Purification and Immunological Analyses of Cylindrical-Inclusion Protein Induced by Papaya Ringspot Virus and Watermelon Mosaic Virus 1

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Abstract
Cytoplasmic cylindrical inclusions induced by papaya ringspot virus (PRV) and watermelon mosaic virus 1 (WMV-1), members of the potyvirus group, were partially purified by differential centrifugation. Subunits of cylindrical inclusions were further purified by preparative polyacrylamide gel electrophoresis. UV absorption spectra of cylindrical inclusion proteins (CIP) of PRV and WMV-1 were typical of protein. Both CIP had MW of about 70,000. Results from sodium dodecyl sulfate (SDS)-immunodiffusion tests with highly specific antisera showed that PRV CIP and WMV-1 CIP were serologically indistinguishable, whereas they were serologically unrelated to coat protein. The antisera did not react to CIP of watermelon mosaic virus 2 (WMV-2) and some other potyviruses tested. An indirect enzyme-linked immunosorbent assay (ELISA) method for detecting purified CIP or CIP in plant extract was described. The method could easily detect as little as 1.6 ng of purified CIP/ml or 3.2 .times. 10-5 dilution of the crude extract, using .gamma.-globulin to CIP at the concentration of 1 .mu.g/ml. Results of indirect ELISA confirmed SDS-immunodiffusion tests that PRV CIP and WMV-1 CIP were serologically identical, but not related to WMV-2 CIP. This indirect ELISA method was considered to be a good alternative serological probe to study potyvirus relationships and for virus diagnosis.