Systematic identification of permissive insertion sites for foreign gene expression in a NADC30-like PRRSV infectious clone and development of dual-fluorescent recombinant viruses.

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen threatening the global swine industry. Infectious clone and recombinant virus platforms serve as vital tools for investigating PRRSV biology and developing genetically engineered vaccines. In this study, we constructed a full-length infectious clone of the NADC30-like PRRSV strain GX-2019 and systematically evaluated the tolerance of various genomic regions to foreign gene insertion. We introduced an EGFP expression cassette into selected genomic junction sites and assessed the rescue efficiency, foreign gene stability, and replication characteristics of the recombinant viruses. Among the structural protein regions, recombinant viruses with EGFP insertions at the ORF1b/ORF2 and ORF4/ORF5a junctions were successfully rescued; among the 12 junction regions within the non-structural protein coding sequences, the NSP1β/NSP2 junction was the only site that supported stable foreign gene expression. The replication kinetics and plaque phenotypes of these viruses were comparable to those of the parental virus. Serial passage analysis demonstrated that both the NSP1β/NSP2 and ORF1b/ORF2 sites maintained good genetic stability for the inserted foreign genes. Furthermore, by utilizing the NSP1β/NSP2 and ORF1b/ORF2 sites as independent insertion points for EGFP and mCherry expression cassettes, respectively, we constructed a dual-fluorescence recombinant PRRSV capable of simultaneously and stably expressing both EGFP and mCherry. This study identifies the NSP1β/NSP2 and ORF1b/ORF2 sites as suitable and genetically stable insertion points for foreign genes within the NADC30-like PRRSV genome, providing an experimental basis for optimizing PRRSV-based foreign gene expression vectors and developing multi-gene expression platforms.