Generation of Recombinant Rotaviruses Expressing Fluorescent Proteins by Using an Optimized Reverse Genetics System

Area of Science:

• Virology
• Molecular Biology

Background:

• Rotavirus is a major cause of severe gastroenteritis in children globally.
• Existing reverse genetics systems for rotaviruses can be complex.

Purpose of the Study:

• To establish a highly efficient, plasmid-based reverse genetics system for rotavirus.
• To engineer infectious rotaviruses expressing reporter genes for enhanced study.
• To provide tools for rotavirus research and next-generation vaccine development.

Main Methods:

• Developed a plasmid-based reverse genetics system using 11 cDNA plasmids for rotavirus gene segments.
• Optimized the system by adjusting the ratio of cDNA plasmids for NSP2 and NSP5 genes.
• Engineered recombinant rotaviruses expressing NanoLuc luciferase, enhanced green fluorescent protein (EGFP), and mCherry.

Main Results:

• Successfully generated infectious recombinant rotaviruses using the improved system.
• The engineered rotaviruses demonstrated genetic stability during serial passages.
• The system efficiently incorporated bioluminescent and fluorescent reporter genes.

Conclusions:

• The developed reverse genetics system is robust, simple, and efficient for rotavirus research.
• Recombinant rotaviruses with reporter genes are valuable tools for studying viral replication.
• This approach can aid in the development of novel rotavirus vaccines and expression vectors.