159 Fourteenth IOCV Conference, 2000—Other Viruses Preliminary Purification and Double Stranded RNA Analysis of Citrus Leprosis Virus A. Colariccio, O. Lovisolo, G. Boccardo, C. M. Chagas, M. d’Aquilio, and V. Rossetti ABSTRACT. Partial purification of citrus leprosis virus (CiLV) from local lesions of Cleopatra mandarin was achieved using an extraction buffer of low molarity, viral precipitation with PEG- NaCl, and ultra-centrifugation in a 10 to 50% linear cesium sulfate gradient. Most of the virions disintegrated during the processing, but a limited number of preserved particles, about 45 to 50 × 80 to 120 nm were seen under the electron microscope. Four main bands of dsRNAs were extracted from CiLV local lesions of Chenopodium quinoa leaves infected with the Cleopatra or sweet orange isolates, and separated by PAGE. Similar dsRNAs were also extracted from local lesions on field infected leaves of sweet orange and analyzed the same way. Values of 6 to 8 MDa were estimated. In all cases, the dsRNA bands were absent in tissue extracts from leaves not infected with CiLV. Citrus leprosis virus (CiLV), as far as we know, causes only local lesions but no systemic infection on susceptible plants, and is unstable in plant sap preparations. As a con- sequence, the virus is difficult to purify (8), and that has hampered investigations on its properties and on the production of antisera, that would be extremely useful for diag- nosis and comparison with other similar viruses. Evidence has been obtained that CiLV can be mechanically transmit- ted from sweet orange and Cleopatra mandarin to herbaceous hosts and, with some difficulty, from sweet orange to Caipira sweet orange (2, 7, 8). Bacilliform virus particles have been seen in thin sections of natu- rally and experimentally infected sweet orange plants (2, 8), and in experimentally infected Chenopo- dium quinoa leaves (7). New investigations on purifica- tion and on dsRNAs extracted from CiLV-infected plants are reported in this paper. MATERIALS AND METHODS The isolate of CiLV used for these investigations was from naturally infected Cleopatra mandarin in Limeira, São Paulo State (Brazil). About 20 g of local lesion tissue from Cleopatra mandarin leaves were pulverized with liquid nitrogen, homogenized in presence of extrac- tion buffer (0.05 M phosphate buffer Fig. 1. Light-scattering band (arrow) formed after ultra-centrifugation in a cesium sulfate linear gradient of PEG- precipitated extract from local lesions of CiLV-infected Cleopatra mandarin leaves.