A novel mycovirus was isolated from a cultivated edible mushroom, Pleurotus eryngii, with severe epidemic symptoms. Purification of the virus was carried out by a sequential procedure of polyethylene glycol precipitation, differential centrifugation, and equilibrium centrifugation in a CsCl gradient. Nuclease digestion assay and protein analysis revealed that the virus consisted of a single-stranded RNA (ssRNA) genome of 7.8 kbp which was encapsulated by a coat protein of 22 kDa. Transmission electron microscope showed that it was spherical with a diameter of 31 nm. Since there was neither a previous report on discovery of a virus in P. eryngii, nor known mushroom viruses with similar characteristics, we concluded that this is a novel virus and thus have named it as P. eryngii Spherical Virus (PeSV). Because of a diagnostic test would be helpful in preventing the PeSV-related disease outbreaks, we developed a triple antibody sandwich-ELISA (TAS-ELISA) system using anti-PeSV mouse monoclonal and anti-PeSV rabbit polyclonal antibodies. The TAS-ELISA system successfully detected less than 0.5 mu g of the virus particles in 1 g diseased mushroom tissue collected from various commercial farms.