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

描述（由申请人提供）：迫切需要更好地了解西尼罗病毒（WNV）的地方性传播周期，特别是与理解宿主基因在确定为什么不同种类和菌株的蚊子对西尼罗病毒感染的易感性不同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。目前对系统鉴定在西尼罗河病毒感染中起重要作用的蚊子宿主基因的一个主要障碍是在蚊子中进行功能遗传筛选的不可行性。该项目的目的是评估在黑腹果蝇中使用无偏倚的功能性基因筛选的效用，作为鉴定对西尼罗河病毒感染重要的宿主基因突变的替代实验方法。这一目标是基于这样一个假设，即使用黑腹虫作为西尼罗河病毒感染的替代宿主将提供一个遗传上可处理的模型系统，以加速发现对感染重要的宿主基因。这一假设是基于初步研究表明，黑腹d.m anogaster可以被西尼罗河病毒感染。成功地利用黑腹切蝇鉴定西尼罗河病毒感染的重要宿主基因，将为鉴定该病毒重要蚊媒中调节感染易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：确定黑腹裂腹杆菌中影响西尼罗河病毒感染苍蝇能力的突变。这一目的是基于一种有效的假设，即与西尼罗河病毒感染有关的黑腹裂腹菌基因突变将导致感染，病毒滴度容易检测到变化。研究人员提出了两种基因筛选方法，通过将病毒注射到含有突变染色体或全基因组P元件插入的苍蝇中，诱导邻近基因的错误表达，并通过RT-PCR量化西尼罗河病毒基因组RNA的产生，来筛选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。拟议的研究很重要,因为他们可以提供的证据——原理证明强大的遗传方法可用在d .腹可以用来克服技术的局限性提出了西尼罗河病毒的蚊子自然宿主以识别宿主基因对西尼罗河病毒感染很重要。

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.