Development of a strand specific real-time RT-qPCR assay for the detection and quantitation of murine norovirus RNA

Abstract

Murine norovirus (MNV), currently the only norovirus that efficiently replicates in cell culture, is often used as a model system to understand the molecular mechanisms of norovirus replication. MNV is a single stranded positive sense RNA virus of the Caliciviridae family. Replication of MNV involves the synthesis of both full length genomic and sub-genomic RNAs. The replication of these RNAs involves the synthesis of negative strand intermediates. To understand the molecular mechanism of RNA replication and the role of viral and host factors in virus replication, it is necessary to quantify accurately both positive and negative sense RNA molecules of the viral RNA during replication. Increasingly, strand specific reverse transcription-quantitative PCR (RT-qPCR) is becoming the method of choice for this kind of quantitation. Many strategies have been developed to avoid the false priming property of reverse transcriptase and to amplify specifically one strand in the presence of excess opposite strand. In this report, a SYBR based, real time RT-qPCR assay was developed to detect and quantify specifically the negative and the positive sense RNAs of MNV genomic RNA. This assay is based on using a tagged RT primer containing a non-viral sequence at the 5' end of the viral strand specific sequence. This non-viral sequence is then used to amplify selectively the strand specific cDNA at the PCR stage. This assay can be used for a range of MNV strains including MNV-1 and 3, as these are now widely accepted for use in molecular studies. The specificity of this assay was determined by its ability to quantify one strand in the presence of up to 10(6) copies of competitor opposite sense RNA. Using this assay, the production of both strands of MNV-1 RNA was monitored during viral single step growth curve.

Figure 1. Schematic illustration of the positions of the primers used in this study

The MNV genome is approximately 7.3 kbases, which encodes four open reading frames that are processed into two structural and seven non-structural proteins as depicted in the genome cartoon. The approximate positions of the PCR primers (Gpos and Gneg) used in this study are shown on the positive and negative strands of RNA. In order to generate the RT primer, a specific tag sequence (table 1b) was added to each respective primer at the 5′ end.

Figure 2. Standard RT-qPCR does not display strand specificity

The specificity of normal RT-qPCR was tested by generating standard curves of either positive (Panel A) or negative strand (Panel B) in presence and absence of a fixed amount of in vitro transcribed opposite polarity RNA. The experiment was performed in triplicate and mean Ct value with standard deviation were plotted against RNA absolute copy number. The correlation coefficients of the qPCRs are presented in a table below each curve.

Figure 3. Strand specific RT-qPCR of MNV genomic RNA

The standard curves were generated by quantifying in vitro transcribed RNA using the strand specific RT-qPCR as described in the text. cDNAs synthesised by reverse transcription of either positive (Panel A) or negative strand (Panel B) in presence or absence of a fixed amount of opposite strand using tagged RT primer were quantified using qPCR. The experiment was performed in triplicate and mean Ct value with standard deviation were plotted against RNA absolute copy number. The correlation coefficients of the qPCRs are presented in table below each curve.

Figure 4. Quantitation of MNV positive and negative sense genomic RNA during replication in cell culture

BV-2 cells were infected at MOI of 5 TCID50 per cell and samples for TCID50 and RNA isolation were harvested in triplicate at stated time points post infection. The virus titre was plotted over time to generate one step growth curve of MNV (A). The production of positive and negative sense genomic RNA during one step growth curve were estimated by extrapolation of standard curves generated in figure 3 and plotted against time-points post infection (B). The ratio of positive and negative strands of genomic RNA during the course of infection(C).