Abstract
The ability to manipulate the genomes of herpesviruses is of eminent importance for obtaining insight into gene function and regulation of gene expression of these complex viruses. Here we report the use of in vivo overlap recombination to generate pseudorabies virus mutants. Cotransfection of up to five overlapping cloned subgenomic fragments, which together constitute the entire genomic information of pseudorabies virus, results in the efficient reconstitution of virus. This allows the efficient introduction of multiple well-defined mutations in herpesvirus genomes in a single step, without any selection or screening for a particular phenotype.