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Updated: Jun 8, 2025
Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins
Published on: April 17, 2020
CRISPR/Cas9 screens identify key host factors that enhance rotavirus reverse genetics efficacy and vaccine production
Yinxing Zhu1, Meagan E Sullender2, Danielle E Campbell2
1Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA.
Abstract:
Rotaviruses pose a significant threat to young children. To identify novel pro- and anti-rotavirus host factors, we performed genome-wide CRISPR/Cas9 screens using rhesus rotavirus and African green monkey cells. Genetic deletion of either SERPINB1 or TMEM236, the top two antiviral factors, in MA104 cells increased virus titers in a rotavirus strain independent manner. Using this information, we optimized the existing rotavirus reverse genetics systems by combining SERPINB1 knockout MA104 cells with a C3P3-G3 helper plasmid. We improved the recovery efficiency and rescued several low-titer rotavirus reporter and mutant strains that prove difficult to rescue otherwise. Furthermore, we demonstrate that TMEM236 knockout in Vero cells supported higher yields of two live-attenuated rotavirus vaccine strains than the parental cell line and represents a more robust vaccine-producing cell substrate. Collectively, we developed a third-generation optimized rotavirus reverse genetics system and generated gene-edited Vero cells as a new substrate for improving rotavirus vaccine production.
Insights
Researchers identified SERPINB1 and TMEM236 as key antiviral factors against rotaviruses. Enhancing cell lines with these gene knockouts improves rotavirus recovery and vaccine production efficiency.
Area of Science:
- Virology
- Genetics
Background:
- Rotaviruses are a major cause of severe diarrhea in infants globally.
- Identifying host factors influencing rotavirus replication is crucial for therapeutic and vaccine development.
Purpose of the Study:
- To discover novel host factors affecting rotavirus replication using genome-wide CRISPR screens.
- To optimize rotavirus reverse genetics systems and develop improved cell substrates for vaccine production.
Main Methods:
- Genome-wide CRISPR/Cas9 screening in African green monkey cells (MA104) with rhesus rotavirus.
- Genetic deletion of identified antiviral factors (SERPINB1, TMEM236) in MA104 and Vero cells.
- Optimization of rotavirus reverse genetics systems using SERPINB1 knockout cells.
- Evaluation of TMEM236 knockout Vero cells for live-attenuated rotavirus vaccine production.
Main Results:
- SERPINB1 and TMEM236 were identified as potent antiviral factors, with their deletion increasing rotavirus titers.
- An optimized third-generation rotavirus reverse genetics system was developed, enhancing recovery of difficult strains.
- TMEM236 knockout Vero cells demonstrated significantly higher yields for live-attenuated rotavirus vaccine strains compared to parental cells.
Conclusions:
- SERPINB1 and TMEM236 are critical host factors for controlling rotavirus infection.
- The developed reverse genetics system and gene-edited Vero cells represent significant advancements for rotavirus research and vaccine manufacturing.
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