Abstract

RNA interference (RNAi) is a potent antiviral approach, outperforming traditional pesticides and broad-spectrum drugs. Its use in animal disease control faces two challenges: inefficient target design relying on computer-predicted small-interfering RNAs (siRNAs) rather than virus-derived siRNAs (vsiRNAs), and the lack of cost-effective siRNA delivery systems. In this study, we address both limitations by engineering a probiotic Bacillus subtilis 168 strain, called the recombinant  B. subtilis  AAD (Anti-AIV-DsRNA, targeted AIV), that constitutively expresses vsiRNA-enriched dsRNA targeting the H9N2 avian influenza virus. Oral administration of AAD leads to the release of double-stranded RNA (dsRNA)-loaded extracellular vesicles (EVs), which efficiently reduce H9N2 viral loads and mitigate pathological lesions. Mechanistically, virus-derived dsRNA is processed by the enzyme Dicer into siRNAs, which then activate RNAi and interferon signaling, resulting in approximately a 70% reduction in viral burden. Overall, these findings demonstrate that integrating the probiotic properties of B. subtilis with EV-mediated dsRNA delivery constitutes a sustainable, effective, and residue-free antiviral strategy for animal disease.