Characterization, microRNA profiling, and immunomodulatory role of plasma-derived exosomes from olive flounder (Paralichthys olivaceus) in response to viral hemorrhagic septicemia virus.

Viral hemorrhagic septicemia virus (VHSV) is a highly pathogenic virus that frequently infects olive flounder (Paralichthys olivaceus), causing viral hemorrhagic septicemia (VHS), and posing a significant threat to global aquaculture. This study characterizes plasma-derived exosomes from olive flounder following VHSV challenge (VHSV-Exo) or phosphate buffered saline (PBS) injection (PBS-Exo), comparing their morphology, physicochemical properties, molecular profiles, and immunomodulatory functions. Both PBS-Exo (118.3 ± 8.6 nm) and VHSV-Exo (82.6 ± 5.9 nm) exhibited the typical cup-shaped morphology of exosomes. The successful isolation and purity of exosomes were confirmed by the presence of exosome markers (CD81, CD9, and CD63) and the absence of albumin. High-throughput sequencing identified 13 differentially expressed (DE) microRNAs (miRNAs) between PBS-Exo and VHSV-Exo, including six upregulated and seven downregulated miRNAs (log fold change ≥1 or ≤ -1). Toxicity assessments revealed that neither PBS-Exo nor VHSV-Exo were toxic to murine macrophage Raw 264.7 cells or zebrafish larvae at tested doses (up to 100 and 400 μg/mL, respectively). The absence of green fluorescence at 96 h post-treatment of VHSV-Exo indicated minimal reactive oxygen species generation, further supporting exosome safety. Functional studies demonstrated that both in vitro (Raw 264.7 cells) and in vivo (adult zebrafish) treatments with exosomes regulated immune-related genes and proteins expression. Notabaly, VHSV-Exo exhibited superior immunomodulatory effects, as evidenced by enhanced immune gene and protein expression. To our knowledge, this is the first study demonstrating the immunomodulatory potential of VHSV-Exo. These findings highlight VHSV-Exo as a promising immunomodulatory agent, with potential applications as a prophylactic vaccine candidate against VHSV infection in aquaculture.