Journal of Virological Methods 194 (2013) 146–153 Contents lists available at ScienceDirect Journal of Virological Methods jou rn al hom epage: www.elsevier.com/locate/jviromet Development of a strand-specific real-time qRT-PCR for the accurate detection and quantitation of West Nile virus RNA Stephanie M. Lim, Penelope Koraka, Albert D.M.E. Osterhaus, Byron E.E. Martina ∗ Department of Viroscience, Erasmus Medical Center, Rotterdam, The Netherlands Article history: Received 8 January 2013 Received in revised form 17 July 2013 Accepted 22 July 2013 Available online xxx Keywords: West Nile virus Strand-specific qRT-PCR Tagged qRT-PCR Virus replication kinetics a b s t r a c t 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. © 2013 Elsevier B.V. All rights reserved. 1. Introduction West Nile virus (WNV) is a neurotropic RNA virus with a positive-sense single-stranded genome that belongs to the genus Flavivirus in the Flaviviridae family. The replication of WNV pro- ceeds through a negative strand RNA intermediate (Westaway, 1987), synthesized by the virus-encoded RNA-dependent RNA polymerase. The negative strand is used as a template for the synthesis of new single stranded positive-sense RNA molecules (Cleaves et al., 1981). Therefore, detection of this negative strand, which is generally significantly outnumbered by the positive strand, signifies viral replication. In vitro experiments have shown that cortical astrocytes are susceptible to infection with WNV while replication in microglia cells was not supported (Cheeran et al., 2005). In vivo, WNV infects mostly neuronal cells and on occasion has also been shown to infect microglia cells (Daffis et al., 2008). In general, however, in vivo tropism of WNV for astrocytes and microglia cells remains mostly speculative. In order to determine permissibility of these cells to infection by WNV, synthesis of the negative strand can be assessed ∗ Corresponding author at: Department of Viroscience, Erasmus Medical Centre, Postbus 2040, 3000 CA Rotterdam, The Netherlands. Tel.: +31 10 7044279; fax: +31 10 7044760. E-mail address: b.martina@erasmusmc.nl (B.E.E. Martina). using strand-specific quantitative RT-PCR. Another issue that could be addressed by determining the presence of negative strand is persistence of WNV, which has been described in several organs and animal species (Appler et al., 2010; Murray et al., 2010; Penn et al., 2006; Wheeler et al., 2012; Wu et al., 2008). Specific strand detection can therefore be useful in the elucidation of the mech- anisms of persistent infection and may contribute to the further understanding of WNV pathogenesis. Even though qRT-PCR is the method used most widely for the quantitation of viral RNA, its major disadvantage is that it provides limited strand specificity. As a result, standard qRT-PCR cannot determine the absolute quantity of viral RNA copies in a given sample due to the presence of both positive and negative strands of RNA. This lack of strand specificity has been attributed to a combination of factors, including self-priming of the RNA due to secondary hairpin structures (Haddad et al., 2006; Lanford et al., 1994; Lerat et al., 1996; Stahlberg et al., 2004; Timofeeva and Skrypina, 2001; Tuiskunen et al., 2010), false priming of the incor- rect strand (Gunji et al., 1994; Lanford et al., 1994; Lin et al., 2002; Sangar and Carroll, 1998) and random priming by contaminating endogenous or exogenous nucleic acids (Gunji et al., 1994; Piche and Schernthaner, 2003; Timofeeva and Skrypina, 2001). Attempts to overcome these problems include performing RT reactions at high temperatures (Haddad et al., 2007; Komurian-Pradel et al., 2004), use of the thermostable RTth enzyme (de Paula et al., 2009; Lanford et al., 1994; Radkowski et al., 2002; Selva et al., 2004), use 0166-0934/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2013.07.050