Efficacy of a Combined Natural Extract of Turmeric ( <i>Curcuma longa</i> ) and Liquorice ( <i>Glycyrrhiza uralensis</i> ) Against Viral Haemorrhagic Septicaemia Virus in Olive Flounders ( <i>Paralichthys olivaceus</i> )

Viral haemorrhagic septicaemia virus (VHSV) causes a high mortality rate in cultured olive flounder, and effective treatment for VHSV infection is urgently required. Turmeric (Curcuma longa ) and liquorice (Glycyrrhiza uralensis) are well-known medicinal plants with a variety of biological activities. Turmeric and liquorice extract combination (TLex), containing 12 g glycyrrhizin and 0.72 g curcumin per litre, exhibited low cytotoxicity (CC₅₀: 7903.08 ± 6.26 μg/mL) and potent antiviral activity (EC₅₀: 149.4 ± 2.34 μg/mL) in hirame natural embryo (HINAE) cells. Plaque reduction assays indicated that post-exposure treatment was most effective, with antiviral efficacy increasing over time. Additionally, TLex at 133.4 μg/mL upregulated antiviral genes (Mx, ISG15, IRF3 and IRF8) at 24 h. Oral administration of TLex conferred substantial protection against VHSV. In in vivo trial I, fish receiving TLex at 3.2, 108, and 207.9 μL/fish/day exhibited relative percent survival (RPS) rates of 42.9%-50% at 2 weeks and 35.7%-42.9% at 6 weeks. In in vivo trial II, the minimum effective dose was 1.0 μL/fish/day, yielding RPS rates of 23.5%-25% at 2 weeks and 30.0%-33.3% at 5 weeks, whereas lower concentrations (0.1 and 0.5 μL/fish/day) provided minimal or no protection (5%-20% RPS). Field trials with TLex (30 μL/g of feed weight) resulted in RPS rates of 25.0%-100% in VHSV-challenged fish. These findings demonstrate that TLex is an effective antiviral agent for controlling VHSV infections in olive flounder farms, enhancing immune responses and exhibiting strong in vivo antiviral activity.