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Abstract Infectious salmon anemia virus (ISAV) is the causative agent of infectious salmon anemia (ISA), a relatively novel disease primarily affecting farmed salmon species, primarily in Salmo salar specimens, causing severe outbreaks in most producer countries. Although ISAV has been extensively studied at the molecular level, not much is known about the host/cell interaction at the small RNA level. MicroRNAs (miRNAs) are small, non-coding RNA that regulate mRNA expression at the post-transcriptional level. In recent years, the putative role of these molecules in host-pathogen interactions has drawn particular attention because of their pivotal involvement as regulatory elements in a number of eukaryotic organisms. Given the importance of the salmon industry in Chile, a deep understanding of the interaction between ISAV and its hosts is of importance. In the present work, we studied the kinetic expression of selected miRNAs during ISAV infection, both in vitro and in vivo. Based on initial experimental data derived from a small RNA-Seq analysis, a group of miRNAs that were differentially expressed in infected cells were selected for analysis. As a result, two miRNAs, miR-462a-5p and miR-125 b-5p, showed increased and decreased expression, respectively, during ISAV infection. Highlights • Expression patterns of particular miRNAs change during in vitro and in vivo ISAV infections. • One of the distinguishable changes in miRNA expression corresponds to miR-462a-5p which significantly increases its expression during both, in vitro and in vivo, ISAV infections. o Other distinguishable change corresponds to miR-125b-5p which diminish its expression during in vitro ISAV infections. [ABSTRACT FROM AUTHOR]

The infectious salmon anemia virus (ISAV) is an aquatic pathogen that is a member of the Orthomyxoviridae family with lethal hemorrhagic potential. Although it affects other species of salmonid fish, ISAV only causes disease in Atlantic salmon ( Salmo salar ) specimens in sea water. In spite of the fact that the virus has been described as enveloped with icosahedral symmetry, viral like particles with anomalous morphology have been observed in field samples, this we have not been able to recover then in adequate quantities for full demonstration. We report a procedure to concentrate and recover these novel forms of the virus, comparing two cell lines from different origins, demonstrating that these forms were preferentially expressed in cells of epithelial origin. [ABSTRACT FROM AUTHOR]

Background: The detection of pathogens at early stages of infection is a key point for disease control in aquaculture. Therefore, accurate diagnostic procedures are a must. Real-time PCR has been a mainstay in diagnostics over the years due to its speed, specificity, sensitivity, reproducibility and throughput; as such, real-time PCR is a target for improvement. Nevertheless, to validate a novel diagnostic tool, correct setup of the assay, including proper endogenous controls to evaluate the quantity and quality of the samples and to detect possible sample degradation, is compulsory. This work aims to design a unique RT-qPCR assay for pathogen detection in the three salmonid species reared in Chile. The assay uses elongation factor 1 alpha as the single endogenous control, thus avoiding the need for multiple endogenous controls, as well as multiple validations and non-comparable quality control parameters. Results: The in vivo and in vitro analyses of samples from Salmo salar, Oncorhynchus mykiss and Oncorhynchus kisutch showed that when primers were accurately selected to target conserved regions of the elongation factor 1 alpha (ELF1α) gene, a single novel RT-qPCR assay yielding similar and reproducible Ct values between the three species could be designed. The opposite occurred when an assay originally designed for Salmo salar was tested in samples from the two species of the genus Oncorhynchus. Conclusions: Here, we report the design and evaluation of an accurate trans-species RT-qPCR assay that uses the elongation factor 1 alpha (ELF1α) gene as an endogenous control and is applicable for diagnostic purposes in samples obtained from the three salmonid species reared in Chile. [ABSTRACT FROM AUTHOR]

The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II α and class II β loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II α and β genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIα gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites. [ABSTRACT FROM AUTHOR]

Viral infections in salmonids represent an ongoing challenge for the aquaculture industry. Two RNA viruses, the infectious pancreatic necrosis virus (IPNV) and the infectious salmon anemia virus (ISAV), have become a latent risk without healing therapies available for either. In this context, antiviral peptides emerge as effective and relatively safe therapeutic molecules. Based on in silico analysis of VP2 protein from IPNV and the RNA-dependent RNA polymerase from ISAV, a set of peptides was designed and were chemically synthesized to block selected key events in their corresponding infectivity processes. The peptides were tested in fish cell lines in vitro, and four were selected for decreasing the viral load: peptide GIM182 for IPNV, and peptides GIM535, GIM538 and GIM539 for ISAV. In vivo tests with the IPNV GIM 182 peptide were carried out using Salmo salar fish, showing a significant decrease of viral load, and proving the safety of the peptide for fish. The results indicate that the use of peptides as antiviral agents in disease control might be a viable alternative to explore in aquaculture. [ABSTRACT FROM AUTHOR]

Plasma creatine kinase (CK) release is a marker of damage in fish; their expression can indicate drug/contaminant exposure. Three CK isoforms have been described in fish: muscle CK (M-CK), brain CK (B-CK) and mitochondrial sarcomeric, and the release of any of them could be detected in the serum. Nevertheless, the expression and properties of M-CK in Atlantic salmon are still unknown. In this study, we evaluated differential M-CK expression in Atlantic salmon ( Salmo salar L) skeletal muscle and serum at 0, 5, 10, and 15 days post-infection (dpi) with the infectious salmon anaemia virus, a primary aquaculture pathogen in Chile. The M-CK isoform was assessed using a Western blot-validated antibody. Results for qPCR (+10.8 AU) and ELISA (1.24, p < 0.05) showed increased skeletal muscle M-CK at 10 dpi (r = 0.9817, p < 0.05). Serum M-CK significantly increased at 5 and 10 dpi (r = 1.26 and 1.17, p < 0.05). In situ M-CK detection in the myofibrils showed co-localization with the pro-inflammatory cytokine IL-1β, particularly at 10 dpi. In summary, serum M-CK detection apparently serves as a non-invasive, functional indicator for Atlantic salmon skeletal muscle damage, which could indicate disease or environmental stress. [ABSTRACT FROM AUTHOR]

Background: Endosomally produced by eukaryotic cells, exosomes are microvesicles involved in cell-tocell communication. Exosomes have shown a wide range of therapeutic potential as a drug or vaccine delivery system, and they are useful as biomarkers in several disease processes. Another biological function described is pathogen dissemination through host-derived molecules released during infection, thus modulating the immune response in the host. Results: This work characterizes the exosomal fraction recovered from serum of Piscirickttesia salmonischallenged Salmo salar specimens and from the corresponding non-challenged controls. Exosomes presented a spherical morphology and particle size distribution within 50-125 nm, showing similar parameters in both groups. The mass spectrometry analysis of exosomes isolated at 14 and 21 d post-challenge showed the presence of peptides corresponding to the three proteins of Hsp70/DnaK chaperone system (DnaK, DnaJ, and GrpE). BLAST search of these peptides showed the specificity to P. salmonis. Data are available via ProteomeXchange with identifier PXD023594. Conclusions: The chaperones were found with >95% identity in the core genome when aligned to 73 genomes of P. salmonis. The proteins also showed a high degree of similarity with other microorganisms, where this system has proven to be vital for their survival under stress conditions. The presence of these three proteins in exosomes isolated from challenged fish sera calls for further study into their potential role in bacterium pathogenicity. [ABSTRACT FROM AUTHOR]

Infectious salmon anemia virus (ISAV) is the etiological agent of the disease by the same name and causes major losses in the salmon industry worldwide. Epizootic ISAV outbreaks have occurred in Norway and, to a lesser degree, in Canada. In 2007, an ISAV outbreak in Chile destroyed most of the seasonal production and endangered the entire Chilean salmon industry. None of the existing prophylactic approaches have demonstrated efficacy in providing absolute protection from or even a palliative effect on ISAV proliferation. Sanitary control measures for ISAV, based on molecular epidemiology data, have proven insufficient, mainly due to high salmon culture densities and a constant presence of a nonpathogenic strain of the virus. This report describes an alternative treatment approach based on interfering peptides selected from a phage display library. The screening of a phage display heptapeptide library resulted in the selection of a novel peptide with significant in vitro antiviral activity against ISAV. This peptide specifically interacted with the viral hemagglutinin-esterase protein, thereby impairing virus binding, with plaque reduction assays showing a significant reduction in viral yields. The identified peptide acts at micromolar concentrations against at least two different pathogenic strains of the virus, without detectable cytotoxic effects on the tested fish cells. Therefore, antiviral peptides represent a novel alternative for controlling ISAV and, potentially, other fish pathogens. [ABSTRACT FROM AUTHOR]

Multiple cellular components are involved in pathogen-host interaction during viral infection; in this context, the role of miRNAs have become highly relevant. We assessed the expression of selected miRNAs during an in vitro infection of a Salmo salar cell line with Infectious Salmon Anemia Virus (ISAV), the causative agent of a severe disease by the same name. Salmon orthologs for miRNAs that regulate antiviral responses were measured using RT-qPCR in an in vitro time-course assay. We observed a modulation of specific miRNAs expression, where ssa-miR-155-5p was differentially over-expressed. Using in silico analysis, we identified the putative mRNA targets for ssa-miR-155-5p, finding a high prevalence of hosts immune response-related genes; moreover, several mRNAs involved in the viral infective process were also identified as targets for this miRNA. Our results suggest a relevant role for miR-155-5p in Salmo salar during an ISAV infection as a regulator of the immune response to the virus. • We assessed the miRNAs expression during infection of a Salmo salar cell line with Infectious Salmon Anemia Virus (ISAV). • We detected an induction of ssa-miR-155-5p during the in vitro ISAV infection. • in silico analysis revealed an enrichment of immune-related mRNA targets for ssa-miR-155-5p. • Our results suggest a relevant role for miR-155 in Salmo salar. [ABSTRACT FROM AUTHOR]

C-Type Lectin Receptors (CTLR) are involved in the activation of innate and adaptative immune responses. Among these receptors, the Dendritic Cell-Specific ICAM-3-Grabbing nonintegrin (DC-SIGN/CD209) has become a hot topic due to its ability to bind and facilitate the infections processes of several pathogens. Although well characterized in mammals, little documentation exists about the receptor in salmonid fishes. Here, we report the sequence and expression analysis of eight DC-SIGN-like genes in Salmo salar. Each receptor displays structural similarities to DC-SIGN molecules described in mammals, including internalization motifs, a neck region with heptad repeats, and a Ca+2-dependent carbohydrate recognition domain. The receptors are expressed in multiple tissues of fish, and fish cell lines, with differential expression upon infection with viral and bacterial pathogens. The identification of DC-SIGN-like receptors in Salmo salar provides new information regarding the structure of the immune system of salmon, potential markers for cell subsets, as well as insights into DC-SIGN conservation across species. • DC-SIGN receptors are involved in the activation of innate and adaptative immune responses in mammals. • We describe eight genes in Salmo salar coding for DC-SIGN-like receptors. • SsSIGNs are expressed in multiple fish tissues, and gene expression is differentially regulated upon infection. [ABSTRACT FROM AUTHOR]