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.

Infectious salmon anemia virus (ISAV) is an orthomyxovirus causing serious disease in Atlantic salmon(Salmo salar L.). This study presents the characterization of the ISAV 50-kDa glycoprotein encoded by segment 5, here termed the viral membrane fusion protein (F). This is the first description of a separate orthomyxovirus F protein, and to our knowledge, the first pH-dependent separate viral F protein described. The ISAV F protein is synthesized as a precursor protein, F 0 , that is proteolytically cleaved to F 1 and F 2 , which are held together by disulfide bridges. The cleaved protein is in a metastable, fusion-activated state that can be triggered by low pH, high temperature, or a high concentration of urea. Cell-cell fusion can be initiated by treatment with trypsin and low pH of ISAV-infected cells and of transfected cells expressing F, although the coexpression of ISAV HE significantly improves fusion. Fusion is initiated at pH 5.4 to 5.6, and the fusion process is coincident with the trimerization of the F protein, or most likely a stabilization of the trimer, suggesting that it represents the formation of the fusogenic structure. Exposure to trypsin and a low pH prior to infection inactivated the virus, demonstrating the nonreversibility of this conformational change. Sequence analyses identified a potential coiled coil and a fusion peptide. Size estimates of F 1 and F 2 and the localization of the putative fusion peptide and theoretical trypsin cleavage sites suggest that the proteolytic cleavage site is after residue K 276 in the protein sequence. Infectious salmon anemia virus (ISAV) is an enveloped virusbelonging to the family Orthomyxoviridae and the genus Isavirus, and it causes serious disease in Atlantic salmon (Salmo salar L.) (51,58,68,71,80). The ISAV genome is composed of eight negative-sense, single-stranded RNA segments, and while nucleotide sequences of all segments have been published (9,43,44,56,66,67,74,75), much remains to be elucidated with respect to protein identification and characterization. A total of 12 proteins have been detected by immunoprecipitation of lysates from radiolabeled infected cells (40), while four major structural proteins have been recognized in purified ISAV particles, including the matrix (M1; 22 to 24 kDa) (7, 24), the nucleoprotein (NP; 66 to 71 kDa) (3, 24), and two membrane glycoproteins (24). The receptor-binding and receptor-destroying activities are associated with the 42-kDa glycoprotein encoded by segment 6, termed the hemagglutininesterase (HE) (24,25,34,42,43,66), while the activities of the second glycoprotein, glycoprotein 50 (gp50), encoded by segment 5 (9), have not been described previously.ISAV pursues a nuclear replication strategy similar to that of the influenza viruses (3, 26) which is initiated by receptor binding and internalization into cellular endosomes, where the viral and cellular membranes fuse in response to low pH (21). The addition of trypsin to the culture medium during ISAV replication has been demonstrated to have a benefici...

Sleeping disease (SD) is currently a matter of concern for salmonid fish farmers in most parts of the world. A viral etiology of SD has recently been suspected, since virus-like particles have been observed in infected rainbow trout cells. In salmonid-derived cell lines, the maximal rate of virus production was observed at 10°C, while little virus was produced at 14°C. Through biochemical, physicochemical, and morphological studies, SD virus (SDV) was shown to be an enveloped virus of roughly 60 nm in diameter. The genome consists of 12 kb of RNA, with the appearance of a 26S subgenomic RNA during the time course of SDV replication. The screening of a random-primed cDNA library constructed from the genomic RNA of semipurified virions facilitated the identification of a specific SDV cDNA clone having an open reading frame related to the alphavirus E2 glycoproteins. To extend the comparison between SDV structural proteins and the alphavirus protein counterparts, the nucleotide sequence of the total 4.1-kb subgenomic RNA has been determined. The 26S RNA encodes a 1,324-amino-acid polyprotein exhibiting typical alphavirus structural protein organization. SDV structural proteins showed several remarkable features compared to other alphaviruses: (i) unusually large individual proteins, (ii) very low homology (ranging from 30 to 34%) (iii) an unglycosylated E3 protein, and (iv) and E1 fusion domain sharing mutations implicated in the pH threshold. Although phylogenetically related to the Semliki Forest virus group of alphaviruses, SDV should be considered an atypical member, able to naturally replicate in lower vertebrates.Sleeping disease (SD) syndrome of farmed freshwater rainbow trout has been observed in France for many years (4). The most characteristic sign of the disease is the unusual behavior of the fish, which stay on their side at the bottom of the tank. Histological observations of diseased fish showed a chronological appearance of lesions in the pancreas, in the heart, and in the muscle at the last stage of the disease (5, 6). Transmission of SD may occur through contact with contaminated tissue from fish that have SD (5). A viral etiology of SD was suspected, since virus-like particles were observed in purified homogenates from kidneys of diseased fish (3). However, all attempts to isolate a viral agent on commonly available fish cell lines by inoculating organ homogenates from diseased fish remained unsuccessful until recently (7). Isolation of SD virus (SDV) in cell culture was successfully achieved by direct inoculation of salmonid cell lines (CHSE-214 and RTG-2) with plasma from infected fish.The characterization of SDV was successfully achieved by optimizing viral production in tissue culture and by studying several physicochemical features of this virus. Data include the type of nucleic acid, size, and organization of the SDV genome. The viral genome has been shown to be an RNA molecule of roughly 12 kb. A cDNA library has been constructed, and the nucleotide sequencing of recombinant cDNA clones de...

No abstract