An innovative and straightforward approach to construct and manipulate viral infectious clones

构建和操作病毒感染性克隆的创新且简单的方法

• 批准号: 10667766
• 负责人: James D Weger
• 金额: $ 7.55万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-28 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10667766
• 关键词: 2019-nCoV; Applied Research; Attenuated Vaccines; Bacteria; Basic Science; Biology; Biomedical Research; Bypass; Cells; Circular DNA; Clonality; Cloning; Communities; Complementary DNA; Coronavirus; DNA; DNA Viruses; DNA biosynthesis; DNA chemical synthesis; Data; Databases; Dengue Vaccine; Dengue Virus; Dengvaxia; Disease; Epithelial Cells; Escherichia coli; Family; Flaviviridae; Flavivirus; Future; Genetic; Genetic Determinism; Genome; Genotype; Goals; Growth; Human; Immune Evasion; Immunity; In Vitro; Kinetics; Link; Measures; Mediating; Methods; Mission; Molecular; Molecular Virology; Mutation; Nose; Pathogenesis; Phenotype; Plasmid Cloning Vector; Plasmids; Point Mutation; Polymerase; Protocols documentation; Public Health; RNA Viruses; Reaction; Recombinants; Reporter; Research; Resource Sharing; Resources; SARS-CoV-2 B.1.1.529; SARS-CoV-2 variant; Scientist; Serotyping; Severity of illness; System; Techniques; Toxic effect; Tube; United States National Institutes of Health; Vaccines; Viral; Viral Genome; Virus; Virus Assembly; Virus Replication; Work; Yeasts; airway epithelium; burden of illness; deep sequencing; diagnostic tool; empowerment; fighting; fundamental research; innovation; mutant; novel; novel strategies; pathogen; promoter; recombinant virus vaccine; reconstitution; reverse genetics; tool; transmission process; vaccine candidate; variants of concern; virology; virus genetics

Project Summary / Abstract. RNA viruses like SARS-CoV-2 (family Coronaviridae) and dengue virus (DENV; family Flaviviridae) cause significant disease globally. The rise of SARS-CoV-2 variants of concern has highlighted the need to understand the viral genetic determinants underlying disease severity, transmission potential, and ability to evade immunity, which requires molecular tools to make viral mutants. However, current approaches to constructing and manipulating viral infectious clones rely on a living host (bacteria or yeast), which can lead to unwanted mutations and deletions in the viral genome, especially for larger viruses like coronaviruses and flaviviruses. Due to these issues, few labs can successfully perform this work. Since the host causes these unwanted mutations, a strategy that removes the need for a living host represents the best way forward. Our long-term goal is to create simple-to-use tools that facilitate fundamental virology research aimed at reducing disease burden, in line with NIH’s mission. This project aims to develop and optimize a paradigm- shifting but straightforward host-free approach for constructing and manipulating viral infectious clones, facilitating studies seeking to identify viral genetic determinants of disease, transmission, and other phenotypes. This approach can also be used to construct recombinant vaccine viruses and diagnostic tools like reporter viruses. We will use an exciting technique—replication cycle reaction (RCR)—which reconstitutes the E. coli DNA replication machinery in a tube. RCR can efficiently amplify a single DNA molecule of up to 1 Mb with high fidelity in a simple-to-use format. Here, we propose developing this RCR-based system to construct new infectious clones (Aim 1) and manipulate existing ones (Aim 2). Aim 1 will use chemically synthesized DNA to create infectious clones for the rapidly spreading SARS-CoV-2 Omicron variant and DENV strain Puo-218, a component of the approved DENV vaccine, Dengvaxia. The impact of this aim will be an innovative system to generate new infectious clones. Aim 2 will use the RCR-based system to make mutations and deletions in the SARS-CoV-2 Omicron variant in an existing infectious clone; we will then study the impact these mutations have on viral replication in primary human cells. The impact of this aim will be a straightforward method to generate viral mutants, facilitating mechanistic studies to understand the viral genetic determinants of pathogenesis, transmission, and immune evasion. These studies will also identify mutations outside Spike that impact viral replication. We expect these studies will have a sustained impact on molecular virology by empowering more labs to construct and manipulate infectious clones. We anticipate the techniques generated here to be broadly applicable to other positive-sense RNA viruses and negative-sense RNA and DNA viruses as well. Finally, we will share all the methods and tools generated here openly and without restriction.

项目摘要 /摘要。 RNA病毒（例如SARS-COV-2（家族冠状科）和登革热病毒（DENV； Family Flaviviridae）导致原因 全球重要的疾病。 SARS-COV-2的兴起的关注变体凸显了需要理解的 病毒遗传决定因素潜在的疾病严重程度，传播潜力和能够证明免疫性史的能力， 这需要分子工具来制造病毒突变体。但是，当前的构造方法和 操纵病毒感染克隆依赖于活宿主（细菌或酵母），这可能导致不必要的突变 病毒基因组中的缺失，尤其是对于冠状病毒和黄病毒等大型病毒。由于这些 问题，很少有实验室可以成功执行这项工作。由于宿主会导致这些不必要的突变，因此 这消除了对活寄主的需求代表了前进的最佳方式。 我们的长期目标是创建易于使用的工具，以促进旨在的基本病毒学研究 与NIH的任务相一致，减少了伯恩的疾病。该项目旨在开发和优化范式 - 转移但直接的无宿主方法来构建和操纵病毒感染克隆， 促进旨在识别疾病，传播和其他表型的病毒遗传决定者的研究。 这种方法也可以用于构建重组疫苗病毒和诊断工具（例如记者） 病毒。我们将使用激动人心的技术 - 复制周期反应（RCR） - 重构大肠杆菌 DNA复制机械在管中。 RCR可以有效扩增高达1 Mb的单个DNA分子 以简单的格式保真。在这里，我们建议开发基于RCR的系统来构建新的 传染性克隆（AIM 1）并操纵现有的克隆（AIM 2）。 AIM 1将使用化学合成的DNA到 为快速扩散的SARS-COV-2 Omicron变体和DENV菌株PUO-218创建传染性克隆，A 这个目标的影响将是一个创新的系统 产生新的感染克隆。 AIM 2将使用基于RCR的系统来制造突变和删除 现有传染性克隆中的SARS-COV-2 OMICRON变体；然后，我们将研究这些突变的影响 原代人细胞中的病毒复制。这个目标的影响将是生成的直接方法 病毒突变体，支持机械研究以了解发病机理的病毒遗传决定因素， 传播和免疫驱动。这些研究还将识别出影响病毒的突变的突变 复制。我们预计这些研究将通过增强能力来对分子病毒学产生持续的影响 实验室来构建和操纵传染性克隆。我们预计这里生成的技术将是广泛的 适用于其他阳性RNA病毒以及阴性的RNA和DNA病毒。最后，我们 将在此处公开生成的所有方法和工具，而无需限制。