Viral hemorrhagic septicemia virus (VHSV) is a major viral pathogen that causes substantial and persistent economic losses in olive flounder aquaculture. In this study, recombinant olive flounder interferon-γ (IFN-γ) was produced in the eukaryotic green microalga Chlorella vulgaris PKVL7422, and its in vitro antiviral activity was evaluated in epithelioma papulosum cyprini (EPC) cells. A codon-optimized olive flounder IFN-γ gene was introduced into C. vulgaris, and Western blot analysis showed that the recombinant protein was detected mainly in the culture supernatant. Cytotoxicity assays showed no detectable cytotoxicity at the concentrations tested in the antiviral assays, and cell viability remained above 85.6% even at 10,000 ng/mL. Antiviral activity was evaluated using recombinant VHSV expressing enhanced green fluorescent protein (EGFP). Plaque assay showed that recombinant IFN-γ reduced VHSV infectivity in a dose-dependent manner, with an IC50 of 15.42 ng/mL and reduced infectivity to approximately 0% at concentrations of 100 ng/mL or higher. Fluorescence microscopy and flow cytometric analysis showed that recombinant IFN-γ reduced the VHSV-positive EPC cell population from 60.22% in the infected control group to 28.47%, 9.57%, and 4.92% at concentrations of 20, 50, and 100 ng/mL, respectively. In addition, recombinant IFN-γ induced the expression of antiviral immune-related genes, including STAT1 and ISG15, in EPC cells. These findings suggest that recombinant olive flounder IFN-γ produced in C. vulgaris retains biological activity after purification and exerts a dose-dependent antiviral effect against VHSV in a fish cell model.
Macrophage colony-stimulating factor (MCSF) is a key hematopoietic cytokine that regulates the proliferation, differentiation, and survival of mononuclear phagocytes through its receptor, MCSFR. In this study, we cloned and characterized the full-length cDNAs encoding MCSF and MCSFR from two commercially important perciform fish, largemouth bass (Micropterus salmoides) and spotted sea bass (Lateolabrax maculatus). The largemouth bass MCSF (MsMCSF) cDNA contains a 792-bp open reading frame (ORF) encoding 263 amino acids, whereas the spotted sea bass MCSF (LmMCSF) cDNA has an 813-bp ORF encoding 270 amino acids. Both MsMCSF and LmMCSF possess a signal peptide, a conserved CSF-1 domain, three cysteine residues required for homodimerization, and a transmembrane region. The corresponding MCSFR cDNAs encode proteins of 1050 (MsMCSFR) and 1025 (LmMCSFR) amino acids, each featuring a signal peptide, four immunoglobulin-like domains, and an intracellular tyrosine kinase domain. Phylogenetic analysis revealed that both MCSF and MCSFR cluster with their teleost counterparts, respectively. Tissue distribution profiling showed that MCSF and MCSFR transcripts are most abundant in the immune-related tissues, especially the headkidney (HK) and spleen. Upon challenge with Poly(I:C), Edwardsiella tarda, or Nocardia seriolae in largemouth bass, and with LPS, Poly(I:C), or E. tarda in spotted sea bass, all the four genes exhibited significant and tissue-specific transcriptional modulation from 6 to 48 h post-injection, suggesting that they may participate in the antiviral and antibacterial immune responses of largemouth bass and spotted sea bass. These results provide a basis for further functional studies on the immunoregulatory roles of MCSF/MCSFR signaling in teleost.
White spot syndrome virus (WSSV) inflicts severe economic losses on global aquaculture and poses a grave threat to the farming of shrimp, crabs, and other invertebrates. This review synthesizes the molecular pathways through which WSSV infection induces reactive oxygen species (ROS) generation in the host; examines the dual role of oxidative stress, wherein moderate ROS contribute to antiviral defense while excessive ROS drive oxidative injury; and, drawing on the threshold effect concept established in mammalian studies, evaluates its applicability and limitations as a working hypothesis in invertebrate WSSV infection models. We consolidate the relatively well-characterized oxidative stress response pathways in penaeid shrimp and, based on the limited evidence available, explore potential differences in crabs, mollusks, and other taxa—highlighting the near absence of research on non-shrimp groups. We further identify key challenges and controversies in the field, including incomplete elucidation of signaling pathways, the disconnect between experimental conditions and aquaculture environments, and pronounced species imbalance in research coverage. Finally, we prospect future research directions encompassing multi-omics dissection of molecular networks, application of antioxidants and ferroptosis modulators, and selective breeding for disease-resistant stocks, thereby providing a reference framework for antiviral research and disease management in aquaculture.
Spore-forming Bacillus probiotics have attracted increasing interest as antibiotic-free functional feed additives in shrimp aquaculture. However, evidence for the probiotic potential of Bacillus safensis remains limited. This study evaluated the effects of dietary B. safensis NPUST1 on growth performance, innate immunity, intestinal microbiota, and resistance to Vibrio alginolyticus in white shrimp (Penaeus vannamei). Shrimp were fed a basal diet (control) or diets supplemented with NPUST1 at 105 (E−5), 106 (E−6), or 107 CFU g−1 (E−7) for 8 weeks. Disc diffusion assays showed measurable inhibition zones against all antibiotics tested. Compared with the control, the E−6 and E−7 diets significantly increased final body weight, weight gain, specific growth rate, and feed efficiency without affecting survival. Probiotic supplementation also enhanced innate immunity, as indicated by increased phenoloxidase activity in the E−6 and E−7 groups, elevated phagocytic activity in all probiotic-treated groups, and higher respiratory burst, superoxide dismutase, and lysozyme activities, particularly in the E−7 group. Expression of prophenoloxidase was upregulated in the E−6 and E−7 groups, whereas TNF-α and astakine were significantly upregulated in all probiotic-treated groups. Following V. alginolyticus challenge, NPUST1 supplementation reduced cumulative mortality in a dose-dependent manner. Intestinal microbiota analysis revealed distinct diet-associated shifts in community composition, characterized by reduced Vibrio, Flavobacterium, and Tenacibaculum abundance together with enrichment of beneficial genera, including Bacillus and Bifidobacterium. Overall, dietary supplementation with B. safensis NPUST1 was associated with improved growth performance, enhanced innate immune responses, reduced mortality following V. alginolyticus challenge, and changes in intestinal microbial composition. The 107 CFU g−1 diet showed the most consistent overall effects, supporting its potential application in white shrimp culture.
