Figure 8. Effect of rCsISG15 and anti-rCsISG15 serum on viral infection. (A) Tongue sole head kidney (HK) lymphocytes were treated with or without (control) rCsISG15, rCsISG15M, or Escherichia coli preparation, and then infected with megalocytivirus. (B) Tongue sole HK lymphocytes were pre-treated with or without (control) anti-rCsISG15 serum or preimmune serum and then infected with megalocytivirus. In both panels, viral numbers in the cells were determined at 2 h and 4 h post-infection. Values are shown as means6SE (N = 5). N, the number of repeated experiments. *P,0.05; **P,0.01. doi:10.1371/journal.pone.0044884.g008 rCsISG15M-treated cells exhibited significantly higher numbers of viral numbers at 4 h post-infection. In contrast, E. coli preparation had no effect on viral infection. Antibody neutralization of CsISG15 and its effect on viral infection With the above results, i.e., CsISG15 was released into the culture supernatant of lymphocytes and able to stimulate lymphocyte immune response against viral infection, we wondered whether antibody blocking of the released CsISG15 would have any effect on viral infection. To examine this question, HK lymphocytes were infected with megalocytivirus in the presence or absence of anti-rCsISG15 serum or preimmune serum for 2 h and 4 h. Subsequent determination of intracellular viral number showed that the presence of anti-rCsISG15 serum, but not the presence of preimmune serum, significantly increased intracellular viral load at both examined time points (Fig. 8B). Interference with CsISG15 expression and its effect on viral infection To examine whether interference with CsISG15 expression would have any effect on viral infection, HK lymphocytes were subjected to RNAi assay by being transfected with pRiCsISG15 and pRiNC1, which expressed CsISG15-specific siRNA and a nonspecific siRNA respectively. As a control, the cells were also transfected with the backbone vector pRNAT-CMV3.1. To Cytokine Property of Tongue Sole ISG15 Figure 9. Effect of CsISG15 RNAi on viral infection. Tongue sole head kidney lymphocytes transfected with pRiCsISG15, pRiNC1, and pRNAT-CMV3.1 (control) were infected with megalocytivirus. Viral numbers in the cells were determined at 4 h post-infection. Data are shown as means6SE (N = 5). N, the number of repeated experiments. Significance between RNAi and the control is indicated with asterisks. **P,0.01. doi:10.1371/journal.pone.0044884.g009 examine the effect of RNAi on CsISG15 expression, the transfectants were infected with megalocytivirus for 4 h, and CsISG15 expression was determined by qRT-PCR and Western blot. qRT-PCR analysis showed that in lymphocytes transfected with pRiCsISG15, CsISG15 mRNA decreased to 0.9% of that of the control cells, while in lymphocytes transfected with pRiNC1, CsISG15 mRNA was comparable to that of the control cells. Consistently, Western blot analysis showed that compared to control cells, cells transfected with pRiNC1 produced a compa- rable amount of extracellular CsISG15, whereas cells transfected with pRiCsISG15 produced no detectable CsISG15 (Figure S4). Taken together, these results indicated that CsISG15 expression was indeed interfered specifically in lymphocytes transfected with pRiCsISG15. To determine the effect of RNAi on viral infection, siRNA- treated and untreated lymphocytes were infected with mega- locytivirus and analyzed for viral infection. The results showed that the viral number in lymphocytes transfected with pRiC- sISG15 was 5.1-fold higher than that in the control cells (Fig. 9). In contrast, the viral number in lymphocytes transfected with pRiNC1 exhibited no apparent difference from that of the control cells. Discussion In this study, we identified CsISG15, an ISG15 homologue from tongue sole, and analyzed its expression and function. Like all known ISG15, CsISG15 possesses two tandem UBL domains and the highly conserved LRGG motif, which categorize CsISG15 as a member of the ISG15 family. In fish, reports have shown that expression of most of the identified ISG15 is enhanced by microbial challenge. For example, viral infection and poly(I:C) treatment activate the expression of the ISG15 of Atlantic cod [28,29,31], Atlantic salmon [27,32], black rockfish [21], channel catfish [23], crucian carp [26], and Japanese flounder [25], while bacterial challenge activates ISG15 expression in Atlantic cod [25], goldfish [22], and red drum [30]. In this study, we observed constitutive CsISG15 expression in a wide range of tissues and drastically enhanced expression of CsISG15 in kidney and spleen following experimental infection with megalocytivirus and V. anguillarum. In both kidney and spleen, the induction patterns caused by viral and bacterial challenges differed, probably due to different activation mechanisms effected by these two pathogens. PLOS ONE | www.plosone.org 6 September 2012 | Volume 7 | Issue 9 | e44884