Fig 2.

WNV does not cause significant activation of caspase-3 until late in infection. A549 cells were infected with WNV at an MOI of 0.1, 1, or 3 or VSV at an MOI of 0.1. At 24, 48, and 72 h postinfection, WNV-infected cells were harvested. VSV-infected cells were harvested only at 24 h postinfection because shortly after this time point, the majority of cells were dead. (A, B) Following fixation, WNV-infected cells were incubated with mouse NS2B/3 (to detect virus antigen) and rabbit active caspase-3 and then anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 647. Samples were then subjected to flow cytometric analysis. Histogram overlays were constructed from representative experiments in which the fluorescent intensity distribution of mock-infected samples (unfilled histograms) is shown together with that of infected samples (gray histograms). An interval gate (vertical line) is indicated in each plot. Events to the right of the interval gate are considered positive for the given antigen. (C) VSV-infected cells were stained with a mouse monoclonal antibody (BW8G65) to VSV glycoprotein (virus antigen) or rabbit anti-active caspase-3, and analyses were performed as described for panels A and B. The results of three independent experiments are summarized in panel D.