Research Article N 2 Gas Plasma Inactivates Influenza Virus by Inducing Changes in Viral Surface Morphology, Protein, and Genomic RNA

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1 iomed Reseach Intenational Volume 2013, ticle ID , 7 pages Reseach ticle N 2 Gas Plasma Inactivates Influenza Vius by Inducing Changes in Vial Suface Mophology, Potein, and Genomic RN kikazu Sakudo, 1,2 Naohio Shimizu, 3 Yuichio Imanishi, 3 and Kazuyoshi Ikuta 1 1 Depatment of Viology, Reseach Institute fo Micobial Diseases, Osaka Univesity, Yamadaoka, Suita, Osaka , Japan 2 Laboatoy of iometabolic Chemisty, School of Health Sciences, Univesity of the Ryukyus, Nishihaa, Okinawa , Japan 3 NGK Insulatos, Mizuho-ku, Nagoya , Japan Coespondence should be addessed to kikazu Sakudo; sakudo@biken.osaka-u.ac.jp Received 28 pil 2013; Revised 4 July 2013; ccepted 25 ugust 2013 cademic Edito: Kevin M. Coombs Copyight 2013 kikazu Sakudo et al. This is an open access aticle distibuted unde the Ceative Commons ttibution License, which pemits unesticted use, distibution, and epoduction in any medium, povided the oiginal wok is popely cited. We have ecently teated with N 2 gas plasma and achieved inactivation of bacteia. Howeve, the effect of N 2 gas plasma on viuses emains unclea. With the aim of developing this technique, we analyzed the viucidal effect of N 2 gas plasma on influenza vius and its influence on the vial components. We teated influenza vius paticles with inet N 2 gas plasma (1.5 kpps; kilo pulses pe second) poduced by a shot high-voltage pulse geneated fom a static induction thyisto powe supply. bioassay using chicken embyonated eggs demonstated that N 2 gas plasma inactivated influenza vius in allantoic fluid within 5 min. Immunochomatogaphy, enzyme-linked immunosobent assay, and Coomassie billiant blue staining showed that N 2 gas plasma teatment of influenza and viuses in nasal aspiates and allantoic fluids as well as puified influenza and viuses induced degadation of vial poteins including nucleopotein. nalysis using the polymease chain eaction suggested that N 2 gas plasma teatment induced changes in the vial RN genome. Scanning electon micoscopy analysis showed that aggegation and fusion of influenza viuses wee induced by N 2 gas plasma teatment. We believe these biochemical changes may contibute to the inactivation of influenza viuses by N 2 gas plasma. 1. Intoduction Infection mediated by medical devices is thought to be a majo contibuto to hospital-acquied infections [1]. Howeve, medical devices and instuments ae often not sufficientlyobusttowithstandepeatedoundsofsteilization by autoclaving o dy-heat teatment [2]. ltenative steilization techniques involve the geneation of γ-ays o electon beams, which equie expensive facilities and ae not appopiate fo outine daily use [3]. lthough ethylene oxide gas (EOG) can be used to steilize heat-sensitive medical instuments, the gas is both toxic and cacinogenic, which limits its usage [4]. Recently, steilization using hydogen peoxide gas plasma was poposed, although it is ineffective against endotoxins and lipopolysacchaides (LPSs) [5, 6]. Residual amounts of endotoxin deived fom bacteia may cause symptoms including feve [7]. gas plasma is geneated by emoving electons fom a gas to poduce a highly excited mixtue of chaged nuclei andfeeelectons[8, 9]. Recently, we succeeded in geneating N 2 gas plasma using a fast high-voltage pulse fom a static induction (SI) thyisto powe supply [6, 8 10]. N 2 gas plasma teatment efficiently inactivates bacteia and bacteial spoes, as well as degading LPS, which showed a moe than 5 log eduction in 30 min [6]. The D value (the decimal eduction time) of Geobacillus steaothemophilus was less than 1.3 minutes, wheeas that of spegillus nige was even smalle [6]. Howeve, the effect of N 2 gas plasma on viuses emains unclea. Theefoe, we teated influenza vius, as aepesentativeenvelopedvius,withn 2 gas plasma and analyzed the steilizing efficiency. We also analyzed the effect of this teatment on vial components such as poteins and RNs.

2 2 iomed Reseach Intenational Pulsed powe supply N 2 gas Dischage aea Cove glass Dischage aea High-voltage electode Eath electode High-voltage electode Exhaust pump Heate (a) (b) Figue 1: Expeimental setup fo N 2 gas plasma poduction. (a) Schematic of N 2 gas plasma instument (LT-TES No.1) used in this study. The distance between the high-voltage electode and the eath electode was 50 mm. N 2 gas flow ate was 10 L/min. Duing N 2 gas plasma geneation, the sample box was kept at 0.5 atmospheic pessue. (b) Photogaph of dischage egion duing geneation of N 2 gas plasma. static induction (SI) thyisto was used as a pulsed powe supply. 2. Mateials and Methods 2.1. Viuses Nasal spiates. Nasal aspiates wee collected fom childen at the aba pediatic clinic (Kadoma, Osaka, Japan) as descibed peviously [11]. iefly, saline was intoduced into the nasal cavity, and then the wash solution was aspiated using elvital (Melisana, Nogent-su-Mane, Fance). In ode to emove cell debis, the nasal fluid was filteed using a stainless steel mesh [200 gids pe inch (25.4 mm)]. The obtained nasal aspiates wee checked by immunochomatogaphy fo influenza viuses and as descibed below. Each aspiate sample (3 influenza and 3 influenza ) was subjected to analysis. The eseach poject fo espiatoy infectious diseases was appoved by the Ethics Committees of the Reseach Institute fo Micobial Diseases in Osaka Univesity, and witten infomed consent was obtained fom the patients N 2 Gas Plasma Instument and Teatment. LP-TES No.1 (NGK Insulatos, Ltd) was used as a device to poduce N 2 gas plasma by geneating a fast high-voltage pulse utilizing a SI thyisto powe supply (Figue 1). The sample was exposed to N 2 gas ( %, Okano, Okinawa, Japan) at about 0.5 atmosphees pio to applying the high-voltage pulse. 20 μl aliquot of each sample solution was dopped onto a cove glass, ai died, and then teated with N 2 gas plasma at 1.5 kpps (kilo pulses pe second). Each sample on the cove glass was subsequently ecoveed by dissolving in 20 μl of distilled wate (Otsuka Phamaceuticals Co., Tokyo, Japan) llantoic Fluid. Influenza vius-infected allantoic fluid was pepaed fom 11-day-old chicken embyonated eggs injected with influenza vius H1N1 (/PR/8/34) o influenza vius (/Gifu/2/73) Puified Influenza and Viuses. llantoic fluids of 11-day-old embyonated eggs infected with eithe influenza vius H1N1 (/Taiwan/1/86), H3N2 (/Panama/2007/99), o influenza vius (/Tokio/53/99) wee collected afte 48 h incubation at 37 C and subjected to factionation using a sucose density gadient geneated by ultacentifugation. The obtained faction was checked by sodium dodecyl sulfate (SDS) polyacylamide gel electophoesis (PGE) followed by Coomassie illiant lue (C) staining as descibed below. The esults showed that the puity of the influenza and viuses was ove 99% Measuement of TCID 50 (Tissue Cultue Infectious Dose 50). Vial tites wee detemined by pefoming 10-fold seial dilutions of samples in 96-well plates containing Madin- Daby Canine Kidney (MDCK) cells with 8 eplicates of each sample. efoe infection, cells wee washed with phosphatebuffeed saline (PS). Infected cells wee incubated at 37 C with 5% CO 2, and fesh typsin was added (final concentation, 0.03%). Vial tites wee ead as TCID 50 calculated by themethodofreedandmuench[12] Immunochomatogaphy fo Influenza and Viuses. The amount of influenza and viuses nucleopotein (NP) inthecollectedsolutionwasanalyzedbyimmunochomatogaphy (ESPLINE Influenza & -N, Fujiebio Inc., Tokyo, Japan) as follows. iefly, the immunochomatogaphy test assembly was developed using anti-influenza vius and antiimmunoglobulin (Ig) antibodies. The anti-influenza and NP antibodies wee immobilized onto a nitocellulose membane fo test lines (lines and, esp.) to captue influenza vius NP. To pepae the efeence line, an anti-ig antibody was immobilized onto a nitocellulose membane to captue Ig. conjugated pad containing anti-influenza vius NPs used fo the test line was labelled with colloidal gold, impegnated onto glass fibes, died, and then placed between the test lines and the sample dopping egion. The nitocellulose membane and glass fibe pad wee assembled with a glass fibe pad on a plastic sheet within a plastic case. This assembled kit was stoed in a bag with desiccant at 4 C until equied. The kit was then used to quantify the amount of influenza vius NPs Polymease Chain Reaction (PCR) fo Influenza Vius. Vial genomic RN was extacted fom unteated and N 2

3 iomed Reseach Intenational 3 gas plasma teated allantoic fluid infected with influenza vius (/PR/8/34) using a QIamp Vial RN mini kit (Qiagen, Hilden, Gemany). The RN was tanscibed with PimeSciptII 1st stand cdn Synthesis kit (Takaa io Inc., Otsu, Japan) and andom pimes to make cdn by using the following tempeatue egime: 65 Cfo5min,4 C fo 5 min, and 42 C fo 60 min. The esultant cdns wee subjected to PCR fo matix potein (M1), hemagglutinin (H), neuaminidase (N), and nonstuctual potein (NS) using Takaa Ex Taq (Takaa io Inc.). The tempeatue cycling conditions used fo the PCR wee 95 Cfo5min,25 cycles of 95 Cfo1min,55 Cfo1min,and72 Cfo1min with one final cycle of 72 C fo 10 min. PCR was caied out using the following pimes modified fom pevious papes [13, 14]: M1-F: 5 -CG G CTT G GT GTC TTT GCT G-3 ; M1-R: GCT CTG TCC TG TT TTT GG TC-3 ;H-F:5 -GC GC GG GG T-3 ; H-R: 5 -GCT TT TCT GGG GTG T CT-3 ;N-F:5 - TTG CTT GGT CGG C GTG C-3 ;N-R:5 -CC GTC CC CC TTT GG TCC-3 ;NS-F:5 -G GGC TTT CC CG G GG-3 ;NS-R:5 -CCC TT CTC TT CT GCT TC-3.Theintensityoftheamplifiedbandsfomthe PCR poducts was semiquantitatively analyzed by agaose gel electophoesis. ands in test samples wee visually compaed to those in unteated contols. The amplified PCR poducts geneated fom each pai of pimes wee veified by DN sequencing. 0 min 5 min Hemagglutination tite Hemagglutination tite <2 1 <2 1 <2 1 <2 1 <2 1 <2 1 Figue 2: Inhibition of the polifeation of influenza vius in embyonated eggs by N 2 gas plasma. Six independent spots of influenza vius (/PR/8/34) infected allantoic fluid (vial tite: TCID 50 /ml) on cove glass wee ai-died, teated with N 2 gas plasma (1.5 kpps, 5 min) using LP-TES No.1, and injected into 6 embyonated chicken eggs. The eggs wee incubated fo 48 h befoe collecting allantoic fluid. The pesence of influenza vius in the allantoic fluid was analyzed by immunochomatogaphy fo influenza vius NP (ESPRINE Influenza & -N, Fujiebio, Inc.). In addition, hemagglutination tites of each allantoic fluid wee measued (see below). and indicate the lines fo NP of influenza vius and influenza vius, espectively. The efeence line () is also indicated. N 2 gas plasma teatment (1.5 kpps) effectively inactivates influenza vius within 5 min. SEM was then pefomed using a JSM-6320F (JEOL Ltd., Tokyo, Japan) instument at a magnification of x50, Hemagglutination ssay. Samples wee seially diluted two- o thee-fold in 25 μl of PS in V-shaped well plates, and an equal volume of 1% chicken eythocytes in suspension was added. The mixtue was then incubated at oom tempeatuefo1h.theagglutinationpattenwasead,and the hemagglutination tite was defined as the ecipocal of the last dilution of sample that showed hemagglutination Enzyme Linked Immunosobent ssay (ELIS) fo Influenza Vius. Seion ELIS antigen influenza vius test (Institut Viion/Seion GmbH, Wüzbug, Gemany) was used fo quantification of influenza vius NP SDS-PGE and C Staining. Samples wee solubilized in an equal volume of 2 SDS gel-loading buffe [90 mm Tis-HCl (ph 6.8), 10% mecaptoethanol, 2% SDS, 0.02% bomophenol blue, and 20% glyceol], boiled fo 5 min, and sepaatedbysds-pge(12%gel).thepoteinbandsinthe gelweevisualizedbycstaining Scanning Electon Micoscopy (SEM). The influenza vius-infected allantoic fluids wee ai-died on cove glass, teated with N 2 gas plasma, and then fixed with 2% glutaaldehyde/0.1 M phosphate buffe (ph 7.4) ovenight at 4 C. The cove glasses wee subsequently teated with 2% osmium tetaoxide at 4 C fo 3 h. Samples wee dehydated though a gaded ethanol seies (50 100% ethanol) at oom tempeatue. Finally, the cove glass was subjected to citical point dying and evapoation coating by osmium plasma ion Influenza Vius ioassay Using Embyonated Eggs. N 2 gas plasma teated samples wee injected into 11-day-old chicken embyonated eggs. The eggs wee cultued at 37 C fo 48 h befoe allantoic fluid was collected. The obtained samples wee then subjected to immunochomatogaphy fo influenza vius and analyzed by hemagglutination assay Statistical nalysis. Results wee compaed by nonpaied Student s t-test. In cases whee P < 0.05, the diffeences wee consideed significant. 3. Results Fist, we investigated the N 2 gas plasma teated influenza vius (/PR/8/34) in infected allantoic fluid and detemined whethe influenza vius was inactivated by N 2 gas plasma teatment (Figue2). Samples including influenza vius (/PR/8/34) at TCID 50 /ml wee teated with N 2 gasplasmafo5minandinjectedinto11-day-old chicken embyonated eggs. fte 48 h incubation at 37 C, the allantoicfluidsweesubjectedtoimmunochomatogaphy fo influenza vius to check whethe the N 2 gas plasma teated influenza vius had polifeated in the embyonated eggs. Influenza viuses deived fom all six independent spots teated with N 2 gas plasma fo 5 min wee unable to polifeate. y contast, influenza viuses deived fom six unteated spots did polifeate all. Moeve, these esults wee consistent with those obtained fom the hemagglutination assay. In addition, an infection assay using MDCK cells showed that vial tites of TCID 50 /ml changed fom

4 4 iomed Reseach Intenational 10 5 Vial tite (TCID 50 /ml) No1 No2 No1 No2 No1 No2 0 min 0.5 min 1 min Figue 3: Decease in vial tite of influenza vius by N 2 gas plasma. Influenza vius (/PR/8/34)-infected allantoic fluid on a cove glass was ai-died, teated with N 2 gas plasma (1.5 kpps; 0, 0.5, and 1 min) using LP-TES No.1, and incubated with MDCK cells. fte 72 h incubation, vial tites of TCID 50 /ml wee calculated as descibed in Section 2.Theassaywasdoneinduplicate(No1andNo2),whichwasdeivedusing diffeent died spots (a) (b) (c) TW1 PM7 (d) TK53 Figue 4: Effects of N 2 gas plasma on nucleopotein (NP) of influenza vius types and. Influenza (a) and influenza (b) viuses in nasal aspiate, influenza vius (/PR/8/34) in allantoic fluid (Vial tite: TCID 50 /ml) (c), and puified influenza viuses (d) wee used. The samples wee ai-died on cove glass and subjected to N 2 gas plasma teatment (1.5 kpps) fo the indicated time using LP-TES No.1. Samples wee subsequently ecoveed in distilled wate and then analyzed by immunochomatogaphy (ESPRINE Influenza & -N, Fujiebio, Inc.). and indicate the lines fo NP of influenza vius and influenza vius, espectively. The efeence line () is also indicated. Influenza vius H1N1 stain TW1: /Taiwan/1/86 and H3N2 stain PM7: /Panama/2007/99 wee used. Influenza vius TK53: /Tokio/53/99 was also used and at 0 min to and at 0.5 min and and at 1 min (Figue 3). These esults also suppoted the inactivation of influenza vius by N 2 gas plasma. Next, the effect of N 2 gas plasma on vial poteins was investigated. The esults fom immunochomatogaphy show that NP of influenza and viuses was decomposed by N 2 gas plasma teatment of (i) nasal aspiates fo 5 min (Figues 4(a) and 4(b)), (ii) allantoic fluid fo 5 min o 15 min (Figue 4(c)), and (iii) puified vius fo 15 min (Figue 4(d)). Specifically, a band coesponding to NP in the test line and was detected at 0 min, but it became less obvious afte N 2 gas plasma teatment fo 5 o 15 min. band in the efeence line was detected at all time points indicating that the immunochomatogaphy was woking as anticipated. Regading the nasal aspiates, all 3 influenza and 3 influenza samples showed a simila tendency. ased on this esult we conclude that NP in the nasal aspiates and allantoic fluid was decomposed by N 2 gas plasma teatment. Next, ELIS using anti-influenza vius NP antibody was caied out to veify degadation of NP in influenza vius deived fom infected allantoic fluid that had been teated with N 2 gas plasma (Figue 5). Within 5 min, the concentation of influenza vius NP was deceased to

5 iomed Reseach Intenational 5 Influenza vius (μg/ml) ( ) (+) Figue 5: Degadation of vial poteins in influenza vius by N 2 gas plasma. Quantity of influenza vius (GF21: /Gifu/2/73) deived fom infected allantoic fluid befoe and afte teatment with N 2 gas plasma. llantoic fluid (20 μl) was died on a cove glass and teated with N 2 gas plasma at 1.5 kpps fo 0 o 5 min. The quantity of NP of influenza vius was measued by an enzyme-linked immunosobent assay against NP. steisk indicates a significant diffeence (P < 0.05) when compaed with0min (a) 31.7 (b) less than 1/6 fo /Gifu/2/73. These esults ae consistent with pevious and pesent immunochomatogaphy findings egading NP of influenza and viuses, which was shown to be degaded by the N 2 gas plasma [15]. SDS-PGE analysis followed by C staining was also used to monito vial poteins and/o induced poteins in the allantoic fluid afte infection with influenza vius (/PR/8/34) (Figue 6(a)).Ou esults show that the poteins wee degaded afte N 2 gas plasma teatment (1.5 kpps) fo eithe 15 o 30 min (Figue 6(b)). pevious study epoted that the majo vial poteins in influenza vius deived fom allantoic fluid ae N oligome (ove 70 kda), H1 (aound 70 kda), N monome and NP (aound 70 kda), and M1 and H2 (aound 30 kda) [16]. Similaly sized bands appeaed to be detected in the pesent SDS-PGE and C stained gels of vius-infected allantoic fluid but faded afte teatment of the fluid with N 2 gas plasma. Theefoe, the bands obseved and degaded by N 2 gas plasma may be mainly influenza vial poteins. Next, mophologies of the influenza viuses wee obseved by SEM (Figue 7). Ou SEM obsevations showed that the N 2 gas plasma teatment (1.5 kpps, 5 min) disupted fibes connecting influenza viuses in the allantoic fluid. Moeove, the N 2 gas plasma teated influenza viuses displayed a shunken appeaance. In addition, fused viuses wee also obseved in the teated samples, suggesting that the N 2 gas plasma may modify the vial envelope. Next, the N 2 gas plasma teated influenza viuses wee subjected to vial genomic RN extaction. We then attempted to amplify vaious influenza vius genes, such as M1, NS, H, and N, by PCR (Figue 8). The esults showed that amplification of each of the genes was geatly epessed by N 2 gas plasma teatment (1.5 kpps; 5 and 30 min), suggesting that the vial genomic RN was damaged following N 2 gas plasma teatment. The magnitude of the obseved decease in the amplified poduct vaied among the diffeent vial genes. Figue 6: Degadation of influenza vius-infection induced poteins by N 2 gas plasma teatment. (a) Poteins in influenza vius (/PR/8/34) infected (+) and uninfected ( ) allantoic fluid wee subjected to sodium dodecyl sulfate (SDS) polyacylamide gel electophoesis (PGE) and stained with Coomassie billiant blue (C) solution. (b) The level of influenza vius-infection induced poteins in allantoic fluid, which include both vial poteins and the othe poteins in allantoic fluid esponding to the infection, was deceased afte N 2 gasplasmateatmentfo15and30mincompaed to the unteated contol (0 min). The aowheads highlight potein bands induced afte influenza vius infection. The positions of the molecula weight makes ae indicated on the left hand side. The inhibition efficiency of H and N was high compaed tothatofm1andns.thismaybeduetothestuctual diffeences and aangements of influenza vius segments encoding each vial gene in the vial paticle. The poducts amplified fo M1 and NS appeaed to be slightly moe intense afte 30 min of teatment than those obseved afte 5 min of teatment, wheeas the amplified poducts fo N appeaed to be slightly moe intense afte 5 min of teatment than those obseved afte 30 min of teatment. Repeated expeiments showed that the band intensities fo M1, NS, H, and N at 5 min and 30 min of teatment wee simila. 4. Discussion Influenza vius can be elatively easily disinfected by chemicals such as alcohol [17, 18]. The pesence of oganic matte intefees with the action of chemical steilants because they can eact, theeby deceasing the efficiency of disinfection. Indeed, disinfection efficiency of alcohol against influenza vius vaies depending on the pesence of coexisting oganic mateial [19]. Most egulatoy authoities equie steilant efficacy testing to be conducted in the pesence of 5% soil [20]. Influenza vius is usually encounteed in the nasal fluid of patients and allantoic fluid of eggs. In the case of

6 6 iomed Reseach Intenational 0 min 5 min Figue 7: Change in mophology of influenza vius afte teatment with N 2 gas plasma. Influenza vius (/PR/8/34) in allantoic fluid was ai-died on cove glass and teated with N 2 gas plasma (1.5 kpps fo 5 min) using LP-TES No.1. n identical sample was also pepaed that was not teated with N 2 gas plasma. The mophologies of influenza vius wee obseved at 5 kv with a magnification of x50,000 by SEM (JSM-6320F, JEOL Ltd.) M1 NS H N Figue 8: N 2 gas plasma teatment causes damage to vial genomic RN. n aliquot of influenza vius (/PR/8/34) infected allantoic fluid was dopped onto a cove glass and ai-died. The died spot was then teated with N 2 gas plasma (1.5 kpps) fo the indicated times. The vial genomic RN of the influenza vius was subsequently extacted and subjected to evese tansciption in ode to geneate complementay DN (cdn). Damage to the genomic RN of influenza vius induced by the N 2 gas plasma teatment was analyzed using the polymease chain eaction (PCR). Namely, PCR was pefomed using cdns as template and specific sets of pimes fo matix potein (M1), nonstuctual potein (NS), hemagglutinin (H), and neuaminidase (N) of influenza vius. DN size make (100 bp DN ladde Dye plus, Takaa io Inc.) was un on the left hand lane of the gel. medical devices, bonchoscopes in paticula may be at isk of influenza vius contamination. In ou study, we used 100% nasal aspiates and 100% allantoic fluid as a souce of oganic mateial. The obtained esults showed that N 2 gas plasma can inactivate influenza vius in these envionments. Cuently, the items/aeas that can be teated by the method aeestictedtothesizeofthechambeboxofthen 2 gas plasma instument. To enable the steilization of medical devices and lage sufaces, it would be necessay to expand thesizeofthedischageaea. NP and genomic RN, which ae both localized to the cental egion of the influenza vius paticle, wee subject to decomposition and/o modification by teatment with N 2 gas plasma. Likewise, lipids localized in the oute envelope of the vius wee also modified and/o degaded afte this teatment. Micoscopic investigations showed that teatment with N 2 gas plasma disupt the fibes between vius paticles in the allantoic fluid, although the significance of the fibes connecting the viuses in the unteated samples in elation to the infectivity of the vius is unclea. Pevious studies have shown that oxidative stess contibutes to the mechanism of action of a gas plasma. Fo example, the addition of oxygen to helium has been found to enhance the efficiency of inactivation in the case of bacteia [21]. In addition, oxidation and peoxidation pocesses on the suface of cells and within cells esult in inactivation [22, 23]. Futhemoe, destuction of the suface stuctue by gas plasma may be the main mechanism undelying the inactivation of bacteia [24], which may also be the case fo viuses. lthough these oxidative factos contibute to the mechanism of action in N 2 gas plasma, futhe studies ae equied to identify the most citical facto(s) fo inactivation. In this study, we analyzed the effect of N 2 gas plasma teatment on influenza vius, which is enveloped. Howeve, the effectiveness of this teatment against vaious othe pathogens, which may diffe in esistance to disinfection, is unknown. Fo example, it would be inteesting to investigate the effect of this teatment on nonenveloped viuses, such as noovius o adenovius as well as the highly esistant pion agents. 5. Conclusion In conclusion, the pesent esults suggest that N 2 gas plasma teatment modifies vial genomic RN and degades vial poteins, including NPs, as well as the vial envelope and fibes elated to allantoic fluid. Taken togethe, the esults

7 iomed Reseach Intenational 7 indicate that N 2 gas plasma teatment may be an effective means of disinfection fo influenza vius. Conflict of Inteests The authos declae that they have no conflict of inteests. cknowledgments The authos ae gateful to D. Koichi aba (aba pediatic clinic, Kadoma, Osaka, Japan) fo sample collection. This wok was suppoted by Gant-in-id fo Pomotion of asic Reseach ctivities fo Innovative iosciences fom io-oiented Technology Reseach dvancement Institution (RIN) and fo Scientific Reseach on Innovative eas fom Japan Society fo the Pomotion of Science and Gantin-id fom the Ministy of Economy, Tade and Industy (METI) of Japan. Refeences [1]. Faise, J. Y. Maillad, and S. Satta, Russell, Hugo and yliffe s Pinciples and Pactice of Disinfection, Pesevation and Steilization, Wiley-lackwell, Hoboken, NJ, US, [2] G.. McCombs and M. L. 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