Method for inactivating herpes simplex virus by plasma-activated water

Abstract

The present application relates to the field of biomedical technology, specifically, a kind of plasma activated water inactivation herpes simplex virus method.The plasma activated water inactivation herpes simplex virus method includes the following steps: constructing plasma jet discharge reaction system;PAW preparation system is constructed, and plasma activated water is prepared using discharge system;Establish PAW inactivation HSV system, and construct the quantitative evaluation system of virus inactivation effect.In the present application, the chemical reaction process of different gas components at gas-liquid interface is studied, and the active substances generated in PAW are characterized by quantitative analysis method, so as to deepen the understanding of the generation mechanism of PAW, and the research basis of PAW generation effect and mechanism is applied to the inactivation research of HSV, and the action mechanism of PAW on HSV inactivation is proposed, which provides a new type of technical scheme with environmental friendliness and broad-spectrum high efficiency for the prevention and control of HSV.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular, to a method for inactivating herpes simplex virus by plasma-activated water. BACKGROUND

[0002] Herpes simplex virus (HSV) is a typical representative of the subfamily of alphaherpesvirinae, mainly invading the skin and nervous tissue system. As a typical enveloped DNA virus, the structure of HSV can be divided into three layers from outside to inside, which are envelope, capsid and linear double-stranded DNA in turn.

[0003] In recent years, studies have shown that the prevention and control strategy of herpes simplex virus (HSV) faces two key challenges.

[0004] One is the problem of viral drug resistance: the current first-line clinical treatment scheme mainly uses nucleoside analogs, among which acyclovir (ACV) is the most widely used. As a prodrug, this drug needs to be phosphorylated by viral-encoded thymidine kinase (TK) to form monophosphate product, and then further phosphorylated to acyclovir triphosphate (ACV-TP) under the action of host cell kinase. ACV-TP can inhibit viral DNA polymerase by competition and interfere with the viral genome replication process. However, this kind of drug is only effective for viruses in the acute infection period, and cannot eliminate the viral genome latent in neurons, leading to persistent infection of HSV. This persistent infection state may promote the generation of viral mutants, eventually leading to the emergence of drug-resistant strains.

[0005] The second is the problem of biological safety protection: the ineffectiveness of traditional disinfectants (such as ethanol, sodium hypochlorite, etc.) on non-enveloped viruses and the irritability to mucosal tissues, although traditional physical disinfection technology (such as ultraviolet irradiation) can destroy viral nucleic acid, it is difficult to penetrate the viral capsid structure, and the inactivation efficiency is limited, at the same time, there is a risk of damaging the genetic material of host cells. Therefore, it is of great clinical significance to develop an efficient and safe HSV-specific inactivation method.

[0006] In recent years, plasma-activated water (PAW) has gradually entered the field of view of scholars due to its unique physicochemical properties (weak acidity, high oxidation-reduction potential, and adjustable active ingredients) and its good local microenvironment adaptability under complex physiological conditions such as mucous membranes and wounds. PAW is a storable mixture produced by the interaction of low-temperature atmospheric plasma and water. Specifically, under the interaction of plasma, water, and gas, high concentrations of reactive oxygen species (ROS, such as ·OH, H2O2, and O3) and reactive nitrogen species (RNS, such as NO· and ONOO⁻) are generated in the system. The types and concentrations of active species in the PAW liquid phase depend on various factors, such as system configuration, gas composition, discharge electrode, applied voltage, discharge gap, and discharge time. The antiviral mechanism of PAW is mainly based on its direct damage to the virus structure. On the one hand, reactive oxygen species can attack the viral protein shell, leading to structural damage and dysfunction. On the other hand, reactive oxygen species can also penetrate the viral coat and damage the internal genome, thereby completely inactivating the virus. In addition, PAW has less irritation to mucosal tissue, which makes it potentially valuable for mucosal or wound treatment. It is worth noting that the genome type has an important influence on the virus inactivation effect of PAW. For example, RNA viruses are more susceptible to PAW attack due to their single-stranded structure, while DNA viruses have a more resistant double-stranded structure. However, current research on virus inactivation mainly focuses 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 effect of DNA viruses, especially HSV.

[0007] The existing technology paper: Plasma-activated water: Candidate hand disinfectant for SARS-CoV-2 transmission disruption (Plasma-activated water: Candidate hand disinfectant for SARS-CoV-2 transmission disruption) DOI: 10.1016 / j.heliyon.2024.e34337. This paper uses an atmospheric pressure plasma jet device (140 kHz / 20 kV, air flow 40 L / min) to treat ultrapure water for 1-5 minutes to prepare plasma-activated water (PAW), and systematically verifies the inactivation effect and mechanism of PAW on SARS-CoV-2 pseudovirus in a pig skin model (simulating human hand contamination).

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

[0009] (1) Gas component research limitations: Existing researches directly use air as the plasma working gas, and do not systematically evaluate the influence of different gas components (such as inert gas Ar, oxygen-rich environment, etc.) on the active species spectrum of PAW and the virus inactivation efficiency. Different gas sources can significantly change the generation ratio of RONS, thereby affecting the yield and stability of the main active species, limiting the universal understanding of the PAW action mechanism.

[0010] (2) Current researches focus on RNA viruses such as SARS-CoV-2 and Newcastle disease, while DNA viruses (such as HSV) may be resistant to the penetration of active oxygen and nucleic acid damage due to their high genomic structural stability. The applicability of PAW technology to complex pathogens still needs to be systematically evaluated. SUMMARY

[0011] The purpose of the present application is to provide a method for inactivating herpes simplex virus by plasma-activated water, in order to solve the problems of gas component research limitations and applicability to complex pathogens raised in the background art.

[0012] To achieve the above-mentioned purpose, the present application provides a method for inactivating herpes simplex virus by plasma-activated water, which comprises the following steps:

[0013] S1, constructing a plasma jet discharge reaction system;

[0014] S2, constructing a PAW preparation system and preparing plasma-activated water using the discharge system;

[0015] S3, establishing a PAW inactivation HSV system and constructing a quantitative evaluation system for virus inactivation effect.

[0016] As a further improvement of the technical solution, the plasma jet discharge reaction system in step S1 comprises a quartz reaction chamber, a high-voltage power supply, a high-voltage electrode, a grounding electrode, a gas cylinder and a glass rotor flowmeter.

[0017] As a further improvement of the technical solution, the specific operation steps of step S2 are as follows: 10 ml of sterile water is added to the quartz reaction chamber, the discharge power is controlled at 35 W, the gas is introduced, and the discharge treatment is carried out for 5 minutes to prepare plasma-activated water (PAW).

[0018] As a further improvement of the technical solution, the specific operation steps of step S3 are as follows: HSV suspension with a titer of 6log 10 PCU / mL is mixed with PAW at a volume ratio of 1:2, treated at 25℃ constant temperature oscillation for 12 minutes, and the HSV suspension treated with deionized water is used as a negative control. The virus titer is detected by plaque assay method, and the inactivation rate is calculated.

[0019] As a further improvement of the technical solution, the gas introduced in step S2 is one of air, oxygen or argon.

[0020] As a further improvement of the technical solution, the PAW in step S3 is stored at 4℃ and has a shelf life of 9 days, and is periodically sampled to detect its inactivation effect.

[0021] As a further improvement of the technical solution, the HSV suspension mixed with PAW at a volume ratio of 1:2 in step S3 is quenched with sodium thiosulfate solution to inactivate active oxygen, and the reaction is terminated by ice bath.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] 1. The present application helps to efficiently utilize characteristic active substances by evaluating the influence of different gas components on the yield of PAW active substances, thereby improving the inactivation effect on HSV. At the same time, through the study of the components of PAW generated by different gas components, the present application can provide guidance for the targeted generation of specific reactive oxygen species (ROS) and reactive nitrogen species (RNS) substances, and through in-depth exploration of the formation mechanism of PAW, it provides theoretical support and practical guidance for further optimization of PAW preparation conditions and improvement of viral inactivation efficiency.

[0024] 2. In the present application, the inactivation effect of PAW with reasonable component regulation on DNA virus (HSV) is verified, which helps to promote the applicability of PAW for viral inactivation, expands the application range of PAW in the field of biomedicine, and especially shows significant potential in the prevention and control of DNA viruses such as HSV.

[0025] 3. The present application provides a safer and more efficient viral inactivation method, which has lower biological safety risk and environmental burden compared with traditional chemical disinfectants and physical disinfection techniques. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a schematic diagram of the plasma jet discharge reaction system of the present application.

[0027] Figure 2 It is a graph showing the relationship between the intensity and wavelength of plasma under different gases of the present application.

[0028] Figure 3 It is a schematic diagram of the change of NO2- concentration in PAW of the present application.

[0029] Figure 4 It is a schematic diagram of the change of NO3- concentration in PAW of the present application.

[0030] Figure 5 It is a schematic diagram of the change of H2O2 concentration in PAW of the present application.

[0031] Figure 6 The figure is a schematic diagram of the change of O3 concentration in the PAW of the present application.

[0032] Figure 7 The figure is a schematic diagram of the change of pH value in the PAW of the present application.

[0033] Figure 8 The figure is a schematic diagram of the inactivation effect of the PAW of the present application on HSV under air condition.

[0034] Figure 9 The figure is a schematic diagram of the inactivation effect of the PAW of the present application on HSV under oxygen condition.

[0035] Figure 10 The figure is a schematic diagram of the inactivation effect of the PAW of the present application on HSV under argon condition.

[0036] Figure 11 The figure is a schematic diagram of the stability experiment of the PAW of the present application.

[0037] Figure 12 The figure is a flow chart of the method for inactivating herpes simplex virus of the present application.

[0038] In the figure: 1, high-voltage power supply; 2, voltage probe; 3, oscilloscope; 4, current probe; 5, grounding electrode; 6, high-voltage electrode; 7, glass rotor flowmeter; 8, gas cylinder; 9, quartz reaction chamber. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0040] In a specific embodiment, as shown in Figures 1-12 The present application provides a method for inactivating herpes simplex virus by using plasma-activated water, which mainly comprises constructing a plasma jet discharge reaction system, constructing a PAW preparation system, preparing plasma-activated water by using the discharge system, establishing a PAW inactivation HSV system, and constructing a quantitative evaluation system for virus inactivation effect. Figure 1 The figure is a schematic diagram of the plasma jet discharge reaction system device.

[0041] (1) Constructing a plasma jet discharge reaction system.

[0042] The plasma jet discharge reaction system comprises a quartz reaction chamber 9, a high-voltage power supply 1, a high-voltage electrode 6, a grounding electrode 5, the quartz reaction chamber 9, a gas cylinder 8 and a glass rotor flowmeter 7. The discharge reaction zone is formed between the high-voltage electrode 6 and the grounding electrode 5. The gas flow rate into 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 oscilloscope 3 is used to record the plasma discharge voltage and current by means of the current probe 4 and the voltage probe 2. As shown in Figure 1 The gas cylinder 8 is connected with the high-voltage electrode 6 inside the quartz reaction chamber 9 through the glass rotor flowmeter 7. The high-voltage electrode 6 and the grounding electrode 5 are located inside the quartz reaction chamber 9, and the high-voltage electrode 6 is connected with the high-voltage power supply 1. The high-voltage power supply 1 is connected with the grounding electrode 5, and the current probe 4 is connected between the high-voltage power supply 1 and the grounding electrode 5. The voltage probe 2 is connected between the high-voltage electrode 6 and the high-voltage power supply 1. The voltage probe 2 and the current probe 4 are connected to the oscilloscope 3.

[0043] (2) Constructing the PAW preparation system, and using the discharge system to prepare the plasma activated water and establish the HSV inactivation system by PAW.

[0044] 10 ml of sterile water is added into the quartz reaction chamber, and the discharge is carried out under different gases to prepare the PAW under different conditions. The discharge power is controlled to be 35 W, and the discharge time is adjusted. Different gases (air, oxygen, argon) are introduced. In order to distinguish, the PAW prepared by discharging into air is marked as PAW-Air, the PAW prepared by discharging into argon is marked as PAW-Ar, and the PAW prepared by discharging into oxygen is marked as PAW-O2.

[0045] (3) Constructing the quantitative evaluation system of virus inactivation effect.

[0046] The HSV suspension with a titer of about 6 log 10 PCU / mL is treated with the preferred PAW (discharge time 5 min, power 35 W, and discharge gas air) for a certain time (mixed at a volume ratio of 1:2). The HSV suspension treated with deionized water is used as a negative control. The plaque assay method is used to detect the virus titer, and the inactivation rate is calculated. In addition, in order to determine the stable period of PAW, the prepared PAW is stored at 4℃, and the effective period is 9 days. The inactivation effect is detected by taking samples regularly.

[0047] The beneficial effects of the present application are illustrated by the specific embodiments as follows:

[0048] In Example 1, the discharge power is controlled to be 35 W, and the discharge time is 0, 1, 3, 7, 9 and 11 min respectively. The discharge gas environment is air, oxygen and argon respectively (the plasma emission spectrum diagrams are shown in Figure 2 , Figure 3 and Figure 4). The concentrations of nitrite (NO2 - ), nitrate (NO3 - ), hydrogen peroxide (H2O2), and ozone (O3) in PAW were detected to provide a reference for the effect of gas components on HSV inactivation by PAW. As shown in Figures 3-7 , the following is a specific analysis and explanation of the relevant results:

[0049] (1) As shown in Figure 3 and Figure 4 , when the gas introduced is argon and oxygen, the concentrations of NO2 - and NO3 - are low and almost undetectable. When the gas introduced is air, the concentrations of NO2 - and NO3 - in PAW increase with increasing activation time. When the activation time is only 1 min, the concentrations of NO2 - and NO3 - in PAW-Air increase from 0 mg / L to 1.94 mg / L and 26.63 mg / L, respectively. When the activation time is 11 min, the content of NO3 - reaches 71.53 mg / L, and the content of NO2 - increases to 9.84 mg / L. The reasons for the generation of NO2 - and NO3 - in plasma-activated water are that plasma discharge generates NO and NO2 at the gas-liquid interface, which is then generated through a series of addition reactions with O2, ·OH, and H2O in the liquid phase;

[0050] (2) The changes in the concentration of H2O2 in this example are shown in Figure 5 . When the activation time is 1 min, the concentrations of H2O2 in PAW-Air and PAW-Ar increase from 0 mg / L to 3.29 mg / L and 13.15 mg / L, respectively. The maximum values are 6.42 mg / L and 17.07 mg / L, respectively, after 5 min of activation time. The concentration of H2O2 in PAW-Ar is higher than that in PAW-Air when the activation time is the same, and it is basically undetectable in PAW-O2;

[0051] (3) The changes in the concentration of O3 in this example are shown in Figure 6 . The concentration of O3 produced under air conditions is relatively high, while the concentrations of O3 produced under the other two gas conditions are basically negligible. Figure 7For the result of pH change, the pH value of PAW under air condition decreased rapidly after 1 min of activation, from 7.07 at the initial time to 4.24; when the activation time reached 11 min, the pH value in PAW decreased to 2.78. The obvious change of pH was derived from the NOx product generated by plasma activation, and the decrease of pH under air condition enabled O3 to exhibit a higher concentration (compared to O2 and Ar gas) because O3 was difficult to decompose under acidic conditions.

[0052] Example 2: In order to comprehensively evaluate the influence of different discharge gas environments on the effect of HSV inactivation by PAW, HSV-1 inactivation experiments were also conducted. The above-mentioned preferred conditions (control discharge time 5 min, power 35 W, and discharge gas air, oxygen, and argon, respectively) were used. HSV-1 was amplified in Vero cells, purified by centrifugation (20% sucrose cushion, 100,000 x g, 2 h), resuspended in PBS (10 6 PFU / mL), and then the HSV-1 virus suspension (initial titer ≥10 6 PFU / mL) was mixed with PAW at a volume ratio of 1:2, treated in a constant-temperature shaker at 25 °C with 150 rpm oscillation (treatment for 0, 3, 6, 9, and 12 min, respectively, counted as PAW-0, PAW-3, PAW-6, PAW-9, and PAW-12); then sodium thiosulfate solution was added to quench active oxygen, and the reaction was immediately terminated by ice bath; the treated virus was inoculated into Vero cell monolayers, covered with 1.5% methyl cellulose, and after 48 hours, crystal violet staining was performed, and plaque assay was used to detect the residual virus titer and calculate the inactivation rate. The relevant results are shown in Figures 8-10 The following is a specific analysis and explanation of the relevant results:

[0053] (1) Under the three gas conditions, PAW produced by air activation showed the best HSV inactivation effect, with an inactivation efficiency of 99.98% at 9 min and further achieving an inactivation efficiency of 99.99% at 12 min, reducing the initial virus load of 10 6 PFU / mL (million level) to <10² PFU / mL (hundred level), which was much lower than the HSV infection threshold (studies have shown that >10³ PFU / mL is infectious). In comparison, PAW produced by oxygen activation also showed good HSV inactivation effect, but was inferior to air. PAW produced by argon activation did not have an ideal HSV inactivation effect;

[0054] (2) Combined with the aforementioned exploration of the components of PAW prepared under different gas conditions, it can be inferred that the inactivation factor of PAW for HSV is derived from lower pH, higher concentration of NOx, and O3.

[0055] Example 3: The stability of PAW in practical application is also considered in the present application. After the preparation of PAW, it was stored at room temperature and 4℃ refrigeration environment respectively, and samples were taken regularly to detect its inactivation effect on HSV. The experimental results show (as shown in Figure 11 ), under the condition of 4℃ refrigeration, the virus inactivation effect of PAW can be maintained for 9 days (inactivation rate remains above 99.99%), and there is a certain degree of decline on the 12th day, but it can still reduce the virus titer by 3log 10 PCU / mL, and the inactivation rate remains above 99.98%. It is speculated that the decrease of the antiviral activity of PAW may be due to the decay of active substances during storage. The long-term storage characteristics of PAW may be related to the long-lived active substances present in the solution and the short-lived active substances continuously produced by certain reactions. This finding also confirms that PAW has a storage property, providing a theoretical basis and technical feasibility for its practical application.

[0056] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the present application is not limited by the above examples, and the above examples and descriptions in the specification are only preferred examples of the present application and are not intended to limit the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for inactivating herpes simplex virus by plasma-activated water, characterized in that, The method includes the following steps: Step S1: Construct a plasma jet discharge reaction system; Step S2: Construct a plasma-activated water preparation system and prepare plasma-activated water using a discharge system; Step S3: Establish a plasma-activated water inactivation system for HSV and construct a quantitative evaluation system for virus inactivation effectiveness; 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 grounding 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 grounding 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 were recorded using an oscilloscope (3) with a current probe (4) and a voltage probe (2); The specific operation steps of step S2 are as follows: add 10ml of sterile water to the quartz reaction chamber, control the discharge power to 35W, introduce gas, discharge for 5 minutes, and obtain plasma-activated water. The gas introduced in step S2 is air or oxygen.

2. The method for inactivating herpes simplex virus with plasma-activated water according to claim 1, characterized in that, The specific steps of step S3 are as follows: The titer is 6log... 10 HSV suspension with PCU / mL was mixed with plasma-activated water, and HSV suspension treated with deionized water was used as a negative control. Virus titer was detected by plaque analysis and inactivation rate was calculated.

3. The method for inactivating herpes simplex virus with plasma-activated water according to claim 2, characterized in that, HSV suspension and plasma-activated water are mixed at a volume ratio of 1:2; and / or The HSV suspension was treated with plasma-activated water with a discharge time of 5 min, a power of 35 W, and the discharge gas was air; and / or HSV suspension was mixed with plasma-activated water and treated with constant temperature oscillation at 25°C for 12 minutes.

4. The method for inactivating herpes simplex virus with plasma-activated water according to claim 3, characterized in that, The plasma-activated water in step S3 is refrigerated at 4°C and has a shelf life of 9 days. Samples are taken periodically to test its inactivation effect.

5. The method for inactivating herpes simplex virus with plasma-activated water according to claim 3, characterized in that, In step S3, the HSV suspension, after being mixed with plasma-activated water at a volume ratio of 1:2, is quenched with sodium thiosulfate solution to quench active oxygen, and the reaction is terminated in an ice bath.

Images