Strategy for generation of replication-competent recombinant rotaviruses expressing multiple foreign genes

Area of Science:

• Virology
• Molecular Biology
• Gene Expression

Background:

• Rotavirus is a leading cause of gastroenteritis in infants.
• Reverse genetics systems enable rotavirus genome manipulation.
• Current limitations exist for inserting large foreign gene sequences into rotavirus.

Purpose of the Study:

• To assess the feasibility of inserting multiple foreign genes into a single rotavirus genome segment.
• To develop a rotavirus-based vector capable of expressing multiple exogenous genes.
• To evaluate the genetic stability of recombinant rotaviruses carrying multiple foreign genes.

Main Methods:

• Engineered a truncated NSP1 segment with a self-cleaving 2A sequence.
• Generated recombinant rotaviruses expressing NanoLuc (Nluc) luciferase.
• Constructed replication-competent rotaviruses expressing three reporter genes (Nluc, EGFP, mCherry) separated by 2A sequences.

Main Results:

• Successfully generated a recombinant rotavirus stably expressing a model foreign gene (Nluc).
• Demonstrated efficient expression of three distinct reporter genes from a single rotavirus genome segment.
• Confirmed genetic stability of the inserted multiple foreign genes through serial passages in cell culture.

Conclusions:

• Rotaviruses can be engineered to efficiently express multiple foreign genes from a single genome segment.
• The developed strategy provides a robust model for creating rotavirus-based expression vectors.
• Rotavirus vectors show significant potential for the expression and delivery of multiple foreign genes.