Influenza A virus RNA synthesis produces full-length and aberrant RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that several hundred unique aberrant RNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Understanding the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for modeling the outcomes of IAV infection. We here first show that reverse transcription and PCR steps can yield imperfect aberrant RNA quantification data in a sequence-dependent manner. Next, we developed an amplification-free LbuCas13a-based detection method to quantify mvRNA amplification kinetics and subcellular distributions. We show that our assay can quantify the copy numbers of 10 specific mvRNA sequences in total RNA from cell culture, animal tissue or clinical nasopharyngeal swab extracts. In addition, we find kinetic and distribution differences between immunostimulatory and non-immunostimulatory mvRNAs, as well as mvRNAs derived from different segments, during infection. Overall, our results reveal a hitherto hidden diversity in the behavior of IAV mvRNAs and they suggest that their production is linked to replication of the individual viral segments. Cas13 is therefore a valuable new tool in our repertoire for investigating the impact of aberrant RNAs on RNA virus infection.

Cas13-based amplification-free quantification of aberrant RNAs during influenza virus infection