Identification of novel viruses and viroids in citrus hosts by high-throughput sequencing: methodologies, opportunities, challenges and impact

Largely improving the capability of sequencing and characterizing nucleic acids, high-throughput sequencing (HTS) technologies have strongly increased the chances of identifying virus- and viroid-derived sequences in plants. On the one hand, HTS offers interesting opportunities in routine diagnosis that, however, must raise to significant challenges, including the need of developing standard protocols, infrastructures and expertise to store and analyze the data. On the other hand, HTS allows the identification of novel viruses and viroids, provided that sequences of related viruses/viroids are available in databases. This means that viruses/viroids with genomes largely divergent with respect to those already known may escape detection, especially if appropriate controls and search methodologies are not applied. The low titer of the viruses, their uneven distribution, as well as the presence of inhibitors in the extracts may also impair the sensitivity of HTS-mediated identification of known and novel viruses, especially in woody plants, such as citrus, thus requiring additional efforts to adjust the extraction methods, the library preparations and/or the pipelines adopted for the bioinformatic analyses of the sequencing data. These issues together with the results of case studies, in which different HTS methodologies have been tested for their capability of detecting citrus viruses and viroids, will be examined. The identification of novel viruses by HTS may have consequences on the taxonomy of viruses that, in some instance, has been already updated to include viruses discovered by HTS. The need of providing biological data on the novel viruses identified by HTS will be also discussed, paying major attention to the relevance of the information requested for assessing the risk these infectious agents may pose.