A genetically stable GFP reporter virus platform for contemporary Chinese PRRSV-1 and its use in fluorescence neutralization testing.

The rapid dissemination of phylogenetically diverse porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) strains in China necessitates advanced research tools tailored to contemporary isolates. To address the scarcity of such resources, we developed a reverse-genetics platform for the prevalent Chinese PRRSV-1 strain GXFS20220129. A full-length infectious cDNA clone was constructed and used to rescue replication-competent virus (rGXFS/F3/ic), Its growth kinetics were showed slightly attenuated compared to the parental strain but maintained a similar overall growth pattern. Utilizing this system, we generated recombinant reporter viruses expressing green fluorescent protein (GFP), driven by a transcription regulatory sequence (TRS) from either a homologous PRRSV-1 or a heterologous PRRSV-2 source, inserted at the ORF1b-ORF2 junction. The virus incorporating the heterologous PRRSV-2 TRS6 element exhibited significantly stronger GFP and viral protein expression, indicating enhanced transcriptional activity. This optimized reporter virus, designated rGXFS-GFP, demonstrated stable replication kinetics and plaque morphology similar to the parental clone and maintained genetic stability over ten serial passages. Furthermore, rGXFS-GFP was successfully applied in a fluorescence-based virus neutralization assay, providing a direct and quantitative readout for neutralizing antibodies. This study establishes a vital reverse-genetics and reporter virus system for a circulating PRRSV-1 strain, offering a robust tool to accelerate research into viral biology, vaccine efficacy, and antiviral strategies against this emerging threat.