Sulfated polysaccharide lambda carrageenan (lambda-CGN) was evaluated for its antiviral effect against IPNV using the in vitro infection model of CHSE-214 salmonid cells. A plaque reduction lysis assay revealed that lambda-CGN has an IC(50 )of 0.9 mu g center dot mL(- 1), CC50 >128 mu g center dot mL(- 1) and a Selectivity Index (SI) > 142. In comparison, iota, kappa carrageenans and lambda oligo-carrageenan (2-OC) were less effective than lambda-CGN against IPNV. 2-CGN showed no virucidal activity when applied directly to viral particles. Time of addition experiments showed that pretreatment, co-treatment, and post-treatment with lambda-CGN significantly reduced viral RNA copies in the cell supernatant. Additionally, a decrease in intracellular viral RNA was observed with pre-treatment and posttreatment, indicating an impact on different stages of viral replication. Polyacrylamide gel electrophoresis of IPNV genomic RNA in presence of lambda-CGN showed a reduction in the level of viral genomic RNA. Confocal microscopy confirmed the intracellular localization of lambda-CGN, suggesting that lambda-CGN may inhibit the synthesis of IPNV genomic RNA. Moreover, cells pre-treated with lambda-CGN showed an increased expression of innate immunity genes CXCL11, IL1 beta, IFNa, and IRF3. These findings highlight the need for further research to confirm the in vivo pharmacological potential of lambda-CGN as a new antiviral agent in salmonids.