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The monoclonal antibody (mAb) IP5B11, which is used worldwide for the diagnosis of viral haemorrhagic septicaemia (VHS) in fish, reacts with all genotypes of VHS virus (VHSV). The mAb exceptionally also reacts with the carpione rhabdovirus (CarRV). Following next generation genome sequencing of CarRV and N protein sequence alignment including five kinds of fish novirhabdoviruses, the epitope recognized by mAb IP5B11 was identified. Dot blot analysis confirmed the epitope of mAb IP5B11 to be associated with the region N219 to N233 of the N protein of VHSV. Phylogenetic analysis identified CarRV as a new member of the fish novirhabdoviruses.

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

Given the lack of antiviral prophylactic measures authorized in aquaculture, vaccination is the most effective method to prevent and control diseases provoked by viral agents. Viral hemorrhagic septicemia virus (VHSV) is an important fish pathogen, known to induce high mortality rates and large economic losses worldwide in many aquaculture fish species, including rainbow trout (Oncorhynchus mykiss). DNA vaccination against VHSV has been proven a very effective method to induce a long-term protection against the virus, yet the mechanisms through which this vaccine induces protection are still unclear. To provide further insight on this matter, in the current study, we have studied the transcriptional response of leukocytes obtained from vaccinated fish when they were exposed in vitro to the virus. For this, fish were intramuscularly (i.m.) injected with the VHSV vaccine or mock-vaccinated with either the empty plasmid or saline solution and 30 days post-vaccination sacrificed to isolate kidney and spleen leukocytes. Isolated leukocytes were then exposed or not to inactivated VHSV and the transcription of a range of genes related to the antiviral response analyzed. Supernatants were also taken from these cultures and tested for antiviral activity. Our results revealed that leukocytes obtained from vaccinated fish, especially those derived from kidney, transcribed some specific antiviral genes and specially MDA5 at higher levels when exposed to the virus, correlating with an increased secretion of antiviral factors to the supernatants. The results obtained provide important insights on how leukocytes from fish effectively vaccinated respond to a posterior viral encounter, information of value to optimize this and other antiviral vaccines for use in aquaculture and establish correlates of protection. •Kidney leukocytes from VHSV DNA vaccinated fish, when exposed to VHSV, transcribe higher levels of ifn1, ifn2 and irf7.•Kidney leukocytes from vaccinated fish, when exposed to VHSV, transcribe higher levels of mda5 than those of mock-vaccinated fish.•The differential response of DNA vaccinated fish to VHSV is more pronounced in kidney than in spleen.•Supernatants from leukocytes obtained from DNA vaccinated fish have a basal antiviral activity.

•Encapsulation of inactivated VHSV in PLGA NPs for preparation of PNPs-IV vaccine.•Olive flounder fingerlings immunized with PNPs-IV through immersion and oral routes.•PNPs-IV vaccinated fish exhibited high RPS (60–73.3%) post VHSV challenged.•PNPs-IV immunized group showed higher specific antibody response in sera and mucus.•Immune genes were significantly upregulated in immunized fish. Viral haemorrhagic septicaemia virus (VHSV), an OIE listed viral pathogen, is the etiological agent of a contagious disease, causing huge economic losses in farmed olive flounder (Paralichthys olivaceus) and significant mortalities among several other marine fish species in Korea, Japan, and China. In continuation with our previous work, where injection vaccination with inactivated VHSV mixed with squalene (as adjuvant) conferred higher protective immunity to olive flounder, the present study focused on replacing the injection route of vaccine delivery by immersion/oral route to overcome the limitations of the parenteral immunization method. Here, we encapsulated the inactivated VHSV vaccine with PLGA (poly lactic-co-glycolic acid) nanoparticles (PNPs-IV) and evaluated its ability to induce protective immunity in olive flounder (12.5 ± 1.5 g) by initially immunizing the fishes by immersion route followed by a booster with the same dose two weeks later with half of the fish through immersion route and other half through oral route (incorporated into fish feed). Cumulative mortalities post-challenge (1 × 106 TCID50 virus/fish) with virulent VHSV-isolate, were lower in vaccinated fish and RPS of 60% and 73.3% were obtained for PNPs-IV (immersion/immersion) and PNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, PNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to controls, in both the systemic (kidney) and mucosal (skin and intestine) immune compartment of the host post immunization as well as post challenge. Thus it can be inferred that the adopted immunization strategy efficiently protected and transported the inactivated viral antigen to target immune organs and positively stimulated the protective immune response against VHSV in olive flounder.

Fish Red-Blood Cells (RBCs) are nucleated cells that can modulate the expression of different sets of genes in response to stimuli, playing an active role in the homeostasis of the fish immune system. Nowadays, vaccination is one of the main ways to control and prevent viral diseases in aquaculture and the development of novel vaccination approaches is a focal point in fish vaccinology. One of the strategies that has recently emerged is the use of nanostructured recombinant proteins. Nanostructured cytokines have already been shown to immunostimulate and protect fish against bacterial infections. To explore the role of RBCs in the immune response to two nanostructured recombinant proteins, TNF and a G-VHSV protein fragment, we performed different and studies. We show for the first time that rainbow trout RBCs are able to endocytose nanostructured TNF and G-VHSV protein fragment , despite not being phagocytic cells, and in response to nanostructured TNF and G-VHSV fragment, the expression of different immune genes could be modulated.

Vaccination procedures can be stressful for fish and can bring severe side effects. Therefore, vaccines that can minimize the number of administrations and maximize cross-protection against multiple serotypes, genotypes, or even different species would be highly advantageous. In the present study, we investigated the cross-protective ability of two types of vaccines – viral hemorrhagic septicemia virus (VHSV) G protein-expressing DNA vaccine and G gene-deleted single-cycle VHSV genotype IVa (rVHSV-ΔG) vaccine - against both VHSV genotype Ia and infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). The results showed that rainbow trout immunized with VHSV genotype Ia G gene- or IVa G gene-expressing DNA vaccine were significantly protected against VHSV genotype Ia, but were not protected against IHNV. In contrast to the DNA vaccine, the single-cycle VHSV IVa vaccine induced significant protection against not only VHSV Ia but also IHNV. Considering no significant increase in ELISA titer and serum neutralization activity against IHNV in fish immunized with single-cycle VHSV IVa, the protection might be independent of humoral adaptive immunity. The scarcity of cytotoxic T cell epitopes between VHSV and IHNV suggested that the possibility of involvement of cytotoxic T cell-mediated cellular adaptive immunity would be low. The role of trained immunity (innate immune memory) in cross-protection should be further investigated.

The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout ( Oncorhynchus mykiss) and sole ( Solea senegalensis ). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture.

Viperin is a prominent antiviral protein found in animals. The primary function of Viperin is the production of 3’-deoxy-3’,4’-didehydro-cytidine triphosphate (ddhCTP), an inhibitory nucleotide involved in viral RNA synthesis. Studies in mammalian models have suggested that ddhCTP interferes with metabolic proteins. However, this hypothesis has yet to be tested in teleost. In this study, the role of Viperin in regulating metabolic alterations during viral hemorrhagic septicemia virus (VHSV) infection was tested. When infected with VHSV, viperin -/- fish showed considerably higher mortality rates. VHSV copy number and the expression of the NP gene were significantly increased in viperin -/- fish. Metabolic gene analysis revealed significant differences in soda , hif1a , fasn , and acc expression, indicating their impact on metabolism. Cholesterol analysis in zebrafish larvae during VHSV infection showed significant upregulation of cholesterol production without Viperin. In vitro analysis of ZF4 cells suggested a considerable reduction in lipid production and a significant upregulation of reactive oxygen species (ROS) generation with the overexpression of viperin . Neutrophil and macrophage recruitment were significantly modulated in viperin -/- fish compared to the wild-type (WT) fish. Thus, we have demonstrated that Viperin plays a role in interfering with metabolic alterations during VHSV infection.

mRNA vaccines are poised to revolutionize disease prevention, following the approval of their administration to humans against SARS-CoV-2. Although they have been extensively studied for human applications, their potential in the veterinary field has not been explored yet. No mRNA vaccines have yet been reported for fish, despite the urgent need for new vaccines against emerging pathogens in aquaculture. As fish are ectotherms, temperature has an impact on their immune response and on many other biological parameters, including the composition of membrane lipids. It is therefore crucial to identify whether mRNA delivery systems are suitable for in vivo expression in fish for vaccine purposes. In the present study, we developed a proof of concept for mRNA vaccination in rainbow trout, a salmonid, demonstrating the efficacy of current vaccine delivery systems in fish. We used lipid nanoparticles (LNPs), which represent the most advanced delivery technology for mRNA. LNPs use a combination of lipid components that form an encapsulating structure offering protection and promote endosome escape of the mRNA to allow its expression. In vitro assays showed that LNPs are a powerful vehicle for mRNA delivery in fish cells without substantial toxicity. In vivo imaging in adult zebrafish (Danio rerio) demonstrated that intramuscular injection of LNP-formulated egfp mRNA resulted in local expression of eGFP for up to 7 days. An LNP-based mRNA vaccine candidate encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein induced neutralizing antibodies in rainbow trout (Oncorhynchus mykiss) and offers almost complete protection against a lethal viral challenge. Our data constitute a first proof of concept of mRNA vaccination in fish, paving the way for new developments in veterinary vaccines for aquaculture.

The use of functional feed additives is an important approach to both, prevent and fight, viral diseases in aquaculture. In this regard, microalgae-derived products, and, more specifically, microalgal exopolysaccharides (EPSs), have attracted attention, since multiple biotechnological applications are being described for these molecules. Furthermore, depending on culture conditions, the composition and, therefore, properties of EPSs can vary. In the present study, the antiviral activity of EPSs from Tetraselmis suecica and Porphyridium cruentum cultured under autotrophic and heterotrophic conditions has been evaluated in vitro against Viral Haemorrhagic Septicaemia Virus (VHSV), an important pathogen in fish farming. Results showed that EPSs from both species have anti-VHSV activity. T. suecica EPSs from autotrophic cultures showed the strongest effect, since both, adsorption and post-adsorption phases of the VHSV multiplication cycle were affected. In contrast, both, autotrophic and heterotrophic P. cruentum EPSs showed anti-VHSV activity only after the adsorption phase. These results pave the way to use these EPSs to fight VHSV infections, and animate to evaluate the EPS antiviral activity against other viral pathogens relevant to the aquaculture industry.