Characterization and mapping of monoclonal antibodies against the Sleeping disease virus, an aquatic alphavirus

et al. 2006

J Virol

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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)...

Section: Vol 80 2006 Protective Recombinant Sdv 4091mentioning

confidence: 99%

Recovery of a Recombinant Salmonid Alphavirus Fully Attenuated and Protective for Rainbow Trout

Moriette

LeBerre

et al. 2006

J Virol

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“…For mammalian alphaviruses, an E2-derived domain B has been shown to be the major target for neutralizing mAbs (Porta et al, 2014;Fox et al, 2015). In a previous study, the domain recognized by mAb 17H23 on the SAV E2 glycoprotein was roughly mapped and included the SAV E2 domain B counterpart (Moriette et al, 2005). In this alanine scanning approach, 10 amino acid changes from E223 to C236 were introduced by sitedirected mutagenesis in the SAV infectious cDNA.…”

Section: Discussionmentioning

confidence: 99%

“…Three main features distinguish SAV from mammalian alphaviruses: (i) SAV isolates are very close phylogenetically but only distantly related to the other alphaviruses; (ii) SAV non-structural and structural proteins are larger than those of mammalian alphaviruses; and (iii) arthropod-independent virus transmission to the host has been demonstrated in cohabitation experiments (Boucher, 1995), a phenomenon that has never been documented for mammalian alphaviruses. In a previous study, the protein domains recognized by a panel of mAbs directed against some of the non-structural and structural proteins of SAV2 were roughly mapped (Moriette et al, 2005). In the current study, we aimed to define and characterize the epitope recognized by the neutralizing 17H23 mAb directed against the E2 glycoprotein of most of SAV isolates and widely used in a number of laboratories to routinely diagnose SAV.…”

Section: Introductionmentioning

confidence: 99%

“…We hypothesized that the 17H23 epitope is located in the major domain B identified previously in the E2 of mammalian alphaviruses as the domain recognized by most of the E2 neutralizing mAbs (Voss et al, 2010;Fox et al, 2015). Indeed, the SAV E2 domain B counterpart is contained in the protein domain previously characterized as being recognized by mAb 17H23 (Moriette et al, 2005). Thus, to precisely characterize the 17H23 epitope, we developed an alanine scanning mutagenesis approach coupled with the generation of the respective recombinant SAV2 (rSAV) using the available infectious cDNA (Moriette et al, 2006;Mérour et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Fine mapping of a salmonid E2 alphavirus neutralizing epitope

Mérour

Journal of General Virology

Biacchesi

et al. 2016

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In this study, we aimed to characterize the epitope recognized by the neutralizing 17H23 mAb directed against the E2 glycoprotein of most of salmonid alphavirus (SAV) subtypes and widely used in several laboratories to routinely diagnose SAV. We hypothesized that the 17H23 epitope was located in the major domain B, previously identified in the E2 of mammalian alphaviruses as the domain recognized by most of the E2 neutralizing mAbs. Indeed, the SAV E2 domain B counterpart is contained in the protein domain previously characterized as being recognized by mAb 17H23. Thus, to precisely characterize the 17H23 epitope, we developed an alanine scanning mutagenesis approach coupled with the generation of the respective recombinant SAV (rSAV) by using the available infectious cDNA. Ten mutant rSAVs termed A-J from E2 aa 223-236 were produced and characterized in vitro using indirect immunofluorescence assays on virus-infected cells with mAbs 17H23, 51B8 (another non-neutralizing anti-E2 mAb) and 19F3 directed against the non-structural protein nsp1. Two of the mutant rSAVs (G and H) escaped neutralization by mAb 17H23. In addition, we showed that when juvenile trout were infected by bath immersion with the rSAV mutants, some of them were either totally (D, E and G) or partially (H) attenuated. Together, the data from the in vitro and in vivo experiments indicated that the putative 17H23 amino acid sequence epitope comprised the short amino acid sequence 227 FTSDS 231 .

“…120 ml) as described previously (Moriette et al, 2005). Four specific anti-ISAV secreting hybridomas were selected by testing their culture supernatants by indirect fluorescent antibody test (IFAT) against mock-and ISAV-infected cells.…”

mentioning

confidence: 99%

Completion of the full-length genome sequence of the infectious salmon anemia virus, an aquatic orthomyxovirus-like, and characterization of mAbs

Mérour

LeBerre

Journal of General Virology

et al. 2010

We report here the first full-length sequence of the eight ssRNA genome segments of the infectious salmon anemia virus (ISAV, Glesvaer/2/90 isolate), a salmonid orthomyxovirus-like.Comparison of ISAV genome sequence with those of others orthomyxovirus reveals low identity values, and a remarkable feature is the extremely long 59 end UTR of ISAV segments, which all contain an additional conserved motif of unknown function. In addition to the genome nucleotide sequence determination, specific mAbs have been produced through mice immunization with sucrose-purified ISAV. Four mAbs directed against the haemagglutininesterase glycoprotein, the nucleoprotein and free or actin-associated forms of the matrix protein have been characterized by (i) indirect fluorescent antibody test; (ii) virus neutralization; (iii) radioimmunoprecipitation and (iv) Western blot assays. These mAbs will potentially be useful for the development of new diagnostic tests, and the nucleotide sequences will help to establish a reverse genetics system for ISAV.Infectious salmon anemia (ISA) was first observed in Norway from Atlantic salmon (Salmo salar L.) in 1984 (Thorud & Djupvik, 1988). ISA outbreaks have since caused considerable economic losses to the Norwegian salmon farming industry and have also been reported in Scotland (Rowley et al., 1999), in the eastern coast of Canada (Bouchard et al., 1999;Lovely et al., 1999) and has been associated with extensive outbreaks in Chile since June 2007(Godoy et al., 2008. The mortality rate in contaminated salmon farms may vary from 15 to 100 % over several months. Although, ISA virus (ISAV) outbreaks were observed only in Atlantic salmon, the virus was detected from apparently healthy animals in other species (Raynard et al., 2001). The only exception was the isolation of ISAV from diseased Coho salmon (Oncorhynchus kisutch) in Chile in 1999 (Kibenge et al., 2001). Experimental infections showed that other species could be susceptible to ISAV infection, especially juvenile rainbow trout (Oncorhynchus mykiss) for which mortality was recorded after both virus injection and bath immersion, which may represent a threat to the trout industry (Biacchesi et al., 2007).The aetiological agent of ISA was identified as an orthomyxovirus-like with a negative-stranded RNA genome consisting of eight segments (Dannevig et al., 1995;Falk et al., 1997;Mjaaland et al., 1997), and constitutes the only member of the genus Isavirus (Kawaoka et al., 2005). ISAV is an enveloped virus composed of four major structural proteins: the nucleoprotein (66-72 kDa), the matrix protein (22-25 kDa) and two envelope glycoproteins that are the haemagglutinin-esterase glycoprotein (42 kDa) and the fusion protein (47-50 kDa) (Clouthier et al., 2002;Falk et al., 2004). Sequence characterization of ISAV genome segments is almost fully achieved, except for several segment end UTRs (Sandvik et al., 2000). It is now evident that the gene coding assignment to each segment and the putative functions of the ISAV proteins are unique and significa...