戊糖片球菌胞外囊泡對巨大細胞病毒的抗病毒活性及其對金目鱸的免疫調節作用

Abstract

金目鱸 (Lates calcarifer) 為臺灣具重要經濟價值之養殖魚種，在高密度養殖條件下易發生傳染性疾病爆發。其中，嘉鱲虹彩病毒 (Red Sea Bream Iridovirus, RSIV) 為全球養殖及野生魚類族群中造成高死亡率的最嚴重病原之一。胞外囊泡 (Extracellular vesicles, EVs) 為細胞 (包含細菌) 所釋放之具膜結構顆粒，可作為細胞間通訊的媒介，調控多種生理與病理過程。先前研究已證實，植物乳桿菌屬的戊糖片球菌Pediococcus pentosaceus 4012菌株 (LAB4012) 可對魚類病毒具有防治效果。然而,LAB4012所分泌之細胞外囊泡 (EV4012) 是否為其主要抗病毒因子，以及其潛在的抗病毒機制，仍尚未釐清。因此，本研究旨在純化EV4012，並評估其是否具有抵抗RSIV感染的能力。本研究利用切向流過濾技術 (Tangential Flow Filtration, TFF) 自LAB4012培養上清液中純化EV4012，並透過穿透式電子顯微鏡 (Transmission Electron Microscopy, TEM) 與奈米顆粒追蹤分析 (Nanoparticle Tracking Analysis, NTA) 觀察到EV4012具有球形與杯狀之型態，大小約為81 nm，顆粒濃度約為3.49 x 1010 particles/ mL。在穩定性方面，EV4012可儲存於 −80°C長達8個月。此外，體外實驗使用金目鱸腎臟細胞株 (Seabass Kidney cell line, SK) 評估EV4012的安全濃度以及對RSIV之抗病毒活性，結果顯示，稀釋128倍以上的EV4012 接種到SK 細胞24小時不會造成顯著的細胞毒性，並且EV4012在細胞中可以有效中和RSIV。將EV4012作為飼料添加劑，連續餵食金目鱸四週，不會對魚隻造成死亡以及各項組織造成損傷。此外，餵食EV4012能夠提升金目鱸頭腎、脾臟的IL-1β、Mx、C3、IgM、IgT 以及 iNOS等免疫相關基因來提升魚體對RSIV感染之抵抗能力。顯示其在金目鱸養殖中具有作為抵抗RSIV感染之潛在應用價值。

Barramundi (Lates calcarifer) is an economically important aquaculture species in Taiwan. However, under intensive farming conditions, it is highly susceptible to outbreaks of infectious diseases. Among these, Red Sea Bream Iridovirus (RSIV) is one of the most devastating pathogens, causing high mortality in both cultured and wild fish populations worldwide. Extracellular vesicles (EVs) are membrane-bound nanoparticles secreted by cells, including bacteria, and serve as important mediators of intercellular communication by regulating various physiological and pathological processes. Previous studies have demonstrated that Pediococcus pentosaceus strain 4012 (LAB4012), a lactic acid bacterium, exhibits antiviral activity against fish viruses. However, whether the extracellular vesicles secreted by LAB4012 (EV4012) are the major antiviral factor and the underlying mechanisms responsible for their antiviral activity remain unclear. Therefore, this study aimed to isolate and characterize EV4012 and evaluate its antiviral potential against RSIV infection. EV4012 was purified from the culture supernatant of LAB4012 using tangential flow filtration (TFF). Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that EV4012 exhibited spherical and cup-shaped morphology with an average particle size of approximately 81 nm and a concentration of 3.49 × 10¹⁰ particles/mL. Stability analysis showed that EV4012 remained stable after storage at −80°C for up to eight months. In vitro, the seabass kidney (SK) cell line was used to evaluate the cytotoxicity and antiviral activity of EV4012. The results showed that EV4012 diluted at least 128-fold exhibited no significant cytotoxicity after 24 h of treatment. Moreover, EV4012 effectively neutralized RSIV infection in SK cells. For the in vivo study, barramundi was fed an EV4012-supplemented diet continuously for four weeks. No mortality or histopathological abnormalities were observed in the experimental fish. Furthermore, dietary supplementation with EV4012 significantly upregulated the expression of immune-related genes, including IL-1β, Mx, C3, IgM, IgT, and iNOS, in the head kidney and spleen, thereby enhancing the host immune response against RSIV infection. These findings demonstrate that EV4012 has considerable potential as a functional feed additive for preventing RSIV infection in barramundi aquaculture.