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

项目总结/摘要 内共生体Wolbachia pipientis被释放到蚊子的种群中， 登革热、寨卡病毒、基孔肯雅病毒和黄热病病毒。在实验室里，沃尔巴克氏体 已被证明可以给病媒“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，沃尔巴克氏体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， 可能会威胁到这种内共生体的长期功效。在这里，我们使用强大的进化和 蚊子、沃尔巴克氏体和病毒中的重测序方法（1）了解可能的 病原体阻断机制，以及（2）确定最可能的遗传途径， 蚊子和病毒就会产生抗药性。首先，我们将选择改进和减少 在沃尔巴克氏体感染的蚊子中，沃尔巴克氏体介导的登革病毒阻断;我们已经 在试点研究中证明这是可能的。在一个完全复制的设计中，我们将跟踪登革病毒载量， 蚊子和沃尔巴克氏体的密度我们将在选择前和选择后进行RNAseq 和DNA测序在整个制度的线以及随机对照，证明 改变封锁。通过SNP和表达分析，我们将确定与这些基因相关的关键基因。 表型转变，在共生体和载体基因组，并开发一个假定的模型，为病原体 阻挡。此外，我们将有证据表明，如何有可能提高强度， 登革热阻断和可能的速度和多样性的进化路径的阻力。通过 在细胞培养的选择实验中，我们将确定病毒基因组的哪些方面可以赋予 防止沃尔巴克氏体效应。通过研究进化后的蚊子和 进化的病毒，我们将解决这样的变种是否具有竞争力， 对自然环境中的进化构成了真实的威胁。该工作在实时性应用方面是新颖的 这种方法不是“坐等”的方法，而是观察实地的关系如何演变。这 积极主动的战略可能有助于设计有针对性的战略，规避最可能的形式， 并证明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.