West Nile Virus: Epidemiology and Mosquito Competence

描述(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，引起了极大的关注。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病毒在脊椎动物和蚊子中传播和传播的潜在机制具有重要意义。值得注意的是，到目前为止，关于西尼罗河病毒在蚊子中的表型变化的研究非常有限。申请人实验室最近的核酸测序研究描述了西尼罗河病毒自引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了序列分析，发现2003年的几个遗传差异分离株在空斑形态、Vero细胞培养中CPE的延迟产生以及温度敏感性(Ts)方面也与2003年在德克萨斯州和1999年在得克萨斯州和纽约收集的分离株不同。此外，具有小斑块(Sp)和/或ts表型的菌株在细胞培养中复制减少，小鼠神经侵袭性研究也表明其中几个菌株的神经侵袭性减弱。我们推测，这些分离株也会因蚊子感染性而减弱。弄清西尼罗河病毒随时间的遗传和表型变化，对于了解北美西尼罗河病毒的进化、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。这项应用的假设是，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/氨基酸替代引起的。因此，这项应用将对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。在确定了潜在的突变之后，将对基于原型WN-NY99,382-99的基因组的感染性克隆进行定点突变，以确认特定的遗传突变的参与。识别与病毒表型相关的核苷酸和推导的氨基酸替换对于了解西尼罗河病毒的减毒和毒力机制是至关重要的，并将对我们理解该病毒未来如何继续影响人类和兽医健康具有重要意义。提出了以下具体目标：1.比较2006-2011年在北美采集的WNV分离株与1999-2005年间收集的WNV分离株的核苷酸和氨基酸序列；2.鉴定2006-2011年传播季节在美国流行的WNV变异株的表型特征；3.研究WNV减毒株神经侵袭性减弱的机制；4.脊椎动物细胞中的WNV表型与蚊媒感染的关系。

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.