Center for Vector-borne Diseases and Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA.
Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, CO, USA.
J Gen Virol. 2011 Nov;92(Pt 11):2523-2533. doi: 10.1099/vir.0.032318-0. Epub 2011 Jul 20.
The presence of West Nile virus (WNV) was first documented in California, USA, during the summer of 2003, and subsequently the virus has become endemic throughout the state. Sequence analysis has demonstrated that the circulating strains are representative of the North American (WN02) genotype that has displaced the East Coast genotype (NY99). A recent study has indicated that enhanced vector competence at elevated temperatures may have played a role in the displacement of the East Coast genotype by WN02. In the current study, four WN02 strains from California, including an initial 2003 isolate (COAV997), were compared to strain NY99 in growth curve assays in mosquito and duck embryonic fibroblast (DEF) cell lines at differing, biologically relevant temperatures to assess the relative temperature sensitivities of these natural isolates. COAV997 was significantly debilitated in viral replication in DEF cells at 44 °C. Full-length sequence comparison of COAV997 against the NY99 reference strain revealed non-synonymous mutations in the envelope glycoprotein (V159A), non-structural protein 1 (NS1) (K110N) and non-structural protein 4A (NS4A) (F92L), as well as two mutations in the 3' UTR: C→T at nt 10 772 and A→G at nt 10 851. These non-synonymous mutations were introduced into the NY99 viral backbone by site-directed mutagenesis. A mutant containing the NS1-K110N and NS4A-F92L mutations exhibited a debilitated growth phenotype in DEF cells at 44 °C, similar to that of COAV997. One explanation for the subsistence of this genotype is that COAV997 was obtained from an area of California where avian host species might not present elevated temperatures. These data indicate that the NS1 and NS4A mutations identified in some WN02 isolates could reduce thermal stability and impede replication of virus at temperatures observed in febrile avian hosts.
西尼罗河病毒 (WNV) 于 2003 年夏季首次在美国加利福尼亚州被记录存在，此后该病毒已在全州流行。序列分析表明，循环毒株代表北美（WN02）基因型，已取代东海岸基因型（NY99）。最近的一项研究表明，在较高温度下增强的媒介能力可能在 WN02 取代东海岸基因型方面发挥了作用。在当前的研究中，对来自加利福尼亚州的 4 株 WN02 株（包括 2003 年的初始分离株 COAV997），在不同的、具有生物学相关性的温度下，在蚊子和鸭胚胎成纤维细胞（DEF）细胞系中进行生长曲线测定，以评估这些天然分离株的相对温度敏感性。COAV997 在 DEF 细胞中的病毒复制能力在 44°C 时明显受损。COAV997 与 NY99 参考株的全长序列比较显示，包膜糖蛋白（V159A）、非结构蛋白 1（NS1）（K110N）和非结构蛋白 4A（NS4A）（F92L）中有非同义突变，以及在 3'UTR 中有两个突变：nt 10772 处的 C→T 和 nt 10851 处的 A→G。这些非同义突变是通过定点诱变引入 NY99 病毒骨架的。含有 NS1-K110N 和 NS4A-F92L 突变的突变体在 44°C 时在 DEF 细胞中的生长表型明显受损，类似于 COAV997。这种基因型持续存在的一种解释是，COAV997 是从加利福尼亚州的一个地区获得的，该地区的鸟类宿主可能不会出现高温。这些数据表明，在一些 WN02 分离株中发现的 NS1 和 NS4A 突变可能会降低热稳定性，并阻碍在发热鸟类宿主中观察到的温度下病毒的复制。
