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定量 PCR 和定量蛋白质组学研究阳光和 UVC 光对人腺病毒失活的机制。

Mechanisms of human adenovirus inactivation by sunlight and UVC light as examined by quantitative PCR and quantitative proteomics.

机构信息

Laboratory of Environmental Chemistry, School of Architecture, Civil and Environmental Engineering (ENAC), École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

出版信息

Appl Environ Microbiol. 2013 Feb;79(4):1325-32. doi: 10.1128/AEM.03457-12. Epub 2012 Dec 14.

Abstract

Human adenoviruses (HAdV) are important pathogens in both industrialized and developing nations. HAdV has been shown to be relatively resistant to monochromatic UVC light. Polychromatic UVC light, in contrast, is a more effective means of disinfection, presumably due to the involvement of viral proteins in the inactivation mechanism. Solar disinfection of HAdV, finally, is only poorly understood. In this paper, the kinetics and mechanism of HAdV inactivation by UVC light and direct and indirect solar disinfection are elucidated. PCR and mass spectrometry were employed to quantify the extent of genome and protein degradation and to localize the affected regions in the HAdV proteins. For this purpose, we used for the first time an approach involving stable isotope labeling by amino acids in cell culture (SILAC) of a human virus. Inactivation by UVC light and the full sunlight spectrum were found to efficiently inactivate HAdV, whereas UVA-visible light only caused inactivation in the presence of external sensitizers (indirect solar disinfection). Genome damage was significant for UVC but was less important for solar disinfection. In contrast, indirect solar disinfection exhibited extensive protein degradation. In particular, the fiber protein and the amino acids responsible for host binding within the fiber protein were shown to degrade. In addition, the central domain of the penton protein was damaged, which may inhibit interactions with the fiber protein and lead to a disruption of the initial stages of infection. Damage to the hexon protein, however, appeared to affect only regions not directly involved in the infectious cycle.

摘要

人腺病毒(HAdV)是工业化和发展中国家的重要病原体。研究表明,HAdV 对单色谱 UVC 光具有相对抗性。相比之下,多色谱 UVC 光则是一种更有效的消毒手段,这可能是由于病毒蛋白参与了失活动力学机制。最后,太阳光消毒 HAdV 的机制仍知之甚少。在本文中,阐明了 UVC 光以及直接和间接太阳光消毒灭活 HAdV 的动力学和机制。采用 PCR 和质谱技术来定量评估基因组和蛋白质降解的程度,并确定 HAdV 蛋白中受影响的区域。为此,我们首次使用了一种涉及细胞培养中氨基酸稳定同位素标记(SILAC)的方法来对人类病毒进行研究。研究发现,UVC 光和全太阳光光谱都能有效地灭活 HAdV,而 UVA-可见光只有在外部敏化剂存在的情况下才会导致间接太阳光消毒的灭活。UVC 光对基因组的损伤很明显,但对太阳光消毒的影响较小。相比之下,间接太阳光消毒则表现出广泛的蛋白质降解。特别是纤维蛋白和纤维蛋白中负责与宿主结合的氨基酸被降解。此外,五邻体蛋白的中心结构域受损,这可能会抑制与纤维蛋白的相互作用,导致感染初始阶段的破坏。然而,六邻体蛋白的损伤似乎只影响到与传染性周期不直接相关的区域。

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