Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study.

BACKGROUND: Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FL-cDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. RESULTS: Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. CONCLUSIONS: The fast and efficient strategies described here should have broad applications, in particular for the study of "difficult" plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents.

Characterization of Prunus-infecting apricot latent virus-like Foveaviruses: evolutionary and taxonomic implications.

The complete genomic sequences of four Prunus-infecting Apricot latent virus (ApLV) like isolates were determined and used to analyze the taxonomic position and variability of these viruses. The results indicate that all isolates show a typical Foveavirus genetic organization. Despite an average 23% nucleotide divergence, they show strong colinearity with only three regions of significant indel variability, in the internal and 3' non-coding regions and variable N-terminal half of the coat protein (CP). Sequence comparisons using the polymerase (Pol) and CP genes provide a conflicting taxonomic picture, with divergence level in the Pol and CP genes suggesting the existence of a single or of two species, respectively. However, a range of considerations argue that all four isolates should likely be considered as belonging to the ApLV species. ApLV is closely related to Apple stem pitting virus and could be considered a sister species to it, with ASPV being specialized to infect members of the Maloideae family and ApLV members of the Prunoideae. Analysis of selection pressures affecting the five open reading frames of ApLV and ASPV identified two regions under strong purifying selection, that coding for the conserved C-terminal half of the CP and the gene coding for the first protein of the triple gene block (TGBp1).