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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.

•Sq + Al-adjuvanted IV is a safe and non-invasive vaccine formulation.•The formulated vaccine can be delivered via either IP or IM route in olive flounder.•It showed persistency for 17–24 wpv at the injection site without causing side effects.•It provides both short- and long-term protective immunity in flounder against VHSV.•The study recommends IM vaccination in small-sized fish and IP vaccination in table-sized fish. The profitability of the olive flounder (Paralichthys olivaceus) aquaculture industry in Korea depends on high production and maintenance of flesh quality, as consumers prefer to eat raw flounders from aquaria and relish the raw muscles as ‘sashimi’. For sustaining high production, easy-to-deliver and efficient vaccination strategies against serious pathogens, such as viral hemorrhagic septicemia virus (VHSV), is very important as it cause considerable losses to the industry. Whereas, a safe and non-invasive vaccine formulation that is free from unacceptable side-effects and does not devalue the fish is needed to maintain flesh quality. We previously developed a squalene–aluminium hydroxide (Sq + Al) adjuvanted VHSV vaccine that conferred moderate to high protection in flounder, without causing any side effects when administered through the intraperitoneal (IP) injection route. However, farmers often demand intramuscular (IM) injection vaccines as they are relatively easy to administer in small fishes. Therefore, we administered the developed vaccine via IP and IM routes and investigated the safety and persistency of the vaccine at the injection site. In addition, we conducted a comparative analysis of vaccine efficacy and serum antibody response. The clinical and histological observation of the IM and IP groups showed that our vaccine remained persistence at the injection sites for 10–17 weeks post vaccination (wpv), without causing any adverse effects to the fish. The relative percentage of survival were 100% and 71.4% for the IP group and 88.9% and 92.3% for the IM group at 3 and 17 wpv, respectively. Thus, considering the persistency period (24 wpv) and both short and long-term efficacy of our vaccine, the present study offers an option to flounder farmers in selecting either IM or IP delivery strategy according to their cultured fish size and harvesting schedule — IM vaccination for small-sized fish and IP vaccination for table-sized fish.

•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.

•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.

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.

Olive flounder ( Paralichthys olivaceus ) is the most valuable aquaculture species in Korea, corresponding to ~60% of its total production. However, infectious diseases often break out among farmed flounders, causing high mortality and substantial economic losses. Although some deleterious pathogens, such as Vibrio spp. and Streptococcus iniae , have been eradicated or contained over the years through vaccination and proper health management, the current disease status of Korean flounder shows that the viral hemorrhagic septicemia virus (VHSV), Streptococcus parauberis , and Miamiensis avidus are causing serious disease problem in recent years. Furthermore, these three pathogens have differing optimal temperature and can attack young fingerlings and mature fish throughout the year-round culture cycle. In this context, we developed a chitosan-poly(lactide-co-glycolide) (PLGA)-encapsulated trivalent vaccine containing formalin-killed VHSV, S. parauberis serotype-I, and M. avidus and administered it to olive flounder fingerlings by immersion route using a prime-boost strategy. At 35 days post-initial vaccination, three separate challenge experiments were conducted via intraperitoneal injection with the three targeted pathogens at their respective optimal temperature. The relative percentages of survival were 66.63%, 53.3%, and 66.75% in the group immunized against VHSV, S. parauberis serotype-I, and M. avidus , respectively, compared to the non-vaccinated challenge (NVC) control group. The immunized fish also demonstrated significantly ( p < 0.05) higher specific antibody titers in serum and higher transcript levels of Ig genes in the mucosal and systemic tissues than those of NVC control fish. Furthermore, the study showed significant ( p < 0.05) upregulation of various immune genes in the vaccinated fish, suggesting induction of strong protective immune response, ultimately leading to improved survival against the three pathogens. Thus, the formulated mucosal vaccine can be an effective prophylactic measure against VHS, streptococcosis, and scuticociliatosis diseases in olive flounder.

The rhabdovirus viral haemorrhagic septicemia virus (VHSV) is the etiological agent of one of the most important salmonid viral diseases. In the present work, the ability of VHSV to infect and replicate in zebrafish at low temperature (15 °C) was demonstrated. Zebrafish was also used to determine the effectiveness of the recombinant virus rIHNV-Gvhsv GFP as a live attenuated vaccine against the virulent VHSV strain. Fish intraperitoneally injected with 3 × 10 6 to 3 × 10 5 TCID 50/ml of the wild type VHSV showed a 100% of cumulative mortality, meanwhile only 57% of mortality was obtained in bath infections. Infected fish showed external clinical signs and histological observations revealed the appearance of small haemorrhages in the muscle, kidney, liver and dermis. Neither mortalities nor clinical signs were recorded in fish infected with a live attenuated recombinant virus. By RT-PCR technique, VHSV was detected in all the organs as early as 24 h, but the recombinant virus was not detected in all the sampled days. VHSV was able to replicate “in vitro” in head kidney cells but the replication capacity of the attenuated viral strain was limited. The recombinant virus rIHNV-Gvhsv GFP was able to protect against VHSV with a survival rate ranging from 20% to 60% depending of the vaccine dose. The increase of TLR3, IFNαβ, Mx, IFNγ and TNFα expression at 72 h post-infection in the kidney of VHSV-infected fish contrasted with the results obtained with the avirulent virus, which did not induce an increment of this expression in infected fish. Zebrafish is a suitable animal model to study VHSV infection and immune (innate and adaptive) responses and, more importantly, we demonstrate for the first time the usefulness of the zebrafish as a vaccination model to viral diseases. In addition, the high protection obtained with the live attenuated virus demonstrates that the zebrafish is able to mount an efficient antiviral immune response at 15 °C.

In the European Union Viral Haemorrhagic Septicaemia (VHS) eradication is still based on stamping out. Due to the lack of effective low cost vaccines immune prophylaxis is currently not used to combat VHS. This paper describes a new oral delivery method for immunisation of trout with attenuated virus. The vaccine consists of lyophilised virus surrounded by polyethylene glycol (PEG) and was extruded under low temperature. In the stomach of trout, the use of additional neutralising and adsorbing bases resulted in a neutral pH around the vaccine pellets, thus protecting the antigen against gastric acid. The in vivo efficacy of this delivery method was examined in three animal challenge experiments using an attenuated VHS virus (VHSV) strain as a vaccine. After vaccination, VHSV mRNA in gut, heart, kidney, spleen and blood was amplified by semi-nested PCR after RT-PCR. Indirect immune fluorescence test detected VHS vaccine virus in the gut. The expression of MHC class II, CD4 and CD8α mRNAs after oral vaccination was measured in gut using real-time RT-PCR. Antibody levels were measured by ELISA one week before vaccination and five weeks after vaccination. Animals were challenged six weeks after vaccination with highly virulent VHSV and mortality was recorded. The experiments showed that orally delivered vaccine virus was released from the vaccine preparation, penetrated the gut mucosa and led to higher expression levels of MHC class II and CD4 mRNAs when compared to control guts. VHSV antibodies were detected after oral vaccination. Immunisation with this new vaccine formulation was followed by a significant protection against VHSV. While the cumulative mortality in the non-vaccinated control group reached 70%, more than 75% of the orally vaccinated fish were protected upon challenge.