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Journal of General Virology (2008), 89, 60–67 Protective effect of low-concentration chlorinedioxide gas against influenza A virus infection Norio Ogata and Takashi Shibata Research Institute, Taiko Pharmaceutical Co. Ltd, 3-34-14 Uchihonmachi, Suita, Osaka 564-0032, Influenza virus infection is one of the major causes of human morbidity and mortality. Betweenhumans, this virus spreads mostly via aerosols excreted from the respiratory system. Currentmeans of prevention of influenza virus infection are not entirely satisfactory because of their limitedefficacy. Safe and effective preventive measures against pandemic influenza are greatly needed.
We demonstrate that infection of mice induced by aerosols of influenza A virus was preventedby chlorine dioxide (ClO2) gas at an extremely low concentration (below the long-term permissibleexposure level to humans, namely 0.1 p.p.m.). Mice in semi-closed cages were exposed toaerosols of influenza A virus (1 LD50) and ClO2 gas (0.03 p.p.m.) simultaneously for 15 min.
Three days after exposure, pulmonary virus titre (TCID50) was 102.6±1.5 in five mice treated withClO2, whilst it was 106.7±0.2 in five mice that had not been treated (P50.003). Cumulativemortality after 16 days was 0/10 mice treated with ClO2 and 7/10 mice that had not been treated(P50.002). In in vitro experiments, ClO2 denatured viral envelope proteins (haemagglutinin andneuraminidase) that are indispensable for infectivity of the virus, and abolished infectivity.
Taken together, we conclude that ClO2 gas is effective at preventing aerosol-induced influenzavirus infection in mice by denaturing viral envelope proteins at a concentration well below the Received 29 August 2007 permissible exposure level to humans. ClO2 gas could therefore be useful as a preventive means Accepted 7 October 2007 against influenza in places of human activity without necessitating evacuation.
As with many respiratory viruses, influenza virus spreads inthe air as aerosols (droplets) expelled from an infected Among the most frequent infections of the upper and human. It needs to attach to and penetrate target cells to lower respiratory tracts in humans are those caused by establish infection (Wagner et al., 2002). The principal influenza A virus, an enveloped, negative-sense, single- route of entry of the virus into target cells takes place by stranded RNA virus (Skehel & Hay, 1978; Ghendon et al., binding to a receptor on the surface of a respiratory-tract 1981; McCauley & Mahy, 1983). In a typical year, the virus epithelial cell, with subsequent transfer of viral genetic infects 15–20 % of the population, causing .500 000 materials into the infected cell (Wagner et al., 2002). The deaths worldwide (Thompson et al., 2003; WHO, 2003), envelope of the influenza virus carries two major surface but the most frightening effects are seen when new strains glycoproteins, haemagglutinin (HA) and neuraminidase of virus emerge, resulting in devastating pandemics (Reid (NA) (EC 3 . 2 . 1 . 18). HA plays a key role in initiating viral & Taubenberger, 2003). Current reports of avian-to- infection by binding to sialic acid-containing receptors on human transmission of influenza A virus, particularly of host cells and mediates viral entry into cells and fusion with the H5N1 subtype, make the prospect of new pandemics the cellular membrane (Tsuchiya et al., 2001; Wagner et al., particularly alarming (Webby & Webster, 2003; Webster 2002; Bentz & Mittal, 2003). At a later stage of infection, et al., 2007). It cannot be overemphasized that novel strains NA also plays a key role by releasing sialic acid residues of influenza virus have the potential to cause devastating from the surface of progeny virus particles and from the pandemics in the near future (Palese, 2004). In the past infected cell, facilitating viral release (Solorzano et al., 2000; century, three outbreaks of influenza virus infection have Wagner et al., 2002; Gong et al., 2007). When influenza caused significant numbers of human fatalities. Among virus is deficient in NA activity, progeny virus particles them, the 1918 strain was particularly notable for its aggregate at the surface of the infected cell, severely infectivity and the severity of the disease (Kong et al., impairing further spread of the virus to other cells. Both HA and NA are indispensable for successful infection andspread of this virus. Several antiviral compounds, such as Published online ahead of print on 15 October 2007 as DOI 10.1099/ developed, but their long-term efficacy is still limited by 0008-3393 G 2008 SGM Printed in Great Britain Antiviral activity of chlorine dioxide gas toxicity and inevitable selection of drug-resistant viral fetal bovine serum. They were purified by velocity density-gradient mutants (Nicholson et al., 2003). Vaccination against centrifugation through a 20–50 % linear sucrose gradient. Virion- influenza virus still has limited efficacy, and complete containing fractions were collected, titrated and stored at 280 uCuntil use. Just before use, a vial of virus was thawed quickly and prevention of the disease is not yet possible (Ge et al., diluted with Dulbecco's PBS to approximately 1 LD50 (one 50 % lethal dose) when delivered as aerosols. Mice were exposed to thispreparation for 15 min. The diluted virus suspension was placed in a Chlorine dioxide (ClO2) is a water-soluble, yellow gas with reservoir of an Aero-Mist nebulizer (CIS-US, Inc.). As a no-virus a characteristic chlorine-like odour and strong oxidizing control, another interchangeable nebulizer holding a reservoir of PBS activity (Moran et al., 1953; Fukayama et al., 1986; Ogata, alone was used in parallel. The virus and no-virus aerosols were 2007). It is commonly generated by adding acid to sodium changed quickly by a converter (Fig. 1). The day of aerosol challenge was termed day 0.
2) solution. ClO2 is a free radical, owing to one unpaired electron in its molecular orbital (ClO2) (Lynch et al., 1997). Possibly due to its strong oxidizing 2 generator. The ClO2 generator was made in our laboratory activity (Moran et al., 1953; Fukayama et al., 1986), when 2 was generated by mixing 250 mM HCl with 28 mM NaClO2; these solutions were delivered into a reaction vessel by dissolved in water, ClO2 has potent antimicrobial activity precision liquid pumps A and B. ClO2 was generated according to the against bacteria, fungi, protozoa and viruses (Taylor & reaction 5NaClO2+4HClA4ClO2+5NaCl+2H2O. ClO2 generated Butler, 1982; Harakeh et al., 1988; Chen & Vaughn, 1990; in the reaction vessel was next bubbled with air to expel it as gas.
Foschino et al., 1998; Eleraky et al., 2002; Schwartz et al., Approximately 50 p.p.m. ClO2 gas came out of the vessel at a flow 2003; Sivaganesan et al., 2003; Li et al., 2004; Loret et al., rate of 0.4 l min21. The ClO2 gas was next diluted by air using airpumps B and C. Finally, ClO 2005; Sy et al., 2005; Wilson et al., 2005; Okull et al., 2006; 2 gas at approximately 0.8 p.p.m. was delivered from the generator into the mouse cage at a flow rate of Simonet & Gantzer, 2006). However, the antimicrobial about 1.8 l min21 (Fig. 1). Concentration and flow rate of ClO2 gas activities of gas-phase ClO2 have not been well studied.
were adjusted finely by a concentration regulator and a flow-rate This is especially true of ClO regulator, respectively, to meet the gas concentration and flow rate 2 gas at very low concentra- tions (subtoxic levels) that are sufficiently safe to use in required for the experiment. The ClO2 gas was finally delivered into places of human activity without evacuation. According to the mouse cage as shown in Fig. 1(b).
the US Occupational Safety and Health Administration, the Set-up of the animal experimental system. For exposure of mice long-term (8 h) permissible exposure level of ClO2 in to ClO2 gas and virus aerosols, experiments were done in a class II environmental air in a human workplace is 0.1 p.p.m. (v/v) biosafety cabinet. A semi-closed mouse cage of 26637618 cm (inner (US Department of Labor, Occupational Safety and Health dimensions) containing 15 mice was placed in the biosafety cabinet (Fig. 1b). A battery-powered electric fan (26666 cm) to circulate airinside the cage was inserted in the cage with the mice and a battery If gas-phase ClO2 is shown to have potent antimicrobial box. The plexiglass cage was airtight except for the top cover, which activity at a subtoxic level, it would be useful to employ it was placed loosely on the cage so that air could seep out from the cage at such levels to prevent transmission of respiratory to prevent build-up of pressure within the cage. One of two infections in public places such as offices, schools, theatres, interchangeable nebulizers, containing either PBS alone or virussuspension in PBS, was connected to an air pump (Fig. 1b). Aerosols hospitals and airport buildings without evacuating occu- made by the nebulizers, either of PBS alone or of virus suspension in pants. The purpose of the present study was to determine PBS, were delivered into the mouse cage. The above-mentioned two whether ClO2 gas at a subtoxic level can protect against kinds of aerosol were interchanged quickly by the converter. ClO2 gas influenza A virus infection by using a mouse–influenza or air (0 p.p.m. ‘gas' as a control) was delivered into the cage through model. The mechanism of the effect of ClO another hole (Fig. 1b, right). A sampling tube of a ClO virus was further substantiated by in vitro biochemical (model 4330-SP; Interscan Corporation) was inserted into the cagethrough another hole. ClO gas concentrations were measured Pathological examination. Mice were sacrificed by an intramus- cular injection of pentobarbital sodium, and their lungs wereremoved carefully and weighed. A portion of the lung was Reagents, animals and virus. Sodium chlorite (NaClO2) was homogenized with PBS and aliquots were assayed for virus titre by obtained from JT Baker. All other reagents were of reagent grade. CD- using MDCK cells. The virus titre was expressed as TCID50. Another 1 male mice, 6–8 weeks of age, were purchased from Charles River portion of the lung was fixed in buffered formalin and stained with Laboratories. They were acclimatized in the laboratory for at least haematoxylin and eosin for histopathological examinations.
1 week before the experiment. For each set of experiments, groupsconsisted of 15 mice. Five were sacrificed on day 3 (72 h) after Assay of in vitro infectivity, HA titre and NA activity of virus.
exposure to virus aerosols (see below) for virus titre determination in Influenza virus (1 mg protein ml21) was treated with ClO2 at various their lungs and pathological examinations of lung tissue. Ten animals concentrations for 2 min at 0 uC in PBS. The reaction was terminated were observed further for mortality until day 21. The animal by adding a twofold molar excess of Na2S2O3. The in vitro infectivity experiment was approved by Taiko Pharmaceutical Experiment of the virus was determined by using MDCK cells as indicator cells.
Committee. Influenza virus strain A/PR/8/34 (H1N1) was used for Briefly, 16106 cells were inoculated in a Petri dish of 6 cm diameter animal experiments, and strain A/New Caledonia/20/99 (H1N1) was using 10 ml Eagle's MEM containing 10 % fetal bovine serum. Cells used for all in vitro experiments. These viruses were grown and were grown until confluent (about 2 days) and then inoculated with propagated by using Madin–Darby canine kidney (MDCK) cells and tenfold serial dilutions of virus, treated or not treated with ClO2, Eagle's minimum essential medium (MEM) supplemented with 10 % suspended in PBS. Cells were next overlaid with freshly prepared N. Ogata and T. Shibata Fig. 1. (a) Schematic structure of a ClO2generator. (b) Experimental set-up for expo-sure of mice to influenza A virus aerosols andClO2 gas.
medium without serum, but supplemented with 0.9 % agar, 2.5 mg portion (100 ml) of the reaction mixture was then loaded for high- trypsin ml21, 100 units penicillin G ml21 and 100 mg streptomycin performance liquid chromatography (HPLC) using a reverse-phase ml21. The culture dish was incubated at 37 uC for 3–4 days in 95 % column (Cosmosil 5C18-AR-300, 4.6 mm inner diameter, 250 mm air/5 % CO2. The cells were then fixed and stained with crystal violet long; Nacalai Tesque). The column was eluted with a solvent of 0.1 % to count the number of plaques. The concentrations of ClO2 and/or (v/v) trifluoroacetic acid for 6 min and then with a linear gradient of Na2S2O3 used in the above experiment had no effect on the growth of acetonitrile from 10 to 50 % in the above solvent over the next 54 min MDCK cells. For the HA titre assay, twofold serial dilutions of treated at a flow rate of 1 ml min21. Peptides were monitored by absorption (0 uC, 2 min, in PBS) virus were prepared in PBS and added to a at 270 nm. Peak materials (peptides) were collected and lyophilized.
round-bottomed 96-well microtitre plate (50 ml per well). Chicken The lyophilized peptides were next analysed by Edman degradation red blood cells (26106 cells in 50 ml) were then added and incubated using a protein sequencer (Procise; Applied Biosystems) to determine for 1 h at 4 uC. End-point HA titres were expressed as the reciprocal their amino acid sequences. Molecular masses of peptides and amino of the last dilution that showed complete haemagglutination. For the acid residues were determined by using a mass spectrometer (model NA assay, virus was diluted to 8 mg protein ml21, and then 2 mM 29- Ultraflex; Bruker Daltonik) in matrix-assisted laser desorption/ (4-methylumbelliferyl)-a-D-N-acetylneuraminic acid (sodium salt) in ionization–time of flight (MALDI-TOF) and MALDI-TOF/TOF calcium-MES buffer [32.5 mM MES buffer (pH 6.5), 4 mM CaCl ] (tandem MS) modes. a-Cyano-4-hydroxycinnamic acid was used as was added to a final concentration of 1 mM. The mixture was incubated for 1 h at 37 uC. The reaction was terminated by adding800 ml glycine buffer (0.1 M, pH 10.7) containing 25 % ethanol.
Statistical analysis. Data were analysed by using Student's t-test or Fluorescence intensity was measured (lex5365 nm, lem5450 nm) by Fisher's exact test. P values ,0.05 were considered statistically a spectrofluorophotometer (model RF-5300PC; Shimadzu).
Sequencing and mass spectrometry (MS) of peptides. Syntheticpeptides HA1 (NPENGTCYPG) and HA2 (RNLLWLTGKN) corre- spond to aa 101–110 and 162–171, respectively, of the HA protein.
Peptides NA1 (FESVAWSASA) and NA2 (SGYSGSFVQH) corre-spond to aa 174–183 and 400–409, respectively, of the NA protein.
Simultaneous exposure of mice to virus aerosols They were obtained from Global Peptide Services. These peptides (2 mM each) were treated with 4 mM ClO2 at 25 uC for 2 min in PBSin a volume of 500 ml. After the reaction, a twofold molar excess ClO2 gas made by a ClO2 generator (Fig. 1a) was delivered (1.6 ml) of 2.5 M Na2S2O3 was added to terminate the reaction. A into the mouse cage for 15 min simultaneously with Journal of General Virology 89 Antiviral activity of chlorine dioxide gas Table 1. Pulmonary virus titres of each mouse challenged with Table 3. Body mass of mice 1 week after challenge with influenza A virus aerosols in the absence or presence of influenza A virus in the absence or presence of 0.03 p.p.m.
0.03 p.p.m. ClO2 gas Virus titre in each mouse (log10)* Body mass (g) at day: *Virus titre, expressed as TCID50, was measured 72 h after challengeby virus aerosols (n55 mice per group).
*Ratio of body mass on day 7 to that on day 0 in each group.
DP50.003 when the means of two groups were compared (Student's DP50.002 when relative body masses of the 0 and 0.03 p.p.m. ClO2 groups were compared (Student's t-test, n55 in each group).
aerosols of PBS alone or of influenza A virus suspended in supports the protection of mice from morbidity caused by PBS (Fig. 1b). The ClO2 gas concentration in the mouse cage of the ClO2-treated group during this period was (0.03 p.p.m., without virus) and PBS aerosols (without 0.032±0.026 p.p.m. (time-weighted mean±SD). As a virus) were delivered into a cage housing another group of ClO2-untreated control, only air (0 p.p.m. ClO2) and 15 mice to know whether ClO2 gas at a concentration of aerosols of influenza virus suspended in PBS were delivered 0.03 p.p.m. has any toxic effect on mice. Mice were into the mouse cage housing another group of 15 mice. In apparently completely healthy for the 21 days of obser- the ClO2-untreated control group on day 3 (72 h), the vation. Microscopic examination of histopathological pulmonary titre (TCID50) of the virus was 106.7±0.2 (n55), whereas it was 102.6±1.5 in the ClO2-treated group 0.03 p.p.m. ClO2 gas and PBS aerosols showed that their (P50.003, Student's t-test) (Table 1), demonstrating lungs were completely normal (data not shown).
clearly that ClO2 gas was effective in decreasing thenumber of infectious viruses in mouse lungs (a similar Delayed gas-delivery experiment result was obtained in another independent experiment).
Cumulative mortality at day 16 was 70 % (7/10) in the Next, we examined the effect of ClO2 gas delivered for ClO2-untreated group and 0 % (0/10) in the ClO2-treated 15 min into the mouse cage at various delay times after group (P50.002, Fisher's exact test) (Table 2). This result commencement of the delivery of influenza virus aerosols.
indicates that ClO2 gas can prevent mortality of mice The purpose of this experiment was to determine whether challenged with influenza A virus aerosols. We confirmed ClO2 gas delivered after the virus aerosols would still be the reproducibility of the above result in another able to prevent viral infection. Mortality of mice was 0 % experiment, in which the mortality was 5/10 mice without (0/10) when ClO2 was delivered simultaneously with the ClO2 gas, and 0/10 with 0.03 p.p.m. ClO2 gas (P50.03).
virus aerosols (0 min delay, P50.022 versus no-ClO2 Relative body mass (body mass at day 7 compared with group) (Table 4), confirming the result shown in Table 2.
that at day 0) was 1.09±0.08 (n55) in the ClO When ClO2 gas was delivered 5 min after the delivery of group and 0.91±0.04 (n55) in the untreated group virus aerosols (5 min delay), mortality was 10 % (1/10) (P50.002, Student's t-test) (Table 3). This result further (P50.081 versus no-ClO2 group). The mortality rate was50 % (5/10) with a 15 min delay, which was the same as inanimals that received no ClO2 gas treatment (Table 4). The Table 2. Mortality of mice exposed to aerosols of influenza A result indicates that ClO2 gas was an effective preventative virus in the absence or presence of 0.03 p.p.m. ClO of influenza virus infection when present in the envir- onment simultaneously with the virus aerosols. When Values are the number of mice that died at each time point after virus delivered after a 5 min delay, it may have been slightly effective (P50.081), but it was completely ineffective whendelivered 15 min after commencement of the delivery of Time after virus challenge (days) the virus aerosols. Taken together, these results indicate that ClO2 gas inactivated the virus before it entered the lungs, but that it lacked the ability to inactivate viruses that had already entered the lungs and established infection. In summary, ClO2 gas, at an extremely low concentration(below the long-term permissible exposure level to *P50.002 when the 0 and 0.03 p.p.m. groups on day 16 were humans), is effective at preventing infection of mice by compared (Fisher's exact test, n510 for each group).
influenza A virus without any harmful effects.
N. Ogata and T. Shibata Table 4. Mortality of mice challenged with influenza A virus influenza virus is attributable to the decrease of biological aerosols in the absence or presence of 0.03 p.p.m. ClO2 activities of the HA and NA proteins on the virus envelope.
gas that was delivered for 15 min at various delay times aftercommencement of the delivery of virus aerosols Denaturation of HA and NA proteins Values are the number of mice that died at each time point after virus We speculated that ClO2 denatured the HA and NA proteins and inactivated their biological activities. Toprovide support for this hypothesis, we selected two model Time after virus challenge (days) decapeptides (HA1 and HA2) from HA and two (NA1 and NA2) from NA (for sequences, see Methods). After treatment of these peptides with ClO2, they were analysed by reverse-phase HPLC. When these four peptides were treated individually with ClO2, there were several novel peptide peaks on the chromatograms that differed completely from the original peptide peaks (data not shown). This indicates that the original peptides weremodified covalently by reaction with ClO2. This hypothesis *P50.022 when compared with the no-ClO2 group (Fisher's exact was supported further by the fact that, upon sequencing test, n510 in each group).
(by Edman degradation) of the peptide peaks recovered DP50.081 when compared with the no-ClO2 group (Fisher's exact from HPLC, some amino acid residues in the peptides were test, n510 in each group).
not identified (Table 6). For example, regarding thepeptide HA2 (RNLLWLTGKN, aa 162–171) treated withClO2, the sequence of the peptide peak recovered from Effect of ClO2 on the infectivity of influenza A HPLC was RNLLXLTGKN; the fifth amino acid residue (Trp166 in the original protein) could not be identified by Influenza A virus was treated in vitro with ClO the conventional protein-sequencing method. This indi- infectivity was assayed by using cultured cells. Infectivity cates strongly that this residue (tryptophan) was modified of the virus decreased markedly after treatment with covalently by ClO2. Likewise, other peptides were also found to be modified at tryptophan and tyrosine residues 2, demonstrating that ClO2 indeed inacti- vates the infectivity of the virus (Table 5). As the HA and (Table 6). It is unclear whether the cysteine residue of HA1 NA proteins on the virus surface (envelope) are indispens- was modified by ClO2, because cysteine residues are not able to the infectivity of the virus, we assayed their positively identifiable by this conventional sequencing biological activities. As shown in Table 5, both HA and NA activities decreased markedly after ClO2 treatment in vitro.
Covalent modification of tryptophan and tyrosine residues This result suggests that the reduction in the infectivity of by ClO2 was confirmed by MS. As shown in Table 7, in themodified HA2 and NA1 peptides, there was an increase ofabout 32 or 48 atomic mass units in the tryptophanresidues, indicating that two or three atoms of oxygen were Table 5. In vitro infectivity of influenza A virus suspensiontreated with ClO2 Influenza A virus was treated with ClO Table 6. Amino acid sequences of ClO subjected to various assays. Virus and HA titres are the means of two peptides derived from the HA and NA proteins of influenza A experiments. NA activity is the mean±SD of five experiments. ND, Not determined.
Each model peptide (2 mM) was treated with 4 mM ClO2 at 25 uCfor 2 min, and then analysed individually by HPLC. Peak fractions of NA activity [units HPLC were recovered and subjected to protein sequencing. X denotes amino acid residues that gave unusual peaks on chromatograms of the protein sequencer and were therefore not identified.
ClO2-treated peptide *Reciprocal of the last dilution that showed complete haemagglutination.
DP,0.05 when compared with 0 mM ClO2 (Student's t-test).
dP,0.0001 when compared with 0 mM ClO2 (Student's t-test).
Journal of General Virology 89 Antiviral activity of chlorine dioxide gas Table 7. MS analyses of ClO2-treated model peptides prevent their infection by influenza A virus and possibly derived from the HA and NA proteins of influenza A virus other related virus infections of the respiratory tract.
Specifically, ClO2 gas could be used in places such as Peptides HA1 (NPENGTCYPG), HA2 (RNLLWLTGKN), NA1 offices, theatres, hotels, schools and airport buildings (FESVAWSASA) and NA2 (SGYSGSFVQH) (each 2 mM) were without evacuating people, thus not interrupting their treated with 4 mM ClO2 at 25 uC for 2 min. They were then analysed normal activities.
individually by HPLC and two peak fractions were recovered fromeach HPLC run. The peak fractions were analysed by MS. ND, Not Current growing concerns about the threat posed by highly pathogenic H5N1 avian influenza virus have promptedinterest in evaluating measures against this virus. ClO2 and Peptide* Parent ion ([M+H]+)D Amino acid residued chlorine have long been used as disinfectants of publicwater supplies. Thus far, chlorine treatment (chlorination) represents the most common form of disinfection used in water treatment. Rice et al. (2007) reported recently that the H5N1 strain of influenza A virus was inactivated by chlorine in an in vitro experiment. In their experiment, the free chlorine concentration typically used in drinking- water treatment was sufficient to inactive the virus by more than three orders of magnitude. Although the strain of influenza virus used in our present experiment (H1N1) differs from that of Rice et al. (2007), it is suggested thatour present method, namely treatment of influenza virus *Peak fractions recovered from HPLC. The name in parentheses by ClO2, provides another effective manoeuvre for the denotes that of the original peptide.
treatment of public water supplies contaminated by the DMass/charge of the [M+H]+ ion of the ClO2-treated and HPLC- virus, and it paves a new way for prevention of pandemic recovered peptides, determined by MALDI-TOF MS.
dMass/charge of each amino acid residue of the peptide determinedby MALDI-TOF/TOF MS. Only the amino acid residue whose mass/ ClO2 gas is very soluble in water, and is in equilibrium charge was significantly different from that of the original residue is between the gas and water phases. In our preliminary shown. The mass of water (18.0) has been added to the mass/charge experiment, ClO2 reached equilibrium between the gas and for clarity.
water phases within 30 s (half-maximal in 20 s) (N. Ogata, §d denotes the difference between expected and found mass/charge unpublished data). Generally speaking, a water-soluble gaseous substance reaches equilibrium between the gas andwater phases according to Henry's law, C5kP, where Cis the concentration of a substance in the water phase, P is incorporated covalently into tryptophan residues. Likewise, partial pressure of the substance in the gas phase and k is there was an increase of about 32 or 48 atomic mass units an equilibrium constant. When the diameter of the aerosol in the tyrosine residues in the modified HA1 and NA2 is in the range 1–10 mm, as in the present experiment, peptides (Table 7), indicating the covalent incorporation of equilibrium is reached within 1 min. We also found that two or three atoms of oxygen into tyrosine residues. Taken Henry's equilibrium gas constant k regarding the ClO2– together, we conclude that amino acid residues in the HA water equilibrium, namely k in the above equation, was and NA proteins, primarily tryptophan and tyrosine 3.961025 mol l21 Pa21 (N. Ogata, unpublished data).
residues, are modified covalently by ClO2. Such modifica- Therefore, the ClO2 concentration in the virus aerosols is tions of amino acid residues appear to denature the HA theoretically 0.12 mM when the aerosols are in equilibrium and NA proteins of influenza A virus, which are with 0.03 p.p.m. ClO2 gas. This suggests further that the indispensable for its infectivity, and consequently abolish influenza A virus is inactivated at 0.12 mM ClO2 in water infectivity of the virus.
(PBS in our present experiment).
We have shown that ClO2 denatures (abolishes thefunctions of) the HA and NA proteins on the envelope of the influenza virus (Table 5). As these proteins are We have demonstrated that ClO2 gas at an extremely low indispensable for the infectivity of this virus, the fact that concentration can prevent influenza A virus infection of they were denatured by ClO2 could explain why infectivity mice caused by aerosols. According to the US Occupational of the virus decreased after treatment with ClO2. However, Safety and Health Administration, the 8 h permissible it is noteworthy that the reduction in infectivity, as exposure level of ClO2 in human workplaces is 0.1 p.p.m.
demonstrated by plaque assay, did not necessarily parallel The level of ClO2 gas (0.03 p.p.m.) used in this study is the reductions in HA and NA activities (Table 5). One well below this level, and our results indicate that ClO2 at possibility is the presence of other protein(s) in the virus this level could be used in the presence of humans to that is/are critical and indispensable for its infectivity and N. Ogata and T. Shibata is/are denatured by ClO Kong, W.-P., Hood, C., Yang, Z.-Y., Wei, C.-J., Xu, L., Garcia-Sastre, A., 2. For example, the M2 protein, a proton channel in the virus envelope, could be a target of Tumpey, T. M. & Nabel, G. J. (2006). Protective immunity to lethal challenge of the 1918 pandemic influenza virus by vaccination. Proc 2. This protein is indispensable for the virus to establish Natl Acad Sci U S A 103, 15987–15991.
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residue (Trp41) in this protein and abolish its function.
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