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West Nile (WN) virus is maintained in a transmission cycle between infected wild birds and mosquitoes (Hayes 1989). Field populations of Culex mosquitoes in California-particularly Culex pipiens pipiens, Cx. pipiens quinquefasciatus, and Cx. tarsalis-have yielded isolates of WN virus or have transmitted it in laboratory vector competence assays (Anderson et al. 1999; Goddard et al. 2002; Sardelis et al. 2001; Turell et al. 2000, 2001). Initial viral dose, source of host blood meal, temperature, and incubation period are extrinsic factors that influence mosquito susceptibility to infection with an arbovirus like WN (Hardy et al. 1983, Reisen et al. 2005). Alternatively, innate susceptibility to infection can be influenced by mosquito midgut physiology, midgut receptor specificity to viral ligands, and tropisms at the level of the salivary gland or other target tissues, such as the fat body or ovaries (Weaver et al. 1990, Bennett et al. 2005). Innate differences frequently vary from one geographic population to another or, in the case of the Cx. pipiens complex, across regions of genetic introgression (Cornel et al. 2003, Urbanelli et al. 1997). Consistent with this pattern, laboratory vector competence assays of Cx. p. pipiens and of Cx. p. quinquefasciatus from California have yielded various WN virus infection and transmission values, depending on geographic origin of mosquito populations.
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To design effective disease prevention strategies it is crucial to understand variation in vector competence among California mosquitoes. Knowledge regarding which mosquito species are most likely to transmit WN virus will facilitate meaningful control activities by targeting and suppressing specific mosquito populations. Successful control strategies based on vector competence will be important for preventing disease in wildlife, horses, and humans.
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I propose to investigate the antiviral genes responsible for refractoriness in California Culex mosquitoes as markers in natural populations for predicting local risk of virus transmission. A genetic probe for field-caught mosquitoes could be applied by public health and veterinary officials and mosquito abatement districts to assess the potential for virus transmission by geographically and temporally different mosquito populations. Based on results, mosquito control personnel could make informed, strategic decisions about where and when to most effectively apply their mosquito and disease control efforts. Such an assay would be immediate and simple, without the biohazard, time, and expense associated with traditional mosquito vector competence and transmission studies.
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My four objectives are:
<OL> <LI> Carry out vector competence assays for populations of Cx. p. quinquefasciatus in a north-south cline of California. <LI> Using mass spectrometry, identify peptides induced by WNV infection in mosquitoes.<LI> Using targeted gene-silencing, determine the function of identified peptides in limiting WNV proliferation and transmission in mosquitoes. <LI>Develop genetic markers for rapid assessment of vector competence based on confirmed antiviral genes.
NON-TECHNICAL SUMMARY: The goal of this project is to identify protein(s) associated with variation in ability of mosquitoes to transmit West Nile virus and to use that knowledge to develop a simple, rapid assay to detect those protein(s) that will help mosquito control personnel make informed, strategic decisions about how to most effectively apply mosquito and disease control efforts. <P>
APPROACH: Testing variability in vector competence of California mosquitoes: Induction of immune peptides likely explains a portion of the geographic variation in vector competence of California Culex mosquitoes. Infection and transmission data on populations collected at different times and locations from throughout California will provide a state-wide picture of WN virus transmission risk and provide the material I will need to identify and characterize immune peptides. Mosquito examined for vector competence will be obtained in collaboration with mosquito abatement personnel from throughout the state. Methods for feeding, maintaining, and testing mosquitoes for WN virus infection and transmission were previously described by Goddard et al. (2002) and are routine in my laboratory. Briefly, female mosquitoes will be allowed to feed on a suspension of WN virus in defibrinated pig blood. Mosquito bodies and salivary expectorate will be collected after 7 and 14 days of incubation. Whole mosquito body homogenates and salivary expectorate will be tested for WN virus by plaque assay in cell culture. Infective dose of virus and insectary conditions (28 deg.C, 85% RH and 16:8 light:dark) will remain constant across species and populations. Transmission will be determined using an in vitro capillary method. For each population the percent of mosquitoes that become infected and percentage that transmit virus 7 and 14 days post infection will be recorded.
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All experimental WN virus infections and manipulations will be carried out at the UC Davis Center for Vector-Borne Disease level-3 containment laboratory on Old Davis Road. I have over 30 years of experience working with level-2 and level-3 arboviral.
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Proteomics and molecular cloning: To identify peptides induced in response to WN virus infection, I will analyze peptides from mosquitoes from the same collections that I assess for vector competence. I will follow a standard protocol of solubilization, 2D gel electrophoresis, and MALDI-TOF mass spectrometry (Levy et al. 2003, Vierstraete et al. 2004). Most of the proteomics analysis can be completed at the UC Davis Molecular Structure Facility. Once identified, antiviral genes will be silenced by targeted double-stranded RNA interference (Hannon 2002). Synthesis of double-stranded RNA for targeted gene-silencing in mosquitoes will follow standard protocols (Blandin et al. 2002, Goto et al. 2003) using Promega kits. Phenotypic loss-of-function mutants will be confirmed by vector competence studies, Northern hybridization to confirm mRNA presence, and mass spectrometry to confirm loss of the antiviral peptide. If knock-out analyses result in refractory mosquitoes becoming susceptible, mRNA will be isolated by triturating WNV-infected mosquitoes, and reverse-transcribed to produce antiviral cDNA. Frequency of the antiviral gene will indicate the relative refractoriness or susceptibility of tested populations. An assay based on genetic markers would not require biocontainment-handling virus is unnecessary-and thus would simplify predicting the potential for local mosquito populations to transmit WN virus.