Cloning of an orange-spotted grouper (Epinephelus coioides) Mx cDNA and characterisation of its expression in response to nodavirus

Molecular cloning and nucleotide sequencing of cDNA encoding an orange-spotted grouper (Epinephelus coioides) homolog of Mx ("OsgMx") was conducted and its possible role in fish immunity was analysed. Similar to mammalian Mx, the OsgMx are members of a family of interferon-inducible genes that are expressed by cells in response to nodavirus and iridovirus naturally-infected. Expression of OsgMx mRNA was noticeably upregulated in all tissues by nodavirus naturally-infected grouper. The transcription of OsgMx gene increased 6 h after intramuscular injection of nodavirus experimentally- infected fish and peaked at 72 h in their brains. Analysis of the 5′-flanking sequence of the gene shows that as in pufferfish and zebrafish, the OsgMx promoter contains two potential interferon-stimulated response element (ISRE) responsible for the induction of interferon-inducer polyinosinic-polycytidylic acid (Poly[I:C]). Transient transfection of grouper cells in gfp-reporter gene assays shows that the activation of the grouper Mx promoter fragment by Poly[I:C] is sufficient to allow the expression of green fluorescent protein (GFP). These results may provide a possible regulated pathway against nodavirus.