutralization negative. To evaluate the potential cause of these false positives, scientists acquired Cache Valley, Rio Grande, Toscana, and Punta Toro viruses for cross reactivity studies. The ABADRL has developed the international cooperative agreements necessary to carry out research on countermeasures for Rift Valley fever and is in the process of evaluating new immunology and nucleic acid based assays for this important insect-transmitted disease.