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The eukaryotic initiation factor 2 alpha subunit (eIF2α) is a key translation regulator that plays an important role in different cellular pressures and stimuli, including virus infection. In the present study, an eIF2α homolog (EceIF2α) from the orange-spotted grouper (Epinephelus coioides) was cloned and its roles during fish viral infection were characterized. EceIF2α encodes a putative protein of 315 amino acid residues, and shares a high degree of similarity with eIF2αs from other species. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that EceIF2α was distributed in all examined tissues. Both of the expression levels of EceIF2α in the spleen and head kidney of E. coioides were up-regulated when challenged with polyinosine-polycytidylic acid (poly[I:C]). EceIF2α was abundantly distributed in both the cytoplasm and nucleus in grouper spleen (GS) cells. Over-expression of EceIF2α improved the expression of red-spotted grouper nervous necrosis virus (RGNNV) genes in GS cells. In addition, EceIF2α depressed the activation of NK-κB and IFN-β. Furthermore, dephosphorylation inhibitor treatment led to a significant decrease of RGNNV gene transcription. Taken together, these results suggest that EceIF2α might be involved in the fish immune response to virus challenge.EceIF2α was abundantly distributed in both the cytoplasm and nucleus in cells.Overexpression of EceIF2α improved the expression of RGNNV genes in GS cells.EceIF2α depressed the activation of NK-κB and interferon-β.