Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA
Journal of Virological Methods , Volume 194 - Issue 1-2 p. 146- 153
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 r. Tth 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.
|Flavivirus infection, RNA analysis, RNA synthesis, West Nile flavivirus, West Nile virus infection, accuracy, animal cell, animal experiment, animal model, article, brain tissue, controlled study, female, in vitro study, in vivo study, microglia, mouse, nonhuman, priority journal, real time polymerase chain reaction, reliability, reproducibility, reverse transcription polymerase chain reaction, sensitivity and specificity, thermostability, viral tropism, virus cell interaction, virus detection, virus load, virus replication, virus virulence|
|Journal of Virological Methods|
|This work was funded by the European Commission 7th Framework Programme; grant id fp7/261466 - Vector-borne Risks for Europe: Risk assessment and control of West Nile and Chikungunya virus (VECTORIE) (VECTORIE)|
|Organisation||Erasmus MC: University Medical Center Rotterdam|
Lim, S.M, Koraka, P, Osterhaus, A.D.M.E, & Martina, B.E.E. (2013). Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA. Journal of Virological Methods, 194(1-2), 146–153. doi:10.1016/j.jviromet.2013.07.050