Bioprocess Engineering, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
J Virol Methods. 2010 Sep;168(1-2):63-71. doi: 10.1016/j.jviromet.2010.04.017. Epub 2010 Apr 28.
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.
一种用于分析哺乳动物细胞培养中流感病毒转录和复制动态的定量实时逆转录 PCR（RT-qPCR）检测方法被开发出来。该检测方法基于一种极性和序列特异性逆转录，用于特异性区分流感病毒生命周期中第 4 段（HA）、第 6 段（NA）、第 7 段（M）和第 8 段（NS）的病毒基因组（vRNA(-)）、复制中间体（cRNA(+)）和病毒信使 RNA（vmRNA(+)）。用于验证和定量的合成病毒 RNA 被制备为每种病毒 RNA 类型和片段的参考标准。检测验证表明线性度超过五个数量级，灵敏度为 1.0 x 10(3) 到 8.9 x 10(3) 的病毒 RNA 分子，重复性和再现性小于 0.8-3.1%的 CV（变异系数）。在贴壁 MDCK 细胞中分析了流感 A 病毒感染的动力学，MDCK 细胞是一种被认为可用于人类流感疫苗制造的基质。通常，在感染的早期阶段约 0.6 hpi 主要合成 vmRNA(+)，随后立即合成 cRNA(+)，在大约 1.9 hpi 后可检测到病毒基因组复制。vRNA(-)以等量和相似的动力学合成，而在早期转录阶段优先合成 NS1 vmRNA(+)，并且发现 M1 vmRNA(+)有延迟。
