Differentiation of cognate dsRNA genome segments of bluetongue virus reassortants by temperature gradient gel electrophoresis.

The analysis of reassortant viruses has been a valuable tool in the investigation of protein interaction and function in double-stranded (ds) RNA virus research. The differentiation of cognate dsRNA genome segments of reassortants is conventionally achieved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). However, due to a high degree of sequence homology among different bluetongue virus (BTV) serotypes, it is not uncommon to find that certain cognate dsRNA segments cannot be differentiated by SDS-PAGE. Temperature gradient gel electrophoresis (TGGE) has been shown to be a much more sensitive method of differentiating RNA or DNA fragments of high sequence homology. Here we report the preliminary application of TGGE in analysis of genomic reassortants of two BTV serotypes, 1 and 23. While six out of ten genome segments between BTV-1 and BTV-23 could not be resolved by SDS-PAGE, all of them were differentiated by TGGE. The ability of TGGE to distinguish between dsRNA segments of high sequence homology may also make it useful in the search for BTV genes responsible for defined characteristics, such as virulence, by differentiating wild-type and mutated gene segments of viruses displaying altered phenotypes.

Major core protein VP7 of Australian bluetongue virus serotype 15: sequence and antigenicity divergence from other BTV serotypes.

Full-length cDNA of the RNA genome segment coding for the major core protein VP7 of Australian bluetongue virus serotype 15 (BTV-15) has been isolated by reverse transcription-PCR cloning. Comparative analysis indicated that the BTV-15 VP7 sequence had diverged significantly from that of other members of the BTV serogroup. At the amino acid level, BTV-15 VP7 exhibited sequence identities of 80 to 84% with VP7 molecules of other serotypes, significantly lower than the sequence identities of between 93 and 100% observed among other serotypes characterized to date. This was consistent with previous observations that there were significant immunological differences between BTV-15 and other BTV serotypes and that monoclonal antibodies raised against BTV-1 VP7 failed to react with BTV-15 VP7. Recombinant BTV-15 VP7 protein produced from Escherichia coli was largely insoluble, but maintained its immunogenicity. Polyclonal mouse sera raised against the recombinant VP7 protein reacted strongly with VP7 of BTV-15, but weakly with that of BTV-1.

The site of bluetongue virus attachment to glycophorins from a number of animal erythrocytes.

Bluetongue virus (BTV) was shown to agglutinate human, ovine and porcine erythrocytes. Removal of neuraminic acid (NA) from erythrocytes by Vibrio cholerae neuraminidase prevented their agglutination. Haemagglutination was also inhibited by N-acetyl neuraminic acid (NANA), N-glycol neuraminic acid (NGNA) and N-acetyl neuramin-lactose. The ability of BTV to agglutinate trypsin-treated human erythrocytes, which lack the amino-terminal domain and the single N-linked oligosaccharide of glycophorin A, suggests that the virus bound to human erythrocytes via NANA-containing, O-linked oligosaccharides. Glycoproteins with NA-containing oligosaccharides of known structure such as mucin, fetuin, alpha 1-acid glycoprotein, ovomucoid and ovine, porcine, human and equine glycophorin were examined for their ability to inhibit BTV-mediated agglutination of human, ovine and porcine erythrocytes. All glycoproteins containing NANA- or NGNA alpha 2-6GalNAc were capable of inhibiting the agglutination of human and porcine erythrocytes. Treatment of human erythrocytes with Newcastle disease virus neuraminidase and of porcine erythrocytes with Clostridium perfringens neuraminidase to cleave preferentially the NANA- and NGNA alpha 2-3Gal linkages respectively, were shown to have little effect on the ability of the erythrocytes to be agglutinated by BTV. The results suggested that BTV binds to NANA- and NGNA alpha 2-6GalNAc residues in the O-linked oligosaccharides of human and porcine glycophorins respectively and indicated the presence of different binding sites on the virus for erythrocytes from other species.