人類冠狀病毒229E在普通觸摸表面上仍具有傳染性
Human Coronavirus 229E Is Still Infectious on Normal Touching Surface
來自動物的呼吸道的新病毒和重新出現的歷史性強毒株的進化，對人類健康構成了重大的威脅。人畜共患病毒株在人與人之間的傳播效率低下，一開始可能會限制傳播的傳播，但接觸受污染的表面可能會感染。包膜病毒通常容易受到環境壓力的影響，但是引起嚴重急性呼吸道綜合症（SARS）和中東呼吸綜合症（MERS）的人類冠狀病毒最近引起了人們對暴發期間接觸傳播的關注。人類冠狀病毒229E在一系列銅合金上迅速失活（幾分鐘內可模擬指尖污染），而Cu / Zn黃銅在較低的銅濃度下非常有效。暴露於銅會破壞病毒基因組並不可逆轉地影響病毒的形態，包括包膜的解體和表面尖峰的散佈。 Cu+和Cu++部分是造成失活的原因，合金表面活性氧的產生會增強這種失活，導致滅活的速度甚至比非包膜病毒在銅上更快。因此，銅合金表面可用於公共區域和任何群眾聚會，以幫助減少呼吸道病毒從受污染表面傳播，並保護公眾健康。
The evolution of new viruses from animal respiratory tracts and re-emergence of historically virulent strains poses a major threat to human health. The transmission of zoonotic virus strains from person to person is inefficient, and may limit the spread of transmission at the beginning, but it may be infected by contact with contaminated surfaces. Enveloped viruses are usually susceptible to environmental pressures, but the human coronaviruses that cause severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) have recently raised concerns about contact transmission during outbreaks. Human Coronavirus 229E (HuCoV-229E) is rapidly inactivated on a series of copper alloys (fingertip contamination can be simulated within a few minutes), and Cu/Zn brass is very effective at lower copper concentrations. Exposure to copper can destroy the viral genome and irreversibly affects the morphology of the virus, including the disintegration of the envelope and the spread of surface spikes. Cu+ and Cu++ are part of the cause of inactivation. The production of active oxygen on the alloy surface will enhance this inactivation, resulting in inactivation even faster than non-enveloped viruses on copper. Therefore, copper alloy surfaces can be used in public areas and any mass gatherings to help reduce the spread of respiratory viruses from contaminated surfaces and protect public health.
重要性
Importance
呼吸道病毒在全球造成的死亡人數超過任何其他傳染因素。動物冠狀病毒
對人的“宿主轉移”會導致高死亡率的嚴重感染，例如嚴重的急性呼吸道綜合症（SARS），以及最近的中東呼吸綜合症（MERS）。我們在這裡展示了一個與人類冠狀病毒密切相關的229E，它會導致健康個體的上呼吸道感染和合併症患者的嚴重疾病，在公共和家庭區域常見的表面材料上仍保持感染性幾天。低傳染劑量意味著對於任何接觸被污染表面的人來說，這都是很大的感染風險。但是，我們在銅和銅合金表面的冠狀病毒中觀察到RNA的大量缺陷以及大量的結構性破壞，造成了病毒不可逆轉的變化性以及快速的滅活。使用銅合金表面並且藉由有效的清潔方式、良好的臨床實踐，可以有效控制住包括MERS和SARS在內的呼吸道冠狀病毒的傳播。
Respiratory viruses cause more deaths globally than any other infectious factor. The “Host switch” of animal coronavirus to humans can lead to serious infections with high mortality, such as severe acute respiratory syndrome (SARS), and more recently Middle East respiratory syndrome (MERS). Here we show a 229E closely related to the human coronavirus, which can cause upper respiratory tract infections in healthy individuals and serious illnesses in patients with comorbidities, it remains infectious for several days on common surface materials in public and domestic areas. The low infectious dose means that this is a great risk of infection for anyone who touches a contaminated surface. However, we have observed a large number of RNA defects and structural damage in the coronaviruses on the surface of copper and copper alloys, resulting in irreversible variability and rapid inactivation of the virus. The use of copper alloy surfaces and effective cleaning methods and good clinical practices can effectively control the spread of respiratory coronaviruses including MERS and SARS.
不同材質、材料表面以及各種環境條件下的抗菌去活效果的結果
The results of antibacterial deactivation effects under different materials, material surfaces and various environmental conditions
把冠狀病毒在共同表面上以感染狀態持續放置數天。103個菌斑(PFU)的接種物在聚氟四乙烯（Teflon; PTFE），聚氯乙烯（PVC），瓷磚，玻璃和不銹鋼以及21°C的環境溫度以及相對濕度30％到40％下，可以存活至少5天（對於矽橡膠則需要3天），然而，人類冠狀病毒會迅速在黃銅表面上失活。21°C的室溫下，銅鎳表面以及含有至少70%銅成分的黃銅表面，對於HuCoV-229E，滅活速率直接與銅的百分比成正比。以103PFU在模擬的濕滴汙染(每平方釐米20微升)中，不到六十分鐘即可滅活。初始時期分析顯示，滅活約10分鐘後，病毒很快喪失了傳染性。對於諾如病毒，鋅與不銹鋼相比（都不含銅），展現出輕微的抗病毒作用。
Place the coronavirus on the common surface in an infected state for several days. The inoculum of 103 plaques (PFU) in Teflon (PTFE), polyvinyl chloride (PVC), ceramic tiles, glass and stainless steel, and an ambient temperature of 21 °C and a relative humidity of 30% to 40 %, can survive for at least 5 days (3 days for silicone rubber). However, the human coronavirus will quickly inactivate on the brass surface. At a room temperature of 21°C, copper-nickel surfaces and brass surfaces containing at least 70% copper will make the inactivation rate of HuCoV-229E directly proportional to the percentage of copper. In the wet drop pollution simulated by 103PFU (20 microliters per square centimeter), it can be inactivated in less than 60 minutes. The initial analysis showed that the virus quickly lost its infectivity after about 10 minutes of inactivation. For norovirus, compared to stainless steel, zinc has a slight antiviral effect, when neither of them contains copper.
銅鎳還可以有效地滅活HuCoV-229E，但需要更高（90％）的銅含量才能產生一定程度等效於含70％黃銅的滅活效果。對於快速乾燥的指尖汙染模型，C26000砲彈筒黃銅，滅活時間進一步減少8倍到5分鐘以內。使用相同的數據，1微升/平方釐米的濕滴汙染，比較黃銅與銅鎳合金，顯示出相同成分比例的銅90%或是70%，黃銅和銅鎳合金都顯示出銅含量比例增加，會提高抗菌滅活的效率。但是，同時都具有90%銅含量的兩種銅鎳合金C72500，其效果比C70600要差。C70600的優異的抗病毒特性
包括先前已經觀察到對去活例如諾如病毒，並且可能和氧化亞銅層，一種明顯可見的可移除層有一定的關係。然而，對於低銅含量的砲彈黃銅，其去活效果卻是遠高於C71500銅鎳合金，達到三倍以上的速度。
Copper nickel can effectively inactivate HuCoV-229E, but higher (90%) copper content is required to produce a certain degree of inactivation effect equivalent to 70% brass. For the fast-drying fingertip contamination model, the C26000 cannon barrel brass, the inactivation time is reduced by 8 times in less than 5 minutes. Using the same data, comparing 1 microliter/cm2 of wet drop pollution, comparing brass and copper-nickel alloys, shows that the copper content ratio of brass and copper-nickel alloys increases with the same composition ratio of copper (90% or 70%), will improve the efficiency of antibacterial inactivation. However, the two copper-nickel alloys C72500, both with 90% copper content, are less effective than C70600. The excellent antiviral properties of C70600 include that it has previously been observed to deactivate such as norovirus, and may have a certain relationship with the cuprous oxide layer, which is a clearly visible removable layer. However, for shell brass with low copper content, its deactivation effect is much higher than that of C71500 copper-nickel alloy, reaching more than three times the speed.
銅離子的釋放造成活性氧(ROS)的產生，啟動HuCoV-229E在銅上的失活反應
和銅合金表面。將HuCoV-229E病毒分別接種到100%的銅和70%含銅量的彈藥筒黃銅表面，刻意加上了在乙二胺四乙酸（EDTA）和浴銅素二磺酸鹽（BCS）兩種螯合劑分別鎖住了Cu++和Cu+。兩種螯合劑最初都將病毒保護至少兩個小時。結果，顯示出病毒的滅活時間都延長到達2小時以上（儘管BCS在與黃銅接觸2小時之後仍然處於保護病毒的狀態）。這個結果證明了銅的兩種離子種類都直接和/或間接地需要病毒來滅活，而且Cu+的影響可能更為長遠與重要。
The release of copper ions causes the production of reactive oxygen species (ROS), which initiates the deactivation reaction of HuCoV-229E on copper and the surface of the copper alloy. Inoculating the HuCoV-229E virus to 100% copper and the brass surface of the cartridge with 70% copper content, deliberately added in two chelating agents, ethylenediaminetetraacetic acid (EDTA) and bathocuproine disulfonic acid disodium salt(BCS), to lock Cu++ and Cu+ respectively. Both chelators initially protect the virus for at least two hours. In the end, the results showed that the virus inactivation time was extended to more than 2 hours (although BCS was still in the state of protecting the virus after 2 hours in contact with brass). This result proves that both copper on species directly and/or indirectly require viruses to inactivate, and the effect of Cu+ may be more long-term and important.
在放入D-甘露醇和Tiron（4,5-二羥基-1,3-苯二磺酸）兩種能抑制羥基自由基和超氧陰離子的化學物質之下，接種冠狀病毒，以確定這兩個部分(羥基自由基和超氧陰離子)是否參與了冠狀病毒的滅活機理。Tiron在接觸的第一個小時內保護了病毒，這表明超氧化物的產生很重要。D-甘露醇對銅的保護作用極小，但在黃銅測試期間卻能保護病毒。增加D-甘露糖醇的濃度不會延長病毒對在銅表面的感染活性時間。 這表明冠狀病毒在銅表面的快速失活主要是由於銅離子的釋放，並且活性氧的影響極小。 但是，隨著合金中銅含量的降低，ROS的產生起著更重要的作用。 EDTA，BCS，D-甘露醇和Tiron對不銹鋼控製表面或懸浮液中的病毒沒有明顯影響。
After putting D-mannitol and tiron (4,5-dihydroxy-1,3-benzenedisulfonic) two chemicals that can inhibit hydroxyl radicals and superoxide anions, inoculate coronavirus to determine whether the two parts (hydroxyl radical and superoxide anion) are involved in the inactivation mechanism of coronavirus or not. Tiron protected the virus within the first hour of contact, which shows that superoxide production is important. D-Mannitol has very little protection effect on copper, but it can protect the virus during the brass test. Increasing the concentration of D-mannitol will not prolong the active time of the virus on the copper surface. This indicates that the coronavirus on the copper surface is mainly due to the release of copper ions, and the effect of active oxygen is minimal. However, as the copper content in the alloy decreases, the generation of ROS plays a more important role. EDTA, BCS, D-mannitol and tiron have no significant effect on the stainless steel control surface or the virus in the suspension.
上圖實驗結果的說明
Explanation of Experimental Results Above
人類冠狀病毒的快速失活發生在黃銅和銅鎳合金的表面。約103 PFU HuCoV-229E（20升感染細胞裂解液）應用於1平方厘米的試片，這些試片包括各種黃銅（A和B [僅早期時間點]），銅鎳（C）和不含銅的對照金屬表面（不銹鋼，鋅和鎳）。如本文所述，在各個時間點除去病毒並測定感染性。冠狀病毒在黃銅上<40分鐘，在銅含量少於70％的銅鎳上120分鐘。分析病毒與黃銅之間最初30分鐘的接觸（圖2B）揭示了最初一段時間的效果落後，然後迅速的失活。不銹鋼和鎳則沒有顯示出任何抗病毒活性，儘管溫和的抗病毒活性是在鋅上觀察到（僅在60分鐘時才有意義[P 0.046]）。 （D）施用相同的接種物至1 微升 / cm2，立即乾燥以模擬指尖的接觸污染，發現滅活病毒的速度快約8倍。誤差線代表SEM，數據來自於多次的實驗。
The rapid inactivation of the human coronavirus occurs on the surface of brass and copper-nickel alloys. Approximately 103 PFU HuCoV-229E (20 liters of infected cell lysate) are applied to 1 square centimeter test strips. These test strips include various brass (A and B [early time points only]), copper nickel © and none copper contrast metal surface (stainless steel, zinc and nickel). As described herein, virus was removed at various time points and the infectivity was determined. Coronavirus is less than 40 minutes on brass and 120 minutes on copper nickel with less than 70% copper. Analysis of the first 30 minutes of contact between the virus and brass (Figure 2B) revealed that the effect was lagging for the first period of time and then quickly inactivated. Stainless steel and nickel did not show any antiviral activity, although the mild antiviral activity was observed on zinc (only significant at 60 minutes [P 0.046]). In figure D, apply the same inoculum to 1μl/cm2, and dry it immediately to simulate the contact contamination of the fingertips. It is found that the virus is inactivated about 8 times faster.
(Error bars represent SEM, data comes from multiple experiments.)
重點結論整理
Summary of key conclusions
● 金屬表面銅的含量愈高，讓病毒失活的效果愈好
The higher the copper content on the metal surface, the better the effect of viral inactivation.
● 鋅只有相對上有相當差距的失活效果。鎳和不銹鋼則是幾乎無效
Zinc only has a relatively different inactivation effect. Nickel and stainless steel are almost ineffective.
● 環境愈乾燥，讓病毒失活的效果愈高
The drier the environment, the higher the effect of viral inactivation.
有關銅質表面滅菌與讓病毒失活機制的說明
Explanation of Mechanism of Sterilizing Copper Surfaces And Inactivating Viruses
這個機制相對複雜，它不僅涉及銅離子對多個目標的直接作用，還涉及破壞性氧自由基的產生，並且引起“代謝性自殺”。然而，在銅的表面上，我們沒有發現諾羅病毒的破壞，這可能是因為這個病毒，缺乏”呼吸機械”所造成。然而，超氧化物羥基自由基的產生可能對造成病毒失活的效果，非常重要。銅合金對冠狀病毒的抑制作用，在100%的銅表面上，主要是銅離子的作用。將濕滴滴到銅金屬的表面上之後，主要的離子物質會從金屬中溶出，表面是Cu++，但還原為Cu+的還原反應，伴隨著Fenton化學氧化反應(是一種會產生氫氧自由基<-OH>的高級氧化反應，僅次於使用氟的強氧化方式)的過程中，含有來自細胞碎片(有機物質)、分子氧的氧化中間體，或是對於病毒包膜可產生劇毒的羥基自由基(-OH)。
This mechanism is relatively complicated, it not only involves the direct effect of copper ions on multiple targets, but also involves the production of destructive oxygen free radicals and causes “metabolic suicide”. However, on the surface of the copper, we did not find norovirus damage, which may be caused by the lack of “respiratory machinery” in this virus. Also, the generation of superoxide hydroxyl radicals may be very important for the effect of causing virus inactivation. The inhibitory effect of copper alloy on coronavirus is mainly the effect of copper ions on 100% of the copper surface. After the wet drops are dropped on the copper surface, the main ionic substances will dissolve out of the metal, appears to be Cu++, but reaction of reducing to Cu+, accompanied by the Fenton chemical oxidation reaction can produce highly toxic hydroxyl radicals (-OH) to cell debris, oxidation intermediates of O2, and virus envelopes.
ROS是在冠狀病毒感染的自然過程中產生的。ROS (Reactive Oxygen Species)活性氧是人類細胞有氧代謝所產生的一類氧的單電子還原產物。因為其產生迅速，性質活潑可與蛋白質、核酸、脂類等大分子發生氧化反應，引起蛋白質變性、核酸(RNA)斷裂、脂類過氧化等等改變。ROS會促進發病與細胞的凋亡。Fujimori et al.指出，在2009年甲型H1N1病毒快速失活涉及到含有羥基的碘化銅奈米顆粒對病毒所產生抑制後的變化。這些含有羥基的碘化銅奈米顆粒會導致血凝素和神經氨酸酶病毒蛋白的降解。Fujimori et al.推測，儘管沒有外援過氧化氫去為Fenton反應提供燃料，一價銅離子和氧分子反應，可以產生超氧化物以及接著產生過氧化氫。過氧化氫可以通過海博魏斯反應(Haber Weiss Reaction)產生羥基自由基，造成病毒的失活(蛋白質變性、RNA斷裂)
ROS is produced during the natural process of coronavirus infection. ROS( Reactive Oxygen Species) — a one-electron reduction product of a type of oxygen produced by aerobic metabolism of human cells. Because of its rapid production, active nature, it can react with macromolecules such as proteins, nucleic acid and lipids, causing protein denaturation, nucleic acid (RNA) fragmentation and other changes. ROS will promote pathogenesis and cell apoptosis. Fujimori et al. pointed out that the rapid inactivation of the H1N1 virus in 2009 involved changes in the suppression of the virus by copper iodide nanoparticles containing hydroxyl groups. These hydroxy-containing copper iodide nanoparticles can cause the degradation of hemagglutinin and neuraminidase viruses. Fujimori et al. speculate that there is no foreign aid of hydrogen peroxide to fuel the Fenton reaction, the reaction of monovalent copper ions with oxygen molecules can produce superoxide and subsequently hydrogen peroxide. Hydrogen peroxide can generate hydroxyl free radicals through Haber Weiss Reaction, causing virus inactivation (protein denaturation, RNA fragmentation).
暴露於銅表面會導致透射電鏡（TEM）可見的人類冠狀病毒顆粒的形態變化。 暴露於不銹鋼的純化HuCoV-229E和暴露於銅表面的HuCoV-229E的外觀存在顯著差異（圖6）。 在不銹鋼上，暴露10分鐘後即可看到均勻的病毒體（圖6A），但是在銅上，可以看到成簇的受損病毒顆粒（圖6B）以及一些完整的顆粒。 進一步暴露於銅後，損壞程度增加（圖6C）。
Exposure to copper surfaces can cause changes in the morphology of human coronavirus particles visible by transmission electron microscopy (TEM). There is a significant difference in the appearance of purified HuCoV-229E exposed to stainless steel and HuCoV-229E exposed to copper (Figure 6). On stainless steel, uniform virions can be seen after 10 minutes of exposure (Figure 6A), but on copper, clusters of damaged virus particles (Figure 6B) and some intact particles can be seen. After further exposure to copper, the degree of damage increased (Figure 6C).
我們已經觀察到暴露於銅表面會導致非包膜諾羅病毒發生明顯的形態變化，在這種情況下，病毒蛋白包膜的解離可能會使病毒基因組暴露於銅表面而被去活。在這項研究中，我們觀察到冠狀病毒顆粒在暴露於銅後會迅速損壞，包括結塊，破裂，膜損壞和表面突刺的損失，同時一些顆粒顯得更小並且似乎失去了剛性，自身折疊起來。而從不銹鋼表面回收的病毒未觀察到這些變化。
We have observed that exposure to copper surfaces can cause significant morphological changes of non-enveloped norovirus. In this case, the dissociation of the viral protein envelope may expose the viral genome to the copper surface and be inactivated. In this study, we observed that coronavirus particles quickly damage after exposure to copper, including clumping, rupture, membrane damage, and loss of surface spurs. At the same time, some particles appeared smaller and seemed to lose rigidly, and folded themselves. These changes were not observed in viruses recovered from the surface of stainless steel.
對暴露於銅和銅合金表面的病毒中冠狀病毒基因組RNA的分析，揭示了整個基因組的非特定片段化的現象。也可以通過在基因層級的nsp4蛋白小片段的拷貝數減少來觀察到，並且隨著接觸時間的增加，而呈現出損傷程度的增加。我們有觀察到當諾羅病毒衣殼的完整性降低時，銅離子會更容易進入，加速去活病毒的基因組。
The analysis of coronavirus genomic RNA in viruses exposed to copper and copper alloy surfaces revealed the phenomenon of non-specific fragmentation of the entire genome. It can also be observed by the decrease in the copy number of small nsp4 protein fragments at the gene level, and with the increase of contact time, the degree of damage increases. We have observed that when the integrity of the norovirus capsid is reduced, copper ions will enter more easily and accelerate the deactivation of the virus genome.
對於冠狀病毒，包膜和核蛋白同樣會受到損害，並且與無包膜的諾羅病毒相比，該過程的發生速度更快，後者具有抗性衣殼與基因組，也會受到銅離子和/或ROS的破壞。 有趣的是，模擬濕滴污染的滅活有10分鐘的延遲，這可能反映了破壞外殼和破壞核蛋白所需的時間，核蛋白允許銅離子進入冠狀病毒基因組。 進一步的研究可能會確定使用增效清潔劑弱化包膜是否可以減少這種延遲。 Sagripanti等人還報導了與無包膜噬菌體相比，包膜病毒對銅離子溶液的敏感性更高。
For coronaviruses, the envelope and nucleoprotein are also damaged, and this process occurs faster than the non-enveloped norovirus, which has a resistant capsid and genome, and is also affected by copper ions and/or the destruction of ROS. Interestingly, there is a 10-minute delay in the inactivation of simulated wet drop pollution, which may reflect the time required to destroy the outer shell and nucleoprotein, which allows copper ions to enter the coronavirus genome. Further research may determine whether weakening the envelope with a booster cleaner can reduce this delay. Sagripanti et al. also reported that enveloped viruses are more sensitive to copper ion solutions than non-enveloped phages.
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Source of References
Title: Human Coronavirus 229E Remains Infectious on Common Touch Surface
Journal: Materials
Article in mBio · November 2015
DOI: 10.1128/mBio.01697–15
Received 1 October 2015 Accepted 13 October 2015 Published 10 November 2015
Citation Warnes SL, Little ZR, Keevil CW. 2015. Human coronavirus 229E remains infectious on common touch surface materials. mBio 6(6):e01697–15.
doi:10.1128/mBio.01697–15.
Editor Rita R. Colwell, University of Maryland
Copyright © 2015 Warnes et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported
license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Address correspondence to C. W. Keevil, cwk@soton.ac.uk.
This article is a direct contribution from a Fellow of the American Academy of Microbiology.
