Elucidating the Mechanism of Ebola Virus Enterotoxigenicity

点击翻译按钮获取中文摘要

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.