MA, USA). The proteins were resolved by SDS-PAGE and, after electrophoresis, transferred to a nitrocellulose membrane (Amer- sham, Cambridge, UK). Immunoblot was performed as described previously [41] using anti-rCsISG15 antibodies or anti-b-actin antibody (Bioss, Beijing, China). To demonstrate that the b-actin antibody used in the study was able to detect b-actin when it was present, cytoplasmic proteins were prepared and blotted with b- actin antibody. Effect of rCsISG15 on macrophage activation (i) Preparation of HK macrophages. HK macrophages were prepared based on the method of Chung and Secombes [43]. Briefly, HK was removed from two healthy tongue sole (,720 g), washed 36 with PBS, and passed through a sterile metal mesh. The cells were resuspended in L-15 medium and placed onto a 34/51% Percoll (Solarbio, Beijing, China) gradient, followed by centrifugation at 4006 g for 30 min. After centrifugation, the cells at the 34/51% interface were recovered, washed twice and resuspended in L-15 medium containing 10% calf serum, 1% penicillin and streptomycin (Sangon, Shanghai, China), and 20 U/ml heparin (Sigma, St. Louis, MO, USA). The cells were added to 96-well tissue culture plates (,26105 cells/well) and incubated at 25uC for 2 h. Non-adherent cells were washed off after the incubation. (ii) Respiratory burst assay. rCsISG15 and rCsISG15M were diluted in L-15 medium to 1000 ng/ml. Macrophages were added with or without rCsISG15, rCsISG15M, or E. coli lipopolysaccharide (LPS) (Sigma, St. Louis, MO, USA) in L-15 to a final concentration of 100 ng/ml. After incubation at 25uC for 2 h, respiratory burst activity was determined as reported previously [43]. In brief, 100 ml of 1 mg/ml nitroblue tetrazolium (Sangon, Shanghai, China) in L-15 was added to macrophages. After incubation at 25uC for 2 h, the reaction was stopped by adding 100% methanol. The plate was washed with 70% methanol, and the reduced formazan was solubilized in 100 ml of 2 M KOH and 120 ml of DMSO. The plate was read at 630 nm with a microplate reader. (iii) Nitrite assay. HK macrophages as described above were added with or without rCsISG15 or rCsISG15M in L-15 to a final concentration of 100 ng/ml. After incubation at 25uC for 2 h, nitric oxide (NO) production was determined as described by Tafalla and Novoa [44]. In brief, the supernatants were removed to a separate 96-well plate (50 ml/well); one-hundred microlitres of 1% sulphanilamide (Sigma, St. Louis, MO, USA) in 2.5% phosphoric acid was added to each well, followed by adding 100 ml of 0.1% N-naphthylethylene-diamine (Sigma) in 2.5% phosphoric acid. The plate was read at 540 nm, and the molar concentration of nitrite was determined from standard curves generated using known concentrations of sodium nitrate. Effect of rCsISG15 and rCsISG15M on immune gene expression HK lymphocytes prepared above were added with or without rCsISG15 or rCsISG15M in L-15 to a final concentration of 100 ng/ml. The plates were incubated at 25uC for 1 h, 2 h, 4 h, and 8 h, and total RNA was prepared from the cells with the Total RNA Kit I of Omega Bio-tek (Beijing, China) and used for qRT- PCR analysis of the expression of the genes encoding toll-like receptor 9 (TLR9), interleukin (IL) 1b (IL-1b), CsIL8 (a homologue of IL-8) [39], CsCCK1 (a CC chemokine) [45], CsCXCe1 (a CXC chemokine) [46], and major histocompatibility complex (MHC) class IIb as described above. These genes were selected because they are the major immune relevant genes that Cytokine Property of Tongue Sole ISG15 are available in sequence. The primers used to PCR the immune genes have been reported previously [46]. Effect of rCsISG15 and rCsISG15M on cellular resistance against viral infection HK lymphocytes prepared above were added with or without rCsISG15 or rCsISG15M in L-15 to a final concentration of 100 ng/ml. As a control, the preparation from DE3/pET259, i.e., E. coli preparation, prepared above under the same conditions as rCsISG15 was also added to lymphocytes at the same volume as rCsISG15. The plates were incubated at 25uC for 2 h. After the incubation, the cells were added with L-15 containing or not containing megalocytivirus (104 copies/well). The plates were incubated at 25uC for 2 h and 4 h respectively, followed by washing 36 with PBS. Viral copy number in the infected cells was determined by absolute quantitative real time PCR as reported previously [40]. Effect of anti-rCsISG15 serum on viral infection Anti-rCsISG15 serum and preimmune serum were diluted 20006in L-15. HK lymphocytes prepared above were added with or without 100 ml of the diluted anti-rCsISG15 serum or preimmune serum, followed by adding 100 ml L-15 containing or not containing megalocytivirus (104 copies/well). The plates were incubated at 25uC for 2 h and 4 h respectively. After incubation, the plates were washed 36 with PBS, and intracellular viral copy number was determined by absolute quantitative real time PCR as reported previously [40]. RNAi and its effect on viral infection RNAi was performed with the DNA vector-based siRNA technology. For this purpose, the siRNA expression vector pRNAT-CMV3.1 (GenScript, Piscataway, NJ, USA) was used, in which the CMV promoter drives the expression of siRNA that can be inserted into the vector between BamH I and Afl II sites. In our study, the CsISG15-specific siRNA (59- GCTCTGAGCACCATT- GAGC -39) and a nonspecific siRNA (59- GCTTGATG- CTCACGACGAC -39) were inserted into pRNAT-CMV3.1, resulting in plasmids pRiCsISG15 and pRiNC1 respectively. RNAi was carried out by transient transfection of HK lymphocytes with pRiCsISG15, pRiNC1, and pRNAT-CMV3.1 (backbone vector) respectively using Lipofect (Tiangen, Beijing, China) according to manufacturer’s instructions. The transfectants were infected with megalocytivirus for 4 h and then examined for CsISG15 expression by qRT-PCR and immunoblot analysis as described above. To examine the effect of RNAi on viral infection, pRiCsISG15-, pRiNC1-, and pRNAT-CMV3.1-transfected cells in 96-well culture plates were infected with megalocytivirus for 4 h and determined for intracellular viral load as described above. The assay was performed independently for five times. Statistical analysis All statistical analyses were performed with SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). Data were analyzed with analysis of variance (ANOVA), and statistical significance was defined as P,0.05. Supporting Information Figure S1 SDS-PAGE analysis of rCsISG15 and rCsISG15M. Purified rCsISG15 (lane 2) and rCsISG15M (lane 3) were analyzed by SDS-PAGE and viewed after staining with Coomassie blue. Lane 1, protein marker. (TIF) PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44884