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.
Crameri
G S
GS
CSIRO, Australian Animal Health Laboratory, Geelong, Victoria, Australia.
Wang
L F
LF
Eaton
B T
BT
D001820:Bluetongue virus / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005814:Genes, Viral / Q000235:genetics; D016679:Genome, Viral; D012330:RNA, Double-Stranded / Q000032:analysis* / Q000235:genetics; D016865:Reassortant Viruses / Q000235:genetics*; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
D012330:RNA, Double-Stranded