The Crystal Structure and RNA-Binding of an Orthomyxovirus Nucleoprotein

Genome packaging for viruses with segmented genomes is often a complex problem. This is particularly true for influenza viruses and other orthomyxoviruses, whose genome consists of multiple negative-sense RNAs encapsidated as ribonucleoprotein (RNP) complexes. To better understand the structural features of orthomyxovirus RNPs that allow them to be packaged, we determined the crystal structure of the nucleoprotein (NP) of a fish orthomyxovirus, the infectious salmon anemia virus (ISAV) (genus Isavirus). As the major protein component of the RNPs, ISAV-NP possesses a bi-lobular structure similar to the influenza virus NP. Because both RNA-free and RNA-bound ISAV NP forms stable dimers in solution, we were able to measure the NP RNA binding affinity as well as the stoichiometry using recombinant proteins and synthetic oligos. Our RNA binding analysis revealed that each ISAV-NP binds ∼12 nts of RNA, shorter than the 24–28 nts originally estimated for the influenza A virus NP based on population average. The 12-nt stoichiometry was further confirmed by results from electron microscopy and dynamic light scattering. Considering that RNPs of ISAV and the influenza viruses have similar morphologies and dimensions, our findings suggest that NP-free RNA may exist on orthomyxovirus RNPs, and selective RNP packaging may be accomplished through direct RNA-RNA interactions.     

MHC polymorphism and disease resistance in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>); facing pathogens with single expressed major histocompatibility class I and class II loci

Few studies have yet addressed the functional aspects of MHC molecules in fish. To lay the foundation for this, we evaluated the association between disease resistance and MHC class I and class II polymorphism in Atlantic salmon. Standardized disease challenge trials were performed on a semi-wild Atlantic salmon population with subsequent MHC typing and statistical analysis. The pathogens employed were infectious salmon anaemia virus (ISAV) causing infectious salmon anaemia and the Aeromonas salmonicida bacteria causing furunculosis. The material consisted of 1,182 Atlantic salmon from 33 families challenged with A. salmonicida and 1,031 Atlantic salmon from 25 families challenged with ISAV. We found highly significant associations between resistance towards infectious diseases caused by both pathogens and MH class I and class II polymorphism in Atlantic salmon. The observed associations were detected due to independently segregating MH class I and class II single loci, and inclusion of a large number of fish allowing an extensive statistical analysis.     

Infectious salmon anaemia virus—molecular biology and pathogenesis of the infection

Aquaculture has a long history in many parts of the world, but it is still young at an industrial scale. Marine fish farming in open nets of a single fish species at high densities compared to their wild compatriots opens a plethora of possible infections. Infectious salmon anaemia (ISA) is an example of disease that surfaced after large-scale farming of Atlantic salmon (Salmo salar) appeared. Here, a review of the molecular biology of the ISA virus (ISAV) with emphasis on its pathogenicity is presented. The avirulent HPR0 variant of ISAV has resisted propagation in cell cultures, which has restricted the ability to perform in vivo experiments with this variant. The transition from avirulent HPR0 to virulent HPRΔ has not been methodically studied under controlled experimental conditions, and the triggers of the transition from avirulent to virulent forms have not been mapped. Genetic segment reassortment, recombination and mutations are important mechanisms in ISAV evolution, and for the development of virulence. In the 25 years since the ISAV was identified, large amounts of sequence data have been collected for epidemiologic and transmission studies, however, the lack of good experimental models for HPR0 make the risk evaluation of the presence of this avirulent, ubiquitous variant uncertain. This review summarizes the current knowledge related to molecular biology and pathogenicity of this important aquatic orthomyxovirus.     

Fate of infectious salmon anaemia virus (ISAV) in experimentally challenged blue mussels Mytilus edulis

In order to investigate the potential role of blue mussels Mytilus edulis as a vector of the fish pathogenic infectious salmon anaemia virus (ISAV), we developed an experimental bioaccumulation system in which mussels can accumulate virus during normal filtration. Detection of virus in mussels was performed by means of real-time RT-PCR. ISAV-RNA was detected in the mussels until 72 h post-challenge. Hepatopancreas homogenate from experimentally challenged mussels was injected into salmon. All the fish injected with homogenate prepared immediately after accumulations were strongly ISAV positive 4 wk post-challenge. In the group injected with homogenate prepared 24 h after the challenge, 1 fish out of 25 was weakly ISAV positive. All of the fish that were challenged with mussel homogenate prepared 96 h after accumulation were ISAV negative. Mussels sampled from a tank with experimentally infected salmon demonstrating clinical signs consistent with ISA (infectious salmon anaemia) and mussels collected on net pen cages during ISA outbreaks in Atlantic salmon were all ISAV negative. The results indicate that the ISAV is rapidly inactivated in mussels and that mussels are not a likely reservoir host or vector for ISAV.     

The Putative Polymerase Sequence of Infectious Salmon Anemia Virus Suggests a New Genus within the Orthomyxoviridae

The infectious salmon anemia virus (ISAV) is an orthomyxovirus-like virus infecting teleosts. The disease caused by this virus has had major economic consequences for the Atlantic salmon farming industry in Norway, Canada, and Scotland. In this work, we report the cloning and sequencing of an ISAV-specific cDNA comprising 2,245 bp with an open reading frame coding for a predicted protein with a calculated molecular weight of 80.5 kDa. The putative protein sequence shows the core polymerase motifs characteristic of all viral RNA-dependent RNA polymerases. Comparison of the conserved motifs with the corresponding regions of other segmented negative-stranded RNA viruses shows a closer relationship with members of the Orthomyxoviridae than with viruses in other families. The putative ISAV polymerase protein (PB1) has a length of 708 amino acids, a charge of +22 at neutral pH, and a pI of 9.9, which are consistent with the properties of the PB1 proteins of other members of the family. Calculations of the distances between the different PB1 proteins indicate that the ISAV is distantly related to the other members of the family but more closely related to the influenza viruses than to the Thogoto viruses. Based on these and previously published results, we propose that the ISAV comprises a new, fifth genus in the Orthomyxoviridae.     

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV^901_09 and rISAVr^S6-NotI-HPR containing a NotI restriction site and rISAV^S6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 10⁵ PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry.     

Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis

The present study describes the use of molecular methods in studying infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Canada, USA and Chile. The nucleotide sequences of the haemagglutinin gene (HA) from 70 ISAV isolates have been analysed for phylogenetic relationship and the average mutation rate of nucleotide substitutions calculated. The isolates constitute 2 major groups, 1 European and 1 North American group. The isolate from Chile is closely related to the North American isolates. The European isolates can be further divided into 3 separate groups reflecting geographical distribution, time of collection, and transmission connected with farming activity. Based on existing information about infectious salmon anaemia (ISA) and new information emerging from the present study, it is hypothesised that: (1) ISAV is maintained in wild populations of trout and salmon in Europe; (2) it is transmitted between wild hosts mainly during their freshwater spawning phase in rivers; (3) wild salmonids, mainly trout, possibly carry benign wild-type ISAV isolates; (4) a change (mutation) in virulence probably results from deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates; (5) ISA emerges in farmed Atlantic salmon when mutated isolates are transmitted from wild salmonids or, following mutation of benign isolates, in farmed salmon after transmission from wild salmonids; (6) farming activity is an important factor in transmission of ISAV between farming sites in addition to transmission of ISAV from wild salmonids to farmed salmon; (7) transmission of ISAV from farmed to wild salmonids probably occurs less frequently than transmission from wild to farmed fish due to lower frequency of susceptible wild individuals; (8) the frequency of new outbreaks of ISA in farmed salmon probably reflects natural variation in the prevalence of ISAV in wild populations of salmonids.     

Detection of infectious salmon anaemia virus (ISAV) by in situ hybridisation

An in situ hybridisation method was developed to detect infectious salmon anaemia virus (ISAV) in fixed tissues from Atlantic salmon Salmo salar L. Three DNA probes detected ISAV in heart, liver, kidney, spleen, caeca, and mid-gut from infected farmed Atlantic salmon obtained from a natural outbreak of ISA. The strongest signals were obtained using Probe S8, from Segment 8 of ISAV. Hybridisation was most prominent in the endothelial cells of heart tissue. The probes reacted specifically with ISAV; no hybridisation was evident in uninfected tissues from Atlantic salmon. Importantly, the probes did not cross react with the pathogens IHNV (haematopoietic necrosis virus), IPNV (infectious pancreatic necrosis virus), SPDV (salmon pancreas disease virus) and VHSV (viral haemorrhagic septicemia virus).     

Identification of infectious salmon anaemia virus in Atlantic salmon from Nova Scotia (Canada): evidence for functional strain differences

Infectious salmon anaemia (ISA) is a serious disease responsible for high morbidity in farmed Atlantic salmon Salmo salar in Norway, Scotland and New Brunswick, Canada. Recent attempts to identify different strains of ISA virus (ISAV) based on nucleotide sequence variation have shown that the Norwegian and Scottish samples are similar to one another but markedly different from New Brunswick samples. These data may suggest the presence of different strains on each side of the Atlantic but no functional difference has been found with either strain. We describe the first identification and characterisation of ISAV in Atlantic salmon from Nova Scotia, Canada. Further, salmon infected with the Nova Scotia ISAV do not show typical ISAV pathology or mortality. Sequencing of this new strain showed it to possess greater similarity to ISAV from Norway and Scotland than to ISAV from New Brunswick. These findings are discussed in terms of a possible origin of the Nova Scotia ISAV strain and the existence of an avirulent ISAV strain. The impact of current strain variation studies on our knowledge of ISAV is also discussed.     

Serological evidence of infectious salmon anaemia virus (ISAV) infection in farmed fishes,using an indirect enzyme-linked immunosorbent assay (ELISA)

Antibody detection tests are rarely used for diagnostic purposes in fish diseases. Infectious salmon anaemia (ISA) caused by ISA virus (ISAV) is an emerging disease of Atlantic salmon Salmo salar L. The virus has also been isolated from diseased coho salmon Oncorhynchus kisutch in Chile. An indirect enzyme-linked immunosorbent assay (ELISA) that should facilitate serodiagnosis of ISAV infection, the study of epidemiology, and the control of ISA in farmed fishes has been developed using purified ISAV as the coating antigen, and monoclonal antibodies that detect fish immunoglobulins bound to the antigen on the plate. Application of the test to a random sample of farmed Atlantic salmon from the Bay of Fundy, New Brunswick, Canada, positively identified 5 of the 7 ISAV RT-PCR-positive fish, and all 10 RT-PCR-negative fish were also negative in the ELISA. Some RT-PCR-negative fish had an elevated non-specific antibody reactivity suggestive of chronic infection or resistance to ISAV. This test was also able to detect 11 of the 14 coho salmon pooled serum samples from a clinically affected farm in Chile that were positive by the virus neutralization (VN) test, and 2 of the 4 VN-negative samples. We conclude that this ELISA would be suitable as a routine test for ISAV infection or for assessing ISAV vaccine efficacy before placing smolts in sea cages, and for testing fishes in sea cages to detect level of resistance to ISA. The assay enables vaccination in combination with depopulation control methods.     

Investigation into the susceptibility of saithe Pollachius virens to infectious salmon anaemia virus (ISAV) and their potential role as a vector for viral transmission

Wild-caught saithe Pollachius virens were experimentally exposed to an isolate of infectious salmon anaemia virus (ISAV) of Norwegian origin. Mortality attributable to ISAV did not occur following exposure by intra-peritoneal (i.p.) injection of virus or by cohabitation with ISAV-infected Atlantic salmon Salmo salar. Despite the individual testing of 120 ISAV-exposed saithe, ISAV was not detectable using RT-PCR, the most sensitive ISAV diagnostic tool demonstrated to date. Furthermore, saithe exposed to ISAV-infected salmon were not capable of transmitting virus when transferred to tanks containing na?ve salmon. Thus saithe appear to be resistant to this Norwegian isolate of ISAV and incapable of supporting its replication. Saithe which co-exist with salmon in and around aqua-culture facilities are considered unlikely to have a significant impact on the epizootiology of ISAV.     

Infectious salmon anaemia virus in wild fish from Scotland.

Following the outbreak of infectious salmon anaemia (ISA) at salmon farms in Scotland, UK, a survey was established to determine the extent of infection in wild fish. All fish tested were free from the clinical symptoms of ISA. Isolations of ISAV were made from 5 sea trout within areas where ISA affected salmon farms were located. Evidence for ISAV in other sea trout was provided by ISA RT-PCR diagnostic tests. Results from ISA RT-PCR tests reveal evidence for ISAV being present in salmon parr, adult salmon and juvenile brown trout in rivers distant from salmon farms and indicate that, at the time of the survey (1998-1999), ISAV may have been widely distributed. Nucleotide sequence analysis of segments 2 and 8 showed that for most sequences from wild fish there was 100% homology with ISAV isolated from clinically affected farmed fish although evidence is presented which indicates variability in ISAV sequences from wild fish. Modelling the RT-PCR findings indicates that ISAV among salmonid fish was spatially non-random. Brown trout, sea trout and salmon (adult and parr) show a pattern of occasionally large numbers of positive samples against a background of very low numbers.     

A salmonid cell line (TO) for production of infectious salmon anaemia virus (ISAV)

A new cell line designated TO which provides a high yield of infectious salmon anaemia virus (ISAV) has been established. The cells originate from head kidney leukocytes isolated from Atlantic salmon and grow well at 20 degrees C in EMEM with 5% CO2 and without CO2 supplement in HMEM. The cells have at present been passed more than 150 times and no changes in morphology, growth or virus production have been observed. The virus infection results in cytopathic effects (CPE) within 9 d, and the virus titre obtained from centrifuged and filtrated cell lysates, measured as TCID50, was about 10(9.1) ml(-1). The virus isolated from lysates of infected cells by a sucrose gradient provided purified ISAV when examined by silver stained SDS-PAGE. Salmon injected with diluted virus supernatant showed mortalities, hematocrit values and clinical signs in accordance with infectious salmon anaemia.     

Nucleotide sequence variation in isolates of infectious salmon anaemia virus (ISAV) from Atlantic salmon Salmo salar in Scotland and Norway

The nucleotide sequences of segments 2 and 8 of infectious salmon anaemia virus (ISAV) isolates from Scotland and Norway were determined and compared. Sequence variations were found in both segments, with segment 2 being more variable. These variations were of the same degree as those found in previous comparisons of the first recorded strain of ISAV in Scotland and Norwegian strains for which sequence information was available. The sequences again demonstrate the separation of European and Canadian strains of ISAV but, as is expected for an RNA virus, reveal that the European ISAV is not homogeneous. These results demonstrate the value of nucleotide sequences as a tool for strain identification and epidemiological investigation of ISAV.