P141 Viral genome sequencing and small RNA detection by next generation sequencing

Abstract

Introduction Next generation sequencing technologies are increasingly used in many domains. Their application to virology opens the door to various investigations such as virus sequencing and genotyping; analysis at quasispecies level; identification of causative agents of undiagnosed diseases; and finally study of host responses induced by viral infections. In this study, we used influenza- and rhinoviruses as models to validate some of these applications. Methods First we used influenza A virus infected cells to compare complete genome coverage after purification and ultra-deep sequencing of small RNAs (20–30 nt) or total fragmented RNAs (150–250 nt). Second, we sequenced small RNA population in rhinovirus (HRB-B14 and HRV-A39) infected HeLa cells and analyzed human micro RNA (miRNA) changes and the distribution of sequence reads on the viral genome. Results As expected the number of viral reads and genome coverage were much higher by sequencing total RNA, despite a lower ratio of viral versus host sequences. In contrast, small RNA purification from rhinovirus infected cells allowed us to point out to miRNA expression changes upon viral infection as well as over-expressed small viral RNAs (svRNAs). Conclusion svRNAs match the rhinovirus 5′UTR cloverleaf region, an important structural element implicated in the regulation of replication. The generation and the functional role of these svRNAs is currently under investigation.