%0 Journal Article
%T Parallel ClickSeq and Nanopore sequencing elucidates the rapid evolution of defective-interfering RNAs in Flock House virus
%A Andrew Routh
%A Elizabeth Jaworski
%J -
%D 2017
%R 10.1371/journal.ppat.1006365
%X Defective-Interfering RNAs (DI-RNAs) have long been known to play an important role in virus replication and transmission. DI-RNAs emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous RNA recombination. However, the principles of DI-RNA emergence and their subsequent evolution have remained elusive. Using a combination of long- and short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs 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 of DI-RNA emergence and evolution. For short-read RNAseq, we employed ¡®ClickSeq¡¯ due to 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 quantifies the relative abundance of different DI-RNAs throughout passaging. We observe the formation of a diverse pool of defective RNAs at each stage of viral passaging. However, many of these ¡®intermediate¡¯ species, while present in early stages of passaging, do not accumulate. After approximately 9 days of passaging we observe the rapid accumulation of DI-RNAs with a correlated reduction in specific infectivity and with the Nanopore data find that DI-RNAs are characterized by multiple RNA recombination events. This suggests that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer ¡®mature¡¯ DI-RNAs with their selective advantage allowing for their rapid accumulation. Alternatively, it is possible that mature DI-RNA species are generated in a single event involving multiple RNA rearrangements. These insights have important consequences for our understanding of the mechanisms, determinants and limitations in the emergence and evolution of DI-RNAs
%K RNA sequencing
%K Genomic libraries
%K Viral replication
%K RNA viruses
%K Viral genomics
%K Invertebrate genomics
%K Nucleotide mapping
%K Viral evolution
%U https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006365