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

Coralie Moriette1, Monique LeBerre1, Soasig Kerbart Boscher2, Jeannette Castric2 and Michel Brémont1

1 Unité de Virologie et Immunologie Moléculaires, INRA, CRJ Domaine de Vilvert, 78352 Jouy en Josas, France
2 Laboratoire d'études et de recherches en pathologie des poissons, AFSSA, BP 70, 29280 Plouzané, France

Correspondence
Michel Brémont
michel.bremont{at}jouy.inra.fr


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sleeping disease in salmonids was first observed in France in 1985 (Anonymous, 1985). Disease in rainbow trout is characterized by the abnormal behaviour of the fish, which stay on their side at the bottom of the tanks, reminiscent of a ‘sleeping state’, and thus giving the terminology of sleeping disease (Boucher & Baudin-Laurencin, 1994). Viral aetiology of this disease has been suspected (Boucher et al., 1994; Castric et al., 1997), and further confirmed in our laboratory as being alphavirus-like (Villoing et al., 2000). Genome sequences of the Sleeping disease virus (SDV) and the Salmon pancreas disease virus (SPDV), another related aquatic alphavirus, have now been entirely determined (Villoing et al., 2000; Weston et al., 2002). The amino acid identity between SDV and SPDV is 95 and 93·6 % for the non-structural and structural proteins, respectively. Moreover, both viruses have been shown to serologically cross-react (Boucher & Baudin-Laurencin, 1996). This close relationship allowed the classification of SDV and SPDV as salmonid alphavirus. This new classification is based on at least three main features: (i) SDV and SPDV are closely related but differ from the other alphaviruses, (ii) non-structural and structural proteins are much larger, and (iii) arthropod-independent virus transmission to the host has been demonstrated in cohabitation experiments (Boucher, 1995), which has not been documented for mammalian alphaviruses. SDV and SPDV genomes consist of a single positive-stranded RNA molecule of 11 900 and 11 919 nt long, respectively, encoding, as for all alphaviruses, two polyproteins, which after processing through proteolytic cleavages produce the viral mature products, the non-structural proteins nsP1, nsP2, nsP3 and nsP4, and the structural proteins capsid (C) and the two external glycoproteins (E2 and E1).

To date, sleeping disease is mainly diagnosed following observation of clinical signs of fishes suspected to be infected and by the implementation of various methods such as virological assay in cell culture or histological studies (Villoing et al., 2000). The use of methods based on specific antibodies against SDV could help in the future to improve and to reduce the time needed for diagnosis. In this paper, we describe the generation and characterization of a panel of monoclonal antibodies (mAbs) directed against some of the non-structural and structural SDV proteins.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viruses and cells.
The SDV strain S49P used in this study has been described previously (Castric et al., 1997). The P42p SPDV strain (Munro et al. 1984) was kindly provided by Dr Castric (AFSSA, France). Salmonid-derived cell line CHSE-214 (Lannan et al., 1984) and Carp-derived cell line EPC (Fijan et al., 1983) were maintained in ‘Glasgow modified Eagle's medium’ (GMEM) (PAA Laboratories) buffered at pH 7·4 with HEPES and supplemented with 10 % fetal calf serum, 50 penicillin IU ml–1 and 50 µg streptomycin ml–1. Recombinant vaccinia virus expressing T7 RNA polymerase, vTF7-3 (Fuerst et al., 1986), was kindly provided by B. Moss (National Institutes of Health, Bethesda, USA).

Cloning of SDV genes in pET expression vectors.
Individual SDV genes, except for nsP3, were amplified by PCR from full-length SDV genomic cDNA (C. Moriette and others, unpublished) using specific primers depicted in Table 1. pET-14b and pET-28a (Novagen) or pIVEX2.3d (Roche) prokaryotic expression vectors were used to insert the respective PCR products following appropriate restriction enzyme digestions (Table 1). Briefly, PCR products of the nsP1, nsP4 and E2 genes were inserted into pET-14b, the nsP2 gene was inserted into pET-28a, the capsid gene was inserted into pIVEX2.3d (a pET-derived vector), while the E1 product was inserted into both pET-14b and pIVEX 2.3d vectors. The full-length SDV genomic cDNA was used to excise a 1179 nt DNA fragment, corresponding to part of the nsP3 gene, using BamHI and Bsu36I restriction enzyme sites. The ends of the DNA fragment were filled-in using the Klenow fragment of T4 DNA polymerase and inserted into the pET-28a, which had been digested with EcoRI and NotI restriction enzymes and the ends of which had been filled-in. Each plasmid construct was checked for the expression of the respective recombinant protein by T7-driven in vitro transcription/translation assay (Promega) using [35S]methionine and analysed on 8 % SDS-PAGE followed by autoradiography. Each plasmid construct was then transfected into the BL21 (DE3) Escherichia coli strain (Novagen) to overexpress the relevant SDV recombinant protein.


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Table 1. Primers used in this study and cloning strategy

Restriction enzyme sites are underlined. R.E., restriction enzyme; R.E.S., restriction enzyme site of insertion; T4 DNA pol, T4 DNA polymerase. SDV nucleotide sequence: GenBank accession no. AJ238578.

 
Expression of truncated SDV-derived proteins.
To generate truncated recombinant proteins, pET or pIVEX plasmid recombinant constructs containing nsP1, nsP3, E2 or E1 genes were engineered by restriction enzyme digestions such that either the carboxy terminus part, the middle part or the amino terminus part of the protein of interest was expressed. The restriction enzymes used and the resulting DNA products are summarized in Table 2. Following restriction enzyme digestions, DNA fragment ends were either filled-in using the Klenow fragment of T4 DNA polymerase or blunt-ended by treatment with S1 nuclease in order to retain the open reading frame, and the digested plasmids were self ligated. Each plasmid construct was checked for the expression of the respective truncated recombinant protein by in vitro transcription/translation assay (Promega) using [35S]methionine and analysed on 8 % SDS-PAGE followed by autoradiography before transfection into EPC cells (see below).


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Table 2. Plasmids encoding truncated proteins

 
Nucleotide sequencing.
All the plasmid constructs used in the present study were checked by sequencing reactions carried out by an external service (MWG Biotech).

DNA transfection.
EPC cell monolayers in 12-well plates (2x106 cells per well) were infected with the recombinant vaccinia virus vTF7-3 (Fuerst et al., 1986) at an m.o.i. of 5 for 1 h at 37 °C. Cell monolayers were washed twice and transfected by using the jetPEI reagent (Qbiogene) according to the supplier's instructions with individual pET plasmid constructs (0·75 µg) either encoding total recombinant protein or truncated forms of recombinant protein. Cells were incubated for 6 h at 37 °C allowing vTF7-3 to replicate, and then the temperature was shifted to 14 °C for 12–16 h, the usual growth temperature for EPC cells (Fijan et al., 1983).

Expression and purification of SDV proteins.
Luria–Bertani medium (100 ml) containing appropriate antibiotic (100 µg ampicillin ml–1 or 70 µg kanamycin ml–1 for pET-14b and pET-28a constructs, respectively) was inoculated with 4 ml culture (OD600 of 0·6) of each recombinant E. coli clone and grown to OD600 of 0·6. The inducer IPTG was added to a final concentration of 1 mM for pET-28a clones and 0·4 mM for pET-14b clones. Cultures were further grown for either 3 or 6 h at 37 °C. Bacteria were pelleted at 3000 g for 15 min at 4 °C. Pellets were resuspended in 10 ml 50 mM Tris/HCl, 2 mM EDTA pH 8 and centrifuged for 20 min at 3000 g at 4 °C. Pellets were resuspended in 10 ml 50 mM Tris/HCl pH 8, 60 mM NaCl, and supplemented with 10 µl aprotinin, 100 µl Triton X-100 and 200 µl lysosyme (10 mg ml–1). Mixtures were incubated on ice for 15 min and then Benzonase (20 µl; Merck) and 1 M MgCl2 (40 µl) were added to each sample. Samples were sonicated twice for 20 s on ice and centrifuged for 45 min at 15 000 g at 4 °C. Pellets were resuspended in 2 ml 20 mM Tris/HCl pH 8 and finally centrifuged for 15 min at 15 000 g at 4 °C. Pellets were stored at –80 °C until use.

Solubilization of the recombinant proteins.
Pellets (see above) that contain the insoluble recombinant proteins were resuspended in 0·5 M urea, incubated overnight at 4 °C under rotation and then centrifuged for 15 min at 15 000 g at 20 °C. After which most contaminant bacterial proteins are solubilized and found in the supernatant. Pellets were washed twice with 250 mM NaCl, and final pellets were resuspended in deionized water and sonicated before SDS-PAGE analysis and immunization of mice.

mAbs generation.
Six-week-old BALB/c mice were injected following different routes every 2 weeks for at least 6 weeks with either each recombinant SDV protein or 1010 p.f.u. of concentrated virus SDV. The first and second intraperitoneal injections were with complete Freund's adjuvant and incomplete Freund's adjuvant, respectively, and the third injection by eye venous sinus was without adjuvant. Mice were bled 2 weeks later and sera were tested by immunofluorescence assay on fixed mock- and SDV-infected cells, as well as to evaluate the responsiveness of immunized mice. Positive mice received an additional boost by eye venous sinus injection and 3 days later splenocytes of mice were fused to Sp2O cells. Sera (diluted from 1 : 100 to 1 : 500) of mice injected with concentrated SDV were tested in neutralization assay (see below). Splenocytes of sera of mice that had a specific neutralizing titre were fused to Sp2O cells. Hybridomas were selected in HAT medium. Specific anti-SDV secreting hybridomas were selected by testing in immunofluorescence assay, using hybridoma supernatants against mock- and SDV-infected cells. After subcloning positive hybridomas, ascites fluids were produced in mice using standard techniques (Anderson & Potter, 1969).

Virus neutralization test.
SDV (100 p.f.u.) was incubated for 1 h at 14 °C with different dilutions (1 : 100, 1 : 250 and 1 : 500) of sera of mice injected with concentrated SDV or with serial dilutions of mAbs. As a control, SDV was incubated with culture medium. The mixtures were then used to inoculate CHSE cells in 24-well plates (5x105 cells per well) and the cells were allowed to absorb for 1 h at 14 °C. Following addition of GMEM supplemented with 2 % fetal calf serum, cells were incubated for 10 days at 10 °C and then stained with neutral red.

Immunofluorescence assay on fixed and live cells.
Cells infected with SDV or SPDV and cells transfected with each pET plasmid construct were fixed with a mixture of alcohol and acetone [1 : 1 (v/v)] at –20 °C for 15 min. Antigen detection was performed by incubation with SDV-specific mAbs diluted in PBS/Tween 20 for 45 min at room temperature. Cells were then washed, incubated with fluorescein-conjugated anti-mouse immunoglobulins (P.A.R.I.S.) for 45 min at room temperature and washed again. For assays on infected live cells, antibodies were diluted in the cell culture medium and directly used on unfixed cells following the same incubations and washing steps as described above. Cells were examined with a UV-light microscope for staining and photographed with a computer-coupled camera (Nikon).

Western blot and immunoprecipitation assays.
Reactivity of the mAbs was assessed by Western blot assays using either SDV- or SPDV-infected CHSE cell lysates, or E. coli expressed recombinant SDV proteins. Cell pellets from infected cells or overexpressed recombinant proteins were disrupted by the addition of 2x sample buffer [100 mM Tris/HCl pH 6·8, 4 % SDS, 20 % (v/v) glycerol, 0·2 % bromophenol blue, 200 mM dithiothreitol]. Approximately 2x105 cell equivalents of each infected cell extract or the 1/300 part of the recombinant proteins visualized on Fig. 1(b) were subjected to electrophoresis on 8 % SDS-PAGE, and electrotransferred onto a PVDF membrane and incubated with anti-SDV mAbs. Immunodetected antigens were visualized with horseradish peroxidase-coupled goat anti-mouse immunoglobulin (Ig) G by using an enhanced chemiluminescence detection system (ECL; Pierce).



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Fig. 1. Expression of SDV recombinant proteins. (a) In vitro synthesis of SDV recombinant proteins: pET or pIVEX constructs (0·5 µg) were used in a 25 µl reaction of TNT T7 Coupled Reticulocyte Lysate system (Promega) containing [35S]methionine. Samples of 10 µl were loaded onto 8 % SDS-PAGE and separated proteins were visualized by autoradiography. (b) Expression in E. coli of the SDV proteins: bacteria transfected with each individual pET encoding an SDV recombinant protein were induced with IPTG. Lysates were loaded onto 8 % SDS-PAGE and recombinant proteins were visualized by Coomassie blue staining. Calculated molecular mass (kDa) of the SDV proteins: nsP1, 63·3; nsP2, 95; {Delta}nsP3, 42; nsP4, 67·9; C, 31·2; E2, 47·3; and E1, 49·2.

 
For the radioimmunoprecipitation assays, mock- and SDV- or SPDV-infected cells were radiolabelled with [35S]methionine [100 µCi (3·7 MBq) ml–1] for 3 days post-infection (p.i.). After PBS washing, cells were lysed using RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 8, 1 % NP40, 0·5 % sodium deoxycholate, 1 % SDS, aprotinin 1 : 1000). Aliquots of the lysates were calibrated by trichloroacetic acid precipitation and were incubated with the mAbs diluted to 1 : 100 and protein A–Sepharose beads were added. After extensive washes in RIPA buffer, samples were resuspended in Laemmli's buffer, boiled and separated by electrophoresis on 10 % SDS-PAGE followed by autoradiography.

Immunohistochemistry (IHC) assay.
Bouin solution-fixed tissues were dehydrated through graded ethanol baths and embedded in paraffin, and then sectioned to produce slices of 5–6 µm thickness and mounted onto 2 % gelatin- or 10 % poly-L-lysine-coated slides. For IHC, sections were dewaxed, rehydrated, followed by blocking of endogenous peroxidase using 3 % (v/v) hydrogen peroxide. Thereafter, sections were washed in PBS and blocked in a blocking solution containing 2·5 % BSA for 10 min. Sections were incubated with primary SDV-specific mAb for 1 h at 37 °C, washed twice in PBS/Tween 20 and once in PBS, and incubated with the second antibody horseradish peroxidase-coupled goat anti-mouse IgG antibodies (P.A.R.I.S.; diluted to 1 : 200) for 30 min at 37 °C. After washes, the peroxidase substrate solution (AEC Chromogen; Sigma) was added for 10 min. Following washes in distilled water, sections were stained for 2 min with haematoxylin at room temperature and observed under the microscope.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Production of recombinant proteins
The genes encoding the non-structural proteins nsP1, nsP2 and nsP4 and the structural proteins C, E2 and E1 were amplified by PCR, using primers described in Table 1, using full-length cDNA of the SDV genome as a template (C. Moriette, unpublished). Each PCR product was then subcloned either into the pET-14b, pET-28a or the pIVEX2.3d vectors using appropriate restriction enzymes. Because PCR amplification of the total nsP3 gene (1691 nt) was unsuccessful, part of the nsP3 gene was excised from full-length cDNA of the SDV genome by using BamHI and Bsu36I restriction enzymes. Subsequently, the DNA fragment spanning nt 454–1641 of the nsP3 gene was cloned into the pET-28a vector. Plasmid constructs, termed pET-nsP1, pET-nsP2, pET-{Delta}nsP3, pET-nsP4, pIVEX-C, pET-E2 and pET-E1 were first validated by sequencing reactions and then checked by in vitro transcription/translation and analysis on SDS-PAGE as shown in Fig. 1(a). The lower band observed for nsP3 probably corresponds to internal initiation or premature termination of the transcription reaction. Following transfection of each individual recombinant pET plasmid into BL21 (DE3) E. coli strain, overexpression of the respective recombinant protein was induced by IPTG. As shown in Fig. 1(b), with the exception of the capsid protein (data not shown), all the SDV recombinant proteins were expressed. After scaled-up production, solubility of the recombinant proteins was investigated by freeze–thaw of culture pellets. It was observed that even using lower growth temperature conditions or lower inducer concentrations, none of the expressed recombinant proteins were found in the soluble fraction (data not shown), and thus recombinant proteins were semi-purified as inclusion bodies (see Methods). To date, despite several attempts, we have not succeeded in producing the SDV capsid as a recombinant protein in E. coli.

SDV-specific mAb generation
Non-neutralizing mAbs.
Mice were immunized using the E. coli insoluble fractions, which contain roughly 70–80 % of overexpressed SDV recombinant proteins. For each immunization, insoluble fractions were thawed and sonicated to disrupt aggregates, and mice were immunized with 100 times the amount of the protein visualized in Fig. 1(b). Following screening of the hybridomas, six mAbs directed against nsP1 (labelled 8A16, E1, 19F3, I13, J4 and 71L2), two mAbs directed against nsP3 (labelled 3E17 and 26J12) and one mAb (labelled 78K5) directed against the E1 glycoprotein were selected. Some of these mAbs were further produced as ascites fluids. Because the first attempt to immunize mice against nsP2, nsP4 and E2 led to an anaphylactic shock of mice, immunizations were repeated with a new batch of the recombinant proteins that were less contaminated with bacterial proteins. To produce this new batch, the time of induction was reduced from 6 to 3 h at 37 °C, and the final pellets containing the insoluble recombinant proteins were resuspended in 6 M urea instead of 0·5 M urea, which solubilized a larger proportion of contaminant bacterial proteins leaving recombinant SDV proteins in the insoluble fraction. By using this method, we avoided anaphylactic shock of the animal, and one mAb directed against the E2 glycoprotein (labelled 51B8) was obtained and produced as ascite fluid. In contrast, even after several repeated immunizations (roughly 12 injections) over a 6 month period, we never succeeded in getting an immune response against nsP2 and nsP4 antigens.

Neutralizing mAbs.
Attempts to purify SDV free of cell contaminants failed, and thus polyethylene glycol (PEG)-concentrated SDV-infected cell supernatant was used to immunize mice. In order to select neutralizing antibodies, immunized mouse sera that had neutralizing activity were selected to fuse their splenocytes to Sp2O cells. Hybridomas were screened on SDV-infected live cells, and two neutralizing mAbs (labelled 17H23 and 40D20) were selected and amplified as ascites fluids. In addition, two non-neutralizing mAbs directed against E2 (4I16 and 49K16) were also selected.

Characterization of SDV-directed mAbs
Reactivity of the selected mAbs (summarized in Table 3) was investigated following four procedures: (i) indirect immunofluorescence (IIF) assays, (ii) Western blot assays, (iii) immunoprecipitation assays, and (iv) IHC assays. The reactivity of all the mAbs was investigated by IIF, whereas Western blot, immunoprecipitation and IHC were performed only with the mAbs produced as ascites fluids.


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Table 3. Reactivity of the SDV mAbs

IIF, Indirect immunofluorescence assay; IP, immunoprecipitation assay; IHC, immunohistochemistry assay; + and –, positive and negative, respectively; ND, not done.

 
Immunofluorescence assay.
All the mAbs were tested by IIF assay on mock- and SDV-infected CHSE cells, and depending on the mAb used the fluorescence pattern observed differed (Fig. 2a). For example, anti-nsP1 (19F3 and J4) mAbs exhibited a punctate pattern of labelling, contrasting with the more dispersed pattern observed with the other anti-nsP1 (8A16 and I13) mAbs. A similarly dispersed staining pattern was observed when the latter anti-nsP1 mAbs were used in IIF assay on vTF7-3-infected EPC cells and transfected with the pET-nsP1 construct (data not shown). The two anti-nsP3 (3E17 and 26J12) mAbs exhibited a similar pattern of staining, appearing as small irregular spots that seem to be concentrated in a particular compartment of the infected cells. The two non-neutralizing anti-E2 (4I16 and 49K16) mAbs exhibited staining at the cellular membrane, even on fixed cells, whereas the 51B8 mAb recognized E2 in the cytoplasm of the infected cells. Finally, the non-neutralizing anti-E1 (78K5) mAb detected E1 in the cytoplasm and the staining was more intense closer to the nucleus. Both 17H23 and 40D20 neutralizing mAbs recognized SDV protein at the cellular membrane on infected live cells, but only the 17H23 mAb was also reactive against fixed cells. All the mAbs produced as ascites fluids cross-reacted against SPDV, a related salmonid alphavirus, with the exception of the two non-neutralizing anti-E2 (4I16 and 49K16), the neutralizing 40D20 mAb and the 3E17 anti-nsP3 mAb.



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Fig. 2. Reactivity of the mAbs in IIF and IHC. (a) Immunofluorescence staining patterns with different mAbs. SDV-infected CHSE cells were fixed with alcohol/acetone (except when noted) and incubated with the indicated mAbs. Cells were then stained with FITC-labelled anti-mouse antibodies. Objective x100 except for live cells (x40). (b) IHC reactivity of 78K5 anti-E1 mAb on SDV-infected fish sections. Experimentally infected fishes were taken at various days p.i. and paraffin-embedded sections of the pancreas (i, ii) and muscle (iii) were processed for IHC assay with 78K5 mAb. (i) IHC staining in the cytoplasm of exocrine pancreas (normal cells), 7 days p.i., magnification x400, (ii) IHC staining in the cytoplasm of exocrine pancreas (necrosis cells), 7 days p.i., magnification x1000, (iii) IHC staining in the sarcoplasm of white fibres of muscle, 42 days p.i., magnification x1000. Arrows indicate some of the IHC-positive cells.

 
Western blot assays.
All the non-neutralizing mAbs generated were tested for their reactivity by Western blot either against E. coli-expressed proteins or SDV- or SPDV-infected cells. Fig. 3(a) illustrates the detection by the respective mAbs of rnsP1, r{Delta}nsP3, rE2 and rE1 recombinant SDV E. coli-expressed proteins. When mAbs were tested against virus-infected cell lysates, all three anti-nsP1 (8A16, 19F3 and 71L2) mAbs were also reactive against SDV and SPDV (Fig. 3b). However, anti-nsP3 (3E17) and two anti-E2 (4I16 and 49K16) mAbs failed to detect a specific viral protein, while anti-E1 (78K5) was reactive (not shown). Finally, mAbs 51B8 and 78K5 directed against the glycoproteins E2 and E1, respectively, were tested by Western blot assay against PEG-concentrated SDV. Fig. 3(c) shows the reactivity of the 51B8 and 75K5 mAbs when used either alone or as a mixture.



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Fig. 3. Western blot detection of SDV proteins. (a) Insoluble fractions of overexpressed recombinant proteins (1/300 of amounts visualized on Fig. 1b) were loaded onto 8 % SDS-PAGE. The gel was electrotransferred onto a PVDF membrane and incubated with the specified mAbs. Immunodetected antigens were visualized with horseradish peroxidase-coupled goat anti-mouse IgG using an ECL detection system (Pierce). (b) SDV- or SPDV-infected cells or mock-infected (NI) cell lysates were loaded onto 10 % SDS-PAGE. The gel was electrotransferred onto a PVDF membrane and detection of specific proteins was achieved as in (a). (c) PEG-concentrated SDV was loaded onto 8 % SDS-PAGE. Viral proteins were immunodetected either with 51B8 mAb or 78K5 mAb or both. mAbs and immunodetected antigens are indicated on the top and the bottom of the figure, respectively.

 
Immunoprecipitation assays.
Some of the mAbs were tested for their ability to immunoprecipitate SDV and SPDV proteins from [35S]methionine radiolabelled virus-infected cell lysates (Fig. 4). mAbs against nsP1 recognized a protein of expected molecular mass (approx. 63 kDa) for both SDV and SPDV-infected cells, while the anti-nsP3 mAb (3E17) immunoprecipitated a faint band of approximately 58 kDa only from SDV-infected cell lysate. Both anti-E2 (4I16 and 49K16) mAbs immunoprecipitated two bands of 47 and 60 kDa, the lowest band being the E2 protein. It may be hypothesized that the upper band (60 kDa) corresponds to the E1 protein, by comparison to the band observed when the anti-E1 78K5 mAb was used. Note that the 78K5 mAb seems able to immunoprecipitate the E1 protein more efficiently from SPDV than from SDV (Fig. 4, panel 78K5), although this mAb has been generated initially using an SDV-derived recombinant protein. No proteins were immunoprecipitated when the non-neutralizing and neutralizing mAbs 51B8 and 40D20, respectively, were used, while the 17H23 neutralizing anti-E2 mAb immunoprecipitated two bands, as also observed with the 4I16 and 49K16 mAbs, but in this case from SDV- and SPDV-infected cell lysates.



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Fig. 4. Immunoprecipitation of 35S-labelled proteins from SDV- or SPDV-infected cell lysates. [35S]methionine radiolabelled lysates of mock-infected (NI) or SDV- or SPDV-infected cells were immunoprecipitated using specified mAbs (top) and separated by 8·5 % SDS-PAGE. Immunoprecipitated viral proteins (bottom) are indicated by an asterisk.

 
IHC.
Reactivity of mAb 78K5 directed against E1 glycoprotein has been tested by IHC assay on sections of red muscle, pancreas, kidney, brain and heart of experimentally SDV-infected fishes. Positive detection was observed on red muscle, between 21 and 42 days p.i., and pancreas, between 7 and 21 days p.i., as shown in Fig. 2(b).

Mapping of the mAbs
Each recombinant E. coli-expressed protein was analysed in four fragments. For that, initial pET SDV-derived constructs were modified in order to express a different part of each protein. Each plasmid construct was checked by analysis on SDS-PAGE of [35S]methionine labelling of in vitro transcription/translation products (data not shown). Then each construct was transfected into vTF7-3-infected EPC cells. Recognition of the expressed product by the corresponding mAb was assayed by IIF: 1A, 1B, 1C, 1D and nsP1 truncated products (Fig. 5) were assayed with anti-nsP1 mAbs (8A16, 19F3 and 71L2); fragments 3A, 3B, 3C, 3D and nsP3 truncated products were assayed with anti-nsP3 3E17 mAb; fragments E2A, E2B, E2C and E2 truncated products were assayed with anti-E2 mAbs (4I16, 49K16 and 51B8) and neutralizing 17H23 mAb; and fragments E1A, E1B, E1C, E1D and E1 truncated products were assayed with anti-E1 78K5 mAb. Results of such experiments are depicted in Fig. 5. We succeeded in determining the regions specifically recognized by 8A16, 19F3, 71L2, 3E17, 51B8, 78K5 and 17H23 mAbs. When 4I16 and 49K16 mAbs were assayed by IIF on cells transfected with pET-E2, no fluorescence was detected and thus mapping of these two mAbs along the SDV genome was not possible.



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Fig. 5. Mapping of the SDV mAbs. Cells infected with vTF7-3 (Fuerst et al., 1986) were transfected with each pET-SDV derivative expressing complete or truncated nsP1, nsP3, E2 and E1 proteins as indicated by schematic white boxes. Domains (black boxes) recognized by the individual mAbs (bracket) were identified by immunofluorescence assay. Numbers on black boxes refer to the amino acid position on each individual SDV protein. At the top of the figure, schematic representation of the SDV proteins along the genome (not to scale). Dotted lines indicate SDV proteins for which no mAbs have been produced.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The generation of mAbs against the non-structural and structural proteins of a new aquatic alphavirus, namely the Sleeping disease virus, is described. Several attempts to purify SDV from infected cell supernatants have been made but failed to produce virus free of cell contaminants, and thus mice immunized with such a material produced reacting antibodies that were most exclusively directed against cell proteins. In order to avoid this problem, individual SDV-derived proteins were expressed in E. coli. All the SDV-derived proteins could be expressed in E. coli with the exception of the capsid protein, even when using pSBetB vector (Stratagene); this vector provides the E. coli argU gene for suppression of rare arginine codon usage (Schenk et al., 1995) or a bacterial strain that overexpresses the Arg codon as described for the expression of the Sindbis and Ross River viruses, two mammalian alphavirus (Tellinghuisen et al., 1999). Mice immunization with E. coli-expressed SDV proteins produced a panel of mAbs directed against nsP1, nsP3, E2 and E1 SDV proteins. However, even after a 6 month period of repeated injections of mice with nsP2 and nsP4, no immune responsive mice were obtained; these two proteins seem to be poorly immunogenic. Results of immunofluorescence assays suggest localization of some SDV proteins in the infected cells (Fig. 2). As has already been observed in IIF assay on SFV-infected cells using an anti-nsP1 antibody (Peranen et al., 1995), mAbs 19F3 and J4 may recognize nsP1 associated with typical type I cytoplasmic vacuoles (CPVIs), the presence of which was revealed in early studies in mammalian alphavirus-infected cells (Acheson & Tamm, 1967; Grimley et al., 1968) and which are the site of viral RNA replication (Grimley et al., 1968) associated with the nsPs (Ahola et al., 1999; Froshauer et al., 1988). The SDV nsP1 protein is recognized by 8A16 mAb to be more dispersed in the cytoplasm. Similarly, nsP3 protein is recognized by 3E17 mAb to also be associated with CPVIs, as previously observed for SFV nsP3 (Peranen, 1991; Peranen & Kaariainen, 1991). E2 and E1 glycoproteins are recognized by mAbs 51B8 and 78K5 in the cytoplasm, whereas 4I16, 79K16 and 17H23 mAbs interact with E2 at the cellular membrane.

Most of the mAbs have been mapped along the SDV genome (Fig. 5). We notice that regions specifically recognized by these different mAbs are in accordance with previous observations on structures and biochemical properties of SDV proteins compared to other alphavirus proteins (Villoing, 2000): (i) 8A16, 71L2 and 19F3 mAbs are specific for conserved regions of nsP1 not implicated in the binding of nsP1 to lipids (Ahola et al., 1999). (ii) 3E17 mAb is specific for the conserved region of nsP3 (aa 1–344). (iii) The region 201–354 aa of E2 protein specifically recognized by 51B8 and 17H23 mAbs comprises the important antigenic epitopes of E2 described by Rumenapf et al. (1995) for several alphaviruses. (iv) The 78K5 mAb is specific for a region (between aa 209 and 383) of E1 that does not comprise either the fusion domain of the protein (Levy-Mintz & Kielian, 1991) or the transmembrane domain, which may be localized between amino acids 430 and 450 (Villoing, 2000). The reactivity of all the mAbs was evaluated against SPDV, a virus known to be closely related to SDV (Weston et al., 2002), and most of the mAbs cross-react with SPDV. Some other mAbs like 4I16, 49K16 and 40D20 allow discrimination between these viruses. These mAbs would be useful tools to diagnose SDV in infected trout. For example, we show here their usefulness in IHC assay. In addition, because SDV-infected fishes share the same histological lesions of fishes infected with the Infectious pancreatic necrosis virus, a birnavirus, these SDV-specific mAbs constitute a powerful tool to discriminate between IPNV- or SDV-infected trout. Little information is available on the molecular events during the replication of the salmonid alphaviruses; these mAbs will help to clarify the time course of SDV protein (non-structural and structural) synthesis in SDV-infected cells, and may also help in elucidating the kinetics of polyprotein processing during the SDV replication cycle.


   ACKNOWLEDGEMENTS
 
This work was supported by the Commission of the European Communities, Fifth PCRD programme QLK2-CT-2001-00970 and in part by the Institut National de la Recherche Agronomique. We thank Dr Ulrich Desselberger (CNRS, France) for helpful suggestions on the manuscript.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 14 March 2005; accepted 22 July 2005.