Trans-complementation of ∆-NS1-WNV by flavivirus NS1. A. Expression of homologous or heterologous flavivirus NS1 in BHK21 cells using VEEV replicons. WNV (wild type or internal stop (IS), DENV-2, JEV, SLEV, or YFV NS1 were cloned and expressed in VEEV replicons and stable cells propagating these were generated. Cells were stained for NS1 expression with an irrelevant control MAb (E16, anti-WNV E), an anti-DENV-2 NS1 MAb (2G6), anti-WNV NS1 MAb (4NS1), a cross-reactive NS1 MAb (9NS1), and anti-YFV NS1 MAb (4E3). The results are representative of several independent experiments. Red arrows indicate positive expression that was over levels of the negative control MAb. B. Growth kinetics of ∆-NS1-WNV in BHK21-VEEV cells expressing WNV, DENV-2, SLEV, JEV, YFV NS1, or no insert (empty). Cells were infected at an MOI of 1, and supernatants were titrated on BHK VEEV WNV NS1 cells by focus-forming assay. The dashed line indicates the limit of detection of the assay. Results are the average of two independent experiments performed in triplicate. Error bards indicate standard deviations (most of which are smaller than the indicated symbols). C. Examples of (top) focus-forming or (bottom) plaque assays with ∆-NS1-WNV trans-complemented in BHK21-VEEV cells expressing WNV, DENV-2, SLEV, JEV, or YFV NS1. One representative experiment of three is shown.