Fig 5.

Effects of dynasore and nocodazole on ISKNV infection. (A and B) Cells were pretreated for 1 h with dynasore (A) or nocodazole (B) with various concentrations of reagents as indicated or were left untreated (as a positive control), and ISKNV was then added and incubated for 4 h. After 72 h of incubation, the cells were processed for IFA or WB with anti-ORF101L antibody. For IFA, viral infections were quantified as the percentage of positive, treated cells relative to the number of untreated control cells. The viral infection rate of cells not treated with reagents (as a positive control) was arbitrarily set as 100%. The data shown are the means and standard deviations of the results from three independent experiments. *, P < 0.05. For WB, endogenous β-tubulin was included as an internal loading control. Lanes marked “+” indicate untreated control cells, and lanes marked “−” indicate the negative controls without ISKNV infection. (C) Internalization of AF488-LDL and ISKNV into MFF-1 cells. Cells grown on coverslips were prechilled and then infected with ISKNV at 4°C for 1 h, and noninternalized viruses were then washed by using TBS, while the uninfected cells were used as a negative control. Cells were incubated with 50 μg of AF488-LDL for 30 min at 4°C for binding, washed, transferred to 27°C for 30 min, and then fixed with methanol. The cells were analyzed by IFA using anti-ORF101L antibodies and were then viewed under a confocal microscope (LSM510; Zeiss). ISKNV particles (red fluorescence), AF488-LDL (green fluorescence), and nuclei (blue fluorescence) were visualized with Hoechst 33342.