Citrus tristeza virus (CTV), a member of the closterovirus group (Agranovsky, 1996 ;Bar-Joseph et al., 1995 ;Dolja et al., 1994 ;Coffin & Coutts, 1993), is one of the most destructive pathogens of citrus, causing a range of diseases such as quick decline and stem-pitting in susceptible cultivars. A large number of CTV strains or isolates has been characterized differing in symptomatology and serological properties (BarJoseph et al., 1989). The CTV virions are heterodimers and, similarly to beet yellows virus (Agranovsky et al., 1995), consist of two segments ; the larger, ca. 2000 nm, is encapsidated by the p25 coat protein (CP) and the shorter, ca. 75-85 nm, is encapsidated by p27, a diverged coat protein (CPd) (Febres et al., 1996). The CTV particles contain a single component positive-stranded genomic RNA, of 19 296 nt for the T-36 strain from Florida and of 19 226 nt for the Israeli strain VT (Mawassi et al., 1996). The genomes of these strains showed considerable Author for correspondence : Moshe Bar-Joseph.Fax j972 3 9604180. e-mail vpmbj!volcani.agri.gov.il sequence deviation within the 5h half, but were found to have similar organization and to encompass 12 ORFs, which potentially code for at least 17 protein products, including the replication associated proteins, a homologue of the HSP70 proteins and the two coat proteins Mawassi et al., 1996). In addition to the genomic and subgenomic RNAs plants infected with CTV contain multiple species of defective RNAs (D-RNAs). These D-RNAs consist of the 5h-and 3h-terminal segments of the genomic RNA, with extensive internal deletions. Hybridization analysis showed that the DRNAs are abundant in infected plants and occur in most CTV isolates (Mawassi et al., 1995 b, c). Two of the characterized CTV VT D-RNAs of 2n7 and 4n5 kb were found to represent different fusions of the genomic termini, whereas the 2n4 kb D-RNA molecule contained a short nonviral segment of 14 nt at the junction site (Mawassi et al., 1995 b, c). The 2n4 kb dsRNA band, previously shown to contain the 2424 nt D-RNA (Mawassi et al., 1995 b), was later found to contain a second molecule, designated D2.3-RNA, of 2379 nt, structurally representing a fusion of 1521 nt and 858 nt from the 5h and 3h ends, respectively, of CTV-VT. The present paper reports the cloning and sequence characterization of a new D-2.3 RNA and its use for the construction of an infectious cDNA clone.CTV strains VT (Mawassi et al., 1996) and GalT (obtained from Mr Yair Oren, Department of Citriculture, Ministry of Agriculture, Israel), were propagated in Alemow (Citrus macrophylla) seedlings. Total single-stranded RNA (ssRNA) was extracted from the leaves and young bark of infected plants using Tri-Reagent (Molecular Research Center Inc.). Double-stranded (ds) RNA was isolated from bark tissue according to Dodds & Bar-Joseph (1983). The dsRNA molecules were separated by PAGE. The 2n3 kb dsRNA segments were isolated by electroelution using Bio-Trap membranes (Schleicher and Schuell), or by elution into 0n5iTBE buffer (Mania...