INTERFERENCE TO DENGUE VIRUS REPLICATION IN MOSQUITOES

登革热(登革热；黄病毒科)病毒继续构成健康问题 对于世界热带地区的人类人口来说。窝点 出血热和登革热休克综合征正在威胁着 亚洲城市和农村人口的比例 美洲。伴随着疾病传播的是疾病的迅速传播 登革热病毒蚊媒埃及伊蚊的繁殖。这个 缺乏针对登革热的有效疫苗，以及出现 对农药有抗药性的媒介，已经对 制定控制该病的常规策略。这个 这项研究的长期目标是开发分子策略， 减少或消除病毒在载体中的复制并预防病毒 变速箱。我们已经证明了正义或反义的表达 新城疫病毒DEN PrM基因的RNA序列分析 媒介在蚊子细胞或成虫体内。埃及伊蚊有效地干扰了 复制超级感染的登革热病毒。在这项提案中，我们 将使用SIN转导病毒表达第二代抗 埃及伊蚊体内的登革热病毒制剂。这些代理将包括： 1)可靶向的改进设计的正义和反义RNA 对所有4个血清型的登革热病毒，2)DEN复制酶基因突变 NS5和3)抗DEN核酶。设计合理，整齐划一 反义RNA应该补充并潜在地增强 RNA介导的抗性策略已经被证明 要有效率。最初，SIN转导病毒将被用于 表达靶向反式切割β-核酶的模型核酶 半乳糖苷酶基因在转化蚊虫细胞中的表达。这个 利用核酶技术降低或消融能力 蚊子对蚊子病毒的反义是自然延伸 已经在使用的策略。基因突变在蚊子中的表达 DEN聚合酶(NS5)基因的形式代表着重要的 消除病毒在载体中复制的策略及其效果 作为一种抗病毒药物需要进行评估。此外，前景看好 第二代抗病毒药物将在转型中进行分析 蚊子细胞培养时研究这些药剂的效果 来自DNA表达系统。最后，前景看好 从SIN病毒转导和 抗病毒药物的DNA表达将在体内进行测试，使用 伊蚊浓核增多症(AeDNV；细小病毒科)病毒。总之，这就是 工作应该让我们对哪些反蚊子有重要的了解 病毒策略对最终产生转基因很重要 对蚊子病毒感染有抵抗力的蚊子。

Dengue (DEN; Flaviviridae) viruses continue to pose health problems for human populations in tropical regions of the world. DEN hemorrhagic fever and DEN shock syndrome are threatening large proportions of the urban and rural populations in Asia and the Americas. Concomitant with the spread of disease is the rapid proliferation of the DEN virus mosquito vector, Aedes aegypti. The lack of effective vaccines against DEN, as well as the emergence of pesticide resistant vectors, has had a major negative impact on developing conventional strategies for controlling the disease. The long term goal of this research is to develop molecular strategies that reduce or eliminate virus replication in the vector and prevent virus transmission. We have shown that expression of sense or antisense RNA of the DEN prM gene from a Sindbis (SIN; Togaviridae) virus vector in mosquito cells or adult Ae. Aegypti effectively interferes with replication of superinfecting DEN virus. In this proposal, we will use SIN transducing viruses to express second generation anti- DEN virus agents in Aedes aegypti. These agents will include: 1)sense and antisense RNAs of improved design that can be targeted to all 4 serotypes of DEN virus, 2) mutated DEN replicase gene NS5, and 3) anti-DEN ribozymes. Rationally designed, DEN sense and antisense RNAs should complement and potentially enhance RNA mediated resistance strategies that have already been shown to be effective. Initially, SIN transducing viruses will be used to express a model ribozyme targeted to trans-cleave a Beta- galactosidase gene expressed in transformed mosquito cells. The utilization of ribozyme technology to reduce or ablate the competency of mosquitoes for DEN viruses is a natural extension of the antisense strategies already in use. The expression in mosquitoes of mutated forms of the DEN polymerase (NS5) gene represents important strategies for ablating virus replication in the vector and its efficacy as an antiviral agent needs to be assessed. Additionally, promising second-generation anti virus agents will be analyzed in transformed mosquito cell culture to study the efficacy of these agents when delivered from a DNA expression system. Finally, promising interference strategies developed from SIN virus transduction and DNA expression of the anti-virus agents will be tested in vivo using Aedes densonucleosis (AeDNV; Parovoviridae) virus. In toto, this work should give us significant understanding of which anti-DEN virus strategies are important for ultimately generating transgenic mosquitoes that are resistant to DEN virus infections.