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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要埃博拉病毒和FILOVIRUS家族的其他成员会引起严重且经常致命的感染。而有些在实验对策和对洞察力的发展方面取得了进展这些病毒的高度致病性，还有更多未解决的问题，进一步需要进步来开发泛膜病毒治疗剂和疫苗。重要的关于fileviruses研究的障碍是与高管，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，我们以前建立了一种复制缺陷的埃博拉病毒基于Zaire Ebolavirus（EBOV）基因组。该病毒缺乏必需的病毒基因VP30（称为） EBOVΔVP30），在生物学上是惰性的，可以安全地使用高度专业的BSL-4遏制。稳定表达EBOV VP30的工程细胞系变得具有复制能力。因此，这是一个鉴于EBOVΔVP30在其生命周期中类似于正宗病毒，因此EBOV替代了体外研究形态，蛋白质组成和生长动力学。经过细胞培养和在 NIH的动物模型，CDC和科学政策办公室分类为BSL-2代理并从选定剂法规中去除病毒。从那以后，研究EBOV的体外系统一直是由并分发给其他几个研究实验室。推进EBOVΔVP30系统的下一步是将EBOV VP30转基因动物模型开发为支持病毒复制。以前，我们生成了一条转基因鼠标线，该线在下面表达EBOV VP30 鸡β-肌动蛋白启动子（CAG）的控制。尽管我们能够检测到键中的VP30 mRNA 器官，例如肝脏和细胞中的功能性VP30蛋白，例如成纤维细胞，我们无法检测到单核细胞衍生的巨噬细胞中的VP30 mRNA和功能蛋白，EBOV感染的第一个靶标和A 细胞类型对于整个体内的病毒传播必不可少。在这里，我们建议生成一个新的转基因在CD45启动子的控制下具有EBOV VP30表达的小鼠线，该启动子是特定于造血细胞中的表达，包括单核细胞，巨噬细胞和树突状细胞。一旦我们确认 VP30在这些细胞类型中的表达和功能，尤其是巨噬细胞，我们将越过我们的两个不同 VP30转基因线（CAG和CD45）生成双敲门转基因小鼠系。建立后，我们将用小鼠适应的EbovΔVP30感染这些转基因小鼠，并表征表型。我们将还用其他丝状病毒的糖蛋白生成EbovΔVP30的嵌合版本并检查这些嵌合病毒在转基因小鼠中的表型。安全测试后，这种新的小动物模型将对于EBOV感染的真实小鼠模型，成为理想的体内替代物。第一次，转基因将有可用的鼠标模型，可以在BSL-4容器之外有效，安全地使用以检查 EBOV的发病机理并加速了对策的发展和评估。