Flow cytometry assay for intracellular rabies virus detection.
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.
Bordignon
Juliano
J
Laboratory of Immunology, Department of Microbiology and Parasitology at Federal University of Santa Catarina, SC Florianópolis, 88040-900 Brazil.
Pires Ferreira
Silvia Cardoba
SC
Medeiros Caporale
Graciane Maria
GM
Carrieri
Maria Luiza
ML
Kotait
Ivanete
I
Lima
Hermênio Cavalcante
HC
Zanetti
Carlos Roberto
CR
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