Quantitative characterization of defective virus emergence by deep sequencing

Area of Science:

• Virology
• Genomics
• Molecular Biology

Background:

• RNA virus populations naturally generate variants with altered genome characteristics.
• Defective interfering particles (DIPs) are non-viable viral variants that rely on coinfection for replication and can hinder virus growth.
• The population dynamics and biological activity of DIPs remain poorly understood.

Purpose of the Study:

• To demonstrate the utility of deep sequencing for characterizing the emergence, diversity, and abundance of truncated virus variants.
• To investigate the population dynamics of defective interfering particles (DIPs) in vesicular stomatitis virus (VSV).
• To explore how adaptation of deep sequencing can illuminate factors influencing virus population stability and persistence.

Main Methods:

• Culturing vesicular stomatitis virus (VSV) over three passages in BHK host cells.
• Performing Illumina deep sequencing on viral populations from each passage.
• Analyzing sequencing read counts, employing normalization and sliding-window approaches to identify truncated genome variants.
• Validating relative quantification of full-length and truncated viral RNA using quantitative reverse transcription-PCR (qRT-PCR).
• Utilizing transmission electron microscopy and infectivity assays to correlate genome size with particle characteristics and interference activity.

Main Results:

• Deep sequencing revealed distinct plateaus in read counts, indicating the emergence and enrichment of medium (approx. 5,900 nt) and short (approx. 4,000 nt) truncated viral genome variants.
• Quantitative reverse transcription-PCR (qRT-PCR) validated the relative abundance of these truncated species.
• Limit-of-detection analysis suggested deep sequencing offers superior sensitivity for detecting and quantifying defective particles compared to other methods.
• Transmission electron microscopy and infectivity assays linked the increase in smaller genomes to a rise in truncated particles and enhanced interference activity.

Conclusions:

• Deep sequencing provides a comprehensive view of viral population heterogeneity by simultaneously revealing the size, location, and relative abundance of truncated-genome variants.
• The observed variation in sequencing coverage offers a powerful tool for understanding the population dynamics of defective interfering particles (DIPs).
• This deep sequencing approach has the potential to enhance our understanding of virus population stability and persistence in natural settings.