Quasispecies dynamics in arbovirus persistence emergence and fitness

节肢动物传播病毒的局部、特定地点特征在很大程度上控制着传播动态 (虫媒病毒)。虫媒病毒反过来适应当地的条件，最大限度地保持和 成为对健康的威胁。虫媒病毒的适应潜力是由容易出错的复制驱动的，这种复制 在每个宿主中创建了一个遗传多样性的竞争病毒基因型池。这份提案考察了 蚊子和鸟类协同行动，塑造了西尼罗河病毒的进化和健康。我们之前的研究已经允许 我们对每个拟议目标的结果做出非常明确的预测，并促进了我们的能力 将我们之前的工作转化为寨卡病毒等新出现的病原体。 在鸟类中，WNV适合度的增加受到易受影响的脊椎动物(如乌鸦)的高MOI环境的限制 在限制复制的鸟类(如知更鸟)中推广。蚊子对西尼罗河病毒的影响也取决于物种。 多样化和健全性。具有讽刺意味的是，蚊子的系统性感染会导致传播疾病的适合性降低。 西尼罗河病毒种群。因此，目标1将试图通过强迫WNV进入 具有不同寄主组合的传递周期。我们预测乌鸦和CX。蚊子会把蚊子 导致西尼罗河病毒种群的适合度与由 罗宾斯和CX。[医]五倍子属 我们的结果有力地表明，西尼罗河病毒在乌鸦体内复制时对适应能力增长的限制是相关的 与知更鸟在这种宿主中出现的高病毒有关。在高MOI时，单个细胞的混合感染 高效和有缺陷(或低适合度)的基因组得到高适合度基因组的补充。这抑制了 人口的整体健康状况。当前提案的目标2通过体内和外部试验验证这一假说 西尼罗河病毒复制的关键部位禽类PBMCs中西尼罗河病毒载量和多样性的活体研究。我们预测，在 高MOI，明显有害的突变(例如，宿主内长度变异)将持续存在，适合度将 减少了。 我们在西尼罗河病毒感染蚊子期间观察到的适合度下降是因为病毒的高变异。 随着病毒从一种病毒传播到另一种病毒，伴随着人口的随机减少(即瓶颈)的比率 蚊子组织传给另一个。因此，了解它们形成的机制基础是至关重要的。 阻止虫媒病毒传播的“屏障”。我们的初步数据表明，一个关键方面有助于 它们是基于RNAi的针对黄病毒sfRNA的靶向。此外，我们有初步数据表明， SfRNA1/2促进病毒逃避解剖屏障。因此，在目标3中，我们将研究蚊子如何 RNAi针对西尼罗河病毒基因组，特别是sfRNA1起始点，以及病毒种群如何变化 由于被“困”在传播屏障内。这一目标也将利用我们广泛的 从事西尼罗河病毒与宿主相互作用的工作经验，以更深入地了解正在出现的寨卡病毒。至 要做到这一点，我们将使用新开发的西尼罗河病毒和ZIKV反向遗传学系统，这些系统缺乏能力 以产生sfRNA1。申请表中提供了有关这方面的初步数据。 这项工作的意义在于，它将提供关于不同传输周期如何影响的新数据 病毒遗传学，以及这如何导致新病毒株的出现。我们提议的工作还将 提供有关不同鸟类和蚊子对病毒有不同影响的重要机制数据 人口。将我们基于WNV的发现转换到ZIKV也是这项工作的关键，因为我们认为我们的工作是 利用我们所学到的知识来应对新的虫媒病毒威胁。最后，我们工作的意义在于我们 提供了广泛有用的技术和分析工具，使我们能够合作 有效地与广泛的调查人员合作。拟议的研究在技术和概念上都是创新的。 例如，由于我们对西尼罗河病毒传播周期的建模，我们使用单细胞方法，以及我们的 能够分析蚊子唾液腺中的小RNA。

Local, site-specific characteristics largely control the transmission dynamics of arthropod-borne viruses (arboviruses). Arboviruses, in turn, adapt to local conditions, maximizing their potential to perpetuate and emerge as health threats. The adaptive potential of arboviruses is driven by error-prone replication, which creates a genetically diverse pool of competing virus genotypes within each host. This proposal examines how mosquitoes and birds act in concert to shape WNV evolution and fitness. Our previous research has allowed us to make very clear predictions about the outcome of each proposed aim and has facilitated our ability to translate our previous work to new emerging pathogens such as Zika virus. In birds, WNV fitness gains are limited by high MOI environments in susceptible vertebrates (e.g. crows) and promoted in birds that limit replication (e.g. robins). Mosquitoes also have species-dependent impacts on WNV diversification and fitness. Ironically, systemic infection of mosquitoes leads to reduced fitness in transmitted WNV populations. Therefore, Aim 1 will attempt to either reduce or increase WNV fitness by forcing it into transmission cycles with different host assemblages. We predict that crows and Cx. pipiens mosquitoes will result in WNV populations that are dramatically reduced in fitness compared to WNV that is maintained by robins and Cx. quinquefasciatus. Our results strongly suggest that the limitations on fitness gains of WNV when it replicates in crows are related to the high viremias that occur in this host relative to robins. At high MOI, coinfection of individual cells is efficient and defective (or low fitness) genomes are complemented by those of high fitness. This suppresses the overall fitness of the population. Aim 2 of the current proposal tests this hypothesis through in vivo and ex vivo studies of WNV loads and diversity in avian PBMCs, a critical site of WNV replication. We predict that at high MOI, clearly deleterious mutations (intrahost length-variants, for example) will persist and fitness will be reduced. The fitness declines that we observed in WNV during mosquito infection occur because of high virus mutation rates coupled with stochastic reductions in the population (i.e. bottlenecks) as the virus moves from one mosquito tissue to another. It is therefore critical to understand the mechanistic basis for the formation of these “barriers” to arbovirus transmission. Our preliminary data suggests that one critical aspect that contributes to them is RNAi-based targeting of the flavivirus sfRNA. In addition, we have preliminary data suggesting that sfRNA1/2 facilitates virus escape from anatomical barriers. Therefore, in Aim 3 we will examine how mosquito RNAi targets the WNV genome, and in particular the sfRNA1 start site, and how the virus population changes as a result of being “trapped” within a transmission barrier. This aim also will leverage our extensive experience working on WNV-host interactions to more deeply understand the emergent Zika virus. To accomplish this we will use newly developed reverse genetics systems for WNV and ZIKV that lack the ability to produce sfRNA1. Preliminary data on this is provided in the application. The significance of this work is that it will provide novel data on how different transmission cycles can impact virus genetics, and how this can lead to the emergence of new virus strains. Our proposed work will also provide important mechanistic data on why different birds and mosquitoes have different impacts on virus populations. Translating our WNV-based findings to ZIKV is also critical to this work, as we think it is our job to use what we have learned to address new arboviral threats. Finally, the significance of our work is that we have provided technical and analytical tools that are broadly useful and have permitted us to collaborate effectively with a wide array of investigators. The proposed studies are technically and conceptually innovative due to, for example, our modeling of the WNV transmission cycle, our use of single-cell approaches, and our ability to profile small RNAs within mosquito salivary glands.