Identification and quantitation of multiple variants in RNA virus genomes

The goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances greater than 5 kb. As proof of concept, high quality viral RNA of the Dengue 2 component of Takeda’s tetravalent dengue vaccine candidate (TDV-2) was used to develop an RT-PCR protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long read sequencing was validated with multiple clone-derived TDV-2 revertant variants and 4 complex revertant mixtures containing various compositions of TDV-2 and revertant viruses. : PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in differentiation and quantification of complex variants of other RNA viruses. Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single genome molecule which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.