Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis.

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties.

Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro.

Cucurbit chlorotic yellows virus (CCYV) is a new member of the genus Crinivirus (family Closteroviridae) with a bi-partite genome. CCYV RNA 1-encoded p22 has recently been reported to be a weak local suppressor of RNA silencing for which an interaction with cucumber SKP1LB1 through an F-box-like motif was demonstrated to be essential. Using a bacterially expressed maltose-binding protein (MBP) fusion of CCYV p22 in electrophoretic mobility shift assays (EMSA), we have examined in vitro its ability to bind different RNA templates. Our experiments showed that CCYV p22 is able to bind to ss and ds long RNAs, in addition to ss and ds small interfering (si) RNA molecules. CCYV p22 deletion mutants (MBP_CCYV DEL1-4) were produced that covered the entire protein, with MBP_CCYV DEL2 corresponding to the F-box motif and its flanking sequences. None of these deletions abolished the capacity of CCYV p22 to bind ss- and dsRNA molecules. However, deletions affecting the C-terminal half of the protein resulted in decreased binding efficiency for either ss- or dsRNA molecules indicating that essential elements for these interactions are located in this region. Taken together, our data add to current knowledge of the mode of action of suppressors of RNA silencing encoded by genes sited at the 3'-terminus of crinivirus genomic RNA 1, and shed light on the involvement of CCYV p22 in the suppression of RNA silencing and/or in another role in the virus life cycle via RNA binding.