Complement-Mediated Neutralization of Dengue Virus Requires Mannose-Binding Lectin

Avirutnan, Panisadee; Hauhart, Richard E.; Marovich, Mary A.; Garred, Peter; Atkinson, John P.; Diamond, Michael S.

Virus neutralization. DENV-2 or WNV (10 2 PFU of DENV-2; 10 3 PFU of WNV) was incubated with naïve mouse or human serum (fresh or heat inactivated at 56°C for 30 min) or purified recombinant human MBL diluted in gelatin veronal buffer with Mg 2ϩ and Ca 2ϩ (GVB ϩϩ ; Complement Technology) for 1 h at 37°C. Samples were added to a monolayer of BHK21-15 cells and incubated for 1 h at 37°C. Cells were washed, overlaid with 1% low-melting-point agarose (SeaPlaque; Lonza) in minimal essential medium (MEM) containing 4% fetal bovine serum (FBS), and cultured for 3 (WNV) or 4 (DENV-2) days at 37°C. Following formaldehyde (10%) fixation and crystal violet staining, plaques were scored visually. For studies with DENV-1, -3, and -4, 10 2 to 10 3 focus-forming units (FFU) of virus were incubated with serum or purified recombinant human MBL for 1 h at 37°C as described above. Samples were added to a monolayer of Vero cells and incubated for 1 h at 37°C. Cells were washed and overlaid with 1% methylcellulose mixed with DMEM containing 5% FBS and incubated for 48 (DENV-1 and -4) or 60 (DENV-3) hours. Monolayers were washed thrice with PBS to remove methylcellulose, fixed with 1% paraformaldehyde in PBS for 10 minutes at room temperature, rinsed, and permeabilized in Perm Wash (phosphate-buffered saline [PBS], 0.1% saponin, and 0.1% BSA). Infected-cell foci were stained by incubating cells with the flavivirus crossreactive MAb WNV-E18 (54) (1 g/ml) and quantitated as described previously (34) . In some experiments, viruses were incubated with serum or MBL in the presence or absence of 100 g/ml mannan or 1 M mannose prior to the addition to cells.