A real-time PCR method for quantification of the total and major variant strains of the deformed wing virus

PLoS One. 2017 Dec 19;12(12):e0190017. doi: 10.1371/journal.pone.0190017. eCollection 2017.

Abstract

European honey bees (Apis mellifera) are critically important to global food production by virtue of their pollination services but are severely threatened by deformed wing virus (DWV) especially in the presence of the external parasite Varroa destructor. DWV exists as many viral strains with the two major variants (DWV-A and DWV-B) varying in virulence. A single plasmid standard was constructed containing three sections for the specific determination of DWV-A (VP2 capsid region), DWV-B (IRES) and a conserved region suitable for total DWV (helicase region). The assays were confirmed as specific and discriminatory with limits of detections of 25, 25 and 50 genome equivalents for DWV-A, DWV-B and total-DWV, respectively. The methods were successfully tested on Apis mellifera and V. destructor samples with varying DWV profiles. The new method determined a more accurate total DWV titre in samples with substantial DWV-B than the method currently described in the COLOSS Beebook. The proposed assays could be utilized for the screening of large quantities of bee material for both a total DWV load overview along with more detailed investigations into DWV-A and DWV-B profiles.

MeSH terms

  • Animals
  • Base Sequence
  • Bees / parasitology
  • Bees / virology
  • RNA Viruses / genetics*
  • Real-Time Polymerase Chain Reaction / methods*
  • Reference Standards
  • Uncertainty
  • Varroidae / physiology
  • Wings, Animal / virology*

Supplementary concepts

  • Deformed wing virus

Grants and funding

ELB was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) EASTBIO Doctoral Training Partnership (http://www.bbsrc.ac.uk) [grant number BB/J01446X/1] and an Eastern Association Regional Studentship (EARS) and The Morley Agricultural Foundation awarded to ASB. CRC was supported by a KTN BBSRC CASE studentship (BB/M503526/1) (http://www.bbsrc.ac.uk), part-funded by the Scottish Beekeeping Association (https://www.scottishbeekeepers.org.uk/) and the Animal Health - Disease Prevention, Scottish Government awarded to ASB CRC. This project received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement no 613960 (SMARTBEES) (http://www.smartbees-fp7.eu/) awarded to ASB. The funders had no role in study design, data collection and analysis decision to publish, or preparation of the manuscript.