NLS of IHNV NV Is Necessary for IHNV Growth Figure 5. Growths of rIHNV-32/87, rIHNV-32/87-DNV-GFP, rIHNV-NVPRT, and rIHNV-NVPRT-DEGDL in RTG-2 cells. A, Schematic representation of the recombinant IHNVs used in this study. rIHNV-NVPRT, and rIHNV-NVPRT-DEGDL were generated as described in the Materials and Methods section, and rIHNV-32/87 and rIHNV-32/87-DNV-GFP were used as controls. NVPRT refers to the NV gene from the IHNV PRT strain. All other genes are from IHNV 32/87 strain. B, Comparative growths of rIHNV-32/87, rIHNV-32/87-DNV-GFP, rIHNV-NVPRT, or rIHNV-NVPRT-DEGDL in RTG-2 cells. The cells were infected with rIHNVs at an MOI of 0.01 PFU/cell, and samples of the supernatant medium were collected at 0, 24, and 48 h p.i. The samples were titrated in duplicate using a plaque assay on EPC cells. The results are presented as the means 6 SD of three independent experiments (**P,0.01). C. Effect of NV expression on the growth of rIHNV-32/87-DNV-GFP in RTG-2 cells. RTG-2 cells were transfected with pcDNA6/V5-NVPRT or empty vector pcDNA6/V5. After incubation for 24 h, the level of NV protein was determined by Western blotting with anti-V5 antibody (top panel). RTG-2 cells transfected with pcDNA6/V5-NVPRT or empty vector were infected with rIHNVs at an MOI of 0.01 PFU/cell and samples of the supernatant medium were collected at 0, 24, and 48 h p.i. The samples were titrated in duplicate using a plaque assay on EPC cells. The results are presented as the means 6 SD of three independent experiments (**P,0.01). doi:10.1371/journal.pone.0022362.g005 assay. Poly I:C pre-treatment dramatically blocked the growth of both rIHNV-32/87 and rIHNV-32/87-DNV-GFP (Figure 6A). These results suggest that pre-existing antiviral responses induced by poly I:C in RTG-2 cells can block the growth of IHNV even with a functional NV. Next, we determined whether poly I:C treatment after viral infection can affect the growth of the IHNV. RTG-2 cells were infected with rIHNV-32/87 or rIHNV-32/87-DNV-GFP and then treated at 24 h p.i. with 25 mg/ml of poly I:C. The culture supernatants were collected at 0 and 24 h after poly I:C treatment and infectious titers were determined by plaque assay. As shown in Figure 6B, while the growth of wild-type rIHNV-32/87 was not significantly affected (1.2-fold), poly I:C treatment post-infection induced a 13-fold decrease in the yield of the NV-knockout mutant rIHNV-32/87-DNV-GFP. This suggests that wild type IHNV may have mechanisms for overcoming the antiviral activity PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e22362