Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA.

The piscine orthomyxovirus called infectious salmon anemia virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral, and mammalian genetic elements included in the pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells.

In silico prediction of B and T cell epitopes of infectious salmon anemia virus proteins and molecular modeling of T cell epitopes to salmon major histocompatibility complex (MHC) class I.

Infectious salmon anemia (ISA) can be devastating in farmed Atlantic salmon (Salmo salar). The disease can evolve into epidemics if it is not contained and controlled. ISA epidemics were seen in Norway in the early 1990s and Chile in 2007-2009. Consequently, there is an urgent need to develop a vaccine to prevent or treat the infection. In this study, an immunoinformatic approach was employed to predict 32 lineal B-cell epitopes based on antigenicity and surface accessibility prediction for ISAV fusion (F), hemagglutinin-esterase (HE), and matrix (M) proteins. On the other hand, twelve conformational B-cell epitopes were also predicted. We further identified six antigenic cytotoxic T lymphocyte (CTL) epitopes and investigated the binding interactions with five salmon MHC-I proteins after docking the peptides to the binding groove of the MHC-I proteins. Our results showed that all the predicted epitopes could bind to salmon MHC-I with high negative ΔG values with medium to high binding affinities. Hence, the predicted epitopes have a high potential of being recognized by Atlantic salmon MHC-I to elicit a CD8+ T cell response in salmon. The predicted and analyzed B and T cell antigenic epitopes in this work might present an initial set of peptides for future vaccine development against ISAV. The ability to model and predict these interactions will ultimately lead to the ability to predict potential binding for MHCs and epitopes that were not studied previously. As current knowledge of salmon MHC specificity is limited, studying and modeling interactions in the peptide/MHC complex is a key to resolving unknown epitope specificity.

Effects of cholesterol on lipid vesicle fusion mediated by infectious salmon anaemia virus fusion peptides.

Studying the variables that affect the membrane fusion mechanism of enveloped viruses is important for developing new strategies to combat viral infections. We analysed the effects of lipid vesicle cholesterol content on membrane fusion that is facilitated by infectious salmon anaemia virus (ISAV) fusion peptides. Lipid mixing assays were performed to study membrane fusion in large unilamellar vesicles (LUV) composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC) and cholesterol. Lipid mixing (%) increased more over time when 0.2 µm LUV contained no cholesterol or when the LUV membranes contained 15 mol% cholesterol. The secondary structure of the ISAV fusion peptides consistently remained a ß-sheet both in water and in the presence of vesicles. Additionally, the dissociation constant (Kd) between the peptides and the lipid vesicles was obtained with different cholesterol contents. In the tests performed with lipid vesicles (0.2 µm or 0.4 µm LUV), cholesterol was found to influence membrane fusion that was facilitated by ISAV fusion peptides; however, the peptides studied did not require cholesterol in their membranes to facilitate membrane fusion in the smallest lipid vesicles (0.2 µm LUV).

Infectious Salmon Anemia Virus Infectivity Is Determined by Multiple Segments with an Important Contribution from Segment 5.

Infectious salmon anemia virus (ISAV) is the etiological agent of infectious salmon anemia. It belongs to the genus isavirus, one of the genera of the Orthomyxoviridae family, as does Influenzavirus A. The ISAV genome comprises eight negative-sense single-stranded RNA segments that code for at least 10 proteins. Although some ISAV strains can reach 100% mortality rates, the factors that determine isavirus infectivity remain unknown. However, some studies suggest that segments 5 and 6 are responsible for the different degrees of virulence and infectivity among ISAV subtypes, unlike the influenza A virus, where most segments are involved in the virus infectivity. In this work, synthetic reassortant viruses for the eight segments of ISAV were generated by reverse genetics, combining a highly virulent virus, ISAV 752_09 (HPR7b), and an avirulent strain, SK779/06 (HPR0). We characterized the rescued viruses and their capacity to replicate and infect different cell lines, produce plaques in ASK cells, and their ability to induce and modulate the cellular immune response in vitro. Our results show that the majority of ISAV segments are involved in at least one of the analyzed characteristics, segment 5 being one of the most important, allowing HPR0 viruses, among other things, to produce plaques and replicate in CHSE-214 cells. We determined that segments 5 and 6 participate in different stages of the viral cycle, and their compatibility is critical for viral infection. Additionally, we demonstrated that segment 2 can modulate the cellular immune response. Our results indicate a high degree of genetic compatibility between the genomic segments of HPR7b and HPR0, representing a latent risk of reassortant that would give rise to a new virus with an unknown phenotype.

Expression of ssa-miR-155 during ISAV infection in vitro: Putative role as a modulator of the immune response in Salmo salar.

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.

Rapid sequence modification in the highly polymorphic region (HPR) of the hemagglutinin gene of the infectious salmon anaemia virus (ISAV) suggests intra-segmental template switching recombination.

The ISAV has a genome composed of eight segments of (-)ssRNA, segment 6 codes for the hemagglutinin-esterase protein, and has the most variable region of the genome, the highly polymorphic region (HPR), which is unique among orthomyxoviruses. The HPR has been associated with virulence, infectivity and pathogenicity. The full length of the HPR is called HPR0 and the strain with this HPR is avirulent, in contrast to strains with deleted HPR that are virulent to varying degrees. The molecular mechanism that gives rise to the different HPRs remains unclear. Here, we studied in vitro the evolution of reassortant recombinant ISAV (rISAV) in Atlantic salmon head kidney (ASK) cells. To this end, we rescued and cultivated a set of rISAV with different segment 6-HPR genotypes using a reverse genetics system and then sequencing HPR regions of the viruses. Our results show rapid multiple recombination events in ISAV, with sequence insertions and deletions in the HPR, indicating a dynamic process. Inserted sequences can be found in four segments of the ISAV genome (segments 1, 5, 6, and 8). The results suggest intra-segmental heterologous recombination, probably by class I and class II template switching, similar to the proposed segment 5 recombination mechanism.

Expression of DC-SIGN-like C-Type Lectin Receptors in Salmo salar.

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.

Chitosan-Based Nanoparticles for Intracellular Delivery of ISAV Fusion Protein cDNA into Melanoma Cells: A Path to Develop Oncolytic Anticancer Therapies.

Oncolytic virus therapy has been tested against cancer in preclinical models and clinical assays. Current evidence shows that viruses induce cytopathic effects associated with fusogenic protein-mediated syncytium formation and immunogenic cell death of eukaryotic cells. We have previously demonstrated that tumor cell bodies generated from cells expressing the fusogenic protein of the infectious salmon anemia virus (ISAV-F) enhance crosspriming and display prophylactic antitumor activity against melanoma tumors. In this work, we evaluated the effects of the expression of ISAV-F on the B16 melanoma model, both in vitro and in vivo, using chitosan nanoparticles as transfection vehicle. We confirmed that the transfection of B16 tumor cells with chitosan nanoparticles (NP-ISAV) allows the expression of a fusogenically active ISAV-F protein and decreases cell viability because of syncytium formation in vitro. However, the in vivo transfection induces a delay in tumor growth, without inducing changes on the lymphoid populations in the tumor and the spleen. Altogether, our observations show that expression of ISAV fusion protein using chitosan nanoparticles induces cell fusion in melanoma cells and slight antitumor response.

Development of an Isavirus minigenome system to study the function of the pocket RNA-binding domain of the viral nucleoprotein (NP) in salmon cells.

The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (-ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs.

Recovering sustainability after a health crisis in aquatic animals.

Chile faced a severe aquatic animal health crisis in 2007 that affected the production of Atlantic salmon (Salmo salar) after an outbreak of infectious salmon anaemia (ISA). The outbreak had a considerable national economic impact. The response was led by the Competent Authority, the National Fisheries and Aquaculture Service (Sernapesca), which immediately implemented surveillance and control actions to mitigate the crisis. At the end of the initial response, the Competent Authority, together with the industry, set out a roadmap to return to sustainable salmon production. The success of the response was due to early detection and the implementation of biosecurity and control measures at all stages of production and control. These measures underpin the sanitary management model for aquaculture. The Chilean Veterinary Service has analysed critical health measures for salmon production and concluded that there has been an improvement in fish health, as evidenced by decreased mortalities, reduced use of antimicrobials, and improved management and control of prevalent diseases, such as salmon rickettsial syndrome (piscirickettsiosis), caligidosis and ISA. Improvements in health have contributed to increased harvests over time, with the largest monthly harvest for Atlantic salmon being achieved in January-February 2018, with 120,000 tonnes. The ISA crisis provided salutary lessons for the continued recovery and sustainability of Chile's salmon sector. The crisis highlighted the importance of strengthened Veterinary Services and public-private links, as well as a collaborative relationship with research entities and training centres. It was also important to enact new regulations to ensure recovery and sustainability. Fundamentally, the response to this crisis was based upon having good baseline surveillance already in place, supported by a Veterinary Service trained to manage emergency disease outbreaks.

Le Chili a fait face à une crise sanitaire majeure chez les animaux aquatiques suite à la survenue en 2007 d'un foyer d'anémie infectieuse du saumon qui a affecté la production de saumons atlantiques (Salmo salar). L'impact économique de ce foyer a été considérable à l'échelle nationale. Les mesures d'intervention d'urgence ont été conduites par l'autorité compétente, le Service national chilien de la pêche et de l'aquaculture (Sernapesca) qui a immédiatement mis en place des mesures de surveillance et de lutte afin d'atténuer la crise. À l'issue de cette réponse initiale, l'autorité compétente a élaboré une feuille de route avec le concours du secteur salmonicole afin de restaurer la durabilité de la production de saumons. La réussite de cette réponse est due aux capacités de détection précoce et à la mise en œuvre de mesures de biosécurité et de suivi à chaque étape de la production et des contrôles. Ces mesures constituent la base du modèle de gestion sanitaire en aquaculture. Les Services vétérinaires chiliens ont analysé les mesures sanitaires critiques applicables aux élevages de saumons et conclu à une nette amélioration de la santé des poissons, attestée par la baisse de la mortalité, la réduction des quantités d'agents antimicrobiens utilisés et une gestion et un contrôle plus efficaces des maladies présentes dans le pays, en particulier la piscirickettsiose des salmonidés, la caligidose et l'anémie infectieuse du saumon. L'amélioration de la situation sanitaire a contribué à l'augmentation progressive des volumes de production, qui pour le saumon atlantique ont atteint leur plus haut niveau mensuel en janvier-février 2018, avec 120 000 tonnes produites. La crise due à l'anémie infectieuse du saumon a permis de tirer de précieux enseignements qui permettront une récupération durable du secteur salmonicole au Chili. La crise a mis en évidence l'importance de Services vétérinaires renforcés et des partenariats public­privé ainsi que des liens de collaboration avec les institutions de recherche et les centres de formation. L'élaboration d'une nouvelle réglementation a également joué un rôle pour assurer la récupération et la durabilité. Essentiellement, la réponse à cette crise a reposé sur la qualité du système de surveillance déjà en place et sur le soutien de Services vétérinaires formés à la gestion des urgences liées à des foyers de maladie.

Chile hizo frente a una importante crisis sanitaria que se presentó en 2007 y afectó a la producción del salmón del Atlántico (Salmo salar) a raíz de un brote de anemia infecciosa del salmón (ISA). El brote tuvo un gran impacto económico para el país. La repuesta fue dirigida por la autoridad competente, el Servicio Nacional de Pesca y Acuicultura (Sernapesca), que implementó de inmediato medidas de vigilancia y control para mitigar la crisis. Una vez abordados todos los aspectos iniciales de la respuesta inmediata, la autoridad competente, en conjunto con la industria, elaboró una hoja de ruta con el objetivo de mantener una producción sostenible. El éxito de la respuesta se debe a las capacidades de detección temprana y a las medidas de bioseguridad y de vigilancia implementadas en cada etapa del proceso de producción y de control. Estas medidas constituyen la base de la gestión sanitaria de la acuicultura. En base al análisis de las medidas sanitarias aplicables al sector de la salmonicultura realizado por el Servicio Veterinario de Chile, se concluyó que la situación sanitaria había mejorado en los distintos aspectos críticos para esta producción, tales como una evidente disminución de la mortalidad, disminución del uso de antimicrobianos, mejora en la prevalencia y el control de las principales enfermedades prevalentes en la salmonicultura, como piscirickettsiosis, caligidosis e ISA. La mejora de la situación sanitaria contribuyó a la tendencia al alza respecto de las cosechas, alcanzando el máximo mensual de cosechas para el salmón del Atlántico en el mes de enero-febrero 2018, con 120 000 toneladas. Como lecciones aprendidas en términos de recuperación y sostenibilidad del sector de producción del salmón, es importante señalar que, ante la presentación de la crisis, se generó una oportunidad de potenciar a los Servicios Veterinarios, estrechar los vínculos público-privados y desarrollar la colaboración con entidades de investigación y centros de formación. También fue importante promulgar nuevas regulaciones para garantizar la recuperación y la sostenibilidad. Fundamentalmente, la respuesta a esta crisis se basó en contar con una buena vigilancia de referencia ya implementada y con el apoyo de un Servicio Veterinario capacitado para manejar las emergencias debidas a brotes de enfermedades.

Sumoylation of nucleoprotein (NP) mediated by activation of NADPH oxidase complex is a consequence of oxidative cellular stress during infection by Infectious salmon anemia (ISA) virus necessary to viral progeny.

The study evaluated the effects of nucleoprotein viral and the infectious virus in SHK-1 cells. The results show a strong respiratory burst activation and the induction of p47phox, SOD, GLURED, and apoptotic genes. Additionally, the cells alter the profile of SUMOylated proteins by the effect of transfection and infection experiments. In silico analyses show a set of structural motifs in NP susceptible of post-translational modification by the SUMO protein. Interestingly, the inhibition of the NADPH oxidase complex blocked the production of reactive oxygen species and the high level of cellular ROS due to the nucleoprotein and the ISAv. At the same time, the blocking of the p38MAPK signaling pathway and the use of Aristotelia chilensis, decreased viral progeny production. These results suggest that the NP triggers a strong production of ROS and modifying the post-translational profile mediated by SUMO-2/3, a phenomenon that favors the production of new virions.

Molecular features associated with the adaptive evolution of Infectious Salmon Anemia Virus (ISAV) in Chile.

Infectious salmon anemia virus (ISAV) is an Orthomyxovirus challenging salmon production, with a particular impact in Chile. During 2007-2010 a devastating and of unexpected consequences epizootic event almost destroyed a blooming industry in the country. The event was caused by an aggressive variant with a distinctive deletion in Segment 6, one of the eight genomic segments of the virus. After the outburst, although the infective viral variant seemed to have disappeared, a non-infective variant, not previously reported, was discovered and is characterized by a complete, non-deleted coding segment 6, which has prevailed in the fish population until now. This variant, known as HPR0, appears to be the ancestor strain of ISAV from which novel infective variants are generated. Additional variations in segment 5 have also been associated with the virulence observed in the field, an analysis of the differences in these two protein coding segments has been performed. It appears to us that a combinatorial effect exists between the features displayed by segments 5 and 6 which modulate the intensity of viral outbursts. As a result, a theoretical integrative model is presented which explains the different degree of virulence observed in the field based only on molecular data, this could help estimating the intensity of damage a given variant might exert over a productive farm.

Involvement of selected cellular miRNAs in the in vitro and in vivo infection of infectious salmon anemia virus (ISAV).

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.

An updated proposal for classification of infectious salmon anemia virus strains.

Biological databases contain a wealth of valuable information that can contribute to the enrichment of virtually any area. However, the exponential growth of information together with its dissemination through virtual networks has become a double-edged sword, promoting synonymy that leads to confusion and chaos. Organization of data is a big effort that must be accompanied by clarity, both in the deposited data and in the publications arising from them. In this report, an effort is made to organize the information related to infectious salmon anemia virus and its classification based on the variability of genomic segment 6.

Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA.

The piscine Orthomyxovirus called Infectious Salmon Anemia Virus (ISAV) is one of the most important emerging pathogens affecting the salmon industry worldwide. The first reverse genetics system for ISAV, which allows the generation of recombinant ISA virus (rISAV), is an important tool for the characterization and study of this fish virus. The plasmid-based reverse genetics system for ISAV includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). The salmon, viral and mammalian genetic elements included in pSS-URG vectors allow the expression of the eight viral RNA segments. In addition to four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex, the eight pSS-URG vectors allowed the generation of infectious rISAV in salmon cells.

Development, Characterisation and Application of Monoclonal Antibodies for the Detection and Quantification of Infectious Salmon Anaemia Virus in Plasma Samples Using Luminex Bead Array Technology.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus that has had a significant economic impact on Atlantic salmon farming in Europe, North America and Chile. Monoclonal antibodies (mAbs) were developed against Segment 3 (encoding the viral nucleoprotein, NP) of the virus. Six of the mAbs were shown to be specific to ISAV and recognised all isolates from Scotland, Norway and Canada. They reacted with ISAV in enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody technique (IFAT) and western blotting. They were also used to develop a novel detection method based on Luminex (Bio-Plex) bead-based flow cytometric technology for the detection of ISAV in the plasma of Atlantic salmon (Salmo salar L.) smolts experimentally infected with ISAV. Fish were challenged by intraperitoneal (i.p.) injection of virus at 50% Tissue Culture Infective Dose (TCID50) = 2.8 x106 per animal. Virus present in plasma of infected fish, collected at 0, 4, 8, 12, 16, 21 and 28 days post infection using a non-lethal sampling method (n = 12 at each time point), was quantified using the optimised Bio-Plex assay. The results obtained with this assay were compared with absolute quantification of the virus by RT-qPCR using SYBR Green I and TaqMan chemistries. The Bio-Plex assay developed using the NP mAbs appears to be a rapid, sensitive method for detecting and quantifying ISAV in small volumes of fish plasma and has the potential to be multiplexed for the detection of other fish pathogens (e.g. during co-infections). To our knowledge this is the first report of the use of Luminex (Bio-Plex) technology for the detection of a fish pathogen.

Protective oral vaccination against infectious salmon anaemia virus in Salmo salar.

Infectious salmon anemia (ISA) is a systemic disease caused by an orthomyxovirus, which has a significant economic impact on the production of Atlantic salmon (Salmo salar). Currently, there are several commercial ISA vaccines available, however, those products are applied through injection, causing stress in the fish and leaving them susceptible to infectious diseases due to the injection process and associated handling. In this study, we evaluated an oral vaccine against ISA containing a recombinant viral hemagglutinin-esterase and a fusion protein as antigens. Our findings indicated that oral vaccination is able to protect Atlantic salmon against challenge with a high-virulence Chilean isolate. The oral vaccination was also correlated with the induction of IgM-specific antibodies. On the other hand, the vaccine was unable to modulate expression of the antiviral related gene Mx, showing the importance of the humoral response to the disease survival. This study provides new insights into fish protection and immune response induced by an oral vaccine against ISA, but also promises future development of preventive solutions or validation of the current existing therapies.

Long noncoding RNAs (lncRNAs) dynamics evidence immunomodulation during ISAV-Infected Atlantic salmon (Salmo salar).

Despite evidence for participation in the host response to infection, the roles of many long non-coding RNAs (lncRNAs) remain unknown. Therefore, the aims of this study were to identify lncRNAs in Atlantic salmon (Salmo salar) and evaluate their transcriptomic regulation during ISA virus (ISAV) infection, an Orthomyxoviridae virus associated with high mortalities in salmonid aquaculture. Using next-generation sequencing, whole-transcriptome analysis of the Salmo salar response to ISAV infection was performed, identifying 5,636 putative lncRNAs with a mean length of 695 base pairs. The transcriptional modulation evidenced a similar number of differentially expressed lncRNAs in the gills (3,294), head-kidney (3,275), and liver (3,325) over the course of the infection. Moreover, analysis of a subset of these lncRNAs showed the following: (i) Most were similarly regulated in response to ISA virus infection; (ii) The transcript subsets were uniquely modulated in each tissue (gills, liver, and head-kidney); and (iii) A subset of lncRNAs were upregulated for each tissue and time analysed, indicating potential markers for ISAV infection. These findings represent the first discovery of widespread differential expression of lncRNAs in response to virus infection in non-model species, suggesting that lncRNAs could be involved in regulating the host response during ISAV infection.

Chemical Synthesis and In Vitro Evaluation of a Phage Display-Derived Peptide Active against Infectious Salmon Anemia Virus.

UNLABELLED: 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. IMPORTANCE: Identifying novel methods for the efficient control of infectious diseases is imperative for the future of global aquaculture. The present study used a phage display heptapeptide library to identify a peptide with interfering activity against a key protein of the infectious salmon anemia virus (ISAV). A piscine orthomyxovirus, ISAV is a continuous threat to the commercial sustainability of cultured salmon production worldwide. The complex epidemiological strategy of this pathogen has made prophylactic control extremely difficult. The identified antiviral peptide efficiently impairs ISAV infection in vitro by specifically blocking hemagglutinin-esterase, a pivotal surface protein of this virus. Peptide synthesis could further modify the primary structure of the identified peptide to improve specific activity and stability. The present results form the foundation for developing a new pharmacological treatment against ISAV.

Area contact networks and the spatio-temporal spread of infectious salmon anemia virus (ISAV) in Chile.

Area management, the coordination of production and biosecurity practices across neighboring farms, is an important disease control strategy in aquaculture. Area management in aquaculture escalated in prominence in response to outbreaks of infectious salmon anemia (ISA) internationally. Successes in disease control have been attributed to the separation achieved through area-level synchronized stocking, fallowing, movement restrictions, and fomite or pest control. Area management, however, is costly; often demanding extra biosecurity, lengthy or inconveniently timed fallows, and localization of equipment, personnel, and services. Yet, this higher-order organizational structure has received limited epidemiologic attention. Chile's National Fisheries and Aquaculture Service instigated area management practices in response to the 2007 emergence of ISA virus (ISAV). Longitudinal data simultaneously collected allowed retrospective evaluation of the impact of component tenets on virus control. Spatiotemporal analyses identified hydrographic linkages, shared ports, and fish transfers from areas with recent occurrence of ISAV as the strongest predictors of virus spread between areas, though specifics varied by ISAV type (here categorized as HPR0 for the non-virulent genotypes, and HPRv otherwise). Hydrographic linkages were most predictive in the period before implementation of enhanced biosecurity and fallowing regulations, suggesting that viral load can impact spread dynamics. HPR0 arose late in the study period, so few HPRv events were available by which to explore the hypothesis of HPR0 as progenitor of outbreaks. However, spatiotemporal patterns in HPRv occurrence were predictive of subsequent patterns in HPR0 detection, suggesting a parallel, or dependent, means of spread. Better data precision, breadth and consistency, common challenges for retrospective studies, could improve model fit; and, for HPR0, specification of diagnostic test accuracy would improve interpretation.