In Vitro Antiviral Properties of Two Recombinant Sendai Virus Vectors Encoding ORFV 011 and ORFV 059 Genes

Orf virus (ORFV) is a globally distributed zoonotic parapoxvirus that causes a highly contagious mucocutaneous disease in small ruminants. Despite the urgent demand for vaccination-based control, no licensed vaccines are currently available universally. In this study, we generated two recombinant Sendai virus (SeV) vectors expressing ORFV 011 (rSeV-GFP-B2L) and ORFV 059 (rSeV-GFP-059) genes and evaluated their ability to stimulate antiviral responses in vitro. Following the transduction, we assessed transgene expression, innate immune activation, induction of interferon-stimulated genes (A3Z1, OBST2, SAMHD1), and antiviral activity. Both vectors significantly upregulated pattern recognition receptors (TLRs, RIG-I) and type I interferon (IFN-β) genes, with rSeV-GFP-059 inducing the strongest response. Remarkably, OBST2 was robustly upregulated, suggesting a potential role in restricting ORFV replication. Antiviral activity assays revealed a marked reduction in ORFV DNA copies and a mild decrease in ORFV RNA transcription in rSeV-GFP-059-transduced cells, particularly at later time points, accompanied by complete abrogation of the typical cytopathic effect. Collectively, these results demonstrate that SeV-based vectors, particularly rSeV-GFP-059, efficiently prime antiviral immunity and suppress ORFV replication, establishing a promising platform for further in vivo vaccine evaluation in sheep.