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（Colony Collapse Disorder，简称CCD）是一种蜜蜂群体的灾难性损失，在过去的40年里蔓延到美国，在过去的15年里蔓延到欧洲。蜜蜂（意大利蜜蜂）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，一种双顺反子病毒的传播或致病性可能会加剧蜜蜂感染瓦螨。现在，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，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，已经显示，将相关禾谷缢管蚜（Rhopalosiphum padi）（小麦/燕麦蚜虫）双顺反子病毒基因组掺入杆状病毒苜蓿银纹夜蛾多核多角体病毒（AcMNPV）的基因组中导致双顺反子病毒病毒颗粒的合成。基本上，双顺反子病毒是由一个更大的病毒携带沿着，因此绕过了建立自己的感染的需要。使用杆状病毒表达单一重组蛋白如病毒蛋白是公认的，并且如果表达的病毒蛋白是病毒结构的一部分，则它们通常组装成病毒颗粒。对于正链RNA病毒，例如小核糖核酸病毒和双链病毒，情况就是这样，其中整个基因组翻译成单个或双聚蛋白，然后其自身加工并组装成病毒颗粒。该系统允许在昆虫细胞中合成真实的双顺反子病毒颗粒，而不需要建立生产性组织培养系统，并提供了一种用于病毒研究的方法，该方法不依赖于从野生环境中分离病毒的能力。本研究拟利用重组杆状病毒技术生产与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来产生一组单克隆抗体，并起诉他们开发一种易于使用的IAPV感染的hive-side测试。此外，我们将研究病毒株和致病性之间的关系，并确定病毒的翻译产物，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定点诱变结合定量感染测定来研究。所述工作将由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，以匹配阅读的病毒学和重组蛋白表达专业知识。我们的工作将彻底鉴定这一新发现的蜜蜂病毒，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.