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Abstract:
To better understand influenza virus replication dynamics in vaccine production using adherent mammalian MDCK cells a structured mathematical model has been developed by Sidorenko & Reichl (Structured model of influenza virus replication in MDCK cells, Biotechnology and Bioengineering, 2004, 88(1), 1-14). However, detailed modeling of gene-specific transcription and replication for each class of intracellular viral RNA was only partially possible due to a lack of experimental data. Therefore, a quantitative realtime-PCR assay was developed to follow transcription and replication of viral gene segments coding for structural or membrane proteins. Differentiation between vRNA, cRNA and vmRNA was possible due to the use of polarity specific primers.
Subsequent application of this assay for the determination of intracellular RNA concentrations in time series infection experiments with MDCK cells showed gene- and RNA-specific dynamics of transcription and replication during virus production. The comparison of the infection of MDCK cells with influenza A/PR/8/34 from two different providers (RKI, NIBSC) also showed differences in replication dynamics. After infection of MDCK cells with influenza A/PR/8/34 from RKI (MOI of 6), the cRNA of gene segment 5 (NP) and 8 (NS) was detected after 30 min, segment 4 (HA) was found after 60 min, 6 (NA) was found after 210 min and segment 7 (M) after 240 minutes. The infection of MDCK cells with influenza A/PR/8/34 from NIBSC (MOI of 6) showed a faster replication dynamic. The cRNA for all 5 gene segments was detected after 30 min. When comparing the detection time points of the different RNA classes, viral mRNA was found clearly before vRNA for all tested gene segments.
Overall, experimental data resulted in a detailed characterization of viral RNA synthesis in mammalian cells supporting validation of structured mathematical models describing virus propagation in mammalian cell cultures.
