PATHOGENESIS OF FILAMENTOUS INFLUENZA VIRUS INFECTION

我们最近发现甲型流感病毒M1和M2蛋白中的特定氨基酸是丝状病毒形态的重要遗传决定因素。此外，我们已经证明宿主细胞细胞骨架复合体是丝状病毒形成的重要细胞决定因素。本提案的目标扩展了这些最近的观察结果，并侧重于确定与丝状流感病毒进入相关的细胞生物学动力学，以及丝状流感病毒的致病潜力，因为它与感染的传播和疾病的严重性有关。在具体目标1中，描述了实验以解决与丝状病毒附着和进入机制有关的具体问题。采用放射性标记和荧光标记的病毒丝，我们将定量检查病毒丝是否通过部分或完全内吞或可能通过吞噬样机制进入。膜融合试验将用于确定pH要求和病毒丝的细胞融合程度。活细胞视频显微镜、共聚焦显微镜和电子显微镜将用于跟踪内化病毒细丝的命运，并检查出芽病毒细丝是否可以介导感染的细胞间传播。在具体目标2中，我们将重点关注丝状流感病毒的致病潜力。使用原代人鼻咽器官和上皮细胞培养，我们将确定感染是否仅限于上皮细胞亚群，并确定丝状病毒株是否具有增强的纤毛病和组织损伤能力。使用基于荧光染料的结合试验，我们将评估病毒丝是否可以增强细菌与粘膜上皮的结合。使用小鼠和雪貂动物模型，我们将比较遗传相似的甲型流感/乌冬病毒变体在体内的致病潜力，这些变体仅在形态上不同。将评估的致病标准包括：1)50%最小感染剂量，2)组织特异性病毒载量，3)组织特异性组织病理学，4)体重减轻的恢复率。在动物研究中，我们将研究丝状菌株是否促进感染的下降传播，从最初的局部感染鼻道到下呼吸道。这些研究将为进一步确定人类流感病毒发病机制和疾病严重程度的决定因素奠定基础。

We have recently shown that the specific amino acids in the M1 and M2 proteins of influenza A virus are important genetic determinants of filamentous virus morphology. In addition, we have demonstrated that the host cell cytoskeletal complex is an important cellular determinant of filamentous virus formation. The objectives of this proposal expand upon these recent observations and focus on determining the cell biological dynamics associated with filamentous influenza virus entry, and on the pathogenic potential of filamentous influenza virus as it relates to spread of infection and severity of disease. In specific aim 1, experiments are described to address specific questions relating to the mechanisms of filamentous virus attachment and entry. Employing radiolabeled and fluorochrome-labeled viral filaments, we will quantitatively examine whether viral filament entry occurs by partial or complete endocytosis or possibly by a phagocytosis-like mechanism. Membrane fusion assays will be used to determine pH requirements and extent of cell fusion of viral filaments. Live cell video microscopy, confocal and electron microscopy will be used to follow the fate of internalized viral filaments and examine whether budding viral filaments can mediate cell-to-cell spread of infection. In specific aim 2, we will focus on the pathogenic potential of filamentous influenza virus. Using primary human nasopharyngeal organ and epithelial cultures we will determine whether infection is restricted to a subpopulation of epithelial cells and determine whether filamentous virus strains have enhanced ciliopathic and tissue damaging capabilities. Using a fluorochrome-based binding assay, we will assess whether viral filaments can enhance bacterial binding to the mucosal epithelium. Using the mouse and ferret animal models, we will compare in vivo the pathogenic potential of genetically similar variants of influenza A/Udorn virus, which differ only in morphology. Pathogenic criteria that will be assessed include: i) the 50 percent minimal infectious dose, ii) tissue specific viral loads iii) tissue-specific histopathology, and iv) rates of weight loss recovery. In the animal studies, we will examine whether filamentous strains promote a descending spread of infection, from an initial localized infection in the nasal tract to the lower respiratory tract. These studies will set the foundation for establishing further determinants of influenza virus pathogenesis and severity of disease in humans.