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.

项目摘要埃博拉病毒疾病（EVD）高度致命，在2014 - 16年西非流行期间报道了20,000例> 20,000例在刚果民主共和国正在进行的流行病期间报告的> 2800例案件。埃博拉病毒（EBOV）属于Filoviridae家族，并编码7种蛋白质，包括单个糖蛋白（EBOV-GP）已经显示出在EVD发病机理中起着至关重要的作用。在EVD期间，通过大的胃肠道损失体积水状腹泻会导致低血容量减震，电解质失衡和增加死亡率。此外，粪便被归类为高度感染力，因此液体损失过多促进环境污染增加了医院病毒传播的风险。病毒的生理机制充当肠毒素并提示大量腹泻的糖蛋白已得到很好的描述。但是，分子触发器和描述EBOV如何刺激高体积水性腹泻的机制在EVD期间从未被研究过。我们在人肠细胞中的初步数据表明肠毒素 - 像EBOV-GP的行为一样，因为它诱导了细胞内Ca2+的迅速和剂量依赖性增加通过抑制磷脂酶C（PLC）来降低浓度。此外，EBOV-GP刺激诱导氯化物通道的激活。工作假设是EBOV-GP诱导细胞内CA2+ 胃肠肌上皮的增加，引发顶部表面离子转运失调，导致增加渗透性和水分分泌。项目目标是确定EBOV-GP是否充当肠毒素，研究它是否触发不良或分泌过程，并完全阐明了领先的机制在EVD期间到大量水状腹泻。为此，该项目具有用于使用的三个特定目标小肠和结肠细胞的吸收和分泌过程有所不同。 AIM 1将研究 Ca2+源在EBOV-GP触发的细胞内Ca2+水平上的贡献和阐明它的上游信号通路。 AIM 2将评估整个细胞膜的Na+和Cl-的动力学 EBOV-GP刺激后的渗透性和流体转运。偏振细胞培养物将用于模仿离子/流体运动和方向性。 AIM 3将具有全细胞贴片夹以识别离子通道被EBOV-GP改变。该项目将使用现代体外测定法实现，并结合在一起显微镜，分子生物学，电生理学和生物物理学。该项目的完成将提供电生理技术的获奖者培训，发展了他的职业生涯并补充了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的技能在电生理学，免疫学和细胞生物学之间相互作用的背景下相互作用。这个项目具有翻译影响，因为它可能导致新型的治疗靶标和EVD大容量腹泻的策略，直接改善患者的预后并减少该领域的医疗保健环境中的病毒传播。