Stage-dependent efficacy of long double-stranded RNA administered after white spot syndrome virus infection in shrimp.

White spot syndrome virus (WSSV) causes catastrophic losses in shrimp aquaculture, yet no antiviral interventions are available once infection has begun. In this study, we evaluated the stage-dependent efficacy of RNA interference (RNAi) by administering long double-stranded RNA (dsRNA) targeting the WSSV ribonucleotide reductase 2 (rr2) gene at defined time points following viral challenge in Penaeus vannamei. Delivery of dsRNA during the peri-infection period (0-12 h post-infection, hpi) resulted in near-complete protection, consistent with interference prior to extensive viral establishment. Importantly, administration during the early post-infection phase (24-48 hpi) significantly reduced mortality and viral loads, demonstrating that RNAi retains therapeutic relevance after viral replication has begun. In contrast, treatment during the late post-infection phase (72 hpi) provided only limited protection, indicating a sharp decline in efficacy once high viral burdens were established. Increasing dsRNA dosage enhanced viral suppression but did not proportionally improve survival, whereas higher viral challenge doses delayed rr2 transcript suppression and reduced therapeutic outcomes. Together, these results define a biologically constrained early post-infection therapeutic window for RNAi-based intervention against WSSV, determined by both infection stage and viral burden at the time of treatment. Although injection-based delivery limits immediate field application, this study provides a realistic framework for developing post-exposure RNAi strategies for disease management in shrimp aquaculture.