RIG-I detects aberrant influenza A virus transcripts

In this study we identified a novel immunostimulatory viral RNA species produced by the IAV polymerase during transcription called capped cRNA (ccRNA). This RNA contains a 5' cap of mRNA and a cRNA 3' terminus and is detected by RIG-I. We identified that the T677A mutation in the PB1 subunit of the viral polymerase enhances ccRNA synthesis and immune activation and impairs viral growth. To investigate how the mutant virus can compensate for this impairment and adapt to propagation in cell culture, we passaged the wild-type and PB1-T677A mutant A/WSN/33 viruses in A549 cells for the total of seven passages in triplicate. Viral RNA was isolated from cell culture supernatants after each passage and viral titre was determined. The PB1-T677A virus stock and passages, where a change in viral titre was observed, were subjected to NGS of all eight viral segments (passages 5 and 7 for replicate 1, passages 3 and 4 for replicate 2, and passages 4 and 5 for replicate 3). For all three replicates of the wild-type virus, only virus stock and passage 7 were sequenced. The sequencing data were analysed to determine the presence of amino acid variants.