Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease

Funding: The Scottish Government funded this work, as part of their global budget on aquaculture research. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

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

Detection of the myxosporean parasite Parvicapsula pseudobranchicola in Atlantic salmon (Salmo salar L.) using in situ hybridization (ISH)

-Parvicapsula pseudobranchicola is a marine myxosporean parasite infecting farmed Atlantic salmon (Salmo salar). A major site for the parasite is the pseudobranch, which may be destroyed in heavily infected fish. Parvicapsulosis may be associated with significant mortality, although the main effect of infections seems to be runting. In situ hybridization (ISH) is, in the absence of specific antibodies, the preferred method for the detection of cell- and tissue tropisms of myxozoans in the early phases of infection of the host, and provides information about the possible association between the pathogen and pathology. A positive diagnosis of parvicapsulosis is based on histopathology and PCR. The aim of the present work was to develop a specific, sensitive and robust ISH assay for the detection of P. pseudobranchicola in tissues

Infeksjoner med parasitten Nucleospora cyclopteri (Microsporidia) i rognkjeks, Cyclopterus lumpus

Source at https://www.vetinst.no/rapporter-og-publikasjoner/rapporter/2019/infeksjoner-med-parasitten-nucleospora-cyclopteri-microsporidia-i-rognkjeks-cyclopterus-lumpus.Nucleospora cyclopteri (Microsporidia) is one of many parasites infecting lumpfish, Cyclopterus lumpus, and has been shown to cause disease and mortality in lumpfish. Infections in fish are often multifactorial and the impact of one agent on the development of disease can be difficult to elucidate. In addition to mortality, infections in lumpfish can lead to diseases with subsequently lowered appetite. This is of particular importance since lumpfish are used as a biological control agent, eating salmon lice, Lepeophtherius salmonis, off the salmon. Knowledge on the different disease agents of lumpfish is therefore of utmost importance. The main aim of this project was to identify how to obtain an infection-free lumpfish in land-based hatcheries and to study the impact that N. cyclopteri has on the health of the lumpfish and thereby its effect as a biological control agent. The project therefore aimed to map the presence of N. cyclopteri and other disease agents in wild caught lumpfish and in eggs/sperm, in fry and in farmed lumpfish stocked in the sea. In addition, we wanted to study the transmission pathways and clinical significance of the parasite. Unfortunately, we were not able to obtain a group of lumpfish fry infected with N. cyclopteri that we intended to follow through the land phase. The study of pathogenesis, infection dynamics, or whether an infection with N. cyclopteri pre-disposes for secondary infections, was therefore abandoned. We studied the presence of co-infections, methods for optimal sampling and tissue tropism in wild caught lumpfish in this project. Nucleospora cyclopteri was present in 60% of the sampled individuals from the waters around Averøy, in county Møre og Romsdal. The fish were analysed with regard to a range of infectious agents (viruses, bacteria and parasites) commonly found in other fish species, or previously recorded in lumpfish. No viral agents or other important pathogens were detected, but supposedly nonpathogenic microparasites, like Kudoa islandica (Myxozoa) in the muscle tissue and coccidians in the intestine, were frequently found. Nucleospora cyclopteri was detected in all tissues examined: anterior, mid and posterior kidney, spleen, heart, gills, brain, muscle liver and blood, thus indicating that the infection is systemic. The density of N. cyclopteri was highest in the anterior kidney, followed by mid and posterior kidney, spleen and gills, while the prevalence was highest in the ventricle of the heart. Observations from this study indicate that the parasite is released through urine and bile. We also show that N. cyclopteri can be detected using swabs from the skin, gill and vent, and by blood samples and gill biopsies, thus demonstrating the possibility of non-lethal detection of N. cyclopteri in lumpfish. Amongst these, the most promising non-lethal samples for detection were gill biopsies and leukocyte fractions from blood samples. Images normal histology and pathological agents from this project is included in an openly available online image database. This image database can be accessed by diagnosticians and researchers and used when evaluating pathological findings in lumpfish. While vertical transmission cannot be excluded, the results from this project indicate that this is not the dominant route. It is in any case advisable to routinely screen broodfish for N. cyclopteri to avoid using positive individuals for the production of eggs and fry. Given that N. cyclopteri undoubtedly destroys leukocytes in high numbers and over large areas of tissue, it is reasonable to assume that the parasite has an effect on the immune competence of the fish

Mutation of N-glycosylation Sites in Salmonid Alphavirus (SAV) Envelope Proteins Attenuate the Virus in Cell Culture

Salmonid alphavirus (SAV) is the cause of pancreas disease and sleeping disease in farmed salmonid fish in Europe. The spread of these diseases has been difficult to control with biosecurity and current vaccination strategies, and increased understanding of the viral pathogenesis could be beneficial for the development of novel vaccine strategies. N-glycosylation of viral envelope proteins may be crucial for viral virulence and a possible target for its purposed attenuation. In this study, we mutated the N-glycosylation consensus motifs of the E1 and E2 glycoproteins of a SAV3 infectious clone using site-directed mutagenesis. Mutation of the glycosylation motif in E1 gave a complete inactivation of the virus as no viral replication could be detected in cell culture and infectious particles could not be rescued. In contrast, infectious virus particles could be recovered from the SAV3 E2 mutants (E2319Q, E2319A), but not if they were accompanied by lack of N-glycosylation in E1. Compared to the non-mutated infectious clone, the SAV3-E2319Q and SAV3-E2319A recombinant viruses produced less cytopathic effects in cell culture and lower amounts of infectious viral particles. In conclusion, the substitution in the N-linked glycosylation site in E2 attenuated SAV3 in cell culture. The findings could be useful for immunization strategies using live attenuated vaccines and testing in fish will be desirable to study the clone’s properties in vivo.</jats:p

Infectious Salmon Anaemia Virus (ISAV) RNA Binding Protein Encoded by Segment 8 ORF2 and Its Interaction with ISAV and Intracellular Proteins

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus infecting salmonid fish. The virus is adapted to low temperature and has a replication optimum between 10–15 °C. In this study the subcellular localization and protein interactions for the protein encoded by the largest open reading frame of gene segment 8 (s8ORF2) were investigated. In ISAV infected cells the s8ORF2 protein was found mainly in the cytosol but a minor fraction of cells expressed the protein in the nucleus as well. Green fluorescent protein-tagged s8ORF2 did not leak out of the cell when the plasma membrane was permeabilized, suggesting interactions with intracellular structural components. The s8ORF2 protein exists both as monomer and homodimer, and co-immunoprecipitation experiments strongly suggests it binds to the ISAV fusion-, nucleo- and matrix proteins. Two versions of s8ORF2 were detected with apparent molecular weights of 24–26 and 35 kDa in lysates of infected cells. The 35 kDa type is an early viral protein while the smaller version appears during the later phases of infection. The 24–26 kDa type was also the predominant form in viral particles. The s8ORF2 protein has previously been shown to bind RNA and interfere with interferon induction and signaling. Here we found that a fraction of the s8ORF2 protein pool in infected cells is likely to be conjugated to the interferon stimulated gene 15 (ISG15) and ubiquitin. Furthermore, several endogenous proteins pulled down by the s8ORF2 protein were identified by liquid chromatography mass spectrometry (LC-MS)