Flow cytometry-based assay for titrating dengue virus

Abstract

Plaque assays for titrating dengue virus (DENV) are time-consuming and not suitable for strains that do not plaque. Fluorescence-activated cell sorting (FACS) has been used to detect DENV-infected cells. Here we describe a FACS-based assay for titrating DENV. We determined that at 24 h postinfection, the number of infected cells detected by FACS represented the first round of infection and therefore could be used as a readout of the number of infectious particles in the inoculum. When the titers of different laboratory and clinical strains of DENV were compared using FACS, plaque, and endpoint dilution assays, for most strains the FACS titers were comparable to titers obtained by plaque or endpoint dilution assays. The FACS assay is an improvement over the plaque assay because the infection period is reduced from 5 to 7 days to 24 h and the assay can be used to titrate clinical isolates that frequently do not form clear plaques on cell monolayers. The novel FACS-based methods described here will facilitate laboratory studies of dengue.

FIG. 1.

Time course of DENV2 and DENV3 infection of C6/36 insect cells. C6/36 cells grown in six-well plates were infected with DENV2 strain NGC and DENV3 strain H87 at an MOI of 0.05. One well was mock infected to serve as a negative control. Cells were harvested from duplicate wells on days 1, 2, 3, 5, and 7 and processed for FACS. The cells were fixed and permeabilized using the Cytofix/Cytoperm kit from BD Biosciences and stained for FACS using monoclonal antibody 4G2, which cross-reacts with all four DENV serotypes. Cells were gated according to their size and granularity (x axis, side-scattered cells; y axis, forward-scattered cells) to identify intact, single cells. The gated cells were displayed on dot plots in which the x axis is the forward scatter and y axis is fluorescence (FITC) intensity. The dot plot was divided into four quadrants based on including 99.9% of the uninfected control cells in the lower left quadrant. The percentage of DENV-infected cells was determined by counting the number of cells in the upper left quadrant and dividing this number by the total number of cells in the dot plot.

FIG. 2.

Infection of C6/36 cells by different strains of DENV. The DENV3 prototype strain H87 as well as four primary DENV3 clinical isolates were used to infect C6/36 cells at an MOI of 0.01. Cells were harvested at 24-h intervals for 7 days, and infected cells were detected by FACS.

FIG. 3.

Time course of cell-to-cell spread of DENV2-infected C6/36 insect cells. C6/36 cells grown in six-well plates were infected with DENV2 strain NGC at an MOI of 2. Cells and supernatants were harvested from wells at 9, 18, 24, 26, 30, and 46 h postinfection. The cells were fixed and permeabilized using the Cytofix/Cytoperm kit from BD Biosciences and stained for FACS using monoclonal antibody 4G2 to determine the percentage of infected cells; the results are expressed as a continuous line with a closed circle. The amount of virus in the supernatant at each time point was determined by plaque assay on Vero cells and is expressed as a dashed line with a closed square.

FIG. 4.

Titration of dengue virus using FACS. C6/36 cells were infected with 10-fold serial dilutions of prototype strains and a clinical isolate. At 24 hpi, cells were harvested, fixed, and stained for FACS analysis. (A) Contour dot plots for DENV2- and DENV3-infected cells were obtained from FACS analysis of infected C6/36 cells at 24 h postinfection. C6/36 cells grown in six-well plates were infected for 24 h using 10-fold serial dilutions of prototype strains NGC and H87. One well served as a mock-infected control. The percentage of DENV-infected cells was determined by counting the number of cells in the upper left quadrant and dividing this number by the total number of cells in the dot plot. At 24 h postinfection, the cells in the upper left quadrant are likely a measure of the input virus, because this time point is too early for cell-to-cell spread of newly assembled virus. The dot plots shown above represent one experiment out of three and one out of six experiments for DENV2 and DENV3, respectively. (B) C6/36 cells grown in six-well plates were infected for 24 h using 10-fold serial dilutions of prototype strains NGC and H87 and clinical isolates UNC 3001, UNC 3017, and UNC 3018.

FIG. 5.

Comparison of FACS titration and plaque assay methods for estimating DENV2 titers. A stock of DENV2 (1 × 10⁷ PFU/ml) was serially diluted fivefold, and each dilution was titrated on Vero cells by plaque assay and on C6/36 cells by FACS. The lower limit of detection by FACS is 10,000 infectious units per milliliter of inoculum. The lower limit of detection by plaque assay is 50 PFU/ml.