West Nile Virus Infection: Novel Genetic Screens to Identify Important Host Genes

描述(申请人提供)：迫切需要更好地了解西尼罗河病毒(WNV)的地方性传播周期，特别是与了解宿主基因在确定不同种类和品系的蚊子对WNV感染的易感性以及这种变异如何影响疾病传播方面所起的作用有关。这项研究的长期目标是了解虫媒病毒与其宿主之间发生的分子相互作用如何决定感染易感性。目前，对西尼罗河病毒感染中重要的蚊子宿主基因进行系统识别的一个主要障碍是，对蚊子进行功能性遗传筛选是不切实际的。这个项目的目的是评估在黑腹果蝇中使用无偏倚的、功能的遗传筛选作为一种替代的实验方法来识别对西尼罗河病毒感染重要的宿主基因突变的实用性。这一目标是基于这样一种假设，即使用黑腹隐翅虫作为西尼罗河病毒感染的替代宿主将提供一个遗传上易于处理的模型系统，以加速发现对感染重要的宿主基因。这一假说是基于初步研究表明，黑腹隐翅虫可以感染西尼罗河病毒。成功地利用黑腹盘藻识别对西尼罗河病毒感染重要的宿主基因，将为在重要的蚊子病毒载体中识别调节感染敏感性的蚊子基因提供一个基于经验的起点。提出了一个具体的目标：识别影响西尼罗河病毒感染苍蝇能力的黑腹果蝇突变。这一目标是基于一个工作假设，即与西尼罗河病毒感染有关的黑腹葡萄球菌基因突变将导致病毒滴度容易检测到变化的感染。提出了两种遗传筛选方法，即通过将含有诱变染色体或全基因组P元件插入的病毒注入果蝇体内，通过RT-PCR定量检测西尼罗河病毒基因组RNA的产量，从而筛选EMS诱导的隐性突变或P元件诱导的错表达突变对西尼罗河病毒感染的影响。这项拟议的研究意义重大，因为它们可能提供一个原则证明，即黑腹小卷蛾可用的强大遗传方法可以用来克服西尼罗河病毒自然蚊子宿主带来的技术限制，以确定对西尼罗河病毒感染重要的宿主基因。 与公共卫生的相关性：拥有一个基因上易于处理的模型系统来研究宿主和虫媒病毒的相互作用将极大地促进识别对西尼罗河病毒感染至关重要的宿主基因，并将为阐明这些基因如何影响地方病传播周期并最终影响疾病传播提供工具。在确定这些宿主基因之前，对宿主与西尼罗河病毒相互作用的充分了解对确定感染易感性的重要性仍将是有限的，这阻碍了制定有效战略以减少西尼罗河病毒疾病出现和传播造成的健康负担的努力。

DESCRIPTION (provided by applicant): There is a critical need to better understand the enzootic transmission cycles of West Nile virus (WNV), particularly as relates to understanding the role that host genes play in determining why different species and strains of mosquito vary in their susceptibility to WNV infection and how such variation impacts disease transmission. The long term goal of this study is to understand how the molecular interactions that occur between arboviruses and their hosts determine infection susceptibility. A major current impediment to the systematic identification of mosquito host genes that are important in WNV infection is the impracticality of doing functional genetic screens in mosquitoes. The objective of this project is to evaluate the utility of using unbiased, functional genetic screens in Drosophila melanogaster as an alternative experimental approach to identifying mutations in host genes important for WNV infection. This objective is predicated on the hypothesis that using D. melanogaster as a surrogate host for WNV infection will provide a genetically tractable model system to expedite the discovery of host genes important for infection. This hypothesis is based on preliminary studies demonstrating that D. melanogaster can be infected by WNV. Successful utilization of D. melanogaster to identify host genes important for WNV infection would provide an empirically based starting point for identifying mosquito genes that modulate infection susceptibility in important mosquito vectors of the virus. One specific aim is proposed: identify mutations in D. melanogaster that affect the ability of WNV to infect flies. This aim is based on the working hypothesis that mutations in D. melanogaster genes involved in WNV infection will lead to infections that have readily detectable alterations in virus titer. Two genetic screens are proposed in which EMS-induced recessive mutations or P element-induced misexpression mutations will be screened for their effects on WNV infection by injecting virus into flies containing either mutagenized chromosomes or genome-wide P-element insertions that induce the misexpression of neighboring genes and quantifying the production of WNV genomic RNA by RT-PCR. The proposed studies are significant, because they may provide a proof-of- principle demonstration that the powerful genetic methodologies available in D. melanogaster can be used to overcome technical limitations presented by natural mosquito hosts of WNV in order to identify host genes important for WNV infection. Public Health Relevance: Having a genetically tractable model system to investigate host:arbovirus interactions would greatly facilitate the identification of host genes important for WNV infection and would provide the tools for the elucidation of how those genes impact enzootic transmission cycles and, ultimately, disease transmission. Until such host genes have been identified, a full understanding of host:WNV interactions that are important for determining infection susceptibility will remain limited, impeding efforts to develop effective strategies for reducing the health burden caused by the emergence and spread of WNV disease.