Construction and biological characteristics analysis of a gene-deleted recombinant virus strain of LSDV-ORF151

Abstract

Lumpy Skin Disease (LSD), caused by the Lumpy Skin Disease Virus (LSDV), is a highly virulent infectious disease that significantly impacts cattle health and economic productivity. The mechanisms underlying LSDV virulence and immune evasion remains poorly understood, and no commercial gene-deletion attenuated vaccine is currently available. This study aims to elucidate the functional role of LSDV genes and identify potential candidate strains for vaccine development. We focused on LSDV-ORF151, a gene potentially involved in immune response and apoptosis. Through amino acid sequence analysis and protein function prediction, we hypothesized that LSDV-ORF151 modulates host immune responses. RT-qPCR results showed that LSDV-ORF151 significantly reduces IFN-β transcription levels, suggesting its role in viral immune evasion. Using homologous recombination and limited dilution techniques, we constructed and purified a recombinant LSDV strain with an ORF151 deletion, using enhanced green fluorescent protein (EGFP) as a marker. PCR amplification and sequencing confirmed the stable inheritance of the rLSDV-ΔORF151-EGFP strain over at least 20 generations. Growth curve analysis revealed a slightly lower replication capacity compared to the wild-type LSDV strain (LSDV-WT), indicating that ORF151 deletion may attenuate viral replication. RT-qPCR showed that rLSDV-ΔORF151-EGFP induces higher IFN-β transcription levels than LSDV-WT after 24 h. Transcriptomic analysis indicated that the rLSDV-ΔORF151-EGFP strain induces heightened inflammatory and immune responses, and increased apoptosis in MDBK cells compared to LSDV-WT. This study provides a promising candidate for an LSDV gene-deletion attenuated vaccine and a theoretical foundation for further exploration of LSDV-ORF151's biological functions.