IMPACT OF RNA INTERFERENCE ON QUASISPECIES EVOLUTION IN VECTOR-BORNE FLAVIVI

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，不一定是研究者的机构。 由于它们的高突变率、大种群规模和快速复制率，RNA病毒种群作为突变基因组群或“准种”存在。 准种的多样性影响病毒的适应性、疾病的严重程度和对抗病毒治疗的反应。确定形成准种多样性的机制对于更好地预防病毒出现和控制病毒疾病是必要的，特别是对于节肢动物传播的病毒，所有这些病毒都是RNA病毒，其中许多是对全球公共卫生的重大新威胁。最近发现的RNA干扰（RNAi），有针对性地破坏RNA与高同源性的双链RNA触发，揭示了第一个一般途径，节肢动物调节病毒复制。拟议的研究将测试这样的假设，即载体的RNAi反应对突变进行选择，使病毒种群能够逃避RNAi，RNAi从而驱动病毒准种的多样化。为了测试降低RNAi活性应导致准种多样性降低的预测，我们将用蚊媒登革热病毒感染假处理的果蝇细胞和其中RNAi已被特异性下调的果蝇细胞。 将使用焦磷酸测序确定所得病毒子代的多样性。然后将在活果蝇中进行类似的实验，通过用登革病毒感染野生型果蝇或缺乏RNAi途径的特定组分的突变果蝇，并对所得病毒后代进行测序。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Due to their high rates of mutation, large population sizes, and rapid rates of replication, RNA virus populations exist as a swarm of mutant genomes, or "quasispecies". The diversity of a quasispecies influences viral adaptation, disease severity, and response to antiviral therapies. Identifying the mechanisms that shape quasispecies diversity is necessary to better prevent virus emergence and control virus disease, particularly for arthropod-borne viruses, all of which are RNA viruses and many of which are significant emerging threats to global public health. The recent discovery of RNA interference (RNAi), the targeted destruction of RNA with high homology to a double-stranded RNA trigger, has revealed the first general pathway by which arthropods regulate viral replication. The proposed research will test the hypothesis that the RNAi response of vectors imposes selection for mutations that enable virus populations to escape RNAi, and RNAi thereby drives diversification of viral quasispecies. To test the prediction that decreasing RNAi activity should result in decreased quasispecies diversity, we will infect sham-treated Drosophila cells and Drosophila cells in which RNAi has been specifically downregulated with mosquito-borne dengue virus. Diversity of the resulting virus progeny will be determined using pyrosequencing. A similar experiment will then be conducted in live Drosophila by infecting wild type flies or mutant flies that lack specific components of the RNAi pathway with dengue virus and sequencing the resulting viral progeny.