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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.

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.

•Two microRNAs (miR-462/-731) are upregulated in fish immunized with a DNA vaccine.•miR-462 and miR-731 were also induced by poly I:C and interferons.•Inhibiting miR-462 and miR-731 reduced the protective effect of poly I:C.•miR-462 and miR-731 directly contribute to interferon-mediated protection. Mechanisms that account for the high protective efficacy in teleost fish of a DNA vaccine expressing the glycoprotein (G) of Viral hemorrhagic septicemia virus (VHSV) are thought to involve early innate immune responses mediated by interferons (IFNs). Microribonucleic acids (miRNAs) are a diverse class of small (18–22 nucleotides) endogenous RNAs that potently mediate post-transcriptional silencing of a wide range of genes and are emerging as critical regulators of cellular processes, including immune responses. We have recently reported that miR-462 and miR-731 were strongly induced in rainbow trout infected with VHSV. In this study, we analyzed the expression of these miRNAs in fish following administration of the DNA vaccine and their potential functions. Quantitative RT-PCR analysis revealed the increased levels of miR-462, and miR-731 in the skeletal muscle tissue at the site of vaccine administration and in the liver of vaccinated fish relative to empty plasmid backbone-injected controls. The increased expression of these miRNAs in the skeletal muscle correlated with the increased levels of the type I interferon (IFN)-inducible gene Mx, type I IFN and IFN-γ genes at the vaccination site. Intramuscular injection of fish with either type I IFN or IFN-γ plasmid construct resulted in the upregulation of miR-462 and miR-731 at the site of injection, suggesting that the induction of these miRNAs is elicited by IFNs. To analyze the function of miR-462 and miR-731, specific silencing of these miRNAs using anti-miRNA oligonucleotides was conducted in poly I:C-treated rainbow trout fingerlings. Following VHSV challenge, anti-miRNA-injected fish had faster development of disease and higher mortalities than control fish, indicating that miR-462/731 may be involved in IFN-mediated protection conferred by poly I:C.

Background & objectives Probiotics are increasingly used in the pet industry to enhance the health and well-being of companion animals. Among them, Lactobacillus strains and their metabolites have demonstrated the ability to maintain immune homeostasis, modulate immune responses, and exhibit antiviral properties. Despite growing interest in postbiotics, non-viable microbial products or metabolic byproducts, scientific literature on their effects remains limited. This study investigates the immunomodulatory and antiviral properties of three postbiotics derived from heat-inactivated Lactobacillus strains using an adult zebrafish ( Danio rerio) model challenged with viral hemorrhagic septicemia virus (VHSV). Methods A total of 330 zebrafish were assigned to five groups: a non-challenged control (C1), a VHSV-challenged control (C2), and three experimental groups supplemented with one of three heat-treated Lactobacillus strains at the same dosage ( Lacticaseibacillus paracasei HA-108; Lactiplantibacillus plantarum HA-119 or Lactobacillus helveticus HA-122). Fish were fed at 4% of biomass for 21 days. Following this period, a subset of the fish was used for immune gene expression profiling and histological examination of the gut and kidney. The remaining fish were challenged with VHSV and monitored for survival over 10 days. Results All postbiotics treatments modulated immune responses, with L. plantarum HA-119 showing the most pronounced effects, including upregulation of key immune genes such as Il1β and Ifn-γ , indicative of anti-inflammatory and antiviral activity. Histological analysis revealed no significant changes in goblet cell density or villi height, supporting the safety of the postbiotics. Survival rates were significantly higher in the L. plantarum HA-119 and L. helveticus HA-122 groups compared to the VHSV control. Conclusions These findings, derived from a well-established zebrafish model, suggest that postbiotics from Lactobacillus strains may enhance antiviral immunity and overall health in vertebrates, supporting their potential as safe, effective microbial-based nutritional interventions in pet nutrition.

Background Despite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV). Results Two-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components ( c3b, c8 and c9 ) or class I histocompatibility antigens ( mhc1 ) and to newly described genes such as secreted immunoglobulin domain ( sid4 ), macrophage stimulating factor ( mst1 ) and a cluster differentiation antigen ( cd36 ). Conclusions The genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

•Intramuscular VHSV DNA vaccination induces the infiltration of B cells in trout.•These B cells are both IgM+ and IgT+ cells.•CXCL11_L1, CK5B, CK6 and CXCR3B transcription is up-regulated in vaccinated fish.•CK5B and CK6 have chemotactic capacities. In fish, intramuscular (i.m) injection of plasmid DNA encoding viral proteins has proved a highly effective vaccination strategy against some viral pathogens. The efficacy of DNA vaccination in teleost fish is based on the high level of viral antigen expression in muscle cells inducing a strong and long-lasting protection. However, the mechanisms through which this protection is established and effectuated in fish are still not fully understood. Moreover, similarities to mammalian models cannot be established since DNA vaccination in mammals usually induces much weaker responses. In this work, we have focused on the characterization of the immune cells that infiltrate the muscle at the site of DNA injection in vaccinated fish and the chemokines and chemokine receptors that may be involved in their infiltration. We have demonstrated through diverse techniques that B lymphocytes, both IgM+ and IgT+ cells, represented a major infiltrating cell type in fish vaccinated with a viral haemorrhagic septicaemia virus (VHSV) glycoprotein-encoding DNA vaccine, whereas in control fish injected with an oil adjuvant mainly granulocyte/monocyte-type cells were attracted. Among twelve chemokine genes studied, only CXCL11_L1, CK5B and CK6 mRNA levels were up-regulated in DNA vaccinated fish compared to fish injected with the corresponding vector backbone. Furthermore, the transcription of CXCR3B, a possible receptor for CXCL11_L1 was also significantly up-regulated in vaccinated fish. Finally, experiments performed with recombinant trout CK5B and CK6 and chemokine expression plasmids revealed that these chemokines have chemotactic capacities which might explain the recruitment of B cells to the site of DNA injection. Altogether, our results reveal that there is an early chemokine-related B cell recruitment triggered by i.m. DNA vaccination against VHSV which might play an important role in the initial phase of the immune response.

Health control is a major issue in animal breeding and a better knowledge of the genetic bases of resistance to diseases is needed in farm animals including fish. The detection of quantitative trait loci (QTL) will help uncovering the genetic architecture of important traits and understanding the mechanisms involved in resistance to pathogens. We report here the detection of QTL for resistance to Viral Haemorrhagic Septicaemia Virus (VHSV), a major threat for European aquaculture industry. Two induced mitogynogenetic doubled haploid F2 rainbow trout (Oncorhynchus mykiss) families were used. These families combined the genome of susceptible and resistant F0 breeders and contained only fully homozygous individuals. For phenotyping, fish survival after an immersion challenge with the virus was recorded, as well as in vitro virus replication on fin explants. A bidirectional selective genotyping strategy identified seven QTL associated to survival. One of those QTL was significant at the genome-wide level and largely explained both survival and viral replication in fin explants in the different families of the design (up to 65% and 49% of phenotypic variance explained respectively). These results evidence the key role of innate defence in resistance to the virus and pave the way for the identification of the gene(s) responsible for resistance. The identification of a major QTL also opens appealing perspectives for selective breeding of fish with improved resistance.

Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profile.

The viral haemorrhagic septicaemia virus (VHSV) is considered a very significant disease affecting fish. The objective of this project is to develop a multi‐epitope vaccine targeting viral hemorrhagic fever using the reverse vaccinology approach. Epitope prediction was conducted using the immunogenic components of VHSVV. A multi‐epitope vaccine was developed by combining the immunogenic proteins’ most potent B‐ and T‐cell epitopes with the adjuvant. Following that, a comprehensive evaluation was carried out on many facets of the formulated vaccine, including its physicochemical properties, antigenic profile, secondary structure and tertiary structure. In addition, the molecular docking methodology was used to investigate the interaction between the suggested vaccination and its Toll‐like receptor 4 (TLR‐4) receptor. The vaccine's nucleotide sequence was later altered to enhance its production in Lactococcus lactis. The present investigation's results suggest that the produced vaccine demonstrated stability by its molecular weight of 58,769.30 Da and antigenicity score of 0.5283. Furthermore, the analysis of the vaccine's composition revealed that it included 12.43% alpha‐helix, with 87.88% of its residues in the desired region. The vaccine under consideration demonstrated successful docking to its TLR‐4, yielding the minimum energy value of −4005.09. Subsequently, the optimum complex was identified as −19.38 (kcal/mol). On the basis of the acquired results, the proposed vaccine has promising efficacy in mitigating fish infection resulting from VHSV. Our research indicates that peptide vaccination might be a beneficial option for preventing VHSV. Viral haemorrhagic septicaemia virus (VHSV) immunogenic epitopes were used to design a multi‐epitope vaccine with adjuvant. The vaccine's properties, structure and TLR‐4 interaction were analysed via computational methods. Optimized nucleotide sequences were created for Lactococcus lactis expression.

•Squalene (5%) and aluminum hydroxide (0.5%) emulsion is effective as adjuvants.•Single dose of VHSV vaccine provided significant protection for 40 weeks.•Vaccine administered at 19°C provides protection at low temperature of 6°C.•The vaccine formulation (IV+Sq+Al) of this study is safe for olive flounder. Viral hemorrhagic septicemia (VHS) in olive flounder (Paralichthys olivaceus) remains an unsolved health problem in Korean aquaculture. Vaccination plays a significant role in modern aquaculture, and the duration of protection provided is of vital importance. Here, we have demonstrated the efficacy, duration of protection and safety of an inactivated vaccine emulsified with squalene (5%) and aluminum hydroxide (0.5%). The inactivated VHS vaccine provided a moderate protection of 37% and 47% relative percent survival (RPS) at 4 and 10 weeks post vaccination (wpv). Addition of squalene and aluminum hydroxide into inactivated VHS vaccine clearly enhanced the level of protection showing 58% and 83% RPS at 4 and 10wpv, respectively, indicating the need for adjuvants to enhance the efficacy. The vaccinated fish showed significant protection at 3, 6, 12, 18, 24, and 40wpv (except week 57) than non-vaccinated fish to an intraperitoneal challenge of 107.1TCID50/fish at 15°C, with RPS of 60%, 64%, 71%, 55%, 52% and 50% (45% at 57 week), respectively, covering the duration of natural outbreak. Fish challenged at 18wpv at 6°C showed 56% RPS and protection at a low temperature. The antibody titer was high at 3wpv with an OD of 1.08±0.13, but decreased gradually and was undetectable by 24wpv. The vaccine formulation was safe without injection site reactions, adhesions, or pigmentation observed at 6, 12, 18, or 24wpv. Inflammatory reactions were observed in the spleen intestine at 6 and 12wpv but were similar as control by 24wpv. These results confirm that this vaccine is efficient and safe for olive flounder and could offer an appropriate strategy to prevent VHS without causing side effects.