Antiviral effects and multi-omics profiling of a Scutellaria baicalensis , Flos Sophorae Immaturus , and Prunella vulgaris herbal compounds against white spot syndrome virus (WSSV) in crayfish

White spot syndrome virus (WSSV) is a highly virulent pathogen associated with mass mortality in crustaceans, for which no reliable measures are currently available to control WSSV infection. Traditional Chinese medicinal herbs, such as Scutellaria baicalensis (SB), Flos Sophorae Immaturus (FSI), and Prunella vulgaris (PV), have demonstrated potential antiviral characteristics. In this research, crayfish ( Procambarus clarkii ) were adopted as a model animal to investigate the anti-WSSV efficacy of a compound herbal formulation composed of these three herbs. Moreover, the effects of WSSV infection and the compound treatment on the intestinal microbiota composition and metabolic profile of crayfish were investigated. Quantitative PCR (qPCR) analysis revealed that the herbal compound formulation reduced the viral load in the hepatopancreas, intestine, and gills of WSSV-infected crayfish. Histopathological examinations showed that WSSV infection caused severe inflammation and tissue damage in these organs, which were significantly alleviated by herbal treatment. RT-qPCR analysis further revealed that the compound upregulated the expression of innate immunity-associated genes in affected tissues. 16S rRNA sequencing revealed that WSSV infection led to severe gut dysbiosis, characterized by a significant increase in Proteobacteria . This dysbiosis was substantially improved following treatment with the compound. Moreover, untargeted metabolomics analysis revealed distinct alterations in gut metabolites resulting from both WSSV infection and herbal intervention. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis identified 20 differentially expressed metabolic pathways between the challenge (CHG) and treatment (TRE) groups. Beneficial pathways, including glycerophospholipid metabolism, pyrimidine metabolism, and CoA biosynthesis, were significantly upregulated in the TRE group. These findings collectively demonstrate that SB-FSI-PV compound effectively mitigates WSSV infection by reducing viral load, modulating immune responses, restoring gut microbial balance, and improving host metabolic function. Overall, this formulation represents a promising therapeutic approach for controlling and preventing WSSV outbreaks in aquaculture systems.