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Defective (D) RNAs were generated in tobacco upon passage of two isolates of Cucumber mosaic virus (CMV) initially derived from RNA transcripts of cDNA clones. In both cases, the D RNA was derived by a single in-frame deletion of either 339 or 411 nt within the 3a gene of Fny-CMV RNA 3 or M-CMV RNA 3, respectively. The generation of D RNAs was rare and occurred with two CMV isolates, the virions of which were known to differ in physico-chemical properties. The Fny-CMV D RNA 3, designated D RNA 3-1, was maintained by passage together with Fny-CMV in tobacco, but was lost by passage in squash. D RNA 3-1 accumulated in the inoculated squash cotyledons but not in upper, systemically infected leaves. Virions purified from infected squash cotyledons or leaf mesophyll protoplasts did not contain D RNA 3-1. Therefore, the failure of D RNA 3-1 to accumulate in squash leaves systemically infected by CMV was due to a lack of encapsidation of the D RNA 3-1 and movement out of the inoculated leaves.
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