Abstract
The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (−) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (−) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR® Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region.
Highlights
The genomes of RNA viruses occur in a variety of conformations, all must be efficiently copied within the infected cell
We have shown that accurate quantification of ONNV (2) strand RNA is inhibited by the presence of relatively higher levels of cDNA generated from the competing (+) strand RNA, when standard unmodified ONNV-specific primers are used for reverse transcription and quantitative real-time PCR (qPCR) (Fig. 2B)
Regardless of mechanism, we demonstrated false priming of viral RNAs during reverse transcription of RNA extracted from ONNV infected cells (Fig. 1A)
Summary
The genomes of RNA viruses occur in a variety of conformations, all must be efficiently copied within the infected cell. Because infection of the vertebrate host is acute and often associated with disease, continual transmission depends on life-long persistent infection of the invertebrate vector host, for many alphaviruses a mosquito. It is presently unclear how persistent alphavirus infections are maintained in the vector host, after (2) strand synthesis terminates in the infected cells. Assays based on reverse transcription (RT) and PCR of cDNA derived from viral (2) strands increase sensitivity at later times after infection, but are semi-quantitative [4]. This weakness can be overcome with quantitative real-time PCR (qPCR), but the specificity of these assays for a particular strand of viral RNA is crucial to obtaining accurate and conclusive measurements
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