In Europe, 2 closely related alphaviruses (Togaviridae) are regarded as the causative agents of sleeping disease (SD) and salmon pancreas disease (SPD): SD virus (SDV) has been isolated from rainbow trout Oncorhynchus mykiss in France and the UK, while SPD virus (SPDV) has been isolated from salmon Salmo salar in Ireland and the UK. Farmed salmonids in western Norway also suffer from a disease called pancreas disease (PD), and this disease is also believed to be caused by an alphavirus. However, this virus has not yet been characterised at the molecular level. We have cultured a Norwegian salmonid alphavirus from moribund fishes diagnosed with cardiac myopathy syndrome (CMS) and fishes diagnosed with PD. The virus has also been found in salmon suffering from haemorrhagic smolt syndrome in the fresh water phase. The genomic organisation of the Norwegian salmonid alphavirus is identical to that in SPDV and SDV, and the nucleotide sequence similarity to the other 2 alphaviruses is 91.6 and 92.9%, respectively. Based on the pathological changes, host species and the nucleotide sequence, we suggest naming this virus Norwegian salmonid alphavirus (NSAV). Together with SPDV and SDV it constitutes a third subtype of salmonid alphavirus (SAV) species within the genus Alphavirus, family Togaviridae. KEY WORDS: Norwegian salmonid alphavirus · Virus characterisation · Pancreas disease Resale or republication not permitted without written consent of the publisherDis Aquat Org 66: [113][114][115][116][117][118][119][120] 2005 pers. obs., Poppe & Rimstad 1989). Alphavirus spp. have also been found, using RT-PCR and sequencing, in moribund farmed salmon in Norway diagnosed with cardiac myopathy syndrome, CMS (A. Nylund pers. obs.) and in salmon suffering from haemorrhagic smolt syndrome (HSS) in the fresh water phase (Nylund et al. 2003).The majority of cases in which Alphavirus spp. have been detected associated with disease in salmon and rainbow trout have been in Hordaland and Sogn og fjordane (western Norway), but recently a few cases have been seen in northern Norway (Nordland, Troms and Finnmark) (A. Nylund pers. obs.). The genomes of the viruses from northern Norway have been partially sequenced and are identical to those from western Norway. This means that Alphavirus spp. have been found in the majority of Norwegian counties with salmon farming.The complete genomes of SPDV from Ireland (Isolate F93-125) and SDV from France (Isolate S49P) have been sequenced and consist of about 12 kb (Weston et al. 2002). No other isolates have been completely sequenced, but partial sequencing of selected nonstructural and structural protein genes indicate that SPDV and SDV from the UK are identical to those sequenced from Ireland and France, respectively . Included in the studies by Weston et al. (2002Weston et al. ( , 2003 are also 2 Norwegian alphavirus isolates, PD97-N2 and PD97-N3 (supplied by K. E. Christie, Intervet, Bergen), originating from diseased salmon and rainbow trout, respectively. Both isolates were partia...

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Infectious salmon anemia virus (ISAV) is an unclassifiedOrthomyxovirus that has been shown to contain a segmented genome with eight single-stranded RNA species coding for 10 viral proteins. Four major structural proteins were characterized in the present study: two glycosylated proteins with estimated molecular masses of 42 and 50 kDa, one 66-kDa phosphoprotein, and one 22-kDa protein. Examination of lysed virions revealed the two glycoproteins and the 22-kDa protein in the soluble fraction, while the 66-kDa phosphoprotein and a minor part of the 22-kDa protein were found in the pelleted fraction. Immunofluorescence staining of infected cells demonstrated that the 22-kDa protein was a late protein accumulating in the nucleus. We conclude that the 66-kDa protein is the nucleoprotein, the 22-kDa protein is the matrix protein, and the 42-and 50-kDa proteins are the surface proteins. Radioimmunoprecipitation analysis of the 42-kDa glycoprotein, which was previously shown to represent the ISAV hemagglutinin, indicated that this protein exists at least as dimers. Further, by labeling of purified ISAV with [1,3-3 H]diisopropyl fluorophosphate, it was also demonstrated that the viral esterase is located with the hemagglutinin. This finding was confirmed by demonstration of acetylesterase activity in affinity-purified hemagglutinin preparations. Finally, the active-site serine residue could be tentatively identified at position 32 within the amino acid sequence of the hemagglutinin of ISAV strain Glesvaer/2/90. It is proposed that the ISAV vp66 protein be termed nucleoprotein, the gp42 protein be termed HE protein, and the vp22 protein be termed matrix protein.

Pancreas disease is responsible for major economic losses in the European salmonid farming industry. It was previously believed that a single subtype (salmon pancreas disease virus) of the virus species Salmonid alphavirus (SAV) was responsible for all outbreaks of pancreas disease in the UK and Norway. However, the recent discovery that pancreas disease in Norway is caused by a new and distinct subtype of salmonid alphavirus, exclusively found in Norway, has advocated the need for better diagnostic tools. In the present paper, three real-time PCR assays for all known subtypes of salmonid alphavirus have been developed; the Q nsP1 assay is a broad-spectrum one that detects RNA from all subtypes, the Q SPDV assay specifically detects the salmon pancreas disease virus subtype, and the Q NSAV assay only detects the new Norwegian salmonid alphavirus subtype.The results demonstrated the assays to be highly sensitive and specific, detecting <0.1 TCID 50 of virus stocks. Regression analysis and standard curves calculated from the C t -values from 10-fold serial dilutions of virus stocks showed that the assays were highly reproducible over a wide range of RNA input. Thirty-nine field samples were tested in triplicate and compared with traditional RT-PCR. Overall, the real-time assays detected 35 positive compared to 29 positives in standard RT-PCR, and were thus shown to be more sensitive for detecting salmonid alphaviruses in field samples.

Salmonid alphavirus (SAV) (family Togaviridae) causes mortality in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss W.) in Norway, France, UK, and Ireland. At least three subtypes of SAV exist: SPDV in UK/Ireland, SDV in France/UK, and the recently reported Norwegian salmonid alphavirus (NSAV) in western Norway. During 2003 and 2004, disease caused by NSAV was reported for the first time in northern Norway, more than 800 km away from the enzootic area in western Norway. The present study has investigated the phylogenetic relationships among 20 NSAV isolates, based on a 1221-nt-long segment covering part of the capsid gene, E3, and part of the E2 gene, collected over a period of eight years. The results revealed genetic homogeneity among NSAV isolates, including those from northern Norway. The SDV or SPDV subtypes were not found in diseased Norwegian fish. A substitution rate of 1.70 (+/-1.03) x 10(-4) nt subst/site/year was obtained for the NSAV subtype by maximum likelihood analysis. The second aim of this study was to clarify whether NSAV changes genotypically in cell culture by culturing a NSAV isolate through 20 passages in CHSE-214 cells. Sequencing of almost the entire genome (11530 nt) after 20 passages revealed four nucleotide substitutions, all resulting in amino acid substitutions. One of these substitutions, serine to proline in E2 position 206, was also found to have occurred in field isolates.