title: Flow cytometry assay for intracellular rabies virus detection.
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.
authors.affiliation: Laboratory of Immunology, Department of Microbiology and Parasitology at Federal University of Santa Catarina, SC Florianópolis, 88040-900 Brazil.
authors.forename: Juliano
authors.initials: J
authors.lastname: Bordignon
authors.forename: Silvia Cardoba
authors.initials: SC
authors.lastname: Pires Ferreira
authors.forename: Graciane Maria
authors.initials: GM
authors.lastname: Medeiros Caporale
authors.forename: Maria Luiza
authors.initials: ML
authors.lastname: Carrieri
authors.forename: Ivanete
authors.initials: I
authors.lastname: Kotait
authors.forename: Hermênio Cavalcante
authors.initials: HC
authors.lastname: Lima
authors.forename: Carlos Roberto
authors.initials: CR
authors.lastname: Zanetti
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D003593:Cytoplasm / Q000821:virology*; D005434:Flow Cytometry / Q000379:methods*; D011818:Rabies / Q000175:diagnosis / Q000821:virology*; D011820:Rabies virus / Q000302:isolation & purification*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors