Search

Fish skin, the mucosal site most exposed to external antigens, requires protection by an efficient local mucosal immune system. The mucosal reserve of IgM is recognized as an immune strategy that blocks pathogen invasion to maintain homeostasis, whereas the mechanism of skin-associated local IgM production induced by mucosal antigens is not well know. In this study, we found that the skin of flounder (Paralichthys olivaceus) was equipped with the immune cellular and molecular basis for processing mucosal antigens and triggering local specific responses, i.e., CD4 + Zap-70 + T cells, CD4 - Zap-70 + T/NK cells, IgM + MHCII + B cells, PNA + MHCII + antigen-presenting cells, UEA-1 + WGA + and UEA-1 + WGA - antigen-sampling cells, as well as secreted IgM and pIgR, as demonstrated by indirect immunofluorescence assay using different antibodies and lectins. After immersion immunization with inactivated Edwardsiella tarda, qPCR assay displayed up-regulation of immune-related genes in flounder skin. Flow cytometry analysis and EdU labeling demonstrated that the mucosal inactivated vaccine induced local proliferation and increased amounts of cutaneous IgM + B cells. Skin explant culture proved the local production of specific IgM in the skin, which could bind to the surface of E. tarda. ELISA, laser scanning confocal microscopy, and Western blot revealed that, in addition to the elevated IgM levels, pIgR protein level was significantly up-regulated in skin tissue and surface mucus containing the pIgR (secretory component, SC)-tetrameric IgM complex, indicating that mucosal vaccine stimulated up-regulation of IgM and pIgR, which were secreted as a complex into skin mucus to exert the protective effects as secretory IgM. These findings deepen the understanding of IgM-based local responses in the mucosal immunity of teleosts, which will be critical for subsequent investigation into the protective mechanism of mucosal vaccines for fish health., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hirame novirhabdovirus (HIRRV) is a highly pathogenic fish virus that poses a significant threat to the farming of a variety of economic fish. Due to no commercial vaccines and effective drugs available, sensitive and rapid detection of HIRRV at latent and early stages is important and critical for the control of disease outbreaks. However, most of the current methods for HIRRV detection have a large dependence on instruments and operations. For better detection of HIRRV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of HIRRV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 40 °C and 32min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15min. The whole detection procedure including can be accomplished within 1 h, with a detection sensitivity of about 8.7 copies/μl. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses IHNV and VHSV, allowing naked-eye color-based interpretation of the detection results through lateral flow (LF) strip or fluorescence under violet light. Furthermore, the proliferation dynamic of HIRRV in the spleen of flounder were comparatively detected by LF- and fluorescence-based RPA-CRISPR/Cas12a assay in comparison to qRT-PCR at the early infection stage, and the results showed that the viral positive signal could be firstly detected by the two RPA-CRISPR/Cas12a based methods at 6 hpi, and then by qRT-PCR at 12 hpi. Overall, our results demonstrated that the developed RPA-CRISPR/Cas12a method is a stable, specific, sensitive and more suitable in the field, which has a significant effect on the prevention of HIRRV. RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for visual and on-site detection of HIRRV, which shows a great application promise for the prevention of HIRRV infections., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nervous necrosis virus (NNV) is a highly neurotropic virus that poses a persistent threat to the survival of multiple fish species. However, its inimitable neuropathogenesis remains largely elusive. To rummage potential partners germane to the nervous system, we investigated the interaction between red-spotted grouper NNV (RGNNV) and grouper brain by immunoprecipitation coupled with mass spectrometry and discerned Nectin1 as a novel host factor subtly involved in viral early invasion events. Nectin1 was abundant in neural tissues and implicated in the inception of tunnel nanotubes triggered by RGNNV. Its overexpression not only dramatically potentiated the replication dynamics of RGNNV in susceptible cells, but also empowered non-sensitive cells to expeditiously capture free virions within 2 min. This potency was impervious to low temperatures but was dose-dependently suppressed by soluble protein or specific antibody of Nectin1 ectodomain, indicating Nectin1 as an attachment receptor for RGNNV. Mechanistically, efficient hijacking of virions by Nectin1 strictly depended on intricate linkages to different modules of viral capsid protein, especially the direct binding between the IgC1 loop and P-domain. More strikingly, despite abortive proliferation in Nectin1-reconstructed CHSE-214 cells, a non-sensitive cell, RGNNV could gain access to the intracellular compartment by capitalizing on Nectin1, thereby inducing canonical cytoplasmic vacuolation. Altogether, our findings delineate a candidate entrance for RGNNV infiltration into the nervous system, which may shed unprecedented insights into the exploration and elucidation of RGNNV pathogenesis.IMPORTANCENervous necrosis virus (NNV) is one of the most virulent pathogens in the aquaculture industry, which inflicts catastrophic damage to ecology, environment, and economy annually around the world. Nevertheless, its idiosyncratic invasion and latency mechanisms pose enormous hardships to epidemic prevention and control. In this study, deploying grouper brain as a natural screening library, a single-transmembrane glycoprotein, Nectin1, was first identified as an emergent functional receptor for red-spotted grouper NNV (RGNNV) that widely allocated in nervous tissues and directly interacted with viral capsid protein through distinct Ig-like loops to bridge virus-host crosstalk, apprehend free virions, and concomitantly propel viral entry. Our findings illuminate the critical role of Nectin1 in RGNNV attachment and entry and provide a potential target for future clinical intervention strategies in the therapeutic race against RGNNV., Competing Interests: The authors declare no conflict of interest.

The lymphocystis disease (LCD), caused by Lymphocystis disease virus (LCDV), is a benign and self-limiting disease described in a many freshwater and marine fish species. Hypertrophic fibroblasts and extensive aggregation of inflammatory cells are characteristics of LCD. In the present study, small animal imaging and ultrastructural investigations were carried out on the lymphocystis nodules of black rockfish (Sebastes schlegelii) naturally infected with lymphocystis iridovirus, to assess pathology, and the exudate with particular attention to the formation of extracellular traps (ETs) in vivo. Ex vivo were examined by nodules sections and primary cells stimulation. By histopathological analysis, the nodules contained infiltrated inflammatory cells and extensive basophilic fibrillar filaments at the periphery of the hypertrophied fibroblasts. ETs were assessed in nodules samples using indirect immunofluorescence to detect DNA and myeloperoxidase. Moreover, LCDV was able to infect peritoneal cells of black rockfish in vitro and induce the formation of ETs within 4 h. In summary, this study proved that ETs are involved in the response to LCDV infection and may be involved in formation of lymphoid nodules. Taken together, the findings provide a new perspective to determine the impact factors on the growth of nodules., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

β-defensin of flounder plays an important role in immunomodulation by recruiting immune cells and has a potential vaccine adjuvant effect in addition to its bactericidal activity. In this study, adjuvant effects of β-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus) were investigated. The bicistronic eukaryotic expression plasmid pBudCE4.1 plasmid vector with two independent coding regions was selected to construct DNA vaccine of p-OmpC which express only the gene for the outer membrane protein of Edwardsiella tarda and the vaccine of p-OmpC-βdefensin which express both the outer membrane protein of the bacterium and β-defensin of flounder. In vitro and in vivo studies have shown that the constructed plasmids can be expressed in flounder embryonic cell lines and injection sites of muscles. After vaccination by intramuscular injection, both p-OmpC and p-OmpC-βdefensin groups showed significant upregulation of immune-response. Compared to the pBbudCE4.1 and the p-OmpC vaccinated groups, the p-OmpC-βdefensin vaccinated group showed significantly more cell aggregation at the injection site and intense immune response. The proportion of sIgM + cells, as well as the CD4-1 + and CD4-2 + cells in both spleen and kidney was significantly higher in the p-OmpC-βdefensin vaccinated group at peak time point than in the control groups. The relative survival rate of the p-OmpC-βdefensin vaccine was 74.17%, which was significantly higher than that of the p-OmpC vaccinated group 48.33%. The results in this study determined that β-defensin enhances the responses in cellular and humoral immunity and evokes a high degree of protection against E. tarda, which is a promising candidate for vaccine adjuvant., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Infections due to pathogens impact global aquaculture economy, where diseases caused by bacteria should be in particular focus due to antibiotic resistance. Hepcidin and NK-lysin are important innate immune factors having potential to be exploited as alternatives to antibiotics due to their antimicrobial activity and immunomodulatory capacity. In this study, the recombinant proteins of hepcidin 2 and NK-lysin (rPoHep2 and rPoNKL) from flounder (Paralichthys olivaceus) were obtained via a prokaryotic expression system. The results exhibited that rPoHep2 and rPoNKL killed both gram-negative and gram-positive bacteria mainly via attachment and disruption of the membrane. Interestingly, both peptides could bind to bacterial DNA. The antiviral assay showed that both peptides have antiviral activity against hirame nonvirhabdovirus. They exhibited no cytotoxicity to the mammalian and fish cell lines. PoHep2 was found localized in G-CSFR-positive peritoneal cells. Moreover, rPoHep2 significantly enhanced the phagocytosis of flounder leukocytes in vitro. These findings suggested that neutrophils contained rPoHep2 and may respond to the immunoreaction of neutrophils. In summary, both rPoHep2 and rPoNKL possess antimicrobial activities and may be exploited to replace traditional antibiotics. rPoHep2 possess immune regulatory functions, that can be further investigated as an immunostimulant in aquaculture., Competing Interests: Declaration of competing interest The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The non-virion (NV) protein is the signature of genus Novirhabdovirus, which has been of considerable concern due to its potential role in viral pathogenicity. However, its expression characteristics and induced immune response remain limited. In the present work, it was demonstrated that Hirame novirhabdovirus (HIRRV) NV protein was only detected in the viral infected hirame natural embryo (HINAE) cells, but absent in the purified virions. Results showed that the transcription of NV gene could be stably detected in HIRRV-infected HINAE cells at 12 h post infection (hpi) and then reached the peak at 72 hpi. A similar expression trend of NV gene was also found in HIRRV-infected flounders. Subcellular localization analysis further exhibited that HIRRV-NV protein was predominantly localized in the cytoplasm. To elucidate the biological function of HIRRV-NV protein, NV eukaryotic plasmid was transfected into HINAE cells for RNA-seq. Compared to empty plasmid group, some key genes in RLR signaling pathway were significantly downregulated in NV-overexpressed HINAE cells, indicating that RLR signaling pathway was inhibited by HIRRV-NV protein. The interferon-associated genes were also significantly suppressed upon transfection of NV gene. This research would improve our understanding of expression characteristics and biological function of NV protein during HIRRV infection process., Competing Interests: Declaration of competing interest The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Interleukin 2 (IL-2) is an immunoregulatory cytokine that plays significant role in the activation and proliferation of immune cells. In teleost, the functions of IL-2 signaling on the proliferation and differentiation of T lymphocytes were well documented. However, there is still unclear about the effects of IL-2 signaling on B cell immunity in fish. Hence, in this study, full-length transcriptome sequencing was performed to investigate the activation of IL-2 on flounder (Paralichthys olivaceus) lymphocytes in vitro, the effects of IL-2 on the immunity of B cells after its receptors (IL-2Rβ or IL-2Rγ) blocked were further investigated. The results shown that the differentially expressed genes in lymphocytes after IL-2 stimulation were annotated to the pathways related to the immune response of B cells. The percentages of mIgM + B cells were increased, and the capacities of antibody secretion and phagocytosis of B cells were enhanced after IL-2 stimulation. However, the function of IL-2 on B lymphocytes immunity was significantly inhibited after IL-2 receptors were blocked, especially after IL-2Rβ was blocked. Collectively, we can conclude that IL-2 is able to promote the proliferation of B lymphocytes, antibody secretion, and enhance their phagocytosis in flounder, and these effects are mediated through IL-2/IL-2R signaling., Competing Interests: Declaration of competing interest The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Nervous necrosis virus (NNV), a formidable pathogen in marine and freshwater fish, has inflicted enormous financial tolls on the aquaculture industry worldwide. Although capsid protein (CP) is the sole structural protein with pathogenicity and antigenicity, public information on immunodominant regions remains extremely scarce. Here, we employed neutralizing monoclonal antibodies (MAbs) specific for red-spotted grouper NNV (RGNNV) CNPgg2018 in combination with partially overlapping truncated proteins and peptides to identify two minimal B-cell epitope clusters on CP, 122 GYVAGFL 128 and 227 SLYNDSL 233 . Site-directed mutational analysis confirmed residues Y123, G126, and L128 and residues L228, Y229, N230, D231, and L233 as the critical residues responsible for the direct interaction with ligand, respectively. According to homologous modeling and bioinformatic evaluation, 122 GYVAGFL 128 is harbored at the groove of the CP junction with strict conservation among all NNV isolates, while 227 SLYNDSL 233 is localized in proximity to the tip of a viral protrusion having relatively high evolutionary dynamics in different genotypes. Additionally, 227 SLYNDSL 233 was shown to be a receptor-binding site, since the corresponding polypeptide could moderately suppress RGNNV multiplication by impeding virion entry. In contrast, 122 GYVAGFL 128 seemed dedicated only to stabilizing viral native conformation and not to assisting initial virus attachment. Altogether, these findings contribute to a novel understanding of the antigenic distribution pattern of NNV and the molecular basis for neutralization, thus advancing the development of biomedical products, especially epitope-based vaccines, against NNV. IMPORTANCE NNV is a common etiological agent associated with neurological virosis in multiple aquatic organisms, causing significant hazards to the host. However, licensed drugs or vaccines to combat NNV infection are very limited to date. Toward the advancement of broad-spectrum prophylaxis and therapeutics against NNV, elucidating the diversity of immunodominant regions within it is undoubtedly essential. Here, we identified two independent B-cell epitopes on NNV CP, followed by the confirmation of critical amino acid residues participating in direct interaction. These two sites were distributed on the shell and protrusion domains of the virion, respectively, and mediated the neutralization exerted by MAbs via drastically distinct mechanisms. Our work promotes new insights into NNV antigenicity as well as neutralization and, more importantly, offers promising targets for the development of antiviral countermeasures.

Hirame novirhabdovirus (HIRRV) is a fish rhabdovirus belonging to family Rhabdoviridae, genus Novirhabdovirus, which is highly contagious and virulent, and causes hemorrhagic disease in many fish species. In the present work, the whole genome sequence of HIRRV strain CNPo2015 that previously isolated from cultured flounders was obtained using high-throughput sequencing. It consists of 10,998 nucleotides and encodes six viral proteins arranged in order of 3'-N-P-M-G-NV-L-5'. Among Novirhabdovirus, L protein of CNPo2015 possessed the lowest amino acid sequence divergence with HIRRV isolate CA 9703 and HIRRV 080113, and the highest with Snakehead rhabdovirus. Furthermore, the immune response of Hirame natural embryo (HINAE) cell line to HIRRV infection was characterized by RNA-seq, and the results showed that 1976 differentially expressed genes (DEGs) including 1219 up-regulated and 727 down-regulated genes were identified in the HINAE cells infected with HIRRV at 48 h post infection (hpi). Several KEGG pathways were significantly enriched in the viral infected cells, such as cytokine-cytokine receptor interaction, JAK-STAT signaling pathway, cell cycle, apoptosis, RIG-I-like receptors signaling pathway and P13K-AKT signaling pathway. Post viral infection, the flow cytometric Annexin V/PI assay found that apoptotic rate of HINAE cells showed a slight increase within 3 days and then the early and late apoptotic rate were significantly increased to 41 ± 2.65% and 12.37 ± 2.61% at day 4, respectively. Meanwhile, qRT-PCR results also showed that six apoptosis-related genes (BCL2L1, CASPASE 3, CASPASE 10, FAS, AKT and CDK1) were significantly upregulated. This investigation has not only enriched our knowledge of sequence difference characteristics between CNPo2015 and other Novirhabdoviruses, but also provided a data basis for deeper understanding of immune responses in flounder cells post viral infection., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hirame novirhabdovirus (HIRRV) infection is characterized by a pronounced viremia, and the high viral load is typically detected in immune-related organs and the circulatory system. In the present study, we demonstrated that HIRRV has the capacity to invade part of flounder membrane-bound IgM (mIgM + ) B lymphocyte. Eight quantitative real-time PCR (qRT-PCR) standard curves involving HIRRV genomic RNA (gRNA), cRNA, and six mRNAs were established based on the strand-specific reverse transcription performed with tagged primers. It was revealed that viral RNA synthesis, especially the replication of gRNA, was inhibited in B cells, and the intracellular HIRRV even failed to produce infectious viral particles. Moreover, a range of genes with nucleic acid binding activity or related to viral infection were screened out based on the transcriptome analysis of HIRRV-infected B cells, and five molecules were further selected because of their different expression patterns in HIRRV-infected B cells and hirame natural embryo (HINAE) cells. The overexpression of these genes followed by HIRRV infection and RNA binding protein immunoprecipitation (RIP) assay revealed that the flounder B cell lymphoma/leukemia 11A (BCL11A), a highly conserved zinc finger transcription factor, is able to inhibit the proliferation of HIRRV by binding with full-length viral RNA mainly via its zinc finger domains at the C terminus. In conclusion, these data indicated that the high transcriptional activity of BCL11A in flounder mIgM + B lymphocytes is a crucial factor for the abortive infection of HIRRV, and our findings provide new insights into the interaction between HIRRV and teleost B cells. IMPORTANCE HIRRV is a fish rhabdovirus that is considered as an important pathogen threatening the fish farming industry represented by flounder because of its high infectivity and fatality rate. To date, research toward understanding the complex pathogenic mechanism of HIRRV is still in its infancy and faces many challenges. Exploration of the relationship between HIRRV and its target cells is interesting and necessary. Here, we revealed that flounder mIgM + B cells are capable of suppressing viral RNA synthesis and result in an unproductive infection of HIRRV. In addition, our results demonstrated that zinc finger protein BCL11A, a transcription factor in B cells, is able to suppress the replication of HIRRV. These findings increased our understanding of the underlying characteristics of HIRRV infection and revealed a novel antiviral mechanism against HIRRV based on the host restriction factor in teleost B cells, which sheds new light on the research into HIRRV control.

The nerve necrosis virus (NNV), a pathogen of viral nervous necrosis disease in several important mariculture economic fish species, causes economic loss. Its nonstructural protein B2 encoded by the sub-genomic RNA3 affects the amplification of the virus. In this study, the B2 protein was recombinantly expressed, the polyclonal antibodies were produced and the dynamics of the B2 protein and genomes were measured in vivo and in vitro after NNV infection. Then, the effects of the overexpressed B2 protein on virus proliferation were investigated. The results showed that the polyclonal antibodies can recognize the B2 protein in both SSN-1 cells and the brain/eye of the grouper. The RNA3 expression significantly increased at 12 h and kept rising till the end of the experiment; it was 10 6.9 copies/μL at 120 h. The B2 protein could be first detected at 3 h post-infection, which was earlier than the capsid protein was first detected (12 h post-infection). The B2 protein can be detected in the brain, eye and heart on day 3 and the copy number of genomes reached a maximum at 6 d post-infection. There was a low expression of NNV genomes in the liver, spleen and kidney, and no virus was detected in the gill, stomach and intestine. In the meantime, the B2 protein was successfully expressed in GF-1 cells and significantly enhanced virus proliferation, which produced an earlier cytopathic effect and higher cell death rates after 3 d post-infection than the control. In conclusion, the B2 protein acts as an early expressed protein during virus replication and proliferation and is involved in the early infection of NNV. The results may provide insight into the early stage of virus infection and prevention of the disease.

Protection is crucial for a vaccine. In this study, flounder was inoculated with an inactivated Edwardsiella tarda for 5 weeks then challenged with E. tarda, after that, the protective cellular and humoral immune responses were studied using specific antibodies against CD4, CD8 and IgM, respectively, previously produced in our lab. And then gene transcription, relative percent survival (RPS), innate enzyme activities and histopathology were investigated. The results showed that, in the vaccine group, the percentage of CD4 + T and CD8 + T lymphocytes increased significantly, peaking on 7th post challenge day. The percentage of IgM + B lymphocytes increased significantly first and peaked at 2nd post challenge week. Total and specific antibodies significantly increased and peaked at 3rd week. The mRNA expressions of MHCIα, MHCIIα, CD4-1 and CD8α were significantly upregulated. In the meantime, alkaline phosphatase, acid phosphatase, superoxide dismutase and lysozyme significantly rose and reached to the maximum. The RPS was 54.76 ± 1.92%. The bacterial load in spleen and head kidney was lower in the vaccine group than that of PBS group. These data illustrated that T lymphocyte subsets, B lymphocytes, immune enzymes were induced strong protection against E. tarda for flounder. These results revealed a strong support protection of the vaccine from four aspects, and provided a new perspective for the evaluation of fish vaccines., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Previous studies imply that peripheral blood leukocytes (PBLs) may play an important role in systemic lymphocystis disease virus (LCDV) dissemination, but whether the PBLs are susceptible and permissive to LCDV infection and the dissemination mechanism need to be clarified. In this study, LCDV was firstly confirmed to infect the PBLs in flounder ( Paralichthys olivaceus ) in vivo, and to replicate in PBLs in vitro. Subsequently, the 27.8 kDa receptor protein (27.8R), a functional receptor mediating LCDV infection in flounder gill cells, was shown to locate on the cell membrane of PBLs and co-localize with LCDV in PBLs, while blocking of the 27.8R via pre-incubation of anti-27.8R MAb with the PBLs could obviously inhibit LCDV infection, revealing the 27.8R as a receptor for LCDV entry into PBLs. Multicolor fluorescence imaging studies verified that IgM + and IgD + B-lymphocyte were involved in LCDV infection. In the sorted IgM + B-cells, 27.8R + and LCDV + signals were simultaneously observed, and LCDV copy numbers increased with time, indicating that IgM + B-cells expressed the 27.8R and were permissive to LCDV infection. Furthermore, the dynamic changes of IgM + , 27.8R + , LCDV + and LCDV + /IgM + PBLs were monitored during the early phase of LCDV infection. It was found that the percentage of IgM + B-cells in PBLs clearly declined first and then increased, suggesting LCDV infection facilitated damage to B-cells, whereas the amounts of 27.8R + and LCDV + PBLs, as well as LCDV-infected IgM + B-cells, showed an opposite trend. These results proved that IgM + B-lymphocytes could be infected by LCDV via a receptor-mediated mechanism and support viral replication, which provided novel insights for the first time into the role of B-lymphocytes in LCDV dissemination and pathogenesis in teleost fish.

The CD80/86 molecule is one of the important co-stimulatory ligands and involves antigen-specific immune responses by ligating with CD28 and then delivering the required second signal to T-cell activation. In this study, a CD80/86 homolog was identified, and its expression characteristics were studied in flounder ( Paralichthys olivaceus ). The open reading frame (ORF) of CD80/86 is 906 bp, encoding 301 aa, and the extracellular amino acid sequence encoded two IgV- and IgC-like structural domains; fCD80/86 is highly expressed in head kidney, peripheral blood leukocytes (PBLs), and spleen, and has relatively high expression in muscle. Antibodies specific for CD80/86 were produced, and CD80/86 was colocalized with MHCII+, CD40+, and CD83+ leukocytes but not with IgM+, CD3+, or CD4+ lymphocytes. The cloned CD80/86 in flounder shares conserved structural features with its mammalian counterparts and is mainly distributed on antigen-presenting cells. Based on these data, CD80/86 as an adjuvant to enhance the immune response of DNA vaccine was investigated. A bicistronic DNA vaccine expressing both CD80/86 and the outer membrane protein (OmpK) of Vibrio anguillarum (p-OmpK-CD80/86) was successfully constructed. After immunization, p-OmpK-CD80/86 could induce the upregulation of the proportion of IgM+ and CD4+ cells in flounder, compared to the p-OmpK- or p-CD80/86-immunized group; CD28 genes were significantly induced in the p-CD80/86 and p-OmpK-CD80/86 groups. Compared to the p-OmpK group, the higher expression of CD83 , MHCI , CD4 , CD8 , and IL-2 was detected at the injection site. The relative percent survival (RPS) produced by p-OmpK-CD80/86 is 66.11% following the V. anguillarum challenge, while the RPS of p-OmpK or p-CD80/86 is 46.30% and 5.56%, respectively. The results revealed that CD80/86 is mainly found in antigen-presenting cells, and could help elicit humoral immune responses in teleost through the CD80/86-CD28 signaling pathway involving CD4+ lymphocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Xing, Tang, Sheng, Chi and Zhan.)

A specialized lymphoepithelial tissue termed the interbranchial lymphoid tissue (ILT) is recently identified in several fish species. However, the structural variation and mucosal immune functions of the ILT remain largely unknown. In this study, the anti-Zap-70 MAb was firstly determined to specifically recognize ZAP-70 protein, and CD4-1 + , CD4-2 + and CD8β + T-cells, but not IgM + B cells, in peripheral blood leucocytes of flounder (Paralichthys olivaceus). Then we found that aggregates of Zap-70 + cells were located in the epithelium covering the bottom of the interbranchial cleft and along the afferent and efferent edges of the filaments in a cross view, where a meshwork of epithelial cells containing diffused lymphoid cells was exhibited, confirming these structures as the ILT; In a sagittal view, Zap-70 + cells were situated at the base of the filaments (here named as proximal ILT, pILT) and in the interlamellar epithelium (named as distal ILT, dILT). Also, a few IgM + B cells were distributed at these sites. The lymphoepithelium within pILT and dILT was very thin with a low number of Zap-70 + cells in premetamorphosis and postclimax larvae of flounder, and got thicker containing much more Zap-70 + cells in juvenile and adult individuals. The aggregates of CD4-1 + /Zap-70 + , CD4-2 + /Zap-70 + , and CD8β + /Zap-70 + T-cell subsets were identified in the ILT. Post bath vaccination with inactivated Edwardsiella tarda and then intraperitoneal injection of EdU, the amounts of EdU + and Zap-70 + cells obviously increased at 3 d and 7 d, and co-localization of EdU + /Zap-70 + cells identified the presence of proliferative T cells; meanwhile, MHC class II-expressing cells were increased. These findings indicated that the ILT in gills of flounder was an important site for the induction of local T cell-mediated immunity, which would lead to a better understanding of mucosal immunity and defense mechanisms of teleost fish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Interleukin-2 receptor (IL-2R), as the specific ligand of interleukin-2 (IL-2), binds to IL-2 and transmits signals and then can induce the proliferation of T lymphocytes in mammals. In this paper, the subunit of IL-2R in flounder (Paralichthys olivaceus), interleukin-2 receptor subunit gamma (IL-2Rγ) was cloned, and polyclonal antibodies (Abs) against its extracellular region were produced, then the expression of flounder IL-2Rγ (fIL-2Rγ) at transcriptional and cellular levels were characterized. Moreover, the interaction of flounder IL-2 (fIL-2) with fIL-2Rγ was investigated, and the variations on CD4+/IL-2Rγ+ cells in flounder after treatment with recombinant IL-2 (rIL-2), anti-IL-2Rγ Abs were detected, respectively. The results showed that fIL-2Rγ protein had a typical fibronectin type III (FN3) domain. The Abs could specifically recognize native fIL-2Rγ molecules at 39.9 kDa. FIL-2Rγ was localized on both T and B lymphocytes, and the percentages of CD4+/IL-2Rγ+ and IgM+/IL-2Rγ+ lymphocytes were high in spleen. In addition, pBiFC-VN173-IL-2Rγ plasmids could bind to pBiFC-VC155-IL-2 plasmids. The percentage of CD4+/IL-2Rγ+ lymphocytes was significantly decreased after blocking with anti-IL-2Rγ Abs both in vivo and in vitro. In the meantime, four T cell markers genes and six IL-2-IL-2R pathway genes were down-regulated in anti-IL-2Rγ Abs group. These results first demonstrated that fIL-2Rγ molecules were expressed on both T and B lymphocytes in flounder, and the bond between fIL-2Rγ and fIL-2 activated the CD4 T lymphocytes. This study gave a new sight into the exploration of IL-2R function on T lymphocytes proliferation in fish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hirame novirhabdovirus (HIRRV) is a severe viral pathogen of flounder resulting in significant losses to the aquaculture industry. However, the mortality due to the disease would be significantly reduced when the water temperature was increased from 10 to 20 °C. In this study, we examined the potentiality of vaccination with live HIRRV under a temperature-controlled culture condition for development of protective immunity in flounder. Flounders were infected with HIRRV at 10 °C and maintained for 2 days, and then the temperature was shift up to 20 °C. When the temperature was further shift down to 10 °C at 7 (S-7 group), 14 (S-14 group) or 21 (S-21 group) days post infection (dpi), mortality rates of 60%, 13.33% and 0 were observed, respectively. To investigate the development of protective immunity of survived flounder, a re-challenge was performed and a highest survival rate of 80% was found in S-21 group, which was significantly higher than S-14 group (65%) and S-7 group (45%). Moreover, it was found that a lower viral load was detected in the flounder maintained at 20 °C for a longer time, and a longer maintaining of survived flounder at 20 °C would also elicit higher percentages of IgM + B lymphocytes and specific antibodies levels. Notably, a significantly higher levels of specific antibodies were detected post re-challenge compared with the first peak level after initial infection. Therefore, these demonstrated that the initial infection with live HIRRV under a temperature-controlled condition elicited an effective protective immune response against HIRRV, and maintaining at 20 °C for a long enough time would allow the HIRRV-infected flounder to eliminate the virus completely and acquired a protective immunity against HIRRV infection. This is the first study showing the possibility of developing an effective preventive measure against HIRRV by vaccination with live virus under controlled water temperature., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The efficacy of DNA vaccine is associated closely with the expression of the antigen and the intensity of local immune responses. In our previous study, a recombinant DNA plasmid expressing the VAA protein (pVAA) of Listonella anguillarum has been proved to have a good protection against the infection of L. anguillarum. In the present study, the local immune responses eliciting by immunizing flounder with intramuscular (I.M.) injection of pVAA was investigated at the cellular and genetic level, the muscle at the injection site at 7th post vaccination day was sampled and analyzed by hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC), flow cytometry (FCM), RNA sequencing (RNA-Seq)-based transcriptomics and RT-qPCR. Then variations on the specific antibodies in serum at 1st-6th post vaccination week and the relative percent survival rate (RPS) at following 14 days after challenge were measured. The H&E results showed that inflammatory cells and immune cells significantly increased at the injection site. The IHC using monoclonal antibody against T cell markers revealed that both CD4-1 + and CD4-2 + T lymphocytes were recruited to the injection site and FCM results showed that the proportion of CD4-1 + cells in pVAA immunized group was 28.6 %, in the control group was 8.7 %, and that of CD4-2 + cells in two groups was 21.2 % and 8.5 %, respectively. These results indicating that the proportion of CD4 + cells in the immune group was significantly increased compared with the control group. Moreover, there were 2551 genes differently expressed in pVAA immunized group, KEGG analysis showed the genes involved in the signal transduction and immune system, and surface markers for B-cells genes, T-cells and antigen presenting cells (APCs) genes were highly upregulated, suggesting the activation of the systemic immune responses. Antibody specific anti-L. anguillarum or anti-rVAA antibodies were significantly induced at 2nd post-immunization week, that reached a peak at 4-5th week. RPS in pVAA group was 53.85±3.64 %. In conclusion, pVAA induced effective local immune responses and then the systematic response. This probably is the main contribution of pVAA to effective protection against L. anguillarum., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hirame rhabdovirus (HIRRV) is one of the most important viruses of fish, posing a great threat to the fish industry in Asia and Europe. The glycoprotein (G) of HIRRV is known to play important roles in virus attachment and entry, making it an ideal target for both diagnosis and therapy. In this study, a truncated G of HIRRV was expressed as a fusion protein in Escherichia coli. Using the recombinant G protein (rG), monoclonal antibodies (mAbs) were prepared by the hybridoma technology. Subsequently, positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA) and further characterized by Western blot and immunofluorescence assay (IFA). ELISA results showed that two mAbs (3E5 and 4D10) could react with the rG, as well as the purified HIRRV. Western blot analysis showed that the mAbs belong to the IgG isotype and could recognize a 60 kDa viral protein, which is consistent with the molecular weight of G protein and determined to be the G protein of HIRRV by mass spectrometry. The virions in HIRRV-infected EPC could also be recognized by two mAbs in IFA. Moreover, neutralization assay showed that mAb 4D10 could significantly inhibit the proliferation of HIRRV and delay the development of cytopathic effect in viral-infected EPC cells, and in vivo neutralization assay also showed that mAb 4D10 could significantly reduce the mortality of HIRRV-infected flounder, indicating that mAb 4D10 can partially neutralize the HIRRV infection. Western blot analysis showed that mAb 4D10 could specifically bind the C-terminal domain of HIRRV-G protein. These results demonstrated that the produced mAbs could specifically recognize the G protein of HIRRV and displayed virus-neutralizing activity in vitro and in vivo, which could serve as effective detection probes and potential neutralizing antibodies for HIRRV., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Hirame novirhabdovirus (HIRRV), as a highly pathogenic fish virus, is frequently prevalent in a variety of aquaculture fish in recent years, which seriously threatens the healthy development of aquaculture industry. Epidemiological studies show that the outbreak of HIRRV is obviously temperature dependent. Virus proliferation experiments in vitro and in vivo at different temperatures indicate the antiviral response of flounder is a main reason affect the replication of HIRRV. The RNA-Seq was used to analyze the different antiviral response in flounder which infected with HIRRV at different temperatures, the experiment set two temperatures of 10 °C and 20 °C. The flounder infected with HIRRV by artificial injection, and the spleens were collected at 24 h after infection. Meanwhile, the fish injected with EPC supernatant at different temperatures were set as control groups. It can obtain four pairwise comparison groups if determine a single variable, and the differentially expressed genes were further selected. The results showed that after infection with HIRRV at 10 °C and 20 °C, the differentially expressed genes in spleen were significantly enriched in inflammatory and immune-related pathways like Arachidonic acid metabolism, Cytokine-cytokine receptor interaction, Toll-like receptor (TLR) signaling pathway, RIG-I-like receptor (RLRs) signaling pathway, NOD-like receptor (NLR) signaling pathway and Cytosolic DNA-sensing pathway etc. In addition, the expression of phagocytes, lysosomes, endocytosis related genes were significantly upregulated at high temperature whether HIRRV positive or not. But compared to the infected flounder at 10 °C, some genes of RLRs signaling pathway were significantly upregulated at 20 °C, it can be speculated that RLRs pathway may be related to the anti-HIRRV response of flounder. Therefore, key genes of RLRs signaling pathway including mda5, lgp2, mita, mavs, irf3, irf7, ifn I-3 and ifn-γ were selected, and the temporal expression patterns of these genes in infected flounder at different temperatures were further detected by qRT-PCR. The results showed that HIRRV infection can significantly stimulate and activate the RLRs pathway of flounder, and the response level of this pathway was significantly higher at 20 °C than 10 °C. In general, this study provides important data for the further study about the pathogenesis of HIRRV infection in flounder., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The magnitude of the immune response induced by DNA vaccines depends on the amount and type of antigen-presenting cells attracted to the injection site. In our previous study, a DNA plasmid encoding the VAA gene of Vibrio anguillarum was constructed and shown to confer moderate protection against V. anguillarum challenge. To augment the protective efficacy of the VAA DNA vaccine and compare the adjuvant effects of CCL3, CCL4, CCL19 and CCL21, four bicistronic DNA plasmids containing the VAA gene of V. anguillarum together with the gene encoding the CCL3/CCL4/CCL19/CCL21 chemokines of flounder were successfully constructed and administered to fish, and the immune response of the animals and the enhancement of immunoprotection by the four chemokines were investigated. Vaccinated with pCCL3-VAA, pCCL4-VAA, pCCL19-VAA and pCCL21-VAA, flounder showed relative percent survivals of 62.16%, 83.78%, 78.38% and 72.97%, respectively, higher than the relative survival of flounder immunized with pVAA (40.54%). Compared with the pVAA group, the percentages of sIgM + , CD4-1 + , and CD4-2 + lymphocytes and the levels of specific antibodies increased in pCCL3-VAA, pCCL4-VAA, pCCL19-VAA and pCCL21-VAA injection groups; CCL4 and CCL19 induced significantly higher levels of these parameters than CCL3 and CCL21 did. The amount of V. anguillarum in liver, spleen and kidney of pCCL3-VAA-, pCCL4-VAA-, pCCL19-VAA- and pCCL21-VAA-immunized flounder after V. anguillarum challenge was reduced compared to that in the pVAA group. Moreover, the co-expression of CCL3/CCL4/CCL19/CCL21 up-regulated immune-related gene expression associated with the local immune response. Our results indicate that CCL4 and CCL19 are promising adjuvants for use in VAA DNA vaccine against V. anguillarum., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In our previous study, a DNA plasmid encoding the VAA gene of Vibrio anguillarum was constructed and demonstrated to confer moderated protection against V. anguillarum challenge. Here, a bicistronic DNA vaccine (pVAA-IRES-IL2), co-expressing the VAA gene of V. anguillarum and Interleukin-2 (IL2) gene of flounder, was constructed to increase the protective efficacy of VAA DNA vaccine. The potential of pVAA-IRES-IL2 to express both VAA and IL2 in transfected HINAE cell lines was confirmed by immunofluorescence assay. Further, the variation of sIgM + , CD4-1 + , CD4-2 + lymphocytes and production of VAA-specific antibodies in flounder, which was intramuscularly immunized with three DNA plasmids (pIRES, pVAA-IRES, pVAA-IRES-IL2), were investigated, respectively. The bacterial burden and relative percentage survival (RPS) of flounder exposed to V. anguillarum infection were both analyzed to evaluate the efficacy of bicistronic DNA plasmid. Our results revealed that the percentages of sIgM + , CD4-1 + , CD4-2 + lymphocytes and antibodies specific to VAA were remarkably increased in pVAA-IRES or pVAA-IRES-IL2 immunized fish. Moreover, the co-expression of IL2 enhanced the immune response in response to VAA DNA vaccination, as shown by the higher percentages of sIgM + , CD4-1 + , CD4-2 + lymphocytes and production of specific antibody. Importantly, the RPS in pVAA-IRES-IL2 and pVAA-IRES groups reached 64.1% and 51.3%, respectively, when compared with the 97.5% cumulative mortality in pIRES group. Furthermore, the number of V. anguillarum in liver, spleen and kidney of pVAA-IRES or pVAA-IRES-IL2 immunized flounder after V. anguillarum challenge was significantly reduced, as compared to that in pIRES group. These suggest that the bicistronic DNA vaccine can be an effective immunization strategy in inducing immune response against V. anguillarum infection and IL2 has the potential as the adjuvant for VAA DNA vaccine., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Interleukin-2 (IL-2) is mainly produced by CD4 + T helper lymphocytes, which is an important immunomodulatory cytokine that primarily promotes activation, proliferation and differentiation of T cells. In the present study, flounder (Paralichthys olivaceus) interleukin 2 homologue (poIL-2) was identified for the first time, and its expression patterns were characterized in healthy, virus- or bacteria-infected flounder. The full-length cDNA sequences of poIL-2 was 989 bp with an open reading frame of 423 bp coding a polypeptide of 140 amino acids (aa). The deduced aa sequences shared low similarities (<53%) with other known fish IL-2s. Multiple alignment of aa sequences revealed that poIL-2 own the classical IL-2 family signature of "C-X(3)-EL-X(2)-(T/V)-(V/M/L)-(K/T/R)-X-EC" and "DS-X-(F/L)Y(A/T/S)P". In healthy flounder, IL-2 mRNA was highly expressed in PBLs, spleen and hindgut, and moderately expressed in gill, trunk kidney and stomach. PHA, LPS and Con-A could effectively induce poIL-2 expression in primary cultured peripheral blood leukocytes in vitro. poIL-2 transcripts were significantly up-regulated in spleen, kidney, gill and hindgut post infections with Edwardsiella tarda and Hirame novirhabdovirus (HIRRV). The eukaryotic expression vector encoding poIL-2 (pcIL-2) was constructed and intramuscularly injected, which could be successfully expressed in flounders and induced significantly higher expressions of six immune related genes including poIL-2, β-defensin, CD4-1, CD8α, IFN-γ and TNF-α compared with the injection with control plasmid. Moreover, pretreatment with pcIL-2 could markedly increase the survival rate of flounder challenged with HIRRV. Our results demonstrated that poIL-2 plays an important role in the induction of immune responses and immune defense against bacterial and virus infection, which indicated its potential use as an immunopotentiator to prevent diseases in flounder., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Hirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV., Results: Glycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0 × 10 9 cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week (p < 0.05). Meanwhile, oral immunization with Ll:pSLC-G could provide 60.7% protection against HIRRV infection and a significantly lower virus load was detected than the controls on the third day post-challenge (p < 0.01). Moreover, on the first day post 1-week feeding, approximately 10 4 -10 5 recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 10 2 -10 3 L. lactis cells could still be recovered., Conclusions: HIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.

In order to investigate the dynamic distribution of antigen in different tissues post vaccination, an absolute real-time quantitative PCR was employed to detect the amount of antigen in flounder (Paralichthys olivaceus) post intraperitoneal (i.p.) injection with three concentrations (10 7 , 10 8 , 10 9  CFU ml -1 ) of formalin-inactivated Edwardsiella tarda bacterin. The results showed that the amount of uptaken antigen quickly increased and then decreased in different tissues. The peak occurred first in the spleen and head kidney at 6-9 h after injection, and in the liver and blood at 9-15 h, then in the gill, intestine and skin at 15-24 h, finally in the muscle at 24-36 h. The amount of antigen was highest in the spleen and head kidney, followed by the blood, liver and gill, and lowest in the intestine, skin and muscle. Among the three concentration groups, the amount of antigen increased with the increasing concentration of the vaccine in the blood, liver, gill, intestine, skin and muscle, except for the spleen and head kidney, in which more antigens were found in the 10 8  CFU ml -1 group than that in 10 9  CFU ml -1 group. Moreover, IIFA and western blotting was performed to examine the tissue distribution of antigen at 9 h after vaccination with 10 8  CFU ml -1 formalin-inactivated E. tarda. The bacteria were mainly observed in the spleen and head kidney, then the liver, gill and blood, and least in the intestine, skin and muscle, which was roughly in accordance with the results of absolute qPCR. Furthermore, the expressions of CD4-1, MHC IIα, CD8α and MHC Iα in different tissues were detected by RT-qPCR, and the expression levels of these genes were highest in the spleen and head kidney, then in the blood, gill, liver, and lowest in the intestine, skin and muscle. All these results provided useful information for dynamic transportation of antigen uptake post vaccination, and also deepened the understanding of immune response to the injection vaccination., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In the present work, the polymeric immunoglobulin receptor-like (pIgRL) from flounder (Paralichthys olivaceus) was firstly cloned and identified. The full length cDNA of flounder pIgRL was of 1393 bp including an open reading frame of 1053 bp, and the deduced pIgRL sequence encoded 350 amino acids, with a predicted molecular mass of 39 kDa. There were two immunoglobulin-like domains in flounder pIgRL. In healthy flounder, the transcriptional level of pIgRL was detected in different tissues by real-time PCR, showing the highest level in the skin and gills, and higher levels in the spleen and hindgut. After flounders were vaccinated with inactivated Vibrio anguillarum via intraperitoneal injection and immersion, the pIgRL mRNA level increased firstly and then declined in all tested tissues during 48 h, and the maximum expression levels in the gills, skin, spleen and hindgut in immersion group, or in the spleen, head kidney, skin and gills in injection group, were higher than in other tested tissues. In addition, recombinant protein of the extracellular region of flounder pIgRL was expressed in Escherichia coli BL21 (DE3), and rabbit anti-pIgRL polyclonal antibodies were prepared, which specifically reacted with the recombinant pIgRL, and a 39 kDa protein confirmed as natural pIgRL by liquid chromatography-mass spectrometry in skin mucus of flounder. Co-immunoprecipitation assay and western-blotting demonstrated that the pIgRL, together with IgM, could be immunoprecipitated by anti-pIgRL antibody in gut, skin and gill mucus of flounder, suggesting the existence of pIgRL-IgM complexes. These results indicated that the flounder pIgRL was probably involved in the mucosal IgM transportation and played important roles in mucosal immunity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Outer membrane protein K (OmpK) is an immunogenic protein that could act as subunit vaccine candidate for Vibrio anguillarum. In this study, a DNA vaccine encoding the OmpK gene of V. anguillarum was constructed and confirmed to express OmpK in vitro and in vivo. To evaluate the potential of pcDNA3.1-OmpK (pOmpK) as vaccine candidate, the humoral and cellular immune responses, and protective effects were analyzed in flounder model. The results showed that the transcription and translation of OmpK gene occurred in both transfected hirame natural embryo (HINAE) cells and injected fish muscles, indicating the functionality of pOmpK to express OmpK. Fish immunized with pOmpK showed significant increase of surface IgM positive (sIgM + ), CD4-1 + , CD4-2 + lymphocytes and production of specific anti-V. anguillarum or anti-rOmpK antibodies, which indicate the activation of humoral and cellular immune responses after vaccination. Moreover, a relative percent survival (RPS) rate of 50.00% against V. anguillarum infection was obtained for flounder immunized with pOmpK. In conclusion, this study indicates that pOmpK is able to induce humoral and cellular immune responses and can be used as a DNA vaccine candidate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

CD40 is a crucial signal mediating factor in T-dependent B cell responses and involved in many aspects of cellular and humoral immunity. In this paper, recombinant protein of CD40 in flounder (Paralichthys olivaceus) and its antibodies (Abs) were produced, native CD40 molecules in flounder tissues were identified, then the CD40 + leukocytes in T/B lymphocytes were characterized, and the variations of CD40 + leukocytes in flounder after Hirame novirhabdovirus (HIRRV) infection and immunization were investigated, respectively. The results showed that the Abs could specifically recognize native flounder CD40 molecule at 32 kDa. The proportions of CD40 + leukocytes were varied by flounder tissues. CD40 + /IgM + B lymphocytes, CD40 + /CD4-1 + T lymphocytes, CD40 + /CD4-2 + T lymphocytes and CD40 + /CD8 + T lymphocytes in peripheral blood leukocytes (PBLs) were 1.18 ± 0.27%, 0.69 ± 0.17%, 0.75 ± 0.14% and 0.25 ± 0.14%; were 2.80 ± 0.32%, 0.71 ± 0.19%, 0.88 ± 0.23% and 0.33 ± 0.17% in spleen; 4.11 ± 0.47%, 0.92 ± 0.18%, 1.09 ± 0.17% and 0.9 ± 0.17% in head kidney; 1.92 ± 0.39%, 1.02 ± 0.23%, 1.33 ± 0.38% and 0.67 ± 0.24% in intestine; 1.24 ± 0.36%, 1.21 ± 0.24%, 1.70 ± 0.3% and 0.97 ± 0.21% in gill, respectively. The percentages of CD40 + leukocytes in PBLs were significantly increased in both HIRRV infection and immunization groups, and reached their peak levels at 3rd day with 5.70 ± 0.16% and 6.40 ± 0.13%, respectively. Concluded with our previous study, these data first reported that CD40 molecules were expressed on both B and T lymphocytes in teleost, and had a coordination with T and B lymphocytes in immune responses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aims: The aim of this study was to screen vaccine candidates from virulence factors of Streptococcus iniae in flounder model., Methods and Results: The immunogenicity of recombinant phosphoglucomutase (rPGM) and rCAMP factor was confirmed by Western blot. The percentage of surface membrane immunoglobulin-positive (sIg + ) lymphocytes in peripheral blood leucocytes, the specific and total serum IgM and the activity of acid phosphatase (ACP) and peroxidase (POD) in flounder were determined with flow cytometry, ELISA and commercial enzyme activity kits, respectively, after intraperitoneal immunization with rPGM and rCAMP factor. The results showed that rPGM and rCAMP factor could induce significant rise in sIg + lymphocytes, specific serum IgM and activities of ACP and POD. Additionally, the relative percent survival rate of the vaccinated flounder was 64 and 54% in challenge experiment using S. iniae, respectively. These results indicated that rPGM and rCAMP factor could evoke humoural and innate immune response in flounder and provide high-efficiency immunoprotection against S. iniae infection., Conclusions: Phosphoglucomutase (PGM) and CAMP factor were promising vaccine candidates against S. iniae in flounder., Significance and Impact of the Study: Phosphoglucomutase and CAMP factor have the potential to be vaccine candidates, which provide important information for us to develop the effective subunit vaccines, especially the multivaccine, against S .iniae in aquaculture., (© 2018 The Society for Applied Microbiology.)

Outer membrane protein (Omp) K, OmpR and VAA have been identified with good immunogenicity from Vibrio anguillarum, and their recombinant proteins showed variable relative percent survival (RPS) in previous study. In order to develop effective bivalent vaccine candidates, recombinant (r) VAA + rOmpK (AK), rVAA + rOmpR (AR), rOmpK + rOmpR (KR) among VAA, OmpK and OmpR, or formalin-killed cells (FKC) of V. anguillarum were immunized in flounder, respectively. Results revealed that AK, AR, KR and FKC could induce the proliferation of surface membrane immunoglobulin-positive B lymphocytes or CD3 + T lymphocytes in peripheral blood lymphocytes, and significantly enhance the total antibodies, specific antibodies and immune-related gene than those of control group. AK, AR, KR or FKC showed RPS of 74.92%, 78.49%, 82.09% and 56.99%, respectively. These results indicated that three bivalent vaccines AK, AR and KR could induce strong cellular and humoral immunity, and had high protection against V. anguillarum infection in flounders., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Hirame novirhabdovirus (HIRRV) causes severe disease in fish cultures, resulting in great economic loss in Asia and Europe. In this study, the matrix protein (M) of HIRRV was recombinantly expressed as the immunogen to produce monoclonal antibodies (MAbs) using hybridoma cell fusion technology, and 3 MAbs were produced and characterized by indirect ELISA, Western blotting and immunofluorescence assay (IFA). Western blotting and mass spectrometric analysis showed that the MAbs could specifically react with the nature M protein of HIRRV. The MAbs were employed to detect virions in HIRRV-infected epithelioma papulosum cyprini (EPC) cells and flounder Paralichthys olivaceus by IFA and immunohistochemistry (IHC). In the virus-infected EPC cells, the virions were mainly located in the cytoplasm, whereas in flounder, HIRRV was present in all 10 tested tissues, and the positive signals in spleen, head-kidney and heart were higher than in other tissues, consistent with the results obtained by RT-PCR. Moreover, strong positive signals were observed in the endothelial cells of blood vessels, but only the leukocytes were infected by HIRRV in the whole blood cells. These results indicate that the high susceptibility to HIRRV of leukocytes and endothelial cells may facilitate the spread of HIRRV and finally cause systemic infection in flounder. This study provides a foundation for further studies on rapid diagnosis of HIRRV and its infection mechanisms.

Cytokines play vital roles in mounting immune responses and activating host defense network. In this study, the expression plasmid pcDNA3.1 (pcN3) encoding four flounder (Paralichthys olivaceus) cytokines including IL-1β, TNF-α, IL-8 or G-CSF (pcIL-1β, pcTNF-α, pcIL-8 and pcG-CSF) were successfully constructed, and their adjuvant potential on an Edwardsiella tarda (E. tarda) subunit vaccine OmpV (rOmpV) were comparatively analyzed in vaccinated flounder model. Results revealed that flounder vaccinated with rOmpV plus pcIL-1β, pcIL-8 or pcG-CSF produced the relative percent survivals (RPS) of 71%, 65% and 49% respectively, which were higher than that in flounder vaccinated with rOmpV plus pcTNF-α (39%) or pcN3 (36%, the control group). Immunological analysis showed that: (1) except pcTNF-α, higher levels of anti-E. tarda serum antibodies and sIg + lymphocytes in spleen, head kidney and peripheral blood were significantly enhanced by pcIL-1β, pcIL-8 or pcG-CSF, however, pcIL-8 and pcIL-1β enhanced higher levels of sIg + lymphocytes and anti-E. tarda antibodies than pcG-CSF; (2) pcTNF-α could promote the up-regulation of genes participated in cellular immunity (MHCIα, IFN-γ, CD8α and CD8β), pcIL-1β could enhance the expression of genes related to humoral immunity (CD4-1, CD4-2, MHCIIα and IgM), and all the detected genes were augmented by pcIL-8 and pcG-CSF; Among the four cytokines, pcIL-8 and pcIL-1β could strengthen the highest levels of genes participated in cellular immunity and humoral immunity, respectively. These results demonstrated that pcIL-8 and pcIL-1β could enhance stronger cellular and/or humoral immunity induced by rOmpV than pcG-CSF and pcTNF-α, and evoked higher RPS against E. tarda challenge in flounder, which indicated that pcIL-8 and pcIL-1β are promising adjuvants of vaccines in controlling E. tarda infection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Streptococcus iniae is a major Gram-positive pathogen that causes invasive disease in fish worldwide. In this study, in order to identify immunogenic proteins for developing highly effective vaccine against S. iniae, whole-cell lysate proteins of S. iniae were analyzed by western blotting using flounder anti-S. iniae antibodies, and two positive protein bands of molecular weight 37 kDa and 40 kDa were screened, which were identified as pyruvate dehydrogenase E1 subunit alpha (PDHA1), BMP family ABC transporter substrate-binding protein (BMP) and L-lactate dehydrogenase (LDH), as well as ornithine carbamoyltransferase (OCT), lactate oxidas (LOx) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by mass spectrometry. Subsequently, the six recombinant proteins were produced and used to immunize healthy flounder, and the relative percent survival (RPS) value was 72.73%, 27.27%, 36.36%, 9.09%, 36.36% and 63.64% respectively after intraperitoneal challenge with live S. iniae, revealing that rPDHA1 and rGAPDH produced higher relative percent survival than formalin-killed S. iniae (36.36%). To further investigate the protective efficacy of rPDHA1 and rGAPDH, the proliferation of surface membrane immunoglobulin-positive (sIg+) lymphocytes in peripheral blood leucocytes, the total serum IgM, specific IgM against S. iniae and RPS were detected. The results showed that rPDHA1, rGAPDH and formalin-killed S. iniae significantly induced the proliferation of sIg+ lymphocytes, the production of total serum IgM and specific IgM as compared with the control group, and rGAPDH and rPDHA1 provide higher RPS (62.5% and 75%, respectively) again. These results demonstrated that rPDHA1 and rGAPDH are promising vaccine candidates against S. iniae infection in flounder., Competing Interests: The authors have declared that no competing interests exist.

T and B lymphocytes are closely related to immunization and pathogen infection. Our previous study confirmed the CD3 + , CD4-1 + , CD4-2 + , CD8β + T lymphocytes and IgM + B lymphocytes presented in the peripheral blood leukocytes (PBLs) of flounder (Paralichthys olivaceus), in this paper, the variations of T and B lymphocytes of flounder after Hirame novirhabdovirus (HIRRV) infection or immunization were investigated. The flounders were injected with live or inactivated HIRRV, then the percentages of T and B lymphocytes in PBLs were analyzed by Flow cytometry (FCM), total antibodies and HIRRV-specific antibodies in serum were detected by Enzyme-linked immunosorbent assay (ELISA), and expression of twelve immune-related genes in the head kidneys were determined using q-PCR. The results showed that the percentages of CD3 + , CD4-1 + , CD4-2 + , CD8β + T lymphocytes and IgM + B lymphocytes significantly increased in both infection and immunization groups, in infection group they decreased rapidly after the peak and significantly lower than control levels at the end of infection, in immunization group they went down steadily to the control levels at the end of immunization. The total antibodies and HIRRV-specific antibodies increased first and peaked on the 7 th day post infection and on the 14 th day post immunization, respectively, then gradually decreased to the control levels. Additionally, twelve immune-related genes were up-regulated in both groups. These results demonstrated that the HIRRV induced both humoral and cellular immunity of flounder, the lymphocytes varied more sharply in infection group than those in immunization group and CD8 + T lymphocytes responded much more than CD4 + T lymphocytes to HIRRV antigen., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Vibrio anguillarum and Edwardsiella tarda are severe aquaculture pathogens shared similar epidemiological characteristics and susceptible to flounder (Paralichthys olivaceus). In our previous studies, recombinant(r) protein heat shock protein 33 (rHsp33) from V. anguillarum and outer membrane protein C (rOmpC) from E. tarda were proved to have protection against V. anguillarum and E. tarda, respectively. In this paper, the cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, and the protection of divalent vaccine candidate (rHsp33 + rOmpC, rHC) against both V. anguillarum and E. tarda were evaluated. RHC, rHsp33, and rOmpC were vaccinated to flounder, respectively, and the percentages of surface immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBLs), serum IgM, specific antibodies against V. anguillarum or E. tarda, specific antibodies against rHsp33, rOmpC or rHC, the expression of immune-related genes and relative percent survival (RPS) against V. anguillarum or E. tarda were measured. The results showed that: RHC could induced the enhancement of sIg + cells and high levels of specific antibodies against both V. anguillarm and E. tarda; Also a significant increase of specific antibodies against rHsp33, rOmpC or rHC, and up-regulation of gene expression of CD3, CD4-1, CD4-2, CD8α, CD8β and IgM in spleen, head-kidney, and hindgut, RPS of 70 ± 3.45% against V. anguillarum and 60 ± 1.48% against E. tarda, respectively. In addition, rHsp33 induced specific antibodies against E. tarda and rOmpC, and had a RPS of 43.3 ± 3.73% against E. tarda; rOmpC could evoke specific antibodies against V. anguillarum and rHsp33, and had a RPS of 44 ± 1.27% against V. anguillarm; The results demonstrated that there was cross protection of rHsp33 against E. tarda and rOmpC against V. anguillarum, rHC as a divalent vaccine can induce significant immune response and efficient protection against both E. tarda and V. anguillarum in flounder., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In our previous study, ten candidate proteins have been identified with immunogenicity and protection against Vibrio anguillarum in flounder (Paralichthys olivaceus). Among them, FlaC is the important outer protein in the flagellum with immunogenicity; VAA, OmpK and OmpR are protection proteins against V. anguillarum. In this paper, FlaC supplemented with VAA, OmpK or OmpR as bivalent subunit vaccine candidates, and their immune response of flounder and protective effects were evaluated, respectively. Recombinant(r) proteins of FlaC were mixed with rVAA, rOmpK and rOmpR, respectively, rVAA + rFlaC (AF), rOmpK + rFlaC (KF) and rOmpR + rFlaC (RF); formalin-killed cells (FKC) or PBS were injected to flounder, respectively. After immunization, the percentages of CD3 + T lymphocytes and surface membrane immunoglobulin-positive (sIg + ) B lymphocytes in peripheral blood lymphocytes (PBLs), total antibodies (TA), specific antibodies against V. anguillarum (VA), specific antibodies against bivalent recombinant proteins (PA), the expression of immune-related genes and relative percent survivals (RPS) were measured, respectively. The results showed that three bivalent vaccines candidates and FKC could induce the proliferation of sIg + B lymphocytes and CD3 + T lymphocytes in PBLs. The TA, VA and PA induced in bivalent vaccines candidates and FKC groups were significantly higher than that of the control group. CD3, IgM, CD4-1, CD4-2, CD8α and CD8β genes were up-regulated. After challenge with V. anguillarum, RPS in AF, KF, RF and FKC groups exhibited 62.6 ± 2.33%, 78.95 ± 3.01%, 75.45 ± 0.97%, and 56.71 ± 2.15% respectively. The results revealed that three bivalent vaccines candidates and FKC could induce the immune response in flounder, and have good protection against V. anguillarum, and KF can be an efficient bivalent subunit vaccine candidate., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lymphocystis disease virus (LCDV) infection may induce a variety of host gene expression changes associated with disease development; however, our understanding of the molecular mechanisms underlying host-virus interactions is limited. In this study, RNA sequencing (RNA-seq) was employed to investigate differentially expressed genes (DEGs) in the gill of the flounder ( Paralichthys olivaceus ) at one week post LCDV infection. Transcriptome sequencing of the gill with and without LCDV infection was performed using the Illumina HiSeq 2500 platform. In total, RNA-seq analysis generated 193,225,170 clean reads aligned with 106,293 unigenes. Among them, 1812 genes were up-regulated and 1626 genes were down-regulated after LCDV infection. The DEGs related to cellular process and metabolism occupied the dominant position involved in the LCDV infection. A further function analysis demonstrated that the genes related to inflammation, the ubiquitin-proteasome pathway, cell proliferation, apoptosis, tumor formation, and anti-viral defense showed a differential expression. Several DEGs including β actin , toll-like receptors, cytokine-related genes, antiviral related genes, and apoptosis related genes were involved in LCDV entry and immune response. In addition, RNA-seq data was validated by quantitative real-time PCR. For the first time, the comprehensive gene expression study provided valuable insights into the host-pathogen interaction between flounder and LCDV., Competing Interests: The authors declare no conflict of interest.

Vibrio ichthyoenteri was an important causative agent of bacterial enteritis in flounder (Paralichthys olivaceus). Outer membrane protein A (OmpA) of Gram-negative pathogen was a major cell surface antigen. In the present study, OmpA of V. ichthyoenteri was recombinantly expressed in Escherichia coli, and the immunogenicity of OmpA was identified by western blotting using flounder anti-rOmpA and anti-V. ichthyoenteri antibodies. The vaccine potential of rOmpA was tested in a flounder model, and a high relative percentage of survival rate was obtained with 73.1% after challenge with V. ichthyoenteri. Meanwhile, the immune response of flounder induced by rOmpA was also investigated, and the results showed that the sIg + lymphocytes in blood, spleen, and pronephros significantly proliferated, and the peak levels occurred at week 4 after immunization. Moreover, rOmpA could induce higher levels of specific serum antibodies than the control group after immunization, and the peak level occurred at week 5 after immunization. Meanwhile, qRT-PCR analysis showed that the expressions of CD4-1, CD8α, IL-1β, IFN-γ, MHCIα and MHCIIα genes were significantly up-regulated after immunization with rOmpA. Taking together, these results demonstrated that rOmpA could evoke highly protective effects against V. ichthyoenteri challenge and induce strong immune response of flounder, which indicated that OmpA was a promising vaccine candidate., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Hirame rhabdovirus (HIRRV) is a rhabdovirus that causes severe disease in fish. The mortality due to HIRRV infection occurs at temperatures below 15 °C, but no mortality is observed over 20 °C. In this study, Japanese flounder (Paralichthys olivaceus) was artificially infected with the HIRRV CNPo2015 strain at 10 °C or 20 °C. Absolute quantitative real-time PCR was employed to examine the viral replication in spleens after HIRRV infection. Expression profiles of four interferon-related genes (type I IFN, Mx, ISG15, MDA5) and two proinflammatory genes (TNF-α and IL-1β) were also investigated by quantitative real-time PCR. Results showed that viral copies in spleens increased gradually over time and peaked at 72 h post infection (hpi) in the 10 °C group, while viral copies in the 20 °C group increased within 24 hpi, but afterwards decreased to very low levels. Moreover, the expressions of IFNs in the 10 °C group reached the highest levels at 72 hpi, whereas their peak levels appeared much earlier in the 20 °C group, at 12 hpi. The expression levels of TNF-α and IL-1β in the 10 °C group peaked at 12 hpi and then quickly declined. However, the two genes were highly expressed during 6-24 hpi in the 20 °C group. Based on these findings, we concluded that HIRRV infection induced an efficient antiviral immune response at 20 °C, which might inhibit the viral transcription at early stages and finally prevent HIRRV infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Interleukin-6 (IL-6) as a pleiotropic cytokine was widely used as an effective adjuvant for vaccines in mammals. In this study, the immune adjuvant effects of two forms of flounder ( Paralichthys olivaceus ) IL-6, including recombinant IL-6 (rIL-6) and pcDNA3.1-IL-6 (pcIL-6), were evaluated and comparatively analyzed on E. tarda subunit vaccine recombinant outer membrane protein V (rOmpV). The results showed that the relative percent survivals of flounder vaccinated with rOmpV plus rIL-6 or pcIL-6 were significantly higher than that in the two control groups, rOmpV plus recombinant 6× histidine-tag (rHis) or empty expression vector pcDNA3.1 (pcN3). The levels of specific serum antibodies and surface membrane immunoglobulin-positive (sIg+) lymphocytes in peripheral blood, spleen, and head kidney in the two adjuvant groups were also much higher than that in the two control groups. Compared with the two control groups, higher upregulated expressions of major histocompatibility complex class Iα ( MHCIα ), cluster of differentiation 8α ( CD8α ), MHCIIα , CD4-1 , interleukin-1β ( IL-1β ), and tumor necrosis factor-α ( TNF-α ) were detected in flounder vaccinated with rOmpV plus rIL-6 or pcIL-6 after challenge. In addition, the rOmpV plus rIL-6 could induce significant higher levels of specific serum antibodies, sIg+ lymphocytes and four genes expressions than rOmpV plus pcIL-6. These results demonstrated that both rIL-6 and pcIL-6 used as adjuvants could enhance the immune response and evoke immune protections against E. tarda infection, which has a significant value in controlling diseases using vaccines in flounder., Competing Interests: The authors declare no conflicts of interest.

Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. In this paper, expression profiles of the turbot (Scophthalmus maximus) TLR22 gene (tbTLR22) were analyzed with RT-PCR and in situ hybridization. Then its expression patterns simulated with ligands or pathogens were investigated. Streptococcus iniae, Edwardsiella tarda, Hirame rhabdovirus virus (HIRRV), polyinosinic: polycytidylic acid (Poly I:C), peptidoglycan (PGN), or lipopolysaccharides (LPS) was injected to turbot; poly I:C, PGN, or LPS was added into cultured peripheral blood leukocytes (PBL); and then the tbTLR22 in liver, spleen, gill, kidney and cultured PBL was measured using Quantitative PCR. The recombinant protein of tbTLR22 (rp-tbTLR22) and its antibody were produced, then the reactions of antibody to tissues were detected by Western-blotting, and the binding of rp-tbTLR22 to all the stimulants was detected using ELISA. The results showed tbTLR22 expression was significantly up-regulated by PolyI: C, but no significant change in PGN and LPS groups; tbTLR22 significantly increased in liver and spleen after S. iniae infection with the maximum of 3.6 times and 3.3 times; in liver and kidney after E. tarda infection with the maximum of 3.4 times and 4.1 times; and then in gill and kidney after HIRRV infection by 4.8 and 4.1 times. Rp-tbTLR22 antibody could recognize the total protein from liver, kidney, gill and spleen at 40 kDa, 90 kDa and 120 kDa, respectively. The rp-tbTLR22 could bind to three ligands and pathogens in vitro. The expression and reaction data gave a clear recognization model of tbTLR22., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vibrio anguillarum is a severe bacterium that causes terminal haemorrhagic septicaemia in freshwater and marine fish. Virulence-associated proteins play an important role in bacterial pathogenicity and could be applied for immunoprophylaxis. In this study, six antigenic proteins from V. anguillarum were selected and the immune protective efficacy of their recombinant proteins was investigated. VirA, CheR, FlaC, OmpK, OmpR and Hsp33 were recombinantly produced and the reactions of recombinant proteins to flounder-anti-V. anguillarum antibodies (fV-ab) were detected, respectively. Then the recombinant proteins were injected to fish, after immunization, the percentages of surface membrane immunoglobulin-positive (sIg+) cell in lymphocytes, total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were analyzed, respectively. The results showed that all the recombinant proteins could react to fV-ab, proliferate sIg + cells in lymphocytes and induce production of total antibodies, specific antibodies against V. anguillarum or the recombinant proteins; the RPS of rVirA, rCheR, rFlaC, rOmpK, rOmpR and rHsp33 against V. anguillarum was 70.27%, 27.03%, 16.22%, 62.16%, 45.95% and 81.08%, respectively. The results revealed that rHsp33, rVirA and rOmpK have good protections against V. anguillarum and could be vaccine candidates against V. anguillarum., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vibrio anguillarum is a severe bacterial pathogen that can infect a wide range of fish species. Identification of immunogenic proteins and development of vaccine are essential for disease prevention. In this study, immunogenic proteins were screened and identified from V. anguillarum, and then protective efficacy of the immunogenic proteins was evaluated. Immunogenic proteins in V. anguillarum whole cell were detected by Western blotting (WB) using immunized flounder (Paralichthys olivaceus) serum, and then identified by Mass spectrometry (MS). The recombinant proteins of four identified immunogenic proteins were produced and immunized to fish, and then percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBL), total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were measured, respectively. The results showed that five immunogenic proteins, VAA, Groel, OmpU, PteF and SpK, were identified; their recombinant proteins, rOmpU, rGroel, rSpK and rVAA, could induce the proliferation of sIg+ cells in PBL and production of total antibodies, antibodies against V. anguillarum and antibodies against the recombinant proteins; their protection against V. anguillarum showed 64.86%, 72.97%, 21.62% and 78.38% RPS, respectively. The results revealed that the immunoproteomic technique using fish anti-V. anguillarum serum provided an efficient way to screen the immunogenic protein for vaccine antigen. Moreover, the rVAA, rGroel and rOmpU had potential to be vaccine candidates against V. anguillarum infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Hirame rhabdovirus virus (HIRRV) is a rhabdovirus that causes acute hemorrhage disease in fish culture, resulting in a great economic loss in parts of Asia and Europe., Methods: In this study, we isolated a virus strain named as CNPo2015 from cultured Japanese flounder in Shandong province, China. Cell isolation, electron microscopic observation, RT-PCR detection and phylogenetic analysis were used for virus identification. Further, artificial infection experiment was conducted for virulence testing., Results: The gross signs included abdominal distension, fin reddening and yellow ascitic fluid in the abdominal cavity. Histopathological examination revealed marked cell degeneration and necrosis in the kidney. The tissue homogenates induced obvious cytopathic effects in EPC, FHM and FG cell lines. Electron microscopic observation showed the virus had a bullet-like shape with a capsule membrane. RT-PCR and sequencing analysis revealed that CNPo2015 belonged to the HIRRV with high sequence identity to HIRRV isolates. Infection experiment confirmed that the HIRRV CNPo2015 strain was virulent to flounder juveniles with a LD 50 value of 1.0 × 10 5.9 TCID 50 /fish., Conclusion: In conclusion, we described the first isolation and characterization of a HIRRV from Japanese flounder in China. This will provide a candidate material for further research on the infection mechanism and preventive strategies of HIRRV.

Outer membrane protein C is a porin that resides in the outer membrane, and it has been identified as an antigenic protein in many bacteria, such as Escherichia coli, Salmonella typhi, Aeromonas hydrophila and Edwardsiella tarda. In the present work, we aimed at evaluating the immune protective potential of E. tarda OmpC as a DNA vaccine. For this purpose, a recombinant DNA plasmid encoding OmpC (pCG-OmpC) gene was constructed and its vaccine potential was analyzed in a flounder (Paralichthys olivaceus) model. The results showed that pCG-OmpC produced a relative percent survival (RPS) of 55% following E. tarda challenge at 6 w post vaccination. Moreover, OmpC transcripts and OmpC-GFP fusion protein could be detected in flounder after immunization with pCG-OmpC, and OmpC-GFP fusion protein could also be detected in HINAE cells after transfection with pCG-OmpC. Meanwhile, the immune responses induced by pCG-OmpC were investigated, and the results showed that pCG-OmpC could significantly induce the production of specific serum antibodies and the proliferation of sIg + lymphocytes in blood, spleen and pronephros. qRT-PCR analysis showed that the expressions of TLR5M, MyD88, NF-κB, MHCIα, MHCIIα, CD4-1, CD8α, IL-1β and TNF-α genes were significantly induced after vaccinated with pCG-OmpC, while there was no significant difference in expressions of TLR2 and IL-6 between pCG-OmpC and PBS injected fish. Taking together, these results indicated that pCG-OmpC could induce strong innate, humorol and cellular immune responses of flounder, and finally evoked highly protective effects, suggesting that pCG-OmpC was a promising DNA vaccine candidate., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Our previous work has demonstrated that the immune response of Japanese flounder was associated with the concentration of formalin-inactivated Edwardsiella tarda and immersion time. In order to further investigate the influence of immersion vaccine dose and bath time on the antigen uptake, formalin-killed Edwardsiella tarda bacterin was prepared and adjusted to four concentrations (10 9 , 10 8 , 10 7 , 10 6  cfu ml -1 ) for 30, 60 and 90 min immersion in Japanese flounder model, respectively. Absolute quantitative real-time PCR was employed to examine the bacterin uptake in gill, skin, spleen and kidney at 3 and 6 h post vaccination. The results showed that the antigen uptaken in gills and skin were significant higher than spleen and kidney, and the antigen amounts in gill and skin both declined from 3 to 6 h, whereas the antigen amounts in spleen and kidney gradually increased. Significant higher antigen amounts were detected in 10 9 -30, 10 9 -60, 10 8 -60, 10 8 -90 and 10 8 -90 groups than other groups (P < 0.05), especially the 10 8 -60min group displayed the highest antigen uptaken. Meanwhile, the expression profiles of antigen recognization and presentation genes (MHCⅡα, TcRα, CD4-1), immunoglobulins (IgM, IgT), inflammatory cytokines (IL-1β, IL-6), heat shock protein 70 (HSP70) and c-type lysozyme were analyzed using real-time PCR. On the whole, the transcription levels of the eight genes exhibited to be higher in 10 7 -90, 10 8 and 10 9  cfu ml -1 groups than other groups (P < 0.05), especially the 10 8 -60 group displayed the highest up-regulation. These results demonstrated that immersion with formalin-inactivated E. tarda, especially under 10 8 -60 min condition could efficiently enhance the antigen uptake and the expression of immune-related genes, which provided evidences for an enhanced vaccination effects under an optimized combination of vaccine dose and immersion time., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the present study, molecular chaperone GroEL of Edwardsiella tarda was found both react with flounder (Paralichthys olivaceus) anti-recombinant GroEL (rGroEL) and anti-E. tarda antibodies, and it was both detected in the outer membrane and the secretome. To evaluate the vaccine potential of GroEL, pCI-neo-GFP-GroEL (pCG-GroEL) was prepared and used to vaccinate healthy flounder, and immune protective effects were investigated. The results showed that pCG-GroEL produced an RPS of 60% following E. tarda challenge at week 5 after immunization. Moreover, GroEL transcripts and GFP-tagged GroEL could be detected in vaccinated flounder after immunization with pCG-GroEL, and the GFP-tagged GroEL could be also detected in HINAE cells after transfection with pCG-GroEL. Meanwhile, the immune response of flounder induced by pCG-GroEL was investigated, and the results showed that: (1) the levels of specific serum antibodies against E. tarda induced by pCG-GroEL were significantly higher than other groups at 3-5 week post vaccination; (2) pCG-GroEL could induce the proliferation of sIg+ lymphocytes, and the levels of sIg+ lymphocytes in blood, spleen and pronephros of pCG-GroEL vaccinated fish were significantly increased compared with the PBS vaccinated fish at 3w, 2w and 2w after immunization, respectively; (3) MHCIα, MHCIIα, CD4-1, CD8α, IL-1β and TNF-α were significantly induced by immunization with pCG-GroEL, and the mRNA levels of CD4-1 and CD8α rapidly increased in pCG-GroEL vaccinated fish after challenge with E. tarda. Taking together, pCG-GroEL could elicit highly protective effects against E. tarda and induce strong immune response of flounder, suggesting that GroEL was a promising vaccine candidate., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders (Paralichthys olivaceus) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30 min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) (P < 0.05), and the highest amount of antigen was detected in flounders immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group (P > 0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine (P < 0.05) compared with the spleen, kidney and liver. Antigen uptake in the gill and skin both peaked at 30 min post immersion, which was significantly higher than the levels of uptake measured in the other tissues (P < 0.05), and then quickly declined. In contrast, antigen uptake in the spleen, kidney and liver gradually increased 3 h post immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group (P < 0.05). In the mucosal-associated tissues, the expression of MHC Iα and CD8α genes peaked at 24 hpi, while the expression of MHC IIα and CD4-1 genes showed up-regulation in the gill and skin and reached the peak in these tissues at 48 hpi. The expressions of the four genes were also up-regulated in spleen, kidney and liver, but reached peak expression in these tissues at 48-72 hpi. The results demonstrated that hyperosmotic immersion, notably 50‰ salinity significantly enhanced antigen uptake and the expression of selected genes associated with antigen presentation, providing evidence for an enhanced immune activation of the fish's immune response by the hyperosmotic immersion treatment prior to vaccination., (Copyright © 2016 Elsevier Ltd. All rights reserved.)