Figure 2. CsISG15 expression in fish tissues. CsISG15 expression in the liver, gill, muscle, kidney, spleen, brain, heart, and gut of tongue sole was determined by quantitative real time RT-PCR. The expression level in liver was set as 1. Five animals were used at each time point, and for each time point the PCR was performed three times. The results are shown as means6SE (N = 5). doi:10.1371/journal.pone.0044884.g002 Figure 3. CsISG15 expression in response to pathogen infection. Tongue sole were infected with megalocytivirus, Vibrio anguillarum, or PBS (control), and CsISG15 expression in kidney (A) and spleen (B) was determined by quantitative real time RT-PCR at various time points. At each time point, the expression level of the control fish was set as 1. Five animals were used at each time point, and for each time point the PCR was performed three times. The results are shown as means6SE (N = 5). Significances between PBS- and pathogen-infected fish are indicated with asterisks. **P,0.01, *P,0.05. doi:10.1371/journal.pone.0044884.g003 PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44884 Cytokine Property of Tongue Sole ISG15 Figure 4. CsISG15 production in kidney and spleen in response to pathogen infection. Tongue sole were infected with megalocy- tivirus for 1 h, 4 h, 12 h, and 24 h, and CsISG15 production in kidney (A and B) and spleen (C and D) was determined by Western blot with antibodies against rCsISG15 (A and C) or b-actin (B and D). doi:10.1371/journal.pone.0044884.g004 Tissue specific expression of CsISG15 under normal physiological conditions and during pathogen infection qRT-PCR analysis showed that under normal physiological conditions, CsISG15 expression was detected, in increasing order, in liver, gill, muscle, kidney, spleen, brain, heart, and gut (Fig. 2). The expression level in gill, muscle, kidney, spleen, brain, heart, and gut were, respectively, 1.1-, 4-, 5.9-, 6-, 11.9-, 20-, and 26.4- fold higher than that in liver. To examine whether CsISG15 expression was affected by pathogen infection, tongue sole were infected experimentally with the viral pathogen megalocytivirus or the bacterial pathogen V. anguillarum, and CsISG15 expression in kidney and spleen was subsequently determine by qRT-PCR at 1 h, 4 h, 12 h, 24 h, and 48 h post-infection. The results showed that both megalocytivirus and V. anguillarum induced CsISG15 expression in time dependent manners (Fig. 3). In kidney, megalocytivirus infection caused significant inductions (3.2-, 38.2-, and 9.3-fold respectively) of CsISG15 expression at 4 h, 12 h, and 24 h post-infection, while V. anguillarum infection caused significant induction (33.4 fold) at 48 h post-infection (Fig. 3A). In spleen, both megalocytivirus and V. anguillarum induced significant CsISG15 expression at 1 h, 4 h, and 12 h post-infection, with peak inductions (20- and 32.6-fold respectively) occurring at 12 h and 4 h post-infection respectively (Fig. 3B). Consistent with the qRT- PCR results, Western blot analysis showed that CsISG15 protein increased with megalocytivirus infection and reached peak levels at 12 h in kidney and spleen (Fig. 4). CsISG15 expression and subcellular localization in HK lymphocytes To examine the effect of pathogen infection on CsISG15 expression at cellular level, tongue sole HK lymphocytes were infected with or without megalocytivirus, and CsISG15 expression was determined by qRT-PCR at 1 h, 2 h, 4 h, 4 h, 8 h, and 16 h post-infection. The results showed that significant inductions of CsISG15 expression were observed at all the examined time points, with maximum induction (75 fold) occurring at 4 h post-infection (Fig. 5). To examine whether CsISG15 was secreted into the