Nikapitiya Chamilani, Jayathilaka E H T Thulshan, Edirisinghe Shan Lakmal, Oh Chulhong, De Zoysa Mahanama
College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon, 34134, Republic of Korea.
Jeju Bio Research Center, Korea Institute of Ocean Science and Technology, Gujwa-eup, Jeju, 2670, Republic of Korea.
Fish Shellfish Immunol. 2025 Jul;162:110316. doi: 10.1016/j.fsi.2025.110316. Epub 2025 Apr 15.
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.
病毒性出血性败血症病毒（VHSV）是一种高致病性病毒，经常感染牙鲆（Paralichthys olivaceus），导致病毒性出血性败血症（VHS），对全球水产养殖业构成重大威胁。本研究对牙鲆在受到VHSV攻击（VHSV-Exo）或注射磷酸盐缓冲盐水（PBS）（PBS-Exo）后血浆来源的外泌体进行了表征，比较了它们的形态、理化性质、分子谱和免疫调节功能。PBS-Exo（118.3±8.6纳米）和VHSV-Exo（82.6±5.9纳米）均呈现出外泌体典型的杯状形态。通过外泌体标志物（CD81、CD9和CD63）的存在以及白蛋白的缺失，证实了外泌体的成功分离和纯度。高通量测序确定了PBS-Exo和VHSV-Exo之间13种差异表达的（DE）微小RNA（miRNA），包括6种上调和7种下调的miRNA（对数倍数变化≥1或≤ -1）。毒性评估显示，在测试剂量下（分别高达100和400μg/mL），PBS-Exo和VHSV-Exo对小鼠巨噬细胞Raw 264.7细胞或斑马鱼幼虫均无毒性。VHSV-Exo处理96小时后无绿色荧光，表明活性氧产生极少，进一步支持了外泌体的安全性。功能研究表明，外泌体在体外（Raw 264.7细胞）和体内（成年斑马鱼）处理均调节免疫相关基因和蛋白质表达。值得注意的是，VHSV-Exo表现出卓越的免疫调节作用，免疫基因和蛋白质表达增强证明了这一点。据我们所知，这是第一项证明VHSV-Exo具有免疫调节潜力的研究。这些发现突出了VHSV-Exo作为一种有前景的免疫调节剂，在水产养殖中作为预防VHSV感染的候选疫苗具有潜在应用价值。
