Characterization of untranslated regions of the salmonid alphavirus 3 (SAV3) genome and construction of a SAV3 based replicon

Salmonid alphavirus (SAV) causes disease in farmed salmonid fish and is divided into different genetic subtypes (SAV1-6). Here we report the cloning and characterization of the 5'- and 3'- untranslated regions (UTR) of a SAV3 isolated from Atlantic salmon in Norway. The sequences of the UTRs are very similar to those of SAV1 and SAV2, but single nucleotide polymorphisms are present, also in the 3' - conserved sequence element (3'-CSE). Prediction of the RNA secondary structure suggested putative stem-loop structures in both the 5'- and 3'-ends, similar to those of alphaviruses from the terrestrial environment, indicating that the general genome replication initiation strategy for alphaviruses is also utilized by SAV. A DNA replicon vector, pmSAV3, based upon a pVAX1 backbone and the SAV3 genome was constructed, and the SAV3 non-structural proteins were used to express a reporter gene controlled by the SAV3 subgenomic promoter. Transfection of pmSAV3 into CHSE and BF2 cell lines resulted in expression of the reporter protein, confirming that the cloned SAV3 replication apparatus and UTRs are functional in fish cells

Immunohistochemical detection of piscine reovirus (PRV) in hearts of Atlantic salmon coincide with the course of heart and skeletal muscle inflammation (HSMI)

Aquaculture is the fastest growing food production sector in the world. However, the increased production has been accompanied by the emergence of infectious diseases. Heart and skeletal muscle inflammation (HSMI) is one example of an emerging disease in farmed Atlantic salmon (Salmo salar L). Since the first recognition as a disease entity in 1999 it has become a widespread and economically important disease in Norway. The disease was recently found to be associated with infection with a novel reovirus, piscine reovirus (PRV). The load of PRV, examined by RT-qPCR, correlated with severity of HSMI in naturally and experimentally infected salmon. The disease is characterized by epi-, endo- and myocarditis, myocardial necrosis, myositis and necrosis of the red skeletal muscle. The aim of this study was to investigate the presence of PRV antigens in heart tissue of Atlantic salmon and monitor the virus distribution in the heart during the disease development. This included target cell specificity, viral load and tissue location during an HSMI outbreak. Rabbit polyclonal antisera were raised against putative PRV capsid proteins μ1C and σ1 and used in immunohistochemical analysis of archived salmon heart tissue from an experimental infection. The results are consistent with the histopathological changes of HSMI and showed a sequential staining pattern with PRV antigens initially present in leukocyte-like cells and subsequently in cardiomyocytes in the heart ventricle. Our results confirm the association between PRV and HSMI, and strengthen the hypothesis of PRV being the causative agent of HSMI. Immunohistochemical detection of PRV antigens will be beneficial for the understanding of the pathogenesis of HSMI as well as for diagnostic purposes

Transcription of reference genes used for quantitative RT-PCR in Atlantic salmon is affected by viral infection

Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections

Immunopathological characterization of red focal changes in Atlantic salmon (Salmo salar) white muscle

Farmed Atlantic salmon (Salmo salar) are prone to various conditions affecting the quality of the fillet. A well-known but so far poorly understood condition is the focal red changes in muscle, often referred to as haemorrhages. Such changes are characterized by muscle necrosis, haemorrhages and acute inflammation. They can progress into focal melanised changes, a chronic inflammatory condition with melanin-producing leukocytes. The initial cause of intramuscular haemorrhages is unknown. In this study, we aimed to reveal some of their key immunological features. Samples of red focal changes were investigated by immunohistochemistry (IHC), in situ hybridization (ISH) and RT-qPCR for various immune markers. The results were compared with samples of melanised changes and control muscle, subjected to the same analyses. In all red changes, infiltrates with mononuclear cells were detected, consisting mostly of MHC class I/II+ cells, but also of CD3+ and CD8+ cells. ISH studies on IgM showed few to moderate amounts of B-cells in red focal changes. Trends in the RT-qPCR showed upregulation of genes related to innate immunity in the red changes, whereas genes related to adaptive immunity were upregulated in the melanised changes. An important result was the significant downregulation of the anti-inflammatory cytokine IL10 in all red changes. Our findings indicate that we can rule out an auto invasive nature of the changes. The downregulation of IL10 at an early phase is a trait for the condition.publishedVersio

Plant-made vaccines against viral diseases in humans and farm animals

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Cellular heterogeneity in red and melanized focal muscle changes in farmed Atlantic salmon (Salmo salar) visualized by spatial transcriptomics

Spatial transcriptomics is a technique that provides insight into gene expression profiles in tissue sections while retaining structural information. We have employed this method to study the pathological conditions related to red and melanized focal changes in farmed Atlantic salmon (Salmo salar). Our findings support a model where similar molecular mechanisms are involved in both red and melanized filet discolorations and genes associated with several relevant pathways show distinct expression patterns in both sample types. Interestingly, there appears to be significant cellular heterogeneity in the foci investigated when looking at gene expression patterns. Some of the genes that show differential spatial expression are involved in cellular processes such as hypoxia and immune responses, providing new insight into the nature of muscle melanization in Atlantic salmon.publishedVersio

Piscine orthoreovirus infection in Atlantic salmon (Salmo salar) protects against subsequent challenge with infectious hematopoietic necrosis virus (IHNV)

Abstract Infectious hematopoietic necrosis virus (IHNV) is endemic in farmed rainbow trout in continental Europe and in various salmonid fish species at the Pacific coast of North America. IHN has never occurred in European Atlantic salmon (Salmo salar) farms, but is considered as a major threat for the European salmon industry. Another virus, Piscine orthoreovirus (PRV), is widespread in the sea phase of Atlantic salmon, and is identified as the causative agent of heart and skeletal muscle inflammation. The aim of this study was to investigate the interactions between a primary PRV infection and a secondary IHNV infection under experimental conditions. A PRV cohabitation challenge was performed with Atlantic salmon. At peak of PRV viremia the fish were challenged by immersion with an IHNV genogroup E isolate. Clinical signs and morbidity were monitored. Target organs were sampled at selected time points to assess viral loads of both pathogens. Antiviral immune response and presence of histopathological findings were also investigated. Whereas the PRV-negative/IHNV positive group suffered significant decrease in survival caused by IHNV, the PRV infected groups did not suffer any morbidity and showed negligible levels of IHNV infection. Antiviral response genes were induced, as measured in spleen samples, from PRV infected fish prior to IHNV challenge. In conclusion, PRV-infection protects Atlantic salmon against IHNV infection and morbidity, most likely by inducing a protective innate antiviral response

Molecular and antigenic characterization of Piscine orthoreovirus (PRV) from rainbow trout (Oncorhynchus mykiss)

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