Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus

Fernandez‐Alonso, M.; Lorenzo, Gema; Pérez, Luis; Bullido, R; Estepa, A.; Lorenzen, Niels; Coll, Julio

doi:10.3354/dao034167

Cited by 32 publications

(22 citation statements)

References 25 publications

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“…Because serum from VHSV-immunized trout strongly reacted with solid-phase frg11 (15,35) by recognizing its linear epitopes (17) and the mutants with mutations in frg11 were expressed in the cellular membrane independently of its conformation, most of the pG fusion-defective mutants described here are likely to induce immune responses in trout. Therefore, some of the mutations described in this work could be used to design attenuated VHSV vaccines, including DNA vaccination with the mutated G gene (2,3,18) or recombinant viruses obtained through reverse genetic methods (5,6).…”

Section: Discussionmentioning

confidence: 94%

“…Therefore, the mutations would be affecting the conformation of pG, which would be the primary reason behind the observed alterations in fusion activity. It is not possible to determine whether the studied mutations have an indirect effect on fusion due to changes in the pG conformation, a direct effect on its fusion capacity, or both, since none of the VHSV mutants were recognized by the C10 or 2F1A12 MAb and there is not yet any other available VHSV neutralizing MAb (17) or any other assay for pG conformation. Furthermore, it is not yet possible to make a direct comparison with the properties of similar VSV fusion-defective mutants described above.…”

Section: Discussionmentioning

confidence: 99%

“…Three of these mutations mapped around two pG locations, showing amino acid variations among VHSV isolates. Because 40% of the VHSV-immunized trout strongly recognized linear epitopes on these regions (15,17,35), the mutants described here might be useful in attempts to design attenuated VHSV strains to be used in DNA vaccines (2,3,18) or in vaccines obtained by reverse genetic methods (5,6).…”

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confidence: 99%

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Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Rocha¹,

Ruiz²,

Tafalla³

et al. 2004

J Virol

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Fourteen single and two double point mutants in the highly conserved region (positions 56 to 159) of the G gene of viral hemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were selected and obtained in plasmids by site-directed mutagenesis. Fish cell monolayers transfected with the mutant plasmids were then assayed for protein G (pG) expression, conformation-dependent monoclonal antibody (MAb) reactivity, and cell-cell fusion. Some mutations located in the phospholipid-binding p2 peptide (positions 82 to 110; mutants P86A, A96E, G98A, and R107A) abolished both MAb recognition and fusion activity, while others (P79A, L85S, and R103A) abolished MAb recognition but retained fusion at similar or lower pHs compared to those for the wild type. Phospholipid-binding assays of p2-derived synthetic peptides suggested that phosphatidylserine binding was not affected by the mutations studied. On the other hand, three (P79A, L85S, and T135E) of the four mutants retaining fusion activity mapped around two locations showing amino acid variation in 22 VHSV isolates and in neutralizing MAb-resistant mutants described previously. Mutations located in the hypothetical fusion peptide (positions 142 to 159; mutants F147K, P148K, and W154K) abolished both MAb recognition and fusion activity. The existence of mutants with altered conformation and defective fusion in both p2 and fusion peptides provides further evidence in favor of the participation of these and adjacent regions in some of the steps of the VHSV fusion processes, as suggested by previous studies. In addition, because the studied region induced strong immunological responses in trout, some of the mutants described here might be used to design attenuated VHSV vaccines.Rhabdoviruses cause highly damaging diseases in fish reared by the worldwide salmon culture industry. Among fish rhabdoviruses (novirhabdoviruses), the viral hemorrhagic septicemia virus (VHSV), which originated in Europe but has recently been found in America (26, 41), affects not only salmonids but also cod, turbot (38), sea bass, eels, flatfish, and shrimp (8,24,27). Despite many efforts, including successful DNA vaccines at the laboratory level, a commercial vaccine against VHSV is not yet available (1,18,25,30).The whole genome of VHSV has been sequenced (23), and the disulfide structure of its protein G (pG) has been elucidated (12). Reverse genetic methods have been developed for the VHSV-related rhabdovirus infectious hematopoietic necrosis virus (5-7), which could allow the design of new vaccines. The study of rhabdovirus fusion in the VHSV model could be important in the design of alternative vaccines to fight these diseases.The pG mutants of vesicular stomatitis virus (VSV), a wellstudied mammalian rhabdovirus, with an altered extent of fusion and/or optimal fusion pH in vitro, had mutations located either in the fusion peptide or in carboxy-terminal regions affecting the low-pH conformational changes required for fusion (39,40,43). Alignment of the pG sequence of VSV with those of 14 o...

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Rocha¹,

Ruiz²,

Tafalla³

et al. 2004

J Virol

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“…Briefly, virus grown on EPC monolayers with complete cytopathic effect (CPE) were clarified by centrifugation and then applied to glycerol cushions (Granzow et al 2001, Betts et al 2003. The resulting pellets were suspended in 100 µl of TNE buffer (10 mM Tris-HCl pH 8.0, 10 mM sodium chloride, and 1 mM EDTA pH 8.0; Granzow et al 2001) applied to a previously prepared 10 to 60% (5% increments) continuous sucrose gradient (Fernandez-Alonso et al 1998, Granzow et al 2001.…”

Section: Methodsmentioning

confidence: 99%

Immunohistochemistry and pathology of multiple Great Lakes fish from mortality events associated with viral hemorrhagic septicemia virus type IVb

Al-Hussinee

Lord²,

Stevenson³

et al. 2011

Dis. Aquat. Org.

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A novel viral hemorrhagic septicemia virus (VHSV) (genotype IVb) has been isolated from mortality events in a range of wild freshwater fish from the Great Lakes since 2005. In 2005 and 2006, numerous new freshwater host species (~90 fish from 12 different species) were confirmed to have VHSV by cell culture and reverse transcriptase polymerase chain reaction. A prominent feature observed in infected fish were the petechial and ecchymotic haemorrhages on the body surface and in visceral organs, as well as serosanguinous ascites; however, many fish had few and subtle, gross lesions. Histologically, virtually all fish had a vasculitis and multifocal necrosis of numerous tissues. Excellent correlation was found between the presence of VHSV IVb antigen detected by immunohistochemistry and the pathological changes noted by light microscopy. Intact and degenerate leukocytes, including cells resembling lymphocytes and macrophages, also had cytoplasmic viral antigen. By contrast, renal tubules and gonadal tissues (ovary and testis), were strongly immunopositive for VHSV IVb, but no lesions were noted.

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“…After incubation, monolayers were fixed for 10 min in cold methanol and air dried. MAb 2C9 directed toward the N protein of VHSV, diluted 1,000-fold in dilution buffer (0.24 mM merthiolate, 5 g of Tween 20/liter, and 50 mg of phenol red/liter in PBS [pH 6.8]), or a cocktail of anti-G MAbs 3F1A12 and I10 (16), diluted 200-fold, was added to the wells (100 l/well) and incubated for 1 h at room temperature. After washing with distilled water, 100 l of peroxidase-labeled (Nordic, Tilburg, The Netherlands) or fluorescein-labeled (Sigma) rabbit anti-mouse IgG Ab was added per well and the incubation was continued for 30 min.…”

Section: Methodsmentioning

confidence: 99%

Reversible Inhibition of Spreading of In Vitro Infection and Imbalance of Viral Protein Accumulation at Low pH in Viral Hemorrhagic Septicemia Rhabdovirus, a Salmonid Rhabdovirus

Más

Rocha

Pérez

et al. 2004

J Virol

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The inhibition of viral hemorrhagic septicemia rhabdovirus (VHSV) in vitro infection by pHs of <7 (low pH) has been previously reported. Nevertheless, the details of the mechanism underlying this effect remain obscure. We present evidence showing that low-pH inhibition occurs during a viral postadsorption step. Thus, while VHSV bound, replicated within single cells, and presented its G protein on the membranes of infected cells at both low and physiological pHs, both cell-to-cell spreading of infection (as estimated by the appearance of foci of infected cells) and fusion (as estimated by a syncytium assay) were inhibited by this low pH. The decreased VHSV titers and the inhibition of both cell-to-cell spreading of infection and fusion could be reversed by adjusting the pH to 7.5 at any time during infection. This effect should be taken into account to avoid false negatives in the diagnosis of VHSV by cell culture. On the other hand, the cell-to-cell spreading of infection at pH 7.5 could be stopped at any time by reducing the pH to 6.5. Since at low pH there were changes in the protein G conformation and smaller and imbalanced amounts of N with respect to M1, M2, and G viral proteins, alterations of the assembly and/or budding of VHSV are most probably involved in the absence of newly released infective virions.In many enveloped viruses, after the virus particle is internalized by a receptor-mediated endocytosis mechanism (6), viral and cell membrane fusions are triggered by the decrease of the endosomal pH (30). Conformational changes are induced by the low pH in the viral glycoproteins (25, 51) to cause fusion. Thus, agents that inhibit the lowering of the pH in the endosomes, such as NH 4 ϩ , also inhibit viral fusion and infectivity (11,44,49). However, it has been shown that a low-pH environment outside the host cells can also inhibit the in vitro viral replication and/or fusion of viruses such as vesicular stomatitis virus (VSV) (32), rabies virus (RV) (21,22,26,35), viral hemorrhagic septicemia rhabdovirus (VHSV) (10, 50, 52), influenza virus (5), poliovirus (18), or retrovirus (4,42,56).It was proposed earlier that the inhibition of VSV infectivity caused by low pH was due not to inhibition of viral RNA or protein synthesis (18) but to some yet-unidentified alterations in the nucleocapsid replication complex (32). Since then, there have been no further references about the mechanisms causing low-pH inhibition of VSV or any other rhabdoviruses, such as RV or VHSV. However, the study of this aspect of rhabdovirus infection might have important implications for the inactivation of virus in biological fluids (29) and for laboratories responsible for proper surveillance of these diseases, which should correctly diagnose possible false negatives.To further study this phenomenon, we choose VHSV (a salmonid rhabdovirus) as a model among rhabdoviruses, because VHSV causes cytopathic effects of established fish cell lines only at pH 7.0 or higher (10, 50, 52), and this might cause problems in cell culture detectio...

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Mapping of linear antibody epitopes of the glycoprotein of VHSV, a salmonid rhabdovirus

Cited by 32 publications

References 25 publications

Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Conformation- and Fusion-Defective Mutations in the Hypothetical Phospholipid-Binding and Fusion Peptides of Viral Hemorrhagic Septicemia Salmonid Rhabdovirus Protein G

Immunohistochemistry and pathology of multiple Great Lakes fish from mortality events associated with viral hemorrhagic septicemia virus type IVb

Reversible Inhibition of Spreading of In Vitro Infection and Imbalance of Viral Protein Accumulation at Low pH in Viral Hemorrhagic Septicemia Rhabdovirus, a Salmonid Rhabdovirus

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