Zinc-finger antiviral protein (ZAP) is a crucial host restriction factor that recognizes CpG dinucleotides in single-stranded RNAs, yet its role in double-stranded RNA (dsRNA) virus replication remains uncharacterized. Here, we demonstrate that ZAP broadly inhibits dsRNA viruses, including bluetongue virus (BTV) serotypes and epizootic hemorrhagic disease virus (EHDV) but not rotavirus (RV). Using BTV as a model, we reveal that ZAP inhibits replication via two mechanisms: (1) ZAP interacts with eukaryotic translation elongation factor 1A to block elongation during viral protein synthesis and (2) it binds preferentially to negative-sense RNA strands to stimulate their degradation. Additionally, BTV-NS1, encoded by segment 5, antagonizes ZAP by impairing its RNA-binding ability. Notably, synonymous CpG enrichment in BTV segment 5 significantly attenuated viral replication both in vitro and in vivo. Together, these findings uncover a dynamic interplay between ZAP and dsRNA viruses and suggest CpG-elevated BTV as a potential live-attenuated vaccine candidate.

Keywords: BTV; CP: microbiology; ZAP; bluetongue virus; dsRNA virus; elongation; negative-strand RNA; zinc-finger antiviral protein.