SRA

Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major global epidemics and pandemics. The replication of IAV is complex, involving both transcription and replication, during which three distinct RNA species, namely mRNA, cRNA, and vRNA are generated for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics has been limited by the ability to detect all three different RNA species in a scalable manner. Here were report the development of a novel pipeline using total stranded RNA-Seq, named Influenza Virus Enumerator of RNA Transcripts (InVERT) which allows for the simultaneous quantification of all three RNA species of IAV. Using InVERT provides a full landscape of the IAV replication kinetics, in which we found that different groups of viral genes followed different traits of kinetics. During a cycle of infection, the RNA-dependent RNA Polymerase (RdRP) produced more vRNA than mRNA while some other genes (NP, NS, HA) consistently make more mRNA than vRNA. vRNA expression levels do not correlate with the cRNA expression, suggesting complex regulations of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports the model that cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA. Overall design: Time-series transcriptome profiling of IAV PR/8/34 in MDCK cells and Delta-HA and Delta-PB1 IAV in A549 cells