Rapid methods for comparing the double-stranded RNA genome profiles of bluetongue virus.
Various double-stranded RNA extraction procedures, gel electrophoresis systems, and methods to detect the RNA bands in the gel were investigated to find the most rapid methods to obtain the genome profiles of bluetongue virus in small volumes (1-25 ml) of infected cell culture fluids. Rapid double-stranded RNA extraction procedures coupled with staining the acrylamide gel slabs with ethidium bromide or silver nitrate resulted in well-defined genome profiles from bluetongue virus infected cell cultures in 6-48 h. Radioactive labelling of viral RNA with 32P was time consuming, cumbersome and expensive. These techniques detect less than 0.5 micrograms of double-stranded RNA which can be obtained from one 1-ml well of a 24-well cluster plate of bluetongue virus infected cell monolayers. The methods were therefore suitable for rapid comparisons of the electropherotypes of multiple virus isolates.
D001820:Bluetongue virus / Q000235:genetics*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004996:Ethidium; D005814:Genes, Viral; D010761:Phosphorus Radioisotopes; D012330:RNA, Double-Stranded / Q000235:genetics*; D012087:Reoviridae / Q000235:genetics*; D012835:Silver Nitrate; D013194:Staining and Labeling / Q000379:methods; D014776:Virus Cultivation / Q000379:methods
D010761:Phosphorus Radioisotopes; D012330:RNA, Double-Stranded; D012835:Silver Nitrate; D004996:Ethidium