Fig. 1.

Determination of infectivity of PUUV by ICW assay. A) Vero E6 cells were infected in triplicates with increasing amounts of PUUV or were left uninfected. At 2 days post infection, cells were fixed and subjected to ICW assay. Infection was monitored by staining against PUUV N protein. N protein was detected on the 800 nm channel and cell number was determined by DRAQ5 and Sapphire700 cell stains on the 700 nm channel. B) For IF, infected Vero E6 cells were stained for N protein with Cy3 fluorescently labeled antibody (red) and nuclei were stained with Hoechst 33342 (blue). C) Quantification of infection by IF and ICW assay. In IF assay, percentage of infected cells was calculated by counting of cells positive for N protein normalized to the number of cells visualized by Hoechst 33342. In ICW assay, relative infectivity was calculated as N protein levels detected in Vero E6 cells normalized to relative cell number as described in A). Three independent experiments were performed for each assay. Data are shown as mean with standard deviation (SD). D) Relationship between quantification of infection by IF and ICW was analyzed using the Pearson correlation.