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Molecular and Antigenic Characterization of Piscine orthoreovirus (PRV) from Rainbow Trout (Oncorhynchus mykiss).

ABSTRACT: Piscine orthoreovirus (PRV-1) causes heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). Recently, a novel PRV (formerly PRV-Om, here called PRV-3), was found in rainbow trout (Oncorhynchus mykiss) with HSMI-like disease. PRV is considered to be an emerging pathogen in farmed salmonids. In this study, molecular and antigenic characterization of PRV-3 was performed. Erythrocytes are the main target cells for PRV, and blood samples that were collected from experimentally challenged fish were used as source of virus. Virus particles were purified by gradient ultracentrifugation and the complete coding sequences of PRV-3 were obtained by Illumina sequencing. When compared to PRV-1, the nucleotide identity of the coding regions was 80.1%, and the amino acid identities of the predicted PRV-3 proteins varied from 96.7% (λ1) to 79.1% (σ3). Phylogenetic analysis showed that PRV-3 belongs to a separate cluster. The region encoding σ3 were sequenced from PRV-3 isolates collected from rainbow trout in Europe. These sequences clustered together, but were distant from PRV-3 that was isolated from rainbow trout in Norway. Bioinformatic analyses of PRV-3 proteins revealed that predicted secondary structures and functional domains were conserved between PRV-3 and PRV-1. Rabbit antisera raised against purified virus or various recombinant virus proteins from PRV-1 all cross-reacted with PRV-3. Our findings indicate that despite different species preferences of the PRV subtypes, several genetic, antigenic, and structural properties are conserved between PRV-1 and-3.

SUBMITTER: Dhamotharan K

PROVIDER: S-EPMC5923464 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss).

Project description:Piscine orthoreovirus (PRV) mediated diseases have emerged throughout salmonid aquaculture. Three PRV subtypes are currently reported as causative agents of or in association with diseases in different salmonid species. PRV-1 causes heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar) and is associated with jaundice syndrome in farmed chinook salmon (Oncorhynchus tshawytscha). PRV-2 causes erythrocytic inclusion body syndrome (EIBS) in coho salmon in Japan. PRV-3 has recently been associated with a disease in rainbow trout (Oncorhynchus mykiss) characterized by anaemia, heart and red muscle pathology; to jaundice syndrome in coho salmon (Oncorhynchus kisutch). In this study, we conducted a 10-week long experimental infection trial in rainbow trout with purified PRV-3 particles to assess the causal relationship between the virus and development of heart inflammation. The monitoring the PRV-3 load in heart and spleen by RT-qPCR shows a progressive increase of viral RNA to a peak, followed by clearance without a measurable change in haematocrit. The development of characteristic cardiac histopathological findings occurred in the late phase of the trial and was associated with increased expression of CD8+, indicating cytotoxic T cell proliferation. The findings indicate that, under these experimental conditions, PRV-3 infection in rainbow trout act similarly to PRV-1 infection in Atlantic salmon with regards to immunological responses and development of heart pathology, but not in the ability to establish a persistent infection.

| S-EPMC6380033 | biostudies-literature

Infection experiments with novel Piscine orthoreovirus from rainbow trout (Oncorhynchus mykiss) in salmonids.

Project description:A new disease in farmed rainbow trout (Onchorhyncus mykiss) was described in Norway in 2013. The disease mainly affected the heart and resembled heart and skeletal muscle inflammation (HSMI) in Atlantic salmon (Salmo salar L.). HSMI is associated with Piscine orthoreovirus (PRV), and a search for a similar virus in the diseased rainbow trout led to detection of a sequence with 85% similarity to PRV. This finding called for a targeted effort to assess the risk the new PRV-variant pose on farmed rainbow trout and Atlantic salmon by studying infection and disease pathogenesis, aiming to provide more diagnostic knowledge. Based on the genetic relationship to PRV, the novel virus is referred to as PRV-Oncorhynchus mykiss (PRV-Om) in contrast to PRV-Salmo salar (PRV-Ss). In experimental trials, intraperitoneally injected PRV-Om was shown to replicate in blood in both salmonid species, but more effectively in rainbow trout. In rainbow trout, the virus levels peaked in blood and heart of cohabitants 6 weeks post challenge, along with increased expression of antiviral genes (Mx and viperin) in the spleen, with 80-100% of the cohabitants infected. Heart inflammation was diagnosed in all cohabitants examined 8 weeks post challenge. In contrast, less than 50% of the Atlantic salmon cohabitants were infected between 8 and 16 weeks post challenge and the antiviral response in these fish was very low. From 12 weeks post challenge and onwards, mild focal myocarditis was demonstrated in a few virus-positive salmon. In conclusion, PRV-Om infects both salmonid species, but faster transmission, more notable antiviral response and more prominent heart pathology were observed in rainbow trout.

| S-EPMC5497981 | biostudies-literature

First Description of a New Disease in Rainbow Trout (Oncorhynchus mykiss (Walbaum)) Similar to Heart and Skeletal Muscle Inflammation (HSMI) and Detection of a Gene Sequence Related to Piscine Orthoreovirus (PRV).

Project description:In fall 2013, anorexia, lethargy and mortalities up to 10-12,000 dead fish per week were observed in rainbow trout Oncorhynchus mykiss in three fresh water hatcheries (salinity 0-1 ‰) on the west coast of Norway. The fish (25-100 g) showed signs of circulatory failure with haemorrhages, ascites and anaemia. The histopathological findings comprised inflammation of the heart and red muscle and liver necrosis. The affected fish had a common origin. Disease and mortalities were also observed up to four months after sea water transfer. Microbiological examination did not reveal presence of any known pathogens. Based on histopathological similarities to heart and skeletal inflammation (HSMI) in Atlantic salmon, associated with piscine orthoreovirus (PRV), extended investigations to detect a virus within the family Reoviridae were conducted. By the use of primer sets targeting the PRV genome, a sequence with 85% identity to a part of segment S1 of PRV was obtained. Further analysis showed that the virus sequence could only be aligned with PRV and no other reoviruses both on amino acid and nucleotide level. Two PCR assays were developed for specific detection of the virus. High amounts of the virus were detected in diseased fish at all affected farms and low amounts were detected in low prevalence at the broodfish farms. Further investigations are needed to determine if the virus is associated with the new disease in rainbow trout and to further characterize the virus with respect to classification, relationship with PRV, virulence, pathology and epidemiology.

| S-EPMC4503464 | biostudies-literature

Molecular characterization of a novel orthomyxovirus from rainbow and steelhead trout (Oncorhynchus mykiss).

Project description:A novel virus, rainbow trout orthomyxovirus (RbtOV), was isolated in 1997 and again in 2000 from commercially-reared rainbow trout (Oncorhynchus mykiss) in Idaho, USA. The virus grew optimally in the CHSE-214 cell line at 15°C producing a diffuse cytopathic effect; however, juvenile rainbow trout exposed to cell culture-grown virus showed no mortality or gross pathology. Electron microscopy of preparations from infected cell cultures revealed the presence of typical orthomyxovirus particles. The complete genome of RbtOV is comprised of eight linear segments of single-stranded, negative-sense RNA having highly conserved 5' and 3'-terminal nucleotide sequences. Another virus isolated in 2014 from steelhead trout (also O. mykiss) in Wisconsin, USA, and designated SttOV was found to have eight genome segments with high amino acid sequence identities (89-99%) to the corresponding genes of RbtOV, suggesting these new viruses are isolates of the same virus species and may be more widespread than currently realized. The new isolates had the same genome segment order and the closest pairwise amino acid sequence identities of 16-42% with Infectious salmon anemia virus (ISAV), the type species and currently only member of the genus Isavirus in the family Orthomyxoviridae. However, pairwise comparisons of the predicted amino acid sequences of the 10 RbtOV and SttOV proteins with orthologs from representatives of the established orthomyxoviral genera and a phylogenetic analysis using the PB1 protein showed that while RbtOV and SttOV clustered most closely with ISAV, they diverged sufficiently to merit consideration as representatives of a novel genus. A set of PCR primers was designed using conserved regions of the PB1 gene to produce amplicons that may be sequenced for identification of similar fish orthomyxoviruses in the future.

| S-EPMC7111338 | biostudies-literature

Molecular Characterization, Nutritional and Insulin Regulation of Elovl6 in Rainbow Trout (Oncorhynchus mykiss).

Project description:Elongation of very long-chain fatty acids protein 6 (Elovl6) is a crucial enzyme in the synthesis of endogenous fatty acids, which participates in the energy balance and metabolic diseases. The main objective of this study was to explore the molecular characterization of Elovl6 and the regulation of elovl6 expression in response to dietary fatty acids and insulin. In the present study, the ORF (open reading frame) of Elovl6 from rainbow trout was cloned and characterized, which showed a high identity (87%) with mammals and other teleost. The results of quantitative PCR showed that the transcriptional levels of elovl6 from rainbow trout that were fed diets containing soybean oil (enriched with 18:2n-6, linoleic acid (LA)) or linseed oil (enriched with 18:3n-3, α-linolenic acid (ALA)) were lower than those in the group that were fed diets containing fish oil (enriched with 20:5n-3, eicosapentaenoic acid (EPA) and 22:6n-3, docosahexaenoic acid (DHA)). Correspondingly, mRNA expression of elovl6 in hepatocytes treated with DHA was dramatically higher than that in LA and ALA groups. The transcriptional expression of elovl6 in hepatocytes treated with insulin was also significantly increased. Moreover, the dual luciferase assay showed the transcription factor CREB1 dramatically up-regulated the promoter activity of elovl6, while FOXO1 significantly down-regulated the elovl6 promoter activity in rainbow trout. The differences in transcriptional expression of crbe1 and foxo1 may contribute to the increase or decrease of elovl6 expression in rainbow trout in response to fatty acids or insulin. These findings revealed the molecular characterization of elovl6 and the regulation of elovl6 expression by CREB1 and FOXO1 in rainbow trout in response to dietary fatty acids or insulin.

| S-EPMC7072538 | biostudies-literature

Molecular characterization and expression analysis of three subclasses of IgT in rainbow trout (Oncorhynchus mykiss).

Project description:As the teleost specific immunoglobulin, IgT plays important roles in systemic and mucosal immunity. In the current study, in rainbow trout, we have cloned the heavy chain (Igτ) genes of a secretory form of IgT2 as well as the membrane and secretory forms of a third IgT subclass, termed IgT3. Conserved cysteine and tryptophan residues that are crucial for the folding of the immunoglobulin domain as well as hydrophobic and hydrophilic residues within CART motif were identified in all IgT subclasses. Through analysis of the rainbow trout genome assembly, Igτ3 gene was found localized upstream of Igτ1 gene, while Igτ2 gene situated on another scaffold. At the transcriptional level, Igτ1 was mainly expressed in both systemic and mucosal lymphoid tissues, while Igτ2 was largely expressed in systemic lymphoid organs. After LPS and poly (I:C) treatment, Igτ1 and Igτ2 genes exhibited different expression profiles. Interestingly the transcriptional level of Igτ3 was negligible, although its protein product could be identified in trout serum. Importantly, a previously reported monoclonal antibody directed against trout IgT1 was able to recognize IgT2 and IgT3. These data demonstrate that there exist three subclasses of IgT in rainbow trout, and that their heavy chain genes display different expression patterns during stimulation. Overall, our data reflect the diversity and complexity of immunoglobulin in trout, thus provide a better understanding of the IgT system in the immune response of teleost fish.

| S-EPMC5701746 | biostudies-literature

Functional characterization of a BCL10 isoform in the rainbow trout Oncorhynchus mykiss.

Project description:The complexes formed by BCL10, MALT1 and members of the family of CARMA proteins have recently been the focus of much attention because they represent a key mechanism for regulating activation of the transcription factor NF-κB. Here, we report the functional characterization of a novel isoform of BCL10 in the trout Oncorhynchus mykiss, which we named tBCL10. tBCL10 dimerizes, binds to components of the CBM complex and forms cytoplasmic filaments. Functionally, tBCL10 activates NF-κB transcription factor and is inhibited by the deubiquitinating enzyme A20. Finally, depletion experiments indicate that tBCL10 can functionally replace the human protein. This work demonstrates the evolutionary conservation of the mechanism of NF-κB activation through the CBM complex, and indicates that the rainbow trout O . mykiss can serve as a model organism to study this pathway.

| S-EPMC4372615 | biostudies-literature

Molecular cloning and functional characterization of two forms of Pax8 in the rainbow trout, Oncorhynchus mykiss.

Project description:We have identified two distinct Pax8 (a and b) mRNAs from the thyroid gland of the rainbow trout (Oncorhynchus mykiss), which seemed to be generated by alternative splicing. Both Pax8a and Pax8b proteins were predicted to possess the paired domain, octapeptide, and partial homeodomain, while Pax8b lacked the carboxy-terminal portion due to an insertion in the coding region of the mRNA. RT-PCR analysis showed each of Pax8a and Pax8b mRNAs to be abundantly expressed in the thyroid and kidney. In situ hybridization histochemistry further detected the expression of Pax8 mRNA in the epithelial cells of the thyroid follicles of the adult trout and in the thyroid primordial cells of the embryo. The functional properties of Pax8a and Pax8b were investigated by dual luciferase assay. The transcriptional regulation by the rat thyroid peroxidase (TPO) promoter was found to be increased by Pax8a, but not by Pax8b. Pax8a further showed synergistic transcriptional activity with rat Nkx2-1 for the human TPO upstream region including the enhancer and promoter. On the other hand, Pax8b decreased the synergistic activity of Pax8a and Nkx2-1. Electrophoretic mobility shift assay additionally indicated that not only Pax8a but also Pax8b can bind to the TPO promoter and enhancer, implying that the inhibitory effect of Pax8b might result from the lack of the functional carboxy-terminal portion. Collectively, the results suggest that for the trout thyroid gland, Pax8a may directly increase TPO gene expression in cooperation with Nkx2-1 while Pax8b may work as a non-activating competitor for the TPO transcription.

| S-EPMC3991817 | biostudies-literature

Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes.

Project description:Piscine orthoreovirus (PRV) belongs to the Reoviridae family and is the only known fish virus related to the Orthoreovirus genus. The virus is the causative agent of heart and skeletal muscle inflammation (HSMI), an emerging disease in farmed Atlantic salmon (Salmo salar L.). PRV is ubiquitous in farmed Atlantic salmon and high loads of PRV in the heart are consistent findings in HSMI. The mechanism by which PRV infection causes disease remains largely unknown. In this study we investigated the presence of PRV in blood and erythrocytes using an experimental cohabitation challenge model. We found that in the early phases of infection, the PRV loads in blood were significantly higher than in any other organ. Most virus was found in the erythrocyte fraction, and in individual fish more than 50% of erythrocytes were PRV-positive, as determined by flow cytometry. PRV was condensed into large cytoplasmic inclusions resembling viral factories, as demonstrated by immunofluorescence and confocal microscopy. By electron microscopy we showed that these inclusions contained reovirus-like particles. The PRV particles and inclusions also had a striking resemblance to previously reported viral inclusions described as Erythrocytic inclusion body syndrome (EIBS). We conclude that the erythrocyte is a major target cell for PRV infection. These findings provide new information about HSMI pathogenesis, and show that PRV is an important factor of viral erythrocytic inclusions.

| S-EPMC4234517 | biostudies-literature

Ontogeny of melanophore photosensitivity in rainbow trout (Oncorhynchus mykiss).

Project description:Migratory species experience morphological and physiological changes during transitions between different life stages. In particular, modification of sensory systems is critical for animals to adapt to new environments. For example, to prepare for entry into seawater, salmonids undergo smoltification with dramatic changes in ultraviolet photoreceptors and polarized vision, which are important for orientation and foraging behaviours. Extraretinal organs are also involved in photoreception; however, the ontogenetic development of extraretinal photoreceptors is not well known, especially in migratory species. Here, we investigated whether rainbow trout dermal photoreceptors, melanophores, undergo change in spectral sensitivity during smoltification and which candidate molecules may account for this ontogenetic alteration. Our results showed that, contrary to parr melanophores which are insensitive to light, smolt melanophores displayed chromatic photoresponses with the emergence of cryptochrome and melanopsin expression. We suggest that these modifications may benefit the active foraging behaviour of smolts and enable adaptation to variable environments.

| S-EPMC4232760 | biostudies-literature

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