Antiviral effects of umbelliferone against viral hemorrhagic septicemia virus in olive flounder (Paralichthys olivaceus).

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to global aquaculture, prompting ongoing efforts to identify potential drug candidates for disease prevention. Coumarin derivatives have recently emerged as a promising class of compounds effective against rhabdoviruses, which severely impact the aquaculture industry. In this study, we assessed the anti-VHSV activity of umbelliferone (7-hydroxycoumarin) in fathead minnow (FHM) cells and olive flounder Paralichthys olivaceus. Umbelliferone exhibited an EC50 of 100 µg/mL by reducing cytopathic effect, with a maximum cytotoxicity of 30.9 % at 750 µg/mL. Mechanistic analyses via a time-course plaque reduction assay revealed that direct incubation with the virus for 1 h resulted in 97.0 ± 1.8 % plaque reduction, showing excellent direct virucidal activity. Pretreatment for 4 h resulted in a 33.5 ± 7.8 % plaque reduction, which increased with longer incubation times. Cotreatment led to a 33.5 ± 2.9 % plaque reduction, suggesting interference with viral binding, whereas postinfection treatment proved less effective. Umbelliferone was prophylactically administered to the olive flounder through short-term (3 days) and long-term (14 days) medicated feeding, followed by a 4-day postinfection period. Short-term administration at 100 mg/kg body weight (bw)/day resulted in the highest relative percent survival (RPS) of 56 %, whereas long-term administration achieved a maximum RPS of 44 % at 30 mg/kg bw/day. Umbelliferone administration delayed mortality at these doses. Additionally, umbelliferone significantly inhibited the expression of the VHSV N gene during viral challenge, with no observed toxic effects in fish up to an administration dose of 30 mg/kg bw/day for 28 days. Our findings suggest that the protective mechanism of short-term administration of 100 mg umbelliferone against VHSV infection may involve the overexpression of TLR2, MDA5, STAT1, and NF-κB at 24 h postinfection (hpi). IL-8 and IFN II expression was upregulated, whereas TNF-α, IL-1ß, and IFN I expression was suppressed at 24 hpi. The upregulation of ISG15 at 48 hpi may contribute to the inhibition of VHSV replication, whereas the downregulation of Caspase 3 expression at 96 hpi suggests a possible inhibition of virus-induced apoptosis at later infection stages. Overall, umbelliferone exhibited anti-VHSV activity through multiple mechanisms, with the added advantage of convenient administration via medicated feed.

Molecular identification and function characterization of four alternative splice variants of trim25 in Japanese flounder (Paralichthys olivaceus).

Trim25 is a member of Tripartite Motif (TRIM) family. Previous studies report that trim25 modulates antiviral activity by activating RIG-I. In this study we explored the four alternative splicing (AS) variants X1-X4 of Japanese flounder trim25. The sequences of the AS variants were highly conserved. Expression levels of trim25 X1-X4 were increased after 12 h of poly I:C treatment in vitro. In vivo expression of X2-X4 in liver, kidney (except X2) and blood was significantly up-regulated in early stages of poly I:C treatment. Subcellular localization analysis showed that Trim25 X1-X4 were distributed in different cellular organelles. The recombinant vector pcDNA3.1-Trim25 X1-X4 were successfully overexpressed in Flounder cells and the samples were collected. Expression patterns of RIG-I pathway genes dhx58, traf6, traf2, nfkbia and il-8 were explored in vitro and in vivo after poly I:C treatment, as well as overexpressed samples. The findings of this study imply that AS variants of trim25 confer antiviral activity in Japanese flounder by modulating innate immune response.

Two CD9 tetraspanin family members of Japanese flounder (Paralichthys olivaceus): characterization and comparative analysis of the anti-infectious immune function.

CD9 is a glycoprotein of the transmembrane 4 superfamily that is involved in various cellular processes. Studies related to the immune functions and activities of CD9 in teleost fish are limited. In this study, we characterized two CD9 homologs, PoCD9.1 and PoCD9.3, from Japanese flounder (Paralichthys olivaceus). Sequence analysis showed that PoCD9.1 and PoCD9.3 possess characteristic transmembrane 4 superfamily (TM4SF) structures. PoCD9.1 shares 70.61% sequence identity with PoCD9.3. The expression of PoCD9.1 and PoCD9.3 in the three main immune tissues was significantly induced in a time-dependent manner by extracellular and intracellular pathogen infection, which indicates that the two CD9 homologs play an important role in the response to pathogenic infection. Following infection with the extracellular pathogen Vibrio anguillarum, the expression profiles of both PoCD9.1 and PoCD9.3 were similar. After infection with the intracellular pathogen Edwardsiella piscicida, the expression levels of PoCD9.1 and PoCD9.3 were different at different stages of infection, especially in the spleen. The spleen was the most important tissue for the PoCD9.1 and PoCD9.3 responses to pathogen infection among the three examined immune tissues. Knockdown of PoCD9.1 and PoCD9.3 attenuated the ability of host cells to eliminate pathogenic bacteria, and PoCD9.1 knockdown was more lethal than PoCD9.3 knockdown for host cells with E. piscicida infection. Overexpression of PoCD9.1 and PoCD9.3 promoted host or host cell defence against E. piscicida infection. These findings suggest that PoCD9.1 and PoCD9.3 serve as immune-related factors, play an important role in the immune defence system of Japanese flounder, and display different functions in response to different pathogens at different stages of infection.

First report of eosinophil peroxidase in starry flounder (Platichthys stellatus): Gene identification and gene expression profiling.

Eosinophils are granular leukocytes that are evolutionarily preserved in the innate immune system of some invertebrates and vertebrates, and these cells can directly remove invading microorganisms and secrete various cytokines, and are also involved in homeostasis. These eosinophils are made up of specific granular proteins that can be differentiated from other cells, and eosinophil peroxidase (EPX) is a peroxidase released only from eosinophils that plays an important role in maintaining the main function and homeostasis of eosinophils. We obtained the sequence information of EPX for the first time from the starry flounder (Platichthys stellatus), and predicted it by amino acid sequencing to confirm sequence alignment and phylogenetic characteristics with other species. Based on analysis of the expression characteristics of PsEPX mRNA in healthy P. stellatus, it was expressed at the highest level in peripheral blood lymphocytes (PBLs) and was also expressed at a relatively high level in the head kidney and intestine, which are immune-related tissues. After artificial infection with Streptococcus parauberis and viral haemorrhagic septicaemia virus, which are the causes of major pathogenic diseases, the expression level of PsEPX was significantly regulated, which showed specific characteristics of pathogens or tissues. These results suggest that PsEPX is an important component of the immune system of P. stellatus and is considered a basic research case for the study of the immunological function of eosinophils in fish.

Molecular cloning and characterization of chemokine C-C motif ligand 34 (CCL34) genes from olive flounder (Paralichthys olivaceus).

Chemokines are a superfamily of chemotactic cytokines that regulate the migration and immune responses of leukocytes. Depending on the arrangement of the first two cysteine residues, chemokines are divided into four groups: CXC (α), CC (ß), C (γ), and CX3C (δ). Chemokine C-C motif ligand 34 (CCL34) is a member of the CC chemokine family and is known as a fish-specific CC chemokine. In this experiment, we analyzed the molecular cloning and characterization of the PoCCL34 gene in olive flounder (Paralichthys olivaceus), including CCL34a.3 (PoCCL34a.3) and CCL34b.3 (PoCCL34b.3). The amino acid sequence of PoCCL34 has four highly conserved cysteine residues and it has a C-C motif. Phylogenetic analysis revealed that PoCCL34 was phylogenetically clustered in the fish CCL34 subcluster. Recombinant PoCCL34 induced chemotaxis of head kidney leukocytes in a dose-dependent manner. Head kidney leukocytes stimulated with PoCCL34 also exhibited significant respiratory burst activity and increased expression of pro-inflammatory cytokines (IL-1ß, IL-6, and CXCL8), but the overall expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15) did not increase. Olive flounder injected with recombinant PoCCL34 demonstrated increased expression of pro-inflammatory cytokines (IL-1ß and IL-6) in the head kidney. However, there was no increase in the expression of interferon-related genes (IFN-α/ß, IFN-γ, Mx, and ISG15). Additionally, recombinant PoCCL34 induced high lysozyme activity in the serum of the flounder. These results indicate that although PoCCL34 is not involved in the antiviral response, it may play a significant role in the overall immune response of the flounder, particularly in mediating the inflammatory response.

RNA-seq transcriptome analysis in flounder cells to compare innate immune responses to low- and high-virulence viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Viral growth and virulence rely on the ability to inhibit the cellular innate immune response. In this study, we investigated differences in the modulation of innate immune responses of HINAE flounder cells infected with low- and high-virulence VHSV strains at a multiplicity of infection of 1 for 12 h and 24 h and performed RNA sequencing (RNA-seq)-based transcriptome analysis. A total of 193 and 170 innate immune response genes were differentially expressed by the two VHSV strains at 12 and 24 h postinfection (hpi), respectively. Of these, 73 and 77 genes showed more than a twofold change in their expression at 12 and 24 hpi, respectively. Of the genes with more than twofold changes, 22 and 11 genes showed high-virulence VHSV specificity at 12 and 24 hpi, respectively. In particular, IL-16 levels were more than two time higher and CCL20a.3, CCR6b, CCL36.1, Casp8L2, CCR7, and Trim46 levels were more than two times lower in high-virulence-VHSV-infected cells than in low-virulence-VHSV-infected cells at both 12 and 24 hpi. Quantitative PCR (qRT-PCR) confirmed the changes in expression of the ten mRNAs with the most significantly altered expression. This is the first study describing the genome-wide analysis of the innate immune response in VHSV-infected flounder cells, and we have identified innate immune response genes that are specific to a high-virulence VHSV strain. The data from this study can contribute to a greater understanding of the molecular basis of VHSV virulence in flounder.

Identification and characterization of Trpm4 gene involved in regulating Japanese flounder (Paralichthys olivaceus) inflammatory response.

The transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed Ca2+ -impermeable cation channel involved in modulating inflammatory and immune responses in mammals. However, the role of TRPM4 channel in fish immunity remains unclear. In this report, from a comparative immunological point of view, we identified and characterized a Trpm4 gene from Japanese flounder (Paralichthys olivaceus) and analysed its potential role in regulating the fish inflammatory response. The Japanese flounder Trpm4 gene is expressed in a wide range of tissues and encodes a 1264-amino acid protein which expresses on the cell surface and shares several conserved domains with its mammalian counterparts. In vitro inflammatory challenge and in vivo bacterial infection experiments revealed that Japanese flounder Trpm4 expression was significantly modulated following different immune challenges, indicating the implication of Trpm4 in the fish immune response. Overexpression of TRPM4 significantly attenuated LPS- and poly(I:C)-induced pro-inflammatory cytokine expression in Japanese flounder FG-9307 cells. In contrast, pharmacological inhibition of the endogenous TRPM4 channel activity in Japanese flounder head kidney macrophages resulted in increased pro-inflammatory cytokine expression following LPS and poly(I:C) stimulations. Taken together, these findings indicate that TRPM4 channels may play a conserved role in regulating inflammatory response(s) in fish.

Elucidating the Functional Roles of Helper and Cytotoxic T Cells in the Cell-Mediated Immune Responses of Olive Flounder (Paralichthys olivaceus).

In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder (Paralichthys olivaceus) CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder's CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.

Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells.

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

Full-length transcriptome sequencing from multiple immune-related tissues of Paralichthys olivaceus.

Olive flounder (Paralichthys olivaceus) is an important economical flatfish in Japan, Korea and China, but its production has been greatly threatened by various of diseases. Although RNA-seq has provided valuable insights into the host-pathogen interaction, there are still some disadvantages, such as a short sequencing length, the incomplete or inaccurate splicing. Therefore, we generated a full-length transcriptome using mixed immune-related tissues of P. olivaceus with PacBio Sequel platform. In this study, 379,671 full-length non-chimeric (flnc) reads were generated with average length of 2482 bp, which is longer than any previously reported in P. olivaceus. A total of 66,420 isoforms of transcript were identified, 46,850 of which were novel isoforms of known genes accounting for 70.54%. In addition, 7720 novel genes, 12,540 alternative splicing (AS) events, 9296 alternative polyadenylation (APA) events, 2298 transcription factors (TFs), 10,270 lncRNAs and 5400 fusion transcripts were identified. Furthermore, functional annotation showed that most of the full-length transcripts were enriched in immune-related signaling pathways. Otherwise, the mRNA-miRNA interacting networks confirmed that 28.5% of mRNAs were predicted to be targeted by more than one miRNA. These results facilitate the understanding of gene structure, post-transcriptional regulatory networks, and subsequently proteomic diversity. In conclusion, our study provides the full-length transcriptome from multiple immune-related tissues of P. olivaceus, which is valuable for exploring its immune responses.

The trypsin-like serine protease domain of Paralichthys olivaceus complement factor I regulates complement activation and inhibits bacterial growth.

In mammals, complement factor I (CFI) is a serine protease in serum and plays a pivotal role in the regulation of complement activation. In the presence of cofactor, CFI cleaves C3b to iC3b, and further degrades iC3b to C3c and C3d. In teleost, the function of CFI is poorly understood. In this study, we examined the immunological property of CFI from Japanese flounder (Paralichthys olivaceus) (PoCFI), a teleost species with important economic value. PoCFI is composed of 597 amino acid residues and possesses a trypsin-like serine protease (Tryp) domain. We found that PoCFI expressions occurred in nine different tissues and were upregulated by bacterial challenge. Recombinant PoCFI-Tryp (rPoCFI-Tryp) inhibited complement activation and degraded C3b in serum. rPoCFI-Tryp exhibited apparent binding capacities to a board-spectrum of bacteria and inhibited bacterial growth. These results provide the first evidence to indicate that CFI in teleost negatively regulates complement activation via degradation C3b, and probably plays a role in host immune defense against bacterial infection.

Japanese flounder Paralichthys olivaceus interleukin 21 induces inflammatory response and plays a vital role in the immune defense against bacterial pathogen.

Interleukin (IL)-21 is a pleiotropic cytokine and plays a vital role in immunity. In the current study, we examined the immune function of Japanese flounder Paralichthys olivaceus IL-21 (PoIL-21). PoIL-21 shares moderate (25.17%-46.25%) sequence identities with other teleost IL-21. PoIL-21 expression occurred in multiple tissues, especially intestine, and was regulated by bacterial infection in a time dependent manner. PoIL-21 was secreted by peripheral blood leukocytes (PBL) upon LPS stimulation. Recombinant PoIL-21 (rPoIL-21) bound to a wide range of Gram-negative and Gram-positive bacteria and inhibited the growth of the fish bacterial pathogen Streptococcus iniae. rPoIL-21 also interacted with PBL, resulting in enhanced cell proliferation, ROS production, and expression of IL-1ß, TNF-α, CD8ß, T-bet, PoIL-21, PoIL-21 receptor, and STAT. Consequently, the presence of rPoIL-21 significantly reduced bacterial infection in PBL. In vivo study showed that rPoIL-21 upregulated the expression of inflammatory cytokines and PoIL-21. Taken together, these results indicate that PoIL-21 is an inducible, secreted cytokine with a broad range of binding capacities and plays an important role in the regulation of anti-bacterial immunity.

Functional characterization of two ecto-nucleoside triphosphate diphosphohydrolase 2 genes in Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) are pivotal regulators of extracellular ATP-mediated purinergic immune signaling. ENTPDase2 is a member of the cell surface-bound ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) protein family that hydrolyzes extracellular nucleoside 5'-triphosphates and nucleoside 5'-diphosphates. However, the immune relevance of ENTPDase2 in fish has not been elucidated. In the present study, from a comparative immunological perspective, we functionally characterized two ENTPDase2 transcript variants (namely ENTPDase2 and ENTPDase2a) from Japanese flounder (Paralichthys olivaceus). Sequence analysis indicates that the deduced Japanese flounder ENTPDase2 and ENTPDase2a proteins possess two conserved transmembrane domains and five apyrase conserved regions that are present in ENTPDase family proteins. However, these proteins only share 54% amino acid sequence identity. Tissue expression analysis revealed that both ENTPDase2 and ENTPDase2a mRNA transcripts are ubiquitously expressed in all examined Japanese flounder tissues, whereas ENTPDase2 is dominantly expressed in blood and ENTPDase2a is abundantly expressed in muscle. Immune challenge experiments showed that ENTPDase2 and ENTPDase2a were significantly upregulated by both inflammatory stimulation and Edwardsiella tarda infection. In addition, the expression of ENTPDase2 and ENTPDase2a was modulated by extracellular ATP (eATP) stimulation in a dose-dependent manner. Furthermore, immunolocalization and functional studies demonstrated that both ENTPDase2 and ENTPDase2a are functional glycosylated plasma membrane proteins. However, ENTPDase2a exhibits greater activity in the hydrolysis of eATP than ENTPDase2 and ENTPDase1 proteins. Finally, knockdown of the ENTPDase2 gene by small interfering RNA significantly upregulated the expression of eATP-induced proinflammatory cytokines IL-1beta, TNF-alpha and G-CSF in Japanese flounder head kidney macrophages, while knockdown of ENTPDase2a only upregulated eATP-induced IL-1beta expression. Taken together, our findings suggest that the two functional Japanese flounder ENTPDase2 isoforms play an essential role in the downregulation of eATP-induced proinflammatory cytokine expression in fish by degrading the available ATP levels in the extracellular milieu.

Skin proteome profiling of tongue sole (Cynoglossus semilaevis) challenged with Vibrio vulnificus.

Vibrio vulnificus is a major pathogen of cultured Cynoglossus semilaevis and results in skin ulceration and haemorrhage, but the proteomic mechanism of skin immunity against V. vulnificus remains unclear. In this study, we investigated the histopathology and skin immune response in C. semilaevis with V. vulnificus infection at the protein levels, the differential proteomic profiling of its skin was examined by using iTRAQ and LC-MS/MS analyses. A total of 951 proteins were identified in skin, in which 134 and 102 DEPs were screened at 12 and 36 hpi, respectively. Selected eleven immune-related DEPs (pvß, Hsp71, MLC1, F2, α2ML, HCII, C3, C5, C8ß, C9 and CD59) were verified for their immune roles in the V. vulnificus infection via using qRT-PCR assay. KEGG enrichment analysis revealed that most of the identified immune proteins were significantly associated with complement and coagulation cascades, antigen processing and presentation, salivary secretion and phagosome pathways. To our knowledge, this study is the first to describe the proteome response of C. semilaevis skin against V. vulnificus infection. The outcome of this study contributed to provide a new perspective for understanding the molecular mechanism of local skin mucosal immunity, and facilitating the development of novel mucosal vaccination strategies in fish.

IL-34 regulates the inflammatory response and anti-bacterial immune defense of Japanese flounder Paralichthys olivaceus.

Interleukin (IL)-34 is a relatively recently discovered cytokine with pleiotropic effects on various cellular activities, including immune response. In fish, the knowledge on the function of IL-34 is limited. In the present work, we investigated the function of Japanese flounder Paralichthys olivaceus IL-34 (PoIL-34) in association with inflammation and immune defense. PoIL-34 possesses the conserved structure of IL-34 superfamily and shares 21.52% sequence identity with murine IL-34. PoIL-34 expression was detected in a wide range of tissues of flounder, in particular intestine, and was regulated to a significant extent by bacterial infection in a time-dependent fashion. In vitro studies showed that recombinant PoIL-34 (rPoIL-34) bound peripheral blood leukocytes (PBLs) and promoted ROS production, acid phosphatase activity, and cellular resistance against bacterial infection. At the molecular level, rPoIL-34 enhanced the expressions of inflammatory cytokines and specific JAK and STAT genes. Similar stimulatory effects of rPoIL-34 were observed in vivo. When PoIL-34 was overexpressed in flounder, the expressions of pro- and anti-inflammatory mediators were significantly affected in a tissue-dependent manner, which correlated with an augmented ability of the fish to eliminate invading pathogens from tissues. Together, these results indicated that PoIL-34 regulated inflammatory response probably via specific JAK/STAT pathways and had a significant influence on the immune defense of flounder against bacterial infection.

Different immune responses of flounder (Paralichthys olivaceus) towards the full-length and N-terminal or C-terminal portion of hirame novirhabdovirus glycoprotein.

Glycoprotein is an important immunogenic protein of Hirame novirhabdovirus (HIRRV). In this study, the full-length and N-/C-terminal portions of glycoprotein were recombinantly expressed (rG, rGn and rGc protein), and the induced immune responses were investigated in flounder (Paralichthys olivaceus) model. The results showed that compared to PBS control, rG, rGn and rGc proteins and inactivated HIRRV suspension (iVS) could all stimulate significant increases of flounder CD4-1+, CD4-2+ T lymphocytes and surface IgM positive (sIgM+) B lymphocytes in peripheral blood, spleen and head kidney (p < 0.05). However, no significant differences of the percentages of CD4-1+ or CD4-2+ T lymphocytes were observed among three protein vaccination groups (p > 0.05). iVS could induce the highest mean levels of CD4+ T lymphocytes in peripheral blood and spleen. For sIgM+ B lymphocytes, the average peak percentages in rG and rGc groups were higher than rGn group. Moreover, significant increases of specific serum IgM against HIRRV or rG protein were observed in iVS, rG, rGn and rGc groups, but rG group exhibited the highest mean level. Furthermore, rG protein induced the highest titer of neutralizing antibodies against HIRRV, followed by iVS. Meanwhile, the challenge test showed that the relative percent survival (RPS) of rG, rGn, rGc and iVS groups were 75.0%, 35.7%, 53.6% and 60.7%, respectively. These results revealed that the full-length G protein would be a more effective subunit vaccine candidate against HIRRV infection.

Two short antimicrobial peptides derived from prosaposin-like proteins in the starry flounder (Platichthys stellatus).

Prosaposin (PSAP) is a precursor of saposin (SAP), which is present in lysosomal and secreted proteins. PSAP is a member of the SAP-like protein families, which comprise multifunctional proteins. In particular, their antimicrobial activity has been reported. We identified PSAP-like (PsPSAPL) sequences from starry flounder and analysed their expression and antimicrobial activity based on cDNA and amino acid sequences. PsPSAPL showed conservation of three saposin B type domains at high levels, and PsPSAPL mRNA was relatively abundantly distributed in the brain and gills of healthy starry founders. PsPSAPL mRNA showed significant expression changes in response to viral haemorrhagic septicaemia virus and Streptococcus parauberis. Synthetic peptides (PsPSAPL-1 and -2), prepared based on amino acid sequences, were used to confirm as well as analyse the antimicrobial activity against bacteria and parasites. Consequently, PsPSAPL-1 and -2 were found to significantly inhibit the growth of various bacteria and kill the Miamiensis avidus. In addition, bacterial biofilm formation was significantly inhibited. Safety was also confirmed by analysing cell haemolysis. These results indicate the immunological function of PsPSAP and the potential antimicrobial activity of the AMPs PsPSAPL-1 and -2.

Probiotic effects of mixture of Groenewaldozyma salmanticensis and Gluconacetobacter liquefaciens on growth and immune responses in Paralichthys olivaceus.

This study was performed to evaluate the effects of dietary probiotics on growth, non-specific immune responses and disease resistance in olive flounder, Paralichthys olivaceus. During 8 weeks, the fish were fed the five experimental diets such as a basal commercial diet (CON), oxytetracycline (OTC) and three basal diets containing Bacillus subtilis (BS), a commercial microbial product (CES) and a mixture of yeast and bacterium (PI), respectively. Fish fed all the probiotics diets and OTC showed a significantly higher growth than fish-fed CON (P < 0·05). Fish-fed PI had a significantly higher nitroblue tetrazolium activity, whereas fish-fed CES showed a higher lysozyme level (P < 0·05). A 7-day challenge test also showed that fish-fed PI had a cumulative survival rate equivalent to that of fish-fed OTC (P < 0·05). Moreover, the diet (PI) appeared to increase the diversity of microbial community in the fish. All these results suggest that the probiotics diet could function as a potential antibiotic replacer in the olive flounder. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is unique in revealing that a diet mixture of yeast, Groenewaldozyma salmanticensis and bacterium Gluconacetobacter liquefaciens can enhance growth, innate immunity and diversity of microbial community including dominant species in the olive flounder. All these indicate that the diet mixture could function as a potential antibiotic replacer in one of the most commercially important fisheries in South Korea.

Transcriptome Analysis of Paralichthys olivaceus Erythrocytes Reveals Profound Immune Responses Induced by Edwardsiella tarda Infection.

Unlike mammalian red blood cells (RBCs), fish RBCs are nucleated and thus capable of gene expression. Japanese flounder (Paralichthys olivaceus) is a species of marine fish with important economic values. Flounder are susceptible to Edwardsiella tarda, a severe bacterial pathogen that is able to infect and survive in flounder phagocytes. However, the infectivity of and the immune response induced by E. tarda in flounder RBCs are unclear. In the present research, we found that E. tarda was able to invade and replicate inside flounder RBCs in both in vitro and in vivo infections. To investigate the immune response induced by E. tarda in RBCs, transcriptome analysis of the spleen RBCs of flounder challenged with E. tarda was performed. Six sequencing libraries were constructed, and an average of 43 million clean reads per library were obtained, with 85% of the reads being successfully mapped to the genome of flounder. A total of 1720 differentially expressed genes (DEGs) were identified in E. tarda-infected fish. The DEGs were significantly enriched in diverse Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially those associated with immunity, disease, and infection. Ninety-one key DEGs involved in 12 immune-related pathways were found to form extensive interaction networks. Twenty-one genes that constituted the hub of the networks were further identified, which were highly regulated by E. tarda and involved in a number of immune processes, notably pathogen recognition and signal transduction, antigen processing and presentation, inflammation, and splicing. These results provide new insights into the immune role of flounder RBCs during bacterial infection.

Characterization of CD4-Positive Lymphocytes in the Antiviral Response of Olive Flounder (Paralichthys oliveceus) to Nervous Necrosis Virus.

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.