Search Results (18)

Metabolomic analysis has been explored to search for disease biomarkers in humans for some time. The application to animal species, including fish, however, is still at the beginning. In the present study, we have used targeted and untargeted metabolomics to identify metabolites in the plasma of Atlantic salmon (Salmo salar) challenged with Piscine orthoreovirus (PRV-1), aiming to find metabolites associated with the progression of PRV-1 infection into heart and skeletal muscle inflammation (HSMI). The metabolomes of control and PRV-1-infected salmon were compared at three time points during disease development by employing different biostatistical approaches. Targeted metabolomics resulted in the determination of affected metabolites and metabolic pathways, revealing a substantial impact of PRV-1 infection on lipid homeostasis, especially on several (lyso)phosphatidylcholines, ceramides, and triglycerides. Untargeted metabolomics showed a clear separation of the treatment groups at later study time points, mainly due to effects on lipid metabolism pathways. In a subsequent multi-omics approach, we combined both metabolomics datasets with previously reported proteomics data generated from the same salmon plasma samples. Data processing with DIABLO software resulted in the identification of significant metabolites and proteins that were representative of the HSMI development in the salmon. Full article

Piscine orthoreovirus (PRV) is a pathogen that causes heart and skeletal muscle inflammation in Salmo salar and has also been linked to circulatory disorders in other farmed salmonids, such as Oncorhynchus kisutch and Oncorhynchus mykiss. The virus has a segmented, double-stranded RNA genome, which makes it possible to undergo genetic reassortment and increase its genomic diversity through point mutations. In this study, genetic reassortment in PRV was assessed using the full genome sequences available in public databases. This study used full genome sequences that were concatenated and genome-wide reassortment events, and phylogenetic analyses were performed using the recombination/reassortment detection program version 5 (RDP5 V 5.5) software. Additionally, each segment was aligned codon by codon, and overall mean distance and selection was tested using the Molecular Evolutionary Genetics Analysis X software, version 10.2 (MEGA X version 10.2). The results showed that there were 17 significant reassortment events in 12 reassortant sequences, involving genome exchange between low and highly virulent genotypes. PRV sequences from different salmonid host species did not appear to limit the reassortment. This study found that PRV frequently undergoes reassortment events to increase the diversity of its segmented genome, leading to antigenic variation and increased virulence. This study also noted that to date, no reassortment events have been described between PRV-1 and PRV-3 genotypes. However, the number of complete genomic sequences within each genotype is uneven. This is important because PRV-3 induces cross-protection against PRV-1, making it a potential vaccine candidate. Full article

Piscine orthoreovirus genotype 1 (PRV-1) is an endemic virus to the Northeastern Pacific that infects both wild and farmed salmon. To better understand PRV-1 prevalence and transmission pathways in the region, we targeted out-migrating juvenile Pacific Salmon in the Strait of Georgia and Johnstone Strait in British Columbia, Canada, for PRV-1 molecular screening with an emphasis on Chinook (Oncorhynchus tshawytscha) and Coho (Oncorhynchus kisutch) salmon. A total of 4031 individuals were screened for PRV-1 and we identified an overall virus prevalence of 3.5% from 2011 to 2020. PRV-1 was absent in enhancement facilities and freshwater catchments and predominately found in the marine environment. The detection of PRV-1 varied greatly between species, year and stock of origin, but cumulatively identified that life history and migratory behaviors likely influenced viral prevalence. Specifically, Chinook salmon, which demonstrate long residence time in the Strait of Georgia relative to other species, had the highest PRV-1 prevalence in this study (7.4%). Varying stock composition and regional detection between year classes within the Strait of Georgia further indicated that the sources for Chinook infection were highly variable between years. These findings enhance our understanding for PRV-1 transmission in the region and more generally between/within salmon species. Full article

Heart and skeletal muscle inflammation (HSMI) caused by Piscine orthoreovirus (PRV) was first described in farmed Atlantic salmon in Chile in 2011. However, as PRV induces long-lasting infections, it is not known when Chilean farmed salmon may have started to show PRV positivity. This study aimed to evaluate the presence/absence of PRV-1 in formalin-fixed, paraffin-embedded Atlantic salmon heart tissues (FFPE) cultured in Chile during 1992 and 1999. The most frequent histopathological findings in the 42 FFPE blocks were mild focal cardiomyocyte degeneration (57.1%) and a mild focal mononuclear inflammatory infiltrate (21.4%) in the ventricular stratum spongiosum of the heart. One of the 42 heart samples analyzed by RT-qPCR was positive for PRV-1 (2.4%). All samples were negative for other viral and bacterial pathogens that can induce similar histological changes in the heart. Taken together, our results show that PRV-1 has been present in Chile—as a low-virulence genogroup—since at least 1994, 17 years before the first HSMI outbreak in 2011. Finally, archaeovirology can be a valid alternative to contribute to the understanding of the epidemiology of diseases in aquaculture. Full article

Piscine orthoreovirus (PRV) infects farmed and wild salmon and trout species in North America, South America, Europe, and East Asia. PRV groups into three distinct genotypes (PRV-1, PRV-2, and PRV-3) that can vary in distribution, host specificity, and/or disease potential. Detection of the virus is currently restricted to genotype specific assays such that surveillance programs require the use of three assays to ensure universal detection of PRV. Consequently, herein, we developed, optimized, and validated a real-time reverse transcription quantitative PCR assay (RT-qPCR) that can detect all known PRV genotypes with high sensitivity and specificity. Targeting a conserved region at the 5′ terminus of the M2 segment, the pan-PRV assay reliably detected all PRV genotypes with as few as five copies of RNA. The assay exclusively amplifies PRV and does not cross-react with other salmonid viruses or salmonid host genomes and can be performed as either a one- or two-step RT-qPCR. The assay is highly reproducible and robust, showing 100% agreement in test results from an inter-laboratory comparison between two laboratories in two countries. Overall, as the assay provides a single test to achieve highly sensitive pan-specific PRV detection, it is suitable for research, diagnostic, and surveillance purposes. Full article

Heart and skeletal muscle inflammation (HSMI), caused by infection with Piscine orthoreovirus-1 (PRV-1), is a common disease in farmed Atlantic salmon (Salmo salar). Both an inactivated whole virus vaccine and a DNA vaccine have previously been tested experimentally against HSMI and demonstrated to give partial but not full protection. To understand the mechanisms involved in protection against HSMI and evaluate the potential of live attenuated vaccine strategies, we set up a cross-protection experiment using PRV genotypes not associated with disease development in Atlantic salmon. The three known genotypes of PRV differ in their preference of salmonid host species. The main target species for PRV-1 is Atlantic salmon. Coho salmon (Oncorhynchus kisutch) is the target species for PRV-2, where the infection may induce erythrocytic inclusion body syndrome (EIBS). PRV-3 is associated with heart pathology and anemia in rainbow trout, but brown trout (S. trutta) is the likely natural main host species. Here, we tested if primary infection with PRV-2 or PRV-3 in Atlantic salmon could induce protection against secondary PRV-1 infection, in comparison with an adjuvanted, inactivated PRV-1 vaccine. Viral kinetics, production of cross-reactive antibodies, and protection against HSMI were studied. PRV-3, and to a low extent PRV-2, induced antibodies cross-reacting with the PRV-1 σ1 protein, whereas no specific antibodies were detected after vaccination with inactivated PRV-1. Ten weeks after immunization, the fish were challenged through cohabitation with PRV-1-infected shedder fish. A primary PRV-3 infection completely blocked PRV-1 infection, while PRV-2 only reduced PRV-1 infection levels and the severity of HSMI pathology in a few individuals. This study indicates that infection with non-pathogenic, replicating PRV could be a future strategy to protect farmed salmon from HSMI. Full article

Piscine orthoreovirus (PRV) belongs to the family Reoviridae and has been described mainly in association with salmonid infections. The genome of PRV consists of about 23,600 bp, with 10 segments of double-stranded RNA, classified as small (S1 to S4), medium (M1, M2 and M3) and large (L1, L2 and L3); these range approximately from 1000 bp (segment S4) to 4000 bp (segment L1). How the genetic variation among PRV strains affects the virulence for salmonids is still poorly understood. The aim of this study was to describe the molecular phylogeny of PRV based on an extensive sequence analysis of the S1 and M2 segments of PRV available in the GenBank database to date (May 2020). The analysis was extended to include new PRV sequences for S1 and M2 segments. In addition, subgenotype classifications were assigned to previously published unclassified sequences. It was concluded that the phylogenetic trees are consistent with the original classification using the PRV genomic segment S1, which differentiates PRV into two major genotypes, I and II, and each of these into two subgenotypes, designated as Ia and Ib, and IIa and IIb, respectively. Moreover, some clusters of country- and host-specific PRV subgenotypes were observed in the subset of sequences used. This work strengthens the subgenotype classification of PRV based on the S1 segment and can be used to enhance research on the virulence of PRV. Full article

Piscine orthoreovirus 1 (PRV-1) is the causative agent of heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus is widespread in Atlantic salmon and was present in Norway long before the first description of HSMI in 1999. Furthermore, in Canada the virus is prevalent in farmed Atlantic salmon but HSMI is not and Canadian isolates have failed to reproduce HSMI experimentally. This has led to the hypothesis that there are virulence differences between PRV-1 isolates. In this study we performed a dose standardized challenge trial, comparing six PRV-1 isolates, including two Norwegian field isolates from 2018, three historical Norwegian isolates predating the first report of HSMI and one Canadian isolate. The Norwegian 2018 isolates induced lower viral protein load in blood cells but higher plasma viremia. Following peak replication in blood, the two Norwegian 2018 isolates induced histopathological lesions in the heart consistent with HSMI, whereas all three historical Norwegian and the Canadian isolates induced only mild cardiac lesions. This is the first demonstration of virulence differences between PRV-1 isolates and the phenotypic differences are linked to viral proteins encoded by segment S1, M2, L1, L2 and S4. Full article

Piscine reovirus (PRV) is the causative agent of heart and skeletal muscle inflammation (HSMI), which is detrimental to Atlantic Salmon (AS) aquaculture, but so far has not been cultivatable, which impedes studying the disease and developing a vaccine. Homogenates of head kidney and red blood cells (RBC) from AS in which PRV-1 had been detected were applied to fish cell lines. The cell lines were from embryos, and from brain, blood, fin, gill, gonads, gut, heart, kidney, liver, skin, and spleen, and had the shapes of endothelial, epithelial, fibroblast, and macrophage cells. Most cell lines were derived from the Neopterygii subclass of fish, but one was from subclass Chondrostei. Cultures were examined by phase contrast microscopy for appearance, and by quantitative polymerase chain reaction (qPCR) for PRV-1 RNA amplification and for the capacity to transfer any changes to new cultures. No changes in appearance and Ct values were observed consistently or transferable to new cultures. Therefore, 31 cell lines examined were unable to support PRV-1 amplification and are described as belonging to the non-supportive PRV-1 invitrome. However, these investigations and cell lines can contribute to understanding PRV-1 cellular and host tropism, and the interactions between virus-infected and bystander cells. Full article

Piscine orthoreovirus (PRV) is a relevant pathogen for salmonid aquaculture worldwide. In 2015, a new genotype of PRV (genotype 3, PRV-3) was discovered in Norway, and in 2017 PRV-3 was detected for first time in Denmark in association with complex disease cases in rainbow trout in recirculating aquaculture systems (RAS). To explore the epidemiology of PRV-3 in Denmark, a surveillance study was conducted in 2017 to 2019. Fifty-three farms, including both flow through and RAS, were screened for PRV-3. Of the farms examined, PRV-3 was detected in thirty-eight (71.7%), with the highest prevalence in grow-out farms. Notably, in Denmark disease outbreaks were only observed in RAS. Additionally, wild Atlantic salmon and brown trout populations were included in the screening, and PRV-3 was not detected in the three years where samples were obtained (2016, 2018, and 2019). Historical samples in the form of archived material at the Danish National Reference Laboratory for Fish Diseases were also tested for the presence of PRV-3, allowing us to establish that the virus has been present in Denmark at least since 1995. Sequence analyses of segment S1 and M2, as well as full genome analyses of selected isolates, did not reveal clear association between genetic makeup in these two segments and virulence in the form of disease outbreaks in the field. Full article

The population of brown trout (Salmo trutta fario) in continental Europe is on the decline, with infectious diseases confirmed as one of the causative factors. However, no data on the epizootiological situation of wild fish in the Czech Republic are currently available. In this study, brown trout (n = 260) from eight rivers were examined for the presence of viral and parasitical pathogens. Salmonid alphavirus-2, infectious pancreatic necrosis virus, piscine novirhabdovirus (VHSV) and salmonid novirhabdovirus (IHNV) were not detected using PCR. Cell culturing showed no viruses as well, and serological analysis of 110 sera did not detect any specific antibodies against VHSV or IHNV. Fish from two rivers were positive for the presence of piscine orthoreovirus-3 (PRV-3), subtype PRV-3b. However, none of the PRV-3-positive fish showed gross pathologies typically associated with PRV infections. By far the most widespread pathogen was Tetracapsuloides bryosalmonae which was confirmed in each of the examined locations, with a prevalence of up to 65% and 100%, as established by immunohistochemistry and PCR, respectively. Furthermore, up to 43.8% of fish showed signs of proliferative kidney disease caused by T. bryosalmonae, suggesting that this parasite is a main health challenge for brown trout in the Czech Republic. Full article

Piscine orthoreovirus-1 (PRV-1) can cause heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar), but the line of events from infection, pathologic change, and regeneration has not been thoroughly described. In this study, the cellular localization and variation of PRV-1 RNA and protein levels were analyzed at different times post-exposure in experimentally infected Atlantic salmon. Immunohistochemistry, flow cytometry, and Western blot were used for assessment of the presence of the PRV-1 σ1 protein, while RT-qPCR and in situ hybridization were performed for viral RNA. Histopathologic evaluation demonstrated that PRV-1 infection induced heart lesions typical of HSMI, such as severe epicarditis and myocarditis with degeneration of cardiomyocytes, necrosis, and diffuse cellular infiltration. PRV-1 infection of erythrocytes and the peak viral plasma level preceded virus presence in cardiomyocytes and hepatocytes. Arginase-2-positive, macrophage-like cells observed in the heart indicated possible polarization to M2 macrophages and the onset of regenerative processes, which may contribute to the recovery from HSMI. The virus was cleared from regenerating heart tissue and from hepatocytes, but persisted in erythrocytes. Full article

For many years, brown trout (Salmo trutta fario) mortalities within the pre-alpine Isar River in Germany were reported by the Bavarian Fisheries Association (Landesfischereiverband Bayern e.V.) and local recreational anglers during August and September. Moribund fish seemed to be affected by proliferative darkening syndrome (PDS). In addition, proliferative kidney disease (PKD) caused by Tetracapsuloides bryosalmonae was discussed. To investigate this phenomenon, the present field study monitored brown trout mortalities by daily river inspection in 2017 and 2018. Moribund brown trout (n = 31) were collected and examined using histology, immunohistochemistry, qPCR, and quantitative stereology. Our investigations identified 29 (93.5%) brown trout affected by PKD. Four brown trout (12.9%) displayed combined hepatic and splenic lesions fitting the pathology of PDS. The piscine orthoreovirus 3, suspected as causative agent of PDS, was not detectable in any of the samples. Quantitative stereological analysis of the kidneys revealed a significant increase of the renal tissue volumes with interstitial inflammation and hematopoietic hyperplasia in PKD-affected fish as compared to healthy brown trout. The identified T. bryosalmonae strain was classified as part of the North American clade by phylogenetical analysis. This study highlights PKD and PDS as contributing factors to recurrent autumnal brown trout mortalities. Full article

Piscine orthoreovirus (PRV-1) can cause heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). The virus targets erythrocytes in the acute peak phase, followed by cardiomyocytes, before the infection subsides into persistence. The persistent phase is characterized by high level of viral RNA, but low level of viral protein. The origin and nature of persistent PRV-1 are not clear. Here, we analyzed for viral persistence and activity in various tissues and cell types in experimentally infected Atlantic salmon. Plasma contained PRV-1 genomic dsRNA throughout an 18-week long infection trial, indicating that viral particles are continuously produced and released. The highest level of PRV-1 RNA in the persistent phase was found in kidney. The level of PRV-1 ssRNA transcripts in kidney was significantly higher than that of blood cells in the persistent phase. In-situ hybridization assays confirmed that PRV-1 RNA was present in erythroid progenitor cells, erythrocytes, macrophages, melano-macrophages and in some additional un-characterized cells in kidney. These results show that PRV-1 establishes a productive, persistent infection in Atlantic salmon and that erythrocyte progenitor cells are PRV target cells. Full article

Heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar) was first diagnosed in Norway in 1999. The disease is caused by Piscine orthoreovirus-1 (PRV-1). The virus is prevalent in farmed Atlantic salmon, but not always associated with disease. Phylogeny and sequence analyses of 31 PRV-1 genomes collected over a 30-year period from fish with or without HSMI, grouped the viral sequences into two main monophylogenetic clusters, one associated with HSMI and the other with low virulent PRV-1 isolates. A PRV-1 strain from Norway sampled in 1988, a decade before the emergence of HSMI, grouped with the low virulent HSMI cluster. The two distinct monophylogenetic clusters were particularly evident for segments S1 and M2. Only a limited number of amino acids were unique to the association with HSMI, and they all located to S1 and M2 encoded proteins. The observed co-evolution of the S1-M2 pair coincided in time with the emergence of HSMI in Norway, and may have evolved through accumulation of mutations and/or segment reassortment. Sequences of S1-M2 suggest selection of the HSMI associated pair, and that this segment pair has remained almost unchanged in Norwegian salmon aquaculture since 1997. PRV-1 strains from the North American Pacific Coast and Faroe Islands have not undergone this evolution, and are more closely related to the PRV-1 precursor strains not associated with clinical HSMI. Full article