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

蜜蜂是全球最重要的授粉昆虫。蜂蜜的生产和授粉都依赖于强大、健康的蜂群，这种情况在上个世纪受到了全球传播的外寄生螨瓦螨的威胁。英国的所有殖民地（除了苏格兰的部分地区和一些岛屿）都有螨虫。瓦螨是一系列蜜蜂病毒的载体，当螨虫以发育中的蛹的血为食时，这些病毒就会被转移。这些病毒中最重要的是变形翼病毒（DWV）。在过去4年的研究中，我们已经证明，螨虫感染优先导致一种特定毒性形式的DWV扩增。在暴露于螨虫的发育蛹中，这种特殊病毒的水平几乎比没有螨虫时高出10,000倍。这是在对感染病毒产生免疫反应的情况下发生的。我们想了解为什么在螨虫感染的菌落或暴露于螨虫的蛹中观察到的致命形式的DWV复制到如此高的水平。是由于病毒获得的途径-直接进入血淋巴-而不是通常的途径-在喂食时口服？这种途径是否允许病毒感染它通常无法接触的组织或器官？它能让病毒绕过蜜蜂的免疫反应吗？DWV是否像许多其他病毒一样，携带可以抑制宿主免疫反应的基因？我们能否在蜜蜂接触病毒之前给它们“接种”疫苗，以防止随后的病毒复制？最后，我们想了解在DWV感染时哪些宿主基因的表达受到抑制，从而导致显性疾病的发展。在隔离的飞行笼中饲养的无瓦罗亚蜂群（来自Andrew Abrahams， Colonsay）或来自我们的研究蜂房的瓦罗亚蜂群将被用作个体蜜蜂幼虫的来源，这些幼虫将在实验室严格控制的条件下收获和饲养。我们可能是英国唯一一家拥有这种方法专业知识的实验室。个体幼虫将被喂食或注射具有特征的病毒种群。病毒复制的组织和器官将在解剖的蛹或切片上使用非常敏感的杂交技术来确定。我们特别渴望确定病毒的毒性形式是否引起全身感染，或者它们是否优先在特定组织或器官中复制，例如腹部和翼芽，两者在明显的DWV感染期间都表现出发育畸形。我们将在幼虫中重复这些研究，我们通过抑制Dicer和Argonaute基因的表达来故意抑制免疫反应的关键成分。这些幼虫现在是否对所有形式的DWV都敏感，而不仅仅是毒性变异？此外，我们还会将幼虫预先暴露（通过喂食；我们从美国路易斯安那州的合作者那里获得的一种技术），以抑制DWV复制的短RNA分子。这些幼虫是否能免受强毒DWV引起的感染和疾病？我们还将使用成熟的技术测试DWV是否携带抑制rna免疫反应有效性的基因。我们研究的第三个组成部分是调查特定宿主基因在免疫反应或发展中对dwv介导的疾病易感性增强的作用。我们在最近的研究中发现了这些基因，但它们的重要性还有待确定。我们将抑制感兴趣的单个基因，然后用已知的病毒种群挑战幼虫。这些研究不仅将确定为什么DWV的特定变体与蜜蜂的显性疾病有关，而且还将证明是否有可能开发出保护发育中的蜜蜂免受感染的治疗方法。

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.