Development and optimization of a hybridization assay for epizootic hemorrhagic disease viruses

Hybridization assays for the double-stranded RNA (dsRNA) orbiviruses causing bluetongue and epizootic hemorrhagic disease are more labor-intensive and less sensitive than enzyme-linked immunosorbent assay (ELISA) and immunoperoxidase assay. Cell-culture EHD virus amplification, rapid extraction, and optimization of RNA blotting conditions were examined to increase sensitivity and decrease labor. Aldehyde RNA denaturations and nylon hybridization membranes resulted in stronger positive hybridization signals. Treatment of infected cells with protease did not increase the yield of viral RNA target. Because RNA extraction is a tedious process, a simple non-phenolic diethyl pyrocarbonate extraction procedure was developed. The sensitivity, versatility, and the reproducibility of hybridization assays are addressed.