No abstract

Although Novirhabdovirus viruses, like the Infectious hematopietic necrosis virus (IHNV), have been extensively studied, limited knowledge exists on the route of IHNV entry during natural infection. A recombinant IHNV (rIHNV) expressing the Renilla luciferase gene was generated and used to infect trout. A noninvasive bioluminescence assay was developed so that virus replication in live fish could be followed hours after infection. We provide here evidence that the fin bases are the portal of entry into the fish. Confirmation was brought by the use of a nonpathogenic rIHNV, which was shown to persist in fins for 3 weeks postinfection.

No abstract

Sleeping disease virus (SDV) is a member of the new Salmonid alphavirus genus within the Togaviridae family.The single-stranded RNA genome of SDV is 11,894 nucleotides long, excluding the 3 poly(A) tail. A full-length cDNA has been generated; the cDNA was fused to a hammerhead ribozyme sequence at the 5 end and inserted into a transcription plasmid (pcDNA3) backbone, yielding pSDV. By transfection of pSDV into fish cells, recombinant SDV (rSDV) was successfully recovered. Demonstration of the recovery of rSDV was provided by immunofluorescence assay on rSDV-infected cells and by the presence of a genetic tag, a BlpI restriction enzyme site, introduced into the rSDV RNA genome. SDV infectious cDNA was used for two kinds of experiments (i) to evaluate the impact of various targeted mutations in nsP2 on viral replication and (ii) to study the virulence of rSDV in trout. For the latter aspect, when juvenile trout were infected by immersion in a water bath with the wild-type virus-like rSDV, no deaths or signs of disease appeared in fish, although they were readily infected. In contrast, cumulative mortality reached 80% in fish infected with the wild-type SDV (wtSDV). When rSDV-infected fish were challenged with wtSDV 3 and 5 months postinfection, a long-lasting protection was demonstrated. Interestingly, a variant rSDV (rSDV 14 ) adapted to grow at a higher temperature, 14°C instead of 10°C, was shown to become pathogenic for trout. Comparison of the nucleotide sequences of wtSDV, rSDV, and rSDV 14 genomes evidenced several amino acid changes, and some changes may be linked to the pathogenicity of SDV in trout.Sleeping disease in salmon was first observed in France in 1985 (1). In the rainbow trout (Oncorhynchus mykiss), the disease is characterized by an abnormal behavior of the fish, staying on their sides at the bottom of the tanks, reminiscent of a "sleeping state" that has provided the name of the disease (1). A related disease in farmed Atlantic salmon (Salmo salar L.) has also been reported (11). A viral etiology of these diseases was suspected (2) and confirmed a few years ago (4,12,14).The viruses responsible for these diseases have been characterized and shown to be like alphaviruses (18,19,22), and the nucleotide sequences of the Sleeping disease virus (SDV) and Salmon pancreas disease virus (SPDV) genomes have been determined (21). Like all alphaviruses, the SDV and SPDV genomes consist of a positive-sense single-stranded RNA molecule of about 12 kb in length. The four nonstructural proteins (nsP1 to nsP4) are involved in virus replication and encoded by the 5Ј-terminal two-thirds of the genome, whereas the structural proteins (C-E3/E2-6K/E1) are encoded by the 3Ј-terminal one-third of the genome (for a review, see reference 17). SDV and SPDV have been classified as a new genus named Salmonid alphavirus. This classification is based on at least three main features. The recovery of infectious virus from cDNA has been described previously for a number of mammalian alphaviruses, including Sindbis virus (13)...

Sleeping disease virus (SDV) is an emerging pathogen in salmonid aquacultures, the impact of which is underestimated to date due to the lack of efficient diagnostic tools. To better characterize this new aquatic alphavirus and to make molecular tools available, a panel of monoclonal antibodies (mAbs) directed against SDV non-structural and structural proteins has been generated by immunizing mice with SDV-specific recombinant proteins overexpressed in Escherichia coli as antigens. So far, mAbs against nsP1, nsP3, E2 and E1 SDV proteins have been produced and their reactivity has been characterized by Western blot, radioimmunoprecipitation and indirect immunofluorescence assays. In addition, protein domains recognized by each mAb have been determined by immunofluorescence assay on truncated expressed SDV-derived proteins. Finally, one mAb directed against the E1 glycoprotein has been evaluated as a potential tool to be used in immunohistochemistry assay on experimentally infected trout.