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.

项目概要/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 全球性的重大疾病。令人担忧的SARS-CoV-2变种的出现凸显了了解 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，目前的建设和 操纵病毒感染性克隆依赖于活宿主（细菌或酵母），这可能导致不必要的突变 以及病毒基因组中的缺失，特别是对于较大的病毒，如冠状病毒和黄病毒。由于这些 然而，很少有实验室能够成功地完成这项工作。由于宿主导致了这些不需要的突变， 不需要活体宿主是最好的方法 我们的长期目标是创建简单易用的工具，以促进基础病毒学研究， 减少疾病负担，符合NIH的使命。该项目旨在开发和优化一种模式- 用于构建和操纵病毒感染性克隆的转变但直接的无宿主方法， 促进寻求确定疾病、传播和其他表型的病毒遗传决定因素的研究。 这种方法也可用于构建重组疫苗病毒和诊断工具，如报告基因， 病毒我们将使用一种令人兴奋的技术-复制循环反应（RCR）-它重建了E。杆菌 试管中的DNA复制机器。RCR可以有效地扩增高达1 Mb的单个DNA分子， 在一个简单易用的格式保真度。在这里，我们建议开发这种基于RCR的系统，以构建新的 感染性克隆（目标1）和操纵现有克隆（目标2）。Aim 1将使用化学合成的DNA， 为快速传播的SARS-CoV-2 Omicron变异株和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的系统， 产生新的感染性克隆。目标2将使用基于RCR的系统在基因组中进行突变和缺失。 SARS-CoV-2 Omicron变体在现有的感染性克隆中;然后我们将研究这些突变的影响 在原代人类细胞中的病毒复制。这一目标的影响将是一个简单的方法来产生 病毒突变体，促进机制研究，以了解病毒遗传决定因素的发病机制， 传播和免疫逃避。这些研究还将确定Spike以外的突变， 复制的我们希望这些研究将对分子病毒学产生持续的影响， 构建和操纵感染性克隆的实验室。我们预计这里产生的技术将广泛地 也适用于其它正义RNA病毒和反义RNA和DNA病毒。最后我们 我们将不受限制地公开分享这里生成的所有方法和工具。