Viral hemorrhagic septicemia caused by a fish rhabdovirus, Viral hemorrhagic septicemia virus (VHSV), results in significant mortality in farmed rainbow trout (Oncorhynchus mykiss Walbaum). Although the disease had been eradicated in Denmark, wildlife marine reservoir of VHSV poses a threat particularly to sea-farmed rainbow trout and thus necessitates strategies to mitigate potential disease outbreaks. A DNA vaccine encoding the glycoprotein gene of VHSV has been developed and shown to elicit protective immune responses in laboratory trials. It is important to identify key factors as biomarkers during infection and vaccination in order to understand the complex web of interactions involved in the underlying host immune response. Micro ribonucleic acids (miRNAs) are a diverse class of small (18-22 nucleotides) endogenous RNAs that potently mediate post-transcriptional silencing of a wide range of genes and are emerging as critical regulators of cellular processes, including immune responses. A microarray experiment in our lab revealed that miR-155, miR-462, and miR-731 were upregulated in fish liver following VHSV infection. Therefore, we analysed the expression of these miRNAs together with that of the type I interferon (IFN)-inducible Mx gene in rainbow trout in response to DNA vaccination. Using quantitative RT-PCR, we found that miR-155, miR-462, and miR-731 were upregulated in the skeletal muscle tissue at the site of injection and the liver of vaccinated fish relative to saline- and empty plasmid-injected controls. The increased expression of these miRNAs in the skeletal muscle correlated with the increased levels of the type I IFN-inducible Mx gene, the vaccine gene, and immune marker genes (CD4, CD8, sec-IgM, TCR, MHCI, and MHCII) at the vaccination site, indicating infiltration with immune cells. Since the expression of these miRNAs correlated with the increased expression of the Mx gene, we then determined whether this induction depends on interferons. Injecting fish with IFN 1-13 (a type I IFN) construct resulted in increased expression of miR-155, miR-462, and miR-731 in the skeletal muscle tissue relative to controls. The same response was obtained from injection with the general IFN stimulator and Toll-like receptor (TLR) 3 agonist, polyinosinic: polycytidylic acid (poly I:C). These suggest that the induction of these miRNAs is elicited by interferons, which are major mediators of immune responses. Regulated miRNAs could serve as molecular signatures of responses to infection and vaccination and could provide suitable selection criteria for identifying disease-resistant fish under production conditions during resistance breeding as fish do not show visible signs of resistance.