∆-NS1-WNV virus does not have an attachment defect. A. Direct binding to BHK21 cells. An MOI of 1 of WNV-WT or ∆-NS1-WNV was incubated at 4°C or 37°C with BHK21 cells for one hour. Unbound virus was removed by centrifugation and washing, and bound and/or internalized virus was quantified after cell lysis and RNA purification using qRT-PCR and a primer and probe set specific for positive strand specific WNV RNA. Values were normalized to 18S rRNA levels to account for possible differences in cell number. Results are the average of three independent experiments, and differences were not statistically significant. B-G. BHK21 (B-C and E-F) or BHK21 VEEV NS1 (D, G) were infected with WNV-WT (B,E) or ∆-NS1-WNV (C-D, and F-G) at an MOI of 5 for one hour and then unattached virus was removed by extensive washing. Two (top panels) or eight (bottom panels) hours later, cells were fixed, permeabilized and stained with anti-E (red) or anti-NS5 (green) antibodies, or a nuclear stain (blue). Yellow arrows indicate E protein in a punctate staining pattern prior to replication, consistent with virus that is entering cells through an endocytic pathway. Magenta arrows denote E and NS5 staining that occurs after viral replication has ensued. The results are representative of several independent experiments.