Fluorescent protein-expressing Modified Vaccinia Ankara encoding T7 RNA polymerase

Area of Science:

• Virology
• Molecular Biology
• Gene Expression Systems

Background:

• Efficient recovery of negative-stranded RNA viruses relies on bacteriophage T7 RNA polymerase, often supplied by Modified Vaccinia Ankara expressing T7 polymerase (MVA T7).
• Challenges exist in generating virus stocks, monitoring MVA T7 infection, and detecting residual helper virus during recombinant virus rescue.

Purpose of the Study:

• To generate and characterize fluorescent protein-expressing MVA T7 viruses (EGFP and mRFP1) for improved visualization and management in reverse genetics.
• To assess if fluorescent MVA T7 viruses maintain growth kinetics and transgene expression comparable to parental MVA T7.
• To evaluate the utility of fluorescent MVA T7 viruses in facilitating recombinant virus rescue and detecting helper virus carryover.

Main Methods:

• Generation of MVA T7 viruses expressing enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein 1 (mRFP1).
• Characterization of fluorescent MVA T7 viruses for plaque morphology, growth kinetics, and transgene expression.
• Assessment of transient protein expression and rescue of recombinant vesicular stomatitis virus (rVSV) using fluorescent MVA T7 viruses.
• Evaluation of helper virus detection during rVSV recovery and amplification.

Main Results:

• Fluorescent MVA T7 viruses (EGFP and mRFP1) exhibited comparable plaque morphology, growth kinetics, and transgene expression to parental MVA T7.
• Fluorescent MVA T7 viruses efficiently supported foreign gene expression and rVSV rescue.
• Direct visualization of MVA T7 infection was enabled by fluorescent proteins.
• Fluorescent MVA T7 viruses facilitated stock generation, titration, and detection of helper virus carryover.

Conclusions:

• EGFP- and mRFP1-expressing MVA T7 viruses are practical tools for monitoring T7-driven rescue of recombinant RNA viruses.
• These fluorescent viruses improve the efficiency, reproducibility, and quality control of reverse genetics systems.
• They simplify stock management and enable rapid identification of helper virus contamination, enhancing viral rescue and amplification processes.