Identification and quantitation of multiple variants in RNA virus genomes

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• Identifying distant mutations in RNA viruses is crucial for vaccine development and understanding viral evolution.
• Existing methods may struggle to characterize complex variants with mutations spread across large genomic distances.

Purpose of the Study:

• To develop and validate a long-read sequencing protocol for identifying and characterizing RNA virus variants with mutations >5 kb apart.
• To assess the utility of PacBio sequencing with unique molecular identifiers for quantifying viral variants.

Main Methods:

• Developed a reverse transcription-polymerase chain reaction protocol to amplify a ~5.3 kb Dengue 2 cDNA segment.
• Incorporated unique molecular identifiers for PacBio long-read library preparation.
• Validated the method using engineered Dengue 2 vaccine revertant variants and complex mixtures.

Main Results:

• The PacBio sequencing method accurately identified and quantified variant compositions in all tested samples.
• Successfully characterized Dengue 2 revertant variants and complex mixtures.
• Demonstrated the method's precision in quantifying variants at attenuation loci.

Conclusions:

• Long-read sequencing is effective for identifying and quantifying complex RNA virus variants with multiple mutations on a single genome.
• This method supports in-depth genetic stability assessment and revertant detection for live-attenuated viral vaccines.
• The approach is valuable for studying virus evolution, immune evasion, and host cell adaptation.