Evolutionary Dynamics of West Nile Virus in the United States, 1999–2011: Phylogeny, Selection Pressure and Evolutionary Time-Scale Analysis

West Nile virus (WNV), an arbovirus maintained in a bird-mosquito enzootic cycle, can infect other vertebrates including humans. WNV was first reported in the US in 1999 where, to date, three genotypes belonging to WNV lineage I have been described (NY99, WN02, SW/WN03). We report here the WNV sequences obtained from two birds, one mosquito, and 29 selected human samples acquired during the US epidemics from 2006–2011 and our examination of the evolutionary dynamics in the open-reading frame of WNV isolates reported from 1999–2011. Maximum-likelihood and Bayesian methods were used to perform the phylogenetic analyses and selection pressure analyses were conducted with the HyPhy package. Phylogenetic analysis identified human WNV isolates within the main WNV genotypes that have circulated in the US. Within genotype SW/WN03, we have identified a cluster with strains derived from blood donors and birds from Idaho and North Dakota collected during 2006–2007, termed here MW/WN06. Using different codon-based and branch-site selection models, we detected a number of codons subjected to positive pressure in WNV genes. The mean nucleotide substitution rate for WNV isolates obtained from humans was calculated to be 5.06×10−4 substitutions/site/year (s/s/y). The Bayesian skyline plot shows that after a period of high genetic variability following the introduction of WNV into the US, the WNV population appears to have reached genetic stability. The establishment of WNV in the US represents a unique opportunity to understand how an arbovirus adapts and evolves in a naïve environment. We describe a novel, well-supported cluster of WNV formed by strains collected from humans and birds from Idaho and North Dakota. Adequate genetic surveillance is essential to public health since new mutants could potentially affect viral pathogenesis, decrease performance of diagnostic assays, and negatively impact the efficacy of vaccines and the development of specific therapies.西尼罗河病毒（West Nile virus, WNV）是一种维持于鸟类-蚊子循环的虫媒病毒（arbovirus），可感染包括人类在内的其他脊椎动物。1999年美国首次报告WNV感染病例，截至目前已报道属于WNV谱系I的3个基因型：NY99、WN02、SW/WN03。本研究报道了2006-2011年美国疫情期间采集的2份鸟类样本、1份蚊虫样本及29份精选人类样本的WNV序列，并对1999-2011年已报道的WNV分离株的开放阅读框（open-reading frame, ORF）进化动态展开分析。本研究采用最大似然法（Maximum-likelihood）与贝叶斯方法（Bayesian methods）开展系统发育分析（phylogenetic analyses），并借助HyPhy软件包完成选择压力分析（selection pressure analyses）。系统发育分析结果显示，人类来源的WNV分离株均分布于美国流行的主要WNV基因型分支中。在SW/WN03基因型内，本研究鉴定出一个由2006-2007年采集自爱达荷州与北达科他州的献血者及鸟类毒株构成的分支，本研究将其命名为MW/WN06。通过多种基于密码子的模型与分支位点选择模型（branch-site selection models），本研究检测到WNV多个基因中存在受正向选择压力作用的密码子。人类来源WNV分离株的平均核苷酸替换率经计算为5.06×10⁻⁴替换位点/年（substitutions/site/year, s/s/y）。贝叶斯天际线图（Bayesian skyline plot）显示，在WNV传入美国并经历一段高遗传变异期后，其种群现已达到遗传稳定状态。WNV在美国的定殖为探究虫媒病毒如何在全新环境中适应与进化提供了独特契机。本研究描述了一个由爱达荷州与北达科他州的人类及鸟类毒株构成的、得到充分支持的新型WNV分支。充分的遗传监测对公共卫生至关重要，因为新型突变株可能会影响病毒致病力、降低诊断试剂的检测效能，并对疫苗效力与特异性治疗手段的开发造成负面影响。