The evolution of two homologues of the core protein VP6 of epizootic haemorrhagic disease virus (EHDV), which correspond to the geographical origin of the virus.

Epizootic haemorrhagic disease virus is a 10-segmented, double-stranded RNA virus. When these ten segments of dsRNA are run on 1% agarose, eastern (Australia, Japan) and western (North America, Africa, Middle-East) strains of the virus can be separated phenotypically based on the migration of genome segments 7-9. In western strains, segments 7-9 are roughly the same size and co-migrate as a single RNA band. In eastern strains, segment 9 is smaller, so while segments 7 and 8 co-migrate, the segment 9 RNA runs faster than its western homologue. Translation experiments demonstrated that these two segment 9 homologues are both functional and produce proteins (VP6) of different sizes-something that has not been reported in any other orbivirus species to date. Sequence analysis suggests that eastern and western versions of segment 9 (VP6) have likely evolved as a response to adaptive selection in different geographical regions via gene duplication and subsequent mutation. These significant findings are considered unusual given the conserved nature of VP6 and its presumed role as the viral helicase. It is not currently known what the biological relevance of each homologue is to the virus.

Development of a nested-PCR test based on sequence analysis of epizootic hemorrhagic disease viruses non-structural protein 1 (NS1).

Two orbiviruses, epizootic hemorrhagic disease (EHD) and bluetongue (BTV) viruses, cause disease in domestic and wild ruminant species. The gene that encodes non-structural protein 1 (NS1) of EHD virus, serotype 1, was sequenced and compared to EHD and BTV NS1 sequences. The NS1 gene was found to be more conserved than the VP3 gene, and was selected as a target for polymerase chain reaction (PCR) amplification. The NS1 genes of several BTV viruses and another orbivirus, African horse sickness (AHS), were compared to the EHD NS1 genes. This information was used to develop a capture nested-PCR for detection and differentiation of EHD from BTV viral RNA.

New generation of African horse sickness virus vaccines based on structural and molecular studies of the virus particles.

African horse sickness virus (AHSV) is a member of the genus Orbivirus, which also includes bluetongue virus (BTV) and epizootic haemorrhagic disease (EHDV) virus. These orbiviruses have similar morphological and biochemical properties, with distinctive pathobiological properties and host ranges. Sequencing studies of the capsid proteins have revealed evolutionary relationships between these viruses. Biochemical studies of the viruses together with the expression of individual proteins and protein complexes have resulted in the development of new generation vaccines. Baculovirus expressed AHSV VP2 provides protection against death caused by AHSV challenge. Similarly, BTV VP2 alone elicits protective neutralising antibodies against BTV in sheep, which is enhanced in the presence of VP5. Recent developments in biotechnology (multiple gene expression baculovirus systems) have made it possible to synthesise orbivirus particles that biochemically and immunologically mimic authentic virions but lack the genetic material. Particle doses as low as 10 micrograms elicit responses that are sufficient to protect sheep 15 months post vaccination, against virulent virus challenge. Moreover, knowledge of the three dimensional structure of these particles enables us to engineer them to deliver multiple foreign peptide components representing other viral epitopes (e.g. foot and mouth disease virus and influenza virus) in order to elicit protective immunity.

Identification of a short domain within the non-structural protein NS2 of epizootic haemorrhagic disease virus that is important for single strand RNA-binding activity.

The role that a conserved amino acid motif, found in the non-structural protein NS2 of orbiviruses, plays in the interaction of this protein with single stranded (ss) RNA was investigated by mutation analysis of the NS2 of epizootic haemorrhagic disease virus. An NS2 mutant in which this motif (amino acids 75 to 83) was deleted was expressed in Spodoptera frugiperda cells by a recombinant baculovirus and found to be unable to bind to poly(U)-Sepharose. The deletion mutant also differed from wild-type NS2 in that it did not appear to be complexed with ssRNA in cells infected with the baculovirus recombinant. Furthermore, the deletion exerted an adverse effect on the ability of NS2 to form inclusion bodies in the cytoplasm of baculovirus-infected insect cells. To further characterize the role of this motif in RNA-binding, specific residues within the region were substituted by site-directed mutagenesis and the mutants were expressed in Escherichia coli as fusion proteins. Analysis of the different mutant proteins indicated that in each case ssRNA-binding was impaired relative to that of the wild-type NS2 control. The degree of impairment corresponded to the number of amino acid substitutions and the largest effects were associated with non-conserved substitutions. It is suggested that the conserved motif is an important structural determinant in the interaction of NS2 with ssRNA.