Figure 8: Modulation of SOCS gene expression in brown trout by VHSV infection. Naïve brown trout were bath challenged for 4 h at 12°C in a suspension of VHSV (isolate J167) at TCID50/mL, or the sterile medium used for suspension of the virus as control. Kidney tissue was collected at 3 days postinfection for total RNA preparation and viral burden assessment, which revealed that 76.7% of fish were VHSV positive after exposure but negative in the control group. Thus, the expression of CISH (a), SOCS1 (b), SOCS3 (c), SOCS5 (d) and SOCS7 (e) was examined by real-time reverse-transcription (RT) PCR in three groups: the unexposed controls, the VHSV-detectable, and VHSV-undetectable challenged fish as described previously [21, 35]. Briefly, tissues were collected and stored in RNAlater (Ambion) or directly used for total RNA isolation using Trizol (Invitrogen). The resulting total RNA was converted into cDNA and quantified by real-time PCRs using a LightCycler 480 system (Roche). The PCRs were performed in duplicate for each sample, and transcript level was calculated using the quantitative fit points method in the integrated LightCycler 480 software. The gene expression was first normalised to that of the housekeeping gene EF-1α and expressed as a fold change relative to the unexposed control. The results are presented as mean + SEM from 5 fish. The value of an LSD post hoc test after a significant one-way analysis of variance between the VHSV exposed and control fish is shown above the bars as , **, and ***. For comparison of the relative expression levels of genes examined, the cp values (the crossing point at which the fluorescence crosses the threshold) in the real-time PCR in 10 unexposed fish are presented as means ± SD (f). Please note that the higher the cp value, the lower the expression level.