Development of an optimized RNA-based murine norovirus reverse genetics system

Area of Science:

• Virology
• Molecular Biology
• Gastroenterology

Background:

• Murine norovirus (MNV) is a key model for human noroviruses, a leading cause of acute gastroenteritis.
• Efficient replication in cell culture makes MNV invaluable for studying norovirus pathogenesis.
• Current reverse genetics methods for MNV can be complex and time-consuming.

Purpose of the Study:

• To develop a novel, rapid, and robust reverse genetics system for genetically defined MNV.
• To optimize RNA transfection and cell culture conditions for efficient MNV recovery.
• To establish a system for accurate infectivity measurements of norovirus RNA mutants.

Main Methods:

• Generation of in vitro transcribed, capped MNV RNA using the ScriptCap system.
• Optimization of Neon electroporation for delivering RNA into RAW 264.7 cells.
• Transfection of kidney cells (human or hamster) to assess virus recovery without re-infection.

Main Results:

• Rapid and robust recovery of infectious MNV was achieved via direct RNA transfection.
• High efficiency of MNV recovery was observed in permissive RAW 264.7 cells.
• Infectious virus was recovered in non-permissive kidney cells, indicating successful RNA transfection and replication.

Conclusions:

• A new, efficient reverse genetics approach for MNV has been established.
• This method facilitates the study of norovirus replication and the impact of mutations.
• The system offers a reproducible platform for quantifying norovirus RNA infectivity.