Siri Mjaaland

Infectious salmon anaemia virus.

Infectious salmon anaemia virus (ISAV) is a commercially important orthomyxovirus causing disease in farmed Atlantic salmon. The cumulative mortality in a net pen during an outbreak may vary from insignificant to more than 90%. The infection is spread by management activity such as well‐boat traffic, but possibly also through contact with wild fish. In many of its aspects, including the structure of the virus particle and replication strategy, the ISAV is similar to the influenza viruses. Variations between ISAV and the influenza viruses can mostly be related to differences in the temperature at which replication occurs and the immune response of their respective host animals. ISAV shows both haemagglutinating and receptor‐destroying activity. The variability of the ISAV haemagglutinin molecule is concentrated around a small domain close to the transmembrane region. The function of this variable region is unknown, but it may be related to a recent or ongoing crossing of a species barrier. Alignment studies based on genetic data indicate that the phylogenetic relationship to the influenza viruses is distant, and that ISAV therefore could possibly warrant a new genus within Orthomyxoviridae.

Characterization of the Infectious Salmon Anemia Virus Genomic Segment That Encodes the Putative Hemagglutinin

ABSTRACT The genomic segment encoding the putative hemagglutinin of infectious salmon anemia virus (ISAV) is described. Expression of the putative hemagglutinin in a salmon cell line demonstrated hemadsorptive properties of the protein for salmon erythrocytes. The polypeptide was recognized by an ISAV-specific monoclonal antibody. Nucleotide sequencing indicated the occurrence of a variable region in the hemagglutinin gene.

Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity

In this study we characterize two proteins encoded by the two smallest genomic segments of the piscine orthomyxovirus infectious salmon anaemia virus (ISAV). Both proteins, encoded by the un-spliced ORF from genomic segment 7 (s7ORF1) and the larger ORF from segment 8 (s8ORF2), are involved in modulation of the type I interferon (IFN) response. The data suggests that the s7ORF1 protein is collinearly encoded, non-structural, contains no nuclear localisation signals, localises mainly to the cytoplasmic perinuclear area and does not bind single- or double-stranded RNA. On the other hand, genomic segment 8 uses a bicistronic coding strategy and the encoded s8ORF2 protein is a structural component of the viral particle. This protein contains two nuclear localisation signals, has a predominantly nuclear localisation, binds both double-stranded RNA and poly-A tailed single-stranded RNA, but not double-stranded DNA. In poly I:C stimulated salmon cells both ISAV proteins independently down-regulate the type I IFN promoter activity. Thus, ISAV counteracts the type I IFN response by the action of at least two of its gene products, rather than just one, as appears to be the case for other known members of the Orthomyxoviridae.

Susceptibility and immune responses following experimental infection of MHC compatible Atlantic salmon (Salmo salar L.) with different infectious salmon anaemia virus isolates.

Summary.Infectious salmon anaemia virus (ISAV) is an aquatic orthomyxovirus causing a multisystemic disease in farmed Atlantic salmon (Salmo salar) where disease development, clinical signs, and histopathology vary to a large extent. Here, an experimental trial was designed to determine the effect of variation in viral genes on virus-host interactions, as measured by disease susceptibility and immune responses. The fish were infected using cohabitant transmission, representing a natural route of infection. Variation caused by host factors was minimized using MHC compatible A. salmon half-siblings as experimental fish. Virus isolates were selected according to HE genotype, as European ISAV isolates can be genotyped according to deletion patterns in their hemagglutinin-esterase (HE) surface glycoprotein, and the course of disease they typically induce, classified as acute versus protracted. The different ISAV isolates induced large variations in death prevalence, ranging from 0–47% in the test-group and 3–75% in the cohabitant fish. The use of MHC compatible experimental fish made it possible to determine the relative contribution of humoral versus cellular response in protection against ISA. Ability to induce a strong proliferative response correlated with survival and virus clearance, while induction of a humoral response was less protective.

Evolutionary mechanisms involved in the virulence of infectious salmon anaemia virus (ISAV), a piscine orthomyxovirus.

Infectious salmon anaemia virus (ISAV) is an orthomyxovirus causing a multisystemic, emerging disease in Atlantic salmon. Here we present, for the first time, detailed sequence analyses of the full-genome sequence of a presumed avirulent isolate displaying a full-length hemagglutinin-esterase (HE) gene (HPR0), and compare this with full-genome sequences of 11 Norwegian ISAV isolates from clinically diseased fish. These analyses revealed the presence of a virulence marker right upstream of the putative cleavage site R267 in the fusion (F) protein, suggesting a Q266-->L266 substitution to be a prerequisite for virulence. To gain virulence in isolates lacking this substitution, a sequence insertion near the cleavage site seems to be required. This strongly suggests the involvement of a protease recognition pattern at the cleavage site of the fusion protein as a determinant of virulence, as seen in highly pathogenic influenza A virus H5 or H7 and the paramyxovirus Newcastle disease virus.

Expression, antigenicity and studies on cell receptor binding of the hemagglutinin of infectious salmon anemia virus

Summary.Infectious salmon anemia (ISA) virus belongs to the proposed genus Isavirus of Orthomyxoviridae and is an emerging pathogen in Atlantic salmon (Salmo salar) farming. The hemagglutinin-esterase (HE) of ISA virus share several characteristics with the influenza virus hemagglutinin. This study reports recombinant expression of different ISA virus HE mutants in fish cell lines. Some introduced mutations, representing minimal differences in single amino acid residues, resulted in remarkable effects on expression efficiency in general and on surface expression specifically. Receptor binding assays showed that amino acid residues in the N-terminal half part are important in receptor binding, either being directly involved in the binding, or by influencing the structure of the binding site. Deletion of the putative N-glycosylation sites of the ISA virus HE, located near the transmembrane region, did not influence expression, receptor binding properties or staining by either a neutralising MAb, or salmon convalescent sera. The humoral immune response of Atlantic salmon after ISA virus infection showed weak neutralising activity and the results indicated that it was directed against HE.

Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus

n Abstractn n Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon (Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser32 to Ala32 amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser32 amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzymes catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure–function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.n n

Analysis of host- and strain-dependent cell death responses during infectious salmon anemia virus infection in vitro

BackgroundInfectious salmon anemia virus (ISAV) is an aquatic orthomyxovirus and the causative agent of infectious salmon anemia (ISA), a disease of great importance in the Atlantic salmon farming industry. In vitro, ISAV infection causes cytophatic effect (CPE) in cell lines from Atlantic salmon, leading to rounding and finally detachment of the cells from the substratum. In this study, we investigated the mode of cell death during in vitro ISAV infection in different Atlantic salmon cell lines, using four ISAV strains causing different mortality in vivo.ResultsThe results show that caspase 3/7 activity increased during the course of infection in ASK and SHK-1 cells, infected cells showed increased surface expression of phosphatidylserine and increased PI uptake, compared to mock infected cells; and morphological alterations of the mitochondria were observed. Expression analysis of immune relevant genes revealed no correlation between in vivo mortality and expression, but good correlation in expression of interferon genes.ConclusionResults from this study indicate that there is both strain and cell type dependent differences in the virus-host interaction during ISAV infection. This is important to bear in mind when extrapolating in vitro findings to the in vivo situation.

Enhanced transfection of cell lines from Atlantic salmon through nucoleofection and antibiotic selection

BackgroundCell lines from Atlantic salmon kidney have made it possible to culture and study infectious salmon anemia virus (ISAV), an aquatic orthomyxovirus affecting farmed Atlantic salmon. However, transfection of these cells using calcium phosphate precipitation or lipid-based reagents shows very low transfection efficiency. The Amaxa Nucleofector technology™ is an electroporation technique that has been shown to be efficient for gene transfer into primary cells and hard to transfect cell lines.FindingsHere we demonstrate, enhanced transfection of the head kidney cell line, TO, from Atlantic salmon using nucleofection and subsequent flow cytometry. Depending on the plasmid promoter, TO cells could be transfected transiently with an efficiency ranging from 11.6% to 90.8% with good viability, using Amaxas cell line nucleofector solution T and program T-20. A kill curve was performed to investigate the most potent antibiotic for selection of transformed cells, and we found that blasticidin and puromycin were the most efficient for selection of TO cells.ConclusionsThe results show that nucleofection is an efficient way of gene transfer into Atlantic salmon cells and that stably transfected cells can be selected with blasticidin or puromycin.

Infectious salmon anaemia virus: An orthomyxovirus causing an emerging infection in Atlantic salmon: review article

Infectious salmon anaemia virus (ISAV) is a commercially important orthomyxovirus causing disease in farmed Atlantic salmon. The cumulative mortality in a net pen during an outbreak may vary from insignificant to more than 90%. The infection is spread by management activity such as well‐boat traffic, but possibly also through contact with wild fish. In many of its aspects, including the structure of the virus particle and replication strategy, the ISAV is similar to the influenza viruses. Variations between ISAV and the influenza viruses can mostly be related to differences in the temperature at which replication occurs and the immune response of their respective host animals. ISAV shows both haemagglutinating and receptor‐destroying activity. The variability of the ISAV haemagglutinin molecule is concentrated around a small domain close to the transmembrane region. The function of this variable region is unknown, but it may be related to a recent or ongoing crossing of a species barrier. Alignment studies based on genetic data indicate that the phylogenetic relationship to the influenza viruses is distant, and that ISAV therefore could possibly warrant a new genus within Orthomyxoviridae.