West Nile Virus: Epidemiology and Mosquito Competence

描述（由申请人提供）：西尼罗河病毒（WNV）传入北美，由于其对人类和兽医健康的持续影响而引起了极大关注。关于西尼罗河病毒基因组中发生的遗传变化的研究对于确定脊椎动物和蚊子病毒传播和传播的潜在机制具有重要意义。值得注意的是，迄今为止，关于蚊子中西尼罗河病毒表型改变的研究非常有限。申请人实验室最近进行的核酸测序研究描述了自西尼罗河病毒传入美国以来的微进化。随后对2003年西尼罗河病毒传播季节在德克萨斯州收集的分离株进行的序列分析显示，2003年的几种遗传上存在差异的分离株在斑块形态、Vero细胞培养中CPE的延迟产生、与2002年在德克萨斯州和1999年在纽约收集的分离株相比，温度敏感性（ts）。此外，具有小斑块（sp）和/或ts表型的分离株在细胞培养中表现出复制减少，小鼠神经侵袭性研究也表明，其中一些分离株的神经侵袭性减弱。我们假设这些分离物也会被蚊子的传染性减弱。阐明西尼罗河病毒随时间的遗传和表型变化对于了解西尼罗河病毒在北美的演变、媒介能力和流行病学及其对人类和兽医健康的影响至关重要。这一应用的假设是，在脊椎动物和蚊子中观察到的病毒表型的变化是由这些分离株基因组中的核苷酸/氨基酸取代所引起的。因此，该应用程序将对2006年至2011年分离株的基因组区域进行测序，以确定核苷酸/氨基酸替换。在确定潜在突变之后，将对基于原型WN-NY99, 382-99基因组的感染性克隆进行定点诱变，以确认特定基因突变的参与。鉴定赋予病毒表型的核苷酸和推断的氨基酸取代对了解西尼罗河病毒的衰减和毒力机制至关重要，对我们了解该病毒未来将如何继续影响人类和兽医健康也很重要。提出了以下具体目标：比较2006-2011年在北美收集的西尼罗河病毒分离物与1999 - 2005年收集的分离物的核苷酸和推断氨基酸序列；2. 2006-2011年传播季节在美国流行的西尼罗河病毒变异表型特征的鉴定3. 探讨小鼠西尼罗河病毒减毒分离株神经侵袭性减弱的机制；4. 脊椎动物细胞中西尼罗河病毒表型与蚊虫媒介感染的相关性

DESCRIPTION (provided by applicant): The introduction of West Nile virus (WNV) into North America has caused great concern due to its continued impact on human and veterinary health. Studies concerning the genetic changes occurring in the genome of WNV have been important in defining potential mechanisms of viral transmission and spread for both vertebrates and mosquitoes. Significantly, to date, there have been very limited studies on alterations of the phenotype of WNV in mosquitoes. Recent nucleic acid sequencing studies by the applicant's laboratory have described the microevolution of WNV since its introduction into the U.S. Subsequent sequence analysis of isolates collected in Texas during the 2003 WNV transmission season has revealed several genetically divergent isolates from 2003 that also differed phenotypically from earlier WNV isolates in plaque morphology, delayed production of CPE in Vero cell culture, and temperature sensitivity (ts) in comparison to isolates collected in Texas in 2002 and New York in 1999. Furthermore, isolates with a small plaque (sp) and /or ts phenotype exhibited reduced replication in cell culture, and mouse neuroinvasiveness studies also indicated that several of these isolates were attenuated in neuroinvasiveness. We hypothesize that these isolates will also be attenuated for mosquito infectivity. Elucidating the genetic and phenotypic changes in WNV over time will be critical to understanding the evolution, vector competence and epidemiology of WNV in North America and its impact on human and veterinary health. It is the hypothesis of this application that changes in the observed viral phenotypes in vertebrates and mosquitoes are conferred by nucleotide/amino acid substitutions in the genomes of these isolates. Thus, this application will sequence genome regions of isolates from 2006 to 2011 to identify nucleotide/amino acid substitutions. Following the identification of potential mutations, site-directed mutagenesis of an infectious clone based on the genome of the prototype WN-NY99, 382-99, will be undertaken in order to confirm the involvement of the specific genetic mutation. Identification of the nucleotides and deduced amino acid substitutions conferring viral phenotypes is essential to understanding the mechanisms of attenuation and virulence of WNV, and will be important to our understanding of how this virus will continue to influence human and veterinary health in the future. The following specific aims are proposed: 1. Compare nucleotide and deduced amino acid sequences of WNV isolate collected in North America during 2006-2011 to those collected between 1999 and 2005; 2. Identification of phenotypic characteristics of WNV variant circulating in the U.S. during the 2006-2011 transmission seasons; 3. Investigate the mechanism of attenuation of neuroinvasiveness of mouse attenuated WNV isolates; 4. Correlate WNV phenotype in vertebrate cells with mosquito vector infection.