Synthetic within-cell flavivirus sensors

这项拟议研究的目的是开发合成生物传感器，这种传感器可以驻留在活细胞中，基本上是惰性的，直到细胞被病毒感染，然后激活并做出反应。在这项提议中，我们重点关注黄病毒，这是一组主要的病毒，包括蚊子传播的病毒登革热、寨卡病毒和黄热病病毒，以及一些通过扁虱传播的病毒。当病毒感染细胞时，它们使用细胞自身的大量机制进行复制，但它们也有自己的蛋白质，编码在自己的小基因组中，而未感染的细胞没有。这些都为特定的检测提供了基础--除非细胞受到感染，否则细胞不会有这些蛋白质和酶。与病毒序列不同的是，病毒的序列对单个病毒类型，甚至是单一病毒的毒株都是非常特异的，而同一一般组中的不同病毒--例如黄病毒或蚊媒黄病毒--之间的这些酶活性非常相似，因此我们希望开发出对一系列病毒中的任何一种都有反应的传感器，可能是对所有蚊媒传播的黄病毒，而不仅仅是对寨卡病毒或一种寨卡病毒。这样的系统有几个潜在的用途。一个是诊断学。基于序列的检测系统，如聚合酶链式反应，需要一些病毒序列的先验知识，并且对该序列中的变异非常敏感(即可能无法检测到变异)。存在更全面的方法，如完全测序，但目前这些方法相对较慢且成本较高。另一种标准方法是将假定的病毒样本应用于培养细胞，并寻找对这些细胞的破坏(细胞病变效应，CPE)，但这有点慢，并不是所有的病毒都会引起明显的病理。我们已经开发了细胞内系统的原型，这些系统似乎具有优越的特性，至少在某些目的上是这样。我们将与项目合作伙伴一起开发这些方法并对其进行详细描述，以提供一套补充现有方法的工具。我们将在蚊子身上使用相同的方法，以培育出对寨卡病毒和其他黄病毒感染有特定反应的蚊子。我们在这里的主要目标是做出反应，降低蚊子传播病毒的能力。与人类不同，蚊子不会受到这些病毒的严重影响，事实上，病毒不能对蚊子造成太大伤害是很重要的，因为它依赖蚊子(总是她：只有雌性蚊子叮咬)才能传播。如果我们能够安排受感染的蚊子对感染异常敏感，例如被病毒杀死，这将大大减少传播。这样的系统还需要传播到野外的目标蚊子种群中。这不是该项目的一部分，该项目完全基于实验室，但如果该项目成功，可能会成为未来的发展。

The aim of the proposed study is to develop synthetic-biology sensors that can sit in living cells, essentially inert until the cell is infected by a virus, and then activate and respond. For this proposal, we focus on flaviviruses, a major group of viruses that includes mosquito-transmitted viruses dengue, Zika and yellow fever viruses, as well as some viruses transmitted by ticks, for example.When viruses infect a cell, they use a lot of the cell's own machinery for their replication, but they also have some proteins of their own, encoded in their own small genomes, that an uninfected cell does not have. These provide the basis for specific detection - the cell does not have these proteins and enzymes unless it is infected. Unlike the virus' sequence, which is very specific to individual virus types, or even strains of a single virus, these enzymatic activities are quite similar between different viruses of the same general group - e.g. flaviviruses, or mosquito-transmitted flaviviruses, so we expect to develop sensors that will respond to any of a range of viruses, perhaps to all mosquito-transmitted flaviviruses, rather than to just, for example Zika virus, or one strain of Zika virus.Such systems have several potential uses. One is in diagnostics. Sequence-based detection systems, such as PCR, need some prior knowledge of the virus sequence, and are very sensitive to variations in this sequence (i.e. may fail to detect a variant). More comprehensive approaches exist, such as complete sequencing, but these are currently relatively slow and expensive. Another standard approach involves applying the putative virus sample to cultured cells and looking for disruption of those cells (cytopathic effect, CPE), but this is a little slow, and not all viruses cause obvious pathology. We have developed prototype within-cell systems that appear to have superior characteristics, at least for some purposes. We will develop these and characterise them in detail, with project partners, to provide a set of tools complementary to current approaches.We will use the same approach in mosquitoes, to develop mosquitoes that respond in specific ways to infection by Zika virus and other flaviviruses. Our main aim here is for that response to reduce the ability of the mosquito to transmit the virus. Unlike humans, mosquitoes are not severely affected by these viruses, indeed it is important for the virus not to harm the mosquito much since it depends on her (always "her": only female mosquitoes bite) for transmission. If we can arrange that infected mosquitoes are unusually sensitive to infection, e.g. are killed by the virus, this will greatly reduce transmission. Such systems would additionally need to be spread into a target mosquito population in the wild. That is not part of this project, which is entirely lab-based, but potentially a future development if this project is successful.