Barcoding of Viruses

Since viruses don’t contain a generic barcode gene, it was decided to sequence the whole genome of viruses in this work package using Next Generation Sequence Technology.

Objectives

The objective of this WP is to develop a DNA barcoding method for identification of high priority viruses present in the EU Plant Health Directive and EPPO Plant Health lists of quarantine pathogens. In order to achieve this aim we will:

  • Develop reliable nucleic acid (DNA&RNA) extraction procedures.
  • Develop standard operating procedures for identification of viruses including extraction, PCR amplification, sequencing and database searching.

Tasks

  • Task 6.1  Selection and acquisition of targets
  • Task 6.2  Nucleic acid extraction
  • Task 6.3  Generic primer sets
  • Task 6.4  Genome sequencing
  • Task 6.5  Generation and validation of Barcode sequences
  • Task 6.6  Development and publication of complete protocols

Deliverables

  • D 6.1 List of selected quarantine viruses (Month 6)
  • D 6.2 A recommended protocol for nucleic acid extraction for virus infected plant material (Month 9)
  • D 6.3 Quarantine viruses (at least two Nepoviruses, Ilarviruses and Criniviruses) acquired by the consortium for primer testing (Month 12)
  • D 6.4 Validation data for published (Nepovirus, Ilarvirus Sadawaviruses, Begomoviruses, Tospoviruses and Crinivirus primer sets) and newly designed (three of Torradovirus, Comovirus or Rhabdovirus) generic primer sets with quarantine viruses (Month 18)
  • D 6.5 Validated barcode and meta data available for Nepovirus, Ilarvirus, Sadawaviruses, Begomoviruses, Tospoviruses and Crinivirus primers and three of Torradovirus, Comovirus or Rhabdovirus on the project/sequence database (Month 30)
  • D 6.6 Complete barcode identification methods written and available for Nepovirus, Ilarvirus, Sadawaviruses, Begomoviruses, Tospoviruses and Crinivirus primers and three of Torradovirus, Comovirus or Rhabdovirus genera (Month 30)
  • D 6.7 Genomic sequence available for the un-sequenced viruses (two of, for example: Potato yellowing virus, Aracacha virus B or Potato black ring spot virus ) (Month 36)
  • D 6.8 Detailed contingency plan for WP6 (Month 6)

Results

Since viruses don’t contain a generic barcode gene, it was decided to sequence the whole genome of viruses in this work package using Next Generation Sequence Technology.

The consortium produced an initial list of viral targets for which little sequence was available at the start of the project. Material was obtained for each of these species and the genome sequences were  produced using 454 and Solexa technology. With the acquisition of a 454 GS-FLX by one of the partners it was possible to optimise the complete sequencing process for virus genome sequencing. Methods have now been developed within the consortium which allow the cheap combining of multiple samples prior to the sequencing processing. These methods, along with an optimum virus RNA specific extraction process have reduced the cost of viral genome sequencing.

Genome sequence data have been produced for a range of viruses including: Arracacha virus B, oca strain, Potato black ringspot virus, Potato virus T, Potato yellowing virus, Tomato infectious chlorosis virus, Chrysanthemum stem necrosis virus, Iris yellow spot virus, Tomato torrado virus, Tomato marchitez virus, Potato yellow vein virus and Tomato chocolate virus. 

A number of different RNA extraction methods have been tested and used to successfully produce virus genome sequence. It was discovered that to maximise virus sequence recovery and thus minimise sequencing cost total RNA extraction of plants containing virus is not the best approach. Methods have been developed within the consortium to purify virus RNA away from plant RNA. These methods have been being compared to determine the optimum method for a particular sample type.

These methods include double stranded RNA isolation, small interfering RNA isolation, partial virus purification prior to RNA isolation, subtractive hybridisation and the use of capture probes. Results suggest that no one method is optimal for all samples.

The optimum methods for viral genome sequencing, along with advice on accessing this technology have now been published as part of the QBOL project. The methods were used to sequence in total 46 viruses and have been instrumental in the diagnosis of a number of novel diseases including the discovery of watercress white vein virus causing damage to the production of watercress and maize lethal necrosis devastating the 2012 Kenyan maize crop.