Elucidating the Mechanism of Ebola Virus Enterotoxigenicity

项目总结 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了2万例病例 在刚果民主共和国目前的疫情期间报告了2800例。埃博拉病毒 (EBOV)属于丝状病毒科，编码包括单一糖蛋白(EBOV-GP)在内的7种蛋白质。 这已被证明在EVD的发病机制中起着关键作用。在EVD期间，胃肠道液体通过大量 容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，因此过度的液体流失会刺激环境 污染增加了医院内病毒传播的风险。病毒的生理机制 糖蛋白作为肠毒素和促进大量腹泻已经被很好地描述过了。 然而，描述EBOV如何刺激高容量水样腹泻的分子触发和机制 在EVD期间从未被研究过。我们在人类肠道细胞中的初步数据表明，一种肠道毒素- 与EBOV-GP相似的行为，因为它诱导细胞内钙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诱导细胞内钙离子 胃肠上皮细胞增多，触发顶端表面离子转运失调，导致 增加通透性和水分分泌。该项目的目标是确定EBOV-GP是否充当 肠毒素，研究它是否会触发吸收不良或分泌过程，并充分阐明导致 在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 由于小肠和结肠细胞在吸收和分泌过程中发散，因此这些细胞的数量和数量也不同。目标1将研究 钙源在EBOV-GP引起的细胞内钙升高中的作用及意义 它的上游信号通路。目标2将评估跨越细胞膜、细胞 EBOV-GP刺激后的渗透率和液体传输。极化细胞培养将被用来模拟 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 正在被EBOV-GP改变。该项目将使用当代的体外测试和联合 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 获奖者接受电生理技术培训，促进他的职业生涯，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寄主病原体的技能。 在电生理学、免疫学和细胞生物学相互作用的背景下的相互作用。这个项目 具有翻译影响，因为它可能导致针对EVD高容量腹泻的新的治疗靶点和策略， 在现场的医疗保健环境中，直接改善患者的预后并减少病毒传播。

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.