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Based on the results of agar gel double immunodiffusion tests with broad spectrum rabbit antisera and narrow spectrum mouse immune ascitic fluids and formalin-fixed purified viruses, a close relationship was established between 3 members of the Cucumovirus group namely Robinia mosaic virus (RoMV), clover blotch virus (CBV) and peanut stunt virus (PSV). The relationship of these 3 viruses to cucumber mosiac virus proved to be more distant. The results also suggested that RoMV and CBV could represent distinct strains of PSV.

Red clover mottle virus was kept in 0.1 M phosphate buffer, pH 7.0, at 4, --20 and --60 degrees C for 1 and 4 weeks and 3 months. The degree of virion disintegration was the highest at --20 degrees C and the lowest at 4 degrees C. Two cycles of freezing and thawing destroyed the virions. In frozen samples (at both --20 and --60 degrees C), the nucleoprotein sedimentation component M was disintegrated more than component B.

The degree of serological relatedness of three isolates of bean yellow mosaic virus (BYMV) was studied. One common antigenic group (m) was demonstrated in all 3 isolates. In addition, another antigenic group (a) was demonstrated in the isolate FvM1. This group was absent from the isolates TpM3 and TpM11, in which the antigenic group n was found. Antisera to the 3 isolates contained the corresponding antibody groups. The antibody group (M) shared by all 3 BYMV isolates was present in the FvM1 antiserum in a level only a little higher than the second antibody group (A). Antisera to TpM3 and TpM11 isolates showed only a low titre of M antibody, while the second antibody group (N) reached titres from 512 to 1024. Cross-absorption tests revealed serological identity of both isolates of the "necrotic type" (TpM3 and TpM11) and allowed to estimate the degree of their serological relatedness to representatives of the "mosaic type" isolates of BYMV (FvM1).

Serological and immunoelectrophoretic properties of 22 RCMV isolates from Czechoslovakia were determined. Based on the differences among the isolates, two electrophoretic types were established: type I--isolates with one non-separable electrophoretic form, and type II--isolates with two electrophoretic forms. Depending on the mobility of the electrophoretic forms these types could be subdivided further into two subtypes each. In all selected isolates representing the individual electrophoretic types and subtypes two isoelectric points were determined at pH 5.2 and pH 5.6.

ELISA was used to determine the degree of serological relationship between red clover mottle (RCMV) and broad bean stain (BBSV) viruses and the optimal conditions for the differentiation of the two viruses by ELISA were established. The titres of homologous and heterologous antibodies determined by ELISA were 100- to 200-fold higher as compared with the ring precipitin test. Antigen concentrations of 0.15 and 0.075 ng/ml, respectively, could be detected. The reactions in ELISA depended on the concentration of antigen, of IgG used for coating and of conjugated IgG. In anti-RCMV IgG, the ratio of group-specific to species-specific antibody as determined by ELISA was from 1:1 to 1:2, while in anti-BBSV IgG this ratio was from 1:4--1:8. The differences between homologous and heterologous reactions in ELISA suggested a similar ratio of antigenic determinants in the two viruses.

Four isolates of broad bean stain virus (BBSV; Comovirus group), representing two serotypes in immunodiffusion tests, were compared by immunoelectrophoresis. Two electrophoretic forms (slow and rapid) migrating from the cathode to the anode were found in virus preparations purified from above-ground plant parts harvested 14 or 21 days after inoculation with any of the four isolates. In virus preparations kept for a year at 4 degrees C in the presence of sodium azide both electrophoretic forms were preserved, while in preparations without a preservative only the rapidly migrating form could be detected. In virus preparations from plant roots, only the rapidly migrating form was demonstrated.

Clover blotch virus (CBV) was purified by differential centrifugation or precipitation with polyethylene glycol 6000 or 20000. Analytical centrifugation of purified CBV preparations revealed 2 components, the proportions of the lighter (96 S) nad heavier (112 S) being 88 and 12%, respectively. RNA isolated from purified CBV represented a micture of several RNA species of different molecular weight and its infectivity was by about 2 log units lower than that of the original nucleoprotein. CBV virions are isometric with a diameter of 28 nm. CBV is tentatively included into the Cucumovirus group.