NLS of IHNV NV Is Necessary for IHNV Growth Figure 6. Requirement of NV for inhibition of poly I:C-induced IFN response in RTG-2 cells. A, Effect of poly I:C pre-treatment on the growth of rIHNVs. RTG-2 cells were pre-incubated with poly I:C at 25 mg/ml for 24 h. The cells were then infected with rIHNV-32/87 or rIHNV-32/87- DNV-GFP at an MOI of 0.01 PFU/cell and samples of the supernatant medium were collected at 0, 24, and 48 h p.i. All data points represent the average of samples taken from duplicate infections. B, Effect of poly I:C treatment after virus infection on the growth of rIHNVs. RTG-2 cells were infected with rIHNV-32/87 or rIHNV-32/87-DNV-GFP at an MOI of 0.01 PFU/cell. After incubation for 24 h, cells were washed three times and treated with 25 mg/ml of poly I:C or serum-free media. At 0 and 24 h after poly I:C treatment, samples of the supernatant medium were collected and titrated in duplicate. The virus titer in the supernatant medium collected at 0 h after poly I:C treatment was defined as one. The results are presented as the means 6 SD of three independent experiments (***P,0.001). ns, not significant. C and D, Analysis of IFN1 and Mx1 expressions in RTG-2 cells treated with poly I:C after virus infection. RTG-2 cells were infected with rIHNV-32/87 or rIHNV-32/87-DNV-GFP at an MOI of 1 PFU/cell. After incubation for 24 h, cells were incubated with culture media containing 25 mg/ml of poly I:C. At 24 h after poly I:C treatment, total RNA was extracted from the cells and analyzed with real-time PCR for IFN1 (C) and Mx1 (D). The negative control was mock-infected RTG-2 cells without poly I:C treatment. The positive control was mock-infected RTG-2 cells stimulated with 25 mg/ml poly I:C. The levels of IFN1 and Mx1 are expressed as mRNA copy number normalized to 1000 copies of ARP mRNA. The results are presented as the means 6 SD of three independent experiments (**P,0.01; ***P,0.001). doi:10.1371/journal.pone.0022362.g006 PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e22362