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

基本信息

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
项目类别：
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

项目摘要

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 病毒如 SARS-CoV-2（冠状病毒科）和登革热病毒（DENV；黄病毒科）引起全球范围内重大疾病的出现凸显了了解 SARS-CoV-2 变种的必要性。疾病严重程度、传播潜力和逃避免疫能力的病毒遗传决定因素，这需要分子工具来制造病毒突变体。然而，目前的构建方法和方法。操纵病毒感染性克隆依赖于活宿主（细菌或酵母），这可能会导致不需要的突变以及病毒基因组的缺失，特别是对于冠状病毒和黄病毒等较大的病毒。由于宿主会导致这些不需要的突变，因此很少有实验室能够成功地完成这项工作。消除对活着的宿主的需要是最好的前进方向。我们的长期目标是创建易于使用的工具，促进基础病毒学研究，旨在减轻疾病负担，符合 NIH 的使命，该项目旨在开发和优化一个范例 - 用于构建和操作病毒感染性克隆的转变但简单的无宿主方法，促进旨在确定疾病、传播和其他表型的病毒遗传决定因素的研究。这种方法还可用于构建重组疫苗病毒和报告基因等诊断工具我们将使用一种令人兴奋的技术——复制循环反应（RCR）——来重建大肠杆菌。 RCR 管中的 DNA 复制机器可以高效地扩增高达 1 Mb 的单个 DNA 分子。在这里，我们建议开发这种基于 RCR 的系统来构建新的系统。感染性克隆（目标 1）并操纵现有克隆（目标 1）将使用化学合成的 DNA 来实现。为快速传播的 SARS-CoV-2 Omicron 变种和 DENV 毒株 Puo-218 创建感染性克隆，已批准的 DENV 疫苗 Dengvaxia 的组成部分这一目标的影响将是一个创新系统目标 2 将使用基于 RCR 的系统来产生新的感染性克隆。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；对原代人类细胞中病毒复制的影响将是一种产生的简单方法。病毒突变体，促进机制研究以了解发病机制的病毒遗传决定因素，这些研究还将确定 Spike 之外影响病毒的突变。我们预计这些研究将通过增强更多的能力对分子病毒学产生持续影响。我们预计这里产生的技术将得到广泛应用。也适用于其他正义RNA病毒以及负义RNA和DNA病毒。将无限制地公开分享这里生成的所有方法和工具。