Human Coronavirus 229E Is Still Infectious on Normal Touching Surface

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.

Importance:

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.

The results of antibacterial deactivation effects under different materials, material surfaces and various environmental conditions:

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.

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.

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.

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.

Explanation of Experimental Results Above:

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 (C) 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.)

Summary of key conclusions:

  • The higher the copper content on the metal surface, the better the effect of viral inactivation.
  • Zinc only has a relatively different inactivation effect. Nickel and stainless steel are almost ineffective.
  • The drier the environment, the higher the effect of viral inactivation.

Explanation of Mechanism of Sterilizing Copper Surfaces And Inactivating Viruses:

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 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).

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).

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.

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.

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.

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.

Other articles related to anti-bacteria information,

please visit ACTife Technology Special Column.

[click here for more]