Exp. Anim.
Virucidal Effect of Laboratory
39(2), 223-229,
1990
of Ozone Treatment Animal Viruses
Hiroshi SATO, Yoji WANANABE, and Hironori MIYATA Laboratory Animal of Medicine,
Center for Biomedical Research, Nagasaki University 12-4 Sakamoto, Nagasaki -shi, Nagasaki 852, Japan
(Received
19 September
1989/Accepted
15 November
School
1989)
An ozonization method was used to inactivate the viral pathogens of laboratory animals. Ozone at a concentration of over 100 ppm with high humidity was highly virucidal against 4 RNA viruses : HVJ, Theiler's murine encephalomyelitis virus (TMEV), Reo type 3 virus (RV) and murine hepatitis virus (MHV). For the ozone tests, 0.1 ml of a virus suspension in deionized water or saline and was placed in 35-mm dishes. The titer of 106 plaque-forming units of TMEV in a liquid-phase, which was highly stable against physical treatments, was reduced within lhr to a level of 0 by 300 ppm of ozone at 80% humidity and 22-25t. HVJ and MHV were more susceptible than TMEV to the ozone treatment. RV was the most resistant of the 4 viruses. The ozonization method may be a good way to disinfect not only for the laboratory animal RNA-viruses (both of enveloped and unenveloped viruses) but also animal rooms, clean rooms and even safety cabinets.
In
our
previous
encephalomyelitis 3 virus
(RV)
chemical
was
virus
hemorrhagic and
[9,13]
and
lymphocytic
treatment units
viruses
were
never
way
results
effective, to
led
syndrome
at (PFU)
22•Ž of
us
the
to
or
the
45•Ž,
former
study
safe
and
RV
109 two
Methods
The
infection
at
cells
(m.
37•Ž
and
treated
applied
supernatants
fetal
Recently, an ozonization method has been developed for the microbial disinfection of water and wastewater [1, 2, 12], and for human viruses in dry-phase virus samples [4], but not for disinfecting laboratory animals. Here we report on an ozone treatment with high virucidal efficacy against the enveloped viruses and even the unenveloped viruses which tend to be resistant to the physicochemical treatment previously reported [13].
at
cells
MHV
were
and Cell
HVJ,
in
C02-air
and
cultures
Calif.
U. by
the
atmosphere.
S. cells
was
cells
were All
essential
heat-inactivated
used.
for
the
and
DBT
stored
and
MA-
propagation
of
[13].
infectivity
assay
LLC-MK
2 cells
Viruses in
titer
TMEV.
collected
were
medium.
monolayer
absorbed
r%
MEM with 5 % FCS. the FCS was reduced
maintenance
Co.,
and
(TMEV)
of
2 was
2 days after
virus
as
respectively
cultures
21 cells grown tion
used
for
harvested
minimal
plus
they
a multiplicity
method
(FCS)
until
with
The
Eagle's
serum
mainly studied physicochemical
in LLC-MK2
same
(MEM)
-80t
104
the
HVJ, propa-
cultured
was
grown
in
calf
were
3 times.
by
(TMEV, TMEV,
was to
virus
RV
medium
animals.
i.)
the
PFU/ml.
develop
viruses studied.
infected
o.
freezing/thawing 108
RNA were
BHK/ 21 cells, and of its resistance
treatment.
to
laboratory
MHV)
in
easily of
and
Four
and
gated because
of
irradia-
inactivated.
this
pathogens
virus UV
completely
convenient,
inactivate
to
Viruses
with virus
renal
Materials
type
physico-
distemper with
exposures
forming
an
to
choriomeningitis
60-min
heat
Reo
compared
canine fever
plaque
Those
resistant
(HVJ),
. After
murine
and
treatments
(CDV), virus
Theiler's
(TMEV)
highly
virucidal
Sendai
tion
studies, virus
were for After
were
The concentrato 2 % for inoculated
35-mm
A.)
BHK/ (RV)
lhr
dishes allowed at
37t
aspiration
onto (Coaster to in of
be 5 % the
224
inoculum, a 1st layer (maintenance medium plus 0.6 % agarose) was overlaid and cultured as above. The infectivity of HVJ and MHV were assayed as previously described [13] . Plaques were counted after staining with neutral red dye. Lyophilization : Twenty five microliters of each virus in vials (Wheaton Co., New Jersey, U. S. A.) with MEM consisting of 0.5 % gelatin and 10% FCS was rapidly frozen in acetone-dry ice and lyophilized at -50t for 6 hr using a freeze-drier (Tokyo Rika Co., Tokyo, Japan). Ozone
treatment
:
1.
Dry-phase
: The
lyophilized
in
the
vials
were
glass
concentrations
of
1 ; ELIOS
Japan).
within
10%
humidity
of
the
controlled
2.
Liquid-phase.
fold
(Nunclon, 0.1-mm
Co.,
thickness
various
ml
to
treatment.
No
portion into
the
a
recorder
of
hundred-
a 35
mm-dish
resulting liquid ozone
as
in
were
in
phase,
of
samples dishes
recorders.
(22-
by
Denmark) of
the
Also,
temperature
put
concentrations
applied
regulated
1. Ozone generation system. The virus samples were put into a glove box through the pass-box, then treated with the gas for selected times. The ozone concentration and humidity were controlled and monitored by
humidifier.
was
Nunc
(Fig. Co.,
was
monitored
A 0.1 sample
box
concentration.
a sonic
Fig.
various
glove Reinetsu
and
were
samples to
generation
90%)
by
virus
a
desired
to
conditions
and
in
; Shinryo
Ozone
(50%
25•Ž)
ozone
Ozonizer
Tokyo,
virus
exposed
gas
the
rocked
a
and were
dry-phase during
the
treatment. Evaluation efficacy
of
was
reduction
by with
(control moisture
; control chamber
with for
same
the
ozone)
time
ozone
treatment by
at
period
treatment)
physicochemical
the by
logo
the
viruses almost as
: Virucidal
calculating
dividing
(treated
the
ozone
evaluated
the as
logo
the virus virus
were 90%
kept humidity
test
viruses
previously
treatments
virus titer titer in
a for
under described
[8,13]
.
Results Effect of relative humidity on dry-phase samples : A preliminary experiment was conducted with two viruses, HVJ and TMEV. Dry-phase samples of the viruses were exposed to 100 or 200 ppm of ozone gas at 50, 70, and 80% humidity (Fig. 2). A zero level of infectivity was obtained within 1 hr (HVJ) and 3 hr (TMEV) when the two virus samples were exposed to 200 ppm of ozone and 80% humidity. To determine precisely the effect of humidity
Fig.
2. Effect samples were or
of (HVJ
treated
80%
(•œ)
were
treated
(•¢)
humidity
humidity.
ozonization on and TMEV).
with
200 ppm
relative
of
humidity.
with and
100
ppm
200 ppm
the HVJ
ozone
dry-phase samples at 50%
TMEV of at
(•›)
samples
ozone 80%
at
70% (•£)
225
Fig.
3.
Effect
of
Lyophilized for
30
60,
70,
kept
min.
in
a
on
moisture
the MEM
10-fold
samples
dilution
MA-104
Control
at
0.1 and
cells.
at
the
* p
Virucidal Effect of Laboratory
39(2), 223-229,
1990
of Ozone Treatment Animal Viruses
Hiroshi SATO, Yoji WANANABE, and Hironori MIYATA Laboratory Animal of Medicine,
Center for Biomedical Research, Nagasaki University 12-4 Sakamoto, Nagasaki -shi, Nagasaki 852, Japan
(Received
19 September
1989/Accepted
15 November
School
1989)
An ozonization method was used to inactivate the viral pathogens of laboratory animals. Ozone at a concentration of over 100 ppm with high humidity was highly virucidal against 4 RNA viruses : HVJ, Theiler's murine encephalomyelitis virus (TMEV), Reo type 3 virus (RV) and murine hepatitis virus (MHV). For the ozone tests, 0.1 ml of a virus suspension in deionized water or saline and was placed in 35-mm dishes. The titer of 106 plaque-forming units of TMEV in a liquid-phase, which was highly stable against physical treatments, was reduced within lhr to a level of 0 by 300 ppm of ozone at 80% humidity and 22-25t. HVJ and MHV were more susceptible than TMEV to the ozone treatment. RV was the most resistant of the 4 viruses. The ozonization method may be a good way to disinfect not only for the laboratory animal RNA-viruses (both of enveloped and unenveloped viruses) but also animal rooms, clean rooms and even safety cabinets.
In
our
previous
encephalomyelitis 3 virus
(RV)
chemical
was
virus
hemorrhagic and
[9,13]
and
lymphocytic
treatment units
viruses
were
never
way
results
effective, to
led
syndrome
at (PFU)
22•Ž of
us
the
to
or
the
45•Ž,
former
study
safe
and
RV
109 two
Methods
The
infection
at
cells
(m.
37•Ž
and
treated
applied
supernatants
fetal
Recently, an ozonization method has been developed for the microbial disinfection of water and wastewater [1, 2, 12], and for human viruses in dry-phase virus samples [4], but not for disinfecting laboratory animals. Here we report on an ozone treatment with high virucidal efficacy against the enveloped viruses and even the unenveloped viruses which tend to be resistant to the physicochemical treatment previously reported [13].
at
cells
MHV
were
and Cell
HVJ,
in
C02-air
and
cultures
Calif.
U. by
the
atmosphere.
S. cells
was
cells
were All
essential
heat-inactivated
used.
for
the
and
DBT
stored
and
MA-
propagation
of
[13].
infectivity
assay
LLC-MK
2 cells
Viruses in
titer
TMEV.
collected
were
medium.
monolayer
absorbed
r%
MEM with 5 % FCS. the FCS was reduced
maintenance
Co.,
and
(TMEV)
of
2 was
2 days after
virus
as
respectively
cultures
21 cells grown tion
used
for
harvested
minimal
plus
they
a multiplicity
method
(FCS)
until
with
The
Eagle's
serum
mainly studied physicochemical
in LLC-MK2
same
(MEM)
-80t
104
the
HVJ, propa-
cultured
was
grown
in
calf
were
3 times.
by
(TMEV, TMEV,
was to
virus
RV
medium
animals.
i.)
the
PFU/ml.
develop
viruses studied.
infected
o.
freezing/thawing 108
RNA were
BHK/ 21 cells, and of its resistance
treatment.
to
laboratory
MHV)
in
easily of
and
Four
and
gated because
of
irradia-
inactivated.
this
pathogens
virus UV
completely
convenient,
inactivate
to
Viruses
with virus
renal
Materials
type
physico-
distemper with
exposures
forming
an
to
choriomeningitis
60-min
heat
Reo
compared
canine fever
plaque
Those
resistant
(HVJ),
. After
murine
and
treatments
(CDV), virus
Theiler's
(TMEV)
highly
virucidal
Sendai
tion
studies, virus
were for After
were
The concentrato 2 % for inoculated
35-mm
A.)
BHK/ (RV)
lhr
dishes allowed at
37t
aspiration
onto (Coaster to in of
be 5 % the
224
inoculum, a 1st layer (maintenance medium plus 0.6 % agarose) was overlaid and cultured as above. The infectivity of HVJ and MHV were assayed as previously described [13] . Plaques were counted after staining with neutral red dye. Lyophilization : Twenty five microliters of each virus in vials (Wheaton Co., New Jersey, U. S. A.) with MEM consisting of 0.5 % gelatin and 10% FCS was rapidly frozen in acetone-dry ice and lyophilized at -50t for 6 hr using a freeze-drier (Tokyo Rika Co., Tokyo, Japan). Ozone
treatment
:
1.
Dry-phase
: The
lyophilized
in
the
vials
were
glass
concentrations
of
1 ; ELIOS
Japan).
within
10%
humidity
of
the
controlled
2.
Liquid-phase.
fold
(Nunclon, 0.1-mm
Co.,
thickness
various
ml
to
treatment.
No
portion into
the
a
recorder
of
hundred-
a 35
mm-dish
resulting liquid ozone
as
in
were
in
phase,
of
samples dishes
recorders.
(22-
by
Denmark) of
the
Also,
temperature
put
concentrations
applied
regulated
1. Ozone generation system. The virus samples were put into a glove box through the pass-box, then treated with the gas for selected times. The ozone concentration and humidity were controlled and monitored by
humidifier.
was
Nunc
(Fig. Co.,
was
monitored
A 0.1 sample
box
concentration.
a sonic
Fig.
various
glove Reinetsu
and
were
samples to
generation
90%)
by
virus
a
desired
to
conditions
and
in
; Shinryo
Ozone
(50%
25•Ž)
ozone
Ozonizer
Tokyo,
virus
exposed
gas
the
rocked
a
and were
dry-phase during
the
treatment. Evaluation efficacy
of
was
reduction
by with
(control moisture
; control chamber
with for
same
the
ozone)
time
ozone
treatment by
at
period
treatment)
physicochemical
the by
logo
the
viruses almost as
: Virucidal
calculating
dividing
(treated
the
ozone
evaluated
the as
logo
the virus virus
were 90%
kept humidity
test
viruses
previously
treatments
virus titer titer in
a for
under described
[8,13]
.
Results Effect of relative humidity on dry-phase samples : A preliminary experiment was conducted with two viruses, HVJ and TMEV. Dry-phase samples of the viruses were exposed to 100 or 200 ppm of ozone gas at 50, 70, and 80% humidity (Fig. 2). A zero level of infectivity was obtained within 1 hr (HVJ) and 3 hr (TMEV) when the two virus samples were exposed to 200 ppm of ozone and 80% humidity. To determine precisely the effect of humidity
Fig.
2. Effect samples were or
of (HVJ
treated
80%
(•œ)
were
treated
(•¢)
humidity
humidity.
ozonization on and TMEV).
with
200 ppm
relative
of
humidity.
with and
100
ppm
200 ppm
the HVJ
ozone
dry-phase samples at 50%
TMEV of at
(•›)
samples
ozone 80%
at
70% (•£)
225
Fig.
3.
Effect
of
Lyophilized for
30
60,
70,
kept
min.
in
a
on
moisture
the MEM
10-fold
samples
dilution
MA-104
Control
at
0.1 and
cells.
at
the
* p
