ArticleJournal of advanced research2026
Combinatorial genome engineering of pseudorabies virus Bartha by developing a reverse genetic system based on three overlapping genomic segments.
Article in Journal of advanced research, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 2 papers.
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2 citing papers in PubMed.
- Article
- Detection of circovirus and herpesvirus co-infection in suddenly dead.Veterinary research communications · 2026Article
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6 authors.
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Abstract
introductionThe 138-kilobase genome of pseudorabies virus vaccine strain Bartha K61 harbors many nonessential genes for replication and exhibits remarkable capacity for incorporating foreign genes for therapeutic applications. However, the large size of the Bartha genome complicates its efficient engineering.
objectivesDevelopment of a reverse genetic system for pseudorabies virus Bartha based on three overlapping genomic segments to facilitate multiplex genome engineering.
methodsThe 138-kb genome of Bartha was split into three overlapping segments (42 kb, 43 kb, and 53 kb), each cloned in a bacterial artificial chromosome (BAC) to facilitate genome engineering. The infectious virus was reconstituted by transfecting the 3 genomic fragments released from the BACs into Vero cells in which a complete virus genome was assembled using 2-kb overlaps between adjacent pieces.
resultsEmploying the reverse genetic system, we individually deleted 15 candidate nonessential genes and confirmed that 10 were dispensable for viral growth in cell culture. Deletion of 7 nonessential genes had no impact on viral growth, whereas UL47 deletion reduced viral growth rate and deletions of UL44, UL47, or US3 resulted in smaller viral plaques. A total of 45 viral genomes with double deletions of nonessential genes were constructed, among which 22 were successfully rescued into infectious virions. Fifteen double-deletion mutant viruses had a viral titer comparable with the wild-type Bartha, while the remaining 7 showed a lower titer. Additionally, expressions of the mNeonGreen reporter gene at nonessential gene loci were evaluated. Cells infected with recombinant viruses carrying mNeonGreen at 8 loci showed strong green fluorescence, whereas those with mNeonGreen at 2 loci exhibited very weak fluorescence.
conclusionThe reverse genetic system developed in this study enables rapid and combinatorial engineering of viruses with the large DNA genome, and will accelerate development of large DNA virus-based therapeutics including live-attenuated vaccines, vector vaccines, and oncolytic herpesviruses.
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