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

蜜蜂主要病毒病原变形翅病毒的生物学和发病机制

基本信息

批准号：
BB/M00337X/1
负责人：
David Evans
金额：
$ 65.8万
依托单位：
University of Warwick
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2014
资助国家：
英国
起止时间：
2014 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FM00337X%2F1
关键词：
biology pathogenesis Deformed Wing Virus

项目摘要

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.

蜜蜂是全球最重要的人工授粉昆虫。蜂蜜生产和授粉都依赖于强壮、健康的蜂群，这种情况在上个世纪受到了外寄生螨狄斯瓦螨全球传播的威胁。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。瓦螨属作为一系列蜜蜂病毒的载体，这些病毒在螨以发育中蛹的血淋巴（血液）为食时转移。这些病毒中最重要的是畸形翼病毒（DWV）。在过去4年的研究中，我们已经证明螨侵染优先导致DWV特定毒性形式的扩增。在螨暴露的发育中的脓疱中，这种特殊的病毒达到的水平几乎是没有螨的情况下的10，000倍。尽管对感染病毒产生了免疫反应，但仍是如此。我们想了解为什么在螨感染的菌落或螨暴露的脓疱中观察到的DWV毒性形式复制到如此高的水平。这是否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，即在喂养过程中口服？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基因，使其能够抑制宿主的免疫反应？我们能在蜜蜂暴露之前给它们接种疫苗以防止随后的病毒复制吗？最后，我们想了解哪些宿主基因，其表达在DWV感染后被抑制，有助于明显疾病的发展。无瓦螨的蜜蜂菌落（来自Andrew Abrahams，Colonsay）保持在隔离的飞行笼中或瓦螨感染的菌落来自我们的研究养蜂场，将被用作个体蜜蜂幼虫的来源，这些幼虫将被收获并在实验室中在仔细控制的条件下保持。我们可能是英国唯一一家拥有这种方法学专业知识的实验室。个体幼虫将被喂食或注射一种特征化的病毒种群。将使用精密灵敏的杂交技术在解剖的脓皮或切片上测定病毒复制的组织和器官。我们特别热衷于确定病毒的毒性形式是否会引起全身感染，或者它们是否优先在特定组织或器官中复制，例如腹部和翅芽，这两种病毒在明显的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和阿尔戈英雄这些幼虫现在对所有形式的DWV都敏感吗？我们还将预先暴露幼虫（通过喂养;我们从美国路易斯安那州的合作者那里获得的技术），以抑制DWV复制的短RNA分子。这些幼虫是否受到保护，不受毒性DWV引起的感染和疾病的影响？我们还将测试DWV是否携带一个基因，抑制RNA为基础的免疫反应的有效性，使用成熟的techniques.我们研究的第三个组成部分是调查特定的宿主基因的作用，参与免疫反应的组成部分或发展，以增强对DWV介导的疾病的易感性。这些基因在我们最近的研究中被发现，但它们的重要性尚未确定。我们将抑制感兴趣的个别基因，然后用已知的病毒种群攻击幼虫。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治疗方法来保护发育中的蜜蜂免受感染。