Elucidating the Mechanism of Ebola Virus Enterotoxigenicity

阐明埃博拉病毒肠毒性机制

基本信息

批准号：
10025919
负责人：
Marcos Javier Ramos-Benitez
金额：
--
依托单位：
U.S. NATIONAL INST ALLERGY & INFECT DIS
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：
2020-09-01 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10025919
关键词：
Abdominal Pain Africa Apical Behavior Biological Assay Biophysics Calcium Case Study Cell Culture Techniques Cell membrane Cells Cellular biology Characteristics Chloride Channels Chlorides Colon Complement Confocal Microscopy Data Democratic Republic of the Congo Development Diarrhea Dose Ebola Hemorrhagic Fever Ebola virus Ebola virus envelope glycoprotein Electrical Resistance Electrodes Electrolytes Electrophysiology (science)Endoplasmic Reticulum Enterotoxins Environmental Pollution Epidemic Epithelial Epithelial Cells Epithelium Family Feces Fever Filovirus Fluids and Secretions G-Protein-Coupled Receptors Gastrointestinal tract structure Genome Glucose Glycoproteins Goals Headache Homeostasis Human Hypovolemic Shock Immunology Intestines Ion Channel Ion Transport Ions Kinetics Lead Liquid substance Literature Measures Mediating Mediator of activation protein Membrane Membrane Glycoproteins Microscopy Molecular Molecular Biology Monitor Movement Myalgia Nonstructural Protein Pathogenesis Pathway interactions Patients Permeability Phenotype Phospholipase C Physiological Play Process Proteins RNA Viruses Regulation Research Risk Role Rotavirus Signal Pathway Small Intestines Sodium Source Stimulus Surface Symptoms System Techniques Testing Training Translating Variant Viral Water absorption apical membrane career design diarrheal disease driving force epithelial Na+ channel extracellular gastrointestinal gastrointestinal epithelium health care settings high throughput screening improved in vitro Assay intestinal epithelium monolayer mortality new therapeutic target novel outcome forecast patch clamp pathogen polarized cell response secretion process skills translational impact transmission process treatment strategy viral transmission voltage

项目摘要

PROJECT SUMMARY Ebola Virus Disease (EVD) is highly lethal with >20,000 cases reported during the 2014-16 West Africa epidemic and >2,800 cases reported during the ongoing epidemic in the Democratic Republic of the Congo. Ebola virus (EBOV) belongs to the Filoviridae family and encodes for 7 proteins including a single glycoprotein (EBOV-GP) that has been shown to play a crucial role in EVD pathogenesis. During EVD, gastrointestinal fluid loss via large volume watery diarrhea leads to hypovolemic shock, electrolyte imbalances, and increased mortality. Furthermore, feces are categorized as highly infectious, thus excessive fluid loss incites environmental contamination increasing the risk of nosocomial viral transmission. The physiological mechanisms of viral glycoproteins acting as enterotoxins and prompting high volume diarrhea have been well described. However, the molecular trigger and mechanisms describing how EBOV stimulates high volume watery diarrhea during EVD have never been studied. Our preliminary data in human intestinal cells suggests an enterotoxin- like behavior for EBOV-GP as it induces a rapid and dose-dependent increase in intracellular Ca2+ concentration that is mitigated by inhibition of Phospholipase C (PLC). Moreover, EBOV-GP stimulation induces activation of chloride channels. The working hypothesis is that EBOV-GP induces intracellular Ca2+ increase in the gastroinstestinal epithelia, triggering an apical surface ion transport dysregulation resulting in increased permeability and water secretion. The project goals are to determine if EBOV-GP acts as an enterotoxin, study if it triggers a malabsorptive or secretory process and fully elucidate the mechanisms leading to high-volume watery diarrhea during EVD. For this, the project has three specific aims conceptualized for using of small intestine and colon cells since they diverge in absorption and secretion processes. Aim 1 will study the contributions of Ca2+ sources in the increased levels of intracellular Ca2+ triggered by EBOV-GP and elucidate its upstream signaling pathway. Aim 2 will assess the dynamics of Na+ and Cl- across the cell membrane, cell permeability and fluid transport after EBOV-GP stimulation. Polarized cell cultures will be used to mimic the ions/fluid movement and directionality. Aim 3 will feature whole-cell patch clamp to identify the ion channels being altered by EBOV-GP. This project will be achieved using contemporary in-vitro assays and combine microscopy, molecular biology, electrophysiology and biophysics. The completion of this project will provide the awardee training in electrophysiology techniques advancing his career and complementing his immunology and cell biology background. The proposed project will provide the awardee the skill set to study host-pathogen interactions in the context of the interplay between electrophysiology, immunology and cell biology. This project has translational impact as it could lead to novel therapeutic targets and strategies for EVD high volume diarrhea, directly improving the patients prognosis and reducing viral transmission in healthcare settings in the field.

项目概要埃博拉病毒病 (EVD) 具有高度致命性，2014-16 年西非疫情期间报告病例数超过 20,000 例刚果民主共和国疫情期间报告了超过 2,800 例病例。埃博拉病毒 (EBOV) 属于丝状病毒科，编码 7 种蛋白质，包括单个糖蛋白 (EBOV-GP) 已被证明在埃博拉病毒病发病机制中发挥着至关重要的作用。在埃博拉病毒病期间，胃肠道液体通过大量流失大量水样腹泻会导致低血容量性休克、电解质失衡和死亡率增加。此外，粪便被归类为高度传染性的，因此过多的液体流失会导致环境污染。污染增加了医院内病毒传播的风险。病毒的生理机制糖蛋白作为肠毒素并引发大量腹泻已被充分描述。然而，描述埃博拉病毒如何刺激大量水样腹泻的分子触发和机制从未研究过 EVD 期间的情况。我们在人类肠道细胞中的初步数据表明，肠毒素与 EBOV-GP 的行为类似，因为它会诱导细胞内 Ca2+ 快速且剂量依赖性增加通过抑制磷脂酶 C (PLC) 可以降低浓度。此外，EBOV-GP刺激诱导氯离子通道的激活。工作假设是 EBOV-GP 诱导细胞内 Ca2+ 胃肠道上皮细胞增加，引发顶端表面离子转运失调，导致增加渗透性和水分分泌。该项目的目标是确定 EBOV-GP 是否充当肠毒素，研究它是否会引发吸收不良或分泌过程，并充分阐明导致的机制 EVD 期间出现大量水样腹泻。为此，该项目具有三个具体目标，旨在使用小肠和结肠细胞的吸收和分泌过程不同。目标 1 将研究 Ca2+ 来源在 EBOV-GP 引发的细胞内 Ca2+ 水平增加中的贡献并阐明其上游信号通路。目标 2 将评估 Na+ 和 Cl- 穿过细胞膜、细胞的动态 EBOV-GP 刺激后的渗透性和液体输送。极化细胞培养物将用于模拟离子/流体运动和方向性。 Aim 3 将采用全细胞膜片钳来识别离子通道被 EBOV-GP 改变。该项目将使用现代体外测定法并结合显微镜、分子生物学、电生理学和生物物理学。该项目的完成将提供获奖者接受电生理学技术培训，促进他的职业生涯并补充他的免疫学和细胞生物学背景。拟议的项目将为获奖者提供研究宿主病原体的技能电生理学、免疫学和细胞生物学之间相互作用的相互作用。本项目具有转化影响，因为它可以为埃博拉病毒病大量腹泻带来新的治疗靶点和策略，直接改善患者预后并减少现场医疗机构中的病毒传播。