Characterizing the evolution of defective-interfering RNA viruses using long-read nanopore sequencing

Defective RNAs are copies of RNA viruses that naturally arise during viral infections due to viral recombination. Despite losing critical portions of the viral genome, defective RNAs retain the functional genomic elements required for replication and encapsidation and as a result can essentially ‘parasitize’ the wild-type viral infection. Defective RNAs have important roles in viral pathology as they can attenuate viral replication, alter the outcome of viral infection, induce viral persistence, and have been proposed as anti-viral therapeutics. Due to their complex genetic structure involving recombination, large insertions, deletions and duplications, nanopore sequencing provides a unique and powerful platform to characterise defective RNAs.

Using a combination of long- and short-read Next-Generation Sequencing, we have characterized defective RNAs that arise during serial passaging of Flock House virus (FHV) in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms defective RNA formation and evolution (Jaworski et al, PLoS Path, 2017). For short-read RNAseq, we employed ‘ClickSeq’ for its ability to sensitively and confidently detect RNA recombination events with nucleotide resolution. In parallel, we used the Oxford Nanopore Technologies’s (ONT) MinION to resolve full-length defective and wild-type viral genomes. Together, these accurately resolve both rare and common RNA recombination events, determine the correlation between recombination events, and quantify the relative abundance of different defective RNAs throughout passaging.

We are extending these studies using direct RNA and cDNA nanopore sequencing to characterize defective RNAs of enteroviruses and alphaviruses. These are important human pathogens, but the role or presence of defective RNAs in their lifecycles and pathology is currently unclear. I will present our on-going work characterising the abundance and genomic-makeup of defective viral genomes of these viruses both in cell-culture and biological specimens.