FIG 1.

IBDV induces autophagic signaling during the late phase of infection. (A) DF-1 cells were infected with IBDV Gt at an MOI of 1; cells were harvested at 12 and 24 h p.i. Cell lysates were separated by SDS-PAGE and analyzed by WB with anti-LC3B, p62, or β-actin for host proteins and anti-(p)VP2 for viral protein. After incubation with IRDye 800CW secondary antibody, proteins were visualized and quantified using the Odyssey system. (B) Intensity band ratio of LC3-II/actin was normalized to the control. Means ± SDs were determined for three independent experiments. t test was used for comparison to the control. (C) DF-1 cells cultured in dishes with coverslip inserts were infected with IBDV Gt at an MOI of 1 for 12 and 24 h p.i. Cells were observed by confocal microscopy for fluorescence puncta. Viral protein is represented by red fluorescence, endogenous LC3 molecule appears green, and the nucleus was stained blue by DAPI. The scale bar is indicated at the bottom right. (D) Puncta per cell were quantified in mock- or IBDV-infected DF-1 cells. Data were analyzed by t test. (E) DF-1 cells were infected with IBDV Gt at an MOI of 1. Cells were fixed at 24 h p.i., and ultrastructure was observed by TEM. Arrows indicate vesicles with morphological characteristics typical of autophagic vacuoles of perinuclear double-membrane vacuoles measuring 0.3 to 2.0 μm. N, nucleus. The scale bar is indicated at the bottom right. (F) Autophagosomes per cell were quantified in mock- or IBDV-infected DF-1 cells. Data were analyzed by t test.