A comprehensive functional landscape of PRV replication and pathogenesis revealed by genome-wide transposon mutagenesis.

Pseudorabies virus (PRV) is a large double-stranded DNA alphaherpesvirus with a genome of approximately 145 kb containing nearly 70 genes. To systematically profile gene functions, we constructed a genome-wide mutant library by performing Tn5 transposon mutagenesis on the PRV infectious clone pBAC-JS2012. Through transposon sequencing, 277 BAC plasmids (pBAC-JS2012) containing a single-copy transposon insertion in the PRV genome were screened from 352 clones, achieving mutations in 52 viral genes. Functional characterization demonstrated that 20 genes were essential for viral replication, while 8 genes, though non-essential, significantly modulated replication efficiency in vitro. Mutations in other 22 genes showed no significant effect on viral replication. In a mouse infection model, eight genes were identified as critical determinants of viral pathogenicity. Collectively, this genome-wide analysis delineates the functional landscape of PRV, classifying genes essential for replication and those contributing to virulence. This study not only advances the understanding of PRV genome organization and function, but also provides a rational basis for developing targeted antiviral interventions and attenuated vaccines.