Detection and Analysis of West Nile Virus Structural Protein Genes in Animal or Bird Samples

Gili Schvartz, Sharon Karniely, Roberto Azar, Areej Kabat, Amir Steinman, Oran Erster*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

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.

Original languageAmerican English
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages127-143
Number of pages17
DOIs
StatePublished - 2023

Publication series

NameMethods in Molecular Biology
Volume2585
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Publisher Copyright:
© 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Keywords

  • Nested PCR
  • Quantitative PCR
  • Sequencing
  • West Nile virus

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