Quantitative Flavivirus Infection Assay

FFA procedures were performed as previously described^{52 (link)}. Tissue samples were homogenized and clarified by centrifugation. Tissue supernatants and sera were diluted serially before infection on BHK cells. BHK cells used in this assay do not express Fcγ receptors. Cells were plated (2.0 × 10⁵ cells/well in a 24-well plate) and incubated overnight at 37 °C, 5% CO₂. Confluent monolayers were inoculated with undiluted or 10-fold serially diluted sera or clarified tissue supernatant, and were incubated for 1 hr at 37 °C. After incubation, the inoculum was removed, and each cell monolayer was overlaid with CMC-media and incubated at 37 °C, 5% CO₂ for 1.5–2 days. Cells were then fixed, permeabilized, and incubated with pan Flavivirus anti-envelope (E) Ab clone 4G2 (BioXCell), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch) and staining with True-Blue peroxidase substrate (Sera Care). Foci were counted, virus levels in the serum were expressed as Focus Forming Units (FFU) per mL, and for most tissues as FFU/g. As it was not technically feasible to weigh sciatic nerves, viral levels in these tissues were expressed as FFU/tissue.