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The genome of the Spanish mild isolate T385 of citrus tristeza virus (CTV) was completely sequenced and compared with the genomes of the severe isolates T36 (Florida), VT (Israel) and SY568 (California). The genome of T385 was 19 259 nt in length, 37 nt shorter than the genome of T36, and 33 and 10 nt longer than those of VT and SY568, respectively, but their organization was identical. T385 had mean nucleotide identities of 81n3, 89n3 and 94 % with T36, VT and SY568, respectively. The 3h UTR had over 97 % identity in all isolates, whereas the 5h UTR of T385 had 67 % identity with VT, 66n3 % with SY568 and only 42n5 % with T36. In the coding regions, the nucleotide differences between T385 and VT were evenly distributed along the genome (around 90 % identity) ; this was not observed between T385 and the other isolates. T385 and T36 had nucleotide identities around 90 % in the eight 3h-terminal ORFs of the genome, but only 72n3% in ORF 1a, a divergence pattern similar to that reported previously for T36 and VT. T385 and SY568 had nucleotide identities close to 90 % in the 5h-and 3h-terminal regions of the genome, whereas the central region had over 99 % identity. Our data suggest that the central region in the SY568 genome results from RNA recombination between two CTV genomes, one of which was almost identical to T385.

We report a phylogenetic study of viroids, some plant satellite RNAs, and the viroidlike domain of human hepatitis 8 virus RNA. Our results support a monophyletic origin of these RNAs and are consistent with the hypothesis that they may be "living fossils" of a precellular RNA world. Moreover, the viroidlike domain of human hepatitis 8 virus RNA appears closely related to the viroidlike satellite RNAs of plants, with which it shares some structural and functional properties. On the basis of our phylogenetic analysis, we propose a taxonomic classification of these RNAs.assumed an intracellular mode of existence sometime after the evolution of cellular organisms.Implicit in this proposal is the possibility that all viroids and viroidlike RNAs may have been derived from a common ancestor. To obtain evidence for or against this proposition, we have conducted a phylogenetic analysis of these small pathogenic RNAs and report here that our results are consistent with a monophyletic origin of viroids and viroidlike satellite RNAs, as well as possibly of the viroidlike domain of HDV RNA.Viroids, subviral pathogens of higher plants, are small (246-375 nucleotide residues), unencapsidated, single-stranded, circular RNAs characterized by highly base-paired, rodlike secondary structures (1). Viroids do not code for any proteins, yet they replicate autonomously (without the assistance of helper viruses) in susceptible cells. Viroidlike satellite RNAs resemble viroids, but they are found within the capsids of specific helper viruses required for their replication (2). Human hepatitis 8 virus (HDV) RNA is a circular RNA requiring hepatitis B virus as a helper virus for packaging and transmission (3). HDV RNA contains a region, termed the viroidlike domain, with significant similarities with viroid and viroidlike satellite RNAs (4). Viroids, viroidlike satellite RNAs, and HDV RNA appear to replicate via oligomeric RNA intermediates by some type of rolling-circle mechanism (2-6).Several hypotheses have been advanced to explain the evolution of viroids. It has been suggested, for example, that viroids may have originated from retroviruses or transposable elements by deletion of interior sequences (7), that they may represent escaped introns (8, 9), or that they have evolved comparatively recently from hypothetical "antenna" or "signal" RNAs, which eukaryotic cells are assumed to interchange (10).With the demonstration that certain RNAs have catalytic properties (11,12), the idea that RNA preceded DNA as a carrier of genetic information has gained support. Most recent models for self-replicating precellular RNAs assume the existence of primitive RNA enzymes with properties that are derived from extant self-splicing introns (13,14). Because one viroid, all known viroidlike satellite RNAs, and the viroidlike domain of HDV RNA are self-cleaving (3, 15, 16), it is equally plausible to consider these RNAs as relics of the RNA world, thus leading to an alternative hypothesis for the evolution of viroids and viroidlike satellite R...

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.

Isolates of citrus tristeza virus (CTV) differ widely in their biological properties. These properties may depend on the structure of viral RNA populations comprising the different isolates. As a first approach to study the molecular basis of the biological variability, we have compared the sequences of multiple cDNA clones of the two terminal regions of the RNA from different CTV isolates. The polymorphism of the 5' untranslated region (UTR) allowed the classification of the sequences into three groups, with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. The variability of an open reading frame (ORF) 1a segment adjacent to the 5' UTR supports the same grouping. Some CTV isolates contained sequences of more than one group. Most sequences from Spanish isolates belonged to group III, whereas a Japanese isolate was composed mostly of sequences of groups I and II. The mildest isolates contained only sequences of group III, whereas the most severe isolates also contained sequences of groups I, II, or both. The most stable secondary structure predicted for the 5' UTR was composed of two stem-loops and remained essentially unchanged as a result of compensatory mutations in the stems and accommodation of most of the variability in the loops. In contrast to the 5'-terminal region, the variability of the 3'-terminal region of CTV RNA was very much restricted, with nucleotide identity values higher than 90%. The presence of a conserved putative "zinc-finger" domain adjacent to a basic region in p23, the predicted product of ORF 11, suggests that this protein might act as a regulatory factor during virus replication.

This comprehensive volume presents indispensable and up-to-date information on viroids and viroid diseases. It provides a single source of information on the properties of viroids, the economic impact of viroid diseases, and methods for their detection and control. It examines the diseases associated with different plant species, the geographic distribution and epidemiology of viroids, diseases of possible viroid etiology, and the future applications of viroids. Viroids examines the biology of viroids, molecular characteristics, localization and movement, replication, pathogenesis, viroids and gene silencing, classification, viroid-like satellite RNAs, detection of viroids using bioamplification hosts, biological indexing, polyacrylamide gel electrophoresis, molecular hybridisation and polymerase chain reaction. The book looks at the geographical distribution and epidemiology of viroids in North America, Australasia, China, Japan, Europe, the Middle East, Africa, South America, and at the global level. It covers the control of viroids including quarantine of imported germplasm, availability of viroid-tested propagation materials, thermotherapy, tissue culture, and other conventional strategies as well as biotechnological control approaches. Special topics such as ribozyme reaction of viroids and economic advantages of viroid infection are also included. Other chapters summarise the current state of knowledge concerning viroid diseases of the crop in question and aspects of the natural history of viroids in horticulture. Among the crops covered are potato, tomato, tobacco, cucumber, pome fruits, stone fruits, avocado, citrus, grapevines, hop, chrysanthemum, coleus, columnea, and coconut palm. The four eminent editors of this watershed volume have assembled an international group of more than 70 scientists who have substantial experience with viroids and viroid diseases. They have produced a cohesive and comprehensive work that can be used by students, researchers, extension agents, and regulators. It may also be of a great value to science managers, policy makers, and industries in formulating policies and products to obtain viroid-free plants and control viroid diseases. The information on plant quarantine and certification programs will help anyone concerned with the safe movement of plant material across international boundaries or within a single country.

SUMMARYA viroid-like RNA was detected in nucleic acid preparations from two of the three commercial hop varieties grown in Spain. It had a size very close to that of avocado sunblotch viroid (ASBV), although dot-blot analysis revealed that it was very different in base sequence from ASBV, coconut cadang-cadang viroid, hop stunt viroid (HSV) and citrus exocortis viroid. In its physical and biological properties, the viroid-like RNA differed from the previously characterized HSV.Viroids are the only class of subviral agents endowed with autonomous replication whose molecular structure is well known. They comprise a single-stranded circular RNA which is highly self-complementary (Diener, 1979) and has a size range from 246 to 375 nucleotide residues (S~inger, 1984;Keese & Symons, 1985;Riesner & Gross, 1985). Viroids cause several diseases of higher plants (Riesner & Gross, 1985), although they can also replicate in other plants without detectable damage to the host (Owens et al., 1978). The last observation led some years ago to the proposal that an increasing number of viroid-like RNAs would be isolated from apparently healthy plants (Diener, 1979). This assumption has been confirmed recently in the case of grapevine (Sano et al., 1985(Sano et al., , 1986Flores et al., 1985). The detection of viroid-like RNAs has been greatly facilitated by the electrophoretic technique of Schumacher et aL (1983), which takes advantage of the very different mobilities of circular and linear RNA molecules of similar size in denaturing polyacrylamide gels. In the present communication we report that by applying this approach to RNA from hops, a new viroid-like RNA has been detected which is different from hop stunt viroid (HSV) described previously .Young leaves and flowers from individual hop plants (Humulus lupulus L.) were taken in the early summer of 1986 from different areas of Le6n (Spain). Samples were collected from the three hop varieties grown in Spain: H-3, H-7 and 'Fino de Alsacia'. Frozen tissue (20 g) was homogenized with a Willems Polytron (Kinematica, Kriens-Lucerne, Switzerland) in an extraction medium: 80 ml of water-saturated phenol, 16 ml 0.2 M-Tris-HC1 pH 8.9, 4 ml 0.1 M-EDTA pH 7, 4 ml 5 ~ SDS and 0.5 ml 2-mercaptoethanol (Semancik & Weathers, 1972). After centrifugation at 6000 g for 15 min the upper aqueous phases were re-extracted with 0.5 vol. of water-saturated phenol. The second aqueous phases were adjusted to a final volume of 40 ml with distilled water and the composition was adjusted to that of STE buffer (50 mM-Tris-HC1 pH 7.2, 100 mM-NaC1 and 1 mM-EDTA) and 35~ ethanol. Following the addition of 2 g of cellulose (CF-11, Whatman) (Franklin, 1966), the mixtures were gently shaken in centrifuge tubes for 10 to 15 min and centrifuged at 1000 g for 5 min. The cellulose pellets were washed three times with 60 ml of 35 ~ ethanol in STE and then with 20 ml of STE. The nucleic acids (and some other accompanying substances) eluted in the last wash were precipitated with 2.5 vol. of ethanol at -20 °C overnight,...

SUMMARYAnalysis by polyacrylamide gel electrophoresis of nucleic acid preparations, obtained from several varieties of grapevine by a procedure designed to isolate and purify viroids, revealed the presence of RNA species with some of the characteristic physical properties of viroids. Under non-denaturing conditions, a band with a mobility faster than that of citrus exocortis viroid (CEV) was detected, and under fully denaturing conditions two bands were observed, one co-migrating with the circular forms of CEV and a second migrating faster than the linear forms of this viroid. This RNA species did not hybridize with a cDNA probe to CEV. Some of the grapevine preparations were infective for Gynura aurantiaca, inducing symptoms similar to those caused by CEV, and the appearance of an RNA which had the same mobility as CEV in denaturing and non-denaturing electrophoretic systems and hybridized with cDNA to CEV. These results suggest that viroid-like and viroid RNAs can be recovered from grapevine, the former (with no detectable sequence homology to CEV) at a concentration sufficient to be observed as a physical entity in gels, and the latter (with close sequence homology to CEV) whose presence could only be revealed by bioassay. The possible involvement of these RNAs in some grapevine diseases of unknown aetiology is discussed.