&EPA
United States
Environmental Protection
Agency
Municipal Environmental Research ^
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S2-82-089 Nov. 1982
Project Summary
Type A Viral Hepatitis:
Effect of Chlorine on
Infectivity
David A. Peterson
A study was conducted to determine
the effects of free residual chlorine on
the infectivity of hepatitis A virus
(HAV). Treatment of HAV with 0.5,
1.5, and 1.5 mg chlorine/L markedly
decreased the development of overt
hepatitis and seroconversion in mar-
moset monkeys when compared with
controls receiving untreated virus.
HAV treated with 2.0 and 2.5 mg
chlorine/L failed to induce hepatitis or
seroconversion in inoculated marmo-
sets. These results suggest that
chlorine treatment levels greater than
or equal to 1.5 mg/L for 30 min (5°C,
pH 7.0) will effectively decrease the
infectivity of HAV by more than four
orders of magnitude.
This Project Summary was developed
by EPA's Municipal Environmental
Research Laboratory. Cincinnati. OH.
to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
Viral hepatitis ranks fourth among
reportable infectious diseases in the
United States, and it is the most
common, serious infectious disease for
which no specific treatment exists. Viral
hepatitis is caused by two distinctly
different viruses — hepatitis A virus
(HAV) and hepatitis B virus (HBV). From
1972 to 1981, about 40,000 to 50,000
cases of HAV and 6,000 to 9,000 cases
of HBV have been reported annually in
the United States. But the actual
incidence of clinical disease and sub-
clinical infection is undoubtedly several
times greater than the number of
reported cases. In addition, 80% to 90%
of patients with post-transfusion hepa-
titis do not develop antibodies to either
HAV or HBV. This fact suggests the
existence of a third type of viral
hepatitis, non-A, non-B (NANB), caused
by two unidentified agents.
The fecal-oral transmission of HAV
by contamination of water supplies,
food, and drink is well documented by
the many detailed reports of hepatitis A
epidemics. HBV transmission generally
occurs by parenteral inoculation of blood
or blood products, or by close family
association. NANB hepatitis is currently
recognized primarily in the post-trans-
fusion situation, and its natural mode of
transmission is probably very similar to
that of HBV.
Waterborne outbreaks of type A
hepatitis could be prevented by treat-
ment of potable water with an effective
virucidal agent. Free residual chlorine is
generally accepted as a universal water
disinfectant and has been shown to be
effective against adenovirus and various
enteroviruses. Although large amounts
of chlorine have been shown to destroy
the infectivity of HAV in crude fecal
filtrates, the effect of chlorine has not
been extensively examined because an
in vitro system for propagating HAV has
only recently been identified. As an
alternative to studies in vitro, the effect
of chlorine on HAV infectivity can be
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studied in a susceptible animal model.
The virologic, serologic, and pathologic
findings that characterize HAV infections
and type A hepatitis in humans can be
reproduced experimentally in marmoset
monkeys.
This study determines the effects of
free residual chlorine on the infectivity
of HAV derived from fecal material.
Methods and Materials
Fecal material shown previously to
induce hepatitis in marmoset monkeys
(Saguinus sp.) was selected as the virus
source. HAV was partially purified from
these infectious feces by precipitation
with polyethylene glycol; it was then
further purified by the sucrose gradient
centrifugation technique.
Partially purified HAV was diluted
1:50 in chlorine-demand-free buffer
and treated for various exposure periods
(15, 30, or 60 min) with 0.5, 1.0, 1.5,
2.0, or 2.5 mg/L free residual chlorine
and gentle agitation at 5°C and pH 7.00.
After the exposure period, the chlorine
was neutralized with sodium thiosulfate,
and the treated preparations were
inoculated into marmosets. Untreated
control preparations of HAV diluted
1:50, 1:500, 1:5,000 1:50,000, and
1:500,000 were processed similarly
and inoculated into marmosets.
The infectivity liter of the polyethylene
glycol/sucrose preparation in marmo-
sets was performed in parts. In each
chlorine treatment experiment, the
untreated control preparation was a
serial tenfold dilution of the untreated
preparation. The undiluted, partially
purfied HAV preparation had 10508
marmoset infective does per milliliter
(MIDso/mL).
Results
The untreated control 1:50 dilution
induced hepatitis and the development
of antibodies to HAV (anti-HAV) in 100%
of the animals inoculated (5 of 5).
Treatment of HAV with 0.5 mg chlorine/L
in three experiments (15-, 30-, and 30-
min exposures) resulted in 14% (2 of 14)
of the animals developing hepatitis
(versus 100% of the controls). In
addition, the incubation period (71 days)
and seroconversion time (85 days) were
longer than those of the controls (40 and
36 days, respectively). HAV treated with
1 mg chlorine/L in three experiments
(30-, 30-, and 60-min exposures)
induced hepatitis in 8% (1 of 12) of the
animals and seroconversion in 33% (4
of 12). Treatment with 1.5 mg chlorine/L
in two experiments (30-min exposures)
resulted in 10% (1 of 10) of the animals
developing hepatitis and seroconverting.
HAV treated with 2.0 mg chlorine/L
(two experiments with 30-min expo-
sures) and 2.5 mg chlorine/L (one
experiment with a 30-min exposure) did
not induce the development of apparent
hepatitis or antibodies to HAV.
The immunogenic effect of chlorine-
treated HAV was investigated with six
animals that had been previously
inoculated with chlorine-treated HAV
(four of them with 2.0 mg/L and two
with 2.5 mg/L) were given two additional
inoculations (at 14-day intervals) of
HAV treated with 2.5 mg chlorine/L
(30-min exposures). None of these
animals developed detectable anti-HAV
antibodies.
Discussion
Treatment of partially purified HAV
with 0.5 to 1.5 mg chlorine/Lfor 30 min
decreased the incidence of overt hepa-
titis (10% to 14% versus 100% in
controls) and seroconversion (10% to
33% versus 100% in controls). Because
of limited numbers of animals and
funds, it was impractical to titrate the
chlorine-treated HAV preparations;
thus, it was not possible to determine
accurately the decrease in infectivity.
The 1:50 dilution used for all chlorine
treatment experiments contained on
the order of 1O3*8 MIDso/mL. In the 0.5
mg chlorine/L treatment experiments,
only 14% (2 of 14) of the animals
developed hepatitis and 29% (4 of 14)
developed antiblodies. Thus both the
incidence of ovftrt hepatitis and sero-
conversion are well below the 50%
level, implying: that the titer after
treatment was le^s than 1 MID5o/mL or a
minimal reduction in titer of 10338
MIDso/mL. The extended incubation
and seroconversjon periods observed in
the treated groups also indicate low
infective doses and have been seen
repeatedly in titration experiments in
marmosets. The brotracted seroconver-
sion periods (70 to 107 days) appear to
be a true reflection of infection and
multiplication of HAV with subsequent
development of gnti-HAV, since immu-
nization of anirr)als three times with
HAV treated with 2.0 and 2.5 mg free
residual chlorine; failed to induce any
antibodies recognized as anti-HAV.
The development of overt hepatitis in
10% (1 of 10) 'of the animals that
received virus treated with 1.5 mg
chlorine/L for 30 min was probably due
to the presence oif an aggregate of HAV
that was not completely penetrated by
the chlorine. The fact that only thij
animal seroconverted suggests that the
vast majority of single or small aggregates
of virus were inactivated by 1.5 mg
chlorine/L.
Though 8% (1 of 12) of the animals in
the 1-mg chlorine/L treatment group
developed hepatitis, 33% (4 of 12)
seroconverted. This serconversion level
is comparable to that of the 0.5-mg
chlorine/L treatment group (29%, or 4
of 14). If we exclude the animal that
developed hepatitis and seroconverted
in the 1.5-mg chlorine/L treatment
group, we see that treatment with 1.5,
2.0, and 2.5 mg chlorine/L essentially
destroys the infectivity of HAV. Thus the
critical treatment level appears to be
between 1.0 and 1.5 mg chlorine/L in
these experiments. The levels of free
residual chlorine required to inactivate
HAV that is protected by fecal material,
aggregated, and dispersed in water are
unknown.
These results (the decrease in disease
incidence, the development of anti-
HAV) indicate that treatment levels of
0.5 to 1.5 mg chlorine/L for 30 min
inactivated most but not all HAV in the
preparations. Concentrations of 2.0 and
2.5 mg chlorine/L destroyed the infec-
tivity.
An American Water Works Association
Committee recently recommended that
appropriate disinfection of drinking
water to ensure viral inactivation could
be achieved by maintaining a free
chlorine residual of 1.0 mg chlorine/L
for at least 30 min at a water pH of less
than 8,0. Data from a number of
investigations reviewed by the National
Academy of Sciences indicate that
under these conditions, enteroviruses
were consistently reduced by two
orders of magnitude in less than 5 min.
The results of this investigation indicate
that HAV may be considerably more
resistant to chlorine than other entero-
viruses.
The full report was submitted in
fulfillment of Grant No. R805986-02-2
by Rush-Presbyterian-St Luke's Medical
Center under the sponsorship of the
U.S. Environmental Protection Agency.
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David A. Peterson formerly with Rush-Presbyterian-St. Luke's Medical Center,
Chicago, IL, is currently with Abbott Laboratories, Inc., North Chicago, IL
60064.
John C. Hoffis the EPA Project Officer (see below).
The complete report, entitled "Type A Viral Hepatitis: Effect of Chlorine on
Infactivity." (Order No. PB 83-115 170; Cost: $8.50, subject to change) will
be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Municipal Environmental Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
U S. GOVERNMENT PRINTING OFFICE: 1982 659.O17/O868
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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