Studying the tropism and replication kinetics of West Nile virus (WNV) in different cell types in vitro and in tissues in animal models is important for understanding its pathogenesis. As detection of the negative strand viral RNA is a more reliable indicator of active replication for single-stranded positive-sense RNA viruses, the specificity of qRT-PCR assays currently used for the detection of WNV positive and negative strand RNA was reassessed. It was shown that self-and falsely-primed cDNA was generated during the reverse transcription step in an assay employing unmodified primers and several reverse transcriptases. As a result, a qRT-PCR assay using the thermostable rTth in combination with tagged primers was developed, which greatly improved strand specificity by circumventing the events of self- and false-priming. The reliability of the assay was then addressed in vitro using BV-2 microglia cells as well as In C57/BL6 mice. It was possible to follow the kinetics of positive and negative-strand RNA synthesis both in vitro and in vivo; however, the sensitivity of the assay will need to be optimized in order to detect and quantify negative-strand RNA synthesis in the very early stages of infection. Overall, the strand-specific qRT-PCR assay developed in this study is an effective tool to quantify WNV RNA, reassess viral replication, and study tropism of WNV in the context of WNV pathogenesis. (C) 2013 Elsevier B.V. All rights reserved.