West Nile Virus: Epidemiology and Mosquito Competence

描述（由申请人提供）：西尼罗河病毒（WNV）进入北美引起了极大的关注，因为它对人类和兽医健康的持续影响。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的病毒传播和传播的潜在机制非常重要。值得注意的是，迄今为止，关于蚊子中WNV表型改变的研究非常有限。申请人的实验室最近的核酸测序研究已经描述了自WNV引入美国以来WNV的微进化。随后对2003年WNV传播季节期间在德克萨斯州收集的分离株的序列分析已经揭示了来自2003年的几种遗传上不同的分离株，其在空斑形态、Vero细胞培养物中CPE的延迟产生、和温度敏感性（ts）与2002年在德克萨斯州和1999年在纽约收集的分离物进行比较。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减少的复制，小鼠神经侵袭性研究也表明，这些分离株中的几个在神经侵袭性方面减弱。我们假设这些分离株也会减弱蚊子的感染性。随着时间的推移，阐明西尼罗河病毒的遗传和表型变化将是至关重要的，以了解北美西尼罗河病毒的进化，载体能力和流行病学及其对人类和兽医健康的影响。本申请的假设是，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物的基因组中的核苷酸/氨基酸取代赋予的。因此，本申请将对2006年至2011年分离株的基因组区域进行测序，以鉴定核苷酸/氨基酸取代。在鉴定潜在突变后，将进行基于原型WN-NY 99，382-99的基因组的感染性克隆的定点诱变，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导的氨基酸取代对于理解WNV的减毒和毒力机制是必不可少的，并且对于我们理解这种病毒将来如何继续影响人类和兽医健康将是重要的。提出了以下具体目标：1。比较2006-2011年和1999 - 2005年在北美收集的西尼罗河病毒分离物的核苷酸和推导的氨基酸序列; 2.鉴定2006-2011年传播季节期间在美国传播的WNV变体的表型特征; 3.探讨小鼠WNV减毒株神经侵袭力减弱的机制; 4.将脊椎动物细胞中的WNV表型与蚊媒感染相关联。

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.