Molecular basis of fish antiviral responses

病毒病原体对养殖鱼和野生鱼都有威胁，但人们对鱼类抗病毒反应的比较方面知之甚少，也不知道鱼类在生命早期如何抵御病毒。我的研究项目使用转录组学（即全局基因表达）和互补方法来研究参与鱼类防御反应的基因和分子途径。鱼类是比较和发育免疫学的重要模型，也是研究免疫基因和基因家族分子进化的重要模型。在接下来的5年里，我们将研究和比较硬骨鱼从胚胎到成鱼的不同生命阶段的抗病毒免疫反应的基因和机制。四种进化上分化的物种——大西洋鲑鱼、大西洋鳕鱼、斑马鱼和斑鱼——都有基因组测序，其中三种对水产养殖/渔业很重要（鲑鱼、鳕鱼、斑鱼），将被用作模型。我们将全面表征这4个物种的关键抗病毒基因，为基因表达、调控和功能的研究提供基础。巨噬细胞在脊椎动物的免疫（如抗病毒）反应中起着核心作用。我假设，通过对4种模式物种的研究，将确定参与巨噬细胞抗病毒反应的进化保守和谱系特异性基因和mirna。为了验证这一假设，将分离鲑鱼、鳕鱼、斑马鱼和疙瘩鱼头部肾巨噬细胞并用pIC（一种合成RNA，可引起抗病毒反应）进行免疫刺激。通过RNA测序（mRNA, miRNA）评估巨噬细胞的抗病毒反应，并使用实时定量聚合酶链反应（qPCR）和原位杂交（ISH）等技术进一步研究选定的抗病毒基因和miRNA的表达。此外，我们将研究鱼类发育过程中抗病毒基因和mirna的表达和功能。根据我们最近的研究结果[例如，irf7、viperin和其他关键干扰素（IFN）通路基因在鳕鱼胚胎发生过程中的动态表达]，我假设IFN通路在鱼类发育过程中发挥重要作用。为了验证这一假设，我们将使用各种技术（如qPCR， ISH）研究IFN通路mrna和mirna在免疫刺激（如pIC）和对照鲑鱼，鳕鱼，斑马鱼和块状鱼胚胎中的表达；Irf7和viperin基因在胚胎发育过程中的功能将通过敲除靶基因和表征所产生的表型来研究。跨物种比较我们的结果将揭示巨噬细胞和鲑鱼，鳕鱼，斑马鱼和块鱼的最后共同祖先（即早期硬骨鱼，约2.5亿年前）的胚胎中的抗病毒防御，以及抗病毒防御的谱系特异性机制（例如存在于鳕鱼而不存在于鲑鱼中）。更全面地了解这些鱼类的基因和分子途径将对包括进化生物学、渔业和水产养殖在内的许多研究领域有价值。

Viral pathogens threaten both cultured and wild fish, yet little is known about comparative aspects of fish antiviral responses, and how early life stage fish defend themselves against viruses. My research program uses transcriptomics (i.e. global gene expression) and complementary methods to study the genes and molecular pathways involved in fish defense responses. Fish are key models for comparative and developmental immunology, and for studying the molecular evolution of immune genes and gene families. Over the next 5 years we will study and compare the genes and mechanisms involved in antiviral immune responses of teleost fish of different life stages from embryo to adult. Four evolutionarily diverged species - Atlantic salmon, Atlantic cod, zebrafish and lumpfish - all with sequenced genomes and 3 of which are important for aquaculture/fisheries (salmon, cod, lumpfish), will be used as models. We will fully characterize key antiviral genes in all 4 species, facilitating research on gene expression, regulation and function. Macrophages play central roles in vertebrate immune (e.g. antiviral) responses. I hypothesize that evolutionarily conserved and lineage-specific genes and miRNAs involved in macrophage antiviral responses will be identified using studies with the 4 model species. To test this hypothesis, salmon, cod, zebrafish and lumpfish head kidney macrophages will be isolated and immune stimulated with pIC, a synthetic RNA that elicits an antiviral response. Macrophage antiviral responses will be assessed by RNA sequencing (mRNA, miRNA), and expression of selected antiviral genes and miRNAs will be further studied using techniques such as real-time quantitative polymerase chain reaction (qPCR) and in situ hybridization (ISH). In addition, we will study the expression and function of antiviral genes and miRNAs during fish development. Based on our recent results [e.g. dynamic expression of irf7, viperin and other key interferon (IFN) pathway genes during cod embryogenesis], I hypothesize that the IFN pathway plays important roles during fish development. To test this hypothesis, we will study the expression of IFN pathway mRNAs and miRNAs in immune-stimulated (e.g. pIC) and control salmon, cod, zebrafish and lumpfish embryos using various techniques (e.g. qPCR, ISH); irf7 and viperin gene function during embryonic development will be investigated using target gene knockdown and characterization of the resulting phenotypes. Comparison of our results across species will shed light on the antiviral defences in macrophages and embryos of the last common ancestor of salmon, cod, zebrafish and lumpfish (i.e. early Teleost, ~ 250 million years ago), as well as lineage-specific mechanisms of antiviral defense (e.g. present in cod but not salmon). A more complete understanding of the genes and molecular pathways involved in these fish species will be valuable to many research areas including evolutionary biology, fisheries, and aquaculture.