Development of a replication-restricted, esterase-deficient influenza D virus as a live vaccine candidate.

Influenza D virus (IDV) is an emerging bovine respiratory pathogen associated with bovine respiratory disease complex (BRDC), highlighting the need for a safe and effective live attenuated vaccine. In this study, we developed a replication-restricted IDV vaccine candidate by introducing a point mutation (S57Q) into the esterase active site of the hemagglutinin-esterase-fusion (HEF) protein, rendering the virus incapable of replication in conventional host cells. To enable efficient virus production, we established a swine testis (ST) cell line expressing wild-type HEF under a doxycycline-inducible Tet-On system, allowing controlled complementation of viral replication in trans. The rescued rD/OK-HEF-S57Q virus replicated exclusively in HEF-induced ST-Tet-On-HEF cells and exhibited no detectable replication in the respiratory tract of mice following intranasal inoculation, indicating a highly attenuated phenotype in vivo. Despite this strict replication restriction, intranasal immunization elicited robust virus-specific systemic IgG and mucosal IgA responses and conferred complete protection against challenge with wild-type IDV, with viral titers reduced below the limit of detection in both nasal turbinates and lungs. These findings highlight the potential of rD/OK-HEF-S57Q as a safe and effective live attenuated vaccine candidate for IDV.