Search Results (7)

Several studies have demonstrated that methionine supplementation in fish diets enhances immune status, inflammatory response, and resistance to bacterial infections by modulating for DNA methylation, aminopropylation, and transsulfuration pathways. However, the immunomodulatory effects of methionine in viral infections remain unexplored. This study aimed to evaluate the effect of methionine supplementation on immune modulation and resistance to the viral haemorrhagic septicaemia virus (VHSV) in rainbow trout (Oncorhynchus mykiss). Two diets were formulated and fed to juvenile rainbow trout for four weeks: a control diet (CTRL) with all nutritional requirements, including the amino acid profile required for the species, and a methionine-supplemented diet (MET), containing twice the normal requirement of DL-methionine. After feeding, fish were bath-infected with VHSV, while control fish were exposed to a virus-free bath. Samples were collected at 0 (after feeding trial), 24, 72, and 120 h post-infection for the haematological profile, humoral immune response, oxidative stress, viral load, RNAseq, and gene expression analysis. In both diets, results showed a peak in viral activity at 72 h, followed by a reduction in viral load at 120 h, indicating immune recovery. During the peak of infection, leukocytes, thrombocytes, and monocytes migrated to the infection site, while oxidative stress biomarkers (superoxide dismutase glutathione S-transferase, and glutathione redox ratio) suggested a compromised ability to manage cellular imbalance due to intense viral activity. At 120 h, immune recovery and homeostasis were observed due to an increase in the amount of nitric oxide, GSH/GSSG levels, leukocyte replacement, monocyte influx, and a reduction in the viral load. When focusing on the infection peak, gene ontology (GO) analysis showed several exclusively enriched pathways in the skin and gills of MET-fed fish, driven by the upregulation of several key genes. Genes involved in recognition/signalling, inflammatory response, and other genes with direct antiviral activity, such as TLR3, MYD88, TRAF2, NF-κB, STING, IRF3, -7, VIG1, caspases, cathepsins, and TNF, were observed. Notably, VIG1 (viperin), a key antiviral protein, was significantly upregulated in gills, confirming the modulatory role of methionine in inducing its transcription. Viperin, which harbours an S-adenosyl-L-methionine (SAM) radical domain, is directly related to methionine biosynthesis and plays a critical role in the innate immune response to VHSV infection in rainbow trout. In summary, this study suggests that dietary methionine supplementation can enhance a more robust fish immune response to viral infections, with viperin as a crucial mediator. The improved antiviral readiness observed in MET-fed fish underscores the potential of targeted nutritional adjustments to sustain fish health and welfare in aquaculture. Full article

The first known outbreak caused by a viral haemorrhagic septicaemia virus (VHSV) strain of genotype III in rainbow trout occurred in 2007 at a marine farm in Storfjorden, Norway. The source of the virus is unknown, and cod and other marine fish around the farms are suspected as a possible reservoir. The main objective of this study was to test the susceptibility of juvenile Atlantic cod to the VHSV isolate from Storfjorden. As the pathology of VHS in cod is sparsely described, an additional aim of the study was to give a histopathological description of the disease. Two separate challenge experiments were carried out, using both intra peritoneal (ip) injection and cohabitation as challenge methods. Mortality in the ip injection experiment leveled at approximately 50% three weeks post challenge. Both immunohistochemical and rRT-PCR analysis of organs sampled from diseased and surviving fish confirmed VHSV infection. No VHSV was detected in the cohabitants. The results indicate that Atlantic cod has a low natural susceptibility to this VHSV genotype III strain. One of the most extensive pathological changes was degeneration of cardiac myocytes. Immunohistochemistry confirmed that the lesions were related to VHSV. In some fish, the hematopoietic tissue of spleen and kidney showed degeneration and immunostaining, classical signs of VHS, as described in rainbow trout. Positive immunostaining of the capillaries of the gills, suggests this organ as a useful alternative when screening for VHSV. Full article

The basis of pathogenicity of viral haemorrhagic septicaemia virus (VHSV) was analysed in the transcriptome of a rainbow trout cell line inoculated with pathogenic and non-pathogenic VHSV isolates. Although both VHSV isolates showed similar viral replication patterns, the number of differentially expressed genes was 42-fold higher in cells inoculated with the non-pathogenic VHSV at 3 h post inoculation (hpi). Infection with the non-pathogenic isolate resulted in Gene Ontologies (GO) enrichment of terms such as immune response, cytokine-mediated signalling pathway, regulation of translational initiation, unfolded protein binding, and protein folding, and induced an over-representation of the p53, PPAR, and TGF-β signalling pathways. Inoculation with the pathogenic isolate resulted in the GO enrichment of terms related to lipid metabolism and the salmonella infection KEGG pathway involved in the rearrangement of the cytoskeleton. Antiviral response was evident at 12hpi in cells infected with the pathogenic isolate. Overall, the data showed a delay in the response of genes involved in immune responses and viral sensing in cells inoculated with the pathogenic isolate and suggest transcriptional shutoff and immune avoidance as a critical mechanism of pathogenicity in VHSV. These pathways offer opportunities to further understand and manage VHSV pathogenicity in rainbow trout. Full article

The viral hemorrhagic septicemia virus (VHSV) is the causative agent of an important disease in freshwater and marine fishes. Its diagnosis officially relies on the isolation of the virus in cell culture and its identification by serological or polymerase chain reaction (PCR) methodologies. Nowadays, reverse transcription real-time quantitative PCR (RT-qPCR) is the most widely employed technique for the detection of this virus and some studies have reported the validation of RT-qPCR procedures for the detection, typing, and quantification of VHSV isolates. However, although the efficacy of this technique is not in doubt, it can be cumbersome and even impractical when it comes to processing large numbers of samples, a situation in which cross-contamination problems cannot be ruled out. In the present study, we have designed and validated a macroarray for the simultaneous detection, typing, and quantification of VHSV strains. Its analytical sensitivity (5–50 TCID₅₀/_mL), analytical specificity (intra and intergroup), efficiency (E = 100.0–101.1) and reliability (repeatability and reproducibility with CV < 5%, and standard curves with R² < 0.95) with strains from any VHSV genotype have been widely demonstrated. The procedure is based on the ‘binary multiplex RT-qPCR system (bmRT-qPCR)’ previously reported by the same team, applied to arrays of 96-well PCR strip tubes plates, which can be stored at −25 °C for three months and up to one year before their use, without significant loss of efficiency. Full article

The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo. Full article

Viral haemorrhagic septicaemia virus (VHSV) is one of the worst viral threats to fish farming. Non-virion (NV) gene-deleted VHSV (dNV-VHSV) has been postulated as an attenuated virus, because the absence of the NV gene leads to lower induced pathogenicity. However, little is known about the immune responses driven by dNV-VHSV and the wild-type (wt)-VHSV in the context of infection. Here, we obtained the immune transcriptome profiling in trout infected with dNV-VHSV and wt-VHSV and the pathways involved in immune responses. As general results, dNV-VHSV upregulated more trout immune genes than wt-VHSV (65.6% vs 45.7%, respectively), whereas wt-VHSV maintained more non-regulated genes than dNV-VHSV (45.7% vs 14.6%, respectively). The modulated pathways analysis (Gene-Set Enrichment Analysis, GSEA) showed that, when compared to wt-VHSV infected trout, the dNV-VHSV infected trout upregulated signalling pathways (n = 19) such as RIG-I (retinoic acid-inducible gene-I) like receptor signalling, Toll-like receptor signalling, type II interferon signalling, and nuclear factor kappa B (NF-kappa B) signalling, among others. The results from individual genes and GSEA demonstrated that wt-VHSV impaired the activation at short stages of infection of pro-inflammatory, antiviral, proliferation, and apoptosis pathways, delaying innate humoral response and cellular crosstalk, whereas dNV-VHSV promoted the opposite effects. Therefore, these results might support future studies on using dNV-VHSV as a potential live vaccine. Full article

Most commercial vaccines offered to the aquaculture industry include inactivated antigens (Ag) formulated in oil adjuvants. Safety concerns are related to the use of oil adjuvants in multivalent vaccines for fish, since adverse side effects (e.g., adhesions) can appear. Therefore, there is a request for vaccine formulations for which protection will be maintained or improved, while the risk of side effects is reduced. Here, by using an inactivated salmonid alphavirus (SAV) as the test Ag, the combined use of two Toll-like receptor (TLR) ligand adjuvants, CpG oligonucleotides (ODNs) and poly I:C, as well as a genetic adjuvant consisting of a DNA plasmid vector expressing the viral haemorrhagic septicaemia virus (VHSV) glycoprotein (G) was explored. VHSV-G DNA vaccine was intramuscularly injected in combination with intraperitoneal injection of either SAV Ag alone or combined with the oil adjuvant, Montanide ISA763, or the CpG/polyI:C combo. Adjuvant formulations were evaluated for their ability to boost immune responses and induce protection against SAV in Atlantic salmon, following cohabitation challenge. It was observed that CpG/polyI:C-based formulations generated the highest neutralizing antibody titres (nAbs) before challenge, which endured post challenge. nAb responses for VHSV G-DNA- and oil-adjuvanted formulations were marginal compared to the CpG/poly I:C treatment. Interestingly, heat-inactivated sera showed reduced nAb titres compared to their non-heated counterparts, which suggests a role of complement-mediated neutralization against SAV. Consistently elevated levels of innate antiviral immune genes in the CpG/polyI:C injected groups suggested a role of IFN-mediated responses. Co-delivery of the VHSV-G DNA construct with either CpG/polyI:C or oil-adjuvanted SAV vaccine generated higher CD4 responses in head kidney at 48 h compared to injection of this vector or SAV Ag alone. The results demonstrate that a combination of pattern recognizing receptor (PRR) ligands, such as CpG/polyI:C, increases both adaptive and innate responses and represents a promising adjuvant strategy for enhancing the protection of future viral vaccines. Full article