Trans-complementation of ∆-NS1-WNV with ectopically expressed WNV NS1. A. Scheme for construction of ∆-NS1-WNV. Nucleotides 87 to 928 of the NS1 gene were deleted after restriction digest with the BstEII enzyme. B. Recovery of WNV RNA in supernatant after transfection of ∆-NS1-WNV or WNV-WT RNA into BHK21 or BHK21-VEEV-WNV NS1 cells. 12 hours post transfection, supernatant was harvested and assessed for levels of viral RNA by qRT-PCR of the E gene. The results are the average of two independent experiments performed in triplicate. C-D. Immunofluorescence (C) or flow cytometry (D) staining of WNV E and NS5 antigen after mock infection or infection of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5, and at 26 hr post infection, cells were fixed, permeabilized, and stained with anti-E (red), anti-NS5 (green), or a nuclear stain (blue). Results are representative of several independent experiments. E. Single-step growth kinetics of ∆-NS1-WNV or WNV-WT in BHK21 or BHK21-VEEV-WNV NS1 cells. Cells were infected at an MOI of 5 and supernatants were titrated on BHK21-VEEV-WNV NS1 cells by focus-forming assay. The dashed line indicates the limit of detection of the assay. Results are the average of several independent experiments performed in triplicate. Error bards indicate standard deviations.