C7: A CpG oligodeoxynucleotide that induces protective immune response against megalocytivirus in Japanese flounder (Paralichthys olivaceus) via toll-like receptor 9-mediated signaling pathway

• Three CpG ODNs that inhibited megalocytivirus replication in flounder were selected.
• ODN C7 was able to promote leukocyte and phagocyte activation.
• Knockdown of toll-like receptor 9 expression blocked C7-mediated immune response.
• C7 exhibited adjuvant property and augmented the protection of a DNA vaccine.
• C7 enhanced the expression of vaccine-induced genes of innate and adaptive immunity.

Megalocytivirus is the causative agent of severe disease outbreaks in farmed fish. Currently there is no effective control against megalocytivirus in aquaculture. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs are known to possess marked immunostimulatory properties. In this study, we investigated the potentials of ten CpG ODNs as antiviral agents in a model of Japanese flounder (Paralichthys olivaceus). We found that, when administered into flounder, three of the ten CpG ODNs inhibited viral replication in kidney, spleen, and liver. ODN C7, which exhibited the strongest inhibitory activity, was able to promote proliferation of peripheral blood leukocytes and enhance activation of head kidney mononuclear adherent phagocytes. When the expression of toll-like receptor 9 (TLR9) was knocked down in vivo by small interfering RNA, C7-mediated immune response and antiviral activity were significantly blocked. Moreover, when C7 was co-administered with pCN86, a DNA vaccine against megalocytivirus, a significant increase in vaccine-induced protection was observed compared to administration with pCN86 alone. Further analysis showed that compared to fish immunized with pCN86, fish immunized with pCN86 plus C7 exhibited significantly upregulated expression of a wide range of genes involved in innate and adaptive immunity. Taken together, these results indicate that ODN C7 activates TLR9-mediated immune response and possesses antiviral and adjuvant potentials that may be exploited for the control of megalocytivirus infection in farmed flounder.