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.     

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).     

Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in western Norway

Studies of infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Ireland, Canada, the USA and Chile, suggest that natural reservoirs for this virus can be found on both sides of the North Atlantic. Based on existing information about ISAV it is believed to be maintained in wild populations of trout and salmon in Europe. It has further been suggested that ISAV is transmitted between wild hosts, mainly during their freshwater spawning phase in rivers, and that wild salmonids, mainly trout, are possible carriers of benign wild-type variants of ISAV. Change in virulence is probably a result of deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates after transmission to farmed salmon. Hence, it has been suggested that the frequency of new outbreaks of ISA in farmed salmon could partly reflect natural variation in the prevalence of ISAV in wild populations of salmonids. The aims of the present study were to screen for ISAV in wild salmonids during spawning in rivers and to determine the pathogenicity of resultant isolates from wild fish. Tissues from wild salmonids were screened by RT-PCR and real-time PCR. The prevalence of ISAV in wild trout Salmo trutta varied from 62 to 100% between tested rivers in 2001. The prevalence dropped in 2002, ranging from 13 to 36% in the same rivers and to only 6% in 2003. All ISAV were nonpathogenic when injected into disease-free Atlantic salmon, but were capable of propagation, as indicated by subsequent viral recovery. However, non-pathogenic ISAV has also been found in farmed salmon, where a prevalence as high as 60% has been registered, but with no mortalities occurring. Based on the results of the present and other studies, it must be concluded that vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking. This information can only be gained by further screening of possible reservoirs, combined with the development of a molecular tool for typing virulence and the geographical origin of the virus isolates.     

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.     

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.     

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.     

Infectious pancreatic necrosis virus infection in Atlantic salmon Salmo salar post-smolts affects the outcome of secondary infections with infectious salmon anaemia virus or Vibrio salmonicida.

Infectious pancreatic necrosis (IPN) virus (IPNV) infection in Atlantic salmon Salmo salar L. post-smolts and its influence on the outcome of secondary infections with infectious salmon anaemia (ISA) virus (ISAV) or Vibrio salmonicida were studied. The infections with ISAV or V salmonicida were performed both in a period of acute IPN and in the following IPNV carrier stage, 3 and 6 to 8 wk after experimental IPNV challenge, respectively. An IPNV carrier condition at low virus titre did not influence the mortality rates after secondary infections. Neither the ISAV infection nor the V. salmonicida infection in experimentally induced IPNV carriers resulted in mortalities different from those observed after challenge of IPNV-free fish. At higher IPNV titres in Atlantic salmon with acute IPN, the outcome of secondary infections was quite different from that observed in IPNV-free fish and in IPNV carriers. In 2 different experiments significantly more fish died when fish with acute IPN were infected with V salmonicida than when fish were infected with V salmonicida alone. Mortality also started earlier in the double-infected group than in the group challenged with V. salmonicida alone, 3 to 4 and 8 d after V salmonicida infection, respectively. Similar results were observed independent of whether mortalities due to IPN alone were registered in the experiments. When Atlantic salmon with acute IPN were infected with ISAV, significantly fewer fish died than when fish were infected with ISAV alone. The ongoing IPNV infection seemed to provide some protection against development of ISA.     

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.     

Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus

Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4-O-acetylated sialic acid (Neu4,5Ac(2)). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs.     

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.     

Salmonid alphavirus-based replicon vaccine against infectious salmon anemia (ISA): Impact of immunization route and interactions of the replicon vector

A salmonid alphavirus (SAV)-based replicon encoding the infectious salmon anemia virus (ISAV) hemagglutinin-esterase (HE), pSAV/HE, is an efficacious vaccine against infectious salmon anemia (ISA). Delivered intramuscularly (i.m.), the replicon vaccine provides high protection against subsequent ISAV challenge in Atlantic salmon (Salmo salar), and induces a strong innate response locally at the injection site. This may be beneficial and could warrant reduced doses and improved efficacy compared to conventional DNA vaccines. In the present study, we found that intraperitoneal (i.p.) administration of the pSAV/HE replicon vaccine did not induce protection, neither alone or in combination with a sub-potent, inactivated low-dose ISAV vaccine given i.p. No significant differences between the two immunization routes regarding systemic immune responses could be observed. I.m. injection of the replicon vector encoding a non-viral gene or the protective glycoprotein (G protein) from the heterologous viral hemorrhagic septicemia virus (VHSV) induced no protection against ISA. Although the replicons without the ISAV HE did induce IFN-signaling pathways at the muscle injection site similar to the pSAV/HE replicon they did not improve the efficacy of a sub-potent inactivated low-dose ISAV vaccine delivered i.p. Moreover, there was a tendency for reduced efficacy of the pSAV/HE replicon vaccine injected i.m. when co-injected with the replicon encoding the VHSV G protein, which previously, after DNA vaccination, have been reported to induce cross-protection against heterologous virus challenge in fish.