The first alphavirus to be isolated from fish was recorded in 1995 with the isolation of salmon pancreas disease virus from Atlantic salmon, Salmo salar L., in Ireland. Subsequently, the closely related sleeping disease virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), in France. More recently Norwegian salmonid alphavirus (SAV) has been isolated from marine phase production of Atlantic salmon and rainbow trout in Norway. These three viruses are closely related and are now considered to represent three subtypes of SAV, a new member of the genus Alphavirus within the family Togaviridae. SAVs are recognized as serious pathogens of farmed Atlantic salmon and rainbow trout in Europe. This paper aims to draw together both historical and current knowledge of the diseases caused by SAVs, the viruses, their diagnosis and control, and to discuss the differential diagnosis of similar pathologies seen in cardiomyopathy syndrome and heart and skeletal muscle inflammation of Atlantic salmon.

Cell culture isolates of salmon pancreas disease virus (SPDV) of farmed Atlantic salmon and sleeping disease virus (SDV) of rainbow trout were compared. Excluding the poly(A) tracts, the genomic nucleotide sequences of SPDV and SDV RNAs include 11,919 and 11,900 nucleotides, respectively. Phylogenetic analysis places SPDV and SDV between the New World viruses of Venezuelan equine encephalitis virus and Eastern equine encephalitis virus and the Old World viruses of Aura virus and Sindbis virus. When compared to each other, SPDV and SDV show 91.1% nucleotide sequence identity over their complete genomes, with 95 and 93.6% amino acid identities over their nonstructural and structural proteins, respectively. Notable differences between the two viruses include a 24-nucleotide insertion in the C terminus of nsP3 protein of SPDV and amino acid sequence variation at the C termini of the capsid and E1 proteins. Experimental infections of Atlantic salmon and rainbow trout with SPDV and SDV confirmed that the disease lesions induced by SPDV and SDV were similar in nature. Although infections with SPDV and SDV produced similar levels of histopathology in rainbow trout, SDV induced significantly less severe lesions in salmon than did SPDV. Virus neutralization tests performed with sera from experimentally infected salmon indicated that SPDV and SDV belonged to the same serotype; however, antigenic variation was detected among SDV and geographically different SPDV isolates by using monoclonal antibodies. Although SPDV and SDV exhibit minor biological differences, we conclude on the basis of the close genetic similarity that SPDV and SDV are closely related isolates of the same virus species for which the name Salmonid alphavirus is proposed.

Pancreas disease (PD) of farmed Atlantic salmon Salmo salar has been recognised in Scotland, Ireland, Norway, the USA, France and Spaln and can cause severe economlc loss Thls paper reports the ~solatlon, flom PD-affected f~s h , of a virus with physicochemlcal characteristics and moiphology resembling members of the Togavirldae When Inoculated into Atlantic salmon post-smolts it causes pathological changes in pancreas h e a~t and muscle tlssues which are ind~stinguishable from those piesent in fleld outbreaks of PD I t is proposed that the virus be named salmon pancreas disease vlrus (SPDV) KEY WORDS. Pancreas d~s e a s e . PD Atlantlc salmon Toga-like SPDV

Experimental pancreas disease in Atlantic salmonSalmo salar post-smolts induced b y salmon pancreas disease virus (SPDV) ' ABSTRACT: Disease-free Atlantic salmon Salrno salarpost-smolts (mean weight 87 g) were maintained in a flow-through ozone-sterilized sea water system at 12 to 15'C and ambient salinity. One hundred fish were intraperitoneally inoculated with 0.1 m1 of salmon pancreas disease virus (SPDV) of a titre 107 TCIDSO ml-' Fifty fin-clipped uninoculated smolts were placed in-contact in the same tank. One hundred fish were kept in another tank as controls and were inoc.ulated with a lysate from un-infected Chinook salmon embryo (CHSE-214) cell cultures. Blood and tissues for virus isolation, serum neutralisation tests and histological examination were taken at intervals up to 42 days post inoculation (dpi). Virus was re-isolated from SPDV inoculated smolts at 7, 10. 15 and 21 dpi and in-contact fish at 14 and 21 dpi. Neutralising antibody was first detected in the inoculated fish at 10 dpi and in the in-contact fish 11 d later. Clinical signs and microscopic lesions indistinguishable from naturally occurring pancreas disease (PD) were observed in SPDV inoculated and in-contact smolts. No lesions were detected in the negative controls. These results provide strong evidence that SPDV is the etiologic agent of PD in farmed Atlantic salmon in Ireland.

A 5.2-kb region at the 3' terminus of the salmon pancreas disease virus (SPDV) RNA genome has been cloned and sequenced. The nucleotide and predicted amino acid sequences show that SPDV shares considerable organizational and sequence identity to members of the genus alphavirus within the family Togaviridae. The SPDV structural proteins encoded by the 5.2-kb region contain a number of unique features when compared to other sequenced alphaviruses. Based on cleavage site homologies, the predicted sizes of the SPDV envelope glycoproteins E2 (438 aa) and E1 (461 aa) are larger than those of other alphaviruses, while the predicted size of the alphavirus 6K protein is 3.2 K (32 aa) in SPDV. The E2 and E1 proteins each carry one putative N-linked glycosylation site, with the site in E1 being found at a unique position. From amino acid sequence comparisons of the SPDV structural region with sequenced alphaviruses overall homology is uniform, ranging from 32 to 33%. While nucleotide sequence analysis of the 26S RNA junction region shows that SPDV is similar to other alphaviruses, analysis of the 3'-nontranslated region reveals that SPDV shows divergence in this region.

A comparative challenge study of six marine isolates representing subtypes 1-6 of salmonid alphavirus (salmon pancreas disease virus, Genus Alphavirus, Family Togaviridae) was conducted in Atlantic salmon in a fresh water cohabitation trial. Histopathological lesions typical of pancreas disease were observed with all subtypes, and virus was re-isolated from serum of cohabitant fish in each case. Using a virus neutralization (VN) test neutralizing salmonid alphavirus (SAV) subtype 1 strain F93-125, VN antibodies were detected in all challenge groups, consistent with serological cross-reactivity between these subtypes. Using real-time RT-PCR, SAV RNA was detected in heart tissue from 2 to 3 weeks post-challenge (wpc) in all cohabitant groups excluding controls. The results obtained suggested differences in the dynamics of infection between strains of SAV and potentially between subtypes. Results for SAV subtypes 1 and 3 suggested essentially synchronous infection of cohabitant fish. These two study groups also had the highest virus load in heart tissue as measured by quantitative RT-PCR and also had the most extensive histopathological changes. In contrast, results for SAV subtypes 2 and 6 strains were consistent with asynchronous infection in the cohabitant fish and were characterized by slow spread, low virus loads and mild histopathological changes. The SAV subtype 4 and 5 strains occupied an intermediate position in this regard. Despite the use of concentration procedures, it was not possible to detect SAV RNA in water samples from selected study tanks. However, testing of faeces from the SAV subtypes 1, 3 and 6 challenge groups found positive signals in each beginning at 1-3 wpc and remaining detectable for a further 2-3 weeks. Parallel testing of mucus samples found these became positive at 2-3 wpc and remained positive for a further 1-3 weeks. These results demonstrate for the first time that shedding and transmission of virus may occur by both these routes and suggest that dispersal in these matrices should be included in any disease transmission models.

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The present paper describes, for the first time, clinical signs and pathological findings of pancreas disease (PD) in farmed Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), in sea water in Norway. Similarities and differences with reports of PD from Ireland and Scotland are discussed. Samples of 68 rainbow trout from disease outbreaks on 14 farms and from 155 Atlantic salmon from outbreaks on 20 farms collected from 1996 to 2004 were included in the present study. The histopathological findings of PD in Atlantic salmon and rainbow trout in sea water were similar. Acute PD, characterized by acute necrosis of exocrine pancreatic tissues, was detected in nine Atlantic salmon and three rainbow trout. Salmonid alphavirus (SAV) was identified in acute pancreatic necroses by immunohistochemistry. Most fish showed severe loss of exocrine pancreatic tissue combined with chronic myositis. Myocarditis was often but not consistently found. Kidneys from 40% and 64% of the rainbow trout and Atlantic salmon, respectively, had cells along the sinusoids that were packed with cytoplasmic eosinophilic granules. These cells resembled hypertrophied endothelial cells or elongated mast cell analogues. Histochemical staining properties and electron microscopy of these cells are presented. SAV was identified by RT-PCR and neutralizing antibodies against SAV were detected in blood samples.