Figure 1.

LC3B is lipidated following WNV Infection. Vero cells were transfected with GFP-LC3 expression plasmid followed by A) mock or WNV (MOI 3) inoculation, harvested at 24 hours post-infection, and labeled with antibody to WNV envelope antigen (cy3:red) and nuclei stained with DAPI. Images shown at 600x and 200x magnification. B) GFP-LC3 positive puncta were counted in WNV antigen negative (uninfected) and antigen positive cells (WNVAg+, *p<0.0001). C) Vero cells were mock or WNV (MOI 3) inoculated and cells were harvested at the specified time points post-infection and analyzed by western blot analysis for the LC3B-II. D) Relative LC3B-II band densities of 4 replicate experiments were measured with Image-J software and corrected for β-actin band density (*p=0.018). E) Vero cells were mock inoculated or inoculated with clone derived WNV or a Kenyan strain (Ken) of WNV (MOI 3) and cells harvested 24 hours post-infection, membrane-cytosol fractions were resolved by SDS-PAGE and analyzed for the presence of LC3B-II in the membrane fraction. F&G) BSCs were inoculated with WNV (10⁴ pfu/well) and tissue harvested at specified time points post-infection, processed for whole-tissue lysate and analyzed for LC3B-II with western blot. 4 replicate experiments were analyzed for relative band density at 24 hrs post-infection as above (*p=0.006).