A synthetic & recombinant approach to the production and characterisation of IAPV an associated agent of honey bee Colony Collapse Disorder

蜂群崩溃障碍(CCD)是指在过去40年里蔓延到美国各地，在过去15年里蔓延到整个欧洲的蜂群灾难性损失。最近，由于感染以色列急性麻痹病毒(IAPV)而导致的蜜蜂(Apis Melvena)的损失被归因于以色列急性麻痹病毒(IAPV)的感染。IAPV是一种双环病毒，其传播或致病性可能会因蜜蜂感染瓦罗螨类而加剧。现在把重点放在IAPV及其控制的可能性上是合适的，但该病毒从未在纯培养中分离出来，因此不可能进行基因操作和生产纯病毒制剂，从而减缓了诊断和治疗的进展。最近的研究表明，相关的麦长管蚜双胞体病毒基因组被整合到杆状病毒的基因组中，导致双胞体病毒颗粒的合成。从本质上讲，双胞菌病毒是由一种更大的病毒携带的，因此绕过了确定自身感染的需要。杆状病毒用来表达单一的重组蛋白，如病毒蛋白，这是众所周知的，如果所表达的病毒蛋白是病毒结构的一部分，它们通常会组装成病毒颗粒。这就是正链RNA病毒的情况，例如小核糖核酸病毒和双链病毒，整个基因组翻译成一个或双个多蛋白，然后该多蛋白进行自我处理并组装成病毒颗粒。该系统允许在昆虫细胞中合成真正的双胞病毒颗粒，而不需要建立有效的组织培养系统，并提供了一种独立于从野生环境中分离病毒的能力的病毒研究方法。在这里，我们建议使用重组杆状病毒技术来生产以色列急性麻痹病毒，该病毒是与CD相关的病毒。我们将使用产生的病毒颗粒来产生一组单抗，并起诉它们来开发一种易于使用的针对IAPV感染的蜂群侧检测。此外，我们还将研究病毒株和致病性之间的关系，并定义病毒的翻译产物，这对于全面了解病毒的作用是必不可少的。还将通过定点突变和定量感染分析相结合的方法来研究特定病毒蛋白在致病性中的作用。所描述的工作将由PI与约克中央科学实验室的国家蜜蜂单位合作完成，后者将提供病毒测量和诊断方面的专业知识，以与雷丁的病毒学和重组蛋白表达专业知识相匹配。我们的工作将彻底描述这种新描述的蜜蜂病毒的特征，并有助于长期控制蜂群疾病。

Colony Collapse Disorder (CCD) is the name given to a catastrophic loss of honey bee colonies that has spread across the United States during the last 40 years and across Europe in the last 15 years. The loss of honey bees (Apis mellifera) through CCD has recently been attributed to infection by Israel acute paralysis virus (IAPV), a dicistrovirus whose spread or pathogenicity may be exacerbated by bee infestation with the Varroa mite. A focus on IAPV and the possibilities for its control is now appropriate but the virus has never been isolated in pure culture so genetic manipulation and production of the pure virus preparations is impossible, slowing progress in diagnostics and treatments. Recently it has been shown that the incorporation of the related Rhopalosiphum padi (wheat/oat aphid) dicistrovirus genome into the genome of the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) leads to the synthesis of dicistrovirus virus particles. Essentially the dicistrovirus is being carried along by a larger virus so bypassing the need to establish its own infection. The use of baculoviruses to express single recombinant proteins such as viral proteins is well established and if the virus protein expressed is part of the structure of the virus they often assemble into virus particles. This is the case for positive strand RNA viruses, such as picornaviruses and dicistroviruses, where the entire genome translates to a single or twin polyprotein which then processes itself and assembles into virus particles. The system allows the synthesis of authentic dicistrovirus virus particles in insect ells without the need to establish a productive tissue culture system and provides a method for virus study that is independent of the ability to isolate the virus from the wild. Here we propose to use the recombinant baculovirus technology to produce Israel acute paralysis virus, the virus associated with CCD. We will use the virus particles produced to generate a panel of monoclonal antibodies and sue them to develop an easy-to-use hive-side test for IAPV infection. In addition we will investigate the relationship between virus strain and pathogenicity and define the translational products of the virus, essential for a full understanding of its action. The role of particular viral proteins in pathogenicity will also be studied through site directed mutagenesis coupled to quantitative infection assays. The work described will be done by the PI in collaboration with the National Bee Unit at the Central Science Laboratory, York who will provide expertise in virus measurement and the development of diagnostics to match the virology and recombinant protein expression expertise at Reading. Our work will thoroughly characterise this newly described bee virus and benefit the long term control of CCD.