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

Emma L. Bradford; Craig R. Christie; Ewan M. Campbell; Alan S. Bowman

doi:10.1371/journal.pone.0190017

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

Emma L. Bradford, Craig R. Christie, Ewan M. Campbell, Alan S. Bowman^* (Corresponding Author)

^*Corresponding author for this work

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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.

Original language	English
Article number	e0190017
Pages (from-to)	1-19
Number of pages	19
Journal	PloS ONE
Volume	12
Issue number	12
DOIs	https://doi.org/10.1371/journal.pone.0190017
Publication status	Published - 19 Dec 2017

Bibliographical note

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.

Acknowledgments
The authors wish to thank Mr W. Thrale, Mr Z. Blackmore, Mr J. Quinlan, and Mr J. Palombo for sample collection from the South East of England and Margie Ramsey for Beinn Eighe National Nature Reserve sample collection.

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10.1371/journal.pone.0190017Licence: CC BY

A real-time PCR method for quantification of the total and major variant strains of the deformed wing virus
© 2017 Bradford et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. https://creativecommons.org/licenses/by/4.0/
Final published version, 4.43 MBLicence: CC BY

Cite this

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title = "A real-time PCR method for quantification of the total and major variant strains of the Deformed wing virus",

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.",

author = "Bradford, {Emma L.} and Christie, {Craig R.} and Campbell, {Ewan M.} and Bowman, {Alan S.}",

note = "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{\textquoteright}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. Acknowledgments The authors wish to thank Mr W. Thrale, Mr Z. Blackmore, Mr J. Quinlan, and Mr J. Palombo for sample collection from the South East of England and Margie Ramsey for Beinn Eighe National Nature Reserve sample collection.",

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ER -

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

Abstract

Bibliographical note

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