Molecular characterization and functional analysis of MyD88 in red-spotted grouper (Epinephelus akaara): Insights into immune regulation and antiviral defense.

Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein involved in the signal transduction pathways of Toll-like receptors (TLRs) and interleukin receptors, playing a pivotal role in modulating inflammatory and immune responses. In this study, we characterized MyD88 from the red-spotted grouper (Epinephelus akaara, EaMyD88) and investigated its immunological functions. The complete open reading frame of EaMyD88 is 870 bp in length, encoding a protein of 289 amino acids with a molecular weight of 33.3 kDa and an isoelectric point of 5.10. The absence of a signal peptide suggests an intracellular function. EaMyD88 contains a death and Toll/interleukin-1 receptor domains. Phylogenetic analysis revealed that the EaMyD88 has the highest sequence identity (97.6 %) with MyD88 from Epinephelus lanceolatus. Expression analysis revealed that EaMyD88 mRNA was most abundantly expressed in the blood, followed by the spleen and head kidney. Temporal expression profiles demonstrated significant upregulation following immune challenges with lipopolysaccharide, polyinosinic: polycytidylic acid, and nervous necrosis virus. The antiviral role of EaMyD88 was validated by analyzing antiviral gene expression and viral copy numbers during viral hemorrhagic septicemia virus infection in Fathead Minnow (FHM) cells. Additionally, EaMyD88 overexpression in FHM cells activated nuclear factor-kappa B and activator protein-1 signaling pathways, and induced apoptosis. Moreover, EaMyD88 enhanced pro-inflammatory cytokine expression and promoted macrophage polarization towards the M1 phenotype. Collectively, these findings underscore the critical role of EaMyD88 in the E. akaara immune regulation and antiviral defense mechanisms.