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

蜂群衰竭失调症（CCD）是指在过去的40年里，蜜蜂群落的灾难性损失在美国蔓延，在过去的15年里传遍了欧洲。蜜蜂（Apis mellifera）通过CCD的损失最近被归因于以色列急性麻痹病毒（IAPV）的感染，这是一种双异病毒，其传播或致病性可能因蜜蜂感染瓦螨而加剧。现在重点关注IAPV及其控制的可能性是适当的，但该病毒从未在纯培养物中分离出来，因此不可能进行遗传操作和生产纯病毒制剂，从而减缓了诊断和治疗的进展。最近有研究表明，将相关的小麦/燕麦蚜虫（Rhopalosiphum padi）双侧病毒基因组整合到加利福尼亚自签名杆状病毒（AcMNPV）多核多角体病毒（AcMNPV）的基因组中，可以合成双侧病毒颗粒。从本质上讲，双链病毒是由一个更大的病毒携带的，因此不需要建立自己的感染。杆状病毒表达单一重组蛋白（如病毒蛋白）的方法已得到广泛应用，如果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。正链RNA病毒（如小核糖核酸病毒和二叉病毒）就是这种情况，它们的整个基因组转化为单个或双多蛋白，然后进行自我加工并组装成病毒颗粒。该系统允许在昆虫细胞中合成真正的双瘟病毒病毒颗粒，而无需建立高效的组织培养系统，并为病毒研究提供了一种独立于从野外分离病毒的能力的方法。本文提出利用重组杆状病毒技术制备与CCD相关的以色列急性麻痹病毒。我们将使用产生的病毒颗粒产生一组单克隆抗体，并利用它们开发一种易于使用的蜂房侧检测IAPV感染的方法。此外，我们将研究病毒株与致病性之间的关系，并定义病毒的转译产物，这对于充分了解其作用至关重要。特定病毒蛋白在致病性中的作用也将通过位点定向诱变与定量感染试验相结合来研究。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开发方面的专业知识，以匹配雷丁的病毒学和重组蛋白表达专业知识。我们的工作将彻底表征这种新描述的蜜蜂病毒，并有利于CCD的长期控制。

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.