Orf virus early genes ORFV113 and ORFV114 are individually dispensable for replication in vitro but required for full virulence in vivo

Orf is a highly contagious zoonotic disease caused by Orf virus (ORFV), resulting in significant economic losses to the livestock industry. ORFV113 and ORFV114 , located in the terminal variable region of the ORFV genome, are putative virulence-associated genes. To elucidate their biological functions, we characterized the transcription kinetics of both genes and generated single-gene deletion mutants by homologous recombination, using transfer plasmids carrying flanking homology arms and an enhanced green fluorescent protein (EGFP) reporter cassette. Lamb testis (LT) cells transfected with the recombinant plasmids were infected with the parental ORFV-JS strain; recombinant viruses were enriched by limiting dilution, purified by consecutive rounds of plaque isolation, and verified by polymerase chain reaction (PCR). Both genes are highly conserved among ORFV isolates (92–100% nucleotide identity) and are transcribed with early kinetics. The genetically stable deletion mutants, designated ORFV-Δ113 and ORFV-Δ114, were successfully generated. Deletion of either gene did not significantly alter viral growth kinetics or cytopathic effects (CPE) in LT cells. In a rabbit skin scarification model ( n = 4 per group), however, both mutants induced markedly milder macroscopic lesions with lower cumulative clinical scores ( P < 0.05), and viral loads in scab tissue were reduced 17-fold (ORFV-Δ113) and 10-fold (ORFV-Δ114) relative to the wild-type strain ( P < 0.01). Thus, ORFV113 and ORFV114 are dispensable for viral replication in vitro but are required for full virulence in vivo. The resulting deletion mutants provide valuable tools for dissecting virus–host interactions and are promising candidates for the rational design of live-attenuated ORFV vaccines.