MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2

McCarthy, Mary K.; Reynoso, Glennys V.; Winkler, Emma S.; Mack, Matthias; Diamond, Michael S.; Hickman, Heather D.; Morrison, Thomas E.

For FRNT assays, Vero cells were seeded in 96-well plates. Serum samples were heat-inactivated and serially diluted in DMEM/F12 medium with 2% FBS in 96-well plates. Approximately 100 focus-forming units (FFU) of virus stock was added to each well and the serum plus virus mixture was incubated for 1 h at 37ËšC. At the end of 1 h, medium was removed from Vero cells and serum sample + virus mixture was added for 2 h at 37ËšC. After 2 h, sample was removed and cells were overlaid with 0.5% methylcellulose in MEM/5% FBS and incubated 18 h at 37ËšC. Cells were fixed with 1% PFA and probed with 500 ng/mL of the anti-CHK-11 mAb [12] diluted in 1X PBS/0.1% saponin/0.1% bovine serum albumin (BSA) for 2 h at RT. After washing, cells were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Southern Biotech, 1:2000) for 1.5-2 h at RT. After washing, CHIKV-positive foci were visualized with TrueBlue substrate (Fisher) and counted using a CTL Biospot analyzer and Biospot software (Cellular Technology). Percent infectivity was calculated compared to a virus only (no serum) control. The FRNT 50 value was defined as the reciprocal of the last dilution to exhibit 50% infectivity. Fold change was calculated by dividing the FRNT 50 of each sample by the average FRNT 50 of the group receiving IgG2b control Ab.