Figure 1. JEV infection leads to accumulation of autophagosomes in host cells. (A–C) Neuro2a cells transfected with GFP-LC3 (green, left panels), were mock-infected (A), infected with JEV (MOI 5, 24 h) (B), or serum-starved for 12 h (C). Cells were fixed and stained with JEV-E antibody (blue, middle panels). The merging of the 2 signals is shown in the right panels. Scale bar: 10 µm. (D and E) Quantification of cells showing punctate distribution of GFP-LC3 (D), and the number of GFP-LC3 puncta per cell (E). (F) Mock-infected, serum-starved and JEV-infected Neuro2a cells were lysed at the indicated times postinfection and poststarvation. Lysates were analyzed by western blotting with LC3, JEV, NS5 (infection control), and GAPDH (loading control) antibodies. (G) Quantitative PCR (qPCR) of autophagy genes in Neuro2a cells that were mock-infected, serum-starved (12 h), or infected with JEV (MOI 5, 24 h). The graph shows the relative increased expression of gene transcription normalized to mock-infected samples. Values represent mean ± SD of 3 independent experiments. (H and I) Vero cells (H), WT and atg5^−/− MEFs (I) were mock-infected, serum-starved (12 h) or infected with JEV (MOI 5, 24 h). Western blots were done using LC3, JEV, NS5, and GAPDH antibodies. The ratio of LC3-I/GAPDH and LC3-II/GAPDH was calculated as shown below the representative blots. The Student t test was used to calculate P values. *P < 0.05, **P < 0.01.