Genomic assembly, rescue, and characterization of a functional pseudorabies virus.

With recent advances in synthetic biology methods, the genomes of several large DNA viruses have been de novo synthesized and assembled, leading to the functional rescue of the respective viruses. Pseudorabies virus (PRV), a large DNA virus belonging to the family Herpesviridae, causes severe diseases in swine, resulting in significant economic losses to the global pig farming industry. Genome editing is crucial for attenuating virulence and developing safer vaccines for PRV. However, its complex repetitive sequences and extremely high GC-rich genome pose significant challenges for genetic manipulation. In this study, we developed a PRV genome assembly platform using yeast-based transformation-associated recombination (TAR) technology. The genome of a prevalent genotype II variant strain, PRV-GX-2011 (GenBank number PV405324.1), was divided into nine A-level fragments and cloned into vectors via TAR. Subsequently, three B-level fragments were generated by recombining three A-level fragments each. In vitro CRISPR/Cas9-mediated editing was introduced to insert an egfp gene into the non-coding intergenic region between UL23 and UL22 genes. Infectious viruses were rescued by co-transfection of linearized B-level fragments in Vero cells, and an isolated virus, PRV-GX-Syn1, was purified via plaque assay. While PRV-GX-Syn1 exhibited reduced viral titer and smaller plaque size compared to the parental strain, its morphological characteristics remained indistinguishable from the parental virus. In BALB/c mice, PRV-GX-Syn1 caused lethal infection, producing lung pathology comparable to the parental strain. This TAR-based platform offers faster and more flexible genomic modification of PRV, facilitating both basic research and PRV-based vaccine vectors.