Antiviral effects and mechanism of fully phosphorothioate-modified CpG-ODN 2216 in fish and human cell lines in vitro.

Viral pathogens cause considerable economic losses to the aquaculture industry. However, effective therapeutic options are limited. CpG oligodeoxynucleotides (CpG-ODNs) are immune-stimulating agents with potential antiviral properties, but their application as therapeutics is underexplored. In this study, we aimed to determine the antiviral effects and underlying mechanisms of fully phosphorothioate-modified CpG-ODN 2216 (dODN 2216) in fish and human cell lines. Gene expression was assessed using semi-quantitative PCR and qPCR, and viral replication was measured using plaque assay and qRT-PCR. dODN 2216 induced a novel "gene lockdown" effect by rapidly suppressing the mRNA expression of both housekeeping and immune-related genes across various cell lines (KTS, EPC, and A549). This effect was specific to dODN 2216 and its analogue dODN 2243 and was not observed in other CpG-ODNs. Lockdown began within 0.5 h, lasted for 6 h and persisted for several days before gene expression gradually recovered. dODN 2216 effectively inhibited VHSV replication in EPC cells in vitro, resulting in significantly reduced PFUs and increased cell viability. In SARS-CoV-2-infected Vero-E6 cells, dODN 2216 showed an IC50 of 7 μM with relatively low cytotoxicity, suggesting broad-spectrum antiviral effects. Structural modifications of CpG-ODNs can generate antiviral mechanisms beyond TLR9-mediated immune stimulation. The gene lockdown effect of dODN 2216 represents a novel antiviral mechanism that demonstrated potent in vitro activity against both fish and human viruses. These findings suggest potential broad-spectrum antiviral applications in aquaculture and human health.