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Protective effect of low-concentration chlorine
dioxide gas against influenza A virus infection
Norio Ogata and Takashi Shibata
Correspondence
Norio Ogata
nogata7@yahoo.co.jp
Research Institute, Taiko Pharmaceutical Co. Ltd, 3-34-14 Uchihonmachi, Suita, Osaka 564-0032,
Japan
Received 29 August 2007
Accepted 7 October 2007
Influenza virus infection is one of the major causes of human morbidity and mortality. Between
humans, this virus spreads mostly via aerosols excreted from the respiratory system. Current
means of prevention of influenza virus infection are not entirely satisfactory because of their limited
efficacy. 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 prevented
by chlorine dioxide (ClO
2
) gas at an extremely low concentration (below the long-term permissible
exposure level to humans, namely 0.1 p.p.m.). Mice in semi-closed cages were exposed to
aerosols of influenza A virus (1 LD
50
) and ClO
2
gas (0.03 p.p.m.) simultaneously for 15 min.
Three days after exposure, pulmonary virus titre (TCID
50
) was 10
2.6±1.5
in five mice treated with
ClO
2
, whilst it was 10
6.7±0.2
in five mice that had not been treated (P50.003). Cumulative
mortality after 16 days was 0/10 mice treated with ClO
2
and 7/10 mice that had not been treated
(P50.002). In in vitro experiments, ClO
2
denatured viral envelope proteins (haemagglutinin and
neuraminidase) that are indispensable for infectivity of the virus, and abolished infectivity.
Taken together, we conclude that ClO
2
gas is effective at preventing aerosol-induced influenza
virus infection in mice by denaturing viral envelope proteins at a concentration well below the
permissible exposure level to humans. ClO
2
gas could therefore be useful as a preventive means
against influenza in places of human activity without necessitating evacuation.
INTRODUCTION
Among the most frequent infections of the upper and
lower respiratory tracts in humans are those cause d by
influenza A virus, an enveloped, negative-sense, single-
stranded RNA virus (Skehel & Hay, 1978; Ghendon et al.,
1981; McCauley & Mahy, 1983). In a typical year, the virus
infects 15–20 % of the population, causing .500 000
deaths worldwide (Thompson et al., 2003; WHO, 2003),
but the most frightening effects are seen when new strains
of virus emerge, resulting in devastating pandemics (Reid
& Taubenberger, 2003). Current reports of avian-to-
human transmission of influenza A virus, particularly of
the H5N1 subtype, make the prospect of new pandemics
particularly alarming (We bby & Webster, 2003; Webster
et al., 2007). It cannot be overemphasized that novel strains
of influenza virus have the potential to cause devastating
pandemics in the near future (Palese, 2004). In the past
century, three outbreaks of influenza virus infection have
caused significant numbers of human fatalities. Among
them, the 1918 strain was particularly notable for its
infectivity and the severity of the disease (Kong et al.,
2006).
As with many resp iratory viruses, influenza virus spreads in
the air as aerosols (droplets) expelled from an infected
human. It needs to attach to and penetrate target cells to
establish infection (Wagner et al., 2002). The principal
route of entry of the virus into target cells takes place by
binding to a receptor on the surface of a respiratory-tract
epithelial cell, with subsequent transfer of viral genetic
materials into the infected cell (Wagner et al., 2002). The
envelope of the influenza virus carries two major surface
glycoproteins, haemagglutinin (HA) and neuraminidase
(NA) (EC 3 . 2 . 1 . 18). HA plays a key role in initiating viral
infection by binding to sialic acid-containing receptors on
host cells and mediates viral entry into cells and fusion with
the cellular membrane (Tsuchiya et al., 2001; Wagner et al.,
2002; Bentz & Mittal, 2003). At a later stage of infection,
NA also plays a key role by releasing sialic acid residues
from the surface of progeny virus particles and from the
infected cell, facilitating viral release (Solorzano et al., 2000;
Wagner et al., 2002; Gong et al., 2007). When influenza
virus is deficient in NA activity, progeny virus particles
aggregate at the surface of the infected cell, severely
impairing further spread of the virus to other cells. Both
HA and NA are indispensable for successful infection and
spread of this virus. Several antiviral compounds, such as
zanamivir, oseltamivir and resveratrol, have been
developed, but their long-term efficacy is still limited by
Published online ahead of print on 15 October 2007 as DOI 10.1099/
vir.0.83393-0.
Journal of General Virology (2008), 89, 60–67 DOI 10.1099/vir.0.83393-0
60 0008-3393
G
2008 SGM Printed in Great Britain
toxicity and inevitable selection of drug-resistant viral
mutants (Nicholson et al., 2003). Vaccination against
influenza virus still has limited efficacy, and complete
prevention of the disease is not yet possible (Ge et al.,
2004).
Chlorine dioxide (ClO
2
) is a water-soluble, yellow gas with
a characteristic chlori ne-like odour and stron g oxidizing
activity (Moran et al., 1953; Fukayama et al., 1986; Ogata,
2007). It is commonly generated by adding acid to sodium
chlorite (NaClO
2
) solution. ClO
2
is a free radical, owing to
one unpaired electron in its molecular orbital (ClO
2
:
)
(Lynch et al., 1997). Possibly due to its strong oxidizing
activity (Moran et al., 1953; Fukayama et al., 1986), when
dissolved in water, ClO
2
has potent antimicrobial activity
against bacteria, fungi, protozoa and viruses (Taylor &
Butler, 1982; Harakeh et al., 1988; Chen & Vaughn, 1990;
Foschino et al., 1998; Eleraky et al., 2002; Schwartz et al.,
2003; Sivaganesan et al., 2003; Li et al., 2004; Loret et al.,
2005; Sy et al., 2005; Wilson et al., 2005; Okull et al., 2006;
Simonet & Gantzer, 2006). However, the antimicrobial
activities of gas-phase ClO
2
have not been well studied.
This is especially true of ClO
2
gas at very low concentra-
tions (subtoxic levels) that are sufficiently safe to use in
places of human activity without evacuation. According to
the US Occupational Safety and Health Administration, the
long-term (8 h) permissible exposure level of ClO
2
in
environmental air in a human workplace is 0.1 p.p.m. (v/v)
(US Department of Labor, Occupational Safety and Health
Administration, 2006).
If gas-phase ClO
2
is shown to have potent antimicrobial
activity at a subtoxic level, it would be useful to employ it
at such levels to prevent transmission of respiratory
infections in public places such as offices, schools, theatres,
hospitals and airport buildings without evacuating occu-
pants. The purpose of the present study was to determine
whether ClO
2
gas at a subtoxic level can protect against
influenza A virus infection by using a mouse–influenza
model. The mechanism of the effect of ClO
2
against this
virus was further substantiated by in vitro biochemical
experiments.
METHODS
Reagents, animals and virus. Sodium chlorite (NaClO
2
) was
obtained from JT Baker. All other reagents were of reagent grade. CD-
1 male mice, 6–8 weeks of age, were purchased from Charles River
Laboratories. They were acclimatized in the laboratory for at least
1 week before the experiment. For each set of experiments, groups
consisted of 15 mice. Five were sacrificed on day 3 (72 h) after
exposure to virus aerosols (see below) for virus titre determination in
their lungs and pathological examinations of lung tissue. Ten animals
were observed further for mortality until day 21. The animal
experiment was approved by Taiko Pharmaceutical Experiment
Committee. Influenza virus strain A/PR/8/34 (H1N1) was used for
animal experiments, and strain A/New Caledonia/20/99 (H1N1) was
used for all in vitro experiments. These viruses were grown and
propagated by using Madin–Darby canine kidney (MDCK) cells and
Eagle’s minimum essential medium (MEM) supplemented with 10 %
fetal bovine serum. They were purified by velocity density-gradient
centrifugation through a 20–50 % linear sucrose gradient. Virion-
containing fractions were collected, titrated and stored at 280 uC
until use. Just before use, a vial of virus was thawed quickly and
diluted with Dulbecco’s PBS to approximately 1 LD
50
(one 50 %
lethal dose) when delivered as aerosols. Mice were exposed to this
preparation for 15 min. The diluted virus suspension was placed in a
reservoir of an Aero-Mist nebulizer (CIS-US, Inc.). As a no-virus
control, another interchangeable nebulizer holding a reservoir of PBS
alone was used in parallel. The virus and no-virus aerosols were
changed quickly by a converter (Fig. 1). The day of aerosol challenge
was termed day 0.
ClO
2
generator. The ClO
2
generator was made in our laboratory
(Fig. 1a). ClO
2
was generated by mixing 250 mM HCl with 28 mM
NaClO
2
; these solutions were delivered into a reaction vessel by
precision liquid pumps A and B. ClO
2
was generated according to the
reaction 5NaClO
2
+4HClA4ClO
2
+5NaCl+2H
2
O. ClO
2
generated
in the reaction vessel was next bubbled with air to expel it as gas.
Approximately 50 p.p.m. ClO
2
gas came out of the vessel at a flow
rate of 0.4 l min
21
. The ClO
2
gas was next diluted by air using air
pumps B and C. Finally, ClO
2
gas at approximately 0.8 p.p.m. was
delivered from the generator into the mouse cage at a flow rate of
about 1.8 l min
21
(Fig. 1). Concentration and flow rate of ClO
2
gas
were adjusted finely by a concentration regulator and a flow-rate
regulator, respectively, to meet the gas concentration and flow rate
required for the experiment. The ClO
2
gas was finally delivered into
the mouse cage as shown in Fig. 1(b).
Set-up of the animal experimental system. For exposure of mice
to ClO
2
gas and virus aerosols, experiments were done in a class II
biosafety cabinet. A semi-closed mouse cage of 26637618 cm (inner
dimensions) containing 15 mice was placed in the biosafety cabinet
(Fig. 1b). A battery-powered electric fan (26666 cm) to circulate air
inside the cage was inserted in the cage with the mice and a battery
box. The plexiglass cage was airtight except for the top cover, which
was placed loosely on the cage so that air could seep out from the cage
to prevent build-up of pressure within the cage. One of two
interchangeable nebulizers, containing either PBS alone or virus
suspension in PBS, was connected to an air pump (Fig. 1b). Aerosols
made by the nebulizers, either of PBS alone or of virus suspension in
PBS, were delivered into the mouse cage. The above-mentioned two
kinds of aerosol were interchanged quickly by the converter. ClO
2
gas
or air (0 p.p.m. ‘gas’ as a control) was delivered into the cage through
another hole (Fig. 1b, right). A sampling tube of a ClO
2
analyser
(model 4330-SP; Interscan Corporation) was inserted into the cage
through another hole. ClO
2
gas concentrations were measured
intermittently.
Pathological examination. Mice were sacrificed by an intramus-
cular injection of pentobarbital sodium, and their lungs were
removed carefully and weighed. A portion of the lung was
homogenized with PBS and aliquots were assayed for virus titre by
using MDCK cells. The virus titre was expressed as TCID
50
. Another
portion of the lung was fixed in buffered formalin and stained with
haematoxylin and eosin for histopathological examinations.
Assay of in vitro infectivity, HA titre and NA activity of virus.
Influenza virus (1 mg protein ml
21
) was treated with ClO
2
at various
concentrations for 2 min at 0 uC in PBS. The reaction was terminated
by adding a twofold molar excess of Na
2
S
2
O
3
. The in vitro infectivity
of the virus was determined by using MDCK cells as indicator cells.
Briefly, 1610
6
cells were inoculated in a Petri dish of 6 cm diameter
using 10 ml Eagle’s MEM containing 10 % fetal bovine serum. Cells
were grown until confluent (about 2 days) and then inoculated with
tenfold serial dilutions of virus, treated or not treated with ClO
2
,
suspended in PBS. Cells were next overlaid with freshly prepared
Antiviral activity of chlorine dioxide gas
http://vir.sgmjournals.org 61
medium without serum, but supplemented with 0.9 % agar, 2.5 mg
trypsin ml
21
, 100 units penicillin G ml
21
and 100 mg streptomycin
ml
21
. The culture dish was incubated at 37 uC for 3–4 days in 95 %
air/5 % CO
2
. The cells were then fixed and stained with crystal violet
to count the number of plaques. The concentrations of ClO
2
and/or
Na
2
S
2
O
3
used in the above experiment had no effect on the growth of
MDCK cells. For the HA titre assay, twofold serial dilutions of treated
(0 uC, 2 min, in PBS) virus were prepared in PBS and added to a
round-bottomed 96-well microtitre plate (50
ml per well). Chicken
red blood cells (2610
6
cells in 50 ml) were then added and incubated
for 1 h at 4 uC. End-point HA titres were expressed as the reciprocal
of the last dilution that showed complete haemagglutination. For the
NA assay, virus was diluted to 8
mg protein ml
21
, and then 2 mM 29-
(4-methylumbelliferyl)-
a-D-N-acetylneuraminic acid (sodium salt) in
calcium-MES buffer
[
32.5 mM MES buffer (pH 6.5), 4 mM CaCl
2
]
was added to a final concentration of 1 mM. The mixture was
incubated for 1 h at 37 uC. The reaction was terminated by adding
800
ml glycine buffer (0.1 M, pH 10.7) containing 25 % ethanol.
Fluorescence intensity was measured (
l
ex
5365 nm, l
em
5450 nm) by
a spectrofluorophotometer (model RF-5300PC; Shimadzu).
Sequencing and mass spectrometry (MS) of peptides. Synthetic
peptides 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.
They were obtained from Global Peptide Services. These peptides
(2 mM each) were treated with 4 mM ClO
2
at 25 u C for 2 min in PBS
in a volume of 500
ml. After the reaction, a twofold molar excess
(1.6
ml) of 2.5 M Na
2
S
2
O
3
was added to terminate the reaction. A
portion (100
ml) of the reaction mixture was then loaded for high-
performance liquid chromatography (HPLC) using a reverse-phase
column (Cosmosil 5C18-AR-300, 4.6 mm inner diameter, 250 mm
long; Nacalai Tesque). The column was eluted with a solvent of 0.1 %
(v/v) trifluoroacetic acid for 6 min and then with a linear gradient of
acetonitrile from 10 to 50 % in the above solvent over the next 54 min
at a flow rate of 1 ml min
21
. Peptides were monitored by absorption
at 270 nm. Peak materials (peptides) were collected and lyophilized.
The lyophilized peptides were next analysed by Edman degradation
using a protein sequencer (Procise; Applied Biosystems) to determine
their amino acid sequences. Molecular masses of peptides and amino
acid residues were determined by using a mass spectrometer (model
Ultraflex; Bruker Daltonik) in matrix-assisted laser desorption/
ionization–time of flight (MALDI-TOF) and MALDI-TOF/TOF
(tandem MS) modes.
a-Cyano-4-hydroxycinnamic acid was used as
a matrix.
Statistical analysis. Data were analysed by using Student’s t-test or
Fisher’s exact test. P values ,0.05 were considered statistically
significant.
RESULTS
Simultaneous exposure of mice to virus aerosols
and ClO
2
gas
ClO
2
gas made by a ClO
2
generator (Fig. 1a) was delivered
into the mouse cage for 15 min simultaneously with
Fig. 1. (a) Schematic structure of a ClO
2
generator. (b) Experimental set-up for expo-
sure of mice to influenza A virus aerosols and
ClO
2
gas.
N. Ogata and T. Shibata
62 Journal of General Virology 89
aerosols of PBS alone or of influenza A virus suspended in
PBS (Fig. 1b). The ClO
2
gas concentration in the mouse
cage of the ClO
2
-treated group during this per iod was
0.032±0.026 p.p .m. (time-weighted mean±
SD). As a
ClO
2
-untreated control, only air (0 p.p.m. ClO
2
) and
aerosols of influenza virus suspended in PBS were delivered
into the mouse cage housing another group of 15 mice. In
the ClO
2
-untreated control group on day 3 (72 h), the
pulmonary titre (TCID
50
) of the virus was 10
6.7±0.2
(n55),
whereas it was 10
2.6±1.5
in the ClO
2
-treated group
(P50.003, Student’s t-test) (Table 1), demonstrating
clearly that ClO
2
gas was effective in decreasing the
number of infectious viruses in mouse lungs (a similar
result was obtained in another independent experiment).
Cumulative mortality at day 16 was 70 % (7/10) in the
ClO
2
-untreated group and 0 % (0/10) in the ClO
2
-treated
group (P50.002, Fisher’s exact test) (Table 2). This result
indicates that ClO
2
gas can prevent mortality of mice
challenged with influenza A virus aerosols. We confirmed
the reproducibility of the above result in another
experiment, in which the mortality was 5/10 mice without
ClO
2
gas, and 0/10 with 0.03 p.p.m. ClO
2
gas (P50.03).
Relative body mass (body mass at day 7 compared with
that at day 0) was 1.09±0.08 (n55) in the ClO
2
-treated
group and 0.91±0.04 (n5 5) in the untreated group
(P50.002, Student’s t-test) (Table 3). This result further
supports the protection of mice from morbidity caused by
influenza A virus. As another control, ClO
2
gas
(0.03 p.p.m., without virus) and PBS aerosols (without
virus) were delivered into a cage housing another group of
15 mice to know whether ClO
2
gas at a concentration of
0.03 p.p.m. has any toxic effect on mice. Mice were
apparently completely healthy for the 21 days of obser-
vation. Microscopic examination of histopathological
specimens of lungs from five mice treated with
0.03 p.p.m. ClO
2
gas and PBS aerosols showed that their
lungs were completely normal (data not shown).
Delayed gas-delivery experiment
Next, we examined the effect of ClO
2
gas delivered for
15 min into the mouse cage at various delay times after
commencement of the delivery of influenza virus aerosols.
The purpose of this experiment was to determine whether
ClO
2
gas delivered after the virus aerosols would still be
able to prevent viral infection. Mortality of mice was 0 %
(0/10) when ClO
2
was delivered simultaneously with the
virus aerosols (0 min delay, P50.022 versus no-ClO
2
group) (Table 4), confirming the result shown in Table 2.
When ClO
2
gas was delivered 5 min after the delivery of
virus aerosols (5 min delay), mortality was 10 % (1/10)
(P50.081 versus no-ClO
2
group). The mortality rate was
50 % (5/10) with a 15 min delay, which was the same as in
animals that received no ClO
2
gas treatment (Table 4). The
result indicates that ClO
2
gas was an effective preventative
of influenza virus infection when present in the envir-
onment simultaneously with the virus aerosols. When
delivered after a 5 min delay, it may have been slightly
effective (P50.081), but it was completely ineffective when
delivered 15 min after commencement of the delivery of
the virus aerosols. Taken together, these results indicate
that ClO
2
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, ClO
2
gas, at an extremely low concentration
(below the long-term permissible exposure level to
humans), is effective at preventing infection of mice by
influenza A virus without any harmful effects.
Table 1. Pulmonary virus titres of each mouse challenged with
influenza A virus aerosols in the absence or presence of
0.03 p.p.m. ClO
2
gas
[
ClO
2
gas
]
(p.p.m.)
Virus titre in each mouse (log
10
)* Mean±SD
0 6.3 6.8 6.8 6.8 6.8 6.7±0.2D
0.03 1.3 2.1 3.6 4.8 1.3 2.6±1.5D
*Virus titre, expressed as TCID
50
, was measured 72 h after challenge
by virus aerosols (n55 mice per group).
DP50.003 when the means of two groups were compared (Student’s
t-test).
Table 2. Mortality of mice exposed to aerosols of influenza A
virus in the absence or presence of 0.03 p.p.m. ClO
2
gas
Values are the number of mice that died at each time point after virus
challenge.
[
ClO
2
gas
]
(p.p.m.)
Time after virus challenge (days) Total
1–10 11 12 13 14 15 16
0 0 300112 7*
0.03 0 0 00000 0*
*P50.002 when the 0 and 0.03 p.p.m. groups on day 16 were
compared (Fisher’s exact test, n510 for each group).
Table 3. Body mass of mice 1 week after challenge with
influenza A virus in the absence or presence of 0.03 p.p.m.
ClO
2
gas
[
ClO
2
gas
]
(p.p.m.)
Body mass (g) at day: Relative
body mass*
07
0 28.4±1.2 25.7±1.3 0.90±0.04D
0.03 26.0±1.8 28.3±2.1 1.09±0.08D
*Ratio of body mass on day 7 to that on day 0 in each group.
DP50.002 when relative body masses of the 0 and 0.03 p.p.m. ClO
2
groups were compared (Student’s t-test, n55 in each group).
Antiviral activity of chlorine dioxide gas
http://vir.sgmjournals.org 63
Effect of ClO
2
on the infectivity of influenza A
virus in vitro
Influenza A virus was treated in vitro with ClO
2
and its
infectivity was assayed by using cultured cells. Infectivity
of the virus decreased markedly after treatment with
40–320
mM ClO
2
, demonstrating that ClO
2
indeed inacti-
vates the infectivity of the virus (Table 5). As the HA and
NA proteins on the virus surface (envelope) are indispens-
able to the infectivity of the virus, we assayed their
biological activities. As shown in Table 5, both HA and NA
activities decreased markedly after ClO
2
treatment in vitro.
This result suggests that the reduction in the infectivity of
influenza virus is attributable to the decrease of biological
activities of the HA and NA proteins on the virus envelope.
Denaturation of HA and NA proteins
We speculated that ClO
2
denatured the HA and NA
proteins and inactivated their biological activities. To
provide support for this hypothesis, we selected two model
decapeptides (HA1 and HA2) from HA and two (NA1 and
NA2) from NA (for sequences, see Methods). After
treatment of these peptides with ClO
2
, they were analysed
by reverse-phase HPLC. When these four peptides were
treated individually with ClO
2
, 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 were
modified covalently by reaction with ClO
2
. This hypothesis
was suppor ted further by the fact that, upon sequencing
(by Edman degradation) of the peptide peaks recovered
from HPLC, some amino acid residues in the peptides were
not identified (Table 6). For example, regarding the
peptide HA2 (RNLLWLTGKN, aa 162–171) treated with
ClO
2
, the sequence of the peptide peak recovered from
HPLC was RNLLXLTGKN; the fifth amino acid residue
(Trp
166
in the original protein) could not be identified by
the conventional protein-sequencing method. This indi-
cates strongly that this residue (tryptophan) was modified
covalently by ClO
2
. Likewise, other peptides were also
found to be modified at tryptophan and tyrosine residues
(Table 6). It is uncle ar whether the cysteine residu e of HA1
was modified by ClO
2
, because cysteine residues are not
positively identifiable by this conventional sequencing
method.
Covalent modification of tryptophan and tyrosine residues
by ClO
2
was confirmed by MS. As shown in Table 7, in the
modified HA 2 and NA1 peptides, there was an increase of
about 32 or 48 atom ic mass units in the tryptophan
residues, indicating that two or three atoms of oxygen were
Table 5. In vitro infectivity of influenza A virus suspension
treated with ClO
2
Influenza A virus was treated with ClO
2
at 0
u
C for 2 min and
subjected to various assays. Virus and HA titres are the means of two
experiments. NA activity is the mean±
SD of five experiments. ND,
Not determined.
Concentration of
ClO
2
(mM)
Virus titre
(p.f.u. ml
”1
)
HA
titre*
NA activity
[
units
(mg protein)
”1
]
0 5.8±10
5
2
9
0.23±0.004
40 5.0±10
4
2
8
0.21±0.009D
80 ,5.0±10
1
2
5
0.19±0.006d
160 ,5.0±10
1
2
5
0.11±0.004d
240
ND 2
4
0.059±0.002d
320
ND 2
2
0.041±0.001d
*Reciprocal of the last dilution that showed complete haemagglutination.
DP,0.05 when compared with 0
mM ClO
2
(Student’s t-test).
dP,0.0001 when compared with 0
mM ClO
2
(Student’s t-test).
Table 6. Amino acid sequences of ClO
2
-treated model
peptides derived from the HA and NA proteins of influenza A
virus
Each model peptide (2 mM) was treated with 4 mM ClO
2
at 25
u
C
for 2 min, and then analysed individually by HPLC. Peak fractions of
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.
Original peptide ClO
2
-treated peptide
Name aa Sequence Sequence found
HA1 101–110 NPENGTCYPG NPENGTCXPG
HA2 162–171 RNLLWLTGKN RNLLXLTGKN
NA1 174–183 FESVAWSASA FESVAXSASA
NA2 400–409 SGYSGSFVQH SGXSGSFVQH
Table 4. Mortality of mice challenged with influenza A virus
aerosols in the absence or presence of 0.03 p.p.m. ClO
2
gas that was delivered for 15 min at various delay times after
commencement of the delivery of virus aerosols
Values are the number of mice that died at each time point after virus
challenge.
ClO
2
gas delay
time (min)
Time after virus challenge (days) Total
8 9 10 11 12 13 14
0 0 000000 0*
5 0 100000 1D
10 2 200000 4
15 2 300000 5
No ClO
2
0 221000 5
*P50.022 when compared with the no-ClO
2
group (Fisher’s exact
test, n510 in each group).
DP50.081 when compared with the no-ClO
2
group (Fisher’s exact
test, n510 in each group).
N. Ogata and T. Shibata
64 Journal of General Virology 89
incorporated covalently into tryptophan residues. Likewise,
there was an increase of about 32 or 48 atomic mass units
in the tyrosine residues in the modified HA1 and NA2
peptides (Table 7), indicating the covalent incorporation of
two or three atoms of oxygen into tyrosine residues. Taken
together, we conclude that amino acid residues in the HA
and NA proteins, primarily tryptophan and tyrosine
residues, are modified covalently by ClO
2
. Such modifi ca-
tions of amino acid residues appear to denature the HA
and NA proteins of influenza A virus, which are
indispensable for its infectivity, and consequently abolish
infectivity of the virus.
DISCUSSION
We have demonstrated that ClO
2
gas at an extremely low
concentration can prevent influenza A virus infection of
mice caused by aerosols. According to the US Occupational
Safety and Health Administration, the 8 h permissible
exposure level of ClO
2
in human workplaces is 0.1 p.p.m.
The level of ClO
2
gas (0.03 p.p.m.) used in this study is
well below this level, and our results indicate that ClO
2
at
this level could be used in the presence of humans to
prevent their infection by influenza A virus and possibly
other related virus infections of the respiratory tract.
Specifically, ClO
2
gas could be used in places such as
offices, theatres, hotels, schools and airport buildings
without evacuating people, thus not interrupting their
normal activities.
Current growing concerns about the threat posed by highly
pathogenic H5N1 avian influenza virus have prompted
interest in evaluating measures against this virus. ClO
2
and
chlorine have long been used as disinfectants of public
water supplies. Thus far, chlorine treatment (chlo rination)
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 magnitu de. Although the strain of
influenza virus used in our present experiment (H1N1)
differs from that of Rice et al. (2007), it is suggested that
our present method, namely treatment of influenza virus
by ClO
2
, provides another effective manoeuvre for the
treatment of public water supplies contami nated by the
virus, and it paves a new way for prevention of pandemic
influenza.
ClO
2
gas is very soluble in water, and is in equilibrium
between the gas and water phases. In our preliminary
experiment, ClO
2
reached equilibrium between the gas and
water phases within 30 s (half-maximal in 20 s) (N. Ogata,
unpublished data). Generally speaking, a water-soluble
gaseous substance reaches equilibrium between the gas and
water phases according to Henry’s law, C5kP, where C
is the concentration of a substance in the water phase, P is
partial pressure of the substance in the gas phase and k is
an equilibrium constant. When the diameter of the aerosol
is in the range 1–10
mm, as in the present experiment,
equilibrium is reached within 1 min. We also found that
Henry’s equilibrium gas constant k regarding the ClO
2
–
water equilibrium, namely k in the above equation, was
3.9610
25
mol l
21
Pa
21
(N. Ogata, unpublished data).
Therefore, the ClO
2
concentration in the virus aerosols is
theoretically 0.12
mM when the aerosols are in equilibrium
with 0.03 p.p.m. ClO
2
gas. This suggests further that the
influenza A virus is inactivated at 0.12
mM ClO
2
in water
(PBS in our present experiment).
We hav e shown that ClO
2
denatures (abolishes the
functions of) the HA and NA proteins on the envelop e
of the influenza virus (Table 5). As these proteins ar e
indispensable for the infectivity of this virus, the fact that
they were denatured by ClO
2
could explain why infectivity
of the virus decreased after treatment with ClO
2
. However,
it is noteworthy that the reduction in infectivity, as
demonstrated by plaque assay, did not necessarily parallel
the reductions in HA and NA activities (Table 5). One
possibility is the presence of other protein(s) in the virus
that is/are critical and indispensable for its infectivity and
Table 7. MS analyses of ClO
2
-treated model peptides
derived from the HA and NA proteins of influenza A virus
Peptides HA1 (NPENGTCYPG), HA2 (RNLLWLTGKN), NA1
(FESVAWSASA) and NA2 (SGYSGSFVQH) (each 2 mM) were
treated with 4 mM ClO
2
at 25
u
C for 2 min. They were then analysed
individually by HPLC and two peak fractions were recovered from
each HPLC run. The peak fractions were analysed by MS.
ND, Not
determined.
Peptide* Parent ion (
[
M+H
]
+
)D Amino acid residued
Expected Found
d§
Expected Found
d§
a (HA1) 1051.1 1096.4 45.3 181.2 (Y)
ND ND
b (HA1) 1051.1 1083.8 32.7 181.2 (Y) 213.0 31.8
c (HA2) 1214.1 1246.7 32.6 204.2 (W) 236.1 31.9
d (HA2) 1214.1 1262.7 48.6 204.2 (W) 252.1 47.9
e (NA1) 1054.1 1102.5 48.4 204.2 (W) 252.1 47.9
f (NA1) 1054.1 1086.4 32.3 204.2 (W) 236.0 31.8
g (NA2) 1068.1 1099.8 31.7 181.2 (Y)
ND ND
h (NA2) 1068.1 1116.5 48.4 181.2 (Y) 229.0 47.8
*Peak fractions recovered from HPLC. The name in parentheses
denotes that of the original peptide.
DMass/charge of the
[
M+H
]
+
ion of the ClO
2
-treated and HPLC-
recovered peptides, determined by MALDI-TOF MS.
dMass/charge of each amino acid residue of the peptide determined
by MALDI-TOF/TOF MS. Only the amino acid residue whose mass/
charge was significantly different from that of the original residue is
shown. The mass of water (18.0) has been added to the mass/charge
for clarity.
§
d denotes the difference between expected and found mass/charge
values.
Antiviral activity of chlorine dioxide gas
http://vir.sgmjournals.org 65
is/are denatured by ClO
2
. For example, the M2 protein, a
proton channel in the virus envelope, could be a target of
ClO
2
. This protein is indispensable for the virus to establish
infection (Tang et al., 2002). A tryptophan residue (Trp
41
)
of this protein protrudes into the proton channel and
works as a ‘gate’ for a proton that enters and passes
through the channel (Tang et al., 2002). As tryptophan
residues were modified by ClO
2
in this study (Tables 6 and
7), it is likely that ClO
2
could also modify the tryptophan
residue (Trp
41
) in this protein and abolish its function.
Tyrosine (Tyr
108
) and tryptophan (Trp
166
) residues in HA
are conserved among many strains of influenza virus and
constitute the binding site of the protein for the receptor
(sialic acid)
[
Tyr
98
and Trp
153
, respectively, in Stevens et al.
(2006)
]
. Therefore, covalent modification of these amino
acid residues (Tables 5–7) explains the reduction in HA
activity caused by ClO
2
treatment. Likewise, tyrosine
(Tyr
402
) and tryptophan (Trp
179
) residues a re conserved
in NA. They constitute an active-site pocket of the protein
and are necessary for its catalytic activi ty
[
Tyr
406
and
Trp
178
, respectively, in Lentz et al. (1987)
]
; this view is
supported by the fact that complete loss of its enzymic
activity occurs by their substitution with other amino acids
(Lentz et al., 1987). Therefore, covalent modification of
these amino acid residues in NA by ClO
2
would exp lain its
inactivation by ClO
2
.
ACKNOWLEDGEMENTS
We thank Dr Philip R.Wyde, Koji Abe, Cholsong Lee and Hirofumi
Morino for their contribution to this work. We also thank Dr
Yoshinobu Okuno for the New Caledonia strain influenza A virus.
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