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.

Project description:Rainbow trout (Oncorhynchus mykiss) is a species of cold-water fish with important economic values, which is widely cultivated around the world. It is susceptible to infectious hematopoietic necrosis virus (IHNV), resulting in a large number of deaths. In this study, we measured the changes of immune parameters in different periods (6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144 hours post infection (hpi)), transcriptome profiles of T48G/C48G and important immune-related genes expression patterns in rainbow trout gill following IHNV challenge through biochemical methods, RNA-seq and qRT-PCR. The results showed that AKP, ACP, T-SOD, ALT and AST activities, as well as MDA and LZM content decreased and then increased during infection, and reached to the lowest value at 48 hpi (P < 0.05), whereas CAT activity showed significant peaks at 48 hpi (P < 0.05). The mRNA and miRNA analysis identified 4343 differentially expressed genes (DEGs) and 11 differentially expressed miRNAs (DEMs), and numerous DEGs involved in immune responses, such as tlr3, tlr7, tlr8, traf3, ifih1, trim25, dhx58, ddh58, hsp90a.1, nlrc3, nlrc5, socs3, irf3, irf7, mx1, vig2, bcl2, casp3 and casp8。Integrated analysis identified four key miRNAs (miR-27d-3p, miR-124-5p, miR-301a-5p and miR-146-5p) that target important immune-related DEGs (tlr3, ifih1, vig2, irf8, socs3 and cox1). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that DEGs and target genes were significantly enriched in various immune-related terms including inflammatory response, DNA replication, cell adhesion and pathways of Toll-like receptor signaling, apoptosis, DNA replication, p53 signaling and MAPK signaling. The expression trends of 15 DEGs and 2 DEMs were further verified by quantitative real-time PCR, which confirmed the reliability of the transcriptome sequencing results. Expression analysis showed that tlr3, tlr7, traf3, ifih1, trim25, dhx58, hap90a.1, irf3, irf7 and mx1 genes increased significantly after 48 hpi during infection. This study contributes to the understanding of the immune response of rainbow trout against IHNV infection, and provides new insights into the immune regulation mechanisms for future studies.

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Transcriptome Analysis of Gene Expression Regulation Mechanisms in the Spleen of Rainbow Trout (Oncorhynchus mykiss) Against Infectious Hematopoietic Necrosis Virus

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