Ozone: Science & Engineering-accepted (Jan 09)
Development Of A Practical Method For Using Ozone Gas
As A Virus Decontaminating Agent
Hudson, JB. Sharma, M. Vimalanathan, S.
Viroforce Systems Inc. Laboratory, Vancouver, Canada.
Affiliations: Drs. James B Hudson, Professor; Manju Sharma, Research Associate;
Selvarani Vimalanathan, Research Associate
Address Correspondence to:
Dr. JB. Hudson, Department of Pathology & Laboratory Medicine, University of
British Columbia, C-360 Heather Pavilion, 2733 Heather Street,Vancouver V5Z 1M5,
Tel.no. I-604-875-4351 Fax no. 1-604-875-4351 e-mail:
Running Head: Ozone Gas: a Practical Antiviral Agent
Our objective was to develop a practical method of utilizing the known anti-viral
properties of ozone in a mobile apparatus that could be used to decontaminate rooms
in health care facilities, hotels and other buildings. Maximum anti-viral efficacy
required a short period of high humidity (> 90% relative humidity) after the attainment
of peak ozone gas concentration (20-25 ppm). All 12 viruses tested, on different hard
and porous surfaces, and in the presence of biological fluids, could be inactivated by at
least 3 log
in the laboratory and in simulated field trials. The ozone was
subsequently removed by a built-in catalytic converter.
The anti-viral and anti-microbial properties of ozone have been well documented,
although the mechanisms of action are not well understood, and several
macromolecular targets could be involved (Carpendale and Freeberg, 1991; Wells et al
1991; Khadre and Yousef, 2002; Shin and Sobsey,2003; Cataldo, 2006; Lin and Wu,
2006; Lin et al 2007). Aqueous solutions of ozone are in use as disinfectants in many
commercial situations, including waste water treatment, laundries, and food
processing (Kim et al, 1999; Shin and Sobsey, 2003; Naitou and Takahara, 2006;
2008; Cardis et al 2007), but the use of the gas on a commercial scale as a
decontamination device has not been exploited. Ozone gas however has a number of
potential advantages over other decontaminating gases and liquid chemical
applications (McDonnell and Russell, 1999; Barker et al 2004). Thus ozone is a
natural compound, is easily generated in situ from oxygen or air, and breaks down to
oxygen with a half life of about 20 minutes (± 10 min depending on the environment).
As a gas it can penetrate all areas within a room, including crevices, fixtures, fabrics,
and the undersurfaces of furniture, much more efficiently than manually applied liquid
sprays and aerosols (Barker et al, 2004; Malik et al, 2006; Hudson et al 2007).
The only significant disadvantages are its ability to corrode certain materials, such as
natural rubber, on prolonged exposure, and its potential toxicity to humans. The
recognition of the risk of pathologic consequences following exposure of people and
experimental animals to ozone gas has led to restrictions in its use in public areas.
However the latter consideration can be offset to some extent by the potential benefits
of ozone therapy in medicine and dentistry (Devlin et al 1996; Bocci, 2004;
Ciencewicki and Jaspers 2007; Huth et al 2007).
The health hazard can be overcome in practice by ensuring that the room to be treated
is temporarily closed to people during the treatment and is sealed to prevent escape of
the gas into the environment. Sensitive materials can be temporarily covered or
removed if necessary. In addition the ozone gas can be removed quickly after
treatment by use of a catalytic converter, which can transform the ozone back into
oxygen within minutes.
We evaluated the feasibility of using ozone gas as an effective means of
decontaminating various hard and porous surfaces containing dry or wet films of
different viruses, in the presence and absence of cell debris and biological fluids.
Following successful laboratory experiments, we then developed an efficient
prototype ozone generator and catalytic converter which could be used in a room
containing viral contaminants. We also examined a role for high humidity in
enhancing the virus inactivation process, and incorporated this feature into the field
Materials and Methods
The laboratory test chamber was a molded polycarbonate box with a transparent
plastic front window that could be lifted to allow access to samples. Within the test
chamber was a small ozone generator (corona discharge system, from Treated Air
Systems, Vancouver) fitted with a control dial that could be pre-set to determine the
approximate ozone dosage in ppm, an ozone sampler tube connected to the exterior
ozone measuring system (for accurately recording ozone concentration, see below),
and the probe of a hygrometer for measuring relative humidity and
temperature. Humidity was provided in the form of a mist of deionized sterile water by
means of a spray bottle, which had been washed out with 70% ethanol
The model 1000 Viroforce ozone generator (Fig 1) was a portable module containing
multiple corona discharge units, a circulating fan, and an efficient catalytic converter
(scrubber) to reconvert ozone to oxygen at the termination of the ozone exposure
period (further details are available in www.viroforce.com). In addition a portable
commercial humidifier (Humidifirst Inc, Florida) was used to provide a burst of water
vapor (at ambient temperature) when required. All the components were controlled
remotely from outside the test room. Ozone concentration was monitored continuously
by means of an Advanced Pollution Instrumentation Inc. model 450 system (from
Teledyne, San Diego), which measured samples of ozonated air passed through a UV
spectrometer. This apparatus was used for all accurate ozone measurements in all test
locations. The input teflon sampling tube was taped in an appropriate location for the
duration of the experiment. Relative humidity and temperature were recorded by a
portable hygrometer (VWR Scientific, Ontario). The probe was taped in a convenient
location inside the test room.
The lids of sterile polystyrene tissue culture trays were used as plastic surfaces. Glass
slides, 75 x 25 mm; stainless steel circular disks, 1.0 cm diameter; and pieces of fabric
and cotton (typical of those used in hospital and hotel rooms) were cleaned in
detergent, washed, dried, and sterilized by autoclaving. Cotton tips (Q-tips) were
heated for 2 min in a microwave oven. Fetal bovine serum and PBS (phosphate
buffered saline) were obtained from Invitrogen (Ontario). Sterile plastic 24 well plates
and other supplies were BD-Falcon brand obtained from VWR Scientific (Ontario).
Cell Lines & Viruses:
All cell lines (Vero monkey kidney cells; MDCK canine kidney cells; H-1 sub clone
of HeLa cells; A549 human lung epithelial cells; feline kidney cells; all acquired
originally from ATCC; mouse DBT cells, from Dr. Pierre Talbot) were passaged
regularly in Dulbecco MEM, in cell culture flasks, supplemented with 5-10% fetal
bovine serum, at 37° C in a 5% CO2 atmosphere, with the exception of the H-1 cells,
which were grown at 35
C. No antibiotics or antimycotic agents were used.
The following 12 viruses were used: influenza, strain H3N2, human isolate (from BC
Centre for Disease Control), propagated in MDCK cells; HSV (herpes simplex virus
type 1, BC-CDC), propagated in Vero cells; rhinovirus types 1A and 14 (RV 1A and
RV 14, from ATCC), propagated in H-1 cells; Adenovirus types 3 and 11 (ATCC), in
A549 cells; mouse coronavirus (MCV, from Dr. Pierre Talbot) in DBT cells. Sindbis
virus (SINV), yellow fever virus (YFV), vesicular stomatitis virus (VSV), poliovirus
(PV,vaccine strain), vaccinia virus (VV), all ATCC strains, were grown in Vero cells.
All the stock viruses were prepared as clarified cell-free supernatants, with titers
ranging from 10
pfu (plaque-forming units) per mL.
Aliquots of virus, diluted when necessary in PBS, usually 100 uL, were spotted onto
the appropriate sterile surface, spread into a film by means of a sterile tip, and allowed
to dry, within a biosafety cabinet (normally 30-40 min). In some experiments the
spread films were left wet for the ozone treatment. The samples were then transported
in sterile containers to the appropriate chamber or room for ozone treatment. Controls
consisted of equivalent samples transported to the test site but not exposed to ozone,
and others retained in the biosafety cabinet for the entire duration of the experiment.
All control samples were contained were contained within sealed sterile plastic boxes
and kept outside the ozone- exposed room or chamber for the duration of the
1. The initial field trials were conducted in an unused laboratory, volume 65 m
which we used three small ozone generators (Treated Air Systems) located in different
parts of the room, together with a circulating fan. These tests were carried out at
ambient humidity (40-45% RH).
2. In most of the subsequent field tests we used an office, volume 35 m
normal office furniture, which was located adjacent to the laboratory. We placed the
prototype ozone generator (Viroforce model 1000) in the centre of the room, together
with the humidifier. Test samples were placed in various locations of the room, and
the probes for the ozone monitor and the hygrometer were taped in convenient
locations. All instruments were controlled remotely from outside the test room. At the
beginning of the test the air vent was covered with plastic and the door was sealed
with duct tape. The standard program adopted for most of the tests involved increasing
the ozone level over a period of 15 min to 25 ppm, maintaining this level for 10 min,
at which point the humidifier was activated to produce a rapid burst of water vapor.
This resulted in the RH increasing to > 95% within 5 min. Following this the
humidifier and generator were switched off and the catalytic converter was switched
on, which resulted in a decrease in ozone to < 1ppm within 15 min. The door was then
opened and the samples retrieved and covered for transport back to the biosafety
cabinet. These samples, and equivalent control samples that had been kept in the
biosafety cabinet for the duration of the test, were then reconstituted in 1.0 mL PBS
and stored at -70
C until assayed by plaque formation (plaque forming units, pfu) in
the appropriate cells. Unless otherwise indicated, results are presented as pfu/mL.
3. A similar protocol was employed for use in the test hotel room, a typical room with
a double bed, furniture and adjacent bathroom, volume 42.5 m
, situated in
Vancouver. Dried samples of the viruses on plastic surfaces were transported in sterile
containers between the laboratory and the hotel room.
Inactivation of viruses by ozone gas on different surfaces: Since we
wanted to evaluate the effect of ozone gas on dried samples of virus we first
examined the ability of several representative viruses to retain significant infectivity
following the drying process. Most of the viruses showed up to 1 log
infectivity as a result of the drying process itself. After this the dried films (of HSV,
influenza virus, FCV, poliovirus, and RV) showed similar decay curves, with a 50%
decrease (T ½) of 3-4 hours at room temperature. Thus in all cases there were more
than adequate amounts of infectious virus remaining after several hours, during which
experiments with ozone gas could be carried out. These decay curves were not
significantly affected by the presence of 10% serum (fetal bovine serum, FBS).
Similar findings on virus drying kinetics were reported recently (Terpstra et al 2007),
and these results confirm the general belief that infectious viruses can persist for long
times on inanimate surfaces.
Several viruses, representing different virus families and structural features, were then
treated with a single mobile ozone generator in the laboratory chamber, as described in
Materials and Methods. All viruses tested, HSV, influenza, MCV, FCV, and RV,
representing DNA and RNA viruses with and without membranes, showed similar
kinetics of virus inactivation on three hard surfaces, plastic, glass and stainless steel.
The T ½ values ranged from 5-8 hours, but there were no consistent differences
between the viruses or the surfaces. Examples for HSV (DNA virus with membrane),
influenza (RNA virus with membrane), and RV (RNA virus without membrane) are
shown in Fig.2. Rhinovirus (Fig 2c) was slightly more resistant than the other two
viruses. Nevertheless these results suggest that all or most viruses should be
susceptible to ozone gas.
Field tests at Ambient Humidity: Following successful inactivation of several
viruses in the laboratory experiments, we conducted tests in a large unoccupied
laboratory, volume 65 m
, with the aid of three portable ozone generators of the kind
used in the previous tests. Peak ozone level attained was 28 ppm, at an ambient RH of
40%, and total time of exposure, including rise and fall periods, was 60 min. The
results from two separate tests are combined in Table 1, and these indicate successful
inactivation of two log
or more infectious virus under these simulated field
conditions. Duplicate samples showed reasonable agreement, and the results were
unaffected by the position of the sample within the room. Thus prolonged exposure to
a fairly high dose of ozone gas at ambient humidity can result in two log’s inactivation
of several viruses; but in practice we would prefer a system giving greater efficacy.
Enhancement of virus inactivation by high humidity: We next examined the
possibility of improving virus killing by treating dried samples of several viruses with
ozone gas in the presence of high relative humidity. Preliminary laboratory
experiments indicated that the maximum enhancing effect was obtained by increasing
the ozone to the maximum level first followed by a burst of water vapour to increase
RH to greater than 70%, preferably >90%. However we did not have the capability of
testing the enhancing effect of graded doses of humidity.
Table 2 shows the effect of RH on the degree of inactivation of 3 different viruses
within a test office, 35.4 m
volume. Under these conditions, which involved much
more restricted exposure than the conditions used for Table 1, the degree of
inactivation was lower and more variable at ambient RH, but in all cases the
combination of ozone gas plus high RH consistently yielded substantial inactivation.
Therefore optimum efficacy of the ozone treatment requires the presence of high RH,
for at least several minutes.
Composition of virus samples: Based on these findings, we next conducted a
number of experiments with different viruses in the test office, which contained
standard office furniture. For this purpose we used a newly developed prototype ozone
generator, containing multiple ozone units, together with a built in catalytic converter
and fan (shown in Fig 1), and an accessory humidifier capable of generating a
humidity of more than 90% within 5 minutes. Details of the protocols are described in
Materials and Methods.
In this test system we were able to examine the effects of sample preparation and
composition, organic load, and sample location within the room. Wet and dry films of
viruses were found to be equally susceptible to the treatment regimen. Also the nature
of the surface on which samples were dried did not affect the result. Thus in addition
to the different hard surfaces mentioned above (glass, plastic and stainless steel, Fig
2), cotton and fabric surfaces gave results similar to plastic (not shown). Inoculum size
(10-1000 uL) and degree of dilution of the virus did not influence the result, nor did
the presence of cellular debris in the sample. For example influenza virus and Sindbis
virus in crude cell extracts and in clarified supernatants were equally susceptible
(more than 3 log inactivation in dried films treated with ozone in high humidity).
We also tested the effect of serum and blood products, since samples in the field, such
as tissues and corpses, and instruments used in dental and hospital clinics, might be
contaminated with such materials (Cristina et al 2008). However, as shown in Table 3,
the presence of whole human blood, or human and bovine serum components, did not
affect the efficacy of virus inactivation, in either dry (data shown) or wet samples of
Viral aerosols: Virus - containing aerosols, a potential problem in certain dental and
medical practices (Cristina et al 2008), were also tested by spraying known volumes of
FCV suspension into the test chamber in the presence or absence of ozone gas, and
collecting samples of condensate for virus assays. In comparison, similar amounts of
virus were sprayed into the chamber without ozone gas, and measured volumes
collected. This experiment was performed twice, resulting in retrieval of
approximately 1% of the sprayed virus each time, and inactivation of more than 99%,
as indicated in Table 4. Thus the ozone gas is also capable of efficiently killing
Field tests with high humidity: A standard hotel room (volume 42.5 m
) was used
for the evaluation of the prototype ozone generator with accessory humidifier, using
influenza- and FCV as examples of viruses with and without membranes, respectively.
Known amounts of virus were dried onto glass slides, which were then transported to
the room for ozone and humidity treatment, using the protocol developed in the office
tests, above. Pairs of samples were placed in three different locations within the room,
including an adjacent bathroom. Treated and control (unexposed) samples were then
returned to the laboratory for reconstitution and assay. The Results are summarized in
table 5. Both viruses were substantially inactivated, and the location of samples within
the room did not affect the outcome.
Susceptible Viruses: Table 6 summarizes the viruses successfully inactivated, by 3
or more log
, and their relevance. As indicated, these viruses represent many different
families with a range of animal virus structures. Some of them have also been
suggested to be suitable surrogates for important viruses that are difficult to cultivate
in vitro or require special containment facilities (eg. Sindbis virus and yellow fever
virus for hepatitis C; These two viruses plus vesicular stomatitis viruses for HIV;
human influenza virus for avian influenza; Steinman, 2004). To date we have not
encountered an ozone- resistant virus.
The objective of this study was to develop a practical and efficient apparatus for
decontamination of confined spaces containing infectious viruses. Such an apparatus
could be very useful in hospitals and health care facilities, and other locations where
outbreaks are relatively common, such as cruise liners (Lawrence 2004). In addition
there are many other public and private buildings that could benefit from an
appropriate antiviral decontamination apparatus. Existing technologies are clearly
inadequate (McDonnell and Russell, 1999; Barker et al 2004; Sattar 2004).
Previous studies with ozone in water have proven its usefulness in commercial
laundries and food processing facilities (Kim et al, 1999; Shin and Sobsey, 2003;
Naitou and Takahara, 2006; 2008; Cardis et al 2007). However, in order to decrease or
eradicate virus contaminants in inaccessible locations, such as crevices, fixtures,
undersides of furniture, etc. it is necessary to utilize the efficient penetrating ability of
a gas. Since ethylene oxide is not considered an acceptable alternative (McDonnell
and Russell,1999), then gaseous ozone should be the best choice available.
A few studies have indicated the feasibility of ozone gas as an antiviral agent
(Carpendale and Freeberg, 1991; Wells et al 1991; Khadre and Yousef, 2002; Shin and
Sobsey, 2003; Cataldo, 2006; Lin and Wu, 2006). We verified this by means of
laboratory studies and several field trials in a large room. We then discovered that the
addition of a burst of high humidity, following the attainment of peak ozone level,
resulted in substantially greater reductions in virus infectivity, under a variety of
conditions. The precise mechanisms of action against virus are not understood;
however the broad oxidizing activity against many macromolecules (Cataldo 2006)
suggest that viral membranes, protein coats and nucleic acids could all be vulnerable.
Nevertheless the requirement of humidity for optimal efficacy indicates that hydroxyl
ions and possibly additional water-derived radicals could be involved, as suggested for
the aqueous environments (Lin and Wu 2006).
We developed the prototype apparatus to take advantage of the desired features based
on these experimental results (Fig 1,Viroforce 1000). The key features are: a battery of
ozone generators enclosed within the machine; a powerful catalytic converter to
convert ozone back to oxygen within minutes, allowing immediate entry to the
decontaminated premises; a circulating fan; built in remote control and programmable
functions. In addition we employ an accessory humidifier, which produces an
immediate cloud or mist of microscopic water droplets, without heating. We
demonstrated that this apparatus was capable of inactivating 3 log’s or more of many
different infectious viruses in rooms such as an office and a hotel room. We also
reported recently that the same apparatus worked efficiently in a cruise liner cabin to
inactivate norovirus (Hudson et al 2007).
To date we have successfully tested the apparatus in laboratory and field conditions
against 12 representative viruses, mostly human pathogens. Some of these viruses
(Table 6 legend) have also been promoted as valid surrogates for viruses that are
difficult or dangerous to cultivate and test by conventional techniques, such as
hepatitis C virus, HIV, avian influenza (Steinman, 2004).
The location of the test virus in the room was not a factor, a result that might be
expected considering the penetrability of the ozone gas, nor was the presence of blood
and serum products. The latter was an important result since the possibility of microbe
protection against ozone by organic films has been suggested (Serra et al 2003). In
addition, the presence of such contaminated materials has been suggested as a risk for
spread of infections in medical and dental practices (Cristina et al 2008). Another
possible factor, which has been shown to play a role in other liquid anti-microbial
applications (Sattar 2004; Malik et al 2006), is the presence of a porous surface such
as fabric or carpet in which the virus or other organism is embedded. This limitation
was not seen however in our experience with ozone gas against viruses or bacteria
(Hudson et al 2007; Sharma and Hudson 2008).
As a result of these studies, we believe that the apparatus we have developed, based on
the use of ozone gas and high humidity, has many potential applications wherever
efficient decontamination of rooms is required.
Abbreviations: DMEM, Dulbecco minimum Eagle medium; PBS, phosphate-
buffered saline; pfu, plaque-forming unit (one pfu = one infectious virus particle); RH,
relative humidity; viruses: Ad 3/11, adenovirus type 3/11; FCV, feline calicivirus;
HSV, herpes simplex virus type 1; MCV, mouse coronavirus; PV, poliovirus type 1
vaccine strain; RV 1A/14, rhinovirus type 1A/14; SINV,Sindbis virus; VSV, vesicular
stomatitis virus; VV, vaccinia virus; YFV, yellow fever virus,vaccine strain.
Key words: ozone, ozone gas, antiviral, decontamination, viruses, humidity, ozone
generator, catalytic converter, field trials
Barker J, Vipond IB, Bloomfield SF. Effects of cleaning and disinfection
in reducing the spread of Norovirus contamination via environmental surfaces. J Hosp
Infect. 58:42-49 (2004)
Bocci V. Ozone as Janus: this controversial gas can be either toxic or medically
useful. Mediat.Inflamm. 13(1): 3-11 (2004)
Cardis D. Tapp C. DeBrum M.and Rice RG. Ozone in the Laundry Industry-Practical
Experiences in the United Kingdom. Ozone: Science and Engineering. 29: 85-99
Carpendale MTF. Freeberg JK. Ozone inactivates HIV at noncytotoxic concentrations.
Antiviral Res. 16(3): 281-292. (1991)
Cataldo F. Ozone Degradation of Biological macromolecules: Proteins, Hemoglobin,
RNA, and DNA. Ozone: Science and Engineering 28: 317-328. (2006)
Ciencewicki J. and Jaspers I. Air Pollution and Respiratory Virus Infection. Inhal.
Toxicol. 19: 1135-1146 (2007)
Cristina ML. Spagnolo AM. Sartini M. Dallera M. Ottria G. Lombardi R. and Perdelli
F. Evaluation of the risk of infection through exposure to aerosols and spatters in
dentistry. Am. J. Infect. Control 36: 304-307 (2008)
Devlin R. McDonnell WF. Becker S. Madden MC. McGee MP. Perez R. Hatch G.
House DE. Koren HS. Time-Dependent Changes of Inflammatory Mediators in the
Lungs of Humans Exposed to 0.4 ppm Ozone for 2 hr. Toxicol. Appl. Pharmacol. 138:
Hudson, JB. Sharma M. Petric M. Inactivation of Norovirus by ozone gas in
conditions relevant to healthcare. J. Hosp. Infect. 66: 40-45. (2007)
Huth KC. Saugel B. Jakob FM. Cappello C. Quirling M. Paschos E. Ern K. Hickel R.
and Brand K. Effect of Aqueous Ozone on the NF-kB System. J. Dent.Res. 86(5):
Khadre MA. Yousef AE. Susceptibility of human rotavirus to ozone, high pressure,
and pulsed electric field. J. Food Prot. 65: 1441-1446 (2002)
Kim JG. Yousef AE. Dave S. Application of ozone for enhancing the microbiological
safety and quality of foods: a review. J. Food Prot. 62: 1071-1087. (1999)
Lawrence DN. Outbreaks of gastrointestinal diseases on cruise ships: lessons from
three decades of progress. Curr Infect Dis Rep;6:115-123. (2004)
Lin Y-C. Wu S-C. effects of ozone exposure on inactivation of intra- and extracellular
enterovirus 71. Antiviral Res. 70: 147-153. (2006)
Lin Y-C. Juan H-C. and Cheng Y-C. Ozone exposure in the culture medium inhibits
enterovirus 71 virus replication and modulates cytokine production in
rhabdomyosarcoma cells. Antiviral Res. 76: 241-251 (2007)
Malik YS, Allwood PB, Hedberg CW, Goyal SM. Disinfection of fabrics and carpets
artificially contaminated with calicivirus: relevance in institutional and healthcare
centres. J Hosp Infect 63:205-210. (2006)
McDonnell G. and Russell D. Antiseptics and Disinfectants: Activity, Action, and
resistance. Clin. Microbiol. Rev. 12(1); 147-179 (1999)
Naito S.and Takahara H. Ozone Contribution in Food Industry in Japan. Ozone:
Science and Engineering 28: 425-429 (2006)
Naito S. Takahara H. Recent Developments in Food and Agricultural uses of Ozone as
an Antimicrobial Agent-Food Packaging Film Sterilizing Machine using Ozone.
Ozone: Science and Engineering. 30:81-87 (2008)
Sattar SA. Microbicides and environmental control of nosocomial viral infections.
J. Hosp. Infect. 56: S64-S69. (2004)
Serra R, Abrunhosa L, Kozakiewcz Z ,Venancio A, Lima N. Use of ozone to reduce
molds in a cheese ripening room. J.Food protection 66: 2355-2358 (2003)
Sharma M. Hudson JB. Ozone gas is an effective and practical antibacterial agent.
Amer. J. Infect Control. 36: 559-563 (2008)
Shin G-A, Sobsey MD. Reduction of Norwalk virus, Poliovirus 1, and Bacteriophage
MS2 by ozone disinfection in water. Appl Environ Microbiol;69:3975-3978. (2003)
Steinman J. Surrogate viruses for testing virucidal efficacy of chemical disinfectants. J
Hosp Infect; 56: S49-S54. (2004)
Terpstra FG. Van den Blink AE. Bos LM. Boots AGC. Brinkhuis FHM. Gijsen E. van
Remmerden Y. Schuitemaker H. and van’t Wout AB. Resistance of surface-dried
virus to common disinfection procedures. J.Hosp. Infect. 66: 332-338 (2007)
Wells KH. Latino J. and Poiesz BJ. Inactivation of human Immunodeficiency Virus
Type 1 by Ozone in vitro. Blood 78(1): 1882-1890 (1991)
TABLE I: INACTIVATION OF VIRUSES BY OZONE GAS IN LARGE
ROOM (AMBIENT HUMIDITY)
Exp #1: HSV
Exp #1: HSV
Exp #1: HSV
Exp #2: HSV
Exp #2: RV
Exp #2: PV
Viruses were dried onto glass slides and transported to a large test room, volume 65
, where they were treated with ozone from small generators, at ambient humidity
(40% RH) and temperature (20
C), for 1 hour; the peak level attained in both
experiments was 28 ppm. Following the treatment the samples were retrieved and
transported back to the laboratory for reconstitution and subsequent infectivity assays.
Control slides were not exposed to ozone. Complete details are described in Materials
TABLE II: EFFECT OF HUMIDITY ON VIRUS INACTIVATION IN TEST
+ 38% RH
+ 70% RH
Viruses were dried onto plastic surfaces and placed in the test office for ozone
treatment at ambient humidity (38% RH) or at elevated humidity (70% RH), in
separate tests on the same day. The prototype generator was programmed to deliver up
to 20 ppm for 20 min, with or without a burst of extra humidity, followed by catalytic
conversion of ozone to oxygen, as described in Materials and Methods. At the end of
the test, samples were retrieved for reconstitution and subsequent assays.
TABLE III: OZONE INACTIVATION OF VIRUS (SINV) IN THE PRESENCE
OR ABSENCE OF BLOOD COMPONENTS
No ozone (pfu)
+ ozone (pfu)
2.0 x 10
3.2 x 10
+ bovine serum albumin 1:1
4.2 x 10
3.1 x 10
+ human serum 1:1
4.2 x 10
4.3 x 10
+ whole human blood 1:1
1.0 x 10
1.3 x 10
Samples of dried SINV on plastic surfaces, containing the supplements indicated, were
treated with ozone in the test office, as described in the legend for Table 2, with a
burst of high humidity (90% RH).
TABLE IV: EFFECT OF OZONE ON VIRUS AEROSOL
Virus titer, no ozone
Virus titer + ozone
Suspensions of FCV were prepared in PBS and sprayed into the laboratory test
chamber with or without 20 ppm ozone produced by a small generator. Samples of the
condensate were collected in 6-well trays, and their volumes and content of infectious
FCV were measured.
TABLE V: INACTIVATION OF VIRUSES (INFLUENZA, FCV) IN HOTEL
Dried samples of the viruses were transported to the hotel, duplicate pairs were placed
in different locations within the test hotel room, and ozone treatment conducted as
described in Table 2, with an accessory humidifier, which gave a maximum RH of
95% . Following the treatment, samples were returned to the laboratory for
reconstitution and assay, together with unexposed controls.
TABLE VI: VIRUSES SUSCEPTIBLE TO OZONE GAS + HIGH HUMIDITY
(> 3 LOG
(+ or -)
Herpes simplex virus (HSV)
Representative herpes virus
Adenovirus types 3 and 11
Vaccinia virus (VV)
Representative pox virus
(human strain H3N2)
Representative of human and
avian influenza viruses
Murine coronavirus (MCV)
Surrogate for SARS virus
Sindbis virus (SINV)
Surrogate for Hepatitis C virus
Yellow fever virus (YFV)
Surrogate for Hepatitis C virus
Vesicular stomatitis virus (VSV)
Rhabdovirus (ubiquitous in
vertebrates, invertebrates, plants)
1A & 14 (RV 1A, 14)
Common cold viruses
Feline calicivirus (FCV)
Surrogate for Norovirus
No ozone-resistant viruses have been found
Sindbis virus and Yellow fever virus are in the same virus family as Hepatitis C virus,
and have been promoted as suitable substitutes in antiviral testing.
Influenza virus, murine coronavirus, Sindbis virus, Yellow Fever virus, and vesicular
stomatitis virus are all RNA viruses with membranes, with structures similar to HIV.
Therefore this combination could be considered as a suitable substitute for HIV in
FIG 1. Prototype Ozone Generator (Viroforce 1000)
The generator contains 8 corona discharge units, a powerful circulating fan,
and a catalytic converter to convert ozone back to oxygen after the treatment.
Part of the control panel is also visible. The unit is shown by itself and located
in the test hotel room.
Not shown is the accessory humidifier.
FIG 2. Kinetics of Inactivation of Viruses on Different Surfaces.
Multiple aliquots of each virus, in separate experiments in the laboratory, were
dried onto the different surfaces, and exposed to ozone gas (10 ppm) at
ambient humidity (45% RH). Periodically duplicate samples were removed for
reconstitution and freezing. They were subsequently thawed and assayed by
plaque formation on the appropriate cell lines.