Experimental evolution of Wolbachia-mediated dengue virus blocking in the mosquito, Aedes aegypti

项目摘要/摘要 内共生沃尔巴克氏菌正在被释放到蚊子媒介的种群中 登革热、寨卡、基孔肯雅热和黄热病病毒席卷整个热带地区。在实验室里，沃尔巴克氏菌 已被证明可以为该媒介接种疫苗，降低或消除其传播病毒的能力。一 对这种生物防治方法的担忧是，沃尔巴克氏菌的作用模式仍然未知。 此外，与所有干预措施(药物、疫苗和杀虫剂)一样，出现抗药性 可能会威胁这种内共生菌的长期疗效。在这里，我们使用强大的进化和 蚊子、沃尔巴克氏菌和病毒中的重测序方法，以(1)了解可能的 病原体阻断的机制，以及(2)确定最可能通过的遗传途径 蚊子和病毒会产生抗药性。首先，我们将选择改进和减少 沃尔巴克氏菌介导的登革热病毒在感染沃尔巴克氏菌的蚊子中的阻断；我们已经 在一项初步研究中表明，这是可能的。在完全复制的设计中，我们将跟踪登革热病毒在 蚊子和沃尔巴克氏菌的密度随着时间的推移而增加。我们将在选择前和选择后执行RNAseq 以及整个制度的DNA测序，以及在随机对照上，证明 阻挡方面的变化。通过SNP和表达分析，我们将确定与该基因相关的关键基因 在共生体和媒介基因组中的表型转变，并开发了一个假定的病原体模型 阻挡。此外，我们将有证据表明如何可能提高 登革热阻断以及抗药性进化路径的可能性和多样性。穿过 在细胞培养中的选择实验中，我们将确定病毒基因组的哪些方面可以赋予 针对沃尔巴克氏菌效应的保护。通过检查进化的蚊子和 进化的病毒，我们将解决这样的变种是否具有竞争性和引起 对自然环境中的进化构成真正的威胁。这项工作在实时方面的应用是新颖的 而不是“静观其变”，而不是“静观其变”，以了解实地的关系是如何演变的。这 积极主动的战略可能有助于设计有针对性的战略，以规避最可能的 并展示了一种提高病原体阻断水平的方法 目前的释放菌株。

PROJECT SUMMARY/ABSTRACT The endosymbiont Wolbachia pipientis is being released into populations of the mosquito vector of dengue, Zika, chikungunya and yellow fever viruses throughout the tropics. In the laboratory, Wolbachia has been shown to “vaccinate” the vector, reducing or eliminating its ability to transmit viruses. One concern for this biocontrol approach is that the mode of Wolbachia’s action remains unknown. Additionally, as with all interventions (drugs, vaccines and insecticides), the emergence of resistance may threaten the long-term efficacy of this endosymbiont. Here, we use powerful Evolve and Resequence approaches in the mosquito, Wolbachia and in the virus to (1) understand the likely mechanism of pathogen blocking, and (2) determine the most likely genetic paths through which resistance will emerge in the mosquito and the virus. First, we will select for both improved and lessened Wolbachia-mediated blocking of dengue virus in Wolbachia-infected mosquitoes; we have already shown this is possible in a pilot study. In a fully replicated design, we will track the dengue virus load in the mosquito and Wolbachia densities through time. We will perform RNAseq pre- and post-selection and DNA sequencing throughout the regime on the lines as well as on random controls, demonstrating changes in blocking. Via SNP and expression analyses, we will identify key genes associated with the phenotypic shifts, in both symbiont and vector genomes, and develop a putative model for pathogen blocking. In addition, we will have evidence of how it might be possible to improve the strength of dengue blocking and the likely rate and diversity of evolutionary paths toward resistance. Through selection experiments in cell culture, we will determine which aspects of the virus genome can confer protection against the Wolbachia effect. By examining the fitness of the evolved mosquitoes and of the evolved viruses, we will address the question of whether such variants would be competitive and pose a real threat to evolution in natural environments. This work is novel in its application of real-time evolution rather than a “sit and wait” approach to see how the relationships evolve in field sites. This proactive strategy may help to design targeted strategies that circumvent the most likely forms of resistance and demonstrate a means to improve upon the level of pathogen blocking exhibited by current release strains.