Requirement of NV for inhibition of IFN systems in IHNV-infected RTG-2 cells.

<p><i>A</i> and <i>B</i>, IFN1 and Mx1 expression in RTG-2 cells infected with rIHNV-32/87 or rIHNV-32/87-ΔNV-GFP. RTG-2 cells were harvested at 24 h p.i., and total RNA was analyzed with real-time PCR for IFN1 (<i>A</i>) and Mx1 (<i>B</i>). 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 µg/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 ± SD of three independent experiments (**P<0.01; ***P<0.001). <i>C</i>, Assay for functional IFN1 released from RTG-2 cells infected with rIHNV-32/87 or rIHNV-32/87-ΔNV-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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022362#s2" target="_blank">Methods</a>. 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 µg/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 ± SD of three independent experiments (*P<0.05; ***P<0.001).