Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• The Reoviridae family comprises nonenveloped viruses with double-stranded RNA genomes of 9-12 segments.
• Reverse genetics systems are crucial for studying viral gene function and developing interventions.
• Existing reverse genetics systems are not available for Reoviridae viruses with 12-segmented genomes.

Purpose of the Study:

• To develop a plasmid-based reverse genetics system for Tarumizu tick virus (TarTV), a Coltivirus species with a 12-segmented genome.
• To enable functional studies of TarTV and other 12-segmented Reoviridae viruses.

Main Methods:

• Development of a complete plasmid-based reverse genetics system for TarTV.
• Generation of recombinant TarTVs by transfecting baby hamster kidney cells with cloned complementary DNAs encoding the TarTV genome.
• Utilized T7 RNA polymerase for efficient viral RNA synthesis.

Main Results:

• Successfully generated recombinant TarTVs using the developed system.
• Created VP12 mutant viruses, confirming VP12 as an N-glycosylated protein.
• Developed a reporter virus expressing HiBiT-tagged VP8 protein.

Conclusions:

• The established reverse genetics system is the first for a 12-segmented Reoviridae virus.
• This system will significantly advance the understanding of Coltivirus biology.
• Facilitates deeper insights into the replication mechanisms of the Reoviridae family.