Inactivation Efficacy of Nonthermal Plasma-Activated Solutions against Newcastle Disease Virus

ABSTRACT In recent years, plasma-activated solutions (PASs) have made good progress in the disinfection of medical devices, tooth whitening, and fruit preservation. In this study, we investigated the inactivation efficacy of Newcastle disease virus by PASs. Water, 0.9% NaCl, and 0.3% H2O2 were excited by plasma to obtain the corresponding solutions PAS(H2O), PAS(NaCl), and PAS(H2O2). The complete inactivation of virus after PAS treatment for 30 min was confirmed by the embryo lethality assay (ELA) and hemagglutination (HA) test. Scanning electron microscopy (SEM) results showed that the morphology of the viral particle changed under PAS treatments. The total protein concentration of virus decreased as measured by a Bradford protein assay due to PAS treatment. The nucleic acid integrity assay demonstrated that viral RNA degraded into smaller fragments. Moreover, the physicochemical properties of PASs, including the oxidation-reduction potential (ORP), electrical conductivity, and H2O2 concentration, and electron spin resonance spectra analysis indicated that reactive oxygen and nitrogen species play a major role in the virus inactivation. Therefore, the application of PASs, as an environmentally friendly method, would be a promising alternative strategy in poultry industries. IMPORTANCE Newcastle disease (ND), as an infectious viral disease of avian species, caused significant economic losses to domestic animal and poultry industries. The traditional chemical sanitizers, such as chlorine-based products, are associated with risks of by-product formation with carcinogenic effects and environmental pollution. On the basis of this, plasma-activated water as a green disinfection product is a promising alternative for applications in stock farming and sterilization in hospitals and public places. In this study, we explored the inactivation efficacy of different plasma-activated solutions (PASs) against ND virus (NDV) and the possible underlying mechanisms. Our results demonstrated that reactive oxygen and nitrogen species detected in PASs, including short-lived OH˙ and NO˙ and long-lived H2O2, changed the morphology, destroyed the RNA structure, and degraded the protein of the virus, consequently resulting in virus inactivation. These lay a foundation for the application of PASs to resolve the issues of public health and environmental sanitation.

strains in different parts of the world (3). From the perspective of the loss of livestock, ND ranked as the fourth important disease (4). Moreover, the number of bird species infected by ND virus (NDV) has reached 236. In addition to poultry species, virulent NDV isolates are usually found in double-crested cormorants and pigeons (5)(6)(7).
The virus strains of low virulence may result in mild enteric infections. Viruses of intermediate virulence primarily bring about respiratory disease, while virulent viruses may lead to 100% mortality (8). The clinical signs observed with an NDV infection will be nonspecific, including open mouth breathing, depression, ruffled feathers, anorexia, and listlessness before death (9). Furthermore, NDV is also pathogenic to humans (10). The most commonly reported clinical symptoms in human infections are eye infections, usually accompanied by unilateral or bilateral reddening, excessive lacrimation, and eyelid or conjunctival congestion. Other infection signs may sometimes occur, causing chills, headaches, and fever. Therefore, it is necessary to develop antiviral agents that can effectively prevent the spread of viruses and eliminate viruses from the environment.
Nonthermal plasma is a complex mixture, which consists of charged particles, reactive species, and UV photons (11). It has been proved effective in biomedical fields, including for decontamination and sterilization (12,13), teeth whitening (14), and food preservation (15,16). Plasma-activated water is prepared by treating distilled water with nonthermal plasma and has been considered a potential disinfectant against a variety of microbes (17)(18)(19)(20). It is generally accepted that the disinfection efficacy of plasmaactivated water depends on the synergistic effects of reactive oxygen and nitrogen species (RONS) such as the hydroxyl radical (OH˙), atomic oxygen (O), hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO˙) and its derivatives formed with water, including nitrites (NO 2 Ϫ ), nitrates (NO 3 Ϫ ), and peroxynitrites (ONOOH) (21-25). As a "green" disinfection product, plasma-activated water is a promising alternative to traditional sanitizers applied in agriculture and for sterilization in hospitals and public places. Recent studies have reported that plasma-activated water could inactivate foodborne microbes on fruits and maintain the postharvest quality of mushrooms (15,16). It was also proposed that the plasma-activated medium could kill cancer cells in a dosedependent manner (26). In addition, the treatment of water with nonthermal plasma discharges could contribute to the change of its physicochemical properties, which in turn may influence the plant growth and agriculture produce quality (27). To our knowledge, a few research studies have verified the inactivation effect of cold plasma on viruses (28)(29)(30)(31). However, there is no information on the inactivation effect of plasma-activated water and the mechanism of interaction, which are well worth investigating.
In this study, three different solutions (H 2 O, 0.9% NaCl, and 0.3% H 2 O 2 ) were excited by nonthermal plasma to obtain the corresponding plasma-activated solutions (PASs), namely, PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ). The inactivation efficacies of the PASs on NDV were investigated by the embryo lethality assay (ELA) and the hemagglutination (HA) test. Scanning electron microscopy (SEM) was used to observe the morphological changes of the viruses after PAS treatment. Furthermore, the biological properties of the virus, including proteins and nucleic acids, were analyzed. Additionally, the physicochemical characteristics of the PASs, including the oxidation-reduction potential (ORP), electrical conductivity, and the concentrations of H 2 O 2 and some other shortlived species were measured.

RESULTS
Inactivation efficacy of PAS. In Fig. 1, the inactivation efficacies of PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) were verified. The ELA results are shown in Fig. 1a, c, and e. In the control group, the survival rate of chicken embryos was 71.13% Ϯ 7.68% at 72 h and decreased to 27.77% Ϯ 6.93% at 96 h. After 120 h of inoculation, all chicken embryos were dead. However, the survival rates of chicken embryos treated with PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) were 100% when the PAS and NDV interacted at ratios of 3:1, 5:1, and 9:1.
Correspondingly, Fig. 1b, d, and f show the results of the hemagglutination test, and the virus titers in the allantoic fluids of chicken embryos after 120 h of inoculation were examined. Compared to the control group with an average HA titer of 9.40 (Ϯ 0.58) log 2 , no HA titers of any of the treatment groups were detected.
To further assess whether NDV was inactivated completely after PAS treatment, ELAs and HA tests were used to record the survival of chicken embryos and test the virulence of allantoic fluids after three passages. According to Table 1, the chicken embryos in the treatment group all survived after serial passages, while the chicken embryos in the tively. Furthermore, the conductivity of PAS(NaCl) was higher than those of PAS(H 2 O) and PAS(H 2 O 2 ).
The pH values of the PASs were also recorded during the experiments. The initial pH value of the sterile water was around 7.1. As presented in Fig. 2d, the pH of PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) decreased to 3.0, 2.7, and 2.9, respectively, after plasma activation. There were significant differences between the water group and PAS groups.
Evaluation of NO radical and OH radical. It is generally realized that RONS of a PAS, such as atomic oxygen (O), the hydroxyl radical (OH˙), and the nitric oxide radical (NO˙), are related to bacterial inactivation. In previous studies, long-lived RONS, including hydrogen peroxide (H 2 O 2 ), nitrite, and nitrate, were proven to contribute to the prolonged antibacterial effects of PASs (32). To better understand the mechanism of PASs, short-lived RONS (OH˙and NO˙) were detected by electron spin resonance (ESR) spectroscopy in this study. As shown in Fig. 3, there were no signals in the water group. In contrast, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH and NO-Fe 2ϩ (N-methyl-D-glucamine dithiocarbamate [MGD]) 2 signals were detected in PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ), demonstrating that OH˙and NOẇ ere generated by plasma activation. Morphology studies of treated NDV. The morphological change was a pivotal factor, which could explain the inactivation mechanism of PASs. SEM was employed to verify the influence of PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) on the morphology of viral particles. As shown in Fig. 4a, the surface morphology of the virus in the control group was smooth and complete. However, after PAS treatment, the virus surface was shrunken and the structure was damaged, as shown in Fig. 4b, c, and d. The majority Evaluation of RNA intensity. The application of an RNA intensity assay may bring more information about the mechanism of PAS inactivation and the interaction between PASs and living organisms. The Agilent 2100 bioanalyzer provides a platform for the electrophoresis of RNA on a disposable chip (33), which could evaluate the degree of degradation of RNA after PAS treatment. In this study, a virtual gel was visually analyzed and the fluorescence data were tabulated and graphed ( Fig. 6a and b). The utilized RNA ladder was from 25 to 5,770 nucleotides (nt). The bioanalyzer analysis revealed the enrichment of RNA of the control group was for RNA of approximately 500 to 2,000 nt in length. After PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) treatments, the size distribution of RNA was 200 to 500 nt. The experimental evidence showed that the degradation of RNA after PAS exposure was detectable.

DISCUSSION
The ELA and HA test results (Fig. 1) showed that different PASs could effectively inactivate NDV at a ratio of not less than 3:1. This further confirmed the effectiveness and completeness of PAS inactivation via three passages in chicken embryos (Table 1).
To have a more in-depth understanding of the inactivation mechanism, follow-up detection and analysis, including of the physicochemical properties of PAS and the biological characteristics of virus after treatment, were used.
ORP was an indicator of the overall redox potential of the different solutions. After nonthermal plasma activation, there were obvious changes in different PASs, which might have an influence on the biological properties of the virus. A high ORP would destroy the surface proteins and the viral envelope, inactivate the viral enzymes, and cause damage to viral nucleic acids (34). In combination with the previous studies, we believed that ORP values in PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) may play a very important role in virus inactivation. Hydrogen peroxide, as a significant long-lived species in PAS, has attracted attention. The changes in H 2 O 2 concentrations were consistent with the ORP results ( Fig. 2a and b), which increased significantly after plasma activation. Several studies have also reported H 2 O 2 in solution after plasmaactivation. Naïtali et al. found that there were many long-lived active species, such as NO 3 Ϫ , NO 2 Ϫ and H 2 O 2 , in plasma-activated water, which exerted a synergistic effect and were responsible for microbial decontamination (35). Golkowski et al. suggested that the disinfection efficacy of low-temperature plasma samples was enhanced by H 2 O 2 , which may be associated with the increased OH concentration (36) There were no significant differences among PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) on the basis of our results, which indicate that H 2 O 2 in PAS(H 2 O 2 ) may convert to other species during the process of plasma activation.
The increase in conductivity might be the interaction between the active substance and water molecules (37). Meanwhile, it has been reported that the increase in conductivity might promote the photolysis of H 2 O 2 and increase the yield of OH (38). The conductivity of PAS(NaCl) was higher than those of the other treatment groups (Fig. 2c). A possible explanation is that the following reactions occurred.
Cl · ϩ Cl · → Cl 2 (2) When plasma jetting was performed in a 0.9% NaCl solution, Cl Ϫ could be converted to a chlorine radical and form HClO according to reaction 3. HClO is known to be the major bactericidal agent in electrochemical inactivation (39). Subsequently, HClO reacts with H 2 O 2 to produce H ϩ (40,41). As a result of the higher ionic mobility of H ϩ than other ions, the electrical conductivity in PAS(NaCl) was higher than in PAS(H 2 O) and PAS(H 2 O 2 ). In consideration of ELA and HA test results, the electrical conductivity of PAS might be responsible for the inactivation of NDV, which was significantly higher than in the control group. Moreover, the role of conductivity in plasma-activated water sterilization has been confirmed (37). On the other hand, the electrical conductivities of different PASs made a great difference. However, there was little difference in the inactivation efficacies of different PASs, which needs further study.
It is generally agreed that the combination of a high ORP and low pH is responsible for the inactivation ability of PASs, which is in agreement with our results. Previous studies have reported that the reactive species effectively inactivate the bacteria at low pH (42). Simultaneously, it was proved that the ORP alters the redox potential and initiates the peroxidation of fatty acids in the cell membrane under acidic conditions (43,44).
In addition to some long-lived species, such as H 2 O 2 , nitrite, and nitrates, several short-lived RONS (OH˙and NO˙) were detected by ESR spectroscopy in this study (Fig.  3). It was speculated that the production of radicals was via the following reactions (45).
OH˙can easily attack unsaturated fatty acids on the cell membrane and interfere with large intracellular molecules (46). NO˙can change ion currents, cause DNA mutation, and inhibit DNA repair and enzyme activities, resulting in cellular necrosis (47). Therefore, OH˙and NO˙existing in PASs may be the vital factors responsible for the virus inactivation.
It was observed that certain proteins of the virus were damaged and degraded after PAS treatment (Fig. 5), which was associated with the morphological change of the virus (Fig. 4). According to a previous study, Wu et al. proposed that nonthermal plasma can destroy surface proteins and related RNA genes of MS2, leading to viral inactivation and a loss of infectivity (48), which depend on the power level and the working gas. These active species could act at the protein level and cause protein peroxidation and degradation, thereby contributing to the destruction of the capsid. On the basis of the results shown in Fig. 6, it was suggested that PASs could result in the breakage of RNA, which was most likely attributed to RONS in the PASs. These reactive species may attack the RNA sugar phosphate backbone. Yasuda et al. also proposed that nonthermal plasma inactivates bacteriophage by affecting the viral coat protein and causing damage to the DNA (49).
Altogether, the inactivation mechanisms of PASs against virus can be speculated, including the following steps (1). Nonthermal plasma could produce a large number of free radicals in the PAS, leading to high ORP and electrical conductivity (2). OH˙, NO˙, and H 2 O 2 , as representative of short-lived and long-lived species in the PAS, are the major species generated by nonthermal plasma. They could react with carbohydrates and initiate lipid peroxidation and cross-linking of the fatty acid side chains, resulting in alterations of the chemical bonds and molecular structure (3). RONS could induce oxidative stress in the virus. On the one hand, they act at the protein level, causing protein peroxidation and inducing the destruction of the virus envelope, ultimately resulting in changes to the viral morphology. On the other hand, they can damage viral nucleic acids encoding enzymes, contributing to reduced gene expression and the elimination of virus replication, thereby leading to virus inactivation. These findings suggest that the treated fluid may carry RONS, which maintain the inactivation properties. However, it is not yet fully known which exact species and products are generated in these fluids (i.e., deionized water, NaCl, and H 2 O 2 ), which need authentication by several techniques for detailed chemical characterizations.
Conclusion. In summary, this study indicates that a PAS has the potential to inactivate NDV. PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) as antiviral solutions can completely inactivate NDV at an appropriate ratio, which was verified by ELA and HA tests. These lay a foundation for the application of PASs to resolve the issues of public health and environmental sanitation. The possible mechanism of PAS inactivation against NDV is that RONS, including short-lived OH˙and NO˙and long-lived H 2 O 2 detected in PAS, might change viral morphology, destroy the viral RNA structure, and degrade viral protein, consequently resulting in virus inactivation. Therefore, the application of PASs, which are environmentally friendly, can be a promising alternative strategy in poultry industries. Further studies are required to verify whether these changes are sufficient to disrupt virus functions and to further shed light on the determinant factors for virus inactivation by PASs.

MATERIALS AND METHODS
Plasma microjet device and PAS generation. The air plasma generator, schematically illustrated in Fig. 7a, was designed on the basis of a dielectric barrier structure with hollow electrodes (HEDBS). The device was composed of a copper electrode and dielectric quartz. The working gas was air with a 260-liter/h flow rate, which was injected into the quartz tube. The high-voltage electrode and a power source (20 kHz) were connected. Homogeneous plasma was produced in the discharge gap of 0.5 mm and was ejected through the end outlet of 0.5 mm, reaching 7 mm long. The specific details about the structure have been described by Yu et al. (50).
As shown in Fig. 7b, the PASs were generated by plasma activation beneath the solution surfaces. The distance between the liquid surface and the plasma microjet (PMJ) end was 20 mm. The 10-ml sterile distilled water, 0.9% NaCl, and 0.3% H 2 O 2 solutions were activated by plasma for 10 min to obtain the PASs, defined as PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ), respectively, which were freshly prepared for inactivating NDV.
PAS treatment. NDV (LaSota strain) was propagated in 10-day-old specific-pathogen-free (SPF) chicken embryos. The embryos that died within 24 h were discarded, and the allantoic fluids were harvested, centrifuged at 1,000 ϫ g, and stored at Ϫ20°C before use. Fifty percent egg infectious dose (EID 50 ) titers were calculated by the method of Reed and Muench after serial titration in eggs (51,52). To assess the inactivation ability of the PASs, the allantoic fluids containing NDV were treated with PAS(H 2 O), PAS(NaCl), and PAS (H 2 O 2 ) at ratios of 1:3, 1:5, and 1:9, respectively, for 30 min. The NDV-allantoic fluid without PAS treatment was used as the control.
Embryo lethality assay. Viruses treated by PAS(H 2 O), PAS(NaCl), and PAS(H 2 O 2 ) at different ratios and the control sample were inoculated into 10-day-old SPF chicken embryos via an intra-allantoic route, with five embryos in each group. Every embryo was injected with 0.1 ml of 10 8.3 EID 50 NDV treated by PAS. According to previous studies (53)(54)(55), the eggs were candled every 24 h, and the embryos that died within 24 h were discarded. The death number beyond 24 h was recorded until 120 h, which was summarized as the ELA results. The SPF chicken embryos were supplied by Beijing Merial Vital Laboratory Animal Technology Co., Ltd.
HA test. The HA assays conform to OIE standard procedures (56). To perform the HA test, 50 l of phosphate-buffered saline (PBS) was added to every well of a 96-well plate. Then, 50 l of the samples was added to the first column of the plates and mixed thoroughly. Subsequently, 50 l of the mixture in the first column was added to the second column accordingly and mixed again. In accordance with the above procedure, the samples were constantly diluted until they were added to the 11th column. After mixing, 50 l of the mixture in the 11th column was discarded. Finally, 50 l of 1% erythrocytes was added to every well. After slight oscillation, the plates were placed for 25 min at 37°C. The HA titer was read as log 2 of the highest dilution of antigen giving complete HA (no streaming).

ACKNOWLEDGMENTS
This work was supported by the Beijing Key Laboratory for Prevention and Control of Infectious Diseases in Livestock and Poultry (grant number Z161100005016028).
We declare that we have no competing interests.