Flow cytometry assay for intracellular rabies virus detection |
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Authors: | Bordignon Juliano Pires Ferreira Silvia Cardoba Medeiros Caporale Graciane Maria Carrieri Maria Luiza Kotait Ivanete Lima Hermênio Cavalcante Zanetti Carlos Roberto |
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Affiliation: | Laboratory of Immunology, Department of Microbiology and Parasitology at Federal University of Santa Catarina, SC Florianópolis, 88040-900 Brazil. |
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Abstract: | Following previous studies reporting microbiological diagnosis by flow cytometry, the possibility of using this method was examined to monitor infection of susceptible cell lines by a fixed rabies virus strain (Pasteur Virus strain-PV) or a wild rabies virus strain (WRS). Suspensions of BHK-21 and C6 cells were infected with viruses and a time course of virus infection was established. Sequentially, at several time points, infected and control uninfected cells were fixed, permeabilized, and stained with a rabies virus-specific antibody conjugate. This was achieved by resuspending cells in a solution containing p-formaldehyde in FACS lysis fluid, which allowed the detection of intracellular virus with flourescein-coupled antibodies by flow cytometry. A Becton Dickinson FACSCalibur flow cytometer was used to analyze the percentage of cells infected and the kinetics of the infection process was determined. As early as 12 h after inoculation with both rabies virus strains, significant levels (P<0.01) of infection (from 4.7 to 7.1%) were detected by flow cytometry. The maximum level of infection was obtained at 48 h in C6 cells (88%) with both viruses. The advantages of this method for examination of intracellular virus infection are discussed. |
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