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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/2
负责人：
David Evans
金额：
$ 55.46万
依托单位：
University of St Andrews
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2016
资助国家：
英国
起止时间：
2016 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FM00337X%2F2
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受控授粉昆虫。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体外寄生蠕虫瓦罗亚破坏者的全球传播的威胁。英国的所有殖民地(苏格兰的部分地区和一些岛屿除外)都有这种螨虫。Varroa作为一种媒介传播蜜蜂的一系列病毒，这些病毒在螨虫以发育中的蛹的血淋巴(血液)为食时传播。其中最重要的病毒是变形翼病毒(DWV)。在过去四年的研究中，我们已经证明，螨类感染优先导致一种特定毒力形式的DWV的放大。在暴露在蠕虫的发育中的蛹中，这种特殊的病毒水平几乎是没有蠕虫时的10,000倍。这是尽管对感染病毒的免疫反应有所发展。我们想了解为什么在蠕虫出没的蜂群或暴露在蠕虫的蛹中观察到的DWV毒力形式复制到如此高的水平。是因为病毒直接进入血淋巴的途径，而不是通常的喂养过程中口服的途径吗？这种途径是否允许病毒感染它通常无法接触到的组织或器官？它是否允许病毒绕过蜜蜂的免疫反应？DWV是否像许多其他病毒一样，携带允许其抑制宿主免疫反应的基因？我们能否在蜜蜂暴露之前为它们接种疫苗，以防止随后复制这种剧毒病毒？最后，我们想要了解哪些寄主基因的表达在DWV感染时被抑制，这些寄主基因有助于显性疾病的发展。在隔离的飞行笼子中保持的无瓦罗亚的蜜蜂群体(来自安德鲁·亚伯拉罕，殖民地)或来自我们研究蜂房的瓦罗亚感染的蜂群将被用作单个蜜蜂幼虫的来源，这些蜜蜂幼虫将在实验室中在严格控制的条件下收获和维护。我们可能是英国唯一一个在这一方法方面拥有专业技术的实验室。每个幼虫都将被喂养或注射特定的病毒种群。病毒在其中复制的组织和器官将使用高度敏感的杂交技术在解剖的蛹或切片上进行确定。我们尤其热衷于确定病毒的强毒形式是否会导致系统性感染，或者它们是否会优先在特定的组织或器官中复制，例如腹部和翼芽，这两个组织或器官在明显的DWV感染期间都会表现出发育畸形。我们将在幼虫中重复这些研究，在这些研究中，我们通过抑制基因DICER和ArgAerte的表达来故意抑制免疫反应的关键成分。这些幼虫现在是不是对所有形式的DWV都敏感，而不仅仅是毒性变种？我们还将预先暴露幼虫(通过喂养；这是我们从美国路易斯安那州的合作者那里获得的一种技术)，以短RNA分子设计来抑制DWV复制。这些幼虫是否受到了强毒DWV的感染和疾病的保护？我们还将使用成熟的技术测试DWV是否携带抑制基于RNA的免疫反应有效性的基因。我们研究的第三部分是调查特定宿主基因在免疫反应或发育中的作用，以增强对DWV介导的疾病的易感性。这些基因是在我们最近的研究中发现的，但它们的重要性尚未确定。我们将抑制单个感兴趣的基因，然后用已知的病毒种群来挑战幼虫。这些研究不仅将确定为什么特定的DWV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疗法。