In vitro inhibitory activity of recombinant olive flounder interferon-γ produced in Chlorella vulgaris against viral hemorrhagic septicemia virus infection.

Viral hemorrhagic septicemia virus (VHSV) is a major viral pathogen that causes substantial and persistent economic losses in olive flounder aquaculture. In this study, recombinant olive flounder interferon-γ (IFN-γ) was produced in the eukaryotic green microalga Chlorella vulgaris PKVL7422, and its in vitro antiviral activity was evaluated in epithelioma papulosum cyprini (EPC) cells. A codon-optimized olive flounder IFN-γ gene was introduced into C. vulgaris, and Western blot analysis showed that the recombinant protein was detected mainly in the culture supernatant. Cytotoxicity assays showed no detectable cytotoxicity at the concentrations tested in the antiviral assays, and cell viability remained above 85.6% even at 10,000 ng/mL. Antiviral activity was evaluated using recombinant VHSV expressing enhanced green fluorescent protein (EGFP). Plaque assay showed that recombinant IFN-γ reduced VHSV infectivity in a dose-dependent manner, with an IC50 of 15.42 ng/mL and reduced infectivity to approximately 0% at concentrations of 100 ng/mL or higher. Fluorescence microscopy and flow cytometric analysis showed that recombinant IFN-γ reduced the VHSV-positive EPC cell population from 60.22% in the infected control group to 28.47%, 9.57%, and 4.92% at concentrations of 20, 50, and 100 ng/mL, respectively. In addition, recombinant IFN-γ induced the expression of antiviral immune-related genes, including STAT1 and ISG15, in EPC cells. These findings suggest that recombinant olive flounder IFN-γ produced in C. vulgaris retains biological activity after purification and exerts a dose-dependent antiviral effect against VHSV in a fish cell model.