Cytopathic effects (CPE) caused by three AF and three AS ZIKV strains in ESCd at 48, 60 and 72 h PI.

<p>Colonies of ESC were grown in 6-well matrigel-coated cell culture dishes and differentiated for 4 days by BAP treatment. After 4 days, cells were infected with each ZIKV strain at 1, 0.1 or 0 (mock) MOI. (A) At 48 h PI, cells were fixed and stained with crystal violet to observe CPE. Cell lysis was evident in cells infected with all three of the AF strains, even at 0.1 MOI, whereas minimal cell lysis was evident in AS ZIKV infected (1 MOI) colonies. (B) Cell viability was measured at 48 h and 60 h PI to demonstrate the extent of lysis induced by each ZIKV strain at 1 MOI. At 48 h post- infection, only the three AF strains of ZIKV (shades of blue) showed a significant reduction in cell viability when compared to the mock infected control (one-way ANOVA, <i>p</i> < 0.001). Conversely, none of the AS strains of ZIKV (shades of red) induced a significant reduction in cell viability. All six ZIKV strains showed a significant reduction in cell viability when compared to the mock infected control at 60 h PI (<i>p</i> < 0.01 (Mexico), <i>p</i> < 0.001 (all other strains)). Data are presented as mean ± SEM (n = 5). (C) The extent of cell lysis at 72 h PI is accentuated by images taken at a higher magnification. At 72 h PI, severe cell lysis was evident in cells infected with all three AF strains and became apparent in cells infected with the AS strains. In the images, areas of deep purple staining highlight the areas of multinucleated syncytia. There were no syncytial areas present in the cultures infected with the AF strains of ZIKV, while small areas remained visible after infection with the AS strains. At 72 h PI, mock infected controls showed non-virus induced cell lysis, as the cells naturally started to die off at this time point post-differentiation. These areas are indicated by black arrows. Scale bars are 3 mm (A) and 1 mm (C).