Consistent with these observations in a live fish model, cellular study showed that megalocytivirus infection induced strong induction of CsISG15 expression in HK lymphocytes in a time- dependent manner. Like human ISG15, which is known to be secreted into the extracellular milieu, CsISG15 protein was detected in the culture medium of HK lymphocytes following viral challenge. These results indicate that viral challenge stimulates CsISG15 expression at both transcription and transla- tion levels. Since, like all ISG15, CsISG15 lacks an apparent signal sequence, extracellular exportation of CsISG15 is probably accomplished through a non-classical secretion pathway. It is known that recombinant human ISG15 purified from E. coli exhibits cytokine-like activity and is able to induce natural killer cell proliferation and IFN-c production by T lymphocytes [15– 18]. In our study, we found that rCsISG15 stimulated the respiratory burst and NO production of HK macrophages and upregulated the expression of immune relevant genes in HK lymphocytes. These results suggest that rCsISG15 possesses immunoregulatory property. Since CsISG15, like some fish ISG15 such as those of Paralichthys olivaceous, Sciaenops ocellatus, and Oncorhynchus mykiss, has a C-terminal extension, which is a seven-residue sequence in CsISG15, beyond the LRGG motif, and this extension was not removed from rCsISG15, it is likely that cleavage of the C-terminal extension, as is observed with mammalian ISG15 during ISGylation, is not necessary for the biological activity of CsISG15 as an extracellular cytokine. Mutational study showed that, in contrast to rCsISG15, the mutant rCsISG15M had no apparent effect on the respiratory burst activity of HK macrophages and, compared to rCsISG15, much reduced though significant effect on NO production. These results suggest that the LRGG motif is required for the optimum activity of CsISG15 as an extracellular cytokine. It is possible that the LRGG motif, besides being participating in conjugate formation with intracellular substrate proteins, has additional functions such as being involved in interactions with cell surface proteins that serve as receptors for extracellular CsISG15. qRT-PCR analysis showed that rCsISG15 drastically upregu- lated the expression of CsIL8, CsCCK1, and CsCXCe1, suggesting that rCsISG15 is a strong inducer of both CC and CXC chemokines. In addition to chemokines, rCsISG15 also upregulated the expression of TLR9 and the proinflammatory cytokine IL-1b. These results indicate that rCsISG15 is a stimulator of innate immunity, which may at least in part account for the anti-microbial property of rCsISG15. Compared to rCsISG15, rCsISG15M exhibited, in most cases, significantly lower stimulatory effects on the expression of immune genes, which is in line with the observation that rCsISG15M was impaired in the ability to activate HK macrophages and to block viral infection. Previous studies have indicated that mammalian ISG15 possess a broad-spectrum antiviral activity against a wide range of DNA and RNA viruses, and in most cases the antiviral activity is dependent on ISGylation of either host or viral proteins [9,14]. However, a recent study of Chikungunya virus infection in a murine model showed that the antiviral effect of mouse ISG15 is independent on its ability to form conjugates with substrate proteins, which suggests a non-classical role for ISG15 during viral infection [33]. In our study, we found that treatment of HK lymphocytes with rCsISG15 before megalocytivirus infection reduced intracellular viral load, whereas interference with CsISG15 expression drastically increased intracellular viral load, suggesting that rCsISG15 is required for optimum resistance against megalocytivirus infection. Since rCsISG15 induced signif- icant inductions of CsIL8, CsCCK1, CsCXCe1, IL-1b, and Cytokine Property of Tongue Sole ISG15 TLR9, which are involved in innate immunity against pathogen infection, it is likely that the anti-megalocytivirus effect of rCsISG15 is the result of augmented cellular immune response rather than a direct interaction between rCsISG15 and the viral pathogen. In line with these observations, the presence of rCsISG15 antiserum in the culture medium of HK lymphocytes during megalocytivirus infection increased the intracellular viral load to significant extents, suggesting that interaction between anti-rCsISG15 antibodies and the released CsISG15 most likely blocked the activity of CsISG15, thus resulting in enhanced viral infection. In conclusion, we showed in this study that CsISG15 is upregulated in expression by microbial pathogens and that, upon viral infection, CsISG15 is released into the extracellular milieu, where the protein probably acts as a cytokine and activates the response of various immune cells, which leads to elevated host defense against viral infection. Hence, it appears that, unlike mammalian ISG15, whose antiviral effects are mediated mostly through intracellular protein modification, CsISG15 exerts its immunoregulatory activity extracellularly as an unconjugated protein. Our results also indicate for the first time that the conserved LRGG motif of a teleost ISG15 is not essential to but required for the optimum activity of ISG15 as an extracellular cytokine, which suggests a role for the LRGG motif other than that in ISGylation. Taken together, these results support the conclusion that CsISG15 functions as an unconjugated extracel- lular cytokine that promotes antiviral immunity in a LRGG motif- dependent manner. Materials and Methods Ethics statement Experiments involving live fish and rats were conducted in accordance with the ‘‘Regulations for the Administration of Affairs Concerning Experimental Animals’’ promulgated by the State Science and Technology Commission of Shandong Province. Fish Tongue sole (Cynoglossus semilaevis) were purchased from a commercial fish farm in Shandong Province, China and main- tained at 22uC in aerated seawater. Fish were acclimatized in the laboratory for two weeks before experimental manipulation. Before each experiment, fish were randomly sampled for the examination of bacterial recovery from internal organs (blood, liver, kidney, and spleen), and no bacteria were detected from any of the examined fish. Fish were euthanized with tricaine methanesulfonate (Sigma, St. Louis, MO, USA) before tissue collection. Cloning of CsISG15 A cDNA library of tongue sole head kidney and spleen was constructed as described previously [34]. One thousand and five hundred clones of the library were randomly selected and subjected to DNA sequence analysis; one clone was found to contain the cDNA of CsISG15 with 59- and 39 - untranslated regions (UTRs). The nucleotide sequence of CsISG15 has been deposited in GenBank database under the accession number JN967757. Sequence analysis The cDNA and amino acid sequences of CsISG15 were analyzed using the BLAST program at the National Center for Biotechnology Information (NCBI) and the Expert Protein Analysis System. Domain search was performed with the simple PLOS ONE | www.plosone.org 7 September 2012 | Volume 7 | Issue 9 | e44884