Method for inactivating herpes simplex virus through plasma activated water

Abstract

The invention relates to the technical field of biomedicine, in particular to a method for inactivating herpes simplex virus through plasma activated water. The method for inactivating the herpes simplex virus by the plasma activated water comprises the following steps: constructing a plasma jet discharge reaction system; constructing a PAW preparation system, and preparing plasma activated water by using a discharge system; a PAW inactivation HSV system is established, and a quantitative evaluation system of the virus inactivation effect is constructed. According to the present invention, the chemical reaction process of different gas components on the gas-liquid two-phase interface is researched, and the quantitative analysis method is adopted to characterize the active substances generated in the PAW so as to deepen the understanding of the PAW generation mechanism, and the research basis of the PAW generation effect and the PAW generation mechanism is applied to the HSV inactivation research so as to provide the important significance for the HSV inactivation research. An action mechanism of PAW on HSV inactivation is provided, and a novel technical scheme with environmental friendliness, broad spectrum and high efficiency is provided for prevention and control of HSV.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, in particular to a method for inactivating herpes simplex virus by using plasma activated water. Background Art

[0002] Herpes simplex virus (HSV) is a representative of the alphaherpesvirus subfamily, primarily affecting the skin and nervous system. As a typical enveloped DNA virus, HSV's structure can be divided into three layers: the envelope, the capsid, and the linear double-stranded DNA.

[0003] Studies in recent years have shown that herpes simplex virus (HSV) prevention and control strategies face two key challenges.

[0004] First, there's the issue of viral resistance. Currently, first-line clinical treatment primarily utilizes nucleoside analogs, with acyclovir (ACV) being the most widely used. As a prodrug, this drug undergoes phosphorylation by virally encoded thymidine kinase (TK) to form a monophosphate product, which is then further phosphorylated by host cell kinases to acyclovir triphosphate (ACV-TP). ACV-TP competitively inhibits viral DNA polymerase, interfering with viral genome replication. However, this class of drugs is only effective against the virus during the acute infection phase and is unable to eliminate viral genomes lurking in neurons, leading to persistent HSV infection. This persistent infection may promote the development of viral mutations, ultimately leading to the emergence of drug-resistant strains.

[0005] Second, there's the issue of biosafety. Traditional disinfectants (such as ethanol and sodium hypochlorite) are ineffective against non-enveloped viruses and can be irritating to mucosal tissues. While traditional physical disinfection techniques (such as ultraviolet irradiation) can destroy viral nucleic acids, they struggle to penetrate the viral capsid, resulting in limited inactivation efficiency and the risk of damaging host cell genetic material. Therefore, developing efficient and safe HSV-specific inactivation methods is of great clinical significance.

[0006] In recent years, plasma-activated water (PAW) has gradually attracted attention due to its unique physicochemical properties (weak acidity, high redox potential, and adjustable active ingredients) and its excellent adaptability to the complex physiological microenvironments of mucosa and wound surfaces. PAW is a storable mixture produced by the interaction of low-temperature atmospheric plasma with water. Specifically, the interaction of plasma with water and gas generates high concentrations of reactive oxygen species (ROS, such as ·OH, H2O2, and O3) and reactive nitrogen species (RNS, such as NO· and ONOO⁻). The types and concentrations of reactive species in the PAW liquid phase depend on various factors, including system configuration, gas composition, discharge electrodes, applied voltage, discharge gap, and discharge time. The antiviral mechanism of PAW is primarily based on its direct destruction of viral structure. On the one hand, reactive oxygen species can attack the viral protein coat, causing structural damage and functional impairment. On the other hand, reactive oxygen species can penetrate the viral capsid and destroy the viral genome, thereby completely inactivating the virus. Furthermore, PAW is minimally irritating to mucosal tissue, making it potentially useful in mucosal or wound treatment. It is worth noting that genome type has a significant impact on the viral inactivation efficacy of PAW. For example, RNA viruses are more susceptible to PAW attack due to their single-stranded structure, while DNA viruses are more resistant due to their double-stranded structure. However, current research on viral inactivation has primarily focused on RNA viruses (such as Newcastle disease virus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and human coronavirus 229E), and there is a lack of systematic research on the inactivation efficacy of DNA viruses (especially HSV).

[0007] Prior art paper: Plasma-activated water: Candidate hand disinfectant for SARS-CoV-2 transmission disruption (DOI: 10.1016 / j.heliyon.2024.e34337). This paper systematically validates the inactivation efficacy and mechanism of plasma-activated water (PAW) against SARS-CoV-2 pseudovirus in a porcine skin model (simulating human hand contamination) by treating ultrapure water with an atmospheric pressure plasma jet device (140 kHz / 20 kV, 40 L / min air flow).

[0008] At the same time, the existing technology has the following defects:

[0009] (1) Limitations of gas component research: Existing studies have mostly used air as the plasma working gas, and have not systematically evaluated the effects of different gas components (such as inert gas Ar, oxygen-rich environment, etc.) on the spectrum of PAW active substances and the virus inactivation efficiency. Different gas sources may significantly change the RONS generation ratio, thereby affecting the yield and stability of the main active substances, limiting the universal understanding of the mechanism of action of PAW.

[0010] (2) Current research focuses on RNA viruses such as SARS-CoV-2 and Newcastle disease. DNA viruses (such as HSV) may be resistant to the penetration and nucleic acid damage of reactive oxygen species due to their high genome structural stability. The applicability of PAW technology to complex pathogens still needs to be systematically evaluated. Summary of the Invention

[0011] The object of the present invention is to provide a method for inactivating herpes simplex virus by using plasma-activated water, so as to solve the problems of limitations in gas component research and applicability to complex pathogens raised in the above-mentioned background technology.

[0012] To achieve the above object, the present invention provides a method for inactivating herpes simplex virus using plasma-activated water, the method comprising the following steps: S1. Construct a plasma jet discharge reaction system; S2. Construct a PAW preparation system and use the discharge system to prepare plasma-activated water; S3. Establish a PAW HSV inactivation system and construct a quantitative evaluation system for the virus inactivation effect.

[0013] As a further improvement of the present technical solution, the plasma jet discharge reaction system in step S1 includes a quartz reaction chamber, a high-voltage power supply, a high-voltage electrode, a ground electrode, a gas cylinder and a glass rotor flowmeter.

[0014] As a further improvement of the present technical solution, the specific operation steps of step S2 are: adding 10 ml of sterile water to the quartz reaction chamber, controlling the discharge power to 35 W, introducing gas, and performing discharge treatment for 5 minutes to produce plasma activated water (PAW).

[0015] As a further improvement of this technical solution, the specific operation steps of step S3 are: 10 The HSV suspension with PCU / mL was mixed with PAW at a volume ratio of 1:2 and treated with constant temperature shaking at 25°C for 12 minutes. The HSV suspension treated with deionized water was used as a negative control. The virus titer was detected by plaque assay and the inactivation rate was calculated.

[0016] As a further improvement of the present technical solution, the gas introduced in step S2 is one of air, oxygen or argon.

[0017] As a further improvement of the present technical solution, the PAW in step S3 is refrigerated and stored at 4° C. with a shelf life of 9 days, and samples are taken regularly to detect the inactivation effect.

[0018] As a further improvement of the present technical solution, the HSV suspension mixed with PAW at a volume ratio of 1:2 in step S3 is quenched with a sodium thiosulfate solution to quench active oxygen, and the reaction is terminated in an ice bath.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. By evaluating the effects of different gas components on the yield of PAW active substances, the present invention helps to efficiently utilize characteristic active substances and improve the inactivation effect on HSV. At the same time, by studying the components of PAW produced by different gas components, the present invention can provide guidance for the targeted promotion of the generation of specific reactive oxygen species (ROS) and reactive nitrogen species (RNS). By deeply exploring the formation mechanism of PAW, the present invention provides theoretical support and practical guidance for further optimizing PAW preparation conditions and improving virus inactivation efficiency.

[0020] 2. In the present invention, the inactivation effect of PAW with rationally regulated components on DNA viruses (HSV) was verified, which helps to promote the applicability of PAW for virus inactivation and expand the application scope of PAW in the biomedical field, especially showing significant potential in the prevention and control of DNA viruses such as HSV.

[0021] 3. The present invention provides a safer and more efficient means of virus inactivation, which has lower biosafety risks and environmental burdens compared to traditional chemical disinfectants and physical disinfection technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the plasma jet discharge reaction system of the present invention.

[0023] Figure 2 Graph showing the relationship between plasma intensity and wavelength under different gases of the present invention.

[0024] Figure 3 Schematic diagram of the change in NO2- concentration in the PAW of the present invention.

[0025] Figure 4 Schematic diagram of the change in NO3- concentration in the PAW of the present invention.

[0026] Figure 5 Schematic diagram of the change in H2O2 concentration in the PAW of the present invention.

[0027] Figure 6 Schematic diagram of the change of O3 concentration in the PAW of the present invention.

[0028] Figure 7 Schematic diagram of pH change in the PAW of the present invention.

[0029] Figure 8 Schematic diagram of the inactivation effect of PAW of the present invention on HSV under air conditions.

[0030] Figure 9 Schematic diagram of the inactivation effect of PAW of the present invention on HSV under oxygen conditions.

[0031] Figure 10 Schematic diagram of the inactivation effect of PAW of the present invention on HSV under argon conditions.

[0032] Figure 11 Schematic diagram of the PAW stability experiment of the present invention.

[0033] Figure 12 The present invention is a flow chart of the method for inactivating herpes simplex virus.

[0034] In the figure: 1. High-voltage power supply; 2. Voltage probe; 3. Oscilloscope; 4. Current probe; 5. Ground electrode; 6. High-voltage electrode; 7. Glass rotor flowmeter; 8. Gas cylinder; 9. Quartz reaction chamber. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] In a specific embodiment, Figures 1-12 As shown, the present invention provides a method for inactivating herpes simplex virus by using plasma activated water. The method mainly includes constructing a plasma jet discharge reaction system, constructing a PAW preparation system, and using the discharge system to prepare plasma activated water and establish a PAW inactivation HSV system, and constructing a quantitative evaluation system for the virus inactivation effect. Figure 1 Schematic diagram of the plasma jet discharge reaction system.

[0037] (1) Construct a plasma jet discharge reaction system.

[0038] The plasma jet discharge reaction system includes a quartz reaction chamber 9, a high-voltage power supply 1, a high-voltage electrode 6, a ground electrode 5, a quartz reaction chamber 9, a gas cylinder 8, and a glass rotor flowmeter 7. A discharge reaction zone is formed between the high-voltage electrode 6 and the ground electrode 5; the gas flow rate entering the quartz reaction chamber 9 is controlled by the gas cylinder 8, and the discharge parameters used in the reaction are adjusted by the high-voltage power supply 1. In addition, an oscilloscope 3 is used to record the plasma discharge voltage and current with a current probe 4 and a voltage probe 2, as shown in FIG. Figure 1 As shown, the gas cylinder 8 is connected to the high-voltage electrode 6 inside the quartz reaction chamber 9 through the glass rotor flowmeter 7; the high-voltage electrode 6 and the ground electrode 5 are located inside the quartz reaction chamber 9, and the high-voltage electrode 6 is connected to the high-voltage power supply 1; the high-voltage power supply 1 is connected to the ground electrode 5, and a current probe 4 is connected between the high-voltage power supply 1 and the ground electrode 5, a voltage probe 2 is connected between the high-voltage electrode 6 and the high-voltage power supply 1, and the voltage probe 2 and the current probe 4 are connected to the oscilloscope 3.

[0039] (2) Construct a PAW preparation system, use the discharge system to prepare plasma-activated water, and establish a PAW inactivation HSV system.

[0040] 10ml of sterile water was added to the quartz reaction chamber, and discharges were performed under different gas conditions to prepare PAWs under different conditions. The discharge power was controlled at 35W, and the discharge time was adjusted to varying degrees. Different gases (air, oxygen, and argon) were introduced. For easy identification, PAWs produced by discharge with air are designated PAW-Air, those produced by discharge with argon are designated PAW-Ar, and those produced by discharge with oxygen are designated PAW-O2.

[0041] (3) Establish a quantitative evaluation system for virus inactivation effects.

[0042] The titer is about 6 log 10 An HSV suspension with a PCU / mL concentration was treated with a preferred PAW (discharge time 5 min, power 35 W, air as the discharge gas) for a specified period of time (mixed in a 1:2 volume ratio). An HSV suspension treated with deionized water served as a negative control. Viral titer was determined using a plaque assay, and inactivation efficiency was calculated. Furthermore, to determine the stability of the PAW, the prepared PAW was refrigerated and stored at 4°C with a shelf life of 9 days. Samples were collected regularly to monitor its inactivation efficacy.

[0043] The beneficial effects of the present invention are described below through specific embodiments: Example 1: The discharge power is controlled to be 35W, the discharge time is 0, 1, 3, 7, 9, 11 min, and the discharge gas environment is air, oxygen, and argon (the plasma emission spectra are shown in Figure 1). Figure 2 、 Figure 3 and Figure 4By detecting nitrite (NO2 - ), nitrate (NO3 - ), hydrogen peroxide (H2O2), and ozone (O3) concentrations to provide a reference for the effect of gas components on PAW inactivation of HSV. Figure 3-Figure 7 The following is a detailed analysis and explanation of the relevant results: (1) If Figure 3 and Figure 4 As shown in the figure, when the gases introduced are argon and oxygen, NO2 - and NO3 - The concentration is low and almost undetectable. When the inlet gas is air, the NO2 in PAW - and NO3 - The concentration of NO2 in PAW-Air increases with the activation time. When the activation time is only 1 min, the concentration of NO2 in PAW-Air increases with the activation time. - and NO3 - The concentration increased from 0 mg / L to 1.94 mg / L and 26.63 mg / L respectively. When the activation time was 11 min, NO3 - The content of NO2 can reach 71.53 mg / L, while NO2 - The content rises to 9.84 mg / L. Plasma activation produces NO2 in water - and NO3 - The reason is that plasma discharge generates NO and NO2 at the gas phase interface, which are then generated through a series of addition reactions with O2, ·OH and H2O in the liquid phase; (2) The change of H2O2 concentration in this embodiment is as follows Figure 5 As shown in the figure, when the activation time is 1 minute, the H2O2 concentration in PAW-Air and PAW-Ar increases from 0 mg / L to 3.29 mg / L and 13.15 mg / L, respectively; after 5 minutes of activation, it reaches its maximum value of 6.42 mg / L and 17.07 mg / L, respectively. At the same activation time, the H2O2 concentration in PAW-Ar is higher than that in PAW-Air, while it is basically undetectable in PAW-O2. (3) The change of O3 concentration in this embodiment is as follows Figure 6 As shown in the figure, only the O3 concentration produced under the air condition is high, while the O3 concentrations produced under the other two gas conditions are basically negligible. Figure 7The pH of the PAW decreased rapidly after 1 minute of activation under air conditions, from an initial pH of 7.07 to 4.24. After 11 minutes of activation, the pH in the PAW decreased to 2.78. This significant change in pH is due to the production of NOx by plasma activation. Since O3 is difficult to decompose under acidic conditions, the decrease in pH under air conditions allows O3 to be present at a higher concentration (compared to O2 and Ar gas).

[0044] Example 2: To comprehensively evaluate the effects of different discharge gas environments on the inactivation of HSV by PAW, an HSV-1 inactivation experiment was also conducted. Under the above-mentioned preferred conditions (discharge time 5 min, power 35 W, discharge gas 50 W, air, oxygen, and argon), HSV-1 was amplified in Vero cells, purified by centrifugation (20% sucrose cushion, 100,000 × g, 2 h), and resuspended in PBS (10 6 PFU / mL), and then HSV-1 virus suspension (initial titer ≥10 6 PFU / mL) was mixed with PAW at a volume ratio of 1:2 and shaken at 150 rpm at 25°C (treatment time 0, 3, 6, 9, and 12 min, respectively, designated as PAW-0, PAW-3, PAW-6, PAW-9, and PAW-12). Sodium thiosulfate solution was then added to quench the active oxygen species, and the reaction was immediately terminated by ice bath. The treated virus was inoculated into a Vero cell monolayer and covered with 1.5% methylcellulose. After 48 hours, the virus was stained with crystal violet, and the residual virus titer was detected by plaque assay and the inactivation rate was calculated. Figures 8-10 The following is a detailed analysis and explanation of the relevant results: (1) Under the three gas conditions, the PAW produced by air activation showed the best HSV inactivation effect, showing an inactivation efficiency of 99.98% at 9 minutes and further achieving an inactivation efficiency of 99.99% at 12 minutes, which reduced the initial viral load to 10 6 The PFU / mL (millions) dropped to <10² PFU / mL (hundreds), well below the HSV infection threshold (research shows >10³ PFU / mL is considered infectious). In comparison, PAW activated with oxygen also demonstrated good HSV inactivation, but was inferior to air. Argon-activated PAW, on the other hand, was less effective at inactivating HSV. (2) Combined with the above research on the PAW components prepared under different gas conditions, it can be inferred that the inactivation factors of PAW for HSV come from lower pH, higher concentrations of NOx and O3.

[0045] Example 3: The present invention also takes into account the stability of PAW in practical applications. After the PAW was prepared, it was stored in a room temperature and 4°C refrigerated environment, and samples were taken regularly to detect its inactivation effect on HSV. The experimental results showed that ( Figure 11 ), under refrigerated conditions at 4°C, the virus inactivation effect of PAW can be maintained for 9 days (the inactivation rate remains above 99.99%), and there is a certain degree of decline on the 12th day, but the virus titer can still be reduced by 3 log 10 PCU / mL and above, with an inactivation rate maintained above 99.98%. It is speculated that the reduced antiviral activity of PAW may be due to the decay of its active substances during storage. The long-term storage properties of PAW may be related to the presence of long-lived active substances in the solution and the continuous production of short-lived active substances by certain reactions. This finding also confirms the storability of PAW, providing theoretical basis and technical feasibility for its practical application.

[0046] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely preferred examples of the present invention and are not intended to limit the present invention. Various changes and improvements may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and improvements fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for inactivating herpes simplex virus using plasma activated water, characterized in that: The method comprises the following steps: S1. Construct a plasma jet discharge reaction system; S2. Construct a PAW preparation system and use the discharge system to prepare plasma-activated water; S3. Establish a PAW HSV inactivation system and construct a quantitative evaluation system for the virus inactivation effect.

2. The method for inactivating herpes simplex virus using plasma-activated water according to claim 1, wherein: The plasma jet discharge reaction system in step S1 includes a quartz reaction chamber (9), a high-voltage power supply (1), a high-voltage electrode (6), a ground electrode (5), a gas cylinder (8) and a glass rotor flowmeter (7); A discharge reaction zone is formed between the high voltage electrode (6) and the ground electrode (5); The gas flow rate entering the quartz reaction chamber (9) is controlled by the gas cylinder (8), and the discharge parameters used in the reaction are adjusted by the high voltage power supply (1); In addition, the plasma discharge voltage and current are recorded using an oscilloscope (3) with a current probe (4) and a voltage probe (2).

3. The method for inactivating herpes simplex virus using plasma-activated water according to claim 1 or 2, wherein: The specific operation steps of step S2 are: adding 10 ml of sterile water into the quartz reaction chamber, controlling the discharge power to 35 W, introducing gas, and performing discharge treatment for 5 minutes to prepare plasma activated water (PAW).

4. The method for inactivating herpes simplex virus with plasma-activated water according to claim 1, wherein: The specific operation steps of step S3 are: 10 PCU / mL HSV suspension was mixed with PAW, and HSV suspension treated with deionized water was used as a negative control. The virus titer was detected by plaque assay and the inactivation rate was calculated.

5. The method for inactivating herpes simplex virus using plasma-activated water according to claim 4, wherein: The HSV suspension is mixed with PAW in a volume ratio of 1:2; and / or When the HSV suspension is treated with PAW, the discharge time is 5 minutes, the power is 35 W, and the discharge gas is air; and / or The HSV suspension was mixed with PAW and shaken at 25°C for 12 minutes.

6. The method for inactivating herpes simplex virus using plasma-activated water according to claim 3, wherein: The gas introduced in step S2 is one of air, oxygen or argon.

7. The method for inactivating herpes simplex virus using plasma-activated water according to claim 4, wherein: The PAW in step S3 is refrigerated and stored at 4° C. with a shelf life of 9 days, and samples are taken regularly to detect the inactivation effect.

8. The method for inactivating herpes simplex virus using plasma-activated water according to claim 4, wherein: The HSV suspension mixed with PAW in a volume ratio of 1:2 in step S3 was quenched with sodium thiosulfate solution to quench active oxygen, and the reaction was terminated in an ice bath.

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