White spot syndrome virus (WSSV) is a highly pathogenic agent that poses a significant constraint on the sustainable aquaculture of the red swamp crayfish (P. clarkii). Thymidylate synthase (TS) and ribonucleotide reductase (RR), two genes involved in viral DNA replication, are potential targets for RNAi-based control, but their functional validation and low-cost use remain limited. Bioinformatics analysis revealed that WSSV TS differs evolutionarily from crustacean TS but shares 64% homology with P. clarkii TS, suggesting potential virus-host substrate competition. In vitro-synthesized dsRNA-TS and dsRNA-RR both significantly suppressed WSSV replication in infected P. clarkii. TS was selected for further study due to its evolutionary profile and potential compatibility with molecular breeding approaches. The dsRNA-TS injection eliminated detectable virus within 3 days and reduced cumulative mortality by 10%. Under simulated transport stress conditions, dsRNA-TS did not enhance survival rates, likely due to immunosuppressive effects; however, it sustained the suppression of WSSV replication from 7 to 14 days post-infection. The dsRNA-TS expressed in Escherichia coli HT115 (DE3) had no significant effect, probably because of low purity, low concentration, and poor delivery. The findings provide a sustainable biological control strategy against WSSV in P. clarkii aquaculture, and lay the foundation for the optimization of prokaryotic dsRNA production systems as well as the integration of RNAi with molecular breeding techniques.
