Murine Norovirus Plaque Assay

RAW 264.7 cells were seeded into six-well plates at a density of 2 × 10⁶ viable cells per well. On the following day, 10-fold dilutions of virus inoculum were prepared in complete DMEM medium and plated in duplicate wells. Plates were incubated for 1 h at room temperature on a rocking apparatus before aspirating the inoculum and overlaying the cells with 2 ml of 37–40 °C 1.5% SeaPlaque agarose in MEM supplemented with 10% low-endotoxin fet al.calf serum, 1% HEPES, 1% penicillin/streptomycin, and 2% glutamine (complete MEM) per well. Plates were incubated at 37 °C and 5% CO₂ for 2 d. To visualize plaques, cells were stained with 2 ml of 56 °C 1.5% SeaKem agarose in complete MEM containing 1% neutral red per well for 6–8 h.
For plaque neutralization assays, differing concentrations of purified mAb (A6.2, anti-MNV-1 capsid; isotype control, 10H2, anti-reovirus μ1c) were incubated with equal plaque-forming units of either MNV-1.CW1 or MNV-1 brain homogenate for 30 min at 37 °C prior to performing the MNV-1 plaque assay.