N2 Gas Plasma Inactivates Influenza Virus by Inducing Changes in Viral Surface Morphology, Protein, and Genomic RNA

We have recently treated with N2 gas plasma and achieved inactivation of bacteria. However, the effect of N2 gas plasma on viruses remains unclear. With the aim of developing this technique, we analyzed the virucidal effect of N2 gas plasma on influenza virus and its influence on the viral components. We treated influenza virus particles with inert N2 gas plasma (1.5 kpps; kilo pulses per second) produced by a short high-voltage pulse generated from a static induction thyristor power supply. A bioassay using chicken embryonated eggs demonstrated that N2 gas plasma inactivated influenza virus in allantoic fluid within 5 min. Immunochromatography, enzyme-linked immunosorbent assay, and Coomassie brilliant blue staining showed that N2 gas plasma treatment of influenza A and B viruses in nasal aspirates and allantoic fluids as well as purified influenza A and B viruses induced degradation of viral proteins including nucleoprotein. Analysis using the polymerase chain reaction suggested that N2 gas plasma treatment induced changes in the viral RNA genome. Scanning electron microscopy analysis showed that aggregation and fusion of influenza viruses were induced by N2 gas plasma treatment. We believe these biochemical changes may contribute to the inactivation of influenza viruses by N2 gas plasma.


Introduction
Infection mediated by medical devices is thought to be a major contributor to hospital-acquired infections [1]. However, medical devices and instruments are often not sufficiently robust to withstand repeated rounds of sterilization by autoclaving or dry-heat treatment [2]. Alternative sterilization techniques involve the generation of -rays or electron beams, which require expensive facilities and are not appropriate for routine daily use [3]. Although ethylene oxide gas (EOG) can be used to sterilize heat-sensitive medical instruments, the gas is both toxic and carcinogenic, which limits its usage [4]. Recently, sterilization using hydrogen peroxide gas plasma was proposed, although it is ineffective against endotoxins and lipopolysaccharides (LPSs) [5,6]. Residual amounts of endotoxin derived from bacteria may cause symptoms including fever [7].
A gas plasma is generated by removing electrons from a gas to produce a highly excited mixture of charged nuclei and free electrons [8,9]. Recently, we succeeded in generating N 2 gas plasma using a fast high-voltage pulse from a static induction (SI) thyristor power supply [6,[8][9][10]. N 2 gas plasma treatment efficiently inactivates bacteria and bacterial spores, as well as degrading LPS, which showed a more than 5 log reduction in 30 min [6]. The value (the decimal reduction time) of Geobacillus stearothermophilus was less than 1.3 minutes, whereas that of Aspergillus niger was even smaller [6]. However, the effect of N 2 gas plasma on viruses remains unclear. Therefore, we treated influenza virus, as a representative enveloped virus, with N 2 gas plasma and analyzed the sterilizing efficiency. We also analyzed the effect of this treatment on viral components such as proteins and RNAs.  The distance between the high-voltage electrode and the earth electrode was 50 mm. N 2 gas flow rate was 10 L/min. During N 2 gas plasma generation, the sample box was kept at 0.5 atmospheric pressure. (b) Photograph of discharge region during generation of N 2 gas plasma. A static induction (SI) thyristor was used as a pulsed power supply.

Viruses Nasal
Aspirates. Nasal aspirates were collected from children at the Baba pediatric clinic (Kadoma, Osaka, Japan) as described previously [11]. Briefly, saline was introduced into the nasal cavity, and then the wash solution was aspirated using Belvital (Melisana, Nogent-sur-Marne, France (NGK Insulators, Ltd) was used as a device to produce N 2 gas plasma by generating a fast high-voltage pulse utilizing a SI thyristor power supply ( Figure 1). The sample was exposed to N 2 gas (99.9995%, Okano, Okinawa, Japan) at about 0.5 atmospheres prior to applying the high-voltage pulse. A 20 L aliquot of each sample solution was dropped onto a cover glass, air dried, and then treated with N 2 gas plasma at 1.5 kpps (kilo pulses per second). Each sample on the cover glass was subsequently recovered by dissolving in 20 L of distilled water (Otsuka Pharmaceuticals Co., Tokyo, Japan). Inc., Otsu, Japan) and random primers to make cDNA by using the following temperature regime: 65 ∘ C for 5 min, 4 ∘ C for 5 min, and 42 ∘ C for 60 min. The resultant cDNAs were subjected to PCR for matrix protein (M1), hemagglutinin (HA), neuraminidase (NA), and nonstructural protein (NS) using Takara Ex Taq (Takara Bio Inc.). The temperature cycling conditions used for the PCR were 95 ∘ C for 5 min, 25 cycles of 95 ∘ C for 1 min, 55 ∘ C for 1 min, and 72 ∘ C for 1 min with one final cycle of 72 ∘ C for 10 min. PCR was carried out using the following primers modified from previous papers [13,14]: The intensity of the amplified bands from the PCR products was semiquantitatively analyzed by agarose gel electrophoresis. Bands in test samples were visually compared to those in untreated controls. The amplified PCR products generated from each pair of primers were verified by DNA sequencing.

Hemagglutination
Assay. Samples were serially diluted two-or three-fold in 25 L of PBS in V-shaped well plates, and an equal volume of 1% chicken erythrocytes in suspension was added. The mixture was then incubated at room temperature for 1 h. The agglutination pattern was read, and the hemagglutination titer was defined as the reciprocal of the last dilution of sample that showed hemagglutination.  SEM was then performed using a JSM-6320F (JEOL Ltd., Tokyo, Japan) instrument at a magnification of x50,000.

Influenza Virus Bioassay Using Embryonated
Eggs. N 2 gas plasma treated samples were injected into 11-day-old chicken embryonated eggs. The eggs were cultured at 37 ∘ C for 48 h before allantoic fluid was collected. The obtained samples were then subjected to immunochromatography for influenza virus and analyzed by hemagglutination assay.

Statistical Analysis.
Results were compared by nonpaired Student's t-test. In cases where < 0.05, the differences were considered significant.

Results
First, we investigated the N 2 gas plasma treated influenza virus (A/PR/8/34) in infected allantoic fluid and determined whether influenza virus was inactivated by N 2 gas plasma treatment (Figure 2). Samples including influenza virus (A/PR/8/34) at 3.16 × 10 14 TCID 50 /mL were treated with N 2 gas plasma for 5 min and injected into 11-day-old chicken embryonated eggs. After 48 h incubation at 37 ∘ C, the allantoic fluids were subjected to immunochromatography for influenza A virus to check whether the N 2 gas plasma treated influenza virus had proliferated in the embryonated eggs. Influenza viruses derived from all six independent spots treated with N 2 gas plasma for 5 min were unable to proliferate. By contrast, influenza viruses derived from six untreated spots did proliferate all. Morever, these results were consistent with those obtained from the hemagglutination assay. In addition, an infection assay using MDCK cells showed that viral titers of TCID 50 /mL changed from  7.5 × 10 4 and 10 × 10 4 at 0 min to 5.6 × 10 3 and 10 × 10 3 at 0.5 min and 1.3 × 10 3 and 1.0 × 10 3 at 1 min (Figure 3). These results also supported the inactivation of influenza virus by N 2 gas plasma.
Next, the effect of N 2 gas plasma on viral proteins was investigated. The results from immunochromatography show that NP of influenza A and B viruses was decomposed by N 2 gas plasma treatment of (i) nasal aspirates for 5 min (Figures 4(a) and 4(b)), (ii) allantoic fluid for 5 min or 15 min (Figure 4(c)), and (iii) purified virus for 15 min (Figure 4(d)). Specifically, a band corresponding to NP in the test line A and B was detected at 0 min, but it became less obvious after N 2 gas plasma treatment for 5 or 15 min. A band in the reference line was detected at all time points indicating that the immunochromatography was working as anticipated. Regarding the nasal aspirates, all 3 influenza A and 3 influenza B samples showed a similar tendency. Based on this result we conclude that NP in the nasal aspirates and allantoic fluid was decomposed by N 2 gas plasma treatment.
Next, ELISA using anti-influenza B virus NP antibody was carried out to verify degradation of NP in influenza B virus derived from infected allantoic fluid that had been treated with N 2 gas plasma ( Figure 5). Within 5 min, the concentration of influenza B virus NP was decreased to less than 1/6 for B/Gifu/2/73. These results are consistent with previous and present immunochromatography findings regarding NP of influenza A and B viruses, which was shown to be degraded by the N 2 gas plasma [15].
SDS-PAGE analysis followed by CBB staining was also used to monitor viral proteins and/or induced proteins in the allantoic fluid after infection with influenza A virus (A/PR/8/34) (Figure 6(a)). Our results show that the proteins were degraded after N 2 gas plasma treatment (1.5 kpps) for either 15 or 30 min (Figure 6(b)). A previous study reported that the major viral proteins in influenza virus derived from allantoic fluid are NA oligomer (over 70 kDa), HA1 (around 70 kDa), NA monomer and NP (around 70 kDa), and M1 and HA2 (around 30 kDa) [16]. Similarly sized bands appeared to be detected in the present SDS-PAGE and CBB stained gels of virus-infected allantoic fluid but faded after treatment of the fluid with N 2 gas plasma. Therefore, the bands observed and degraded by N 2 gas plasma may be mainly influenza viral proteins.
Next, morphologies of the influenza viruses were observed by SEM (Figure 7). Our SEM observations showed that the N 2 gas plasma treatment (1.5 kpps, 5 min) disrupted fibers connecting influenza viruses in the allantoic fluid. Moreover, the N 2 gas plasma treated influenza viruses displayed a shrunken appearance. In addition, fused viruses were also observed in the treated samples, suggesting that the N 2 gas plasma may modify the viral envelope.
Next, the N 2 gas plasma treated influenza viruses were subjected to viral genomic RNA extraction. We then attempted to amplify various influenza virus genes, such as M1, NS, HA, and NA, by PCR (Figure 8). The results showed that amplification of each of the genes was greatly repressed by N 2 gas plasma treatment (1.5 kpps; 5 and 30 min), suggesting that the viral genomic RNA was damaged following N 2 gas plasma treatment. The magnitude of the observed decrease in the amplified product varied among the different viral genes. The inhibition efficiency of HA and NA was high compared to that of M1 and NS. This may be due to the structural differences and arrangements of influenza virus segments encoding each viral gene in the viral particle. The products amplified for M1 and NS appeared to be slightly more intense after 30 min of treatment than those observed after 5 min of treatment, whereas the amplified products for NA appeared to be slightly more intense after 5 min of treatment than those observed after 30 min of treatment. Repeated experiments showed that the band intensities for M1, NS, HA, and NA at 5 min and 30 min of treatment were similar.

Discussion
Influenza virus can be relatively easily disinfected by chemicals such as alcohol [17,18]. The presence of organic matter interferes with the action of chemical sterilants because they can react, thereby decreasing the efficiency of disinfection. Indeed, disinfection efficiency of alcohol against influenza virus varies depending on the presence of coexisting organic material [19]. Most regulatory authorities require sterilant efficacy testing to be conducted in the presence of 5% soil [20]. Influenza virus is usually encountered in the nasal fluid of patients and allantoic fluid of eggs. In the case of medical devices, bronchoscopes in particular may be at risk of influenza virus contamination. In our study, we used 100% nasal aspirates and 100% allantoic fluid as a source of organic material. The obtained results showed that N 2 gas plasma can inactivate influenza virus in these environments. Currently, the items/areas that can be treated by the method are restricted to the size of the chamber box of the N 2 gas plasma instrument. To enable the sterilization of medical devices and larger surfaces, it would be necessary to expand the size of the discharge area.
NP and genomic RNA, which are both localized to the central region of the influenza virus particle, were subject to decomposition and/or modification by treatment with N 2 gas plasma. Likewise, lipids localized in the outer envelope of the virus were also modified and/or degraded after this treatment. Microscopic investigations showed that treatment with N 2 gas plasma disrupt the fibers between virus particles in the allantoic fluid, although the significance of the fibers connecting the viruses in the untreated samples in relation to the infectivity of the virus is unclear. Previous studies have shown that oxidative stress contributes to the mechanism of action of a gas plasma. For example, the addition of oxygen to helium has been found to enhance the efficiency of inactivation in the case of bacteria [21]. In addition, oxidation and peroxidation processes on the surface of cells and within cells result in inactivation [22,23]. Furthermore, destruction of the surface structure by gas plasma may be the main mechanism underlying the inactivation of bacteria [24], which may also be the case for viruses. Although these oxidative factors contribute to the mechanism of action in N 2 gas plasma, further studies are required to identify the most critical factor(s) for inactivation.
In this study, we analyzed the effect of N 2 gas plasma treatment on influenza virus, which is enveloped. However, the effectiveness of this treatment against various other pathogens, which may differ in resistance to disinfection, is unknown. For example, it would be interesting to investigate the effect of this treatment on nonenveloped viruses, such as norovirus or adenovirus as well as the highly resistant prion agents.

Conclusion
In conclusion, the present results suggest that N 2 gas plasma treatment modifies viral genomic RNA and degrades viral proteins, including NPs, as well as the viral envelope and fibers related to allantoic fluid. Taken together, the results indicate that N 2 gas plasma treatment may be an effective means of disinfection for influenza virus.