Key result
An optimized 11-plasmid reverse genetics system with increased amounts of NSP2 and NSP5 plasmids significantly improved the rescue efficiency of recombinant rotaviruses, enabling the generation of viruses expressing full-length fluorescent proteins.
p-value: p=<0.05
An optimized reverse genetics system enables the generation of stable recombinant rotaviruses expressing reporter genes, providing a tool for studying rotavirus virology and vaccine development.
May aid rotavirus research tools; leaves open translation to vaccines or therapies.
An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors. IMPORTANCERotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant group A rotaviruses expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.
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Komoto et al. (2018) studied Rotavirus infection (in vitro). Optimized 11-plasmid reverse genetics system (increased ratio of NSP2 and NSP5 plasmids) vs. Original 11-plasmid or 12-plasmid system was evaluated on Efficiency of recombinant rotavirus rescue (virus titer) (p=<0.05). An optimized 11-plasmid reverse genetics system with increased amounts of NSP2 and NSP5 plasmids significantly improved the rescue efficiency of recombinant rotaviruses, enabling the generation of viruses expressing full-length fluorescent proteins.
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