Department of Microbiological and Gynaecological Sciences, University of Catania, Via Androne 81, 95124 Catania, Italy.
Antiviral Res. 2011 Jan;89(1):83-8. doi: 10.1016/j.antiviral.2010.11.010. Epub 2010 Nov 21.
Our previous study demonstrated that Melaleuca alternifolia (tea tree) oil (TTO) had an interesting antiviral activity against Influenza A in MDCK cells. In fact, when we tested TTO and some of its components, we found that TTO had an inhibitory effect on influenza virus replication at doses below the cytotoxic dose; terpinen-4-ol, terpinolene, and alfa-terpineol were the main active components. The aim of this study was to investigate the mechanism of action of TTO and its active components against Influenza A/PR/8 virus subtype H1N1 in MDCK cells. None of the test compounds showed virucidal activity nor any protective action for the MDCK cells. Thus, the effect of TTO and its active components on different steps of the replicative cycle of influenza virus was studied by adding the test compounds at various times after infection. These experiments revealed that viral replication was significantly inhibited if TTO was added within 2h of infection, indicating an interference with an early step of the viral replicative cycle of influenza virus. The influence of the compound on the virus adsorption step, studied by the infective center assay, indicated that TTO did not interfere with cellular attachment of the virus. TTO did not inhibit influenza virus neuraminidase activity, as shown by the experiment measuring the amount of 4-methylumbelliferone, cleaved by the influenza virus neuraminidase from the fluorogenic substrate 2'-O-(4-methylumbelliferyl)-N-acetylneuraminic acid. The effect of TTO on acidification of cellular lysosomes was studied by vital staining with acridine orange using bafilomycin A1 as positive control. The treatment of cells with 0.01% (v/v) of TTO at 37°C for 4h before staining inhibited the acridine orange accumulation in acid cytoplasmic vesicles, indicating that TTO could inhibit viral uncoating by an interference with acidification of intralysosomal compartment.
我们之前的研究表明，互叶白千层（茶树）油（TTO）对 MDCK 细胞中的甲型流感病毒具有有趣的抗病毒活性。事实上，当我们测试 TTO 及其某些成分时，我们发现 TTO 在低于细胞毒性剂量时对流感病毒复制具有抑制作用；萜品-4-醇、松油烯和α-松油醇是主要的活性成分。本研究旨在探讨 TTO 及其活性成分对 MDCK 细胞中甲型流感病毒/PR/8 亚型 H1N1 的作用机制。测试化合物均无杀病毒活性，也对 MDCK 细胞没有保护作用。因此，通过在感染后不同时间加入测试化合物，研究了 TTO 及其活性成分对流感病毒复制周期不同步骤的作用。这些实验表明，如果在感染后 2 小时内加入 TTO，则病毒复制会受到显著抑制，表明其干扰了流感病毒复制周期的早期步骤。通过感染中心测定法研究了化合物对病毒吸附步骤的影响，表明 TTO 不干扰病毒与细胞的附着。TTO 不抑制流感病毒神经氨酸酶活性，如通过测量从荧光底物 2'-O-(4-甲基伞形基）-N-乙酰神经氨酸裂解的 4-甲基伞形酮的量来测定流感病毒神经氨酸酶活性的实验所示。通过使用巴弗洛霉素 A1 作为阳性对照，用吖啶橙对细胞进行活体染色来研究 TTO 对细胞溶酶体酸化的影响。在用 0.01%（v/v）TTO 处理细胞 4 小时后，在染色前于 37°C 处理细胞可抑制吖啶橙在酸性细胞质囊泡中的积累，表明 TTO 可以通过干扰内溶酶体腔内的酸化来抑制病毒脱壳。
