Fig 5.

Endosomal acidification is required for BEFV entry at a low pH. (A) MDBK and Vero cells were pretreated with different concentrations of bafilomycin A1 for 1 h or at different time points (before, during, and after virus absorption), followed by infection with BEFV at an MOI of 2. Each value represents the mean of three independent experiments ± standard deviation. (B) MDBK and Vero cells were pretreated with different concentrations (upper panel) of NH₄Cl for 1 h or at different time points (lower panel), followed by infection with BEFV at an MOI of 2. The cell lysates and supernatants of BEFV-infected cells were collected at 24 hpi. The synthesis of M protein was detected by Western blotting. The progeny virus titer of BEFV was determined by plaque assay. (C and D) Unbound virus was removed by washing cells with cold MEM, and virus-bound cells either were directly treated with low-pH solutions at different pHs for 10 min (C) or were preincubated at 37°C for 1 h in the presence of bafilomycin A1 and then treated with low-pH solutions at different pHs for 10 min (D). Cell lysates were collected at 24 hpi, and the level of M protein was detected by Western blotting. The β-actin was used as an internal control. The results are from triplicate experiments.