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Double Infection, Interference and Superinfection in Protoplasts Exposed to Two Strains of Raspberry Ringspot Virus

Scottish Horticultural Research Institute, Invergowrie, Dundee, U.K.

The interaction in mesophyll protoplasts of two strains of raspberry ringspot virus, RRV-S and RRV-E, was studied using fluorescent antibody to detect strain-specific antigen. Staining with fluorescent antibody was weak and generalized unless the protoplasts were also infected with tobacco rattle virus (TRV), which induces RRV to form antigen aggregates and was therefore used routinely to make RRV antigen more easily detectable.

When tobacco protoplasts were inoculated simultaneously with equal amounts of the two RRV strains, both strain-specific antigens were produced in more than half the protoplasts. The proportion of protoplasts producing antigen aggregates of both strains depended on the ratio of particles of the two strains in the inoculum, but RRV-S had a greater specific infectivity than RRV-E and tended to dominate unless this difference was compensated for. Adding an equal amount of RRV-S to an inoculum of RRV-E decreased the proportion of protoplasts producing RRV-E antigen aggregates. When one RRV strain and TRV were inoculated before the other strain, the strain inoculated second produced antigen aggregates in fewer protoplasts than when it followed a first inoculation with TRV only. Exclusion of the second strain increased with increasing interval between inoculations and was not overcome by increasing the inoculum virus concentration. It was less strong in RRV-E-inoculated than in RRV-S-inoculated protoplasts, in which it was total by 12 h.

Nicotiana benthamiana plants systemically infected with RRV-S did not develop additional symptoms after their recovered leaves were inoculated with RRV-E, but some RRV-S-infected protoplasts from recovered leaves produced RRV-E antigen after inoculation with RRV-E and TRV. This shows that some stages of RRV-E replication can occur in cells long infected with RRV-S. However, RRV-E antigen aggregates were produced in only 11% of the challenge-inoculated protoplasts, 99% of which became infected with TRV from the same inoculum, suggesting that partial protection exists and is virus-specific.

The phenomena of interference and cross protection seem best explained by competition in cells for virus-specific sites or material, possibly RNA polymerase, that can be used in the replication of either strain.

Received 30 March 1978; accepted 2 May 1978.