Green Bees: Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism

Olesya N. Gusachenko; Luke Woodford; Katharin Balbirnie-Cumming; Ewan M. Campbell; Craig R. Christie; Alan S. Bowman; David J. Evans

doi:10.3390/v12050532

Green Bees: Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism

Olesya N. Gusachenko^*, Luke Woodford, Katharin Balbirnie-Cumming, Ewan M. Campbell, Craig R. Christie, Alan S. Bowman, David J. Evans

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

31 Citations (Scopus)

4 Downloads (Pure)

Abstract

Environmental and agricultural pollination services by honey bees, Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses.

Original language	English
Article number	532
Number of pages	17
Journal	Viruses
Volume	12
Issue number	5
Early online date	12 May 2020
DOIs	https://doi.org/10.3390/v12050532
Publication status	Published - May 2020

Bibliographical note

Funding: This work was supported by grant funding from Biotechnology and Biological Sciences Research Council (BBSRC): BBSRC BB/M00337X/2 and BB/I000828/1. C.R.C. was supported by a KTN BBSRC CASE studentship BB/M503526/1 (http://www.bbsrc.ac.uk). C.R.C was part-funded by the Scottish Beekeeping Association (https://www.scottishbeekeepers.org.uk/) and the Animal Health and Welfare program by the Scottish Government. E.M.C. was supported by the Veterinary Medicines Directorate, Department for Environment Food & Rural Affairs (Project # VM0517) (https://www.gov.uk/government/organisations/veterinary-medicines-directorate).
Acknowledgments: We would like to express our gratitude to Robert J Paxton (Martin Luther University Halle-Wittenberg) for providing a reference sequence for the construction of DWV-B RG system, Marcus Bischoff and Gill McVee (University of St Andrews) for helping with microscopic imaging set up, Javier Tello (University of St Andrews) for help and advice on sample cryosectioning, Ivan Gusachenko (University of St Andrews) for obtaining a photo of EGFP-expressing pupa, Ashley Pearson (University of St Andrews) for assistance in molecular biology assays, and Kirsten Bentley (University of St Andrews) for the high practical and moral support throughout the project.

Keywords

insect viruses
honey bee
pollination
virus vector
Varroa
RNA viruses
DWV
reverse genetics
Reverse genetics
Pollination
Insect viruses
Virus vector
Honey bee

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.3390/v12050532Licence: CC BY

Gusachenko_et_al_Viruses_GreenBees_VoR
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Final published version, 3.97 MBLicence: CC BY

http://www.mdpi.com/1999-4915/12/5/532/s1Licence: CC BY

Cite this

@article{3cceec8be7df471fa7a9c80b86b19bea,

title = "Green Bees: Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism",

abstract = "Environmental and agricultural pollination services by honey bees, Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses. ",

keywords = "insect viruses, honey bee, pollination, virus vector, Varroa, RNA viruses, DWV, reverse genetics, Reverse genetics, Pollination, Insect viruses, Virus vector, Honey bee",

author = "Gusachenko, {Olesya N.} and Luke Woodford and Katharin Balbirnie-Cumming and Campbell, {Ewan M.} and Christie, {Craig R.} and Bowman, {Alan S.} and Evans, {David J.}",

note = "Funding: This work was supported by grant funding from Biotechnology and Biological Sciences Research Council (BBSRC): BBSRC BB/M00337X/2 and BB/I000828/1. C.R.C. was supported by a KTN BBSRC CASE studentship BB/M503526/1 (http://www.bbsrc.ac.uk). C.R.C was part-funded by the Scottish Beekeeping Association (https://www.scottishbeekeepers.org.uk/) and the Animal Health and Welfare program by the Scottish Government. E.M.C. was supported by the Veterinary Medicines Directorate, Department for Environment Food & Rural Affairs (Project # VM0517) (https://www.gov.uk/government/organisations/veterinary-medicines-directorate). Acknowledgments: We would like to express our gratitude to Robert J Paxton (Martin Luther University Halle-Wittenberg) for providing a reference sequence for the construction of DWV-B RG system, Marcus Bischoff and Gill McVee (University of St Andrews) for helping with microscopic imaging set up, Javier Tello (University of St Andrews) for help and advice on sample cryosectioning, Ivan Gusachenko (University of St Andrews) for obtaining a photo of EGFP-expressing pupa, Ashley Pearson (University of St Andrews) for assistance in molecular biology assays, and Kirsten Bentley (University of St Andrews) for the high practical and moral support throughout the project.",

year = "2020",

month = may,

doi = "10.3390/v12050532",

language = "English",

volume = "12",

journal = "Viruses",

issn = "1999-4915",

publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",

number = "5",

}

TY - JOUR

T1 - Green Bees

T2 - Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism

AU - Gusachenko, Olesya N.

AU - Woodford, Luke

AU - Balbirnie-Cumming, Katharin

AU - Campbell, Ewan M.

AU - Christie, Craig R.

AU - Bowman, Alan S.

AU - Evans, David J.

N1 - Funding: This work was supported by grant funding from Biotechnology and Biological Sciences Research Council (BBSRC): BBSRC BB/M00337X/2 and BB/I000828/1. C.R.C. was supported by a KTN BBSRC CASE studentship BB/M503526/1 (http://www.bbsrc.ac.uk). C.R.C was part-funded by the Scottish Beekeeping Association (https://www.scottishbeekeepers.org.uk/) and the Animal Health and Welfare program by the Scottish Government. E.M.C. was supported by the Veterinary Medicines Directorate, Department for Environment Food & Rural Affairs (Project # VM0517) (https://www.gov.uk/government/organisations/veterinary-medicines-directorate). Acknowledgments: We would like to express our gratitude to Robert J Paxton (Martin Luther University Halle-Wittenberg) for providing a reference sequence for the construction of DWV-B RG system, Marcus Bischoff and Gill McVee (University of St Andrews) for helping with microscopic imaging set up, Javier Tello (University of St Andrews) for help and advice on sample cryosectioning, Ivan Gusachenko (University of St Andrews) for obtaining a photo of EGFP-expressing pupa, Ashley Pearson (University of St Andrews) for assistance in molecular biology assays, and Kirsten Bentley (University of St Andrews) for the high practical and moral support throughout the project.

PY - 2020/5

Y1 - 2020/5

N2 - Environmental and agricultural pollination services by honey bees, Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses.

AB - Environmental and agricultural pollination services by honey bees, Apis mellifera, and honey production are compromised by high levels of annual colony losses globally. The majority are associated with disease caused by deformed wing virus (DWV), a positive-strand RNA virus, exacerbated by the ectoparasitic mite Varroa destructor. To improve honey bee health, a better understanding of virus transmission and pathogenesis is needed which requires the development of tools to study virus replication, transmission, and localisation. We report the use of reverse genetic (RG) systems for the predominant genetically distinct variants of DWV to address these questions. All RG-recovered viruses replicate within 24 h post-inoculation of pupae and could recapitulate the characteristic symptoms of DWV disease upon eclosion. Larvae were significantly less susceptible but could be infected orally and subsequently developed disease. Using genetically tagged RG DWV and an in vitro Varroa feeding system, we demonstrate virus replication in the mite by accumulation of tagged negative-strand viral replication intermediates. We additionally apply a modified DWV genome expressing a fluorescent reporter protein for direct in vivo observation of virus distribution in injected pupae or fed larvae. Using this, we demonstrate extensive sites of virus replication in a range of pupal tissues and organs and in the nascent wing buds in larvae fed high levels of virus, indicative of a direct association between virus replication and pathogenesis. These studies provide insights into virus replication kinetics, tropism, transmission, and pathogenesis, and produce new tools to help develop the understanding needed to control DWV-mediated colony losses.

KW - insect viruses

KW - honey bee

KW - pollination

KW - virus vector

KW - Varroa

KW - RNA viruses

KW - DWV

KW - reverse genetics

KW - Reverse genetics

KW - Pollination

KW - Insect viruses

KW - Virus vector

KW - Honey bee

UR - http://www.scopus.com/inward/record.url?scp=85084786541&partnerID=8YFLogxK

U2 - 10.3390/v12050532

DO - 10.3390/v12050532

M3 - Article

C2 - 32408550

SN - 1999-4915

VL - 12

JO - Viruses

JF - Viruses

IS - 5

M1 - 532

ER -

Green Bees: Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism

Abstract

Bibliographical note

Keywords

UN SDGs

Access to Document

Other files and links

Fingerprint

Centre for Genome-Enabled Biology and Medicine

Cite this

Green Bees: Reverse Genetic Analysis of Deformed Wing Virus Transmission, Replication, and Tropism

Abstract

Bibliographical note

Keywords

UN SDGs

Access to Document

Other files and links

Fingerprint

Equipment

Centre for Genome-Enabled Biology and Medicine

Cite this