Kimron Veterinary Institute, Israel Ministry of Agriculture, Bet Dagan, Israel.
Koret School of Veterinary Medicine, The Robert H. Smith, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel.
Methods Mol Biol. 2023;2585:127-143. doi: 10.1007/978-1-0716-2760-0_13.
West Nile virus (WNV) is an important zoonotic pathogen, which is detected mainly by identification of its RNA using PCR. Genetic differentiation between WNV lineages is usually performed by complete genome sequencing, which is not available in many research and diagnostic laboratories. In this chapter, we describe a protocol for detection and analysis of WNV samples by sequencing the entire region of their structural genes capsid (C), preM/membrane, and envelope. The primary step is the detection of WNV RNA by quantitative PCR of the NS2A gene or the C gene regions. Next, the entire region containing the structural protein genes is amplified by PCR. The primary PCR product is then amplified again in parallel reactions, and these secondary PCR products are sequenced. Finally, bioinformatic analysis enables detection of mutations and classification of the samples of interest. This protocol is designed to be used by any laboratory equipped for endpoint and quantitative PCR. The sequencing can be performed either in-house or outsourced to a third-party service provider. This protocol may therefore be useful for rapid and affordable classification of WNV samples, obviating the need for complete genome sequencing.
西尼罗河病毒（WNV）是一种重要的人畜共患病原体，主要通过使用 PCR 鉴定其 RNA 来检测。WNV 谱系之间的遗传分化通常通过完整基因组测序来进行，但许多研究和诊断实验室无法进行这种测序。在本章中，我们描述了一种通过对结构基因衣壳（C）、前 M/膜和包膜的整个区域进行测序来检测和分析 WNV 样本的方案。主要步骤是通过 NS2A 基因或 C 基因区域的定量 PCR 检测 WNV RNA。接下来，通过 PCR 扩增包含结构蛋白基因的整个区域。然后，在平行反应中再次扩增初级 PCR 产物，并对这些二级 PCR 产物进行测序。最后，生物信息学分析可检测突变并对感兴趣的样本进行分类。本方案旨在供任何配备终点和定量 PCR 的实验室使用。测序可以在内部进行，也可以外包给第三方服务提供商。因此，该方案可能有助于快速且经济地对 WNV 样本进行分类，避免了进行完整基因组测序的需要。
