中性電解水與微波共振對病毒去活性效果之研究

Abstract

病毒是生活中常見造成人類致病的微生物，在人與人之間的傳播會造成疾病的流行。因此找出能廣效殺死病毒的方法一直是一個重要的議題。目前常用將病毒去活性的方法，化學法的部分有界面活性劑、有機溶劑及氧化劑等三類方法，各類方法因其作用機制的不同，而無法達到廣效將病毒去活性的效果，或是對人體具有毒性，因此更好的化學去活性方法仍有待開發。次氯酸是已知能有效將病毒去活性的氧化劑，目前常用的形式是次氯酸鈉水溶液(漂白水)，但因其腐蝕性而在使用上有所限制。物理方法的部分，紫外線與放射線較常用於殺菌，也有用微波加熱的方式達到殺菌的效果。近年來科學家發現不產熱的微波，能將波的能量轉換成熱能以外的形式傳遞，例如動能，並且在使用特定頻率照射下能達到更好的殺菌效果，但是否對病毒也有類似的效果則屬未知。 Envirolyte公司的電極所製造之中性電解水，主成分為次氯酸且不含鈉。實際測試結果發現，此電解水(410 ppm)對於日常生活中常見的9種病毒(3種A型流感病毒、B型流感病毒、單純皰疹病毒、3種腸病毒、腺病毒)都具有相當好的去活性效果。接著進行濃度與作用時間之相關研究，發現此電解水對不同病毒的有效作用濃度有所不同，在最高作用濃度下(410 ppm)，1分鐘內就完成去活性的作用。 電解水的作用機制方面，從流感病毒血球凝集試驗發現次氯酸會影響病毒之血球凝集素。與細胞結合能力試驗結果發現，次氯酸能破壞病毒與細胞膜結合，阻止病毒感染細胞達到去活性之效果，而破壞程度與病毒種類及次氯酸濃度有關。最後，以SDS PAGE分析兩種流感病毒之蛋白質受到次氯酸破壞的程度，發現HA、NA與NP蛋白質較容易受次氯酸影響，而B型流感病毒蛋白質較A型流感病毒H3N2蛋白質不易受破壞。 微波去病毒活性之研究，在與臺大醫工所劉子銘老師的合作下，探討微波對A型流感病毒H3N2之共振頻率。經過實驗後發現A型流感病毒H3N2的微波共振頻率約在8 GHz左右，而對頻率7 ~ 11 GHz的微波亦都有吸收。實際測試此共振能量的吸收是否能轉換成將病毒去活性的能量，結果發現，病毒感染力在非吸收頻率範圍內，有下降之趨勢；反之在有共振之特定頻率範圍內，病毒感染力有上升之現象。 總結而言，化學法之中性電解水，對於日常生活常見的致病病毒有很好的去活性效果，加上作用時間短及中性的特點，可廣泛運用於日常生活環境中的清潔消毒工作，達到預防疾病傳播的目的。而物理法微波共振方面，雖然得到的結果不顯著，但其研究數據仍可供未來相關研究之重要參考。

Viruses are common pathogenic microorganism, and the transmission of virus between human could cause outbreak or epidemic infections. Several chemical and physical methods have been used to inactivate viral infectivity in the environment and to terminate the chain of viral transmission. Chemically, detergents, organic agents and oxidants were used to inactivate viruses, but those cannot reach broad range of effectiveness due to various acting mechanisms or toxicity to human. To overcome these disadvantages, some inactivation methods have been developed. Hypochlorous acid is a well-known oxidant which could effectively inactivate viruses, and sodium hypochlorite solution (bleach) was used widely in our daily life. However, due to the corrosiveness, its usage is limited. Physically, UV, radiation and microwave heating are usually used for sterilization. Recently, scientists discovered non-thermal microwave, which can transfer the energy directly to other energy modes, usually kinetic energy, rather than thermal energy. Expose to microwave at specific frequency may have the sterilizing activity against bacteria. The application of this strategy to viral inactivation is worth of exploration. Neutral electrolyzed water made by Envirolyte electrodes containing hypochlorous acid without sodium. Neutral electrolyzed water (410 ppm of hypochlorous) has good inactivating effectiveness to nine common viruses (3 kinds of influenza A virus, influenza B virus, herpes simplex virus, adenovirus and 3 kinds of enterovirus). After testing for the various concentrations of hypochlorous acid and reaction time, we determined the effective concentrations to inactivate different viruses, and the inactivation reactions reach the end point in one minute with the highest concentrations (410 ppm). To understand the mechanism of inactivation by neutral electrolyzed water , we performed hemagglutination test after treating virus suspension with neutral electrolyzed water. The results indicated that hypochlorous acid may affect virus hemagglutinin protein. As result of cell binding assay, hypochlorous acid treatment may block virus binding to cells in a dose-dependent manner. Hypochlorous acid had less effects on the level of protein degradation of Influenza B virus than proteins of Influenza A virus H3N2 using SDS PAGE. The HA, NP/NA proteins were the most effective proteins of both viruses. In the microwave study cooperated with Dr. T.M. Liu, we studied the effect of microwave on influenza A virus H3N2. Microwave resonance spectrum (the virus absorbs microwaves) ranged 7 ~ 11 GHz, and the peak of absorption as resonant frequency was 8 GHz. In order to know the association between the absorption of microwave energy and the inactivation of virus, the residual viral infectivity after exposing microwave was analyzed. The results demonstrated that the absorption spectrum were not coincidence with the reduction of the virus infectivity. Instead, the viral infectivity increased at the absorption spectrum regions and decreased at the non-absorption spectrum region. The potential mechanism of this finding requires further investigation. In conclusion, neutral electrolyzed water can be used for the environmental and instrument cleaning works because of its good inactivating ability, short reaction time and neutral. Although the inactivating ability of microwave resonance is not clear, the observation in this study can provide further exploration of the microwave expose and virus inaction.