title: Real-time RT-qPCR assay for the analysis of human influenza A virus transcription and replication dynamics.
abstract: A quantitative real-time reverse transcriptase PCR (RT-qPCR) assay was developed for the analysis of influenza A virus transcription and replication dynamics in mammalian cell culture. The assay is based on a polarity- and sequence-specific reverse transcription used to distinguish specifically between viral genomes (vRNA(-)), replicative intermediates (cRNA(+)) and viral messenger RNAs (vmRNA(+)) of segments 4 (HA), 6 (NA), 7 (M) and 8 (NS) during the life cycle of influenza virus. Synthetic viral RNAs used as reference standards for validation and quantitation were prepared for each viral RNA type and segment. Assay validation demonstrated linearity over five orders of magnitude, sensitivity of 1.0 x 10(3) to 8.9 x 10(3) of viral RNA molecules, repeatability and reproducibility of less than 0.8-3.1% CV (coefficient of variation). Dynamics of influenza A virus infection in adherent MDCK cells, a substrate considered for human influenza vaccine manufacturing, were analyzed. In general, mainly vmRNA(+) were synthesized during early phases of infection at about 0.6 hpi, followed immediately by cRNA(+) synthesis and after a short delay of about 1.9 hpi viral genome replication could be detected. The vRNA(-)s were synthesized in equimolar amounts and similar dynamics whereas preferential synthesis of NS1 vmRNA(+) in early transcription phases and a delay for M1 vmRNA(+) was found.
authors.affiliation: Bioprocess Engineering, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany. vester@mpi-magdeburg.mpg.de
authors.forename: Diana
authors.initials: D
authors.lastname: Vester
authors.forename: Antje
authors.initials: A
authors.lastname: Lagoda
authors.forename: Diana
authors.initials: D
authors.lastname: Hoffmann
authors.forename: Claudius
authors.initials: C
authors.lastname: Seitz
authors.forename: Stefan
authors.initials: S
authors.lastname: Heldt
authors.forename: Katja
authors.initials: K
authors.lastname: Bettenbrock
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Genzel
authors.forename: Udo
authors.initials: U
authors.lastname: Reichl
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D020869:Gene Expression Profiling; D009980:Influenza A virus / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014779:Virus Replication
chemical_list: D012367:RNA, Viral