Fluorescent protein-expressing Modified Vaccinia Ankara encoding T7 RNA polymerase.

Efficient recovery of negative-stranded RNA viruses from plasmid cDNA requires robust bacteriophage T7 RNA polymerase expression, commonly supplied by Modified Vaccinia Ankara expressing T7 polymerase (MVA T7). Here, we describe the generation and characterization of two fluorescent MVA T7 viruses expressing either enhanced green fluorescent protein (EGFP) or monomeric red fluorescent protein 1 (mRFP1). Both fluorescent protein-expressing MVA T7 viruses retain plaque morphology, growth kinetics, and transgene expression comparable to parental MVA T7. Importantly, both fluorescent protein-expressing MVA T7 viruses support transient protein expression of foreign genes and rescue of replication-competent recombinant vesicular stomatitis virus (rVSV), while enabling direct visualization of MVA T7 infection. Fluorescent protein expression from MVA T7 viruses facilitates stock generation and titration and allows detection of the remaining helper virus during rVSV recovery and amplification. Altogether, EGFP- and mRFP1-expressing MVA T7 viruses provide a practical tool for monitoring T7-driven rescue of recombinant RNA viruses and identifying helper virus carryover. IMPORTANCE: Reverse genetics systems for negative-stranded RNA viruses rely on robust bacteriophage T7 RNA polymerase expression, commonly provided by a Modified Vaccinia Ankara virus expressing T7 polymerase (MVA T7). However, generation of virus stocks, monitoring MVA T7 helper virus infection, and detecting residual virus during rescue of negative-stranded RNA virus can be challenging. We generated fluorescent protein-expressing MVA T7 viruses that enable direct visualization of helper virus infection without compromising viral growth kinetics, transgene expression, or efficient rescue of recombinant vesicular stomatitis virus (rVSV), while enabling direct visualization of MVA T7 infection. MVA T7 viruses expressing fluorescent proteins simplify stock generation and titration, facilitate optimization of negative-stranded RNA rescue conditions, and allow rapid identification of helper virus carryover during generation and amplification of recombinant virus, improving the efficiency, reproducibility, and quality control in reverse genetics systems relying on T7 expression, widely used across virology.