Six ways to "kill" viruses: Which one is best for the new coronavirus?

This article summarizes several ways to inactivate viruses and their inactivation principles, so that everyone can find a suitable disinfection method based on this. At the same time, we will also see that there may be more room for the development of the new crown inactivated vaccine.

Written by Peng Cheng (PhD student in Li Sai's lab at Tsinghua University), Li Sai (Researcher at Tsinghua University's Advanced Innovation Center for Structural Biology)

The history of human development is also a history of fighting against viruses. Looking back at history, viruses have launched countless fierce attacks on humans. It is speculated that the smallpox virus appeared more than 10,000 years ago and was rampant around the world until 1980. Smallpox has a high mortality rate, causing about 300 million deaths worldwide in the 20th century alone [1]. Influenza viruses have also ravaged the world many times in the past 100 years, and the Spanish flu in 1918 alone claimed tens of millions of lives [2]. In addition, human immunodeficiency virus (commonly known as "HIV"), Ebola virus, and hepatitis virus are still eyeing humans, and the current new coronavirus continues to show strong transmission characteristics due to the continuous emergence of mutant strains. So far, the death toll from this new coronavirus epidemic has risen to the third place in the number of deaths caused by viral plagues in human history (from Wikipedia). Faced with this invisible enemy, humans seem to be somewhat passive. Can't we take the initiative to "kill" the virus?

In fact, before we knew about viruses, humans had already crossed the river by feeling the stones and found some ways to "kill" viruses. Ancient Chinese medicine also recorded this, and some methods are still of scientific significance today. For example, Li Shizhen recorded in "Compendium of Materia Medica" that "when there is a plague, take out the patient's clothes and steam them in a steamer, and the whole family will not be infected." This is an example of using high temperature to "kill" viruses. With the progress of biology, physics, modern medicine and chemistry, human cognition of viruses has also changed from the original "evil spirit" and "God's punishment" to the current "a kind of tiny biological particles that specialize in parasitism", which also provides a biological basis for the development of means to "kill" viruses.

Nowadays, people have a variety of ways to "kill" viruses. Some can destroy the virus structure and quickly "kill" the virus, such as alcohol (destroying the capsule) and high temperature (protein denaturation). These are also common methods of disinfection. Some methods can retain the immunogenicity or structural integrity of the virus on the basis of "killing" the virus, such as formaldehyde (fixing protein) and β-propiolactone (destroying nucleic acid). After the virus is "killed" by them, it is often used to prepare vaccines or for structural biology research of the virus.

How can we “kill” the virus?

In fact, the expression "killing the virus" is not accurate. The virus can be said to be one of the organisms designed by nature with "minimalist" style. Its structure is so simple that only nucleic acid wrapped in protein/lipid membrane remains. In vitro, it does not replicate itself, has no energy consumption, and no synthesis and decomposition of substances. It seems to be in a "dead" state, but once it invades the cell, it can turn the entire cell into its own production factory and reproduce more virus particles. Therefore, the topic of whether viruses are alive has never had a clear answer. The "killing" mentioned in the previous article actually refers to making the virus lose the ability to reproduce, that is, to prevent the virus from "coming back to life" even if it encounters a host. In vitro, this process is called "inactivation" in the virology community.

If you want to block the reproduction of the virus, you must first understand the life cycle of the virus. Virus particles use the protein on their surface as a "key" to identify the host and enter the cell, using the raw materials and energy of the cell to replicate their own genome and synthesize their own proteins. These newly synthesized genomes and structural proteins will be assembled into progeny virus particles and released outside the cell. According to the life activities of the virus, there are generally three ways to inactivate it. One is to destroy the overall structure of the virus or the protein structure of the virus, so that it loses the ability to invade cells. The other is to "lock" the protein of the virus so that it cannot function. The last is to destroy the genome of the virus so that its genome cannot replicate in the cell.

The life cycle of the new coronavirus and influenza virus[3].

The life cycle of both the new coronavirus (left) and influenza virus (right) goes through the following steps:

① Entering the cell. The surface protein of the virus particle recognizes the receptor, membrane fusion occurs and the genetic material is released;

② Genome replication. The new coronavirus induces cells to produce double-membrane vesicles (DMVs) [4] as the site for its genome replication, while the influenza virus replicates its genome in the cell nucleus;

③Viral protein translation;

④ Virus particle assembly and budding. The assembly of the new coronavirus occurs in the endoplasmic reticulum-Golgi intermediate (ERGIC), while the influenza virus assembles on the cell membrane;

⑤ Release of progeny viruses.

Based on the above ideas, various methods of virus inactivation have been born. According to the inactivation method, it can be divided into chemical inactivation and physical inactivation. Different inactivation methods have different effects on viruses. Some methods can quickly inactivate viruses, while others can retain the protein structure or immunogenicity of the virus on the basis of inactivating the virus. Scientists are also conducting downstream scientific research based on different inactivation methods, such as the establishment of rapid disinfection methods, structural analysis of viruses, and the development of inactivated vaccines. This article will briefly introduce several major virus inactivation methods and principles.

Chemical inactivation

formaldehyde

Everyone should be familiar with formaldehyde. Its aqueous solution is the famous formalin, which is a preservative and fixative. Formaldehyde contains a central carbon atom with a missing electron and is electrophilic, so it can undergo nucleophilic addition reactions with nucleophiles. It can monohydroxylate the amino group at the nitrogen end of proteins and the nitrogen-containing amino acids in the side chains (such as lysine, arginine, tyrosine, etc.), and further dehydrate to form imine intermediates. These intermediates can react again with arginine and tyrosine residues to form methylene bridges and cross-link. In addition, formaldehyde can also monohydroxylate adenine, preventing gene reading[5]. Therefore, formaldehyde can not only achieve a double blow to the protein and genome of the virus, but also fix the structure of the viral protein through cross-linking. Because formaldehyde can "lock" the protein structure, formaldehyde inactivation is not only a major candidate for the development of inactivated vaccines, but also a common method of virus inactivation used by structural biologists in virus structure analysis[6].

Principle of the reaction of formaldehyde with adenine and lysine[5]

β-Propiolactone

β-Propiolactone is an alkylating agent that mainly targets guanine and is also considered to be an inactivating agent that tends to damage nucleic acids. Electrophilic β-propiolactone can undergo nucleophilic substitution reactions with guanine, causing β-propiolactone ring opening and guanine alkylation, resulting in inactivation of the viral genome[5]. Currently, β-propiolactone is the most widely used inactivating agent in the development of inactivated vaccines, but studies have shown that β-propiolactone can also modify some amino acids, resulting in acylation and cross-linking reactions[7].

Principle of the reaction between β-propiolactone and guanine[5]

Ethanol

Ethanol is alcohol. Ethanol inactivates viruses very quickly, especially enveloped viruses. Enveloped viruses are a type of virus that is encapsulated by a lipid bilayer. Since ethanol is both lipid and water-philic, it can enhance the membrane's affinity for water while reducing the interaction between non-polar amino acid residues, thereby destroying the overall structure of the virus and denaturing viral proteins[8].

In 2021, Das et al. described the destructive effect of ethanol on enveloped viruses through molecular dynamics simulation. After the virus was immersed in a 75% ethanol solution, its envelope would disintegrate and release its contents[9]. The new coronavirus is also an enveloped virus. In 2020, Annika Kratzel found through experiments that the infectivity of the new coronavirus can be reduced to the background level after being immersed in an alcohol solution higher than 30% for 30 seconds[10]. In addition to ethanol, there are many alcohol compounds that can also show a faster inactivation rate, such as n-propanol and isopropanol[10]. Because these compounds have the characteristics of fast inactivation rate and low toxicity, they are often used as the main component of hand sanitizers and surface disinfectants of objects.

Schematic diagram of the mechanism by which alcohol compounds destroy the new coronavirus[8]

Physical inactivation

temperature

High temperature is also a common and frequently used inactivation method. Under the action of high temperature, the chemical bonds that maintain the secondary and tertiary structures of viral proteins will be destroyed, resulting in protein denaturation, which will cause the virus to lose its ability to infect cells and replicate. Didac Martí and others from the Polytechnic University of Catalonia used molecular dynamics simulation to find that high temperature can cause structural changes and hydrogen bond rearrangements in the surface proteins of the new coronavirus, especially in the receptor binding region of the surface proteins [11]. In addition, high temperature can also cause the backbone of nucleic acids to break.

It is worth mentioning that it is currently believed that "relatively low" temperatures (below 41°C) can break the viral genome without affecting protein structure and function [5]. In 2020, Christophe Batéjat of the Pasteur Institute subjected nasopharyngeal swab samples of the new coronavirus to high-temperature treatment at 56°C, 65°C, and 95°C, and found that the new coronavirus would be inactivated after 20 minutes, 10 minutes, and 3 minutes, respectively [12]. High-temperature inactivation is now also a necessary step in the treatment of biosafety waste because of its stable effect and broad spectrum.

Ultraviolet light

Ultraviolet light is invisible light with a higher frequency than blue-violet light. According to the standards of the International Organization for Standardization (ISO), ultraviolet light can be further divided into UVA band (320–400 nm), UVB band (280-320 nm) and UVC band (200-280 nm) according to wavelength. Among them, UVC is the most destructive to viruses, followed by UVB and UVA[13]. Therefore, UVC is also the main band used to inactivate viruses.

The main way that ultraviolet light destroys viral nucleic acid is to induce the formation of pyrimidine dimers inside the nucleic acid. When ultraviolet light is absorbed by the viral nucleic acid, two adjacent or opposite pyrimidines in the viral genome will undergo photochemical fusion to form covalently linked pyrimidine dimers. Due to the covalent action, pyrimidine dimers will introduce tension to the skeleton of the genome, causing the genome skeleton to break[5]. For some viruses, ultraviolet light can retain the fusion activity of the virus on the basis of complete inactivation of the virus[14]. For the new coronavirus, after 9 minutes of UVC irradiation (cumulative dose of 1048 mJ/cm2), the new coronavirus with a high infection titer (5 * 106 TCID50/mL) can be completely inactivated[13]. Although UVC has a good inactivation effect, its penetration ability is poor. Ordinary transparent glass, clothes, plastics, etc. can block most of the UVC. Therefore, UVC is often used to disinfect the surface of instruments and objects and the indoor space of hospitals and biosafety level laboratories.

Schematic diagram of the formation of pyrimidine dimers by adjacent uracils under ultraviolet light irradiation[5]

Ionizing radiation

Ionizing radiation is a type of energy that can cause atoms or molecules of matter to ionize. The three main ionizing radiation technologies are gamma radiation, electron beam, and X-ray. Gamma rays are a type of radiation emitted when atoms decay and break apart. Electron beams (eBeams) are high-energy electron beams generated by electron beam accelerators. X-rays are radiation generated by the transition or stimulation of electrons outside the nucleus of an atom[15]. Although the sources are different, the mechanisms of inactivating viruses are basically similar.

The destructive effects of ionizing radiation on viruses can be divided into direct and indirect effects. Ionizing radiation can directly break the chemical bonds of molecules in the virus body, and can also ionize water molecules. The radiolysis of water molecules will produce a variety of highly active free radicals. Although these free radicals exist for a very short time, they can react with surrounding proteins and nucleic acids, causing great destructive effects and indirectly causing damage to the virus[15]. However, some people believe that ionizing radiation mainly attacks the genome of the virus rather than its protein[16]. The inactivation effect of ionizing radiation is related to the radiation dose, and there are large differences between different viruses[16]. With the update and advancement of radiation source technology, ionizing radiation inactivation technology has also begun to be used in vaccine development, cold chain packaging disinfection in importing countries and other fields.

Is an inactivated virus an inactivated vaccine?

When talking about virus inactivation, we naturally cannot avoid the topic of inactivated vaccines. The purpose of vaccines is to enable our bodies to acquire antigen-specific adaptive immunity. With the help of adjuvants, the specific immunogenicity of vaccines allows us to establish specific immune memory. When the same pathogen invades next time, the body can quickly activate the corresponding immune system and quickly eliminate the pathogen[17]. In the establishment of vaccine-mediated immune memory, the immunogenicity of vaccines plays a key role, and inactivated vaccines are no exception.

Although inactivated viruses have certain safety guarantees, not all inactivated viruses still have the correct immunogenicity. From a molecular level, the retention of immunogenicity means the retention of the integrity and integrity of the virus surface protein. As can be seen from the above, the protein structure will inevitably be affected during the inactivation of the virus. Therefore, in the process of inactivated vaccine research and development, it is necessary to find a balance point, that is, to increase the immunogenicity of the virus as much as possible while ensuring the stable and complete inactivation of the virus. Since the beginning of the last century, scientists have been constantly trying to prepare inactivated vaccines with immunogenicity in different ways, and there are many vaccines that are already available and popular, including polio virus vaccine, hepatitis A virus vaccine and rabies vaccine.

At present, the inactivation method of inactivated vaccines is still mainly chemical reagents, and its preparation process is roughly as follows: virus amplification - virus inactivation - chemical reagent detoxification - virus purification - adjuvant addition - bottling. Although the process looks relatively traditional, there are still several major difficulties that need to be solved in the research and development process:

1. Before preparing the vaccine, it is necessary to obtain a stable seed strain with good immune effect;

2. It is necessary to select appropriate inactivating agents, dosages and treatment methods to ensure the immunogenicity of the vaccine;

3. The inactivated virus needs to be purified to high standards;

4. A high level of industrial competence is required to support ultra-large-scale rapid mass production.

These factors mean that it usually takes about 10 years for a general vaccine to be developed and marketed. However, due to the advancement of scientific research and technology and the urgency of time, less than a year and a half after my country isolated the first strain of the new coronavirus, its independently developed and marketed inactivated vaccine has obtained emergency use authorization from the WHO.

Compared with other vaccines, inactivated vaccines have the advantages of short R&D cycle, relatively mature technology, relatively guaranteed safety, and easy storage and transportation. However, inactivated vaccines also have some weaknesses. For example, in order to respond to the epidemic situation of rapid mutation of pathogens, it is often necessary to update vaccines against mutant strains. The update speed of nucleic acid vaccines may be faster than that of inactivated vaccines. In addition, for some viruses with poor antigenic stability, traditional inactivation methods will inevitably damage immunogenicity. Studies have found that after the new coronavirus was incubated with 0.05% β-propiolactone at 4°C for 36 hours, 74% of the protein conformations on its surface no longer had the correct immunogenicity [18]. This also indirectly shows that the development of inactivated vaccines still has great development potential.

The structure of the new coronavirus inactivated by paraformaldehyde[6] and β-propiolactone[18] and electron micrographs of adenovirus vaccine-induced antigen expression on the cell surface[19].

Figure A and Figure B: Compared with the virus fixed with paraformaldehyde, β-propiolactone inactivation may cause a huge conformational change in the spike protein, from an inverted triangle (blue in the pie chart) to a long column (orange in the pie chart). However, it does not rule out that this drastic change occurred during virus purification, because the spike protein treated with β-propiolactone has a higher degree of freedom than that treated with formaldehyde. However, this does not mean that the formaldehyde-inactivated virus is more immunogenic. Studies have shown that formaldehyde fixation may reduce the exposure of the receptor binding region on the spike protein, which in turn affects the induction of immune response [20].

Figure C: A type of nucleic acid vaccine - adenovirus vaccine can directly induce host cells to express antigens and stimulate the body's immune response. The antigens introduced by this method will not be damaged by the outside world. The picture shows a large number of antigens expressed on the surface of cells after being vaccinated with adenovirus vaccine.

Outlook

Inactivation is our weapon to proactively fight against viruses. In addition to direct disinfection, we can also use inactivated viruses to add an immune wall to ourselves. Finding an inactivation method that only destroys nucleic acids without changing the antigen structure, is low-cost, high-throughput, and non-toxic is the goal of improving the preparation process of inactivated virus vaccines. At present, a variety of physical inactivation methods have also shown great potential in vaccine development, and some even have advantages. First, physical inactivation does not introduce new toxicity and can eliminate the detoxification operation during preparation; in addition, physical inactivation has a broad spectrum and is more applicable to different types of viruses or viruses that can mutate rapidly. In addition, physical inactivation has the potential for research and development of retaining good immunogenicity, low preparation cost, and high throughput. At present, there are examples of using ionizing radiation to develop inactivated vaccines, such as influenza virus inactivated by gamma rays[15] and polio virus[21], which have shown certain development potential.

With the continuous accumulation of immunological theories and the continuous maturity of various inactivation technologies, I believe that in the near future, humans will be able to respond immediately when facing an epidemic, and will be able to quickly disinfect and prepare vaccines, actively fight back, and prevent the spread and transmission of the virus.

References

[1] Henderson, DA, The eradication of smallpox--an overview of the past, present, and future. Vaccine, 2011. 29 Suppl 4: p. D7-9.

[2] Lemon, SM and AA Mahmoud, The threat of pandemic influenza: are we ready? Biosecur Bioterror, 2005. 3(1): p. 70-3.

[3] Li, S., Cryo-electron tomography of enveloped viruses. Trends Biochem Sci, 2022. 47(2): p. 173-186.

[4] Wolff, G., et al., A molecular pore spans the double membrane of the coronavirus replication organelle. Science, 2020. 369(6509): p. 1395-1398.

[5] Delrue, I., et al., Inactivated virus vaccines from chemistry to prophylaxis: merits, risks and challenges. Expert Rev Vaccines, 2012. 11(6): p. 695-719.

[6] Yao, H., et al., Molecular Architecture of the SARS-CoV-2 Virus. Cell, 2020. 183(3): p. 730-738 e13.

[7] Uittenbogaard, JP, et al., Reactions of beta-propiolactone with nucleobase analogues, nucleosides, and peptides: implications for the inactivation of viruses. J Biol Chem, 2011. 286(42): p. 36198-214.

[8] Basak, D. and S. Deb, Sensitivity of SARS-CoV-2 towards Alcohols: Potential for Alcohol-Related Toxicity in Humans. Life (Basel), 2021. 11(12).

[9] Das, S., et al., The role of the envelope protein in the stability of a coronavirus model membrane against an ethanolic disinfectant. J Chem Phys, 2021. 154(24): p. 245101.

[10] Kratzel, A., et al., Inactivation of Severe Acute Respiratory Syndrome Coronavirus 2 by WHO-Recommended Hand Rub Formulations and Alcohols. Emerg Infect Dis, 2020. 26(7): p. 1592-1595.

[11] Marti, D., et al., Temperature effect on the SARS-CoV-2: A molecular dynamics study of the spike homotrimeric glycoprotein. Comput Struct Biotechnol J, 2021. 19: p. 1848-1862.

[12] Batejat, C., et al., Heat inactivation of the severe acute respiratory syndrome coronavirus 2. J Biosaf Biosecur, 2021. 3(1): p. 1-3.

[13] Heilingloh, CS, et al., Susceptibility of SARS-CoV-2 to UV irradiation. Am J Infect Control, 2020. 48(10): p. 1273-1275.

[14] van Duijl-Richter, MKS, et al., Chikungunya virus fusion properties elucidated by single-particle and bulk approaches. J Gen Virol, 2015. 96(8): p. 2122-2132.

[15] Bhatia, SS and SD Pillai, Ionizing Radiation Technologies for Vaccine Development - A Mini Review. Front Immunol, 2022. 13: p. 845514.

[16] Abolaban, FA and FM Djouider, Gamma irradiation-mediated inactivation of enveloped viruses with conservation of genome integrity: Potential application for SARS-CoV-2 inactivated vaccine development. Open Life Sci, 2021. 16(1): p. 558-570.

[17] Vetter, V., et al., Understanding modern-day vaccines: what you need to know. Ann Med, 2018. 50(2): p. 110-120.

[18] Liu, C., et al., The Architecture of Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM and Cryo-ET. Structure, 2020. 28(11): p. 1218-1224 e4.

[19] Watanabe, Y., et al., Native-like SARS-CoV-2 Spike Glycoprotein Expressed by ChAdOx1 nCoV-19/AZD1222 Vaccine. ACS Cent Sci, 2021. 7(4): p. 594-602.

[20] Bewley, KR, et al., Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques. Sci Adv, 2021. 7(37): p. eabg7996.

[21] Tobin, GJ, et al., A novel gamma radiation-inactivated sabin-based polio vaccine. PLoS One, 2020. 15(1): p. e0228006.

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