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Nervous necrosis virus (NNV) results in high mortality rates of infected marine fish worldwide. Interferons (IFNs) are cytokines in vertebrates that suppress viral replication and regulate immune responses. Heterologous overexpression of fish IFN in bacteria could be problematic because of protein solubility and loss of function due to protein misfolding. In this study, a protein model of the IFN-&#945; of <i>Epinephelus septemfasciatus</i> was built based on comparative modeling. In addition, PelB and SacB signal peptides were fused to the N-terminus of <i>E. septemfasciatus</i> IFN-&#945; for overexpression of soluble, secreted IFN in <i>Escherichia coli</i> (E-IFN) and <i>Bacillus subtilis</i> (B-IFN). Cytotoxicity tests indicated that neither recombinant grouper IFN-&#945; were cytotoxic to a grouper head kidney cell line (GK). The GK cells stimulated with E-IFN and B-IFN exhibited elevated expression of antiviral Mx genes when compared with the control group. The NNV challenge experiments demonstrated that GK cells pretreated or co-treated with E-IFN and B-IFN individually had three times the cell survival rates of untreated cells, indicating the cytoprotective ability of our recombinant IFNs. These data provide a protocol for the production of soluble, secreted, and functional grouper IFN of high purity, which may be applied to aquaculture fisheries for antiviral infection.