induced by poly I:C 24 h after IHNV infection, and that the NV may be the key factor for this function. To test if NV is involved in inhibition of the IFN response induced by poly I:C, 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. Cells were collected at 24 h after poly I:C treatment and the expression levels of IFN1 and Mx1 were determined using real-time PCR. Results were expressed as mRNA copy number and normalized per 1000 copies of a housekeeping gene mRNA (ARP). Cells infected with wild-type rIHNV-32/87 or NV-knockout mutant rIHNV-32/87-DNV-GFP produced decreased levels of IFN1 (12 and 19 copies, respectively) and Mx1 (327 and 625 copies, respectively) compared with mock- infected poly I:C treated control (IFN1, 31 copies; Mx1, 801 copies). However, cells infected with rIHNV-32/87-DNV-GFP produced 1.4- and 1.8-fold higher amount of IFN1 and Mx1, respectively, than those infected with rIHNV-32/87 (Figure 6C, 6D). This suggests that NV is required for effective inhibition of poly I:C-induced IFN responses in RTG-2 cells, but virus without NV is also capable of significant inhibition. NV is required for the inhibition of the IHNV-induced IFN response in RTG-2 cells We also determined whether NV is involved in the blocking of the IHNV-induced IFN system. RTG-2 cells were infected with rIHNV-32/87 or rIHNV-32/87-DNV-GFP, after which total RNA was extracted at 24 h p.i., and the induction of IFN1 was analyzed using real-time RT-PCR. While wild-type rIHNV-32/87 induced a 1.5-fold increase in IFN1 expression (6 copies per 1000 copies of ARP mRNA) compared with mock-infected controls (4 copies), the NV-knockout mutant rIHNV-32/87-DNV-GFP induced a 6-fold increase in IFN1 expression (25 copies) (Figure 7A). If the NV is involved in the inhibition of IFN1 expression in NLS of IHNV NV Is Necessary for IHNV Growth IHNV-infected cells, the NV-knockout mutant rIHNV should induce higher levels of interferon-stimulated genes (ISGs) than the wild-type rIHNV. To test this, we analyzed the expression level of the Mx1 gene, one of the ISGs, in the rIHNV-infected RTG-2 cells using real-time PCR. While wild-type rIHNV-32/87 induced a 6-fold increase in Mx1 gene expression (64 copies) relative to mock infected controls (10 copies), the NV-knockout mutant rIHNV-32/87-DNV-GFP induced a 48-fold increase of in Mx1 gene expression (478 copies) (Figure 7B). Taken together, these data indicate that the NV may support the growth of IHNV through inhibition of the IFN system in IHNV-infected RTG-2 cells. In addition, we tested the effect of NV on the secretion of functional IFN1 from IHNV-infected RTG-2 cells. Supernatants were collected from RTG-2 cells infected with rIHNV-32/87 or rIHNV-32/87-DNV-GFP at 24 h p.i. and IFN1 activities in the supernatants were assayed using RTG-P1 cells. RTG-P1 cells are RTG-2 derivatives stably transfected with a luciferase reporter vector controlled by a rainbow trout Mx1 promoter [36]. RTG-P1 cells were treated with supernatants of RTG-2 cells for 48 h, cell extracts were prepared and luciferase activity was measured. RTG-P1 cells treated with supernatant from mock-infected RTG- 2 cells were used as a negative control and those treated with 25 mg/ml of poly I:C were used as a positive control. While the supernatant of wild-type rIHNV-32/87-infected RTG-2 cells showed a 1.1-fold increases in luciferase activity compared with that of mock-infected RTG-2 cells, that of NV-knockout mutant rIHNV-32/87-DNV-GFP induced a 2.5-fold increase in luciferase activity (Figure 7C). This result is similar to the IFN1 gene expression pattern obtained using real-time PCR (Figure 7A). Together, these data revealed that the NV plays an important role in the inhibition of IFN1 expression in IHNV-infected RTG-2 cells. Figure 7. Requirement of NV for inhibition of IFN systems in IHNV-infected RTG-2 cells. A and B, IFN1 and Mx1 expression in RTG-2 cells infected with rIHNV-32/87 or rIHNV-32/87-DNV-GFP. RTG-2 cells were harvested at 24 h p.i., and total RNA was analyzed with real-time PCR for IFN1 (A) and Mx1 (B). 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). C, Assay for functional IFN1 released from RTG-2 cells infected with rIHNV-32/87 or rIHNV-32/87-DNV-GFP. The culture supernatants were collected at 24 h p.i. and IFN activities were measured by adding the supernatants to RTG-P1 cells and analyzing luciferase activity in RTG-P1 cells as described in the Materials and Methods. 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 luciferase activity in the RTG-P1 cells treated with supernatants from the negative control cells was defined as one. The results are presented as the means 6 SD of three independent experiments (*P,0.05; ***P,0.001). doi:10.1371/journal.pone.0022362.g007 PLoS ONE | www.plosone.org 9 July 2011 | Volume 6 | Issue 7 | e22362