EPA/600/D-88/042
March 1988
INACTIVATION OF HEPATITIS A VIRUS AND MODEL VIRUSES
IN WATER BY FREE CHLORINE AND MONOCHLORAMINE
by
Mark D. Sobsey, Takashi Fuji and Patricia A. Shields
University of North Carolina
Chapel Hill, NC 27599-7400
CR-813024
Project Officer
John C. Hoff
Drinking Water Research Division
Water Engineering Research Laboratory
Cincinnati, OH 45268
WATER ENGINEERING RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OH 45268
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
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I
INACTIVATION OF HEPATITIS A VIRUS AMD MODEL VIRUSES
IN WATER BY FREE CHLORINE AND MONOCHLORAMINE
Mark D. Sobsey, Takashi Fuji and Patricia A. Shields
Dept. of Environmental Sciences and Engineering, School of Public Health
University of North Carolina
Chapel Hill. North Carolina 27599-7400. U.S.A.
ABSTRACT
The kinetics end extent of inactivation of hepatitis A virus (HAV) as well as three other
viruses, coxsackievirus B5 (CB5) and coliphages MS2 and $X174. by 0.5 mg/1 free chlorine. pH
6-10, and 10 mg/1 monochloramine. pH 8. at 5 C in 0.01 M phosphate buffer were determined.
HAV was relatively sensitive to 0.5 mg/1 free chlorine but relatively resistant to 10 mg/1
monochloramine. Compared to HAV. CBS was quite resistant to inactivation by free chlorine but
similar in resistance to inactivation by monochloramine. Inactivation of ^)X174 by free
chlorine was rapid at pH 6-9 and intermediate between that of HAV and CB5 at pH 10. <$X174 was
inactivated most rapidly of all viruses tested by 10 mg/1 monochloramine. Inactivation of MS2
by free chlorine was somewhat more rapid than HAV at low pH but less rapid than HAV at high
pH. MS2 inactivation by 10 mg/1 monochloramine was slowest of all viruses tested. These
results indicate that HAV is inactivated relatively rapidly by free chlorine but relatively
slowly by monochloramine. Coliphage MS2 is a reasonable model to predict inactivation of HAV
by free chlorine and inactivation of HAV and CB5 by monochloramine. It is poor model for
predicting free chlorine inactivation of CB5 and perhaps some other human enteric viruses.
KEY WORDS
Hepatitis A virus, coxsackievirus, enteroviruses, coliphages, disinfection, inactivation, free
chlorine, monochloramine, water.
INTRODUCTION
Hepatitis A virus (HAV) is an important waterborne enteric virus that has caused outbreaks of
hepatitis A due to consumption of treated and untreated drinking water in the United States
(Lippy and Waltrip, 1984) and elsewhere. In some reported outbreaks the drinking water was
chlorinated and met coliform bacteria and other quality standards (Hejkal ct al., 1982). The
growing number of reports on the isolation of viruses, including HAV, from treated drinking
water (Bitton et al.. 1986) suggests that some viruses may survive treatment under certain
conditions. Establishing reliable water treatment practices and water quality standards to
insure the virological safety of water supplies requires knowledge of the response of HAV and
other waterborne viruses to disinfection. However, there have been few reports on HAV
inactivation by chlorine. Early studies by Neefe et al. (1945, 1947) suggested that HAV is
relatively resistant to chlorine. Total and free chlorine concentrations of 1.1 and 0.4 mg/1.
respectively, in purified effluent were needed to prevent infectious hepatitis in volunteers.
More recently, Peterson et al. (1983) reported that the marmoset infectivity of a partially
purified preparation of HAV containing about 1500 infectious units/ml was only partially
reduced by treatment with up to 1.5 mg/1 of free residual chlorine at neutral pH for 30
minutes. These results, along with observations from the outbreak of hepatitis in Georgetown,
Texas (Hejkal et al. . 1982) , suggest that HAV is more resistant to water chlorination
processes than other enteroviruses and indicator bacteria.
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In contrast to the studies cited above, those by Grabcw et al. (1983) indicated that HAV may
be more sensitive to free chlorine than suggested by previous studies and epidemiological
evidence. Using HAV infectivity assays in cell cultures, Grabow and co-workers found that HAV
was quite sensitive to low levels of free chlorine relative to selected indicator viruses and
bacteria. However, further studies indicated that HAV vac relatively resistant to combined
forms of chlorine in tap water and sewage effluent (Grabow et al., 1984),
Considering the limited data and inconsistent findings of previous reports, there is a need
for further studies of HAV inactivation by free and combined forma of chlorine under
controlled conditions. Such studies are now feasible using new methods for the cultivation
and enumeration of HAV in cell cultures (Daemer et al,, 1981; Lemon et al., 1983). The
purpose of this present study is to determine the kinetics and extent of HAV inactivation in
water by free chlorine and combined chlorine in the form of monochloramine at defined pH
levels and temperature. Inactivation of HAV is compared to the inactivation of model viruses
including coxsackievirus B5 and bacteriophages MS2 and $X174.
METHODS AND MATERIALS
Viruses. Cell Cultures and Virus Purification
HAV. The HM175 strain of HAV, originally isolated from feces of an infected human in
Australia (Daemer et al., 1981; Gust et^ al.. 1985), is produced in persistently infected BS-C-
1 cells grown at 37 C. HAV infectivity is assayed by the radioimmunofocus assay (RIFA) in BS-
C-l cells as previously described (Lemon et al., 1983; Sobsey et^ al. , 1985). Persistently
infected cells and culture fluid harvests are centrifuged at ca. 3,000 * £, resuspended in
small volumes of phosphate-buffered saline (PBS), pH 7.5, and extracted by homogenizing in an
equal volume of chloroform. The HAV-containing PBS is recovered by low speed (5-10,000 x £)
centrifugation. The cell debris and chloroform are further extracted four to six more times
with equal volumes of PBS and then twice more with equal volumes of 0.1% SDS in PBS to obtain
additional virus. All PBS and SDS-PBS extracts are recovered by low speed centrifugation at
room temperature or 4 C, and SDS is removed by precipitation and centrifugation at 4°C.
HAV in cell culture fluids is concentrated by precipitation with polyethylene glycol 6000
(PEG) (122 w/v, pH 7.2) overnight at 4 C. Precipitates are recovered by low speed
centrifugation, resuspended in a small volume of PBS and extracted with an equal volume of
chloroform. PBS extracts are cleared of chloroform and PEG by low speed centrifugation. HAV
in pooled PBS extracts of cells and PEG precipitates is pelleted by ultracentrifugation at
105,000 x £ for 4 hours at 5 C. HAV pellets are resuspended in small volumes of 0.01M
phosphate-buffered, halogen demand-free (PBKDF) water, supplemented with CsCl to give a
density of 1.33 g/nil, and ultracentrifuged to equilibrium in self-generated gradients at
90,000 x £ and 5 C for 3 days. Peak fractions of HAV from CsCl gradients are desalted by
ultrafiltration and washed with PBHDF water using Centricon 30 ultrafiltration tubes (Amicon
Inc). Desalted fractions are layered onto 10-30% sucrose gradients in PBHDF water, pH 7.5,
and ultracentrif uged in the SW27 rotor (Beckman Instruments) at 90,000 x jg and 5°C for 5
hours. Harvested gradient fractions corresponding to single virions are then pooled and mixed
with appropriate amounts of single virions of the other test viruses. The titer of each virus
in the mixture is about 1-5 x 10 infectious units/ml.
Coxsackievirus B5. CB5 (Faulkner strain) is grown and assayed by the plaque technique in BGM
cells as previously described (Sobsey et al, 1978). Virus in cell lysates is liberated from
cell debris by freezing and thawing, and then centrifuging at low speed for 15-30 minutes.
Viruses in resulting supernatants are supplemented to 0.1% SDS and pelleted by
ultracentrifugation (105,000 x £ and 5 C for 4 hours). Resulting virus pellets are
resuspended in PBHDF water, homogenized 1 minute, and centrifuged at 5,000 x £ and 5°C for 20
minutes to remove additional debris and precipitated SDS. After supplementing with CsCl to a
density of 1.33 g/ml, viruses are banded to equilibrium as for HAV. CsCl gradient fractions
containing the virus peak are desalted and subjected to rate-zonal centrifugation in 10% to
30% sucrose gradients as for HAV. Sucrose gradient fractions corresponding to single virions
are added to HAV samples to give the desired virus titer.
Bacteriophages. Coliphages MS2 (ATCC 15597-B1) and ^)X174 (ATCC 13706-B1) are grown and
assayed by the top agar plaque technique (Adams, 1959) in E. coli C3000 (ATCC 15597) and E.
coli C (ATCC 13706) hosts, respectively, using nutrient agar #2. Crude virus is harvested""
from the top agar of plaque assay plates having confluent lysis by scraping into small volumes
(3-5 ml/plate) of PBS. Harvests are extracted with chloroform and centrifuged at 10,000 x £
or 10 minutes. Viruses in the resulting supernatant are pelleted by ultracentrifugation for
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4 hours at 105,000 x £ and 5°C. Pellets are resuspended in PBKDF water, supplemented with
CsCl to give a density of 1.44-1.45 g/ml^ and the viruses are banded to equilibrium in CsCl
gradients for 3 days at 90,000 x a and 5 C. Gradient fractions with the virus peak are
desalted using Centricon 30 ultrafilters and then filtered successively through Tween-80-
treated, 0.2 and 0.08 pm pore size polycarbonate filters (Nuclepore) to remove virus
aggregates. The filtrates, containing almost exclusively single virions, are combined with
single virions of HAV and CBS.
Glassware. Chlorine Reagents and Chlorine Analysis
Glassware for disinfection experiments is soaked >4 hours in a 10-50 mg/1 free chlorine
eolation and then rinsed thoroughly with HDF water. HDF water and buffer solutionss are
prepared from twice deionized, activated carbon-filtered water which is then passed through a
macroreticular scavenging resin bed (Rohm and Haas). HDF, phosphate-based buffers, 0.01 M,
are used to prepare test solutions for disinfection experiments. Household bleach (5.25%
sodium hypochlorite; Clorox) is diluted in HDF water to prepare a 100 mg/1 stock solution.
Monochloraoine stock solution of about 100 mg/1 is prepared after Berman and Hoff (1984) by
combining equal volumes of a 200 mg/1 free chlorine solution and ar. 800 mg/1 NH^Cl solution,
both in 0.01 M phosphate buffer, pli 9.5. Stock chlorine solutions are then diluted in test
water (PBHDF water, pH 6-10) to give the target chlorine concentrations (0.5 mg/1 free
chlorine and 10 mg/1 monochloramine).
Chlorine concentrations are measured by DPD colorimetric methods, and these procedures are
standardized by the DPD ferrous titrametric method (American Public Health Association, 19855,
Chlorine samples from the U.S. Environmental Protection Agency are analyzed regularly as check
samples.
Protocol for Disinfection Experiments and Data Analysis
Test samples are placed in 25 mm dia. x 150 an long test tubes and kept in a water bath at
5°C. A 0.24 ml volume of purified, monodispersed virus mixture (HAV, CBS, HS2 and $X174) is
added to 11.76 ml of a chlorine solution containing 0.51 tag/1 free chlorine (or 10.2 mg/1
monochloramine) and then briefly mixed. A second test tube containing only chlorine solution
serves as a halogen control. A third tube containing a 1:50 dilution of stock virus in PBHDF
water serves as a virus control. Samples of 0.7 ml are withdrawn from the reaction tube
(chlorine solution plus added virus) for viral analysis at 0.33, 1, 3, 10, 30 and 60 minutes
after virus addition. These samples are immediately diluted two-fold in 2X Eagle's MEM
containing 1% Na S,Q_ and stored at 4 C for subsequent virus assays. For virus assay, samples
are further diluted serially 10-fold in separate diluents for HAV. CBS and the two phages.
After the 60 minute reaction period, the remaining test mixture (halogen plus added virus) and
the chlorine control sample are re-analyzed for free and combined chlorine. Samples from the
virus control (virus in PBHDF water) are diluted serially 10-fold at the beginning and the end
of the 60 minute reaction period for subsequent virus assays.
Virus disinfection data, as plaque forming units (PFU) per ml for CBS, MS2 and |X174 or
radioimaunofocus forming units (RFU) per ml for HAV, are average values frem triplicate
cultures. For each experiment, the virus concentrations of the virus control sample at time =
0 are computed and taken as N , the initial virus concentration. Virus concentrations in
control samples did not change appreciably over 60 minutes. For each test sample (samples
taken from the test mixture at 0.33, 1. 3. 10, 30 and 60 minutes), the average concentration
of each virus is computed. The proportion of initial viruses remaining at each test time (t)
is computed by dividing the virus concentration at each test time (N ) by the initial virus
concentration (N ). These values are then log^-transf ormed (log,Q IN /NQ]), and the values
of duplicate or triplicate experiments are averaged. These mean data for log1£j H /Nq are then
paired with the data for sampling time (t) and analyzed by linear regression to obtain the
correlation coefficient, slope of the regression line, and estimated tiae for 99.99%
inactivation of the initial viruses.
RESULTS AND DISCUSSION
The mean results of duplicate or triplicate experiments at 5°C in PBHDF water using 0.5 mg/1
free chlorine, pH 6 to 10, and 10 mg/1 monochloramine, pH 8, are susinarized in Table 1 as
times for 99.99% inactivation of the initial viruses (T-99.99).
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TABLE 1 1 reactivation of HAV, CBS and ColiphaRes MS2 and $X174 at 5°C in 0.01 M Buffer
by 0.S fflg/1 Free Chlorine at pH 6. 7, 8. 9 and 10 and 10 mg/1 Monochloramine at pH 8
Chlorine
Form
pH
Time
(Min.) for
99.99% Inactivation
HAV
CBS
MS2
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0
1
2
3
4
0 2 4 6 8 10 12
Be—&
-2
-3
-4
30
20
40
10
50
60
70
0
TIME (min)
Figure 1. Inactivation of HAV (—<=>—), QS5 ( Q-—), MS2 (•—*—and 0X174 (-—)f--) by
0.5 mg/1 Free Chlorine at 5oC and pH 6 (Panel A), pH 8 (Panel B) and pH 10 (Panel C).
Arrow (^ ) denotes the limit of virus detection.
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HAV and MS2 are generally consistent with those of Grabov et al. (1984). In tapwater
containing 0.1 mg/1 free chlorine and about 11 mg/1 chloramines at pH 8, they obtained 99.99%
inactivation of HAV in AO minutes and only 97% inactivation of MS2 in 60 minutes.
L
0
G
N
/ ~2
N
o
-3
-4
10
0
20
30
40
50
60
70
TIME (min)
Figure 2. Inactivation of KAV (—• G35 ( Q—) . MS 2 (-—*—•) and 0X174 (— ) by
10 mg/1 Monochloramine at pH 8 and 5 C. Arrow ( f ) denotes the limit of virus detection.
Considering that monochloraaine was tested at a 20-fold higher concentration than free
chlorine, it was a relatively poor virucide. Because monochloramine is typically used in
drinking water at only 1-2 mg/1, it appears to be a poor primary disinfectant for HAV and
other viruses in drinkling water, unless contact tines are extremely long.
Factors Influencing Virus Disinfection
A factor which may influence virus disinfection at different pH levels is the degree of virus
aggregation. Aggregation was controlled in stock virus preparations by using only gradient
fractions or filtrates containing single particles. However, the addition of monodispersed
viruses to reaction mixtures at different pH levels may have caused virus aggregation, thus
resulting in slower inactivation kinetics. Previous studies have shown that acid pH levels
can induce virus aggregation and decrease inactivation rates (Young and Sharp, 1985).
Another factor which may influence virus inactivation rates at different pH levels is the
conformational form of the virus. A form of the virus existing at one pH may be more
resistant to disinfection and/or less infectious than another form existing at another pH.
Both polio-virus 1 and echovirus 1 can exist in at least two different, pH-dependent
conformational forms (Young and Sharp, 1985). Different conformational forms of HAV have not
been established, but preliminary evidence from this laboratory suggests possibly two
conformational forms of HAV HM175 (unpublished results).
Sensitivity to disinfection sometimes differs among strains of the same virus type. Studies
on the disinfection of other strains of HAV by free and combined chlorine are needed in order
to determine if the response of strain HM175 is typical or representative of other strains.
SUMMARY
Results of this study indicate that HAV strain HM175 is relatively sensitive to free chlorine
and more sensitive than CB5. Coliphages MS2 and $X174 also were relatively sensitive to free
chlorine, thus making them poor indicators for free chlorine disinfection of enteric viruses
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such as CB5. Both phages were more resistant than or similar in resistance to HAV at some pH
levels- MS2 was more like HAV than was $X174 with respect to inactivation by free chlorine.
This suggests that MS2 and similar phages may be useful indicators of HAV disinfection by free
chlorine. All four viruses were relatively resistant to monochloramine. HAV and CB5 had
similar resistance to monochloramine, while KS2 vas most resistant and (j)XI74 was rnobi.
sensitive. These results suggest that MS2 and similar coliphages may be useful indicators of
enteric virus disinfection by monochloramine. The considerable resistance of test viruses to
monochloramine makes it a poor choice as a primary disinfectant for drinking water.
ACKNOWLEDGEMENT
This research was supported by Cooperative Agreement No. CR813024 from the U.S. Environmental
Protection Agency.
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Neefe, J.R., J. Stokes, J.B. Baty and J.G. Reinhold (1945) Disinfection of water containing a
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TECHNICAL REPORT DATA
(Please read Instructions on ihe reverse before completing)
1. REPORT NO. 2.
EPA/600/D-88/042
3. RECIPIENT'S ACCESSION NO.
PRSi. m o ft
4. TITLE AND SUBTITLE
INACTIVATION OF HEPATITIS A VIRUS AND MODEL VIRUSES
IN WATER BY FREE CHLORINE AND MONOCHLORAMINE
5. REPORT DATE
March 1988
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Mark D. Sobsley, Takashi Fuji, and Patricia A. Shields
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
University of North Carolina
Chapel Hill, North Carolina 599-7400
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
CR-
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