Realtime-PCR-assay for the quantitative analysis of human influenza A virus replication: validation of an assay for vRNA, cRNA \& mRNA polarity specific primers

Influenza A virus is a negative-strand RNA virus with a genome comprising of eight individual segments coding for 11 proteins. For a detailed understanding of intracellular replication dynamics in mammalian cells polarity specific primers for the vRNA, cRNA and mRNA are needed for representative segments, i.e. segments coding for structural or membrane proteins. By realtime-PCR each class of intracellular RNA can be quantified. To follow the time course of viral RNA synthesis and to obtain information on the control of viral RNA synthesis a thorough analysis by a validated RRT-PCR-Assay (reverse transcription realtime PCR) is essential. Here, we present the validation of a RRT-PCR-Assay for different segments of human influenza A/PR/8/34 (e.g. HA, NA, M, NS) using in vitro transcribed RNA reference standards. To differentiate between the intracellular vRNA, cRNA and mRNA, polarity specific primers were used in reverse transcription. Quantification of the intracellular RNAs was done by subsequent realtime-PCR. As part of the validation protocol repeatability of the intraassay, interassay and the whole method was analyzed. Additionally, melting curve analysis and negative controls were performed to check for specificity of the polarity specific primers. The sensitivity of this assay showed a quantification limit of 103 RNA copies. Subsequent application of this assay for the determination of intracellular RNA concentrations in time series infection experiments with MDCK cells showed dynamics of transcription and replication of the viral segments during virus production. After infection of MDCK cells with a MOI of 6, the cRNA of gene segment 8 (NS) was detected after 30 min, segment 4 and 7 (HA, M) were found after 120 min and segment 6 (NA) after 150 minutes. When comparing the detection time points of the different RNA classes, viral mRNA was found clearly before cRNA for all tested gene segments. Overall, experimental data allowed detailed characterization of viral RNA synthesis in mammalian cells. Quantitative data obtained supports validation of a structured mathematical model of influenza virus replication in single cells (Sidorenko, Y. and Reichl, U. (2004): Structured model of influenza virus replication in MDCK cells, Biotechnology and Bioengineering, 88(1), 1-14)