Protective efficacy and immune responses of largemouth bass (Micropterus salmoides) immunized with an inactivated vaccine against the viral hemorrhagic septicemia virus genotype IVa.

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to the aquaculture industry, prompting the need for effective preventive measures. Here, we developed an inactivated VHSV and revealed the molecular mechanisms underlying the host's protective response against VHSV. The vaccine was created by treating VHSV with 0.05 % formalin at 16 °C for 48 h, which was determined to be the most effective inactivation method. Compared with nonvaccinated fish, vaccinated fish exhibited a remarkable increase in survival rate (99 %) and elevated levels of serum neutralizing antibodies, indicating strong immunization. To investigate the gene changes induced by vaccination, RNA sequencing was performed on spleen samples from control and vaccinated fish 14 days after vaccination. The analysis revealed 893 differentially expressed genes (DEGs), with notable up-regulation of immune-related genes such as annexin A1a, coxsackievirus and adenovirus receptor homolog, V-set domain-containing T-cell activation inhibitor 1-like, and heat shock protein 90 alpha class A member 1 tandem duplicate 2, indicating a vigorous innate immune response. Furthermore, KEGG enrichment analysis highlighted significant enrichment of DEGs in processes related to antigen processing and presentation, necroptosis, and viral carcinogenesis. GO enrichment analysis further revealed enrichment of DEGs related to the regulation of type I interferon (IFN) production, type I IFN production, and negative regulation of viral processes. Moreover, protein-protein interaction network analysis identified central hub genes, including IRF3 and HSP90AA1.2, suggesting their crucial roles in coordinating the immune response elicited by the vaccine. These findings not only confirm the effectiveness of our vaccine formulation but also offer valuable insights into the underlying immunological mechanisms, which can be valuable for future vaccine development and disease management in the aquaculture industry.

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

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

The extracellular matrix protein EFEMP2 is involved in the response to VHSV infection in the olive flounder Paralichthys olivaceus.

The EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) is involved in connective tissue development, elastic fiber formation, and tumor growth. In this study, we characterized the cDNA of EFEMP2 (PoEFEMP2), a member of the fibulin family of ECM proteins, in the olive flounder Paralichthys olivaceus. The coding region of PoEFEMP2 encodes a protein that contains six calcium-binding EGF-like (EGF-CA) domains and four complement Clr-like EGF-like (cEGF) domains. PoEFEMP2 shows 67.51-96.77 % similarities to orthologs in a variety of fish species. PoEFEMP2 mRNA was detected in all tissues examined; the highest levels of PoEFEMP2 mRNA expression were observed in the heart, testis, ovary and muscle. The PoEFEMP2 mRNA level increases during early development. In addition, the PoEFEMP2 mRNA level increased at 3 h post-infection (hpi) and decreased from 6 to 48 hpi in flounder Hirame natural embryo (HINAE) cells infected with viral hemorrhagic septicemia virus (VHSV). Disruption of PoEFEMP2 using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system resulted in a significant upregulation of VHSV G mRNA levels and immune-related genes expression in knockout cells. These findings implicate PoEFEMP2 in antiviral responses in P. olivaceus.

A single amino acid variation of NV protein of viral hemorrhagic septicemia virus increases protein stability and decreases immune gene expression.

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV-S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.

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

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

Kinetics of CD4-1+ lymphocytes in brown trout after exposure to viral haemorrhagic septicaemia virus.

T-helper cells express CD4 as a co-receptor that binds to major histocompatibility complex class II to synchronize the immune response against upcoming threats via mediating several cytokines. We have previously reported the presence of CD4 homologues in brown trout. The study of cellular immune responses in brown trout is limited by the availability of specific antibodies. We here describe the generation of a polyclonal antibody against CD4-1 that allows for the investigation of CD4+ cells. We used this novel tool to study CD4+ cells in different tissues during viral haemorrhagic septicaemia infection (VHSV) using flow cytometric technique. Flow cytometric analyses revealed an enhanced level of surface CD4-1 expression in the infected group in major lymphoid organs and in the intestine. These results suggest an important role for the T-helper cells within the immune response against viruses, comparable to the immune response in higher vertebrates.

Generation of VHSV replicon particles carrying transmembrane and C-terminal cytoplasmic region-deleted G gene (rVHSV-GΔTM) and comparison of vaccine efficacy with G gene-deleted VHSV (rVHSV-ΔG).

Vaccines based on viral replicon particles would be advantageous to induce immune responses compared to inactivated viruses in that they can infect host cells (only once) and can produce viral proteins in the infected cells like live viruses. Furthermore, as viral replicon particles are replication-defective, they are safer than live attenuated viruses. Previously, we had rescued viral hemorrhagic septicemia virus (VHSV) replicon particles lacking full ORF of G gene (rVHSV-ΔG). In the present study, to enhance the immunogenicity of VHSV replicon particles, we newly generated another form of VHSV replicon particles that can produce the transmembrane and C-terminal cytoplasmic region-deleted G protein in host cells (rVHSV-GΔTM), and compared the protective efficacy of rVHSV-GΔTM with that of rVHSV-ΔG through immunization of olive flounder (Paralichthys olivaceus). In addition, we evaluated the safety of rVHSV-GΔTM by the analysis of effects on wild-type VHSV replication. In the vaccine experiment, olive flounder immunized with rVHSV-GΔTM showed significantly higher titers of serum neutralization activity than fish immunized with rVHSV-ΔG suggesting that the G protein that is not only spiked on the viral envelop but also secreted extracellularly can contribute to the enhancement of adaptive humoral immunity. Moreover, fish immunized with rVHSV-GΔTM showed higher survival rates than fish immunized with rVHSV-ΔG, suggesting that the amount of G protein provided to hosts is an important factor for the enhancement of vaccine efficacy against VHSV disease. In a safety aspect, rVHSV-GΔTM could not replicate in infected cells, and significantly inhibited the replication of wild-type VHSV when co-infected, suggesting that rVHSV-GΔTM would not worsen disease progression caused by wild-type VHSV infection.

In vitro transcribed dsRNA limits viral hemorrhagic septicemia virus (VHSV)-IVb infection in a novel fathead minnow (Pimephales promelas) skin cell line.

The farming of baitfish, fish used by anglers to catch predatory species, is of economic and ecological importance in North America. Baitfish, including the fathead minnow (Pimephales promelas), are susceptible to infection from aquatic viruses, such as viral hemorrhagic septicemia virus (VHSV). VHSV infections can cause mass mortality events and have the potential to be spread to novel water bodies through baitfish as a vector. In this study, a novel skin cell line derived from fathead minnow (FHMskin) is described and its use as a tool to study innate antiviral immune responses and possible therapies is introduced. FHMskin grows optimally in 10% fetal bovine serum and at warmer temperatures, 25-30 °C. FHMskin is susceptible and permissive to VHSV-IVb infection, producing high viral titres of 7.35 × 107 TCID50/mL after only 2 days. FHMskin cells do not experience significant dsRNA-induced death after treatment with 50-500 ng/mL of in vitro transcribed dsRNA for 48 h and respond to dsRNA treatment by expressing high levels of three innate immune genes, viperin, ISG15, and Mx1. Pretreatment with dsRNA for 24 h significantly protected cells from VHSV-induced cell death, 500 ng/mL of dsRNA reduced cell death from 70% to less than 15% at a multiplicity of infection of 0.1. Thus, the novel cell line, FHMskin, represents a new method for producing high tires of VHSV-IVb in culture, and for studying dsRNA-induced innate antiviral responses, with future applications in dsRNA-based antiviral therapeutics.

Production of a monoclonal antibody against of muskellunge (Esox masquinongy) IgM heavy chain and its use in development of an indirect ELISA for titrating circulating antibodies against VHSV-IVB.

This study reports the development of a monoclonal antibody (designated 3B10) against the muskellunge (Esox masquinongy) IgM. The 3B10 monoclonal antibody (mAb) belongs to the IgG3 kappa isotype. Western blotting demonstrated that 3B10 mAb reacted primarily to muskellunge IgM heavy chain. 3B10 also reacted strongly with the IgM heavy chain of other esocids, including the northern pike (Esox lucius), tiger muskellunge (E. masquinongy x E. lucius), and, to a much lesser extent, the chain pickerel (E. niger). The 3B10 mAb did not bind to IgM from 10 other fish species resident in the Great Lakes basin. Using the 3B10 mAb, it was possible to determine the muskellunge Ig ability to bind to antigens. Using trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH) as the eliciting antigen, muskellunge Ig subclasses exhibited a range of affinities with log aK values 5.56-6.25 that is considered intermediate compared to other fish species. 3B10 mAb was used to develop and evaluate an indirect ELISA for the detection and quantitation of circulating antibodies against the viral hemorrhagic septicemia virus genotype IVb (VHSV-IVb). Using the newly optimized assay, anti-VHSV-IVb antibodies were detected in sera of VHSV-IVb vaccinated muskellunge as well as from those of wild muskellunge sampled from an endemic waterbody. In addition to its use in immunoassays, the developed 3B10 mAb will enable future investigation aiming at deciphering immune mechanism of this important fish species to pathogens.

Immune response modulation upon sequential heterogeneous co-infection with Tetracapsuloides bryosalmonae and VHSV in brown trout (Salmo trutta).

Simultaneous and sequential infections often occur in wild and farming environments. Despite growing awareness, co-infection studies are still very limited, mainly to a few well-established human models. European salmonids are susceptible to both Proliferative Kidney Disease (PKD), an endemic emergent disease caused by the myxozoan parasite Tetracapsuloides bryosalmonae, and Viral Haemorrhagic Septicaemia (VHS), an OIE notifiable listed disease caused by the Piscine Novirhabdovirus. No information is available as to how their immune system reacts when interacting with heterogeneous infections. A chronic (PKD) + acute (VHS) sequential co-infection model was established to assess if the responses elicited in co-infected fish are modulated, when compared to fish with single infections. Macro- and microscopic lesions were assessed after the challenge, and infection status confirmed by RT-qPCR analysis, enabling the identification of singly-infected and co-infected fish. A typical histophlogosis associated with histozoic extrasporogonic T. bryosalmonae was detected together with acute inflammation, haemorrhaging and necrosis due to the viral infection. The host immune response was measured in terms of key marker genes expression in kidney tissues. During T. bryosalmonae/VHSV-Ia co-infection, modulation of pro-inflammatory and antimicrobial peptide genes was strongly influenced by the viral infection, with a protracted inflammatory status, perhaps representing a negative side effect in these fish. Earlier activation of the cellular and humoral responses was detected in co-infected fish, with a more pronounced upregulation of Th1 and antiviral marker genes. These results reveal that some brown trout immune responses are enhanced or prolonged during PKD/VHS co-infection, relative to single infection.

Effect of miR-155 as a molecular adjuvant of DNA vaccine against VHSV in olive flounder (Paralichthys olivaceus).

Rhabdoviral G protein-based DNA vaccines have been recognized as a useful way to protect cultured fish from rhabdoviral diseases. In Korea, viral hemorrhagic septicemia virus (VHSV) genotype IVa has been the primary culprit of high mortalities of cultured olive flounder (Paralichthys olivaceus). In this study, we inserted a miR-155-expressing cassette into the VHSV's G protein-based DNA vaccine, and analyzed the effects of miR-155 on the antiviral activity and on the vaccine efficacy in olive flounder. Olive flounder fingerlings were intramuscularly (i.m.) immunized with 10 µg/fish (1st experiment) or 1 µg/fish (2nd experiment) of DNA vaccine plasmids. However, there were no significant differences in mortalities and serum neutralization titers between fish immunized with 1 µg and 10 µg plasmids/fish, suggesting that i.m. injection with 1 µg plasmids/fish would be enough to induce effective adaptive immune responses in olive flounder fingerlings. In survival rates, as fish immunized with just G protein expressing plasmids showed no or too low mortalities, the adjuvant effect of miR-155 was not discernible. Also, in the serum neutralization activities, although G gene or G gene plus miR-155 expressing DNA vaccines induced significantly higher activities than control vaccines (PBS and vacant vector), no significant differences were found between G gene alone and G gene plus miR-155 expressing DNA vaccines. In the serum virucidal activity, fish immunized with G gene plus miR-155 expressing DNA vaccine showed significantly higher activity against hirame rhabdovirus (HIRRV) at 3 days post-immunization (d.p.i.) compared to other groups, suggesting that miR-155 produced from the vector can enhance innate immune responses in olive flounder. The significantly enhanced serum virucidal activities against VHSV especially at 28 d.p.i. in the groups immunized with G gene alone and G gene plus miR-155 expressing DNA vaccines reflect the increased antibodies against G protein, which could activate the classical complement pathway and subsequent viral inactivation. As the available information on the DNA vaccines in olive flounder is not sufficient, more diverse researches on the protective efficacy of DNA vaccines are needed to make more practical use of DNA vaccines in olive flounder farms.

Effect of CRISPR/Cas9-mediated knockout of either Mx1 or ISG15 gene in EPC cells on resistance against VHSV infection.

Although the type I interferon-mediated increase of Mx1 and ISG15 gene expression in Epithelioma papulosum cyprini (EPC) cells has been reported, the antiviral role of Mx1 and ISG15 in EPC cells has not been investigated. In this study, to know the anti-viral hemorrhagic septicemia virus (VHSV) role of Mx1 and ISG15 of EPC cells, either Mx1 or ISG15 gene was knocked-out using a CRISPR/Cas9 system, and the progression of cytopathic effects (CPE) and viral growth were analyzed. Mx1 gene and ISG15 gene knockout EPC cells were successfully produced via CRISPR/Cas9 coupled with a single-cell cloning. Through the sequence analysis, one clone showing two heterozygous indel patterns in Mx1 gene and a clone showing three heterozygous indel patterns in ISG15 gene were selected for further analyses. Mx1 knockout EPC cells did not show any differences in VHSV-mediated CPE progression, even when pre-treated with polyinosinic:polycytidylic acid (poly I:C), compared to control EPC cells. These results suggest that Mx1 in EPC cells may be unfunctional to cytoplasmic RNA viruses. In contrast to Mx1, ISG15 knockout cells showed clearly hampered anti-VHSV activity even when pre-treated with poly I:C, indicating that ISG15 plays an important role in type I interferon-mediated anti-viral activity in EPC cells, which allowed VHSV to replicate more efficiently in ISG15 knockout cells than Mx1 knockout and control cells.

Effect of CXCL12-expressing viral hemorrhagic septicemia virus replicon particles on leukocytes migration and vaccine efficacy in olive flounder (Paralichthys olivaceus).

Viral replicon particles are single-cycle viruses defective for function(s) needed for viral replication, which allow them to be recognized as a safer form for the vaccination of animals compared to attenuated live viruses. However, deletion of genes that are critical for the induction of protective immunity can diminish the vaccine potential of viral replicon particles. Therefore, the manipulation of viral replicon particles to produce a molecular adjuvant can be a way to increase immunogenicity of vaccines based on viral replicon particles. Chemokines are a class of chemotactic cytokines that control the migration of diverse cells of vertebrates. CXC chemokine ligand 12 (CXCL12) binds to a receptor CXCR4, and CXCL12-CXCR4 signaling plays an important role in the migration of hematopoietic cells during embryogenesis and the attraction of leukocytes. In the present study, to evaluate the possible use of CXCL12 as a molecular adjuvant for an rVHSV-ΔG vaccine and to know differences between CXCL12a and CXCL12b in the adjuvant ability, we rescued VHSV replicon particles that are expressing olive flounder CXCL12a, CXCL12b, or eGFP (rVHSV-ΔG-CXCL12a, rVHSV-ΔG-CXCL12b, or rVHSV-ΔG-eGFP), and compared the ability to attract olive flounder leucocytes and to induce protection against a VHSV challenge. In the leukocytes migration assay, supernatants collected from cells infected with rVHSV-ΔG-CXCL12a and rVHSV-ΔG-CXCL12b showed significantly higher ability to attract olive flounder leukocytes than the supernatant of cells infected with rVHSV-ΔG-eGFP. Moreover, the significantly higher number of leukocytes were attracted to rVHSV-CXCL12a supernatant compared to rVHSV-CXCL12b supernatant, suggesting that CXCL12a would be more appropriate for the induction of immunity than CXCL12b in olive flounder. In the immunization experiment, olive flounder immunized with rVHSV-ΔG-CXCL12a showed significantly higher survival rate than fish immunized with rVHSV-ΔG-CXCL12b or rVHSV-ΔG-eGFP. In addition, fish immunized with rVHSV-ΔG-CXCL12a showed the highest serum neutralization activity. These results suggest the availability of CXCL12a for a molecular adjuvant of vaccines based on VHSV replicon particles.

Molecular characterization and expression analysis of rockfish (Sebastes schlegelii) viperin, and its ability to enervate RNA virus transcription and replication in vitro.

Viperin, also known as RSAD2 (Radical S-adenosyl methionine domain containing 2), is an interferon-induced endoplasmic reticulum-associated antiviral protein. Previous studies have shown that viperin levels are elevated in the presence of viral RNA, but it has rarely been characterized in marine organisms. This study was designed to functionally characterize rockfish viperin (SsVip), to examine the effects of different immune stimulants on its expression, and to determine its subcellular localization. SsVip is a 349 amino acid protein with a predicted molecular mass of 40.24 kDa. It contains an S-adenosyl l-methionine binding conserved domain with a CNYKCGFC sequence. Unchallenged tissue expression analysis using quantitative real time PCR (qPCR) revealed SsVip expression to be the highest in the blood, followed by the spleen. When challenged with poly I:C, SsVip was upregulated by approximately 60-fold in the blood after 24 h, and approximately 50-fold in the spleen after 12 h. Notable upregulation was detected throughout the poly I:C challenge experiment in both tissues. Significant expression of SsVip was detected in the blood following Streptococcus iniae and lipopolysaccharide challenge, and viral hemorrhagic septicemia virus (VHSV) gene transcription was significantly downregulated during SsVip overexpression. Furthermore, cell viability assay and virus titer quantification with the presence of SsVip revealed a significant reduction in virus replication. As with previously identified viperin counterparts, SsVip was localized in the endoplasmic reticulum. Our findings show that SsVip is an antiviral protein crucial to innate immune defense.

Functional nutrition modulates the early immune response against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout.

Although viruses represent a major threat for cultured fish worldwide, the commercialization of vaccines capable of providing effective and long-lasting protection is still lacking for most of these viral diseases. In this situation, the use of supplemented diets could be a suitable strategy to increase the immune status of the fish and reduce the impact of viral pathogens. Among possible immunostimulants that could be included in these functional feeds, some studies have previously shown that certain ß-glucans can significantly increase certain immune parameters of fish and reduce the impact of viral diseases. However, the mechanisms through which ß-glucans exert their activity have not been fully elucidated yet. In the current study, we have studied the immune response of different tissues to viral haemorrhagic septicaemia virus (VHSV) in rainbow trout fed with a non-supplemented control diet as well as in fish fed a commercial functional aquafeed (Protec™, Skretting) containing ß-glucans, vitamin C, vitamin E and zinc. For this, after 30 days of feeding the fish with one of the two diets, they were subsequently infected with VHSV by bath or mock-infected. After 2 or 6 days post-infection, fish were sacrificed and the levels of transcription of different immune genes such as IgM, IgT, IgD, Mx, interferon γ (IFN γ) and perforin studied in different tissues (kidney, gut and gills). Additionally, the levels of natural IgMs in serum were also determined. Our results demonstrate that fish fed the functional diet were capable of mounting an increased IgM, IgT, IgD and Mx transcriptional response to the virus. Additionally, these fish also showed increased levels of natural IgMs in serum. These results reveal a previously undescribed effect of functional diets on fish Ig production and point to Protec™ as an adequate diet to be incorporated in holistic programs aimed at mitigating the effect of viral diseases.

Time-course study of the protection induced by an interferon-inducible DNA vaccine against viral haemorrhagic septicaemia in rainbow trout.

The highly effective DNA vaccines against diseases caused by fish rhabdoviruses in farmed fish consist of a DNA plasmid vector encoding the viral glycoprotein under the control of a constitutive cytomegalovirus promoter (CMV). Among others, attempts to improve efficacy and safety of these DNA vaccines have focused on regulatory elements of plasmid vectors, which play a major role in controlling expression levels of vaccine antigens. Depending on the context, use of a fish-derived promoter with minimal activity in mammalian cells could be preferable. Another aspect related to the CMV promoter is that constitutive expression of the vaccine antigen may lead to rapid elimination of antigen expressing cells in the fish and thereby potentially reduce the long-term effects of the vaccine. In this study, we compared DNA vaccines with the interferon-inducible Mx promoter from rainbow trout and the CMV promoter, respectively. Plasmid constructs encoding the enhanced green fluorescent protein (EGFP) were used for the in vitro analysis, whereas DNA vaccines encoding the glycoprotein (G) of the viral haemorrhagic septicaemia virus (VHSV) were applied for the in vivo examination. The in vitro analysis showed that while the DNA vaccine with the CMV promoter constitutively drove the expression of EGFP in both fish and human cell lines, the DNA vaccine with the Mx promoter inducibly enhanced the expression of EGFP in the fish cell line. To address the impact on protection, a time-course model was followed as suggested by Kurath et al. (2006), where vaccinated fish were challenged with VHSV at 2, 8 and 78 weeks post-vaccination (wpv). The DNA vaccine with the CMV promoter protected at all times, while vaccination with the DNA vaccine containing the Mx promoter only protected the fish at 8 wpv. However, following induction with Poly (I:C) one week before the challenge, high protection was also evident at 2 wpv. In conclusion, the results revealed a more fish host dependent activity of the trout Mx promoter compared to the traditionally used cross species-active CMV promoter, but improvements will be needed for its application in DNA vaccines to ensure long term protection.

Efficacy of algal Ecklonia cava extract against viral hemorrhagic septicemia virus (VHSV).

The inhibition efficacy of an extract from Ecklonia cava (E. cava) was studied to determine whether the extract and compounds exhibited inhibitory activity against VHSV in the fathead minnow (FHM) cell line and following oral administration to the olive flounder. Based on its low toxicity and effective concentration, the E. cava extract (Ext) and compounds (eckol and phlorofucofuroeckol A) were selected for further analysis. In the plaque reduction assay, simultaneous co-exposure of VHSV to Ext, eckol and phlorofucofuroeckol A showed a higher level of inhibition than the pre- and post-exposure groups. The antiviral activity in the FHM cell line was time-dependent and increased with the exposure time with the virus and Ext or the compounds. In the in vivo experiments, different Ext concentrations were orally administered to the olive flounder. In trial I, the relative percent survival (RPS) following oral administration of 500 and 50 µg/g/day of Ext was 31.25% and 12.50%, respectively. In trial II, the RPS for 1000, 500 and 50 µg/g/day of Ext was 31.57%, 0% and 0%, respectively. In trial III, the RPS after 1 and 2 weeks (1000 µg/g/day) of exposure to Ext was 26.31% and 31.57%, respectively. Oral administration of Ext (1000 µg/g/day) significantly induced inflammatory cytokine responses (IL-1ß, IL-6 and IFN-γ) at 1 and 2 days post-oral administration (dpa). Additionally, IFN-α/ß (7-12 dpa), ISG15 (2, 7 and 10 dpa) and Mx (7-12 dpa) were significantly activated in the olive flounder. In conclusion, we demonstrated an inhibitory ability of the E. cava extract and compounds against VHSV in the FHM cell line. Moreover, oral administration of the E. cava extract to the olive flounder enhanced antiviral immune responses and the efficacy of protection against VHSV, resulting in an anti-viral status in the olive flounder.

Comparative susceptibilities and immune reactions of wild and cultured populations of Caspian trout Salmo trutta caspius to VHSV.

Caspian trout Salmo trutta caspius is an endangered subspecies of brown trout Salmo trutta which is native to the Caspian Sea. Restocking programmes have been established, but recent introduction of the rhabdovirus viral haemorrhagic septicaemia virus (VHSV) into Iranian rainbow trout farms connected to waterbodies supporting wild Caspian trout may represent an additional threat to the declining stock. The susceptibility of wild and cultured populations of this endemic subspecies was demonstrated by performing controlled VHSV infection experiments (both by bath and injection challenges). Subsequently, VHSV infection in exposed fish was confirmed (CPE and quantitative PCR), virus levels were measured, and regulation of immune genes in exposed fish was investigated with a focus on the genes encoding IL-8, IFNγ, TGFß, TNFα, SAA, C3-4, CD8α, IgM, MHC I, MHC II, iNOS and IGF-1. The presence of IgM-, CD8α- and MHC II-positive cells in host organs was visualized by immunohistochemistry. Both wild and cultured trout strains proved to be VHSV-susceptible following experimental challenge, but the mortality curves and associated regulation of immune-related genes differed between the 2 trout types. Implications of the results for future management of Caspian trout populations are discussed.

Development of a monoclonal antibody against the CD3ε of olive flounder (Paralichthys olivaceus) and its application in evaluating immune response related to CD3ε.

The T cell receptor (TCR) is the binding site of antigen and is responsible for specifically activating the adaptive immune response. CD3, an essential component of the CD3-TCR complex, is known to be composed of γδ and ε chains in teleost. However, there are few monoclonal antibodies (mAb) available to identify these molecules on T cells, so we aimed to produce a mAb against CD3ε to improve our understanding of T cell immune response in olive flounder (Paralichthys olivaceus). CD3ε recombinant protein was expressed in yeast, the expression of which was confirmed by SDS-PAGE, MALDI-TOF/TOF MS and Western blot analysis. A CD3ε-specific mAb 4B2 was selected, the specificity of which was examined by confocal microscopy, flow cytometry and RT-PCR, and the mAb was subsequently used to examine the CD3ε lymphocyte population in several different immune organs, with relatively high percentages of these cells seen in trunk-kidney and spleen, while lower percentages were seen in the liver and peripheral blood of olive flounder. During a viral hemorrhagic septicemia virus (VHSV) infection in olive flounder, the number of CD3ε lymphocytes was seen to gradually increase in the liver, spleen and trunk-kidney of infected fish until 7 days post infection (dpi). In peripheral blood, on the other hand, the increase in CD3ε lymphocyte numbers peaked by 3 dpi. These results suggest that CD3ε lymphocytes might be involved in the immune response against VHSV.

Integrated profiling of global metabolomic and transcriptomic responses to viral hemorrhagic septicemia virus infection in olive flounder.

Viral hemorrhagic septicemia virus (VHSV) is one of the most serious viral pathogen that infects farmed fish. In this study, we measured the replication of VHSV increased steadily at 9, 24, 72, and 120 h after infection and progression of necrosis was observed at 72 hpi. We performed transcriptomic and metabolomics profiling of kidney tissues collected at each infection time using Illumina HiSeq2000 and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectroscopy to investigate the mechanisms of VHSV infection in the kidneys of olive flounder. A total of 13,862 mRNA molecules and 72 metabolites were selected to identify the mechanisms of infection and they were integrated using KEGG pathway database. Six KEGG metabolic pathways, including carbohydrate metabolism, amino acid metabolism, lipid metabolism, transport and catabolism, metabolism of cofactors and vitamins, and energy metabolism, were significantly suppressed, whereas the immune system was activated by VHSV infection. A decrease in levels of amino acids such as valine, leucine, and isoleucine, as well as in their derivative carnitines, was observed after VHSV infection. In addition, an increase in arachidonic acid level was noted. Integrated analysis of transcriptome and metabolome using KEGG pathway database revealed four types of responses in the kidneys of olive flounder to VHSV infection. Among these, the mechanisms related to the immune system and protein synthesis were activated, whereas ATP synthesis and the antioxidant system activity were suppressed. This is the first study describing the mechanisms of metabolic responses to VHSV infection in olive flounder. The results suggest that the suppression of ATP synthesis and antioxidant systems, such as glutathione and peroxisome signaling, could be the cause of necrosis, whereas the activation of the immune system could result in the inflammation of kidney tissue in VHSV-infected olive flounder.