INTERFERENCE TO DENGUE VIRUS REPLICATION IN MOSQUITOES

登革热（DEN;黄病毒科）病毒继续造成健康问题 对世界热带地区的人口来说。 DEN 出血热和登革出血热休克综合征是威胁大 亚洲和太平洋区域城市和农村人口比例 美洲. 伴随着疾病的传播， 登革病毒蚊媒埃及伊蚊的增殖。 的 缺乏针对DEN的有效疫苗，以及 对杀虫剂有抗药性的病媒， 制定控制疾病的常规策略。 的 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， 传输 我们已经表明，表达正义或反义 来自辛德毕斯（SIN;披膜病毒科）病毒的DEN prM基因的RNA 载体在蚊子细胞或成年Ae.埃及伊蚊有效地干扰了 与重复感染DEN病毒的复制有关。 在本提案中，我们 将使用SIN转导病毒表达第二代抗- 埃及伊蚊中的DEN病毒因子。 这些代理人将包括： 1)可以靶向的改进设计的正义和反义RNA 对DEN病毒的所有4种血清型，2）突变的DEN复制酶基因 NS 5和3）抗DEN核酶。 设计合理，DEN sense 反义RNA应该能补充并潜在地增强 RNA介导的抗性策略已经被证明 要有效率。 最初，SIN转导病毒将用于 表达靶向反式切割β- 半乳糖苷酶基因在转化的蚊子细胞中表达。 的 利用核酶技术降低或消除 蚊子对登革病毒的反应是反义核酸的自然延伸， 已经在使用的战略。 在蚊子中的表达 DEN聚合酶（NS 5）基因的形式代表了重要的 消除载体中病毒复制的策略及其效力 作为抗病毒药的效果需要评估 此外，有前途的 第二代抗病毒药物将在转化的 蚊子细胞培养，以研究这些药剂的功效， 从DNA表达系统传递。 最后，有希望 从SIN病毒转导开发的干扰策略， 抗病毒剂的DNA表达将在体内使用 伊蚊浓核症病毒。 总的来说，这 这项工作应该让我们对哪种反DEN有重要的了解， 病毒策略对于最终产生转基因植物是重要的。 对登革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.