Molecular cloning and characterization of Toll-like receptor 3 in Japanese flounder, Paralichthys olivaceus

► We cloned the Japanese flounder TLR3 (JfTLR3) gene. ► Gene expression of JfTLR3 was up-regulated in PBLs after stimulation with poly I:C and CpG ODN 1668. ► JfTLR3 gene was induced in different tissues following infection with VHSV. ► Intracellular poly I:C in JfTLR3-overexpressing YO-K cells activates IFN and NF-κB pathways. ► In JfTLR3-overexpressing HINAE cells, extracellular poly I:C activates IFN and NF-κB pathways. Mammalian Toll-like receptor 3 (TLR3) recognizes extracellular and intracellular viral dsRNA, and then initiates signaling cascades leading to NF-κB activation and interferon (IFN) production. To understand the roles of TLR3 in the fish immune system, TLR3 gene (JfTLR3) was identified from Japanese flounder (Paralichthys olivaceus), which consisted of 4 exons and 3 introns. Its expression in peripheral blood leukocytes increased upon stimulation with poly I:C and CpG ODN 1668. Exposure to viral hemorrhagic septicemia virus increased expression of JfTLR3 in the blood, liver, head kidney and spleen. Intracellular poly I:C stimulation in JfTLR3-overexpressing YO-K cells significantly induced IFN-inducible and NF-κB-regulated genes. NF-κB activity in JfTLR3-overexpressing YO-K cells was significantly induced by intracellular poly I:C while expression of IFN-inducible genes and NF-κB reporter activity in JfTLR3-overexpressing HINAE cells increased upon stimulation by extracellular poly I:C. These results suggest that JfTLR3 plays an important role in the induction of antiviral immune response.