USE OF FITC-LABELED INFLUENZA-VIRUS AND FLOW-CYTOMETRY TO ASSESS BINDING AND INTERNALIZATION OF VIRUS BY MONOCYTES-MACROPHAGES AND LYMPHOCYTES

The binding of influenza virus to the surface of cells and the interna lization of virus particles by all or a subset of cells are key points in the pathogenesis of viral infection. The current studies establish ed a method for discrimination of surface-bound from internalized infl uenza virus. Fluorescein isothiocyanate (FITC) was attached to the vir al hemagglutinin and neuraminidase proteins; the fluorescent virus ret ained infectivity. A flow cytometric technique was then adapted for st udy of virus-cell interactions, with addition of ethidium bromide to q uench green fluorescence associated with FITC-labeled virus that was c ell-bound but remained external. Ethidium bromide was excluded by inta ct cell membranes, and internalized virions retained green fluorescenc e. Cells could be examined by fluorescence microscopy or flow cytometr y, with flow cytometry allowing rapid, kinetic assessment of large num bers of cells and subsets of virus-exposed cells. The data showed that , whereas a majority of both monocytes-macrophages and lymphocytes bou nd influenza virus, a large percentage of monocytes-macrophages but on ly a very small percentage of lymphocytes internalized the virus. This procedure provides a simple and effective method to distinguish surfa ce-bound from internalized influenza virus, and allows precise kinetic analyses on large numbers of cells.