Abstract: 【Objective】The purpose of the study was to screen drugs against Singapore grouper iridovirus(SGIV) in vitro and to provide theoretical basis for the prevention and control of Singapore grouper iridoviral diseases.【Method】The effective in vitro drugs against SGIV were screened using a model of grouper spleen(GS) tissue cell line-SGIV infection from four drugs amantadine hydrochloride(AMA), ganciclovir(GCV), chlorogenic acid(CGA) and baicalin(BI).The toxicity of different drugs on GS cells was determined by microscopic observation and cell viability assay. After determining the safe working concentrations of different drugs on GS cells,the regulatory effects of the drugs on SGIV infection and replication were further explored using real-time fluorescence quantitative PCR, protein blotting and other tests.【Result】The results of cell viability assay showed that the highest safe working concentrations of the four drugs AMA, GCV, CGA and BI on GS cells were 200, 200, 1600 and 40 μg/mL, respectively. Microscopic observation indicated that AMA and GCV treatment of cells had obvious inhibitory effect on the cytopathic effect(CPE) process of SGIV infection, whereas CGA and BI did not have obvious inhibitory effect. Fluorescence quantitative PCR analysis showed that AMA and GCV treatments significantly reduced the gene transcription of major capsid protein(MCP) and viral protein VP088of SGIV. Protein blotting analysis showed that AMA and GCV had obvious inhibitory effects on the MCP protein synthesis of SGIV. Inverted fluorescence microscopy observation revealed that AMA and GCV treatments greatly reduced the formation of viral processing factories during infection, suggesting that AMA and GCV might affect viral assembly. The results of viral titer assays showed that AMA and GCV treatments significantly reduced the progeny virus production during SGIV infection(P<0.05). 【Conclusion】AMA and GCV,two drugs screened by applying the SGIV in vitro infection model, have certain anti-SGIV activity, and this antiviral ability mainly affects virus assembly and production by inhibiting gene transcription and protein synthesis of the virus.