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Figure 6 Phylogenetic associations of BTV serotypes from different geographic locations of the world. Equivalent regions of each VP2 gene (from nucleotide 975 to 1190; relative to BTV1AUS VP2) was used to calculate the interrelationships between each BTV serotype. (Data kindly provided by Allan Gould.)

be distinguished by complement fixation, agar gel immunodiffusion, fluorescent antibody staining, ELISA and competition ELISA, using reactions with 'reference' preparations of group specific antibodies. In several of these test systems, VP7(T13), which is the protein on the surface of the virus core of BTV, has been shown to be immunodominant and of major significance. However, RNA sequence data indicate that the gene coding for VP7(T13) is more variable within a serogroup (species) than those encoding some of the other core and nonstructural proteins. This may indicate some partial exposure of VP7(T13) to antibody selective pressure. It has been reported that some VP7 specific monoclonal antibodies can react with whole virus particles, leading to suggestions that this core surface protein is partially exposed in the intact virion. However, more recent studies indicate that this reaction may result from damage to the outer capsid during sample preparation and that the VP7(T13) core surface layer is not normally exposed in the intact virion.

From the practical point of view, sequences from genome segment 5 (coding for nonstructural protein NSl(TuP)) may be most suitable for use as serogroup specific nucleic acid probes or in group specific PCR-based test systems. This is partly because this genome segment is transcribed into the most abundant viral mRNA in infected cells but also because it is reported to be highly conserved within an orbivirus species

(serogroup), while displaying >40% sequence variation between species.

The most variable but serotype-specific of the BTV genome segments and proteins are segment 2 and VP2 (the major neutralization antigen), respectively. From the limited data that are currently available for BTV (for example, between bases 975 and 1190 of segment 2; Fig. 6), viruses within the same serotype, regardless of geographic origin or 'topotype', appear to contain <19% RNA sequence variation in genome segment 2. In contrast, viruses that are classified as different serotypes appear to contain >26% RNA sequence variation in the same region. However, additional studies using crosshybridization of nucleic acid probes generated from genome segment 2 have demonstrated that at high washing stringencies, although such probes can be used to detect some isolates of the same serotype, they do not detect them all, while at lower stringencies they will also bind to RNA from other distinct but closely related serotypes. It may not therefore be possible to produce 'serotype specific' nucleic acid probes from segment 2 that are capable of simultaneously detecting all and only members of each individual serotype.

The variation in segment 2 that is observed within a serotype may reflect mutations, like those between 'topotypes', that have accumulated in geographically separated viruses. There is also a wide range of variation in the percentage difference in segment 2

nucleotide sequence that is observed between viruses from different serotypes. This provides evidence for relatively closer grouping of some serotypes (for example BTV 3, 13 and 16, with <32% RNA sequence difference; Fig. 6), as well as and relatively more distant relationships between 'nucleotype' groups, with higher levels of variation (>34%, <46%). The limited data presented in Fig. 6 suggest that each genome segment 2 from viruses within a single serotype may have a common ancestry. These data also suggest relatively closer ancestral relationships for genome segment 2 from virus serotypes within a single 'nucleotype' and may have important implications for the origins and spread of different virus serotypes found in different parts of the world.

Data from the analysis of reassortant viruses demonstrate that variation in genome segment 6 (coding for VP5, the other major outer coat protein of BTV) can also have a significant influence on virus serotype. In viruses containing relatively similar VP2 proteins, the influence exerted by VP5 on serotype may further complicate attempts to develop serotype specific nucleic acid probes based on the RNA sequence of genome segment 2 alone.

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