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Characterization of Toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss)

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Abstract

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.

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Abbreviations

rtTLR3:

Rainbow Trout Toll-like receptor 3

BAC:

Bacterial Artificial Chromosome

EST:

Expressed Sequence Tag

TIR:

Toll/IL-1 Receptor

LRR:

Leucine-Rich Repeat

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Acknowledgements

The authors would like to thank Dr. Scott Gahr for his valuable contributions, Dr. Scott Lankford for providing EF-1α primers, Dr. Erica Leder for real-time analysis assistance, and Dr. Scott LaPatra for providing fish. We also kindly acknowledge Katherine Hovatter and Renee Fincham for their technical assistance in this project. The experiments performed in this study comply with current US laws.

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Authors and Affiliations

  1. Agriculture Research Service, National Center for Cool and Cold Water Aquaculture, United States Department of Agriculture, Kearneysville, WV, 25430, USA

    M. F. Rodriguez, G. D. Wiens & Y. Palti

  2. School of Aquatic and Fishery Sciences, University of Washington, Box 355020, Seattle, WA, 98195, USA

    M. K. Purcell

  3. NCCCWA/ARS/UDSA, 11876 Leetown Rd., Kearneysville, WV, 25430, USA

    M. F. Rodriguez

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  1. M. F. Rodriguez
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  4. Y. Palti
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Correspondence to M. F. Rodriguez.

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Rodriguez, M.F., Wiens, G.D., Purcell, M.K. et al. Characterization of Toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics 57, 510–519 (2005). https://doi.org/10.1007/s00251-005-0013-1

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