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

Emma L Bradford,Alan S Bowman,Craig R Christie,Ewan M Campbell,Olav Rueppell

doi:10.1371/journal.pone.0190017

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.

Highlights

The European Honeybee (Apis mellifera) is the most economically important global pollinator, estimated to pollinate crops worth $10billion in the United States of America every year, while in the European Union insect pollination is worth around 14.6billion annually [1,2]
When the deformed wing virus (DWV)-A-specific primers were tested with the plasmid construct containing the DWV-B variant of that VP2-capsid region, there was no detection of any product (Ct > 35 cycles, no determinable melting point)
The DWV strain (DWV-A) specific primers readily amplified a product from the plasmid containing the DWVA variant of the VP2-capsid region and the DWV-B primers amplified a product from the plasmid containing the DWV-B variant of the IRES region

Summary

Introduction

The European Honeybee (Apis mellifera) is the most economically important global pollinator, estimated to pollinate crops worth $10billion in the United States of America every year, while in the European Union insect pollination is worth around 14.6billion annually [1,2]. V. destructor is an ectoparasite of A. mellifera which jumped host from the Asian Honeybee (Apis cerana) to A. mellifera in the 1960s and subsequently rapidly spread globally [6,7,8]. V. destructor infestation has been shown to cause immunosuppression and weight loss due to the mites haemolymph feeding activity [9,10]. High individual DWV titres have been found to be associated with overwinter colony losses [12,13]

Methods

Results

Discussion

Conclusion