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Olive flounder (Paralichthys olivaceus) is one of the most valuable marine aquatic species in South Korea and faces tremendous exposure to the viral hemorrhagic septicemia virus (VHSV). Given the growing importance of flounder, it is therefore essential to understand the host defense of P. olivaceus against VHSV infection, but studies on its immune mechanism are hindered by the lack of genomic resources. In this study, the P. olivaceus was infected with disease-causing VHSV isolates, ADC-VHS2012-11 and ADC-VHS2014-5 which showed moderate virulent (20% mortality) and high virulent (65% mortality), in order to investigate the effect of difference in pathogenicity in head kidney during 1, 3, 7 days of post-infection using Illumina sequencing. After removing low-quality sequences, we obtained 144,933,160 high quality reads from thirty-six libraries which were further assembled into 53,384 unigenes with an average length of 563 bp with a range of 200 to 9605 bp. Transcriptome annotation revealed that 30,475 unigenes with a cut-off e-value of 10 -5 were functionally annotated. In total, 10,046 unigenes were clustered into 26 functional categories by searching against the eggNOG database, and 22,233 unigenes to 52 GO terms. In addition, 12,985 unigenes were grouped into 387 KEGG pathways. Among the 13,270 differently expressed genes, 6578 and 6692 were differentially expressed only in moderate and high virulent, respectively. Based on our sequence analysis, many candidate genes with fundamental roles in innate immune system including, pattern recognition receptors (TLRs & RLRs), Mx, complement proteins, lectins, and cytokines (chemokines, IFN, IRF, IL, TRF) were differentially expressed. Furthermore, GO enrichment analysis for these genes revealed gene response to defense response to virus, apoptotic process and transcription factor activity. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of novel gene discovery, gene expression and regulation studies and lays the foundation for fish immunology especially in P. olivaceus against VHSV., (Copyright © 2018. Published by Elsevier Ltd.)

The piscidin family consists of antimicrobial peptides (AMPs) that are mainly found in fish and are crucial effectors of fish innate immune responses. The piscidin family typically has broad-spectrum antimicrobial activity and can modulate immune responses. In this study, we cloned rock bream piscidin (Rbpisc) and investigated its gene expression and biological activity (including antimicrobial and cytotoxic activities). The coding region of Rbpisc consisted of 213 base pairs (bp) encoding 70 amino acid residues. The tertiary structure predicted for Rbpisc includes an amphipathic helix-loop-helix structure. The Rbpisc gene was highly expressed in the gills of healthy fish. The gene expression of Rbpisc increased in the gills after pathogen infection, while the expression was down-regulated in other tissues. A synthetic peptide based on the AMP 12 domain amino acid sequence of Rbpisc appeared to have broad-spectrum antimicrobial activity against various bacteria. However, the synthetic peptide exhibited weak haemolytic activity against fish erythrocytes. These results suggest that Rbpisc might play an important role in the innate immune responses of rock bream., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Atypical chemokine receptor 4 (ACKR4) is regulated by cytokines, binds chemokines and regulates the chemokine gradient. We verified the cDNA sequence by confirming ACKR4 from red sea bream (PmACKR4) by next generation sequencing (NGS) and analysed the molecular characteristics and gene expression profile. In the analysis using the predicted amino acid sequence of PmACKR4, a highly conserved G protein-coupled receptor 1 region and two cysteine residues were identified and included in the ACKR4 teleost cluster in the phylogenetic analysis. In healthy red sea bream, PmACKR4 mRNA was expressed at the highest levels in head kidney and was upregulated in all immune -related tissues used in the experiment after challenges with Streptococcus iniae (S. iniae) and red sea bream iridovirus (RSIV). These results suggest that ACKR4 is highly conserved in red sea bream and may play an important role in the immune system as previously reported. It is thought that ACKR4 acts as a regulator of immune -related cells via immune reactions after pathogenic infection. • An atypical chemokine receptor 4 (ACKR4) was identified in red sea bream (Pagrus major). • PmACKR4 share high conserved structural characteristics with that of other species. • PmACKR4 mRNA was highly expressed in head kidney and spleen from healthy condition. • PmACKR4 mRNA was significantly up-regulated after challenge with fish pathogens. [ABSTRACT FROM AUTHOR]

Abstract: Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α factor (LITAF) plays an important role controlling the expression of TNF-α and the other cytokine genes in the presence of LPS. However, two LITAF homologues have not been characterized in fish. In this study, we cloned two distinct LITAF (RbLITAF1 and RbLITAF2) cDNAs from rock bream (Oplegnathus fasciatus) and characterized their expression profiles after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding regions of RbLITAF1 and RbLITAF2 cDNAs were 492 bp and 417 bp, encoding 153 and 138 amino acid residues, respectively. The genes consisted of a LITAF domain. RbLITAF1 was highly expressed in the spleen and heart of healthy rock bream, whereas RbLITAF2 was highly expressed in the gill, intestine and stomach. In spleen, the gene expression of RbLITAF1 and RbLITAF2 were increased until 5 days post-infection (dpi), and then decreased at 7 dpi. In kidney, E. tarda and RSIV infection led to induction of the RbLITAF1 gene at 1 dpi, RbLITAF2 gene was down-regulated after pathogen infection. These results suggest that RbLITAFs may be involved in the LITAF-mediated immune response and regulate systemic immune responses against pathogen infection. [Copyright &y& Elsevier]

Abstract: CD200 plays an important role in delivering an immunoregulatory signal to the immune system through interaction with its receptor. However, CD200 has not been characterized and its function in teleosts is unknown. In this study, the rock bream (Oplegnathus fasciatus) CD200 gene (RbCD200) was cloned and its expression profile was analyzed after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding region of RbCD200 cDNA was 855 bp, encoding 284 amino acid residues. The gene consisted of two extracellular Ig-like domains and a transmembrane domain. RbCD200 was highly expressed in the brain, erythrocytes, intestine and stomach of healthy rock bream. In the spleen, RbCD200 gene expression was down-regulated until 48 h after E. tarda exposure, except at 12 h RbCD200 gene expression was down-regulated then up-regulated at 12 h and 24 h after infection with S. iniae and RSIV, respectively. In the whole kidney, the RbCD200 gene was down-regulated in response to infection with E. tarda and S. iniae. However, RSIV infection increased RbCD200 gene expression in whole kidney until 48 h. These results suggest that RbCD200 is differentially expressed in the spleen and whole kidney after infection with different pathogens. [Copyright &y& Elsevier]

Abstract: Toll-like receptor (TLR) 5 is responsible for the bacterial flagellin recognition in vertebrates. Synergistic role of TLR 5 membrane form (TLR 5M) and TLR 5 soluble form (TLR 5S) have been reported from the study on rainbow trout. This system is regarded as the unique system in teleost fish. However, systemic response of TLR 5 genes in teleost fish has not been fully understood. Hence, we cloned Japanese flounder (Paralichthys olivaseus) TLR 5M and TLR 5S genes and their expressions were analyzed. The coding region of Japanese founder TLR 5M and TLR 5S cDNA were 2670 bp and 1923 bp, encoding 889 and 640 amino acid residues, respectively. The Japanese flounder TLR 5M was composed of an extracellular leucine rich repeats (LRRs), a transmembrane and an intracellular Toll/interleukin-1 receptor (TIR) domains, whereas TLR 5S possessed only the LRR domain. TLR 5M was highly expressed in the gill, head kidney, heart and liver. TLR 5S was highly expressed in the brain, head kidney and heart. Flagellin stimulation (1 and 5 μg/ml) led to strong gene expression of TLR 5S in peripheral blood leukocytes (PBLs) and liver cells. In contrast to TLR 5S, TLR 5M was down-regulated until 3 h after flagellin stimulation in PBLs and liver cells. The flagellin stimulation also resulted in the production of the flounder IL-1β and IL-6 from the liver cells and PBLs. The gene expression of TLR 5M was highly induced in the liver, while TLR 5S gene expression was drastically increased in the intestine following challenge with Edwardsiella tarda. Increased number of TLR 5M- and 5S-expressing cell populations were detected by in situ hybridization in the lamina propria of the intestine and liver after E. tarda infection, respectively. These results imply that the expression of these TLR 5 paralogs in Japanese flounder are differently regulated in the whole body and play important roles in the immune response against bacterial pathogens. [ABSTRACT FROM AUTHOR]

Abstract: Cathepsins are lysosomal cysteine proteases belonging to the papain family, whose members play important roles in normal metabolism for the maintenance of cellular homeostasis. Rock bream (Oplegnathus fasciatus) cathepsin H (RbCTSH) cDNAs were identified by expressed sequence tag analysis of a lipopolysaccharide-stimulated rock bream liver cDNA library. The full-length RbCTSH cDNA (1326 bp) contained an open reading frame of 978 bp encoding 325 amino acids. The presence of an ERFNIN-like motif was predicted in the propeptide region of RbCTSH. Furthermore, multiple alignments showed that the EPQNCSAT region was well conserved among other cathepsin H sequences. Phylogenetic analysis revealed that RbCTSH is most closely related to Nile tilapia cathepsin H. RbCTSH was expressed significantly in the intestine, spleen, head kidney and stomach. RbCTSH mRNA expression was also examined in several tissues under conditions of bacterial and viral challenge. All examined tissues of fish infected with Edwardsiella tarda, Streptococcus iniae and red sea bream iridovirus (RSIV) showed significant increases in RbCTSH expression compared to the control. In the kidney and spleen, RbCTSH mRNA expression was upregulated markedly following infection with bacterial pathogens. These findings indicate that RbCTSH plays an important role in the innate immune response of rock bream. Furthermore, these results provide important information for the identification of other cathepsin H genes in various fish species. [Copyright &y& Elsevier]