A Transgenic Mouse Model to Study Ebolaviruses and other Filoviruses

研究埃博拉病毒和其他丝状病毒的转基因小鼠模型

• 批准号: 10038454
• 负责人: YOSHIHIRO KAWAOKA
• 金额: $ 20.66万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-22 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10038454
• 关键词: African; Animal Model; Antiviral Agents; Biological; Breeding; Bundibugyo virus; Case Fatality Rates; Cell Culture Techniques; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chickens; China; Chiroptera; Collaborations; Containment; Defective Viruses; Democratic Republic of the Congo; Dendritic Cells; Development; Disease; Disease Outbreaks; Disseminated Intravascular Coagulation; Ebola; Ebola virus; Engineering; Evaluation; Failure; Family; Fibroblasts; Filovirus; Future; Genome; Glycoproteins; Goals; Growth; Guinea; Hematopoietic; Histopathology; Human; Immune response; Immunologics; In Vitro; Infection; Kenya; Kinetics; Knock-in; Laboratories; Laboratory Research; Life Cycle Stages; Liver; Messenger RNA; Modeling; Morphology; Mus; Nature; Organ; PTPRC gene; Pathogenesis; Pathogenicity; Phenotype; Property; Proteins; Public Health; Regulation; Research; Research Personnel; Resistance; Reston Ebola virus; Risk; Science Policy; Sierra Leone; Spain; Sudan Ebola virus; System; Tail; Therapeutic; Therapeutic Agents; Time; Training; Transgenic Animals; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; Universities; Vaccines; Viral; Viral Genes; Viral Genome; Virulence Factors; Virus; Virus Replication; Wild Type Mouse; Wisconsin; Zaire Ebola virus; base; beta Actin; cell type; cost; disease phenotype; forest; genome editing; human disease; in vivo; insight; macrophage; member; monocyte; mouse model; pathogenic virus; promoter; research and development; safety testing; stable cell line; therapeutic vaccine

PROJECT SUMMARY Ebola viruses and other members of the filovirus family cause severe and often lethal infections. While some progress has been made in regards to the development of experimental countermeasures and insights into the highly pathogenic nature of these viruses, there are still many more unanswered questions, and further advancement is needed towards the development of pan-filovirus therapeutic agents and vaccines. A significant hurdle to research on filoviruses is the accessibility and cost associated with high-containment, biosafety level- 4 laboratories. To partially alleviate this issue, we previously established a replication-defective Ebola virus based on the Zaire ebolavirus (EBOV) genome. This virus, which lacks the essential viral gene VP30 (termed EBOVΔVP30), is biologically inert and safe to use outside of highly specialized BSL-4 containment. In engineered cell lines that stably express EBOV VP30, the virus becomes replication-competent; thus it is a perfect EBOV surrogate for in vitro research given that EBOVΔVP30 resembles authentic virus in its life cycle, morphology, protein composition, and growth kinetics. After extensive safety testing both in cell culture and in animal models, the CDC and the Office of Science Policy at the NIH classified EBOVΔVP30 as a BSL-2 agent and removed the virus from Select Agent regulations. Since then, this in vitro system to study EBOV has been requested by and distributed to several other research laboratories. The next step to advance the EBOVΔVP30 system is to develop an EBOV VP30 transgenic animal model to support virus replication. Previously, we generated a transgenic mouse line that expressed EBOV VP30 under the control of the chicken beta-actin promoter (CAG). Although we were able to detect VP30 mRNA in key organs, such as the liver, and functional VP30 protein in cells, such as fibroblasts, we were unable to detect VP30 mRNA and functional protein in monocyte-derived macrophages, the first target of EBOV infection and a cell type essential for virus dissemination throughout the body. Here, we propose to generate a new transgenic mouse line with EBOV VP30 expression under the control of the CD45 promoter, a promoter specific for expression in hematopoietic cells, including monocytes, macrophages, and dendritic cells. Once we confirm the expression and function of VP30 in these cell types, particularly macrophages, we will cross our two different VP30 transgenic lines (CAG and CD45) to generate a double knock-in transgenic mouse line. Once established, we will infect these transgenic mice with mouse-adapted EBOVΔVP30 and characterize the phenotype. We will also generate chimeric versions of EBOVΔVP30 with glycoproteins from other filoviruses and examine the phenotype of these chimeric viruses in the transgenic mice. After safety testing, this new small animal model will be an ideal in vivo surrogate for the authentic mouse model for EBOV infection. For the first time, a transgenic mouse model will be available that can be used efficiently and safely outside of BSL-4 containment to examine EBOV pathogenesis and accelerate the development and evaluation of countermeasures.

项目总结 埃博拉病毒和丝状病毒家族的其他成员会导致严重的、往往是致命的感染。虽然有些人 在制定试验性对策和对 这些病毒的高致病性，仍然有很多悬而未决的问题，而且进一步 需要在开发泛丝状病毒治疗剂和疫苗方面取得进展。一个重要的 丝状病毒研究的障碍是与高遏制、生物安全水平相关的可及性和成本- 4个实验室。为了部分缓解这个问题，我们之前建立了复制缺陷埃博拉病毒 基于扎伊尔埃博拉病毒(EBOV)基因组。这种病毒缺乏必要的病毒基因VP30(称为 EBOVBSL VP30)是生物惰性的，在高度专业的Δ-4安全壳外使用是安全的。在……里面 稳定表达EBOV VP30的工程细胞系，病毒变得具有复制能力；因此，它是一种 鉴于eBOVΔVP30在其生命周期中与真实病毒相似，因此是体外研究的完美替代品， 形态、蛋白质组成和生长动力学。经过广泛的安全测试，在细胞培养和 动物模型、疾病预防控制中心和国立卫生研究院科学政策办公室将EBVΔVP30归类为BSL-2毒剂 并从选择代理条例中删除了该病毒。从那时起，这种体外系统一直在研究EBOV 应其他几个研究实验室的要求并分发给其他几个研究实验室。 EBOVVP30系统的下一步进展是建立一种EBOVVP30转基因动物模型，以 支持病毒复制。此前，我们建立了一个表达EBOV VP30的转基因小鼠系 鸡β-肌动蛋白启动子(CAG)的调控。尽管我们能够在KEY中检测到VP30基因 器官，如肝脏，以及细胞中的功能性VP30蛋白，如成纤维细胞，我们无法检测到 单核细胞来源的巨噬细胞中VP30的mRNA和功能蛋白，EBOV感染的第一个靶点和 病毒在全身传播所必需的细胞类型。在这里，我们建议产生一种新的转基因 EBOV VP30小鼠品系在CD45启动子的控制下表达，是一种特异性的启动子 在造血细胞中表达，包括单核细胞、巨噬细胞和树突状细胞。一旦我们确认了 VP30在这些细胞类型中的表达和功能，特别是巨噬细胞，我们将跨越我们的两个不同的 用VP30转基因系(CAG和CD45)建立双敲入转基因小鼠系。一旦建立起来， 我们将用小鼠适应的EB病毒ΔVP30感染这些转基因小鼠，并对其表型进行鉴定。我们会 还用来自其他丝状病毒的糖蛋白生成嵌合版本的EBVΔVP30，并检查 这些嵌合病毒在转基因小鼠中的表型。经过安全测试，这种新的小动物模型将 可作为EBOV感染小鼠模型的理想体内替代物。第一次，转基因生物 将提供可在BSL-4安全壳外高效、安全地使用的鼠标模型，以进行检查 并加速EBOV发病机制的制定和评价对策。