Project description:The Infectious Hematopoietic Necrosic Virus (IHNV) DNA vaccine is based on the viral glycoprotein gene (G gene) and induces a non-specific anti-viral immune response and long-term specific immunity against IHNV in salmonid fishes. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression at the I.M. site was evaluated using a 16,000 feature salmon cDNA microarray. Eighty different transcripts were significantly modulated in the vector DNA group while 910 transcripts were modulated in the IHNV DNA vaccinated group relative to control group. Quantitative reverse transcriptase PCR was used to examine expression of selected genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were higher in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen) evaluated with RT-PCR of specific genes, the main difference was the up-regulation of the type I IFN-related genes in only the IHNV DNA vaccinated group. Keywords: disease vaccine response Gene expression at the site of I.M injection was evaluated 7 days post-injection in clonal rainbow trout. Gene expression was compared across three treatments, with replicate clonal rainbow trout individuals (Hot Creek strain) within each treatment. Treatments included injection with 1X PBS (control group), plasmid vector only (without IHNV G gene; hereafter referred to as vector group), and the pIHNV vaccine (vaccine group). Three clonal individuals from the control, three individuals from the vector group, and four individuals from the vaccine group were labeled with Cy3 and used for individual hybridizations to ten slides, comparing hybridization of each individual to a major reference RNA pool. The major reference pool consisted of three unhandled clonal rainbow trout and constituted a pool of RNA from the anterior kidney, spleen, liver and muscle and was labelled with Cy5. Total RNA was extracted from samples, amplified, and hybridized to 16,000 feature Atlantic salmon cDNA arrays developed by the Genomic Research for Atlantic Salmon project. Differential expression was compared among control, vector, and vaccine groups.

Project description:Rainbow trout (Oncorhynchus mykiss) is an important economical cold-water fish worldwide. However, infection with infectious hematopoietic necrosis virus (IHNV) has severely restricted the development of aquaculture and caused huge economic losses. Currently, little is known about the immune defense mechanisms of rainbow trout against IHNV. In this study, we detected the changes of immune parameters over different post-infection periods (6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144 hours post-infection (hpi)), mRNA and miRNA expression profiles under 48 hpi (T48L) compared to control (C48L), and key immune-related genes expression patterns in rainbow trout liver following IHNV challenge through biochemical methods, RNA-seq, and qRT-PCR, and the function of miR-330-y was verified by overexpression and silencing in vitro and in vivo. The results revealed that AKP, ALT, CAT, and T-SOD activities, and LZM content showed significant peaks at 48 hpi (P < 0.05), whereas MAD content and AST activity decreased continuously during infection (P < 0.05), and ACP activity varied slightly. From RNA-seq, a total of 6844 genes and 86 miRNAs were differentially expressed, and numerous immune-related differentially expressed genes (DEGs) involved in RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, and antigen processing and presentation were significantly upregulated in T48Lm group, including IFIH1, DHX58, MAVS, TRAF3, IRF3, IRF7, MX1, TLR3, TLR8, MYD88, NOD1, NOD2, IL-8, CXCR1, CD209, CD83, and TAP1. Integrated analysis identified seven miRNAs (miR-425-x, miR-185-x, miR-338-x, miR-330-y, miR-361-x, miR-505-y, and miR-191-x) that target at least three key immune-related DEGs. Expression analysis showed that IFIH1, DHX58, IRF3, IRF7, MX1, TLR3, TLR8, and MYD88 showed a marked increase after 24 hpi during infection. Further research confirmed TAP1 as one of the targets of miR-330-y, overexpression of miR-330-y with mimics or agomir significantly reduced the expression levels of TAP1, IRF3, and IFN, and the opposite effects were obtained by inhibitor. These results facilitate in-depth understanding of the immune mechanisms in rainbow trout against IHNV and will provide valuable information for disease resistance breeding.

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Underlying mechanisms for selected disease resistance and enhanced non-specific resistance in rainbow trout

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