λ-Carrageenan inhibits African swine fever virus infection potentially through its interaction with the viral envelope protein CD2v.

African swine fever (ASF) is a highly pathogenic disease threatening the global swine industry and animal welfare, as there are no effective vaccines to prevent ASF virus (ASFV) infection. This study aims to identify carbohydrates that target ASFV proteins and inhibit infection, supporting alternative antiviral strategies. CD2v is an outer envelope of ASFV that mediates virulence and affects virus replication. Here, carbohydrate microarrays, SPR, and antiviral assays revealed that λ-carrageenan, but not κ- or ι-carrageenan, exhibited binding activity to CD2v derived from the HB01 strain of genotype II ASFV and has an anti-ASFV effect. Structural analysis using NMR and electrospray mass spectrometry revealed that the major difference of λ-carrageenan from its κ- and ι-analogs is the presence of the trisulfated disaccharide units, and the feature is attributed to the binding ability of λ-carrageenan on CD2v. Moreover, we found that λ-carrageenan exerts its antiviral effect through three pathways: (1) disrupting CD2v-mediated hemadsorption, a key viral immune evasion strategy; (2) inhibiting viral attachment and entry, potentially via CD2v interaction; and (3) suppressing viral factory formation. Collectively, the results revealed a possible structure-activity relationship of λ-carrageenan and identified it as a polysaccharide with inhibitory activity against the ASFV-HB01 strain.