Rodriguez M F, Wiens G D, Purcell M K, Palti Y
Agriculture Research Service, National Center for Cool and Cold Water Aquaculture, United States Department of Agriculture, Kearneysville, WV 25430, USA.
Immunogenetics. 2005 Aug;57(7):510-9. doi: 10.1007/s00251-005-0013-1. Epub 2005 Sep 29.
Antiviral immunity in fish is not well understood. In mammals, Toll-like receptor (TLR) 3 is involved in double-stranded RNA recognition and host immune response activation. Here, we report the first identification of a rainbow trout TLR3 ortholog (rtTLR3), its genomic structure, and mRNA regulation. Six exons and five introns were identified from bacterial artificial chromosome (BAC) and expressed sequence tag (EST) sequencing, and this genomic organization is similar to mammalian and fish TLR3 genes. The putative 913 amino acid protein has a Toll/interleukin (IL)-1R (TIR) domain, a transmembrane domain, and leucine-rich repeats. In healthy trout, rtTLR3 is highly expressed in the liver, pyloric ceca, intestine, spleen, and anterior and trunk kidney tissues. To investigate whether rtTLR3 is involved in antiviral immunity, transcriptional regulation in vivo was examined by quantitative real-time polymerase chain reaction (PCR) after poly inosinic:cytidylic (I:C) and infectious hematopoietic necrosis virus (IHNV) treatments. TLR3 mRNA expression peaked 1 day after poly (I:C) injection of live animals, while the peak of gene expression after live IHNV challenge was observed on day 3. In vitro stimulation of rainbow trout anterior kidney leukocytes with poly (I:C) also enhanced rtTLR3 expression. Up-regulation was specific to viral challenge as there was no significant up-regulation of rtTLR3 mRNA levels in the spleen and a modest down-regulation in the anterior kidney after bath challenge with a gram-negative bacterial trout pathogen, Yersinia ruckeri. The sequence conservation of trout TLR3 and mRNA regulation after poly (I:C) or RNA virus exposures strongly suggest a role for trout TLR3 in antiviral immunity.
鱼类的抗病毒免疫尚未得到充分了解。在哺乳动物中，Toll样受体（TLR）3参与双链RNA识别和宿主免疫反应激活。在此，我们首次鉴定了虹鳟鱼TLR3直系同源物（rtTLR3）、其基因组结构和mRNA调控。通过细菌人工染色体（BAC）和表达序列标签（EST）测序鉴定出六个外显子和五个内含子，这种基因组组织与哺乳动物和鱼类的TLR3基因相似。推测的913个氨基酸的蛋白质具有Toll/白细胞介素（IL）-1受体（TIR）结构域、跨膜结构域和富含亮氨酸的重复序列。在健康的鳟鱼中，rtTLR3在肝脏、幽门盲囊、肠道、脾脏以及前肾和躯干肾组织中高度表达。为了研究rtTLR3是否参与抗病毒免疫，在注射聚肌苷酸:胞苷酸（I:C）和感染造血坏死病毒（IHNV）后，通过定量实时聚合酶链反应（PCR）检测体内转录调控。在对活体动物注射聚（I:C）后1天，TLR3 mRNA表达达到峰值，而在活体IHNV攻击后，基因表达峰值出现在第3天。用聚（I:C）体外刺激虹鳟鱼前肾白细胞也增强了rtTLR3表达。上调是病毒攻击所特有的，因为在用革兰氏阴性细菌性鳟鱼病原体鲁氏耶尔森菌进行浸浴攻击后，脾脏中rtTLR3 mRNA水平没有显著上调，前肾中则有适度下调。鳟鱼TLR3的序列保守性以及聚（I:C）或RNA病毒暴露后的mRNA调控强烈表明鳟鱼TLR3在抗病毒免疫中发挥作用。
