A functional and comparative approach for the study of Wolbachia-mediated virus blocking in Aedes aegypti and Aedes albopictus

项目摘要/摘要 由于气候和气候的变化，埃及伊蚊在全球范围内的范围继续扩大 城镇化程度不断提高。作为Ae.埃及伊蚊是登革热、寨卡病毒、黄热病和 基孔肯雅病毒，这些范围的变化也增加了人类疾病的风险。几乎没有什么有效的 无论是疫苗还是抗病毒药物，病媒控制仍然是对付蚊子的最好武器。最近， 将一种昆虫内共生菌--沃尔巴克氏菌释放到蚊子种群中，已经成为一种新的 生物防治策略。沃尔巴克氏杆菌是母系遗传的，具有内在的遗传动力，往往会传播 通过昆虫种群。在蚊子体内，这种细菌限制了病毒的复制，这是一个特征 它被称为“病毒拦截”。在沃尔巴克氏杆菌释放到野生种群后，病毒的阻断作用是 导致登革热在人类中的发病率大幅下降。从长远来看，一个问题是- 沃尔巴克氏菌的长期疗效是在蚊子或病毒中出现抗药性。去设计 为了应对这种可能的情况，我们需要了解病毒阻断的遗传基础。 虽然已经提出了许多理论，但没有一个是完全令人满意的。最近，麦格劳 实验室利用选择实验创造了Ae。携带沃尔巴克氏杆菌的埃及伊蚊品系 阻挡。在对这些基因进行测序的过程中，她的团队确定了一组新的候选基因座和一组新的 封堵基础的假设。这项研究也证明了阻挡的强度很可能 在不同的蚊子遗传背景下有所不同。在这里，我们建议提供功能测试 对于与细胞黏附、转录暂停和神经元有关的候选阻断基因 功能，对登革热和基孔肯雅病毒都使用RNA沉默。在这些实验中，我们将 另外，白纹伊蚊与伊蚊相比，白纹伊蚊一直携带着自然感染的沃尔巴克氏菌。 埃及伊蚊和最近人工引入的沃尔巴克氏菌。Ae.中的阻塞性质。白纹伊蚊是一种 这可能预示着在人工感染的物种中阻断病毒的未来。我们还将开展大规模的 常见的园艺实验，剖析蚊子和蚊子的区域差异的相对贡献 病毒多样性要有阻挡的力量。这个项目将为社区提供新的资源。 包括一种新的沃尔巴克氏菌感染Ae。埃及伊蚊，一组抗键阻断关联的抗体 改良的荧光报告病毒对载体研究的蛋白质和数据效果 能力。更广泛地说，这项研究将有助于测试沃尔巴克氏杆菌介导的病毒的修订模型。 阻断-设计延长沃尔巴克氏菌田间药效的策略的基础知识。 它还将揭示阻止蚊子和病毒遗传多样性的健壮性，这将告诉我们 未来沃尔巴克氏菌株在全球部署的战略。

PROJECT SUMMARY/ABSTRACT The global range of the mosquito, Aedes aegypti continues to expand due to a changing climate and increasing urbanization. As Ae. aegypti is the primary vector of dengue, Zika, yellow fever and chikungunya viruses, these range shifts also increase the risk of human disease. With few effective vaccines or antiviral drugs, vector control remains the best weapon against mosquitoes. Recently, the release of an insect endosymbiont, Wolbachia, into mosquito populations has emerged as a novel biocontrol strategy. Maternally inherited and with inbuilt genetic drive, Wolbachia tends to spread through insect populations. Inside the mosquito, the bacterium limits the replication of viruses, a trait called ‘viral blocking’. Following release of Wolbachia into wild populations, the viral blocking effect is leading to substantial reductions in the incidence of dengue fever in humans. One concern for the long- term efficacy of Wolbachia is the emergence of resistance in mosquitoes or viruses. To design strategies to counter this likely scenario, we need to understand the genetic basis of viral blocking. While many theories have been proposed, none have been entirely satisfying. Recently, the McGraw lab used a selection experiment to create Ae. aegypti lines carrying Wolbachia with differences in blocking. In sequencing those lines, her group identified a set of novel candidate loci and a new set of hypotheses for the basis of blocking. This study also demonstrated that the strength of blocking is likely to vary across diverse mosquito genetic backgrounds. Here we propose to provide functional testing for the candidate blocking genes involved with cell adhesion, transcriptional pausing and neuronal function, for both dengue and chikungunya viruses using RNA silencing. In these experiments we will also contrast Aedes albopictus, that has long harbored a natural infection of Wolbachia, against Ae. aegypti with a recently artificially introduced Wolbachia. The nature of blocking in Ae. albopictus is a likely harbinger of the future of blocking in the artificially infected species. We will also carry out large common garden experiments to dissect the relative contribution of regional differences in mosquito and virus diversity to the strength of blocking. This project will produce novel resources for the community including a new Wolbachia infected Ae. aegypti line, a set of antibodies against key blocking association proteins and data on the efficacy of improved fluorescent reporter viruses for the study of vector competence. More broadly, this research will help to test a revised model for Wolbachia-mediated viral blocking – knowledge fundamental for designing strategies to prolong Wolbachia’s efficacy in the field. It will also reveal the robustness of blocking to mosquito and viral genetic diversity, that will inform strategies for future Wolbachia strain deployment globally.