Development of Stable Rotavirus Reporter Expression Systems.

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Recombinant viruses expressing reporter genes are crucial for monitoring viral replication and screening antiviral drugs.
• Previous development of reporter rotaviruses (RVs) using reverse genetics faced challenges with transgene instability and expression as fusion proteins.

Purpose of the Study:

• To develop stable reporter RVs capable of expressing luciferase and fluorescent proteins.
• To improve the genetic stability of reporter genes within the RV genome.
• To utilize the improved reporter RV for screening neutralizing monoclonal antibodies (MAbs).

Main Methods:

• Insertion of reporter genes into the NSP1 open reading frame, expressed as a fusion with an NSP1 peptide.
• Modification of untranslated regions to restore RNA secondary structure for native reporter protein expression.
• Sequence analysis of escape mutants to identify viral protein changes affecting MAb neutralization.

Main Results:

• Stable reporter RV strains were generated, with shorter chimeric NSP1-reporter genes showing enhanced foreign gene stability.
• The improved reporter RV successfully screened for neutralizing MAbs, with escape mutants revealing specific VP4 protein substitutions.
• Modified 5' and 3' terminal sequences restored RNA secondary structure, enabling expression of native reporter proteins.

Conclusions:

• The developed reporter RVs are valuable tools for live monitoring of RV infections and antiviral drug screening.
• This strategy for generating stable reporter RVs can be applied to diverse transgenes, potentially creating RV transduction vectors.
• The findings enhance understanding of the role of 5'/3' terminal sequences in RV genome replication, assembly, and packaging.