The expression profiles of five immune-related genes modulated in the microarray were determined at 3 different times from vaccinated and non-vaccinated fish by using reverse transcriptase real-time quantitative qPCR. Specific PCR primers were designed using the Nifuratel Primer3 program and their amplification efficiency was calculated using seven serial five-fold dilutions of head kidney cDNA from unstimulated turbot with the Threshold Cycle slope method. Dauricine Primer sequences are listed in the Table S1. Individual reactions were carried out in 25 ml reaction volume using 12.5 ml of SYBR GREEN PCR Master Mix, 10.5 ml of ultrapure water, 0.5 ml of each specific primer and 1 ml of five-fold diluted cDNA template in MicroAmp optical 96-well reaction plates. All reactions were performed using technical triplicates in a 7300 Real-Time PCR System thermocycler with an initial denaturation followed by 40 cycles of a denaturation step and one hybridization-elongation step. An analysis of melting curves was performed for each reaction. Relative expression of each gene was normalized using the Elongation Factor-1 alpha as reference gene, which was constitutively expressed and not affected by the experimental treatments, and calculated using the Pfaffl method. The correlation between microarray and qPCR data were analyzed by the Spearman��s Rho test. In order to assess the viral replication success of VHSV strain UK-860/94 in head kidney cells from vaccinated and nonvaccinated fish infected with VHSV, the expression of the five viral genes was analyzed. The evolution of the VHSV replication throughout the tested sampling points in the different experimental groups is shown in Fig. 3. Those fish that were previously inoculated intramuscularly with PBS or with the empty plasmid and one month later infected with VHSV showed an increasing viral replication from 8 hours to 72 hours post-challenge, being the five proteins already detected at 8 hours. On the other hand, turbot previously injected with the DNA vaccine encoding the G glycoprotein showed a limited quantity of viral transcripts after VHSV challenge.