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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...

Infectious hematopoietic necrosis virus (IHNV) is aNovirhabdovirus and is the causative agent of a devastating acute, lethal disease in wild and farmed rainbow trout. The virus is enzootic throughout western North America and has spread to Asia and Europe. A full-length cDNA of the IHNV antigenome (pIHNV-Pst) was assembled from subgenomic overlapping cDNA fragments and cloned in a transcription plasmid between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme. Recombinant IHNV (rIHNV) was recovered from fish cells at 14°C, following infection with a recombinant vaccinia virus expressing the T7 RNA polymerase (vTF7-3) and cotransfection of pIHNV-Pst together with plasmids encoding the nucleoprotein N (pT7-N), the phosphoprotein P (pT7-P), the RNA polymerase L (pT7-L), and the nonvirion protein NV (pT7-NV). When pT7-N and pT7-NV were omitted, rIHNV was also recovered, although less efficiently. Incidental mutations introduced in pIHNV-Pst were all present in the rIHNV genome; however, a targeted mutation located in the L gene was eliminated from the recombinant genome by homologous recombination with the added pT7-L expression plasmid. To investigate the role of NV protein in virus replication, the pIHNV-Pst construct was engineered such that the entire NV open reading frame was deleted and replaced by the genes encoding green fluorescent protein or chloramphenicol acetyltransferase. The successful recovery of recombinant virus expressing foreign genes instead of the NV gene demonstrated that the NV protein was not absolutely required for viral replication in cell cultures, although its presence greatly improves virus growth. The ability to generate rIHNV from cDNA provides the basis to manipulate the genome in order to engineer new live viral vaccine strains.

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are two salmonid rhabdoviruses replicating at low temperatures (14 to 20°C). Both viruses belong to the Novirhabdovirus genus, but they are only distantly related and do not cross antigenically. By using a recently developed reverse-genetic system based on IHNV (S. Biacchesi et al., J. Virol. 74:11247-11253, 2000), we investigated the ability to exchange IHNV glycoprotein G with that of VHSV. Thus, the IHNV genome was modified so that the VHSV G gene replaced the complete IHNV G gene. A recombinant virus expressing VHSV G instead of IHNV G, rIHNV-Gvhsv, was generated and was shown to replicate as well as the wild-type rIHNV in cell culture. This study was extended by exchanging IHNV G with that of a fish vesiculovirus able to replicate at high temperatures (up to 28°C), the spring viremia of carp virus (SVCV). rIHNV-Gsvcv was successfully recovered; however, its growth was restricted to 14 to 20°C. These results show the nonspecific sequence requirement for the insertion of heterologous glycoproteins into IHNV virions and also demonstrate that an IHNV protein other than the G protein is responsible for the low-temperature restriction on growth. To determine to what extent the matrix (M) protein interacts with G, a series of chimeric pIHNV constructs in which all or part of the M gene was replaced with the VHSV counterpart was engineered and used to recover the respective recombinant viruses. Despite the very low percentage (38%) of amino acid identity between the IHNV and VHSV matrix proteins, viable chimeric IHNVs, harboring either the matrix protein or both the glycoprotein and the matrix protein from VHSV, were recovered and propagated. Altogether, these data show the extreme flexibility of IHNV to accommodate heterologous structural proteins.Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are two rhabdoviruses mainly, but not exclusively, infecting trout. Like those of mammalian rhabdoviruses such as rabies virus (RV) and vesicular stomatitis virus (VSV), the IHNV and VHSV negative-strand RNA genomes encode five structural proteins: nucleoprotein (N), polymerase-associated phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L). In contrast to those of mammalian rhabdoviruses, the IHNV and VHSV genomes contain an additional cistron, localized between the G and L genes, which encodes a small nonstructural protein of unknown function, NV (2, 21). Due to the presence of the NV gene, IHNV and VHSV have been classified as novirhabdoviruses. Mammalian rhabdoviruses are generally separated into two major genera, Lyssavirus (prototype, RV) and Vesiculovirus (prototype, VSV), based mainly on the migration pattern of the viral proteins in polyacrylamide gels. Although they are only distantly related and antigenically distinct, IHNV and VHSV have both been considered more related to Lyssavirus than to Vesiculovirus, in contrast to other fish rha...

Three monoclonal antibodies (MAbs) generated against rainbow trout gonad cells (RTG-2) have been selected for their ability to protect cells from the viral hemorrhagic septicemia virus (VHSV) infection, a salmonid rhabdovirus. Protection from infection was restricted to the salmonid-derived cell lines indicating species specificity of the blocking MAbs. Surprisingly, the blocking activity of these MAbs was also effective against other nonantigenically related fish rhabdoviruses. Indirect immunofluorescence and immunoelectron microscopy observations demonstrated that the three MAbs were all directed against an abundant cell plasma membrane component, and immunoprecipitation studies indicated that the target consisted of a heterodimeric complex with molecular masses of 200 and 44 kDa. Biochemical data provided the following evidence that fibronectin is part of this complex and that it could represent the main receptor for fish rhabdoviruses. (i) An antiserum generated against the 200-kDa protein reacted against the recombinant rainbow trout fibronectin expressed in Escherichia coli. (ii) The purified rainbow trout fibronectin was able to bind specifically to VHSV. To our knowledge, this is the first identification of a cellular component acting as a primary receptor for a virus replicating in lower vertebrates and, more interestingly, for viruses belonging to theRhabdoviridae family.

%orresponding author E t b 7~fgnres and 2 tab&d (Receiued~orpnbLcation Mq 26, 1997) summary Iridovirus-like agents isolated from systemic infected fish (Sifwrnsghnis, SFIR IrtaAUruJ mch, CFIR I, CFIR 11, CFIR III) and from frogs ( h n a esrw&nh, REIR) in Europe, Epizootic Haematopoietic Necrosis Virus (EHNV) isolated in Australia from redfin perch (Pmapllviari~), and Frog Virus 3 (FV 3) isolated from frogs (Ram pipicns) in the USA were investigated by electron microscopy, polypeptide composition, immunofluorescence, restriction endonuclease digestion, Southern-blot hybridization and polymerase chain reaction (PCR) amplification. AU virus isolates proved to be similar in morphology and in size and reacted with EHNV polydonal antiserum in the immunofluorescence. Whilst DNA restriction profiles of the European piscine isolates deaved by BamH I were similar, they differed clearly from those of EHNV, REIR and FV 3. Southern-blot analysis of viral BamH I-digested DNA using an EHNV DNA probe revealed cross-hybridization with the DNA of the investigated iridoviruses. Using a set of primers designed for an open reading frame of the EHNV genome, PCR products of about 250 bp were obtained with the DNA of systemic piscine and amphibian iridoviruses. The data suggest that the systemic piscine and amphibian iridoviruses should be regarded as members of the genus Ranavirus within the family Inahindue.

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