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For the identification and analysis of new RNA plant viruses infecting fruit trees, an initial step often involves the laborious procedure of isolation and cDNA synthesis and cloning from purified viral dsRNA. For subsequent RT-PCR detection of these and other viruses from tissue with high phenolic and polysaccharide concentrations, a simple and efficient extraction protocol for viral nucleic acid is also important. A method for rapid cDNA cloning from small amounts of purified dsRNA using a modification of degenerate oligo primed polymerase chain reaction mbox(DOP-PCR), and a modification of a protocol for effective extraction of viral RNA for use in RT-PCR are presented. Both methods were used to analyze a number of mottling diseases described in cherry. The causal agents for two of these diseases have been previously described, Cherry green ring mottle virus, a tentative member of the foveaviruses, and Cherry mottle leaf virus, a member of the trichoviruses. For the diseases cherry rusty mottle and cherry necrotic rusty mottle, data are presented identifying viruses associated with each disease. Viruses associated with cherry rusty mottle, cherry necrotic rusty mottle and European isolates of cherry mottle leaf diseases, are closely related to Cherry green ring mottle virus and can be tentatively included in the foveavirus genus. An additional virus, related to cherry green ring mottle virus, was discovered by RT-PCR cloning and appears to be a common latent virus of cherry. Finally, isolates of cherry necrotic mottle disease could be assayed positive by RT-PCR for a virus

The complete genomic sequence of variant USA6b of Little cherry virus-2 (LChV-2), has been determined and is 15045 nucleotides in length, coding for 11 open reading frames (ORFs). The sequence shares 77.2% identity with a previously published, ca. 6 kb partial replicase sequence of LChV-2 (variant USA6a). Both LChV-2/USA6a and LChV-2/USA6b were obtained from the same tree infected with little cherry disease, and would suggest a mixed infection. LChV-2/USA6b is more closely related to the partially determined genomic sequence of a Canadian isolate of LChV-2, strain LC5 (92.9% identity). LChV-2/USA6b has an unusual genomic organization compared to other members of the Closteroviridae. The LChV-2/USA6b genome is potentially ambi-sense, with a negative sense ORF0 at the 5' terminus, from which an 18.1 kDa protein of unknown function can be expressed in vitro. The N-terminal region of the LChV-2/USA6 ORF1a translation product does not code for a papain-like protease motif. ORF1 codes for a novel motif, of unknown function, also present in isolates of the Grapevine leafroll associated virus-3, (genus Ampelovirus) as well as viruses of the family Flexiviridae. ORF3 lacks an AUG start codon, but could potentially be expressed via read-through of the ORF2 stop codon. At the 3' end, there is a re-organization of encoded genes compared with other members of the Closteroviridae including separation of the coat protein and coat protein duplicate genes by 4 other genes as found for LChV-2/LC5.

New insights into the genetic structure and variability of grapevine leafroll-associated viruses (GLRaVs) gained through worldwide efforts in the last decade or so, and the production and use of new sets of serological reagents, have provided the solid foundation on which the present revision of the taxonomic structure of the family Closteroviridae, and the genus Ampelovirus in particular, is based. A comparative examination of the amino acid sequence divergence of three taxonomically relevant genes [RNA-dependent RNA polymerase (polymerase), heat shock protein 70 homologue (HSP70h) and coat protein (CP)] disclosed a difference among Grapevine leafroll-associated virus 4 (GLRaV-4), -5, -6 and -9 and a group of more recently described viruses (GLRaV-Pr, GLRaV-De and GLRaV-Car) below the 25% limit recently set by the International Committee on Taxonomy of Viruses (ICTV) as a discriminating criterion for the identification of species in the family Closteroviridae. This, plus the recognition that GLRaV4, -5, -6 and -9 are serologically related, have similar biological and epidemiological traits, and that these viruses and GLRaV-Pr, GLRaV-De, GLRaV-Car have a genome with the same structure and size, supports the notion that they are all genetically divergent variants of a single species, GLRaV-4. The genus Ampelovirus is split into two subgroups designated I and II in recognition of the wide difference in the size and structure of the genome of the present members. Finally, the establishment of a fourth genus within the family Closteroviridae, comprising the unassigned putative species Grapevine leafroll-associated virus 7 (GLRaV-7), Little cherry virus 1 (LChV-1) and Cordyline virus 1 (CoV-1), is justified based on their molecular and biological characteristics that differ from those of members of the other three genera of the family.