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

Overproduction of a P450 that metabolizes diazinon is linked to a loss-of-function in the chromosome 2 ali-esterase (Md alpha E7) gene in resistant houseflies

Up-regulation of detoxifying enzymes in insecticide-resistant strains of the house fly is a common mechanism for metabolic resistance. However, the molecular basis of this increased insecticide metabolism is not well understood. In the multiresistant Rutgers strain, several cytochromes P450 and glutathione S-transferases are constitutively overexpressed at the transcriptional level. Overexpression is the result of transregulation, and a regulatory gene has been located on chromosome 2. A Gly137 to Asp point mutation in alpha E7 esterase gene, leading to the loss of carboxylesterase activity, has been associated with organophosphate resistance in the house fly and the sheep blowfly. We show here that purified recombinant CYP6A1 is able to detoxify diazinon with a high efficiency. We also show that either the Gly137 to Asp point mutation In alpha E7 esterase gene or a deletion at this locus confer resistance and overproduction of the CYP6A1 protein. Based on these findings, we propose it is the absence of the wild-type Gly137 allele of the alpha E7 gene that releases the transcriptional repression of genes coding for detoxification enzymes such as CYP6A1, thereby leading to metabolic resistance to diazinon

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