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An intramuscular DNA vaccine encoding the VP2 protein of infectious pancreatic necrosis virus (IPNV) has developed.The vaccine encodes the VP2 gene of an IPNV strain with the P217T221A247 (PTA) motif (Genogroup5, Serotype Sp).Intramuscular delivery of the vaccine elicited high protection against lethal challenge in rainbow trout fry.The vaccine elicited high level of circulating antibodies alongside with significant up-regulation of key immune genes.The vaccine significantly reduced viral load in the internal organs post challenge.IPNV in Atlantic salmon is represented by various strains with different virulence and immunogenicity linked to various motifs of the VP2 capsid. IPNV variant with P217, T221, A247 (PTA) motif is found to be avirulent in Atlantic salmon, but virulent in rainbow trout, and other salmonid species. This study describes a DNA vaccine delivered intramuscularly encoding the VP2 protein of infectious pancreatic necrosis virus (IPNV) with PTA motif that confers high protection in rainbow trout (Oncorhynchus mykiss). Intramuscular injection of 2, 5 and 10μg of DNA (pcDNA3.1-VP2) in rainbow trout fry (4–5g), confers relative protection of 75–83% in the different vaccine groups at 30days post vaccination (450° days). The VP2 gene is expressed in spleen, kidney, muscle and liver at day 30 post-vaccination (RT-PCR), and IFN-1 and Mx-1 mRNA are upregulated at early time post vaccination, and so also for IgM, IgT, CD4 and CD8 in the head kidney of vaccinated fish compared to controls, 15 and 30days post vaccination. Significant increase of serum anti-IPNV antibodies was found 30–90days post-vaccination that was correlated with protection levels. Mortality corresponded with viral VP4 gene expression were significantly decreased in vaccinated and challenged fish. This shows for the first time that a VP2-encoding DNA vaccine delivered intramuscularly elicits a high level of protection alongside with high levels of circulating antibodies in rainbow trout and a lowered viral replication.