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

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.

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.

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.