β-defensin 2 enhances antiviral immunity via autophagy regulation during Micropterus salmoides rhabdovirus (MSRV) infection and promotes survival in largemouth bass.

β-defensins (BDs) are a class of cysteine-rich cationic host defense peptides (HDPs) with broad-spectrum antimicrobial properties. This study identified and functionally characterized β-defensin 2 from largemouth bass (Micropterus salmoides), termed as MsBD-2, as a potent novel antiviral agent. Sequence analysis revealed that MsBD-2 shares 96.8 % amino acid identity with its orthologs in barred knifejaw (Oplegnathus fasciatus) and mandarin fish (Siniperca chuatsi), clustering phylogenetically with teleost BD2 isoforms and showing closest evolutionary relationship to barred knifejaw BD. Structurally, MsBD-2 consists of a 20-amino acid signal peptide and a 43-amino acid mature peptide featuring six conserved cysteine residues forming three disulfide bridges (C1-C5, C2-C4, and C3-C6). Tissue distribution analysis revealed that MsBD-2 was constitutively expressed most highly in the liver of largemouth bass, with significant upregulation observed in the liver, spleen, gill, and muscle following M. salmoides rhabdovirus (MSRV) infection. To functionally validate its antiviral role, recombinant MsBD-2 (rMsBD-2) was produced via prokaryotic expression. In vitro and in vivo assays demonstrated that rMsBD-2 exhibits potent antiviral activity against MSRV. Mechanistically, the antiviral mechanism of action of rMsBD-2 was mediated through the enhancement of autophagy via the AMPK/ULK1 signaling pathway. These findings provide the first evidence that fish BD combats viral infection through autophagy modulation, offering new insights into the functional diversification of teleost defensins.