Molecular basis of fish antiviral responses

病毒病原体威胁养殖和野生鱼类，但很少有人知道鱼类抗病毒反应的比较方面，以及早期生命阶段的鱼类如何抵御病毒。我的研究计划使用转录组学（即全球基因表达）和互补方法来研究鱼类防御反应中涉及的基因和分子途径。鱼类是比较免疫学和发育免疫学以及研究免疫基因和基因家族的分子进化的关键模型。在接下来的5年里，我们将研究和比较硬骨鱼从胚胎到成体不同生命阶段的抗病毒免疫反应所涉及的基因和机制。将使用四种进化上不同的物种-大西洋鲑鱼、大西洋鳕鱼、斑马鱼和块状鱼-作为模型，这些物种都有基因组测序，其中三种对水产养殖/渔业很重要（鲑鱼、鳕鱼、块状鱼）。我们将充分表征所有4个物种中的关键抗病毒基因，促进对基因表达，调控和功能的研究。 巨噬细胞在脊椎动物免疫（例如抗病毒）应答中起核心作用。我假设，进化上保守的和谱系特异性的基因和miRNA参与巨噬细胞抗病毒反应将被确定使用的4个模型物种的研究。为了检验这一假设，将分离鲑鱼、鳕鱼、斑马鱼和圆鱼头肾巨噬细胞，并用pIC（一种引发抗病毒应答的合成RNA）进行免疫刺激。将通过RNA测序（mRNA、miRNA）评估巨噬细胞抗病毒应答，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抗病毒基因和miRNA的表达。 此外，我们还将研究鱼类发育过程中抗病毒基因和miRNAs的表达和功能。根据我们最近的研究结果[例如，动态表达的irf 7，蝰蛇蛋白和其他关键干扰素（IFN）途径基因在鳕鱼胚胎发育]，我假设，IFN途径在鱼类发育过程中起着重要的作用。为了验证这一假设，我们将使用各种技术（例如qPCR、ISH）研究免疫刺激（例如pIC）和对照鲑鱼、鳕鱼、斑马鱼和圆鳞鱼胚胎中IFN途径mRNA和miRNA的表达;将使用靶基因敲低和所得表型的表征研究胚胎发育期间irf 7和蝰蛇蛋白基因功能。 跨物种比较我们的结果将揭示鲑鱼，鳕鱼，斑马鱼和圆鳞鱼最后共同祖先（即约2.5亿年前的早期硬骨鱼）的巨噬细胞和胚胎中的抗病毒防御，以及抗病毒防御的谱系特异性机制（例如存在于鳕鱼中，但不存在于鲑鱼中）。更完整地了解这些鱼类物种中涉及的基因和分子途径将对许多研究领域，包括进化生物学，渔业和水产养殖业有价值。

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.