The properties of two laboratory variants of raspberry bushy dwarf virus (RBDV), D1 (derived from the Scottish type isolate, D200) and Can-S (derived from an isolate infecting 'Canby' red raspberry in Canada), were compared with those of their parental sources and with two naturally occurring variants, RB (a natural variant able to overcome RBDV resistance genes) and M (a serological variant from black raspberry). In agarose gel double-diffusion tests using a polyclonal antiserum to isolate D200, all isolates were indistinguishable except D1 and M that were each spurred over by the other isolates. All 6 isolates reacted strongly with this polyclonal antiserum in DAS and plate-trapped antigen (PTA) forms of ELISA and in Western blotting (WB) and when each of four monoclonal antibodies (Mabs) to an unnamed red raspberry isolate was used to detect antigen trapped by the polyclonal antiserum. However, the virus isolates differed in their reactions to the 4 Mabs in PTA-ELISA and in WB. In herbaceous test plants, variants D1 and Can-S were distinguished readily from their parental sources and from the other two isolates by producing either no symptoms (D1) or very severe symptoms (Can-S) in hosts. Unlike all other isolates, Can-S failed to infect Chenopodium quinoa systemically and induced severe necrotic local lesions in Nicotiana species. The nucleotide sequences of the coat protein genes and parts of the polymerase gene of the isolates were >95% identical.