Expression of micro-RNAs and immune-relevant genes in rainbow trout (Oncorhynchus mykiss Walbaum) upon vaccination with a Viral Haemorrhagic Septicemia Virus

Abstract

Development of strategies to alleviate potential disease outbreaks in sea-farmed rainbow trout (Oncorhynchus mykiss Walbaum) due to wildlife marine reservoir of Viral hemorrhagic septicemia virus (VHSV) remains imperative. A DNA vaccine expressing VHSV glycoprotein (G) gene has been developed and shown to protect fish in VHSV challenge experiments. Identifying key factors as biomarkers during infection and vaccination will allow understanding of the complex web of interactions involved in the underlying host immune response. These molecular signatures of immune responses may also provide suitable selection criteria for identifying disease-resistant fish under production conditions during resistance breeding as fish do not show visible signs of resistance. 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. In a previous microarray experiment, we observed upregulation of miR-155, miR-462, and miR-731 in fish liver following VHSV infection. Therefore, we analysed the expression of these miRNAs and those of immune-related genes in rainbow trout in response to DNA vaccination. Quantitative RT-PCR analysis revealed the increased levels of miR-155, miR-462, and miR-731 in the skeletal muscle tissue at the site of injection and in the liver of vaccinated fish relative to salineand empty plasmid-injected controls. The increased expression of these miRNAs in the skeletal muscle correlated with the increased levels of the type I interferon (IFN)-inducible Mx gene, the vaccine gene (G), type I IFN and IFN-γ genes, and immune cell marker genes (CD4, CD8, sec-IgM, TCR, MHCI, and MHCII) at the vaccination site. The increased expression of immune cell markers indicates infiltration of the vaccination site with activated immune cells. Since the expression of these miRNAs correlated with increased levels of the type 1 IFN gene, IFN-γ gene, and the type I IFN-inducible Mx gene, we then determined whether this induction depends on interferons. Injecting fish with IFN 1-13 (a type I IFN) and IFN-γ constructs resulted in the 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. These regulated microRNAs could potentially be used as biomarkers of immune responses and as suitable selection markers to identify VHSV-resistant fish. Future work will involve identifying the specific cells that express these microRNAs, as well the genes that they regulate.