Elucidating the Mechanism of Ebola Virus Enterotoxigenicity

阐明埃博拉病毒肠毒性机制

• 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
• 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

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 埃博拉病毒病（EVD）具有高度致命性，在2014-16年西非疫情期间报告了超过20，000例病例 在刚果民主共和国持续的疫情期间报告了超过2，800例病例。埃博拉病毒 EBOV（EBOV）属于丝状病毒科，编码7种蛋白质，包括单一糖蛋白（EBOV-GP）， 已经显示在EVD发病机制中起关键作用。在EVD期间，胃肠道液体通过大的 大量水样腹泻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，因此过多的体液流失会引起环境污染。 污染增加了医院内病毒传播的风险。病毒的生理机制 作为肠毒素并引起大量腹泻的糖蛋白已被充分描述。 然而，描述EBOV如何刺激大量水样腹泻的分子触发和机制， 从未被研究过。我们对人类肠细胞的初步研究表明一种肠毒素- EBOV-GP的类似行为，因为它诱导细胞内Ca 2+的快速和剂量依赖性增加 通过抑制磷脂酶C（PLC）而减轻的浓度。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Ca 2 + 胃肠道上皮细胞增加，触发顶端表面离子转运失调，导致 增加渗透性和水分泌。该项目的目标是确定EBOV-GP是否作为 肠毒素，研究它是否触发吸收不良或分泌过程，并充分阐明导致 EVD期间出现大量水样腹泻为此，该项目有三个具体的目标概念化，以使用 小肠和结肠的细胞，因为他们在吸收和分泌过程中分歧。目标1将研究 EBOV-GP触发的细胞内Ca 2+水平升高中Ca 2+来源的贡献，并阐明 它的上游信号通路目的2将评估Na+和Cl-穿过细胞膜、细胞膜和细胞膜的动力学。 EBOV-GP刺激后的渗透性和流体转运。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技术来识别离子通道 被EBOV-GP改变。该项目将使用当代体外试验和联合收割机来实现 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 获奖者在电生理学技术方面的培训，促进了他的职业生涯，补充了他的免疫学， 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 在电生理学、免疫学和细胞生物学之间相互作用的背景下，这个项目 具有转化影响，因为它可能导致EVD大量腹泻的新治疗靶点和策略， 直接改善患者预后并减少该领域医疗机构中的病毒传播。