PATHOGENESIS OF FILAMENTOUS INFLUENZA VIRUS INFECTION

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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.