Antibody recognition of the glycoprotein g of viral haemorrhagic septicemia virus (VHSV) purified in large amounts from insect larvae

<p>Abstract</p> <p>Background</p> <p>There are currently no purification methods capable of producing the large amounts of fish rhabdoviral glycoprotein G (gpG) required for diagnosis and immunisation purposes or for studying structure and molecular mechanisms of action of this molecule (ie. pH-dependent membrane fusion). As a result of the unavailability of large amounts of the gpG from viral haemorrhagic septicaemia rhabdovirus (VHSV), one of the most dangerous viruses affecting cultured salmonid species, research interests in this field are severely hampered. Previous purification methods to obtain recombinant gpG from VHSV in <it>E. coli</it>, yeast and baculovirus grown in insect cells have not produced soluble conformations or acceptable yields. The development of large-scale purification methods for gpGs will also further research into other fish rhabdoviruses, such as infectious haematopoietic necrosis virus (IHNV), spring carp viremia virus (SVCV), hirame rhabdovirus (HIRRV) and snakehead rhabdovirus (SHRV).</p> <p>Findings</p> <p>Here we designed a method to produce milligram amounts of soluble VHSV gpG. Only the transmembrane and carboxy terminal-deleted (amino acid 21 to 465) gpG was efficiently expressed in insect larvae. Recognition of G21-465 by ß-mercaptoethanol-dependent neutralizing monoclonal antibodies (N-MAbs) and pH-dependent recognition by sera from VHSV-hyperimmunized or VHSV-infected rainbow trout (<it>Oncorhynchus mykiss</it>) was demonstrated.</p> <p>Conclusions</p> <p>Given that the purified G21-465 conserved some of its most important properties, this method might be suitable for the large-scale production of fish rhabdoviral gpGs for use in diagnosis, fusion and antigenicity studies.</p

A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout

Viral haemorrhagic septicaemia (VHS) is a fish rhabdovirus infection of world-wide importance. Control policies have been established but the disease still causes heavy losses in fish farming. The development of a recombinant subunit vaccine was initiated to produce a safe and effective vaccine to protect fish against VHS. The VHS virus (VHSV) glycoprotein, which induces neutralizing antibodies in rainbow trout, was chosen for expression in insect cells using a baculovirus vector. The Mr of the recombinant protein estimated by SDS-PAGE was slightly lower than that of the native viral protein. The recombinant protein displayed different degrees of glycosylation and was recognized in ELISA by neutralizing antibodies. It was transported to the plasma membrane of insect cells where its ability to induce membrane fusion was preserved. The efficacy of the recombinant protein as a vaccine was compared with those of an inactivated and an attenuated vaccine. When injected intraperitoneally into rainbow trout, the baculo-virus-encoded protein was shown (i) to induce the synthesis of VHSV-neutralizing antibodies and (ii) to confer protection against virus challenge. Immunization performed by immersion failed. This is the first report of a recombinant vaccine that protects fish against VHSV

The glycoprotein G of rhabdoviruses

Rhabdoviruses show an RNA-containing helically-wound nucleocapsid either enclosed by or enclosing a membrane M protein, surrounded by a lipid bilayer through which dynamic protein trimers made up of noncovalently associated monomers of glycoprotein G (G) project outside. Mature monomeric rhabdoviral G has more than 500 amino acids, 2-6 potential glycosylation sites, 12-16 highly conserved cysteine residues, 2-3 stretches of a-d hydrophobic heptad-repeats, a removed amino terminal hydrophobic signal peptide, a close to the carboxy terminal hydrophobic transmembrane sequence and a carboxy terminal short hydrophylic cytoplasmic domain. Association-dissociation between monomers-trimers and displacement of the trimers along the plane of the lipid membrane, are induced by changes in the external conditions (pH, temperature, detergents, etc.). Throughout conformational changes the G trimers are responsible for the virus attachment to cell receptors, for low-pH membrane fusion and for reacting with host neutralizing monoclonal antibodies (MAbs). Antigenic differences could exist between monomers and trimers, which may have implications for future vaccine developments. The family Rhabdoviridae is made up of the Lyssavirus (rabies), the Vesiculovirus (vesicular stomatitis virus, VSV) and many rhabdoviruses infecting fish, plants, and arthropod insects. All these reasons make the G of rhabdoviruses an ideal subject to study comparative virology and to investigate new vaccine technologies. © 1995 Springer-Verlag