Purification of virus particle

Isolation and purification of virus particle were performed as previously described with minor modifications [12, 18]. Briefly, mycelia of SsBRV2-infected strain and SsBRV2-free strain, respectively, were inoculated in conical flasks containing 150 ml PDB and shake-cultured at 200 rpm for 6 days. Next, mycelia were harvested through 4-layer sterilized gauze and then washed several times with sterilized water. After that, the harvested mycelia (30–40 g) were homogenized in the presence of 3 volume phosphate buffer (0.1 M sodium phosphate, pH 7.0, containing 0.2 M KCl and 0.5 % mercaptoethanol) in a Waring blender, followed by centrifugation at 10,000 rpm, 4 °C for 15 min to remove the hyphal cell debris. After two cycles of ultracentrifugation, virus particles were purified with a gradient sucrose concentration of 20–40 % (W/V) and then collected by a new sterile syringe. After ultracentrifugation to remove the sucrose solution, the virus particle pellets were re-suspended in 200 μl sodium phosphate buffer (0.1 M, pH7.0) and the concentration of the final 200 μl of virus particles was determined by spectrometry reading. dsRNA was isolated from the purified viral particles using phenol-chloroform extraction and detected on a 1 % agarose gel. The Structure proteins of the purified viral particles were separated on a 10 % (wt/vol) polyacrylamide gel amended with 1 % (wt/vol) sodium dodecyl sulfate (SDS).

For negative staining, a drop of about 5 μl of the purified virus particle suspension was loaded on the hydrophobic surface of a parafilm square. Several 200-mesh carbon-formvar coated copper grids were floated onto this drop for 5 min and the excess liquid was removed from each of the grids by touching its border with a cut piece of filter paper. Next, the grids loaded with virus particles were immediately re-floated in a drop of 2 % (W/V) phosphotungstic acid solution for 5 min, and the excess virus particle suspension was removed with filter paper and the prepared grids were left to dry for a few minutes. Finally, the prepared samples were visualized under transmission electron microscopy (TEM) as previously described [12].