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Viroids are the smallest autonomous infectious nucleic acids known so far. With a small circular RNA genome of about 250-400 nt, which apparently does not code for any protein, viroids replicate and move systemically in host plants. Since the discovery of the first viroid almost forty-five years ago, many different viroids have been isolated, characterized and, frequently, identified as the causal agents of plant diseases. The first viroid classification scheme was proposed in the early 1990s and adopted by the International Committee on Taxonomy of Viruses (ICTV) a few years later. Here, the current viroid taxonomy scheme and the criteria for viroid species demarcation are discussed, highlighting the main taxonomic questions currently under consideration by the ICTV Viroid Study Group. The impact of correct taxonomic annotation of viroid sequence variants is also addressed, taking into consideration the increasing application of next-generation sequencing and bioinformatics for known and previously unrecognized viroids.

Eleven pospiviroid isolates from ornamental plants and one from pepper were successfully transmitted to potato and tomato by mechanical inoculation. All isolates with characteristic predominant genotypes were inoculated to a series of potato and tomato plants and propagated for up to four passages. In total, 385 nucleotide sequences were determined, in which 17 new predominant genotypes were identified with minimal differences to the original predominant genotype. In addition, in the original ornamental hosts, only one of eleven predominant pospiviroid genotypes had changed during the experiments over a period of 2 years. These results confirm the high stability of predominant pospiviroid genotypes.

The usefulness of various suggested species demarcation criteria was compared in attempts to determine the taxonomic status of ten new tombusvirus isolates. Five of them (Lim 1, 2, 3, 5 and 6) were obtained from different sources of commercially grown statice (Limonium sinuatum), two (Gyp 1 and 2) from different sources of commercially grown Gypsophila paniculata and three from water samples, i.e. from a small river (Schunter) in Northern Germany, from a brook (near Dossenheim) in Southern Germany and from the groundwater in a Limonium production glasshouse in the Netherlands (Lim 4). The immunoelectron microscopical decoration test allowed a quick preliminary assignment of various isolates to several known tombusviruses. A more precise analysis of the relationships was achieved by comparing the deduced amino acid sequences of the coat proteins. Sequence as well as serological data suggested that eight of the isolates should be classified as strains or variants of either Carnation Italian ringspot virus, Grapevine Algerian latent virus, Petunia asteroid mosaic virus or Sikte waterborne virus, respectively, whereas the 9th isolate (Lim 2) appears to represent a distinct new tombusvirus species. The case of the 10th isolate (Lim 5) illustrates the classification problems experienced when the properties of a virus place it close to the more or less arbitrary man-made borderline between virus species and virus strains. The coat protein gene sequences were also determined for some viruses for which these data had not yet been available, i.e. Neckar river virus, Sikte waterborne virus and Eggplant mottled crinkle virus. The sequences of the coat protein gene and also of ORF 1 of the latter virus proved to be almost identical to the corresponding genome regions of the recently described Pear latent virus, which for priority reasons should be renamed. Criteria which have been suggested in addition to serology and sequence comparisons for tombusvirus species demarcation, i.e. differences in natural and in experimental host ranges, in cytopathological features and in coat protein size, appear to be of little value for the classification of new tombusviruses.

The complete genome sequence of chrysanthemum stem necrosis virus (CSNV) was determined using Roche 454 next-generation sequencing. CSNV is a tentative member of the genus Tospovirus within the family Bunyaviridae, whose members are arthropod-borne. This is the first report of the entire RNA genome sequence of a CSNV isolate. The large RNA of CSNV is 8955 nucleotides (nt) in size and contains a single open reading frame of 8625 nt in the antisense arrangement, coding for the putative RNA-dependent RNA polymerase (L protein) of 2874 aa with a predicted Mr of 331 kDa. Two untranslated regions of 397 and 33 nt are present at the 5' and 3' termini, respectively. The medium (M) and small (S) RNAs are 4830 and 2947 nt in size, respectively, and show 99 % identity to the corresponding genomic segments of previously partially characterized CSNV genomes. Protein sequences for the precursor of the Gn/Gc proteins, N and NSs, are identical in length in all of the analysed CSNV isolates. [ABSTRACT FROM AUTHOR]