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Pergamon
0043-1354(95)00070-4
War. Res. Vol. 29, No. 11, pp. 2548 2553, 1995
Copyright ~ 1995 ElsevierScienceLtd
Printed in Great Britain. All rights reserved
0043-1354/95 $9.50 + 0.00
S U R V I V A L OF THE E N T E R I C A D E N O V I R U S E S 40 A N D 41
IN TAP, SEA, AND WASTE WATER
C A R L O S E. E N R I Q U E Z ~*, C H R I S T O N J. H U R S T 3@ and C H A R L E S P. G E R B A 1"2®
~Department of Microbiology and Immunology, and -'Department of Soil and Water Science, University
of Arizona, Tucson, AZ 85721 and 3U.S. Environmental Protection Agency, Cincinnati, OH 45268,
U.S.A.
(First received September 1994; accepted in revised form February 1995)
Abstract--The
enteric adenoviruses types 40 (Ead 40) and 41 (Ead 41) have emerged as a leading cause
of viral gastroenteritis in children, second in importance only to the rotaviruses. The role of the enteric
adenoviruses as waterborne pathogens has not been evaluated. This study compared the survival of these
agents with poliovirus type 1 (polio I) and the hepatitis A virus (HAV) in tap water at 4°C, and at room
temperature, with polio 1 in primary and secondary wastewater at 4, and 15°C, and in sea water at 15°C.
Assays were conducted at regular intervals by the TCIDs0 method in PLC/PRF/5 cells. The survival of
Ead 40 and Ead 41 in primary and secondary wastewater was slightly greater than that of polio 1.
However, in tap, and sea water, the enteric adenoviruses were substantially more stable than either polio
1 or HAV. These results suggest that the enteric adenoviruses may survive for prolonged periods in water,
representing a potential route of transmission.
Key words--survival, tapwater, wastewater, seawater, hepatitis A, poliovirus, adenovirus
INTRODUCTION
Diarrheal diseases are the leading cause of childhood
morbidity and mortality in developing countries
(Uhnoo et al., 1986). It has been estimated (LeBaron
et al., 1990) that infectious gastroenteritis in the
U.S.A. alone, causes more than 210,000 children of
5 years of age or younger to be hospitalized, at a
yearly cost of nearly 1 billion dollars. Throughout
the world, 3-5 billion cases of diarrhea occur, with
approx. 5-10 million deaths every year (LeBaron
et al., 1990).
Adenoviruses 40 and 41 (Ead 40 and Ead 41) have
been recognized as important etiological agents of
gastroenteritis in children (Uhnoo et al., 1986; Cruz
et al., 1990), second in importance only to rotavirus.
Greater numbers of adenoviruses than enteroviruses
have been consistently found in raw sewage around
the world (Irving and Smith, 1981; Hurst et al., 1988;
Krikelis et al., 1985a, b), and it has been estimated
(Hurst et al., 1988) that 80% of infectious adenoviruses shed in the feces, and present in raw sewage
may be enteric adenoviruses. No etiologic agents
have been identified in half of the reported waterborne disease outbreaks in the U.S.A. (Sobsey et al.,
1993). Therefore, in spite of lack of evidence, the
possibility of th enteric adenoviruses as the cause of
undocumented waterborne disease exists. Furthermore, the transmission of waterborne conjunctivitis
*Author to whom all correspondence should be addressed.
by adenovirus types 3 and 4 has been established
(Rao and Melnick, 1986; Grabow, 1990).
Current priorities in enteric viral research have
been identified by The Centers for Disease Control,
these include: (1) improving diagnostic capabilities;
(2) identifying the etiology of the 50% of gastroenteritis outbreaks that are still of unknown origin; and
(3) determining the modes of transmission, and the
means to prevent disease (LeBaron et al., 1990).
Outbreaks caused by enteric adenovirus have occurred mostly in hospitals and day-care centers,
affecting only children (Chiba et al., 1983). The
enteric nature of Ead 40 and Ead 41, their presence
only in the gastroenteric tract, and their extensive
geographical distribution suggest that water may play
a role in the transmission of these agents. The study
of the enteric adenoviruses in water has been hindered because these agents propagate poorly, or not
at all, in conventional human cell lines in which other
adenoviruses can be propagated effectively (deJong
et al., 1983). However, recently, the successful propagation of both, Ead 40 and Ead 41 in P L C / P R F / 5
human liver cell line was reported (Grabow et al.,
1992). The availability of this cell line provides a
practical method for the study of these viruses in the
aquatic environment.
This study compared the survival of Ead 40 and
Ead 41 with poliovirus type 1, in tap, sea, and waste
water, at 4, 15°C, and room temperature (23:C), and
with the hepatitis A virus (HAV) in tap water, at 4:C,
and room temperature (23 C).
2548
Survival of enteric adenovirus in water
MATERIALS AND METHODS
Production o f viruses and tissue culture assay
Poliovirus type 1 (strain LSc2ab) (polio 1) was propagated in the Buffalo green monkey (BGM) kidney continuous cell line. The cells were grown and maintained in
minimum essential medium (MEM) (Flow Laboratories,
McLean, Va) containing 5 and 2% fetal bovine serum
(FBS), respectively. Hepatitis A virus (HAV) (strain
HMI75), and the enteric adenoviruses types 40 (Ead 40)
(strain Dugan) and 41 (Ead 41) (strain TAK) were propagated on human primary liver carcinoma cells (PLC/PRF/5)
(Grabow et al., 1983). Cell debris from virus suspensions
were removed by low-speed centrifugation (2000g for
10 min), followed by extraction twice with freon (Trichlorotrifluoroethane, Fisher Scientific, Pa). These preparations
were frozen at 20':C before use. Virus titration was carried
out by the tissue culture infectious dose fifty (TCIDs0)
procedure, on PLC/PRF/5 cells in 96-well plates (Becton
Dickinson Labware, N.J.), modified from Precious and
Russell (1985). Survival is reported as the logloNt/No
TCIDs0/ml, which expresses the reduction in viral numbers
at each time interval, where N o and N, are the initial and
final viral titers respectively. Viral rate of inactivation,
correlation coefficient of the data (time vs viral inactivation),
and time for 99% virus inactivation (T99) were calculated
using linear regression analysis (Yates et al., 1985). The
t-test statistic was used to determine significant differences
in survivability between viruses at given conditions.
Source o f water
Tap water was obtained directly from the tap at the
University of Arizona, Tucson. The water is supplied from
a well located on the University of Arizona campus. Absence of total and free chlorine in tap water was determined
by the N,N-diethyl-p-phenylenediamine (DPD) method
(Hach Company, Loveland, Colo.). Settled primary and
secondary sewage samples were collected in sterile 1-1 plastic
bottles, at the Roger Road wastewater treatment facility in
Tucson, Ariz., and sea water was obtained from the coastal
area of California, near Los Angeles. Chemical analysis of
tap, sea, and waste water were adapted from Standard
Methods Jbr the Examination of Water and Wastewater, and
are listed in Tables 1 and 2, respectively.
A total of 248 samples were examined in this study.
Samples of tap and sea water, and primary and secondary
wastewater effluents, were kept in a BOD incubator (LabLine Instruments, Melrose Park, I11.), at either 4 or 15°C,
and individually seeded, with approx. 1 × 104 TCIDs0/ml of
Ead 40, Ead 41, and polio 1. Samples of tap water, kept at
room temperature, were additionally seeded with approx.
1 × 104 TC1Ds0/ml of HAV. Samples were aliquoted in
1.5-ml polypropylene tubes, incubated from 50 to 60 days,
at the indicated temperatures, and withdrawn for assay at
regular intervals.
RESULTS
Overall, the enteric adenoviruses were more stable
t h a n either polio l or HAV. In tap water, at 4°C Ead
41 lost less t h a n 0.5 logt~, and Ead 40 lost nearly one
logl0 during a 55-day period [Fig. l(a)], while the titre
of H A V and polio 1 decreased approx, i.6 and
2.7 log~0, respectively [Fig. l(a)]. The predicted time
for 99% inactivation of these viruses varied with virus
type a n d experimental conditions. The 7"99values for
polio 1, HAV, Ead 40, a n d Ead 41, u n d e r these
conditions, were 41, 56, 92 and 304 days, respectively
(Table 3). U n d e r these conditions, t-test statistic
analysis of the data did not show significant differWR 29 [ I - - H
2549
Table I. Tap water characteristics
Parameter
Value
Free chlorine (mg/I)
0
Total chlorine (mg/l)
0
pH
7.7
Alkalinity (mg/I)
90
Total hardness (mg/l)
134
Ca hardness (mg/l)
132
Mg hardness (mg/l)
2
Nitrogen (nitrate) (mg/I)
3.5
Nitrogen (ammonia) (mg/l)
0.05
Turbidity (NTU)
0.35
ences between the survival o f polio i a n d HAV; H A V
a n d Ead 40; a n d Ead 40 and Ead 41. However, a
significant difference in the survivability was observed
between polio 1 a n d Ead 40 (0.05 significance); polio
1 a n d Ead 41 (0.01 significance); and H A V a n d Ead
41 (0.025 significance).
In tap water at 15°C, the titer of polio 1 declined
nearly 3 lOgl0 after 42 days, whereas, by day 60, the
titers of b o t h Ead 40 a n d Ead 41 were only reduced
1.4 a n d 1.21og~0, respectively [Fig. I(b)]. The estim a t e d T99 value for polio I was of 24 days, whereas
for Ead 40 a n d Ead 41 was of 87 a n d 124, respectively
(Table 3). Using t-test analysis, the difference in
survival between polio 1 and b o t h Ead 40 a n d Ead 41
was significant at the 0.05 level.
In tap water at r o o m temperature, Ead 40 a n d Ead
41 also were more stable t h a n b o t h polio 1 and HAV.
The titers of Ead 40 a n d Ead 41, after 55 days,
decreased nearly 2 logl0 [Fig. l(c)]. Polio 1 and HAV,
on the o t h e r hand, lost approx. 4, a n d 3.5 log~0 after
30 and 50 days, respectively [Fig. l(c)]. The predicted
n u m b e r of days required for 9 9 % viral inactivation at
r o o m temperature in tap water were 60 a n d 84 for
Ead 40 and Ead 41, respectively, whereas the 7"99
value for H A V was 27 a n d t h a t for polio 1 was 11
(Table 3). Significant differences in the viral survivability in tap water held at room t e m p e r a t u r e were
detected, using t-test analysis, between polio 1 and
Ead 40 and Ead 41 (significance of 0.05 a n d 0.01,
respectively), but not between H A V a n d Ead 40 or
Ead 41.
In sewage effluents, the survival of the Ead 40 a n d
Ead 41 was slightly superior to that of polio 1.
However, no significant difference was found using
t-test analysis. In primary wastewater, after 50 days
at 4°C, the titer o f Ead 41 diminished by a b o u t
2 log~0, t h a t of Ead 40 nearly 2.5 log~0, a n d that of
polio 1 2.2 logt0 [Fig. 2(d)]. In primary effluent after
60 days at 15°C, b o t h enteric adenoviruses survived
Table 2. Primary and secondary sewage effluents, and
characteristics
Primary
Secondary
Parameter
effluent
effluent
BOD5 (mg/l)
195
16
pH
8.0
7.7
Turbidity (NTU)
40
18
Salinity (ppt)
ND
ND
ND = not determined.
sea water
Sea
water
ND
8.1
3.4
35
2550
Carlos E. Enriquez et al.
(a) Tap water at 4°C
(b) Tap water at 15°C
0"
-1.0
-1.5
-2.0
~'~..
-2.5
'
~
-3"00
~.0
~
....
II~'ll
5
2
''ll'll'll'l
10
(c) Tap water at 23°C
o
.d
''1
'''''~
15 20 25
....
I ,,I
30
35
,I,
,,I
IIL~IIIIII
40 45
50
Illrl
55 60
(d) Sea water at 15°C
0
-0.5
-1.0
-1.5
-2.0
-2.5
if....................,,....................
0
5
10
15
20
25
30
35
40
45
50
55
-3.0
-3.5
0
5
10
15 20 25
30
35 40 45
50 55 60
Days
Fig. 1. Survival of HAV ( + ), Poliovirus 1 (0), enteric adenovirus 40 (*), and enteric adenovirus 41 (D).
slightly longer than polio l, with a decrease of about
2.6 and 2.8 logj0 for Ead 41 and Ead 40, respectively
[Fig. 2(a)]. Under similar conditions, the titer of polio
1 decreased by approx. 2.7 log~0, but after 42 days
[Fig. 2(a)]. The estimated T99 for polio 1, Ead 40 and
Ead 41, held at 4 C in primary sewage was of 36, 44,
and 48 days respectively. At 15°C, slightly shorter T99
values were calculated, with 28 days for polio 1, 40
for Ead 40, and 43 for Ead 41 (Table 3).
In secondary sewage held at 4°C for 50 days, the
enteric adenoviruses 40 and 41 showed a similar
pattern of inactivation as that of polio 1, with a titer
reduction of 1.9, 2.2 and 2.51og10, respectively
[Fig. 2(b)]. However, at 15°C after 60 days, Ead 40
and Ead 41 survived longer than polio 1, with a titer
reduction of 2.4 and 2.91og10, respectively, and
3.2 log~0 after 35 days for polio 1 [Fig. 2(c)]. The
predicted T99 values for polio 1, and Ead 41 incubated
at 4°C in secondary sewage were very similar, 49 and
47 days respectively, while Ead 40 showed a 7"99value
of 58 days under the same conditions (Table 3). At
15°C, however, polio 1 showed a T99 value of 19,
which, was less than half of those of Ead 40 and Ead
41 (43 and 45, respectively) (Table 3).
Survival of polio 1 in sea water kept at 1 5 C was
shorter than survival of Ead 40 and Ead 41. The polio
1 titer was reduced by 3 log~0 after 28 days, whereas,
after 60 days, the titer of Ead 40 and Ead 41 dropped
only 1.4 and 1.6 log10, respectively [Fig. l(d)]. In sea
water under the described conditions, the T99 values
of 85 days for Ead 41, and 77 for Ead 40 were nearly
4 times larger than the 7"99value of 18 days for polio
1 (Table 3). Analysis by t-test statistic showed a
significant survivability difference between polio 1,
and Ead 40 or Ead 41 (significance 0.05).
All experimental treatments for viral survival
showed a significant correlation between inactivation
rate, and incubation time, with P values of 0.002 or
lower (Table 3).
DISCUSSION
Although, the enteric adenoviruses are a major
cause of viral gastroenteritis in children (Unhoo et al.,
1986), information on their stability in water is not
available. This study demonstrated that these agents
are more stable than polio 1 in tap water, sewage, and
sea water, and more stable than H A V in tap water.
Our results are in agreement with those of Bagdasar
and Abieva (1971), who reported that adenovirus
type 5 survived longer than both poliovirus 1 and
echovirus 7 in tap water, at either 4 or 18 C , and with
those of Sobsey et al. (1986), who found that HAV
in groundwater was more stable than polio 1 at
different temperatures. In this study, H A V was more
resistant than polio I at both 4:C [Fig. l(a)], and
Survival of enteric adenovirus in water
room temperature [Fig. l(c)]. However, it was less
stable than the enteric adenoviruses in tap water at
either temperature [Fig. 1(a, c)]. It has been suggested
(Sobsey et al., 1986) that hepatitis-waterborne outbreaks caused by HAV may be related to the higher
survivability of this agent in water. The longer survival of the enteric adenoviruses in tap and sea water
in comparison to HAV and polio 1, observed in this
study, suggests that waterborne transmission of enteric adenoviruses is a possibility.
As with other viruses (O'Brien and Newman, 1977;
Yates et al., 1985), temperature seems to play a key
role in the survival of enteric adenoviruses in water.
In particular, in the case of tap water held at 4°C
[Fig. l(a)]. After 55 days, the Ead 41 and Ead 40 lost
nearly 0.5 and 1.0 log~0, respectively, with an estimated T99 value of 304 days for Ead 41, which was
nearly 8 times larger than that of polio 1, and a T99
value of 92 days for Ead 40, which was more than
twice the T99 value of polio 1 (Table 3). In contrast,
in tap water at room temperature during the same
period, these agents lost almost 2 log~0 [Fig. l(c)].
Some evidence of the higher thermal stability of the
enteric adenoviruses in comparison to polio 1 was
obtained in an experiment in which both, Ead 40, and
polio 1 were held at 50 and 65°C in phosphate
buffered saline. After incubation at 50°C for 6 min,
Table 3. Viral inactivation rate, and T99 values under different
incubation media and temperatures
Virus type
Inactivation
rate
(Iogt0/d)
T a p water at 4 C
Polio I
0.04571
HAV
-0.03246
Adeno 40
-0.02125
Adeno 41
-0.00653
T a p water at 15"C
Polio 1
0.06209
Adeno 40
-0.02412
Adeno 41
-0.01578
T a p water at 23"C
Polio 1
-0.12461
HAV
-0.07376
Adeno 40
0.03080
Adeno 41
-0.02639
Secondary sewage effluent at 4 ' C
Polio 1
0.04379
Adeno 40
-0.03636
Adeno 41
-0.04425
Secondary sewage effluent at 15 C
Polio 1
-0.08143
Adeno 40
-0.04633
Adeno 41
-0.03784
Primary sewage effluent at 4 C
Polio I
-0.04901
Adeno 40
-0.04705
Adeno 41
0.04483
P r i m a r y sewage effluent at 15 C
Polio 1
-0.05908
Adeno 40
-0.04674
Adeno 41
-0.04520
Sea water at 1 5 C
Polio 1
-0.11106
Adeno 40
-0.02570
Adeno 41
-0.02528
r
p
T99
-0.971
-0.944
-0.943
-0.844
~<0.0001
~<0.0001
~<0.0001
~<0.0001
41
56
92
304
-0.949
0.988
-0.911
0.001
~<0.0001
0.002
24
87
124
-0.955
0.985
-0.900
-0.873
0.001
~<0.0001
~<0.0001
~<0.0001
11
27
60
84
-0.938
-0.974
0.964
0.001
~<0.0001
~<0.0001
49
58
47
-0.965
-0.983
0.958
0.0021
~<0.0001
~<0.0001
19
43
45
-0.972
-0.982
-0.961
~<0.0001
~<0.0001
~<0.0001
36
44
48
-0.965
-0.987
-0.986
~<0.0001
~<0.0001
~<0.0001
28
40
43
-0.996
0.994
-0.975
~<0.0001
~<0.0001
~<0.0001
18
77
85
r = correlation coeffcient, p = level o f significance, T99 = time in
days for 9 9 % inactivation o f the original titer.
2551
polio 1 lost 0.88 log10, whereas Ead 40 lost less than
0.2 log~0. Similarly, at 65°C, polio 1 lost more than
2.5 log~0 in 30 s, while Ead 40 lost only 1 log~0 after
the same incubation time (data not shown).
The fact that temperature plays an important role
in viral survival is further supported by LaBelle and
Gerba (1982), who reported that the titer of polio 1
in sea water at 9°C dropped only 2 log~0 in 35 days,
while in our experiment at 15°C in sea water we
observed a similar reduction, but in approximately
half the time [Fig. l(d)]. In contrast, at a higher
temperature, Loh et al. (1979), and Fujioka et al.
(1980), showed that incubation of polio 1 in sea water
at 24°C, resulted in a rapid viral inactivation with
nearly 3 log~0 in 4 days. Similarly, in situ and laboratory inactivation experiments of polio 1 in sea water
at 20°C (Jofre et al., 1986) have resulted in fast
inactivation rates of 3.5 and 4 log~0 in 5 days, respectively. Although, viral survival studies are difficult to
compare, as they are carried out under different
conditions, Callahan et al. (1994) demonstrated
that polio 1 inactivation in sea water was very
similar using three geographically diverse types of sea
water.
This rapid inactivation suggests that not only
temperature, but other factors are associated with
viral survival in ocean waters. The poor survival
of polio 1 in sea water could have been related
to salinity, which has been negatively associated
with viral survival in sea water, due to viral aggregation, and marine microflora (Gerba and Schaiberger
1975; Fujioka et al., 1980; La Belle and Gerba,
1982).
The enteric adenoviruses did not survive significantly longer than polio 1 in primary and secondary
sewage at either 4 or 15°C, with an inactivation rate
of approx. 2.5 log~0 in 50 days [Fig. 2(a~t)]. This
result was unexpected, as organic matter present in
water samples is known to protect enteroviruses from
inactivation (La Belle and Gerba, 1982). The increased survivability of the enteric adenoviruses in
tap and sea water, and the unexpected faster inactivation in sewage, may indicate that these viruses are
inactivated by different mechanisms than those affecting the enteroviruses.
The increased resistance showed by enteric adenoviruses may be associated to the double stranded
nature of their DNA, which if damaged, may be
repaired by the host cell DNA-repair mechanisms,
which in human cells, in addition to repairing pyrimidine dimers, can also repair a wide range of DNA
damages (Bernstein and Bernstein, 1991). In addition,
both adenovirus DNA strands may serve as template
for replication (Kelly, 1984). Thus, if one strand is
damaged by environmental factors, the other may
still serve as template for replication of progeny
DNA. This mechanism would not be effective on
ssRNA-genome viruses like polio 1 or HAV. It has
been suggested (Enriquez et al., 1993), that nucleic
acids from DNA viruses would persist longer in the
2552
Carlos E. Enriquez et al.
(a) Primary sewage at 15°C
(b) Secondary sewage at 4°C
0 ~
-0.5
-0.5
-1.0
-1.0
-1.5
-1.5
-2.0
-2.0
-2.5
-3.0
2
~o
O
,.d
-2.5 l
3
i [ I I ....
5
~ h~
i i i i knt
nnl
nl
~JI
FI~
nl~
nun
Eli
kn
I In
Irl
~lldlnl
J]
~
n IL
tO 15 20 25 30 35 40 45 50 55 60
-
" 0
5
0
--
10
15
20
25
30
35
40
45
50
(d) Primary sewage at 4°C
(C) Secondary sewage at 15°C
0
-0.5
0.5 ~
-1.0
1.0
-1.5
-2.0
1.5
-2.5
2.0
-3.0
~ 3 , 5
n n i l h i l i l l l l 4
0
5
l0
I l l i [ l l
Ilil~
[ l l l i i l n n l l l l ' i b i i l i l l t
k l l l l l l l l
_ 2 , 5
n[I
0
15 20 25 30 35 40 45 50 55 60
nil
5
e n [ l l n
10
NPI
hit
15
[il
20
I n ~ ' i l i l l l
25
30
b l i l ~
J l i l l
35
40
I l l l
li[
45
50
Days
Fig. 2. Survival poliovirus l (O), enteric adenovirus 40 (*), and enteric adenovirus 41 ([B).
aquatic environment, as indigenous D N A a s e s require
cofactors to be active, and are denatured by temperature more readily than RNAases. It could be speculated then, that the longer survival o f the enteric
adenoviruses in tap and sea water might have been
associated to D N A damage that could have been
repaired by the host cell, while the faster inactivation
in sewage might have resulted from protein capsid
damage, rendering the virions unable to enter the
host cell. This hypothesis could be partially tested by
conducting inactivation experiments as described, but
assaying the samples on both normal cells, and cells
lacking the ability to repair D N A .
From the results obtained in this study, it can be
concluded that the enteric adenoviruses are more
stable in water than polio 1. As this agent has been
used extensively as a model for viral inactivation in
water (Sobsey et al., 1986), its use to assess enteric
adenovirus survival in water, therefore, would not be
appropriate. The greater stability o f the enteric adenovirus, in tap water, in comparison to H A V is o f
concern, as the waterborne transmission o f this agent
has been d o c u m e n t e d (Sobsey et al., 1993). In order
to assess the public health risk associated with the
exposure to enteric adenoviruses in water, it would be
necessary to know the occurrence of these agents in
the aquatic environment. Unfortunately, this data is
not available. Therefore, future research should be
implemented to determine the occurrence o f enteric
adenoviruses in water.
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Bagdasar G. A. and Abieva R. M. (1971) Survival of
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Bernstein C. and Bernstein H. (1991) Aging, Sex, and DNA
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Callahan K. M., Taylor D. J. and Sobsey M. D. (1994)
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