West Nile Virus Genotypic Affect on Host Competence

描述（由申请人提供）：西尼罗河病毒（WNV）已成为美国虫媒病毒性脑炎的主要原因。与西尼罗河病毒迅速出现相关的最显着的流行病学因素是鸟类体内病毒滴度高以及与传播有关的广泛蚊媒。尽管利用鸟类死亡率作为西尼罗河病毒活动的前哨，但人们对西尼罗病毒在鸟类中致病性的潜在病毒遗传基础以及媒介能力增强在西尼罗河病毒传播中的作用知之甚少。基于西尼罗河病毒鸟类宿主和昆虫媒介内复制的基础数据的生成将填补我们对西尼罗河病毒传播知识的关键空白，并有助于改进现有的监测策略和预防人类和兽医疾病的预测出现模型。单个基因改变可能影响西尼罗河病毒的传播潜力，这一事实表明，当西尼罗河病毒进入可能选择额外基因变化的新生态位时，强烈需要对西尼罗河病毒进行基因型监测和毒力表征。我们的总体假设是，入侵病毒必须进化并改变其毒力以适应新的生态位，这将通过以下三个具体目标来解决：（1）确定引入的 NY99 基因型与 1999 年以来从北美鸟类和蚊子中分离的毒株以及来自澳大利亚、肯尼亚和南非的其他 WNV 毒株之间的 WNV 适应性差异。高度敏感的病毒适应度测定将被用作使用体内测定检测标准化高、中、低易感禽类和蚊子宿主表型差异的标记。 (2) 通过将基因变化工程化到由北美引入后的早期 WNV 分离株制成的感染性 cDNA 中，来表征为宿主适应而确定的基因替代的具体作用 (3) 确定越冬期间禽类或蚊子宿主中的长期持续存在是否会导致一致的基因变化，从而改变春季终止后的病毒发生潜力。这些数据可以帮助解释为什么西尼罗河病毒流行在进入新地理区域后的第二年似乎最为广泛。从慢性感染的鸟类中分离出的病毒或从受感染的蚊子中垂直传播的病毒将被检查是否存在与避免宿主免疫系统允许持续存在相关的基因变化。这些具体目标的完成将为调节病毒出现的选择性机制提供新的见解，并具体描述与鸟类/蚊子感染性相关的病毒基因变化，这些变化促进了西尼罗河病毒在北美的跨大陆流行。

Description (provided by applicant): West Nile virus (WNV) has become the leading cause of arboviral encephalitis in the United States. The most notable epidemiological factors associated with the rapid emergence of WNV have been the development of high viral titers within birds and the wide range of mosquito vectors implicated with transmission. Despite the use of bird mortality as a sentinel for WNV activity, the underlying viral genetic basis for WNV pathogenicity in birds and the role of increased vector competence in the transmission of WNV are poorly understood. Generation of fundamental data on the basis of replication within the WNV avian reservoir and insect vector will fill critical gaps in our knowledge of WNV transmission and aid in the improvement of existing surveillance strategies and predictive emergence models for the prevention of human and veterinary disease. The fact that a single gene alteration can impact on the transmission potential of WNV indicates the strong need for genotypic monitoring and virulence characterization of WNV as it moves into new ecological niches that may select for additional genetic change. Our overall hypothesis that that an invading virus must evolve and alter its virulence to adapt to new ecological niches will be addressed by the following three specific aims: (1) Identify WNV fitness differences between the introduced NY99 genotype and strains isolated from North American birds and mosquitoes since 1999 as well as other WNV strains from Australia, Kenya and South Africa. Highly sensitive viral fitness assays will be employed as a marker for the detection of phenotypic differences in standardized high, medium and low susceptible avian and mosquito hosts using in vivo assays (2) Characterize the specific role of genetic substitutions identified for host adaptation by engineering the genetic changes into an infectious cDNA made from an early WNV isolate following North American introduction (3) Determine if long term persistence in avian or mosquito hosts during the overwintering period results in consistent genetic change that alter viremogenic potential after vernal termination. These data could help explain why WNV epidemics seem most extensive the year following introduction into new geographical areas. Virus isolated from chronically infected birds or passed vertically from infected mosquitoes will be examined for genetic changes associated with the avoidance of the host immune systems that allow for persistence. Completion of these specific aims will provide new insight into the selective mechanisms that modulate viral emergence and specifically delineate Iviral genetic changes associated with avian/mosquito infectivity that have facilitated the transcontinental epidemic of West Nile virus in North America.