In Vitro and In Vivo Toxicity and Antiviral Activity of Rhein Derivatives Against Nervous Necrosis Virus

ABSTRACT Rhein has been demonstrated to exhibit antiviral activity against nervous necrosis virus (NNV). As rhein coexists in plants with various structurally analogous natural derivatives and may acquire improved biochemical properties through chemical modification, this study investigated the potential of several natural and synthetic derivatives for anti‐NNV applications. The results revealed that multiple derivatives inhibited NNV replication in infected SSN‐1 cell lines more effectively than rhein. Notably, derivative 8 exhibited an IC 50 of 15.2 µM, whereas that of rhein exceeded 25 µM. However, in vivo experiments indicated high susceptibility of zebrafish larvae and grouper juveniles to anthraquinones, as rhubarb extract and almost all their derivatives caused nearly 100% mortality at 25 µM. Therefore, the direct use of plant extracts containing anthraquinones is not advised for juvenile fish treatment in aquaculture. In addition, an infection model using infectious tissue homogenate was established to simulate natural transmission. Immersion treatment with 10 µM rhein, a novel administration approach, effectively reduced viral load by 76.56% and improved the survival rate of grouper juveniles from 0% to 41.8%. These findings support the potential of rhein for NNV control in aquaculture practices. Although chemical modification represents a promising strategy for enhancing anti‐NNV efficacy, further structural optimization is necessary to reduce toxicity.