The biology and pathogenesis of Deformed Wing Virus, the major virus pathogen of honeybees

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Honeybees are the most important managed pollinating insects globally. Both honey production and pollination depend upon strong, healthy colonies, a situation that has been threatened over the last century by the global spread of the ectoparasitic mite Varroa destructor. All colonies in the UK (excepting parts of Scotland and some islands) have the mite. Varroa acts as a vector for a range of viruses of honeybees which are transferred when the mite feeds on haemolymph (blood) from the developing pupa. The most important of these viruses is Deformed Wing Virus (DWV). In studies over the last 4 years we have demonstrated that mite infestation preferentially leads to the amplification of a specific virulent form of DWV. In mite-exposed developing pupae this particular virus reaches levels almost 10,000 times higher than seen in the absence of the mite. This is despite the development of an immune response to the infecting virus.

We want to understand why the virulent form of DWV observed in mite-infested colonies or mite-exposed pupae replicates to such elevated levels. Is it due to the route by which the virus is acquired - directly into the haemolymph - rather than the usual route which is orally during feeding? Does this route allow the virus to infect tissues or organs it normally does not have access to? Does it allow the virus to bypass the immune response of the honeybee? Does DWV, like many others viruses, carry genes that allow it to suppress the host immune response? Can we 'vaccinate' honeybees before they are exposed to prevent subsequent replication of the virulent virus? Finally we want to understand which host genes, expression of which are suppressed upon DWV infection, contribute to the development of overt disease.

Varroa-free honeybee colonies (from Andrew Abrahams, Colonsay) maintained in isolated flight cages or Varroa-infested colonies from our research apiary will be used as a source of individual honeybee larvae which will be harvested and maintained in the laboratory under carefully controlled conditions. We are perhaps the only lab in the UK with expertise in this methodology.

Individual larvae will be either fed or injected with a characterised virus population. The tissues and organs in which the virus replicates will be determined using exquisitely sensitive hybridisation techniques on either dissected pupae or sections. We are particularly keen to determine whether virulent forms of the virus cause systemic infections, or whether they preferentially replicate in particular tissues or organs, for example the abdomen and wing buds, both of which exhibit developmental deformities during overt DWV infection.

We will repeat these studies in larvae in which we have deliberately suppressed key components of the immune response by inhibiting expression of the genes Dicer and Argonaute. Are these larvae now susceptible to all forms of DWV, not just the virulent variant? We will additionally pre-expose larvae (via feeding; a technique we have acquired from collaborators in Louisiana, USA) to short RNA molecules designed to inhibit DWV replication. Are these larvae protected from infection and disease caused by virulent DWV? We will additionally test whether DWV carries a gene that inhibits the effectiveness of RNA-based immune responses using well-established techniques.

The third component of our study is to investigate the role of specific host genes implicated in components of the immune response or development to enhanced susceptibility to DWV-mediated disease. These genes were identified in our recent studies but their importance has yet to be determined. We will suppress individual genes of interest and then challenge larvae with known virus populations.

These studies will not only determine why a particular variant of DWV is associated with overt disease in honeybees but will also demonstrate whether it is possible to develop therapies to protect developing honeybees from infection.

Technical Summary

We will study the tropism, pathogenesis and control of a virulent variant of deformed wing virus (DWV) that replicates to elevated levels after transmission by the parasitic mite Varroa (or after parenteral transmission in vitro). There are three objectives of this study:
+ To determine the tropism of the virus following different routes of acquisition. We will use Stellaris riboprobes to detect the virus genome in dissected and/or sectioned larvae and pupae, conducting time course studies to investigate virus spread during honeybee development.
+ To investigate the role and importance of the host RNAi response in control of virus replication. We will suppress Dicer and Argonaute expression, so inhibiting effective RNAi responses to DWV and then investigate the tropism and replication of low- and high-virulence variants of DWV. We will use an RNAi sensor approach to investigate whether DWV encodes an RNAi suppressor that acts late in the pathway (post RNAi generation, which we have already quantified). We will additionally prime the RNAi response pre-exposure to DWV to determine whether it is protective.
+ A number of host genes are suppressed upon infection with a virulent form of DWV, including components of the immune response and genes possibly implicated in development. We will investigate the role of these genes by suppressing them prior to challenge with DWV, and subsequently quantify the level and distribution of DWV.
We will use a combination of in vitro studies, including maintenance of honeybee larvae in vitro, coupled with qRT-PCR and next generation sequence analysis of virus (or RNAi) populations. We have considerable expertise with all these methods.

Planned Impact

Honeybees are of global importance for pollination services and honey production (and other hive products). There is considerable scientific, commercial and public awareness of the "plight of the honeybee" and we have already engaged with relevant stakeholders in these three areas, and expect to extend these interactions in the future.
We anticipate that the research outcomes will include:

+ A mechanistic explanation for the high levels of virulent DWV observed in mite-associated pupae, including an appreciation of the tissue distribution of virulent and avirulent strains of the virus.

+ An understanding of the contribution of the host RNAi response in controlling DWV replication. Is the RNAi response effective? Does the virus encode a suppressor of the RNAi response? Can the RNAi response be boosted pre-infection to ameliorate the replication and consequent pathogenic outcome of subsequent infection with virulent strains of DWV?

+ An appreciation of the role individual genes - particularly those involved in Toll signaling pathways in the immune response and potential developmental pathways - in virus replication and pathogenesis. These may be useful genetic markers for future informed breeding of honeybees.

Academic beneficiaries are primarily the UK and international laboratories involved in honeybee research, those studying virus-host interactions or virus pathogenesis and groups attempting to exploit primary research in these areas for applied outcomes. Because of the economic and agricultural importance of honeybees 'academic' research is also directly supported in government agencies such as the National Bee Unit (UK) and the USDA Honeybee Research laboratory in Louisiana (USA). Our research has already defined a virulent form of deformed wing virus. The goal of the studies described in this application includes determining why this variant is virulent - does it have access to different tissues, does it suppress the host immune response? These mechanistic insights will benefit honeybee virus researchers and will also likely provide paradigms for enhanced understanding of how viruses exploit their hosts.

Large and small scale beekeepers (both commercial and hobbyists) are a distinct group of beneficiaries from this research. Although they are less likely to benefit directly from the primary research outcomes there is nevertheless considerable interest from them. Keeping these groups informed - through print, presentations and visits - is an important component of our research and one that we will continue. These 'end users' are more likely to directly benefit from the applied outcomes of our research. For example, diagnostic tests to detect and quantify the virulent form of DWV which might inform timing of miticide treatments. These groups will also benefit from further development of applied aspects of our research, for example the potential exploitation of the RNAi response to generate 'vaccines' capable of inhibiting replication and/or pathogenesis of virulent variants of DWV.

The third group of beneficiaries are the agrichemical industries, some of which have a significant interest and investment in honeybee diseases and therapies. For example, Beeologics, a company with expertise of RNAi treatment for colony collapse disorder have recently been bought by Monsanto. We expect our results from all three objectives of this proposal - in particular the second and third - will be of interest commercially. To this end we have already filed a patent on the identity of virulent strains of DWV, on their detection, quantification and on a wide range of potential methods by which they could be controlled.

We already have extensive links with individuals and groups in academia, government-funded research, commercial and hobbyist beekeepers and are exploring potential opportunities with industry.

Related Projects

Project Reference Relationship Related To Start End Award Value
BB/M00337X/1 30/11/2014 31/07/2015 £516,405
BB/M00337X/2 Transfer BB/M00337X/1 01/08/2016 30/11/2018 £435,239
 
Description Deformed Wing Virus (DWV) is the most important global viral pathogen of honey bees. DWV is transmitted between bees through feeding and by the ectoparasitic mite, Varroa.
In this study we studied the consequences of transmission by feeding or the mite on the virus population, and the responses of the bee.
We demonstrate that:
1. Virus populations are biphasic - either low diversity and high levels when transmitted by the mite, or high diversity and low levels when transmitted by feeding from bee to bee.
2. Direct injection of a mixed virus population leads to the low diversity/high level population, recapitulating the situation with Varroa transmission.
3. There are characteristic changes in host (bee) gene expression when either fed or injected with DWV. This suggests that there are a cascade of host responses that try and 'control' the virus (and the mite?) but that are overcome by virus replication.
4. Historically Varroa-free bees still carry DWV - at high diversity/low levels - and it is apathogenic under these situations.
5. There are quantitative, though possibly not qualitative, immune responses of the bee to the high levels of virus.

Tools were developed to define the diversity in the virus population using bioinformatics and next generation sequencing. Tools were developed to study the replication of the virus in the laboratory. We additionally now better understand the rate at which the virus population changes (detrimentally) after exposure of naive populations to Varroa-infested populations. Understanding this allows the timing and choice of rational treatment to be optimised.
Exploitation Route 1. Diagnostics - use defined tests to identify pathogenic strains of the virus and/or quantify pathogenic levels of the virus
2. Bioinformatics - use software to analyse virus diversity
3. Control - use comprehension of the consequences of Varroa transmission to control virus spread and disease.

We are studying point 3 in further BBSRC-funded studies.
Sectors Agriculture, Food and Drink,Education,Environment

URL https://www.theapiarist.org/
 
Description Our understanding of DWV has been incorporated in numerous talks to beekeeping associations and on my website in an article "When to Treat" which was accessed 12,000 times in 2018. Although the article was written in early 2016 it now has considerable traction and is accessed through internet searches and other links up to 2,000 times per month. Of course, it's not possible to tell how many readers actually implement the recommendations made in the article, but going by responses from talks given to beekeeping associations there is a marked change in understanding and practice. This page, and the website on which it is hosted, is widely cited on internet discussion forums.
First Year Of Impact 2017
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description BBSRC response mode project grant
Amount £920,000 (GBP)
Funding ID BB/R00305X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2018 
End 09/2021
 
Description DWV biology and pathogenesis
Amount £610,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2014 
End 10/2017
 
Description EASTbio PhD. studentship
Amount £75,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description Follow on Fund
Amount £165,588 (GBP)
Funding ID BB/M013685/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 04/2017
 
Description Multiple articles on a website for beekeepers 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I maintain a website for beekeeping which regularly discusses honey bee health. In particular there is a focus on the rational combination of science and practical beekeeping to improve honey bee health. The website was started in late 2014 and involves about 50 posts per year. 2017 access statistics are ~145,000 'real' page views (i.e. not robots or search engines) from ~75,000 visitors spread across ~120 countries. 2018 statistics show about 4,000 visitors per week visit the site. ~66% of site visits are from the UK and USA. My writing results in many invitations to speak at national beekeeping events. Access statistics show a significant increase from 2014 (~12,000), 2015 (~38,000), 2016 (~71,000) to 2017 (~145,000). Several beekeeping associations reproduce the content attributed to thrown members.
Year(s) Of Engagement Activity 2014,2015,2016,2017,2018
URL http://www.theapiarist.org
 
Description TV and radio 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview for South Korean TV on honey bee viruses
Interview for Spanish TV/video media about honey bee viruses

Both were documentaries on honey bee viruses - interview length was 3-5 hours each.
Year(s) Of Engagement Activity 2016,2017
URL http://one.lavanguardia.com
 
Description Talks - many 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Talks
Evening and weekend talks to beekeeping association in England, Scotland and Wales - total number of talks over 2013-2017 is at least 30. Audiences range from ~20 to ~200. Total reached well in excess of 500.
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
 
Description Talks at National/International beekeeping events 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Talk sparked questions afterwards and resulted in further invitations to talk, newsletter articles and collaborations

Collaborations, newsletter articles
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Talks to Beekeeping Associations - evening talks and weekend National conventions 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Numerous talks to beekeeping associations - Perth and District, Nairn, Peebles, Edinburgh and Midlothian, South Devon Beekeepers, Welsh Beekeeping Association and others - either in evenings or weekends. The purpose of the talks is to mix science and practical beekeeping to improve honey bee colony health. Evening talks might be attended by 30-50 people, weekend talks (like the Welsh beekeepers convention) attract an audience in excess of 300. Most of the talks are associated with extensive Q&A sessions and follow up correspondence.
Year(s) Of Engagement Activity 2017,2018
 
Description Talks to beekeeping associations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Very extensive Q&A from interested groups

Sponsorship was provided, though this wasn't the primary reason for the presentation.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015
 
Description Talks to commercial beekeepers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk resulted in extensive Q&A and supplementary invitations, article in newsletters etc.

Beefarmers agreed to provide letters of support for further research
Year(s) Of Engagement Activity 2013,2014,2015
 
Description The Apiarist website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact This website is a science-rich site for beekeepers. It carries weekly posts of relevance to practical beekeeping. In 2018 access statistics showed approaching 4000 pages a week were being read, over 200,000 a year by over 100,000 visitors. Visitors were from over 50 countries. Some posts generated comments from visitors, with ~500 comments/discussions over the 2018.

Readership (visitor numbers - not bots etc) has been increasing year on year, 17,000 in 2015, 34,000 in 2016, 75,000 in 2017 and 102,000 in 2018. 2019 page views and visitors at the time of writing are 34,000 and 16,000 respectively.

The website generates numerous invitations to speak at beekeeping associations and conventions. Many of these talks are on the science underlying practical beekeeping. Many audiences report changes in their beekeeping and improvements in bee health as a result of the information in my talks and on the website. Several articles and posts have been reproduced on beekeeping association sites around the world and in printed beekeeping magazines.
Year(s) Of Engagement Activity 2014,2015,2016,2017,2018,2019
URL http://www.theapiarist.org