A single point mutation in the VP7 major core protein of bluetongue virus prevents the formation of core-like particles.

To understand the assembly process of bluetongue virus (BTV), we have established a functional assay which allows us to produce and manipulate BTV core-like particles (CLPs) composed of the viral VP7 and VP3 proteins. A cDNA clone encoding the 349-amino-acid VP7 protein has been manipulated to generate deletion, extension, and site-specific mutants. Each mutant was coexpressed with the BTV VP3 protein to generate CLPs. Deletion and extension mutants involving the VP7 carboxy terminus prevented CLP formation, while an extension mutant involving an 11-amino-acid rabies virus sequence added to the amino terminus of VP7 allowed CLP formation. Substitution of either of two cysteine residues of VP7 (Cys-15 or Cys-65) by serine also did not prevent CLP formation; however, substitution of the single lysine residue of VP7 (Lys-255) by leucine abrogated CLP formation, indicating a critical role for this lysine.

The expressed VP4 protein of bluetongue virus binds GTP and is the candidate guanylyl transferase of the virus.

A minor core protein, VP4, of bluetongue virus serotype 10 (BTV-10) has been synthesized in insect cells infected with a genetically manipulated recombinant baculovirus. When insect cells were coinfected by this recombinant virus and a recombinant baculovirus expressing the two major core proteins (VP3 and VP7) of the virus, core-like particles (CLPs) consisting of all three proteins were formed. Purified CLPs reacted with [32P]GTP which was covalently bound to VP4 only. Similarly reconstituted CLPs with VP1 or VP6 did not form covalent complexes with [32P]GTP. The virion-derived VP4 was also shown to have GTP-binding activity. The covalent binding of GTP indicates that expressed VP4 not only is biologically active but also is the candidate guanylyl transferase of the virus. The optimum reaction conditions for GTP binding by VP4 have been investigated.

Expression of the major core antigen VP7 of African horsesickness virus by a recombinant baculovirus and its use as a group-specific diagnostic reagent.

The major core protein, VP7, of African horsesickness virus serotype 4 (AHSV-4), the aetiological agent of a recent outbreak of the disease in southern Europe, was expressed in insect cells infected with a recombinant baculovirus containing a cloned copy of the relevant AHSV gene (S7). Analyses of its biochemical and antigenic properties confirmed the authenticity of the protein expressed. The high-level expression of VP7 under the control of the strong polyhedrin promoter of Autographa californica nuclear polyhedrosis virus induced disc-shaped crystals in infected insect cells. This enabled us to purify the protein by a one-step ultracentrifugation procedure and to utilize it for the detection of antibodies raised in horses to various serotypes of AHSV. A serological relationship between AHSV and two other orbiviruses, bluetongue virus and epizootic haemorrhagic disease virus, was also demonstrated.

Synthesis and characterization of chimeric particles between epizootic hemorrhagic disease virus and bluetongue virus: functional domains are conserved on the VP3 protein.

A functional assay has been developed to determine the conservative nature of the interacting sites of various structural proteins of orbiviruses by using baculovirus expression vectors. For this investigation, proteins of two serologically related orbiviruses, bluetongue virus (BTV) and the less studied epizootic hemorrhagic disease virus (EHDV), were used to synthesize chimeric particles. The results demonstrate that the inner capsid protein VP3 of EHDV-1 can replace VP3 protein of BTV in formation of the single-shelled corelike particles and the double-shelled viruslike particles. Moreover, we have demonstrated that all three minor core proteins (VP1, VP4, and VP6) can be incorporated into the homologous and chimeric corelike and viruslike particles, indicating that the functional epitopes of the VP3 protein are conserved for the morphological events of the virus. This is the first evidence of assembly of seven structural proteins of the virus by a baculovirus expression system. Confirmation at the molecular level was obtained by determining the EHDV-1 L3 gene nucleic sequence and by comparing it with sequences available for BTV. The analysis revealed a high degree homology between the two proteins: 20% difference, 50% of which is conservative. The consequences for Orbivirus phylogeny and the possibility of gene reassortments are discussed.

Oral immunization of foxes with avirulent rabies virus mutants.

SAG1, a rabies virus strain bearing one mutation which abolishes virulence for adult animals, was constructed from the SADBern strain of rabies virus which has previously been used as live vaccine for oral immunization of foxes. SAG1 also bears an antigenic mutation which serves as an additional marker of the strain. Studies on mice and four species of wild rodents showed that SAG1 is totally avirulent whereas SADBern is still pathogenic after intracerebral, intramuscular or oral inoculation and thus could cause cases of rabies. Trials of oral vaccination performed on foxes with SAG1 indicate that it is as effective as SADBern. The SAG1 strain represents a significant progress in the search for an efficient and safe live rabies for the oral immunization of wild animals.