Assessing the roles of viral mutations and host factors in the transmission of Mayaro virus and other alphaviruses by urban mosquitoes

项目总结/摘要 甲病毒是代表全球健康威胁的人类病原体。马亚罗病毒和基孔肯雅热 CHIKV病毒（CHIKV）是一种甲病毒，可引起急性疾病，包括发热、头痛、肌痛、寒战和长- 术语衰弱性关节痛。CHIKV谱系对城市蚊子埃及伊蚊和伊蚊的适应 白纹伊蚊促成了其在世界范围内的分布，并导致了大规模暴发。MAYV被认为是 仅限于由嗜血蚊属的森林蚊子传播，但实验室研究表明， MAYV也可以通过伊蚊传播，这表明需要加强监测， 对策我发现MAYV可以感染来自萨尔瓦多（巴西）或加尔维斯顿的伊蚊， 存在于人类病毒血症中的滴度。该项目旨在了解适应性突变和 与宿主的相互作用可能导致甲病毒爆发。我的核心假设是 MAYV nsP 3基因的突变可以通过促进蚊子的传播而导致伊蚊的有效传播。 病毒与宿主的相互作用我将通过三个具体目标来阐述这一假设。在具体目标1中，我将确定 MAYV基因组中在伊蚊的载体能力中起作用的现有和潜在突变 蚊子我对受感染的伊蚊的唾液腺进行了下一代测序， 发现了17个pupillary适应性突变。我将找到并验证蚊子身上出现的MAYV少数变异 使用竞争测定、50%口服感染剂量实验和双宿主模型进行感染。作为证据 根据这一原理，我在病毒非结构蛋白3（nsP 3）中发现了一个适应性突变。我将决定在 蚊子感染的哪一步是重要的，通过一系列的解剖，然后滴定。在 具体目标2，我将揭示nsP 3在蚊子感染和载体能力中的作用。我会召集 不同MAYV毒株的nsP 3嵌合体，以鉴定对于增加适应性是必要且足够的区域 在伊蚊中，研究了一种在MAYV毒株中至少进化了两次的自然插入，这表明它 是适应性的，其提出的蛋白质磷酸化的变化。在具体目标3中，我将评估m6 A如何 MAYV和CHIKV RNA上的修饰可以促进免疫逃避，以及这些修饰在细胞中是如何发生的。 RNA调节免疫系统。这些修饰被认为在免疫系统中具有关键功能 调节和免疫逃避。我发现MAYV在其RNA上有m6 A修饰， 并促进病毒复制。这一项目的完成将对 通过刺激针对病毒宿主的新的监测策略和对策来控制甲病毒 交互.这个项目和职业发展奖与我目前的技能和职业目标非常吻合， 成为独立的首席研究员。它将帮助我填补我的知识空白的载体和发展 成为独立研究员所需的关键行政和写作技能。

Project Summary/Abstract Alphaviruses are human pathogens that represent a global health threat. Mayaro virus (MAYV) and chikungunya virus (CHIKV) are alphaviruses that can cause acute disease with fever, headache, myalgia, chills, and long- term debilitating arthralgia. Adaptation of CHIKV lineages to the urban mosquitoes Aedes aegypti and Aedes albopictus has contributed to its worldwide distribution and led to large outbreaks. MAYV is thought to be restricted to transmission by sylvatic mosquitoes of the genus Haemagogus, but laboratory studies show that MAYV can also be transmitted by Aedes mosquitoes, suggesting the need for improved surveillance and countermeasures. I found that MAYV can infect Aedes mosquitoes from Salvador (Brazil) or Galveston when present at titers found in viremia in humans. This project aims to understand how adaptive mutations and interactions with hosts may lead to the emergence of alphaviruses outbreaks. My central hypothesis is that mutations in the MAYV nsP3 gene can lead to efficient transmission by Aedes mosquitoes through promoting viral-host interactions. I will address this hypothesis through three specific aims. In specific aim 1, I will determine existing and prospective mutations in the genome of MAYV that function in vector competence of Aedes mosquitoes. I have performed next generation sequencing of salivary glands of infected Aedes mosquitoes and found 17 putatively adaptive mutations. I will find and validate MAYV minority variants that arise upon mosquito infection using competition assays, 50% oral infectious dose experiments and dual host models. As a proof-of- principle, I discovered an adaptive mutation in the virus non-structural protein 3 (nsP3). I will then determine at which step of mosquito infection they are important through a series of dissections followed by titrations. In specific aim 2, I will uncover the roles of nsP3 in mosquito infections and vector competence. I will assemble nsP3 chimeras of different MAYV strains to identify regions that are necessary and sufficient for increased fitness in Aedes mosquitoes and study a natural insertion that has evolved at least twice in MAYV strains, suggesting it is adaptive, and its proposed changes in protein phosphorylation. In specific aim 3, I will assess how m6A modifications on MAYV and CHIKV RNA may promote immune evasion and how these modifications in cellular RNAs modulate the immune system. These modifications are thought to have key functions in immune system regulation and immune evasion. I showed that MAYV has m6A modifications on its RNA which are concentrated in the sub genomic RNA and promote viral replication. Completion of this project will have a major impact in the control of alphaviruses by spurring novel surveillance strategies and countermeasures targeting virus-host interactions. This project and career development award aligns well with my current skills and career goals to become an independent principal investigator. It will help me fill gaps in my knowledge of vectors and to develop key administrative and writing skills required to become an independent researcher.