Quantitative real time-PCR-assay for the analysis of gene-specific human influenza A virus transcription & replication dynamics

Abstract

For design and optimization of influenza virus production in mammalian cells details on human influenza A virus transcription and replication dynamics are essential. In addition to the characterization of cell growth and product formation in bioreactors, the use of mathematical models describing relevant aspects of virus replication is advantageous. In a previously developed, structured mathematical model for influenza A virus replication in Madin-Darby canine kidney (MDCK) cells [1] there was a considerable lack of quantitative experimental data. In particular, information regarding dynamics and control of the synthesis of each class of intracellular viral RNA, namely vRNA(-), cRNA(+) and vmRNA(+) was missing. Therefore, polarity specific primers were used in reverse transcription to quantify corresponding RNA species by real time-PCR (RRT-PCR). Here, we present results for establishment and validation of a RRT-PCR-assay for different segments of human influenza A/PR/8/34 (HA, NA, M, NS). As part of the validation protocol repeatability, reproducibility, specificity of the polarity specific primers and sensitivity of this assay was checked using dilution series of RNA reference standards. This validated assay was then used for monitoring vmRNA(+), cRNA(+) and vRNA(-) during infection of adherent MDCK cells. Determination of the intracellular RNA concentrations showed significant differences in viral transcription and replication dynamics. Based on the high quality of quantitative data obtained, validation of mathematical models describing virus-host cell interactions is facilitated significantly. [1] Sidorenko, Y. and Reichl, U. (2004): Structured model of influenza virus replication in MDCK cells, Biotechnology and Bioengineering, 88(1), 1-14