Presented at the WISA 2000 Biennial Conference, Sun City, South Africa, 28 May to 1 June 2000

VIRUSES IN DRINKING WATER

W O K Grabow, M B Taylor, C G Clay and J C de Villiers
Department of Medical Virology, University of Pretoria
Abstract
Viral infections have a long history of association with drinking water supplies. Evidence of water-
borne transmission is predominantly based on epidemiological data. Water-borne transmission has
only in exceptional cases been confirmed by direct detection of viruses in drinking water supplies. This
is because the majority of viruses typically transmitted by water (enteric viruses) are not detectable by
conventional methods. However, molecular techniques based on the reverse transcriptase-polymerase
chain reaction (RT-PCR) available now, have made it possible to detect low levels of a wide variety
of enteric viruses. This preliminary study deals with 411 analyses of drinking water supplies carried out
over a period of two years. The drinking water supplies were derived from acceptable quality surface
water sources using generally accepted treatment and disinfection processes. Glass wool filters were
used for the on-site and in-line recovery of viruses from 100 to 1000 litre volumes of water. Viruses
eluted from the glass wool were inoculated onto combinations of cell cultures including the BGM
monkey kidney, PLC/PRF/5 human liver and CaCo-2 human colon carcinoma cell lines, as well as
primary vervet monkey kidney cells. The purpose of cell culture inoculation was to isolate
cytopathogenic viruses, to amplify the nucleic acid of non-cytopathogenic viruses, and to confirm
viability of viruses. After two passages cell cultures were homogenised and analysed by RT-PCR for
a variety of enteric viruses. Positive results were recorded for 24 % of the samples. Enteroviruses
were detected in 17 % of the samples, adenoviruses in 4 % and hepatitis A virus in 3 %. None of the
viruses were cytopathogenic, which implies that they would not be detected by conventional cell culture
propagation techniques. These findings are in agreement with reports on the detection of viruses in
drinking-water supplies in other parts of the world. The results also support epidemiological studies
which indicate low level transmission of viral infections by drinking water supplies which have been
treated and disinfected by acceptable procedures and meet quality specifications for indicators such as
coliform bacteria. All the water supplies analysed in this study had heterotrophic plate counts of less
than 100/1 ml, total and faecal coliform counts of 0/100 ml, and negative results in qualitative presence-
absence tests for somatic and F-RNA coliphages on 500 ml samples. These findings support earlier
evidence on shortcomings of conventional indicators for assessment of the virological quality of drinking
water. The results have various implications. For instance, the water supplies analysed here, and
probably many others, would fail national and international quality guidelines which state that drinking
water should be free of viruses. However, since these guidelines are based on outdated viral detection
methods, they may be considered due for revision to accommodate new molecular technology. This
may not be an easy decision because the viruses detected by the technology described here are at least
potentially infectious and may be regarded to constitute a health risk. Retention of the existing
guidelines would have major cost and technical implications for the water industry. The results of this
investigation underline the need for more detailed studies.
Introduction
Drinking water supplies have a long history of association with a wide spectrum of viral infections.
Evidence of water-borne transmission is predominantly based on epidemiological data. Water-borne
transmission has only in exceptional cases been confirmed by direct detection of viruses in drinking
water supplies. This is because the majority of viruses typically transmitted by water (enteric viruses)
are not detectable by conventional methods. However, molecular techniques based on the reverse
transcriptase-polymerase chain reaction (RT-PCR) available now, have made it possible to detect low
levels of a wide variety of enteric viruses (Grabow, 1996).
Materials and Methods
A total of 411 samples of drinking water collected at regular intervals over a period of two years were
analysed. The supplies were derived from acceptable quality surface water sources using treatment
processes which conform to international specifications for the production of safe drinking water.
Disinfection was by chlorination based on corresponding specifications for concentration levels and
conditions of application. Glass wool filters were used for the on-site and in-line recovery of viruses
from 100 to 1000 litre volumes of water (Vilagins et al. 1997). Viruses eluted from the glass wool
were inoculated onto combinations of cell cultures including the BGM monkey kidney, PLC/PRF/5
human liver and CaCo-2 human colon carcinoma cell lines, as well as primary vervet monkey kidney
cells. The purpose of cell culture inoculation was to isolate cytopathogenic viruses, to amplify the
nucleic acid of non-cytopathogenic viruses, and to confirm viability of viruses. After two passages cell
cultures were homogenised and analysed by RT-PCR for a variety of enteric viruses (Grabow et al
1999). Corresponding samples of water were analysed for microbial indicators of water quality using
internationally accepted techniques and principles (Grabow, 1996).
Results
Viruses were detected in 24 % of the samples. Entero viruses were detected in 17 %, adeno viruses
in 4 % and hepatitis A virus in 3 %. None of the viruses were cytopathogenic, which implies that they
would not be detected by conventional cell culture propagation techniques. All the water supplies had
heterotrophic plate counts of less than 100/1 ml, total and faecal coliform counts of 0/100 ml, and
negative results in qualitative presence-absence tests for somatic and F-RNA coliphages on 500 ml
samples.
Discussion
The findings are in agreement with reports on the detection of viruses in drinking water supplies
elsewhere in the world (Payment et al 1997, Grabow 1996, Rose et al. 1986). The results also
support epidemiological data which indicate low level transmission of viruses by drinking water supplies
which have been treated and disinfected by standard procedures and meet quality specifications for
indicator bacteria. The results for heterotrophic plate counts, coliform bacteria and coliphages are in
agreement with earlier evidence on shortcomings of conventional indicators for assessment of the
virological quality of drinking water (Grabow, 1996).
Implications of the results include:
1. The water supplies analysed here, and probably many others likewise derived from acceptable
sources using standard procedures of treatment and disinfection, would fail quality guidelines which
specify the absence of viruses from drinking water (WHO 1997, 1996, EPA 1989, EEC 1980).
However, since these guidelines are based on outdated viral detection methods, they may be
considered due for revision to accommodate new technology for the detection of viruses. Decisions in
this regard would have to take into consideration that the viruses detected by the technology used here
are at least potentially infectious and may constitute a health risk. Retention of the existing guidelines
would require substantial improvement in treatment and disinfection to eliminate viruses detectable by
molecular techniques.
2. One option to accomplish existing goals for the absence of viruses from drinking water would be to
intensify chlorination, which may escalate potential risks of health effects associated with disinfection
by-products.
3. The failure of conventional indicator tests to prove the absence of viruses from drinking water
supplies has implications for water quality monitoring procedures. The failure of even sensitive tests for
somatic and F-RNA coliphages in 500 ml samples to reflect the presences of viruses, suggests that the
shortfall of indicator procedures may be substantial. This suggests the sensitivity of microbial indicator
tests may have to be upgraded significantly to reflect the virological safety of drinking water supplies.
4. Improving the efficiency of water treatment and disinfection processes to eliminate viruses
detectable by molecular techniques, and increasing the sensitivity of microbial indicator tests to reflect
the absence of viruses detectable by these techniques, are likely to have major financial implications for
the water industry.
References
EEC (1980) Council Directive of 15 July 1980 relating to the quality of water intended for human
consumption (80/778/.EEC). Official J European Commun No L229:11-22.
EPA (1989) Maximum contaminant level goals for microbiological contaminants. Federal Register,
Part II, Environmental Protection Agency, Part 141.52, p 27527.
Grabow W O K (1996) Waterborne diseases: Update on water quality assessment and control.
Water SA 22:193-202.
Grabow W O K , Botma K L, de Villiers J C, et al. (1999) An assessment of cell culture and PCR
procedures for the detection of polio viruses in waste water. Bull WHO (in press).
Payment P, Siemiatycki J, Richardson L, et al. (1997) A prospective epidemiological study of
gastrointestinal health effects due to the consumption of drinking water. Int J Environ Hlth Res 7:5-31.
Rose JB, Gerba CP, Singh SN, et al. (1986) Isolating viruses from finished water. Res Technol
78:56-61.
Vilagins P, Sarrette B, Champsaur H, et al. (1997) Round robin investigation of glass wool method
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WHO (1996) Guidelines for drinking-water quality. Vol 2: Health criteria and other supporting
information. Second edition. World Health Organization, Geneva, pp 10-99.
WHO (1997) Guidelines for drinking-water quality. Vol 3: Surveillance and control of community
supplies. Second edition. World Health Organization, Geneva. 238 pp.
Prof W O K Grabow
T : (012) 319-2351
F : (012) 325-5550
E : wgrabow@medic.up.ac.za