United States Office of Science EPA/822/R/98/043
Environmental Protection and Technology January 15,1999
Agency Washinnton, DC
&EPA Office of Water
DRINKING WATER CRITERIA DOCUMENT FOR
ENTEROVIRUSES AND HEPATITIS A;
AN ADDENDUM
Prepared for
Health and Ecological Criteria Division
Office of Science and Technology
401 M Street, SW
Washington, DC 20460
Prepared by
NENA NWACHUKU, Ph.D.
Health and Ecological Criteria Division
Office of Science and Technology
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United States Office of Science EPA/822/R/98/043
Environmental Protection and Technology January 15,1999
Agency Washington, DC
&EPA Off ice of Water
DRINKING WATER CRITERIA DOCUMENT FOR
ENTEROVIRUSES AND HEPATITIS A:
AN ADDENDUM
Prepared for
Health and Ecological Criteria Division
Office of Science and Technology
401 M Street, SW
Washington, DC 20460
Prepared by
NENA NWACHUKU, Ph.D.
Health and Ecological Criteria Division
Office of Science and Technology
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TABLE OF CONTENTS
LIST OF TABLES vii
LIST OF FIGURES viii
1.0 Executive Summary 1-1
2.0 General Information and Properties 2-1
2.1 Introduction 2-1
2.2 History and Taxonomy — 2-1
2.2.1 Poliovirus 2-3
2.2.2 Coxsackievirus Group A 2-3
2.2.3 Coxsackievirus Group B 2-5
2.2.4 Echovirus 2-5
2.2.5 Enterovirus Types 68,69,70, and 71 2-5
2.2.6 Hepatitis A Virus 2-6
2.3 Viruses in Water ..: 2-6
2.3.1 Sources of Viruses in Water 2-6
2.3.2 Physical Description of the Viruses in Water 2-7
2.3.3,, Host Range 2-7
2.4 Epidemiology ........... 2-7
2.4.1 Epidcmiological Evidence for Waterborne Transmission of Viruses2-7
2.4.2 Seasonal Distribution of Viruses in Water 2-8
2.5 Waterborne Outbreaks of Viral Diseases in the United States 2-9
2.5.1 Disease Outbreak Surveillance System Criteria 2-9
2.5.2 Outbreak Reports 2-10
2.5.2.1 Etiologic Agent-Associated Outbreaks .............. 2-10
2.5.2.2 Water System-Associated Outbreaks 2-12
2.5.2.3 Water Source-Associated Outbreak 2-13
2.5.2.4 Treatment Deficiency-Associated Outbreaks 2-18
2.5.2.5 Outbreaks Associated With Water and
Etiological Agents 2-18
2.5.2.6 Outbreaks Associated with HAV 2-23
2.5.2.7 Recreational Waters-Associated Outbreaks 2-27
2.5.2.8 Cases of Illness, Hospitalization, and Deaths in
Waterborne Outbreaks .... 2-29
2.5.2.9 Waterborne Outbreaks Worldwide 2-29
2.6 Summary 2-31
3.0 Occurrence in Water 3-1
3.1 Viruses in Environmental Waters 3-1
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5.3.6.2 Seasonality 5-17
5.3.6,3 Immunity 5-17
5.3.6.4 Vaccine 5-17
5.4 Minimal Infective Dose 5-18
5.5 Summary 5-22
6.0 Mechanisms of Disease , 6-1
6.1 Factors That Affect Disease Occurrence 6-1
6.1.1 Virulence 6-1
6.1.2 Susceptibility of Host Cells 6-1
6.1.2.1 Cell Eeceptors . 6-1
6.1.3 Secondary Spread 6-2
6.1.4 Sensitive Subpopulations 6-2
6.2 Chronic Sequelae 6-4
6.3 States of Disease 6-5
6.3.1 Apparent Infections 6-6
6.3.2 Inapparent Infections 6-6
6.4 Host Defense Systems 6-6
6.4.1 Antibodies 6-7
6.4.2 Cell-Mediated Immunity 6-8
6.4.3 Nonspecific Factors in Immunity 6-9
6.5 Summary 6-9
7.0 Risk Assessment 7-1
7.1 Introduction 7-1
7.2 NAS Risk Assessment Framework Document 7-1
7.3 Ecological Risk Assessment Framework 7-1
7.4 Microbial Risk Assessment Framework 7-2
7.5 Transmission of Viruses by Drinking Water 7-7
7.5.1 Endpoints 7-8
7.5.2 Epidemiological Evidence for Viral Transmission in Water 7-8
8.0 Methodology 8-1
8.1 Introduction 8-1
8.1.1 Virus Concentration and Recovery 8-1
8.2 Detection Methods for Viruses in Water .... ... 8-5
8.2.1 Cell Culture Assays 8-5
8.3 Molecular Methods 8-7
8.3.1 PCR Assays 8-7
8.3.2 PCRMethod Studies 8-8
8.4 Immunoassay Technique 8-13
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LIST OF TABLES
Table 2-1 Characteristics of Enteroviruses and Hepatitis A Virus 2-4
Table 2-2a Etiology of Waterborne Outbreaks in System Categorized by
Source of Water, 1971-1996 „ 2-17
Table 2-2b Etiology of Waterborne Outbreaks by System Type, 1971-1996 2-17
Table 2-3a Causes of Waterborne Outbreaks in Drinking Water Systems
by Source Type, 1971-1996 ,. 2-26
Table 2-3b Causes of Waterborne Outbreaks in Drinking Water Systems
by System Type, 1971-1996 2-26
Table 2-4 Outbreaks Associated with Water Intended for Drinking in the United
States in 1989-1990 by Etiologic Agent and Type of Water System 2-28
Table 2-5a Cases of Illness, Hospitalizations, and Deaths in Waterborne Outbreaks
in Water Systems Using Surface and Groundwater Sources, 1971-1996 . 2-30
Table 2-5b Cases of Illness, Hospitalizations, and Deaths in Waterborne Outbreaks
in Water Systems by System Type, 1971-1996 2-30
Table 5-1 Coxsackievirus Group A-Associated Diseases 5-7
Table 5-2 Coxsackievirus Group B-Associated Diseases 5-9
Table 5-3 Echovirus-Associated Diseases 5-11
Table 5-4 Enterovirus Type 68-, 69-, 70-, and 71-Associated Diseases 5-13
Table 5-5 Infectious Doses of Enteric Pathogens in Normal Hosts 5-21
Table 6-1 Sensitive Subpopulations in the United States 6-3
Table 8-1 Methods for Concentrating Viruses from Water 8-2
Table 8-2 Commonly Used Cell Cultures for Propagating Human Enteric Viruses .. 8-6
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1.0 Executive Summary
The Office of Science and Technology (OST) has prepared and revised the Drinking
Water Criteria documents that will support the Office of Water's Ground Water Rule (GWR) and
Surface Water Treatment Rule (SWTR). Waterborne pathogenic enteric viruses are among the
microorganisms to be regulated by these rules. The SWTR requires water systems that use
surface or ground water that is under the direct influence of surface water to (a) disinfect their
water and (b) filter their water or meet criteria for avoiding filtration. Under this rule viruses
must be removed or inactivated at a-99.99% (4 logs) level by meeting the residual concentration
and disinfectant contact time values in the rule.
Four of the enteric viruses, namely, coxsackievirus, echoviras, calicivirus, and
adenovirus, have also been included among the microorganisms of concern on the Environmental
Protection Agency (EPA) Drinking Water Contaminant Candidate List (CCL), The Safe
Drinking Water Act (SDWA) amendments of 1996 require EPA to publish a list of contaminants,
which at the time of publication are not subject to any proposed or promulgated national primary
drinking water regulation (NPDWR), that are known or anticipated to occur in public water
systems and which may require regulations under the SDWA [section 1412(b)(l)],
The enteric viruses are viruses that multiply in the gastrointestinal (GI) tract of man.
These viruses have been shown to cause a variety of diseases in humans, ranging from
poliomyelitis,, to heart disease, encephalitis, aseptic meningitis, hepatitis, hand-foot-and-mouth
disease (HFMD), gastroenteritis, and diabetes mellitis. Enteric viruses are excreted in the feces
of infected humans in numbers as high as 106— 1012/gram of feces.
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A drinking water draft criteria document on enteric viruses was originally developed in
1985. The document now has a 15-year gap in information on the current scientific knowledge
concerning waterborne pathogenic viruses. An updated virus criteria document is essential for
the preparation of EPA's notice of availability to the stakeholders, states, and the general public,
since this document will support the GWR and SWTR mentioned above.
Two drinking water criteria documents for viruses (EPA/822/R/9 8/042;
EPA/822/R/98/043) have been developed by EPA to update information in the original criteria
document. These documents contain new and updated information on various aspects of our
current knowledge of waterborne enteric viruses, including their occurrence in source waters and
sewage, outbreaks, health effects, minimum infectious dose, risk assessment, recovery and
detection methods, and treatment control. The first of these documents (EPA/822/R/98/043)
addresses the enteroviruses including: poliovirus, coxsackievirm group A, coxsackievirus
group B, echovirus, enterovirus types 68, 69, 70, 71, and hepatitis A (formerly enterovirus type
72), which recently has been transferred to a newly created genus, called Hepatovirus. The
second virus document (EPA/822/R/98/042) addresses eight other waterborne enteric viruses:
adenovirus, astrovirus, reovirus, rotavirus, calicivirus, including Norwalk virus, small round
structured viruses (SRSVs), and hepatitis E virus.
The present document (EPA/822/R/98/043) addresses enteroviruses and hepatitis A and
has been organized in 11 chapters. The table of contents outline from the 1985 document was
followed for ease of cross-reference, although a few redundant topics were eliminated. A new
chapter on water treatment has been added. The reader should note the difference between two
terms, enterovirus and enteric virus, used throughout this document. The terms are not
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interchangeable, i.e., all enteric viruses are not enteroviruses. An enteric virus is defined,
functionally, as a virus that multiplies in the GI tract of humans. All of the 12 waterbome viruses
which are the subject of the two drinking water criteria documents are enteric viruses. An
enterovirus belongs to a subgroup of enteric viruses in the genus Enterovirus that share similar
morphological and genetic properties.
All of the enteroviruses, along with hepatitis A viruses, are shed in human feces and
therefore occur in domestic sewage. There are numerous reports of their occurrence in both
waste water and waste water-contaminated surface water. Outbreaks and epidemics have been
associated with the presence of enteroviras in water with serious worldwide consequences. Both
surface and ground water contamination have been linked to many of these outbreaks involving
gastroenteritis and other illnesses. Reports indicate that most of the reported waterborne
outbreaks have been associated with ground water even though this source had been believed to
be relatively free from contamination due to natural filtration by soil layers, which act as barriers
to microbial pollutants. Virus migration has been demonstrated in the soil subsurface for
distances of 1,000 m or more, facilitating virus contamination of aquifers that provide drinking
water to the public. EPA studies, as well as several others, indicate that a significant number of
ground water sources show evidence of fecal contamination. This is the principal rationale for
the requirement for ground water disinfection under the GWR.
The discussion on outbreaks addresses those occurring primarily in the United States.
Many of these outbreaks have been shown to be associated with waterborne transmission.
Waterborne disease outbreaks in the United States associated with treatment deficiencies in water
supply have also been reported. When such deficiencies lead to EPA "boil water" advisories for
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sensitive subpopulations, as happened in the Washington, DC, area recently, consumer
confidence in our water supply can be eroded, thereby increasing the number of consumers who
turn to bottled water as a drinking water source, even after the treatment deficiencies are
corrected.
There is a worldwide distribution of waterborne disease outbreaks. Some devastating
outbreaks occurring outside the United States, and outbreaks in countries with treatment systems
similar to those of the United States, are also discussed. Outbreak reports are not comparable as
there were numerous reports retrieved for this document concerning waterborne outbreaks
occurring in developing countries having insufficient or no treatment control systems. It has
been estimated that the occurrence of enteric viruses in sewage in developing countries may
average 100 to 1,000 times higher than levels seen in the United States. This document therefore
notes only a few of the outbreaks from developing countries, but discusses the health effects
known to occur worldwide regardless of treatment control systems.
It is important, however, that we remain cognizant of the fact that outbreaks outside of
the United States can have worldwide implications, particularly in light of increased global
cooperation and interactions. International travel is increasing, and it is conceivable that viruses
can be exported rapidly across country borderlines by infected travelers. In addition, the
escalating influx of immigrants from developing and war-ravaged countries having inadequate
treatment systems is an important factor in the spread of imported waterborne viral diseases.
With the United States the only superpower remaining in the world, American troops are being
sent on peacekeeping missions around the world. A global partnership and collaboration with
developing countries regarding waterborne outbreaks is needed to rapidly identify emerging or
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reemerging strains of infectious pathogens that could pose a threat both to the United States and
to the world at large.
The problem of waterborne diseases continues to be exacerbated by the high percentage
of acute gastrointestinal illness (AGI) of unknown etiology. It is of significant concern that close
to 50% of all waterborne disease outbreaks in the United States are due to AGI caused by
unknown agents. Given isolation method limitations, it is reasonable to speculate that some of
the AGI of unknown origin may very well be due to viruses. There is a speculation that the
unknown etiological agents may be of viral origin, because the disease patterns support this
speculation. But the evidence for this is inconclusive. Technological methods for bacteria are
well established, and bacteria are well known and can be easily detected. The detection of
viruses, on the other hand, is difficult and complex.
The U.S. Centers for Disease Control and Prevention (CDC) indicates that the number of
reported waterborne disease outbreaks represents only a fraction of the total number. It is not
surprising that waterborne disease outbreaks are grossly under-reported, especially when one
examines the CDC criteria for an outbreak. In order to be recorded an outbreak, two or more
persons must experience a similar illness after the consumption of or use of water intended for
drinking. Epidemiological evidence must implicate water as the source of illness. Factors that
have been listed as contributing to the nonreporting of outbreaks include budget and laboratory
resources, lack of physician interest, and consumer awareness. Another factor to consider is
embarrassment. Many affected people may be unwilling to talk about a little "diarrhea" episode
that may disappear in a few days. Since only two people need manifest symptoms to be
considered an "outbreak," it is likely that embarrassment may account for a significant number of
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cases that go unreported. Therefore, a decrease in reported outbreaks may not be an actual
decrease. A better surveillance system obviously is needed at the local level to accurately track
outbreaks.
The awareness of virus occurrence in water has increased with the improvements in
technology for viral recovery. This in turn has led to greater concerns regarding implications of
virus presence in water. In monitoring waterborne viruses, a major problem has been the
concentration and enumeration of large volumes of virions in raw and finished water. Because of
their small size and low numbers, accurate assessments have been difficult. Detection of viruses
in water sample volumes ranging from a few to 100 liters has remained a major challenge. Vims
recovery methods in existence prior to 1985 include filter adsorption-elution, adsorption to
inorganic precipitates, polyelectrolytes, minerals, clays, glass beads, ultrafiltration,
hydroextraction, and reverse osmosis. Since that time, continuous imrnunomagnetic capture,
continuous flow centrifugation, cross-flow filtration, and vortex flow filtration have emerged as
new technologies for improved virus recovery. The efficiency of these methods, however, varies
from 20% to 80%, even when relatively high concentrations of virions are present in water
samples. With those percentages of variability in recovery, human risk associated with finished
drinking water sources becomes more daunting in light of the fact that infective doses for human
enterovirus infection could be as low as one to four infectious particles.
Selective and sensitive immunological methods for virus detection have emerged
recently, but they are frequently time consuming, require specialized training, and are labor
intensive. Cell culture methods, although available for several decades and a proven way for
determining the infectivity of viral particles, are also slow, require specialized training, and are
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labor intensive. In addition, some waterborne viruses such as coxsackieviruses and Norwalk
vims still cannot be cultivated or grow poorly in cell culture. New cell lines need to be
investigated and developed for noncultivatable viruses.
The greatest improvements in environmental virology during the past 15 years have been
in the development of virus detection methods. Polymerase chain reaction (PCR) reverse
transcriptase (RT) methods in combination with other molecular technologies, however, have
been developed with high specificity and sensitivity, and are proving to be very useful in the
detection of all known pathogenic, waterborne viruses. Previously identified and classified
microorganisms are being reassessed by molecular methods and reclassified into new genera, and
unidentified microorganisms are being identified and classified based on their genomic
sequences. However, the PCR method is very difficult to use with environmental samples
because of inhibitory substances that interfere with the detection of viral nucleic acid, PCR,
unlike the cell culture method, cannot distinguish between infectious and noninfectious particles.
As we approach the next millennium,, a rising world population and its increasing demand
for water have led to greater use of recycled waste water. The use of this resource, which may
contain inactivated viruses, for agricultural purposes and for other human activities, has
increased the risk of viral contamination of drinking water supplies. Enteroviruses have a low
infectivity and it has been shown that 1—4 tissue culture infective doses can infect a person with a
high probability. If this is the case, there is reason for great concern for the hazard posed by the
occurrence of infectious pathogenic virus in drinking water.
The disease states of enterovirus infections are varied. They include poliomyelitis,
infectious hepatitis, aseptic meningitis, heart diseases (pericarditis, myocarditis, myopericarditis,
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cardiomyopathy, ischemic heart disease), hand-foot-and-mouth disease (HFMD), gastroenteritis,
and insulin-dependent diabetes mellitus. It is important to understand the health effects of these
viruses and the resulting implications for public health. Therefore, the health effects chapter of
this document presents as much evidence as is available on the general disease profiles of all the
entero viruses.
The manifestations of disease caused by waterborne viruses reflect the virulence of the
particular pathogenic viral strain and the corresponding susceptibility of the infected host.
Individuals with a depressed immune system, such as immune-suppressed patients (cancer
patients, organ transplant patients, AIDS patients), the elderly, and very young children, are
generally at a higher risk than the normal population to infections and are consequently prone to
more severe attacks and manifest the most severe symptoms. Apparent (showing clinical
symptoms) and inapparent (lacking clinical symptoms) infections by enteric viruses have been
demonstrated, and both must be recognized as asymptomatic individuals may continue to shed
viruses in their feces and consequently infect others. The host defense systems are directly
involved in determining whether the infection becomes clinical or subclinical and whether the
individual may be subject to reinfection.
New approaches to microbial risk assessment by ILSI have been developed within the last
few years that differ significantly from the National Academy of Sciences (NAS) framework for
chemical risk assessment. Differences include pathogen-host interactions, secondary spread of
microorganisms, short-term and long-term immunity, the carrier state, host animal reservoirs,
zoonotic transmission, person-to-person transmission, and conditions that lead to survival, and
multiplication of microorganisms (bacteria) in the environment. Various available risk models
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assume a random distribution of pathogenic microorganisms in water. The risk assessment of
enteric viruses is limited because of lack of information on dose-response, occurrence, and
exposure data. This document identifies a more quantitative risk approach for coxsackievirus
type B4.
There is a question as to whether the standard bacterial indicator of fecal contamination in
drinking water has outgrown its usefulness. This is because there have been numerous instances
in which bacteriological drinking water standards have been met and yet gastroenteritis outbreaks
due to viruses have occurred. The best indicator for the presence of pathogenic microorganisms
is the pathogenic microorganism itself. However, testing for every pathogenic microorganism of
concern is not feasible because pathogenic human viruses are not always easy to detect, and
methods for their detection may be expensive and require specialized equipment and skilled
technicians. Over the years, various alternative indicators have been proposed, such as
bacteriophages, heterotrophic bacteria, Clostridium, Klebsiella, and Bifldobacteria. There is as
yet no evidence that any one of the alternatives can effectively replace E. coli as the indicator of
human fecal contamination. A surrogate for human viruses has not as yet been identified. The
role that bacteriophages will play as viral indicators in the future is not clear at this time. Various
studies show little correlation between the presence of bacteriophage indicators and the human
viruses of concern. More research is needed to assess indicators for human viruses.
Viruses have been shown to be more resistant to treatment than bacteria. Chlorination
has been the disinfection method of choice in the United States for the past several decades
because of its effectiveness in destroying pathogenic microorganisms, but we now know that not
all waterborne viruses are killed or inactivated by chlorine residuals commonly used for drinking
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water (up to 3.75 mg/L). At the same time, cancer risks associated with disinfectant byproducts
such as trihalomethane have become a public health concern. Lower chlorine levels will
decrease the risk posed by chlorination byproducts, but will increase the risk posed by pathogenic
viruses. Conversely, an increase in chlorine concentration will reduce the risk posed by resistant
pathogenic viruses but will greatly increase the risk posed by cancer-causing disinfectant
byproducts. The question then becomes, Which risk do we trade for the other?
Some of the effective alternative disinfection methods include chloramine, chlorine
dioxide, ozonation, and UV light. Ozonation and UV light do not leave residuals to protect
against recontamination events. However, chlorine continues to be the disinfection of choice in
the United States.
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2,0 General Information and Properties
2.1 Introduction
Viruses are obligate intracellular parasites that cannot replicate outside a host cell.
Enteric viruses, however, have the ability to survive in the environment for extended periods of
time. Of all the classified viruses, over 120 of them including all the enteroviruses and hepatitis
A, multiply in the human GI tract. These enteric viruses are excreted by infected individuals into
domestic sewage (Metcalf et al., 1995). The discharge of treated and untreated sewage into
rivers and streams impacts surface*waters, recreational waters, water intakes, lakes, oxidation
ponds, and even shellfish beds in estuaries. Studies have shown that sewage discharge onto land
can result in virus contamination of ground water. Viruses have been recovered from rivers,
water intakes, and ground water that were miles away from where the initial release into water or
on land had occurred. As a consequence of these discharges, disease outbreaks associated with
viruses occur at frequent intervals. The type and concentration of enteric viruses present in the
sewage are dependent on the community, disease incidence, water treatment, seasonality, and
socioeconomic factors.
2.2 History and Taxonomy
Enteroviruses are classified as a genus within the family Picornaviridae by the
International Committee on the Taxonomy of Viruses (ICTV) (ICTV, 1995). The criteria used
by the ICTV for the official taxonomy of all classified viruses include morphology (shape, size,
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and presence or absence of envelope), nucleic acid type (RNA or DNA), and host range (human,
animal, plant, fish, or bird).
The name enterovirus is derived from "entero" (intestine), the primary site of attack for
these viruses. The enterovirus genus is made up of poliovirus; coxsackievirus group A;
coxsackievirus group B; echovirus; enterovirus types 68, 69, 70, and 71; and several
entero viruses of lower animals, such as pigs, mice, monkeys, and cattle. Over 100 serotypes of
enterovirases have been recognized (Melnick, 1996a).
HAV, provisionally classified as enterovirus type 72, has now been transferred into a
newly created genus called Hepatovirm (ICTV, 1995). The basis for this transfer involves
differences in the amino acid sequence of the protein coat and the increased resistance of
hepatitis A to thermal inactivation (Melnick, 1996a).
The enteroviruses share similar properties. They reside in the same habitat, the intestinal
tract of humans, and are resistant to laboratory disinfectants such as alcohol and phenol. Various
solvents and detergents known to destroy other viruses such as ether and deoxycholate are
ineffective against enteroviruses (Melnick, 1996a).
The antigens of the enteroviruses are used to identify specific serotypes (Melnick, 1996b).
However, Prabhakar et al. (1982) reported that antigenic mutations of enteroviruses are frequent,
and as high as 1 per 10,000 virions. All known enteroviruses are resistant to all known
antibiotics and chemotherapeutic agents (Melnick, 1996a). Enteroviruses are thermolabile and
are rapidly destroyed when exposed to a temperature greater than 50°C. Thermal inactivation of
enteroviruses has been shown to be inhibited by magnesium chloride (Melnick, 1996a,b).
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Polio virus is protected against thermal inactivation in the presence of magnesium chloride, and
the property has been used to stabilize oral poliovirus vaccines (Melnick, 1992).
The morphological characteristics of all the enteroviruses and hepatitis A are similar by
electron microscopy (EM). As a result, an electron micrograph of poliovirus can be used to
represent the morphology of any enterovirus member (Williams, 1998). The characteristics of all
the enteroviruses and hepatitis A are summarized in Table 2-1.
2.2.1 Poliovirus
Poliovirus is the best known and the first recognized member of the enteroviruses. It has
also been one of the most studied enteroviruses in part because it produces poliomyelitis, a
devastating paralytic disease of humans. The history of poliovirus is a long one, and recently was
reviewed chronologically by Melnick (1996a), one of the pioneers in the elucidation of, this virus
since its recovery from New York City sewage in the 1940s (EPA, 1985). The work of Melnick
and Sabin has contributed to our understanding of poliovirus (Melnick, 1996a).
Three serotypes have been recognized, and poliovirus type 1 is the type species for the
enterovirus genus (ICTV, 1995).
2.2.2 Coxsackievirus Group A
Coxsackievirus group A is one of two groups of coxsackieviruses that have been
described. Coxsackievirus group A was first discovered by Dalldorf in 1948, and it derives its
name from Coxsackie, a town in New York, where it was first isolated from a patient (Melnick,
1996a). There are 23 recognized serotypes of Coxsackievirus group A (ICTV, 1995).
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TABLE 2-1
Characteristics of Enteroviruses and Hepatitis A Virus
Enteroviruses
Virus
Number of serotypes
Genome
Size
Capsid
Virion
Buoyant density in CsCl
Morphology
Poliovirus
3
ssRNA
27-30 nm
60 subunit
icosahedron
unenveloped
1.33-1.45
g/cm"3
featureless
Coxsackie
group A
23.
ssRNA
27-30 nm
60 subunit
icosahedron
unenveloped
1.33-1.45
g/cm"3
featureless
Coxsackie
group B
6
ssRNA
27-30 nm
60 subunit
icosahedron
unenveloped
1.33-1.45
g/cm"3
featureless
Echovirus
31
ssRNA
27-30 nm
60 subunit
icosahedron
unenveloped
1.33-1.45
g/cm"3
featureless
Enterovirus
types 68, 69, 70, 71
4
ssRNA
27-30 nm
60 subunit
icosahedron
unenveloped
1.33-1.45
g/cm"3
featureless
Hepatovirus
Hepatitis A
virus
1
ssRNA
27 nm
60 subunit
icosahedron
unenveloped
1.33-1.45
g/cm"3
featureless
Source: Williams, 1998; ICTV, 1995; Melnick, 1992,1985.
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2.2.3 Coxsackievirus Group B
Coxsackievirus group B was discovered by Melnick and was first isolated from a patient
in Connecticut (Melnick, 1996a). It was described in 1949, a year after the discovery of
Coxsackievirus group A. Coxsackievirus group B has six recognized serotypes (ICTV, 1995).
2.2.4 Echoviru_s
Echovirus derives its name from the acronym of its full name, Enteric Cytopathogenic
Human Orphan virus. Thirty-one echovirus serotypes have been described, and they are
numbered sequentially from 1 through 31, Three of the serotypes have been reclassified.
Echovirus type 10 has been reclassified as a reovirus, and type 28 has been reclassified as a
rhinovirus. Echovirus type 34 is reclassified as a variant type of Coxsackievirus A 24 (Melnick,
1996a).
2.2.5 Enterovirus Types 68.69.70. and 71
New members of the Enterovirus genus are no longer subclassified as Coxsackievirus or
echovirus but instead numbered sequentially because of the variability in the biological
properties such as the production of pathological changes in newborn mice. This numerical
numbering system will be retained for these enteroviruses until sufficient and definitive data
become available to place them into an appropriate subgroup (Melnick, 1996a; Kibrick, 1964).
Only one serotype has been recognized for each numbered enteroviras (ICTV, 1995).
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2.2.6 Hepatitis A Virus
HAV was formerly classified as enterovirus type 72 in the genus Enterovirus. It is now
classified in the genus Hepatovirus. HAV shares many properties with all the viruses in the
name from Coxsackie, a town in New York, where it was first isolated from a patient (Melnick,
1996). There are 23 recognized serotypes of coxsaekievirus group A (ICTV, 1995). Enterovirus
genus (see Table 2-1). It is, however, more temperature and acid stable than the enteroviruses.
An HAV particle is 27 nm in diameter, is nonenveloped, and has an icosahedral symmetry. It has
a single-stranded RNA genome that contains 7,500 nucleotides. The KNA strand is positive and
thus serves as its own messenger RJJA (Levinthal and Ray, 1966). Only one serotype has been
described for hepatitis A (ICTV, 1995).
2.3 Viruses in Water
2.3.1 Sources of Viruses in Water
Human enteric viruses are excreted in high numbers (108-1012 particles/g of feces) by
infected individuals and consequently are present in waters contaminated by fecal material
(Abbaszadegan et al., 1998; Abbaszadegan and DeLeon, 1997; Payment, 1993). Treated waste
water effluent from sewage treatment plants contains inactivated as well as infectious viruses that
are discharged into surface water (Tani et al., 1995; Black and Finch, 1993; Bosch et al., 1986;
Dahling and Safferman, 1979). The appearance of viruses in recreational or drinking water has
also been linked with sludge disposal (Rao et al.5 1986). Enteroviruses have been shown to be
associated with solids that aid in the transport of these viruses in ocean sediment and in soils
following land disposal of sludge. These solids- associated enteroviruses can then be dislodged
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from the substrate by rainwater or by water turbulence. Once the viruses are dislodged, the
original aggregate of viral particles can then contaminate drinking water or recreational water
(Raoetal, 1986).
2.3.2 Physical Description of the Viruses in Water
The enteroviruses share physical characteristics; these characteristics have been
summarized in Table 2-1 and also discussed in subsections under each member of the enterovirus
group.
2.3.3 Host Range
Man is the natural host for the human enterovirases and hepatitis A, although some
reports indicate domestic animals such as dogs as well (Grew et al., 1970; Clapper, 1970). In
laboratory studies however, polioviruses can infect monkeys and chimpanzees by the oral,
intraspinal and intracerebral routes. Coxsackievirus group A and group B can infect suckling
mice but will produce different distinctive lesions. Echovirus can infect rhesus monkeys and
newborn mice. HAV can infect chimpanzees and some monkey species (Melnick, 1996a; EPA,
1985).
2.4 Epidemiology
2.4.1 Epidemiological Evidence for Waterborne Transmission of Viruses
According to Every and Dawson (1995), a microorganism has to meet two criteria to be
implicated as the etiological agent. It must be found in significantly higher numbers in sick
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individuals than in normal individuals. The microorganism should also be found in the source
(water), or there should be an appropriately timed event that would allow the agent to bypass the
treatment system.
Numerous reported waterborne outbreaks have been associated with gastroenteritis due to
a viral agent (CDC, 1996a). Contaminated drinking water was implicated as the source of
infection. These outbreaks are discussed in detail in the outbreak section of this chapter. The
specific water systems identified in the reported outbreaks such as community, noncommunity,
and individual systems and water source such as ground water and surface water are also
discussed in detail in the outbreak section.
2-4.2 Seasonal Distribution of Viruses in Water
The prevalence of enteroviruses in the United States is seasonal, occurring in late summer
and fall (Melniek, 1996b). Poliovirus, however, can occur year round, particularly in
communities that have active vaccination programs. The seasonality of HAV has been reported
by Hedberg and Osterholm (1993). A high incidence of HAV has also been reported to occur in
autumn. Coxsackievirus has been reported to be more prevalent in the late summer and fall
(KoganetaL, 1969).
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2.5 Waterborne Outbreaks of Viral Diseases in the United States
2.5.1 Disease Outbreak Surveillance System Criteria
A waterborne disease outbreak as defined by the CDC is an incident in which:
1) Two or more persons experience a similar illness after the consumption of
drinking water or after exposure to water used for recreational purposes.
2) Epidemiologic evidence must implicate water as the probable source of the
illness (CDC, 1996a).
The surveillance system for a waterborne disease outbreak is similar to that of a food-
borne disease outbreak. In both systems, the unit of analysis is an outbreak and not an individual
case of a particular disease as in other systems. Two persons or more must experience an
illness after ingesting drinking water. However, the criterion for two persons is waived for single
cases of laboratory confirmed, primary meningoencephalitis and for single cases of chemical
poisoning if water quality data indicate contamination by the chemical. In addition, when
primary and secondary cases are distinguished in an outbreak report, only the primary cases are
included in the outbreak report form. Outbreaks that are due to contamination of water or ice at
the point of use are not classified as waterborne disease outbreaks (CDC, 1996a).
Waterborne disease outbreak information has been collected since 1920 by the United
States Public Health Service. This responsibility was transferred to CDC in 1966 (Lippy and
Waltrip, 1984). In 1971, EPA and CDC joined in a collaborative effort to improve the reporting
of waterborne illness (CDC, 1996a; Lippy and Waltrip, 1984), It is important to note that the
reporting of a waterborne disease outbreak to the Federal Government is voluntary (Calderon and
Craun, 1998). Since 1971, EPA and CDC have maintained a cooperative effort in the
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surveillance and reporting of waterborne outbreak occurrence and their causes and this
information is made available annually. The health departments of individual states in the
United States are required to report water related disease outbreaks to CDC. In addition, the
Health Effects Research Laboratory of EPA contacts all the state water supply agencies to obtain
information on waterborne disease outbreaks annually. CDC however, indicates that the number
of reported waterborne disease outbreaks represents only a fraction of the total number of
occurrences (CDC, 1996a).
2.5.2 Outbreak Reports ,,
The CDC surveillance reports from the collaborative effort's inception in 1971 to the
present reveal that the highest number of drinking water associated outbreaks in the United States
consistently has been due to AGI of unknown etiological agent. This section of the document
examines the CDC surveillance reports from inception to 1994. Some specific years 1991-1992,
and 1992-1993 are also examined. The outbreak associations are considered separately by
etiologic agent, water system, water supply, and type of deficiency. The most recent surveillance
report for 1995-1996 has been included. Cases of illness, hospitalization, and death from
outbreaks for all etiological agents are also presented.
2.5.2.1 Etiologic Agent-Associated Outbreaks
The CDC national surveillance data (1996a) reveal that for a period spanning 24 years,
from 1971 to 1994, the highest number of waterborne disease outbreaks associated with drinking
water was due to AGI of unknown etiology (see Figure 2-1). Only in about 50% of the outbreaks
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Figure 2-1.
Waterborne-disease outbreaks associated with drinking water,
by year and etiologic agent, United States, 1971-1994
1971 1973 1975 1977 1979 1981 1983 1985 1987 1989 1991 1993
*acute gastrointestinal illness of unknown etiology.
Adapted from: CDC, 1996a.
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was the etiological agent identified. The 24-year profile also shows that the highest AGI disease
peak (67%) was reported in 1981 and the second highest (40%) in 1983. The agent that caused
the second highest number of disease outbreaks was parasitic. Bacterial agents came third. Viral
agents only ranked higher than chemical agents out of the five etiological agents associated with
waterborne outbreaks in drinking water from 1971 to 1994.
All the etiological agents, viral, bacterial, parasitic, chemical, and AGI, showed a
consistent outbreak peak pattern for the years 1981,1983, and 1994 (CDC, 1996a). It was not
possible to ascertain what was happening in the years of the peaks from the data examined to
cause the across the board peaks throughout the nation. Figure 2-1 is a representation of reported
waterborne disease outbreaks associated with drinking water for these 24 years and the
responsible etiologic agents (parasitic, bacterial, viral, chemical, and unknown).
2.5.2.2 Water System-Associated Outbreaks
Water systems are classified by EPA as community, noncommunity, and individual. The
community water systems are the public municipal systems and can be owned by investors. They
serve an estimate of 180 million people in large or small communities or subdivisions. The
noncommunity systems are semipublic, such as the systems for institutions, industries, hotels,
camps, and parks, and they serve 20 million people. The individual systems are private, and they
are wells and springs used by one to several residences (Highsmith and Crow, 1992).
The CDC national surveillance data from 1971 to 1994 (CDC, 1996a) also indicates that,
the water system responsible for the highest number of waterborne disease outbreaks associated
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with drinking water was the individual system followed by the noncommunity systems. Figure
2-2 represents water system-associated outbreaks during that period by water system,
Outbreak reports can be variable. An examination of outbreak cases reported for specific
years indicate that for the year 1993-1994 (Figure 2-3), the highest number (46.7%) of
waterborne disease outbreaks associated with drinking water were associated with community
water systems. The noncommunity system was reported to be responsible for the second highest
(30%) outbreak incidence. The individual system ranked third (23.3%) (CDC, 1996a). Table 2-
2b presents more recent data (1971-1996) on etiology of waterborne outbreaks by system type.
2.5.2.3 Water Source-Associated Outbreak
An evaluation of the surveillance data by water source (Figure 2-4) indicates that for the
year 1993-1994, wells (ground water) were associated with more waterborne disease outbreaks
(66.7%) than surface water (23.3%) and other sources (10%) (CDC, 1996a). The most recent
surveillance report (1995-1996) confirms this trend. Groundwater was shown to be associated
with 50.5% outbreaks while surface water was responsible for 40.4% (CDC, 1998). This is a
clear evidence that ground water systems are not protected from contamination and that the
barriers that may be created by surrounding subsurface layers are not sufficient to prevent
microbial contamination. The source water for the highest waterborne outbreaks from 1971-
1996 was ground water (CDC, 1998). See Table 2-2a.
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Figure 2-2.
Waterborne-disease outbreaks associated with drinking water,
by year and type of water system, United States, 1971-1994
70
60
in
_*:
1
.Q
•«—«
ZJ
o
M—
o
i_
CD
JD
E
3
z
^individual
ESnoneommunity
Hlcommunitv
1971 1973 1975 1977 1979 1981 1983 1985 1987 1989 1991 1993
Adapted from: CDC, 1996a.
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Figure 2-3.
Waterborne-disease outbreaks associated with drinking water,
by water system, United States, 1993-1994
46.7%
23.3%
30.0%
community
individual
noncommunity
Adapted from: CDC, 1996a.
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Figure 2-4.
Waterborne-disease outbreaks associated with drinking water,
by source, United States, 1993-1994
66.7%
23.3%
10.0%
Adapted from: CDC, 1996a.
well
other
surface
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TABLE 2~2a
Etiology of Waterborne Outbreaks in System Categorized by
Source of Water, 1971-1996
Etiology
Bacterial
Chemical
Parasitic
Unknown
Viral
Total
Ground
66 (16%)
32 (8%)
32 (8%)
232 (58%)
39 (10%)
401 (100%)
Surface
9 (6%)
15 (9%)
82 (51%)
50(31%)
6 (4%)
162(100%)
Unknown
11(10%)
28 (24%)
15(13%)
56 (49%)
5 (4%)
115(100%L
Total
86 (13%)
75(11%)
129 (19%)
338 (50%)
50 (7%)
678
Source: Calderon and Craun, 1998.
TABLE 2-2b
Etiology of Waterborne Outbreaks by System Type, 1971-1996
Etiology
Bacterial
Chemical
Parasitic
Unknown
Viral
Total
Community
35 (12%)
48 (17%)
91 (32%)
91 (32%)
19(7%)
284 (100%)
Noneommunity^
38 (12%)
9 (3%)
28 (9%)
218(69%)
22 (7%)
315(100%)
Individual
13 (16%)
18(23%)
10(13%)
29 (37%)
9(11%)
79(100%!
Source: Calderon and Craun, 1998.
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2,5,2.4 Treatment Deficiency-Associated Outbreaks
Ground water is generally considered to be protected from contamination because of the
natural filtration of the soil that acts as barrier to pathogenic microorganisms. However, ground
water has been associated with water system deficiencies that led to the highest number (43.2%)
of disease outbreaks in the United States between 1920 and 1983 (Geldreich, 1989). In
1991-1992, the highest number of outbreaks (50%) was caused by drinking water treatment
deficiency, by untreated ground water (29%), by distribution system problems (15%), and by
unknown causes (6%) (Figure 2-5). The CDC surveillance report for 1993-1994 also shows that
the highest number (36.7%) of waterborne disease outbreaks associated with drinking water were
caused by untreated ground water (Figure 2-6). The most current CDC information indicates that
for 1995-1996, treatment deficiency caused more outbreaks (36%) than distribution (32%) (see
Figure 2-10).
2.5.2.5 Outbreaks Associated with Water and Etiological Agents
The outbreaks for 1991-1992 were evaluated by etiological agent, water system water
source, water supply, and deficiency. The CDC surveillance data showed that the highest
number (53.4%) of waterborne disease outbreaks associated with drinking water for the year
1991-1992 were due to unknown etiological agents (Figure 2-7). Viral agent was responsible for
only 11.9%, E. coll 0157:H7 was responsible for 4%, and Giardia, 26.7%.
In 1991-1992, by water source, the highest percentage of outbreaks (50%) associated
with drinking water source was due to wells (ground water). Surface water was responsible for
38%, while spring water as a source caused 12% of the outbreaks (Figure 2-8). The same
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Figure 2-5.
Waterborne-disease associated with drinking water, by deficiency,
United States, 1991-1992 (N=34)
6.0%
29.0%
^ 15.0%
50.0%
untreated groundwater
unknown
treatment deficiency
distribution system
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Figure 2-6.
Waterborne-disease outbreaks associated with drinking water,
by deficiency, United States, 1993-1994
26.7% h
23.3%
13.3%
36.7%
Adapted from: CDC, 1993: 42(ss-5): 1-22.
untreated groundwater
unknown
treatment deficiency
distribution system
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Figure 2-7.
Waterborne-disease outbreaks associated with drinking water,
by etiologic agent, United States, 1991-1992 (N=34)
11.9%
4.0%
26,7%
4.0%
Eco!iO157:H7
Giardia
AGI
CLB
viral
AGI = acute gastrointestinal illness
CLB = cyanobacteria (blue-green algae)-like bodies
Adapted from: CDC, 1993: 42(ss-5): 1-22.
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Figure 2-8.
Waterborne-disease outbreaks associated with drinking water,
by water source, United States, 1991-1992 (N=34)
38.0%
12.0%
50.0%
well
spring
surface water
Adapted from: CDC, 1993: 42(ss-5): 1-22,
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trend was observed in 1995-1996, when ground water was associated with 50.5%, while surface
water was responsible for 40.4% (see Figure 2-10).
In 1991-1992, by water supply, the community water supply caused almost as many
outbreaks (42%) as noncommunity supply (46%) while individual supply was responsible for
12% of the outbreaks (CDC, 1993) (Figure 2-9). A similar trend was observed for 1995-1996,
when the community water supply caused the same number of outbreaks as the noncommunity
water supply (see Figure 2-10).
The causes of outbreaks in drinking water systems for 1971-1996 are given for water
source type in Table 2-3a and for w^ater system type in Table 2-3b.
The outbreaks for 1995-1996 were also evaluated by etiological agent, deficiency, water
supply, and water system water source (Figure 2-10), These data indicate that by etiological
agent, viruses were responsible for 9%, chemical 32%, bacterial 18%, unknown 32%, and
parasitic 9%. By deficiency, outbreaks were caused as follows: treatment 36%, distribution
32%, untreated source 23%, and unknown 9%. By water supply, outbreaks occurred at the same
rate in noncommunity and community water systems (45.5%), while 9.1% of outbreaks occurred
in individual systems. By water system water source, 50.5% of outbreaks occurred from ground
water, 40.4% from surface water, and 9.1% from other sources.
2.5.2.6 Outbreaks Associated with HAV
Of all the viruses that are the subject of this document, HAV is the virus most often
associated with outbreaks. It has been associated with 60 outbreaks from 1946 to 1980
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Figure 2-9.
Waterborne-disease outbreaks associated with drinking water,
by water supply, United States, 1991-1992 (N=34)
42.0%
12.0%
46.0%
community
individual
noncommunity
Adapted from: CDC, 1993: 42(ss-5):l-22.
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Figure 2-10.
Waterborne-disease outbreaks, United States, 1995-1996 (N=22)
Etiology
Viral 9.0%
Bacterial 18.0%
Unknown 32.0%
Chemical 32.0%
Parasitic 9.0%
Deficiency
Treatment 36.0%
Untreated Source 23.0%
Unknown 9.0%
Water Supply
Noncommunity 45.5%
Individual 9.1%
Community 45.5%
Water Source
Surface 40.4%
Groundwater 50.5%
Other 9.1%
Distribution 32.0%
Source: CDC, 1998 (In press).
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TABLE 2-3a
Causes of Waterborne Outbreaks in Drinking Water Systems by
Source Type, 1971-1996
Cause
Untreated Source
Treatment Deficiency
Distribution
Miscellaneous
Total
Ground
199 (50%)
143 (36%)
43(11%)
16 (5%)
401 (100%)
Surface
30 (19%)
102 (63%)
19 (12%)
11(7%)
162 (100%)
Unknown
18 (16%)
22 (19%)
52 (45%)
23 (20%)
115(100%)
Total
247 (36%)
267 (39%)
114(17%)
50 (7%)
678
Source: Calderon and Craun, 1998,
TABLE 2-3b
Causes of Waterborne Outbreaks in Drinking Water Systems by
System Type, 1971-1996
Cause
Untreated Source
Treatment Deficiency
Distribution
Miscellaneous
Total
Community
39 (14%)
143 (50%)
84 (30%)
18(6%)
284 (100%)
Noncommunity
147 (470/0)
122 (39%)
23 (7%)
23 (7%)
315(100%)
Individual
61 (77%)
2 (3%)
7 (9%)
9(11%)
79(100%)
Total
247 (36%)
267 (39%)
114(17%)
50 (7%)
678
Source: Calderon and Craun, 1998.
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(Highsmith and Crow, 1992). The 1989-1990 U.S. outbreak report indicates that a total of 26
outbreaks were reported that led to 4,288 eases. Table 2-4 shows that of the 4288 outbreak cases
reported in 1989-1990, HAV was responsible for 25 (Herwaldt et al., 1992). A total of 26
outbreaks were reported that led to 4288 cases. The etiological agent in 2402 cases was not
identified. However, hepatitis A was identified as the etiological agent in 25 cases (Herwaldt et
al., 1992).
2.5.2.7 Recreational Waters-Associated Outbreaks
Disease outbreaks associated with recreational waters meet the same criteria used for
waterbome outbreaks associated with drinking water. However, recreational water-associated
outbreaks involve exposure to or unintentional ingestion of fresh or marine water (CDC, 1990).
Most of the reported waterbome outbreaks generally focus on drinking water. But there are also
outbreaks of gastroenteritis associated with exposure to recreational waters (CDC, 1998). In
1993-1994,26 outbreaks were associated with recreational activities. Of these, 14 (71.4%)
resulted in gastroenteritis and were reported to be due to protozoan parasites such as
Cryptosporidium. In fact, 4 of the 14 outbreaks associated with recreational waters were due to
Cryptosporidium and were identified in Wisconsin after the famous Milwaukee outbreak of
1993. In addition, 11 of the 26 outbreaks resulted in dermatitis and 8 occurred in cold months
(November to March) and were associated with swimming pools, hot tubs, and whirlpools. One
of the outbreaks resulted in meningoencephalitis in a child and resulted in death. A 1992 report
identified six outbreaks due to recreational activities which had a similar pattern (CDC, 1996a),
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TABLE 2-4
Outbreaks Associated with Water Intended for Drinking in the United States
in 1989-1990 by Etiologic Agent and Type of Water System
Etiologic
agent
AGP
Giardia
Hepatitis A
virus
Norwalk-like
E,coliQ157:H7
CLB (possible)11
Total
Percentage0
Type of water system
Community
Out-
break
4
4
1
0
1
1
11
42
Cases
894
503
"3
0
243
21
1664
39
Noneommunity
Out-
break
8
3
0
1
0
0
12
46
Cases
1402
194
0
900
0
0
2496
58
Individual
Out-
break
2
0
1
0
0
0
3
12
Cases
106
0
22
0
0
0
128
3
Total
Out-
break
14
7
2
1
1
1
26
100
Cases
2402
697
25
900
243
21
4288
100
aAGI: acute gastrointestinal illness of unknown etiology.
bCLB: cyanobacteria-Iike bodies
The percentage of 26 outbreaks, or of 4288 cases.
Source: Herwaldt et al. (1992).
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Recreational water outbreaks due to viruses including coxsackievirus and adenovirus have also
been reported by Dufour (1986).
2.5.2.8 Cases of Illness, Hospitalization. and Deaths in Waterborne Outbreaks
The cases of illness, hospitalization, and deaths associated with waterbome outbreaks in
the United States from 1971 to 1996 have been reported by Calderon and Craun (1998). These
cases are summarized in Tables 2-5a and 2-5b. Ground water was shown to be associated with
the highest outbreaks (59%) and the highest deaths (95%), Surface water, however, was
responsible for the highest cases of, illness (83%) and hospitalization (82%).
In water systems, community systems were associated with highest cases of illness (91%)
and hospitalization (87%). The community system caused almost as much outbreak (42%) as did
the noncommunity system (46%). These data are summarized in Table 2-5b.
2.5.2.9 Waterborne Outbreaks Worldwide
Outbreaks of viral diseases due to enteroviruses and hepatitis A have been reported
worldwide. Many of the outbreaks occurred in countries with insufficient water treatment but
some of them were reported in countries with treatment technologies similar to those in the
United States. Only a few of these are discussed below.
A devastating enterovirus outbreak in Taiwan, Republic of China, was recently reported
beginning in April 1998 by CDC and the Taiwan government (CDC, 1998a). To date, over 69
fatalities have been reported. The population mostly affected were infants and children who
contracted HFMD, meningitis, and encephalitis. Investigators reported the isolation of
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TABLE 2-5a
Cases of Illness, Hospitalizations, and Deaths in Waterborne Outbreaks* in
Water Systems Using Surface and Groundwater Sources, 1971-1996
Water Source
Ground
Surface
All Systems
Outbreaks
401 (59%)
162 (24%)
678 (100%)
Cases of Illness
85,938(15%)
471,222(83%)
564,754 (100%)
Hospitalized
882 (16%)
4,554 (82%)
5,556 (100%)
Deaths
18(95%)
0
19** (100%)
*Waterborne outbreaks reported in systems using ground or surface water sources includes
outbreaks due to miscellaneous and unknown causes and distribution/storage contamination in
addition to those caused by source contamination and treatment deficiencies.
**Does not include deaths from Milwaukee and Las Vegas outbreaks. Reports from Milwaukee
suggest 100 premature deaths.
i f
Source: Calderon and Craun, 1998.
TABLE 2-5b
Cases of Illness, Hospitalizations, and Deaths in Waterborne Outbreaks* in
Water Systems by System Type, 1971-1996
Water Source
Community
Noncommunity
Individual
All Systems
Outbreaks
284 (42%)
315(46%)
79 (12%)
678 (100%)
Cases of Illness
515,519(91%)
51,366(9%)
1,136 (<1)
568,021 (100%)
Hospitalized
4,856 (87%)
678 (12%)
49 (1%)
5,583 (100%)
Deaths
14(16%)
2 (2%)
3 (3%)
19** (100%)
*Waterborne outbreaks reported in systems using ground or surface water sources includes
outbreaks due to miscellaneous and unknown causes and distribution/storage contamination in
addition to those caused by source contamination and treatment deficiencies.
**Does not include deaths from Milwaukee and Las Vegas outbreaks. Reports from Milwaukee
suggest 100 premature deaths.
Source: Calderon and Craun, 1998.
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enterovirus type 71 from the stools and cerebrospinal fluid of affected infants and children (CDC,
1998a). There is no conclusive evidence that the original source of the outbreak was water.
Kukkula et al. (1997) reported a drinking water-associated outbreak of viral
gastroenteritis in Finland. They also indicated that 58% of outbreaks in Finland were due to
unknown agents.
Divizia et al. (1993) reported an outbreak due to hepatitis A in a college in Rome, Italy.
HAV, which caused this outbreak, was identified in the well water by the PCR method. Nasser
(1994) reported that HAV was the most commonly reported causative agent of waterborne
disease, and that its prevalence is related to the socioeconomic level of the population.
2.6 Summary
Enteroviruses have similar physical and biochemical properties. They are single-
stranded unenveloped RNA viruses. Man is the only natural host for enteroviruses, although
some reports indicate domestic animals as well. These viruses have been shown to occur in
sewage, surface water, and ground water as a consequence of fecal contamination and can be
transmitted by water. The CDC surveillance reports indicate that since the inception of CDC-
EP A collaborative outbreak reporting, AGI has been associated with the highest number of
outbreaks in the United States. Ground water contamination has been the leading cause of water
supply outbreaks. This provides evidence that ground water is not protected against microbial
contamination. Ground water has been reported to be associated with the highest number of
deaths while surface water was associated with more cases of illness and hospitalization.
Recreational water outbreaks of gastroenteritis have also occurred due to accidental ingestion of
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contaminated water, CDC indicates that waterborne outbreaks in the United States are grossly
underreported.
Outbreaks due to AGI of unknown agents have been reported to be as high as 50%. It is
generally believed that these unknown agents may be viral for two reasons: first the symptoms
indicate a viral agent pattern, and second, current viral detection methods cannot detect all
pathogenic viruses and water treatment facilities do not routinely test for viruses but only for
indicator organisms, HAV appears to be the virus most commonly identified in viral-associated
outbreaks. HAV has been implicated in 60 outbreaks between 1946 and 1980 (Highsmith and
Crow, 1992).
Only a small percentage of outbreaks have been associated with viral agents. There is a
strong speculation that viral agents may be responsible for a high percentage of the unknown
etiological agents associated with outbreaks due to AGI. The evidence, however, is not
conclusive.
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3.0 Occurrence in Water
3.1 Viruses in Environmental Waters
Viruses excreted in human and animal feces find their way into the sewage
(Abbaszadegan et al., 1998; Dahling et al., 1989; EPA, 1985). They are then discharged with
waste water effluent into surface waters. The enteroviruses and HAV have been shown to occur
in environmental waters including ground water and surface water (Craun, 1990; Payment and
Armon, 1989; Toranzos et al., 1986,1988).
3.1.1 Viruses of Man
The enteric viruses by definition are viruses that infect the GI tract of man, and these
viruses include the human enteroviruses and HAV. The enteric viruses can be excreted in feces
of infected individuals in high numbers of 106-1012 per gram of feces, and all the human viruses
excreted in feces are present in sewage (Abbaszadegan et al., 1998; Abbaszadegan and DeLeon,
1997; Metcalf et al., 1995; EPA, 1985). Not all of the viruses present in sewage are successfully
inactivated by waste water treatment and disinfection. Many viruses present in sewage can be
present in drinking water, ground water, recreational waters, and sludge as a result of
contamination (Abbaszadegan et al., 1998; Payment, 1993; Gerba et al., 1989; Payment, 1985).
3.1.2 Viruses of Animals
There are several enteroviruses of lower animals such as monkeys, pigs, mice, cattle, and
dogs (Melnick, 1996a; Grew, et al., 1970; Clapper, 1970), which are shed when animals defecate
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in fields, farms, zoos, and the streets. These viruses are then transported by storm water runoff
into municipal sewage or combined sewer overflows and consequently can contaminate
environmental waters (EPA, 1985). Humans are not at risk for the animal enteroviruses, as there
is as yet no direct evidence of zoonotic transmission of these viruses to humans,
3.1,3 Bacterial Viruses
Viruses have a varied host range that includes humans, animals, plants, fish, and bacteria.
A bacteriophage is a virus that specifically infects bacteria. Bacteriophages do not infect
humans, but they can genetically impact disease control through gene transfer. Bacteriophages
can be a vehicle for gene transfer from one bacterium to another. They can confer drug
resistance by transferring a drag-resistance gene from one resistant bacterium to another
bacterium that is susceptible to the drug in question.
Human viruses in water are the focus of this document. Bacteriophages occur in
environmental waters. The relevance of the bacteriophage in this document is in its potential use
as an indicator of viral or fecal pollution of drinking water. Bacteriophages that infect E. coli can
be used as indicators of fecal contamination of water and as such can serve as a warning for the
possible presence of human viruses. The use of bacteriophages as potential indicators for viral
pollution is discussed in Chapter 9.
3.2 Viruses in Sewage
The principal source of all the viruses of human origin found in water is domestic
sewage. According to Abbaszadegan et al, (1998), enteric viruses can be excreted in high
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numbers of 1010-1012 per gram of feces of infected individuals. There are more than 120 known
human enteric viruses, and these viruses have been reported to be excreted into domestic sewage
(Abbaszadegan et al., 1998; Metcalf et al, 1995; EPA, 1985; Melnick and Gerba, 1982). Metcalf
et al. (1995) indicate that virus concentration in raw sewage can range from 5000 to 28,000 PFU.
Waste water treatment processes such as primary with sedimentation and flocculation, filtration,
and disinfection inactivate or remove a high percentage of viruses in sewage. However, some of
the viruses are not effectively removed by sewage treatment and consequently are discharged in
treated effluent and may possibly cause outbreaks (Yeager and O'Brien, 1977).
f'
3.2.1 Factors That Affect the Numbers of Viruses in Sewage
Several factors affect the number and type of viruses in sewage, and these factors are
location-specific for each community. Ten factors which influence viral number and type in
sewage (EPA, 1985) are summarized below,
» Virus introduction into the community
» Age distribution in the community
* Immunological status of the community
• Sanitary level of the community
• Microbial flora of the sewage
• Chemical composition of the sewage
• Temperature of sewage
* Treatment methods used
» Climate
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• Solids
Virus introduction in a community. Communities with active polio vaccination programs will
have the poliovirus vaccine strain being shed in the feces, facilitating its entrance into the
municipal sewage system,
Age distribution. Children are more susceptible to viral infections than adults. Communities
with many day care centers and preschool children will have higher numbers of virus being shed
than other communities. Also, children are the major source of the vaccine strain of poliovirus.
Immunological status of community. An attack by poliovirus confers lifetime immunity.
Communities with a majority of their citizens carrying circulating antibody to poliovirus will be
resistant to subsequent poliovirus introduction and infection.
Sanitary level of a community. The overall sanitary level impacts the type and number of
viruses in a community (EPA, 1985). Hepatitis A has been shown to be contracted by secondary
spread and is more prevalent in lower socioeconomic groups living in tight quarters and
practicing poor hygiene.
Microbialflora of sewage. The activated sludge (secondary treatment) process in waste water
treatment has a variety of bacterial flora that may aggregate and create a protective shield for
viruses allowing their absorption and eventual escape from inactivation.
Chemical composition of sewage. The chemical composition of a waste water influent affects
the pH of sewage. Enteroviruses are stable over a wide pH range (3.5-10.0) and therefore will
survive well over this pH range. However, Hain and O'Brien (1979) have shown that ammonia
is virucidal to enterovirus at an alkaline pH of over 7.5.
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Temperature of sewage. Increased temperature has been shown to inactivate some viruses.
Enterovirases are thermolabile (Melnick, 1996a). However, hepatitis A is very resistant to
thermal inactivation, and this is one of the reasons for transferring hepatitis A from the
Enterovirus genus to the Hepatovirns genus.
Hepatitis A has also been shown to survive at a low temperature and can persist for
months at temperatures below 10°C. At ambient temperatures of 20°- 25 °C, HAV can persist
for at least one month (Nasser, 1994). Therefore, elevated temperature may decrease the number
of some viruses but may not have any significant effect on others.
Treatment methods for sewage. Waste water treatment methods can decrease the number of
viruses in sewage. However, primary treatment of sewage using sedimentation will concentrate
viruses from the influent into the settled solids. Anaerobic digestion of solids, which is a thermal
process, will inactivate and reduce the number of viruses in sewage.
Climate. HAV is reported to be seasonal and to be recovered mostly in cold seasons, and
therefore will have an increase in number during cold seasons. Poliovirus on the other hand, will
be recovered year-round in communities with active poliovirus vaccination programs (EPA,
1985). Rainfall will increase the concentration of viruses as solid-absorbed viruses are floated
free and ran off into source waters.
Solids. Virus particles in domestic sewage are trapped in settleable solids (Yeager and O'Brien,
1977). Viruses associated with solids are protected from inactivation by the treatment processes
in waste water treatment and water treatment (Melnick, 1996a). Oliver (1987) indicates that
virus concentrations are four times greater in solids than in the effluents of domestic sewage.
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3.2.2 Recovery of Viruses in Sewage
As stated earlier, sewage is a major source of waterborne, enteric viruses. The number of
viruses recovered from raw sewage in the United States ranges from 50 to 250 plaque-forming
units (PFU) per liter (EPA, 1985), Enteroviruses have been recovered in sewage. Detection of
HAV in sewage and ground water was reported by Hejkal et al. (1982). Recently, a reverse
transcriptase (RT) seminested PCR and restriction fragment length polymorphism (RFLP) assay
was used by Dubois et al. (1997) to demonstrate the presence of viruses in 42% of raw sewage
samples and in 67% of treated effluent samples from a sewage treatment plant in France. These
viruses can migrate underground into ground water and aquifers, or into surface waters, thereby
contaminating intake water at water treatment plants. Recently, Jothikumar et al, (1998) reported
the detection of HAV in raw sewage using an immunomagnetic capture PCR method.
The various waste water treatment methods in existence 14 years ago remain the same
today; these methods include primary treatment, activated sludge secondary treatment, trickling
filter, stabilization ponds, and advanced waste water treatment (nitrification). The waste water
treatment methods listed are discussed in greater detail in the 1985 draft document (EPA, 1985).
Virus removal efficiency varies for each method. Primary treatment can remove 60% of viruses
(EPA, 1985), and activated sludge treatment can remove 90% of viruses (Rao et al., 1987).
3.3 Viruses in Surface Waters
The major source of viruses, including enteroviruses, in surface waters is waste water
effluents (Dubois et al., 1997; EPA, 1985). Most waste water treatment plants are usually
located along rivers and streams. As an example, the Blue Plains Wastewater Treatment Plant in
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Washington, DC, has an outfall that discharges directly into the Potomac River. Many of our
nation's rivers serve as receiving bodies of water for the effluents routinely discharged from
treatment plants. Septic tanks and sewage discharged by farms and small towns also add to the
total viral load in surface waters. Consequently, enteroviruses of human and animal origin can
be expected to be present in source waters that are destined to become drinking water.
Payment (1989) evaluated the presence of enteric viruses in surface water and ground
water from several sites in Quebec, Canada. He showed the presence of human and animal
enteric viruses in both surface and ground water contaminated by discharges from municipal
waste water effluent and runoffs from land application.
3.3.1 Survival of Viruses in Surface Waters
Viruses have the ability to survive for long periods of time in surface waters. HAV can
survive for more than 4 months at a temperature of 5 °C to 25 °C in water, wastewater, and
sediments (Sobsey et al., 1988a). Another report indicates that HAV can persist for months at
temperatures below 10°C and for at least one month at ambient temperatures of 20°-25°C
(Nassar, 1994). The survival of viruses in surface waters is controlled by several factors such as
the seven summarized below.
• Adsorption to suspended materials (solids)
• Temperature
• pH
• Sunlight
• Biological activity (e.g., proteolytic enzymes)
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• Salt concentration and
• Microbial activity
Enteroviruses, such as poliovirus type 1, have been shown to survive for prolonged periods in
fresh waters under laboratory and field conditions (Kutz and Gerba, 1988; Pancorbo et al., 1987),
Solids-associated viruses have been shown to concentrate in shellfish. McDonnell et al. (1997)
has shown greater virus concentration in shellfish than in surrounding water. The relative
survival of the enteric viruses, however, depends on the virus type and the environmental
conditions. But the most important factor in survival is reported to be temperature (Kutz and
Gerba, 1988; Salo and Cliver, 1976).
3.3.2 Recovery of Viruses from Surface Waters
Several studies have documented the recovery of viruses from surface waters (EPA,
1985; Payment, 1989). Using the nested PCR method, Girones et al. (1995) identified
enteroviruses and HAV from a river in Barcelona.
3.4 Viruses in Ground Water
The EPA Office of Ground Water and Drinking Water (OGWDW) reports that there are
more than 158,000 public ground water systems in the United States (OW/OGWDW, 1998).
Almost 89 million people are served by community ground water systems. Of the total 158,000
systems, 157,000 (99%) serve fewer than 10,000 people. The systems that serve more than
10,000 people serve 55% of the population, which is more than 60 million of the total number of
people whose drinking water source is public ground water systems (OW/OGWDW, 1998).
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Ground water is a major source of drinking water in the United States, It is the source
water for over 100 million people in the United States, and 95% of the water used in rural
America is ground water (Bitton and Gerba, 1984). According to the U.S. Geological Survey,
ground water use increased from 35 billion gallons a day in 1950 to about 87 billion gallons a
day in 1980. Approximately one-fourth of all fresh water used in the United States comes from
ground water (EPA, 1990).
Ground water use varies by state. Arkansas, Nebraska, Colorado, and Kansas use 90% of
their ground water for agricultural purposes. Hawaii, Mississippi, Florida, Idaho, and New
Mexico rely on ground water for 7,5% of their household water use. Colorado and Rhode Island
rely on ground water for only 25% of their water needs (EPA, 1990). ~
Before the 1970s, ground water had been presumed to be relatively free from
contamination because of soil layers, especially those containing clay, sand particles, gravel,
crushed rocks, and large rocks, which act as natural filters that trap contaminants in the water and
prevent them from reaching the ground water (EPA, 1990). Various studies now show that
ground water can be contaminated with viruses (Alhajjar et al., 1988; Yates and Yates, 1988a,b;
Thurman and Gerba, 1987; Yates, 1985; Keswick and Gerba, 1980). Septic tanks, broken sewer
lines, land application of waste water effluents, sludges, and also leachates from landfills are
sources of viral contamination of underlying ground water (Sobsey et al., 1986).
Another source of viral contamination of ground water is the application of sludge for
agricultural irrigation or for disposal. CDC surveillance data indicate that ground water is
associated with the highest number of waterborne disease outbreaks in the United States (CDC
1996a; CDC, 1998; Calderon and Craun, 1998). Rao et al. (1986) state that over 50% of
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waterborne outbreaks reported in the United States each year are caused by contaminated ground
-water.
Hejkal et al. (1982) reported the viral contamination of a community ground water supply
that had met microbial water quality standards but was susceptible to sudden contamination after
a heavy rainfall. Subsequently, HAV, coxsackievirus B2 and B3, and' a rotavirus were isolated
from this drinking water supply.
Goyal et al. (1984) monitored the presence of viruses and indicator bacteria in ground
water and soil samples from three slow-rate operational land treatment sites at Lubbock and San
Angelo, Texas, and at Muskegon, Michigan. In each of these cities, waste water was used for
agricultural purposes, and slow-rate sewage irrigation of cropland was practiced. The sewage —
received secondary treatment by aeration before the effluent was used for land application at all
sites tested. Enteric viruses were isolated from water wells located beneath all these sites. The
lowest frequency of virus isolation was seen in wells beneath the site that practiced chlorination
before application by spray irrigation. Viruses were detected in wells as deep as 27.5 m.
Contamination of ground and surface waters with enteric viruses is a worldwide problem.
Payment (1989) isolated human and animal (porcine) enteroviruses and reoviruses at a high
frequency (up to 70%) from a Quebec river in Canada. Two of 22 samples of ground water from
the same region were contaminated with animal enteroviruses. The porcine origin of
enteroviruses was attributed to a massive pig-raising activity in the area, and reoviruses were
assessed as of either avian or human origin, coming from broiler chicken farms. The presence of
human viruses was expected because there were several centers of concentrated human activity
where direct discharge of municipal or private waste was practiced.
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3,4.1 Survival .of Viruses in Ground Water
3.4.1.1 Fate and Transport of Viruses in Ground Water
tr
The fate and transport of microbes in ground water are controlled by the physicochemical
characteristics of the specific microbe and of the ground water and aquifer media (Robertson and
Edberg, 1997). Key characteristics of microbes include size, inactivation (die-off) rate, and
surface electrostatic properties. Key properties of ground water and aquifer systems include
t
aquifer pore size, flow velocity, porosity, solid organic carbon content, temperature, pH,
chemical characteristics of water, and mineral composition.
In order to define the factors that control virus survival in ground water, Yates and»Q||foa
1'
(1985) collected ground water samples across the United States, inoculated the samples with the —
MS-2 coliphage, and determined the rate of phage inactivation. Samples were incubated at the
temperature of native ground water and were analyzed for pH, nitrates, ammonia, turbidity, total
dissolved solids, calcium, magnesium, and total hardness. Multiple regression analysis of the
chemical variates showed that temperature and calcium hardness were significantly correlated
with the rate of virus die-off. Temperature accounted for 60% of the variation in the decay rate.
Using all variates, 94% of the variation could be predicted.
The survival of viruses in the subterranean environment is believed to be influenced by
three interacting factors: the type of soil, the nature of the virus, and climate. Yates and Gerba
(1985) considered various factors and noted their effect on virus survival to be as follows:
• Virus survival is prolonged with increase in soil adsorption.
• Virus survival increases as levels of exchangeable aluminum increase.
• Virus survival decreases as pH and resin-extractable phosphorus increase.
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• Virus-Survival decreases as temperature increases.
• Aerobic soil microorganisms adversely affect virus survival, while anaerobic
microorganisms have no effect on virus survival.
Yates and Gerba (1985) also indicate that surface charge influences virus adsorption to
soil particles. Protein coat differences among viruses also determine the differences in their
susceptibility to denaturation by proteolytic enzymes, temperature, and chemicals. Dowd et al.
(1998) recently reported virus isoelectric point to be the predetermining factor controlling virus
adsorption within aquifers. A similar finding was made by Redman et al. (1997).
Another important factor in determining virus survival is climate. Temperature
influences virus persistence in surface water and in soils. Exposure to sunlight is an important *~
factor in the inactivation of viruses.
Yates et al. (1985) found temperature to be the single most important predictor of virus
persistence in well water because they found a high correlation (77.5%) between the decay rate o§
enteric viruses and ground water temperature.
3.4.1.2 Mobility of Viruses in Ground Water
Viruses exhibit greater mobility in ground water than do bacteria because of their small
size and surface electrical properties. The inactivation or die-off rate is a very important factor
affecting how far microbes can migrate in significant numbers in ground water. Typical half-
lives of microbes in ground water range from only a few hours to a few weeks. Several studies
have demonstrated the migration of viruses in soils (Robertson and Edberg, 1997; Straub et al.,
1995; Alhajjar et al., 1988; Bitton et al., 1979; Schaub and Sorber, 1977). Migration distances of
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viruses are reported to be 1,000-1,600 m in channeled limestones, and 250-408 m in glacial slit-
"sand aquifers. Investigations have shown that distances of 210-325 m from septic tanks will
achieve reduction in virus concentrations by a factor of 1011 (Robertson and Edberg, 1997).
Yahya et al, (1993) studied the survival of two baeteriophages, MS-2 and PRD-1, in
ground water collected from Arizona, Massachusetts, and Canada. The phages tested, MS-2
from E. coli is 23 nm and similar in size to enteroviruses, and PRD-1 from S. typhimurium is 65
nm. Neither phage is adsorbed efficiently onto soils. The results indicate that at 7 °C the phages
showed similar inactivation rates and survived well for 80 days. At a temperature of 23°C,
however, the MS-2 phage was more quickly inactivated than the PRD-1 phage. PRD-1- also
persisted 10 times longer in water samples than did MS-2. Based on the long survival rate results
of PRD-1, the investigators suggested that this phage could be used as a model to evaluate
ground water movement over a long period of time.
Powelson et al. (1993) studied virus transport and removal in the field, using a recharge-
recovery site near Tucson, Arizona. Baeteriophages were used as tracer viruses. After
percolating through more than 4 m, 37%-99% of the viruses were removed. Slower infiltration
rates increased the removal of viruses. Powelson et al. (1993) concluded that human
enteroviruses would be removed more efficiently than the phages; however, it has been
demonstrated that the removal of enteroviruses is largely dependent on the organic content of the
soil, and that the phages behave quite differently from the human viruses. Thus the usefulness of
the phages as a model for human viruses is dependent on the composition of the soil in the
recharge basins.
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Septic tanks are often implicated in the contamination of ground water. In areas where
ground water is used in individual wells, no disinfection is applied and the microbiological
£
quality of the drinking water is not monitored on a regular basis. In order to assure a safe
drinking water source, it is proposed that the setback of septic tanks to an appropriate (specified)
distance be required, in order to virtually assure the absence of viruses from the drinking water.
A-mathematical model was proposed by Yates and Yates (1988a) to determine the appropriate
well setback distance. The model can be applied either to determine the probability of
maintaining a safe drinking water quality, given the setback distances prescribed by local laws, or
to calculate the appropriate setback,distance required to assure the virtual safety of the drinking
water.
Setback distances for septic systems, soil quality, and pumping rates for ground water
wells were discussed by Yates and Yates (1988b). Mathematical and geological models and
considerations are applied to determine whether virus elimination is sufficient to provide a safe
water supply. The authors concluded, however, that a standardized setback distance may not be
adequate to protect the ground water from virus contamination.
Funderburg et al. (1981) studied the removal and adsorption of poliovirus, reovirus, and
phage <&X 174 in several types of soils in simulated laboratory experiments using soil columns.
A high organic soil content and a high cationic exchange capability of the soil significantly
increased the adsorption of poliovirus and reovirus, whereas the phage was more efficiently
removed by soils with low organic content. They concluded that agricultural soils generally
would favor the removal and adsorption of the enteroviruses since they have the characteristics
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that increase the removal of viruses. The depth of the soil necessary to remove sufficient virus
was not determined.
Bitton et al. (1979) compared the filtration and removal of viruses in field applications
and soil columns. Removal of viruses in different soils and under different circumstances varies
greatly, ranging from complete removal to significant contamination of ground water.
Experiments using soil columns were suggested as good predictors of virus removal, provided
the soil used in the columns and other parameters, such as flow rate, closely mimic the conditions
of the field site intended for waste water disposal.
Wang et al. (1981) percolated waste water, seeded with poliovirus and echovirus, through
different soil in columns of I m length, and found that the type of soil seemed to influence the ~~
degree of virus adsorption. However, the flow rate was the most important variable for removing
viruses. At low flow rates, removal was fairly efficient, whereas at higher rates, 300 cm/day, the
removal was poor. It also was shown that adsorption was more efficient in the upper part of the
column. Thus, longer columns are required for modeling virus removal through soil filtration
and adsorption.
Herbold-Paschke et al. (1991) conducted research suggesting that the behavior of bacteria
and viruses in sand columns provides valuable information concerning the movement of these
organisms through the soil layer before they reach the ground water level. In this study, sand
columns of 1 m length were not capable of removing several bacteriophages. They, however,
retained more than 50% of the simian rotaviras SA-11.
The removal of poliovirus from tertiary-treated waste water was dependent on the
infiltration rate used in an operational ground water recharge basin by Vaughn et al. (1981). At
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an infiltration rate of 75-100 cm/h, considerable numbers of seeded poliovirus reached the
ground water, whereas at an infiltration rate of 6 cm/h the removal of virus was considerably
improved. The authors suggested that ground water recharge systems should be individually
tested for virus removal rates at different infiltration rates to determine their capability to remove
enteroviruses effectively,
Yates et al. (1986) studied the disappearance of virus in the environment using MS-2
- V
coliphage as a model virus. The virus was seeded into the environment, and resolution attempts
from 71 drinking water supply wells were performed. Based on the number of viruses isolated, a
spatial model was developed that allowed the estimation of the probability of isolating visuSj|j,at
•'
any location in the environment, including those not actually sampled. According to the authors^
this process would allow valid predictions of the likelihood of finding contaminated ground
water at certain distances from the contamination source, e.g., septic tank systems.
Jorgensen and Lund (1995) assessed the stability of enteric viruses in sludge, soil, and
ground water by applying the municipal sludge to a sandy soil in a forest plantation. Poliovirus,
coxsackievirus, and adenoviruses could be isolated from the environment up to 21 weeks after
application. Thus it was demonstrated that vims survival in the environment is rather good and a
rapid inactivation cannot be assumed. Experimental soil columns were tested for their efficiency
to remove/adsorb viruses from treated sewage. Two phages, MS-2 and PRD-1, and poliovirus
were used as model viruses. The results show that poliovirus was more effectively removed than
the phages, and unsaturated conditions resulted in greater virus removal than saturated flow
conditions.
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Schaub and Sorbet (1977) conducted a field and bench scale study at Fort Devens,
Massachusetts, to evaluate adsorption capacity of various soils and the penetration of poliovirus
and f2 bacteriophage and indicator bacteria in soils and ground water. Coliphage £2 was used as
a tracer virus and the migration of indigenous enteroviruses and indicator bacteria was also
followed. The £2 tracer virus migrated rapidly through the soil levels and appeared in probes
together with the front of the applied waste water. Results obtained showed that both the
indigenous viruses and the tracer virus migrated vertically and horizontally in the application site.
The indicator bacteria, in contrast, were retained in the application site but only occasionally
reached the ground water in the observation wells. The results were corroborated in laboratory
tests, showing that the soil at this site poorly adsorbed viruses from waste water.
Brown et al. (1979) studied the filtration and removal capacity of several soils for 2 years.
Sand content of soils studied ranged from 7.6% to 80%. At 120 cm below the septic lines,
neither coliforms or coliphages were detected, except on a very few occasions that were
attributed to the experimental design of the study. The authors concluded that under these
conditions, the filtration capacity of 1,2m soil was sufficient for the removal of coliforms and
coliphages.
Models for following virus movement, especially in ground water, have been suggested.
Coliphage f2 has been suggested as a model by Wang and Gerba (1981) for tracing virus
movement in the environment because of its similarity in size to enteroviruses and also because it
is not pathogenic to humans, animals, or plants.
Wellings et al. (1975) conducted a study in which secondary effluent from waste water
was discharged into a cypress dome, and the ground water was analyzed for the presence of
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viruses by using-tissue culture techniques. The data showed that vertical (3 m) as well as
•horizontal movement of viruses occurred and also showed a virus survival period of 28 days.
3.5 Viruses in Prinking Waters
Several reports have demonstrated the presence of pathogenic viruses in disinfected
drinking water that met microbiological standards for safety (Payment, 1989; Bitton et al., 1986;
EPA, 1985). Gerba et al. (1984) detected coxsackieviruses in treated drinking and well water.
They also detected enteric viruses in drinking water that met the safety standards, which were
zero coliforms/lOOmL, < 1 NTU, and >0.5mg/L free chlorine level.
Keswick et al. (1984) conducted a study on water samples collected during the dry seasorT
and also during the rainy season. Their results indicate that enteroviruses were detected in 56%
of finished water samples that were collected during the dry season.
3.6 Summary
Enteric viruses occur in environmental waters and sewage. Over 120 human viruses are
excreted in feces and subsequently are transported into sewage. Enteroviruses, HAV, and other
waterborne enteric viruses occur in both pound and surface waters and can be responsible for GI
illnesses that occur in various communities. A great deal of evidence has shown that ground
water is no longer safe from contamination. Ground water is associated with over 50% of
waterborne outbreaks in the United States. The re-use of waste water, use of waste water effluent
for agricultural irrigation, improperly placed septic tanks, and dumping of feces into surface
waters lead to viral contamination of ground water. Viruses can travel vertically and horizontally
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through soil. Viruses can migrate long distances, 1,000 m or more, and reach aquifers and wells
"that are destined to be used as drinking water sources. Numerous reports have shown that even
drinking water that met the standards for microbiological safety by traditional monitoring
methods for fecal coliforms can be contaminated by viruses.
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4.0 Health Effects in Animals
r
4.1 Diseases of Animals Produced by Viruses of Humans
Man is the only natural host of the human enteroviruses. Some enteroviruses infect lower
animals but their infections do not seem to be communicable to man.
4.1.1 Poiiovirus
Chimpanzees and monkeys have been used as animal models in the experimental
laboratories for poliovirus. Poiiovirus infections can be induced in chimpanzees and monkeys
through intracerebral, intraspinal, and oral routes, and these animals can develop antibodies to
the virus (Melnick, 1996; Memiek, 1992; EPA, 1985).
4.1.2 Coxsackieviruses A and B
Coxsackievirus A (1-24) and coxsackievirus B can produce flaccid paralysis in
laboratory-infected (intracerebral inoculation) newborn mice and rhesus monkeys (Dalldorf,
1957; Melnick, 1992). Coxsackievirus B (1-6) can infect infant mice brain and foot pads.
Cultured primate cells are also susceptible to infection by both coxsackievirus A and B (Melnick,
1992).
4.1.3 Echovirus
Echovirus has a wider host range. Over 14 serotypes (types 1—4, 6-9,13-14, 16-18, and
20) can produce laboratory-induced infections in rhesus monkeys and newborn mice. The
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echovirus also grows well and produces cytopathic effects routinely in cultured cells of African
green monkey and rhesus kidney (EPA, 1985).
c
4.1.4 Enterovirus Types 68. 69. 70. 71
The diseases produced by the numbered enteroviruses in animal models are variable but
there is no known zoonotic disease produced in man.
4.1.5 Hepatitis A Virus
Chimpanzees can be infected experimentally by HAV in the laboratory. Some morrfaf
species and marmosets are also experimental hosts of HAV. The virus can produce cytopathic
effects in some cells (Gerba, 1984).
4,2 Minimal Infective Dose
As already stated above, man is the only natural host of enteroviruses. The initiation of
infection in animals with human enteroviruses experimentally has also been discussed.
However, it should be noted that Gerba and Rose (1993) suggest that it may not be feasible to
extrapolate infectious dose data from laboratory animals to human beings.
4.3 Summary
Man is the only natural host of human enteroviruses, but other animals can be infected
experimentally with human enteroviruses to produce diseases such as flaccid paralysis. The
diseases produced by enteroviruses in animals do not appear to be communicable to man. There
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exist a few reports on the detection of enteroviruses in domestic pets. However, the impact of
this on public health is unknown.
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5.0 Health Effects in Humans
5.1 Introduction
The disease commonly associated with enterovirases in humans is gastroenteritis.
However, the range of health effects attributed to enteroviruses goes beyond GI diseases.
According to Melnick (1996a)., the most serious disease caused by the entero viruses is
poliomyelitis. There are numerous studies worldwide that implicate enteroviruses in various
diseases including such debilitating and life-threatening conditions such as paralytic polio, heart
disease, encephalitis, hemorrhagie,conjunctivitis, HFMD, and diabetes mellitis (CDC, 1998b;
Modlin, 1997; Melnick, 1996; Modlin, 1995; Cherry, 1995; Berlin and Rorabaugh, 1993; Smith,
1970; Dalldorf and Melnick, 1965).
A literature search and critical review of available information on the health effects of
enteroviruses and hepatitis A show that many publications present the health effects of
enteroviruses as a group. Some publications also present diseases caused by coxsackieviras and
echovirus together as infections caused by "nonpolio enteroviruses." This chapter on health
effects, therefore, is organized in three parts to reflect prior study interpretations. The first part
presents a general disease profile of all enteroviruses including the diseases due to "nonpolio
enteroviruses," The second part presents all the diseases caused by each enterovirus member by
specific serotype in a table form. Part 3 is a discussion of the clinical symptoms of diseases
caused by each member of the enterovirus and hepatitis A groups. The reader should therefore
note that any reference to nonpolio enterovirus in this document indicates coxsackievirus groups
A and B and echovirus.
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5.2 Disease Profile of the Enteroviruses
Enteroviruses, and specifically coxsackievirus group B, are believed to be the most
common cause of viral-induced heart disease (Smith, 1970). Enteroviruses account for
approximately 10-20% of encephalitis cases with proven viral etiology. These cases are
manifested by changes in mental status and coma (Modlin, 1997). According to Modlin (1995),
over 90% of the infections caused by nonpolio enterovirases (coxsackievirus and echovirus) are
either asymptomatic or they result in undifferentiated febrile illness. The severity of disease is
generally determined by identified factors such as gender (with a prevalence in males over
females), physical exertion, age (thp very young and the elderly), and lack of serum
immunoglobulin production.
A majority of enteroviral infections caused by nonpolio enterovirases as stated above are
asymptomatic, but the persistent cases of enteroviral infections are manifested in bone marrow
transplant recipients and in children with severe X-lmked agammaglobulinemia or combined
immunodeficiency syndrome (Modlin, 1997).
Enteroviral infections occur in all age groups. It has been reported, however, that infants
and young children experience the highest rates of enteroviral infection and disease. In fact,
infants less than 1 year old are infected at a higher rate than older children and adults (Modlin,
1997). Enteroviruses have been reported as a leading cause of acute febrile illness among young
children and infants (Dagan, 1996). Enterovirus infections have a gender preference. Males are
attacked at a 50% higher rate than females (Modlin, 1997).
Infants of low socioeconomic status are at a higher risk of contracting an enteroviras
infection; this is attributed to overcrowding and poor hygiene. Modlin (1997) reports that
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30-80% of all adults have antibodies to the common enterovirus serotypes and that the 15 most
common enterovirus serotypes reported to CDC between 1970-1983 were the following:
Echovirus 11 (12.2%) Echovirus 6 (5.5%) Coxsackie B4 (4.36%)
Echovirus 9 (11.3%) Coxsackie B2 (4.8%) Echovirus 3 (3.2%)
Coxsackie B (8.7%) Coxsackie B3 (4.5%) Echovirus 7 (3.0%)
Echovirus 4 (6.3%) Coxsackie A9 (4.5%) All others (31.4%)
Approximately 50% of infants infected with enterovirus manifest aseptic meningitis. Most of
these infants recover within 10 days, but 10% of them develop acute central nervous system
(CNS) complications that include seizures and increased intracranial pressure (Modlin, 1997).
Cherry (1995) reports that GI illness occurs in 7% of all enteroviral infections of infancy and that
in one study, 81% of neonates with nonpolio entero viral infections had diarrhea and 33% had
vomiting.
The mortality rate in myocarditis cases of neonates is reported to be 30-50% and even
higher when other organs (besides the heart) are involved (Modlin, 1997). Neonatal hepatitis has
been reported in 2% of neonates with clinically severe enteroviral disease, and 80% of infants
with neonatal hepatitis die within 1 to 3 weeks while survivors may develop cirrhosis and
chronic hepatitis insufficiency (Modlin, 1997; Cherry, 1995).
Enterovirus infection late in pregnancy is common and often goes unrecognized because a
majority of the women are asymptomatic (Modlin and Kinney, 1987). Based on serological
surveillance of both mothers and infants, 29% of documented cases of mothers with echo virus 17
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and coxsackievirus B2 and B5 infections transmitted the virus to their infants (Modlin and
Kinney, 1987). The secondary infection rate for enteroviruses is reported to exceed 50% in
household contacts (Modlin, 1997),
5.3 Specific Disease Profiles for Each Member of Enterovirus and Hepatitis A
Specific diseases and the specific member of enterovirus associated with each disease and
the implicated serotypes are presented in Tables 5-1, 5-2, 5-3, and 5-4.
5.3.1 Poliovirus Health Effects
t '•
The polio virus is the most renowned member of the enteroviruses because it produces
poliomyelitis, a devastating paralytic disease of humans. There are three distinct serotypes of
polioviras, type 1 through 3, All three of these serotypes produce paralytic disease in man
(Melnick, 1996).
According to CDC (1998b), since 1980 a total of 143 out of 145 confirmed cases of
indigenously acquired paralytic poliomyelitis in the United States have been associated with oral
polio vaccine. The remaining two cases were classified as indeterminate. In September 1996,
CDC adopted the ACIP recommendations for a sequential vaccination schedule of inactivated
poliovirus vaccine followed by two doses of oral poliovirus vaccine (CDC, 1998b).
5.3.1.1 Poliomyelitis Eradication by the Year 2000?
The incidence of paralytic poliomyelitis has declined substantially worldwide, and the
WHO believes that polio will be eradicated from the world by the year 2000 (WHO, 1998;
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Hovi et al., 1996), Even though the incidence of polio has substantially declined, poliomyelitis
remains a significant cause of illness and death in war-ravaged areas of the world where health
systems have been destroyed and immunization services are either unavailable or insufficient
(WHO, 1998).
A recent survey conducted in Afghanistan showed that the most common cause of
disability among children under 15 years old was polio. WHO believes that Afghanistan
represents a major remaining focus of continuing wild type poliovirus transmission in the world
and therefore is of key importance in the global eradication of polio (WHO, 1998).
Another reason for the concern about the remaining polio pocket is the transmission of
wild type poliovirus from Afghanistan to other countries. WHO indicates that the Afghanistan
wild type poliovirus has spread to Pakistan, Tajikistan, Albania, Greece, Iran, and Russia. The
wild type poliovirus found in Europe and Iran was linked epidemiologically to Pakistan and
Afghanistan. Because of the evidence supporting cross-border transmission of wild type
poliovirus, there is fear that polio-free countries are at risk of reimportation of poliovirus from
the remaining infected war-ravaged countries of the world.
WHO and the United Nations Children's Fund (UNICEF) now have a coordinated
strategy for global eradication of polio. This strategy includes a mass immunization during
national immunization days and a monetary appeal for $35 million to fight poliomyelitis in war-
torn countries such as Afghanistan and Rwanda. Over 110 countries worldwide have
participated in the mass immunization campaigns for polio. In 1996,420 million children under
the age of 5 were immunized worldwide during national immunization days (WHO, 1998).
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5.3.1.2 Polioviriis Transmission
Poliovirus is transmitted via the fecal-oral route. It can be detected in the blood several
days before the onset of clinical signs of CNS involvement seen in patients who develop
paralytic or nonparalytic poliomyelitis (Melnick, 1996a,b). Antibodies to the virus appear early
in .the infection, even before the paralysis appears. In an infection, poliovirus will multiply first
in the tonsils, lymphoid nodes of the neck, Peyer's patches, and small intestine. The CNS is
finally invaded when the virus circulates in the blood of a patient (Melnick, 1996a).
5.3.1.3 Immunity
An infection with poliovirus confers immunity for life. Antibodies to poliovirus infection
are developed 7 days after infection.
5.3.2 Cozsacidevirus GrougjV Health Effects
Coxsackievirus A is associated with several diseases including aseptic meningitis,
infantile diarrhea, encephalitis, acute hemorrhagic conjunctivitis, and upper respiratory diseases.
CDC (1998) indicates that the most common cause of HFMD is coxsackievirus A serotype 16.
HFMD is discussed in detail under enterovirus type 71 because it killed over 69 children recently
in July 1998 in Taiwan. Melnick (1996a,b) stated that the pathogenesis of the nonpolio
enteroviruses (coxsackievirus A and B and echovirus) is similar in the initial stages of infection
except that the target organs differ (CNS or heart muscle). Kogan et al. (1969) reported the
seasonality of coxsackievirus infection in late summer and fall. The reported diseases associated
with coxsackievirus group A are summarized in Table 5-1.
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TABLE 5-1 -
Coxsaekievirus Group A-Associated Diseases
Disease/condition
Aseptic meningitis
Hand-foot-and-mouth
disease
Infantile diarrhea
Acute hemorrhagic
conjunctivitis
Encephalitis
Heart disease (pericarditis)
Myocarditis
Guillain-Barre" syndrome
Epidemic myalgia
Upper respiratory illness
Herpangina
Acute lymphatic or nodular
pharyngitis
Paralytic disease
Hepatitis/hepatic necrosis
Common cold
Undifferentiated febrile
illness
Croup
Ocular disease
CoxsacMevirus
group A serotypes
Al, 2,3,4,5,6,7,
8,9,10,14,16,17,
18,22,24
A5, 10, 16
A 18, 20, 21, 22, 24
A 24
A'2,5,6,7,9 .
Al
A 4, 9, 16
A 2, 5,6,9
A 4, 6, 10
A 21, 24
A 2, 3, 4, 5, 6, 8, 9,
10,22
A 10
A 7, 9
A 4, 9
A 21, 24
A 5, 6
A9
A 9, 10, 16
Reference
Cherry, 1995; Berlin & Rorabaugh,
1993; Dalldorf & Melnick, 1965;
Kibrick, 1964
Dalldorf & Melnick, 1965
Dalldorf & Melnick, 1965
Dalldorf & Melnick, 1965
Cherry, 1995; Kibrick, 1964
Melnick, 1996; Kibrick, 1964
Melnick, 1996; Kibrick, 1964
Kibrick, 1964
Kibrick, 1964
Kibrick, 1964
Modlin, 1997; Cherry, 1995; Dalldorf
& Melnick, 1965; Kibrick, 1964;
Dalldorf & Melnick, 1965
Cherry, 1995; Dalldorf & Melnick,
1965
Dalldorf & Melnick, 1965
Dalldorf & Melnick, 1965
Dalldorf & Melnick, 1965
Kibrick, 1964
Kibrick, 1964
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5.3.3 Coxsackie Group B-Associated Health Effects
About 5% of all symptomatic coxsackievirus B infections include heart disease (Melnick,
1996a,b). Enteroviruses are associated with aseptic meningitis. Leonard! et al. (1993) suggest
that aseptic meningitis disease outbreaks can vary by location and season and that the frequency
of association of aseptic meningitis is greater for coxsackievirus B and some serorypes of
echovirus (9, 12, 30) than for other enterovirus serorypes.
Some studies suggest that coxsackievirus group B can be an etiological agent for juvenile
diabetes mellitus. Yoon et al. (1979) reported the isolation of coxsackievirus group B4 from the
pancreas of a 10-year-old boy who,died of diabetic ketoacidosis. Homogenate from the patient's
pancreas was inoculated into human embryonic kidney cells, mouse and monkey cell cultures
produced diabetes in mice. The clinical profile and the results obtained from the animal studies
show that the diabetes onset was virus induced. The authors suggest that if coxsackievirus B was
the etiological agent that produced the disease, it is possible that juvenile diabetes is caused by
more than one virus type or group.
Table 5-2 summarizes the coxsackievirus group B-associated diseases.
5.3.4 Echovirus-Associated Health Effects
As discussed in the general disease profile for enteroviruses, various reports associate
aseptic meningitis with enteroviruses. The frequency of this association, however, is greater in
some serotypes of echovirus (9, 12, 30) and coxsackievirus B (Leonardi et al., 1993). The
serotype associated with an aseptic meningitis disease outbreak can vary by location and season
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TABLE 5-2
Coxsackievirus Group B-Associated Diseases
Disease/condition
Aseptic meningitis/
meningoencephalitis
Hand-foot-and-mouth
disease
Infantile diarrhea
Acute hemorrhagic
conjunctivitis
Diabetes mellitus
Infantile pneumonitis
Meningoencephalitis
Heart disease (pericarditis)
Myocarditis
Pneumonia, upper
respiratory illness
Epidemic myalgia
Acute lymphatic or nodular
pharyngitis
Paralytic disease
Hepatic necrosis/hepatitis
Croup
Undifferentiated febrile
illness
Pleurodynia
Encephalitis
Coxsackievirus
group B serotype
B 1,2,3,4,5,6
Bl
B2
Bl
B4
B9, 16
B 1,2,3, 4,5
81,2,3,4,5
81,2,3,4,5
84
Al,2,3,4,5
B2
B 2, 3, 4, 5
B2,3,4,5
B5
Bl,3
81,2,3,4
81,2,3,4
Reference
Modlin, 1997; Cherry, 1995; Berlin &
Rorabaugh, 1993; Dalldorf &Melnick,
1965
Cherry, 1995; Dalldorf & Melnick, 1965
Dalldorf & Melnick, 1 965
Dalldorf & Melnick, 1965
Melnick, 1996
Melnick, 1996
Melnick, 1996
Melnick, 1996; Kibrick, 1964
Gerba, 1996; Melnick, 1996; Cherry, 1995;
Kibrick, 1964
Melnick, 1996
Kibrick, 1964
Dalldorf & Melnick, 1965
Cherry, 1995; Dalldorf & Melnick, 1965;
Kibrick, 1964
Cherry, 1995; Dalldorf & Melnick, 1965
Kibrick, 1964
Dagan, 1996; Dalldorf & Melnick, 1965
Dalldorf & Melnick, 1965
Cherry, 1995
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and can be influenced by the socioeconomic conditions of a community. Leonardi et al. (1993)
report an outbreak of echoviras type 30 infection in Nassau County in New York.
Kogan et al. (1969) conducted a 2-year virus surveillance watch for echovirus and
coxsackievirus infections in New York families. They reported that echovirus infections
occurred in all months except May and June while coxsackievirus was not encountered in the
first 5 months of the year. They also showed that the seasonality of echovirus was longer than
that of coxsackievirus. All of the diseases associated with echoviruses are summarized in Table
5-3.
5.3.5 Enterovirus Types 68.69. 70. and 71
The information on the numbered enterovirases overall is limited, but an evaluation of
existing literature reveals that the numbered enteroviruses 68,69,70, and 71 are associated with
respiratory illness, hemorrhagic conjunctivitis, encephalitis, meningitis, and paralytic disease.
Enterovirus type 71 is the second most common cause of HFMD. It is also associated
with aseptic meningitis, encephalitis, and a poliolike paralysis (CDC, 1998c). Enterovirus type
71 has recently received worldwide attention because of the devastating Taiwan outbreak that
claimed the lives of more than 69 infants and young children. The Taiwan outbreak caused
HFMD, aseptic meningitis, and encephalitis among young children (CDC, 1998c,d).
HFMD occurs worldwide and is a common childhood rash illness. It is characterized by
fever, sores in the mouth, and a rash with blisters. The sores usually begin within 2 days of the
initial fever and first appear as red spots that blister and then become ulcers. The associated rash
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TABLE 5-3
Echovirus-Associated Diseases
Disease/syndrome
Aseptic meningitis
Aseptic meningitis
Aseptic meningitis
Paralytic disease
Encephalitis
Guillan-Barre syndrome
Exanthematous disease
Pericarditis
Myocarditis
Diarrheal disease
Respiratory disease
Lymphadenopathy
Neonatal infection
Epidemic myalgia
Serotype
type 4, 6, 9, 11, 14, 16,
30
type 30
type 9, 5, 21, 12
type 3, 4, 6, 7, 9, 11,
14,18,19
type 3, 4, 6, 7, 9, 11,
14,18,19
type 6, 22
type 4, 9, 16
type 1, 9, 19
type 6, 9
type 18
type 4, 9, 11,19,20,25
type 9
type 11
type 1,6, 9
Reference
Melnick, 1996; Berlin &
Rorabaugh, 1993; Melnick, 1965
Rice et al., 1995; Helfand et al.,
1994; Kaplan, 1970
CDC, 1997; Leonardi et al,
1993
Melnick, 1996, 1965
Melnick, 1996, 1965
Melnick, 1996, 1965
Melnick, 1996, 1965
Melnick, 1996, 1965
Melnick, 1996, 1965
Melnick, 1996, 1965
Melnick, 1996, 1965
Melnick, 1996, 1965
Modlin & Kinney, 1987
Melnick, 1996
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is usually found on the palms of the hands and soles of the feet. Not all the above symptoms may
be manifested in every infected person (CDC, 1998d).
HFMD infection is spread by person-to-person contact through nose and throat secretions
such as saliva and nasal mucus and by the stool of infected persons. The highest risk of
contagion is in the first week of illness. There is usually a 3- to 7-day period from infection to
manifestation of symptoms (CDC, 1998d).
International travelers are at risk of exposure to HFMD if they travel to a country
experiencing an outbreak, as in the case of Taiwan. The greatest risk of death is for children
younger than 3 years old. CDC indicates that the risk of death associated with the outbreak was
1/10,000, comparable to the annual death rate from vehicle accidents of 2/10,000 persons of all
ages (CDC, 1998d).
Entero virus type 70 has a different mode of transmission from other enteroviruses
because it is transmitted by fomites (inanimate objects) and by direct inoculation of the
conjunctiva from infected fingers (Melnick, 1996b). The incubation period of enterovirus type
70 is reported to be only 12-72 hours, which is very short compared to the average incubation
period of 7-14 days for other entero viruses. Enterovirus type 70 replicates preferentially at
33-35 °C. This is a much lower temperature than the gut of humans where other entero viruses
multiply. Melnick (1996b) indicates that the lower replication temperature could be an
adaptation to conjunctiva! temperature.
A summary of the diseases associated with the numbered enteroviruses is presented in
Table 5-4.
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TABLE 5-4
Enteroviruses Type 68-, 69-, 70-, and 71-Associated Diseases
Disease/syndrome
Respiratory illness
Hemorrhagic
conjunctivitis
Hand-foot-and-mouth
disease
Encephalitis
Meningitis
Paralytic disease
Enterovirus
type
68, 69
70
71
,- 71
71
71
Reference
Metcalf etal., 1995;Lederberg, 1992
Metcalf et al, 1995; Lederberg, 1992;
Zaoutis and Klein, 1998
CDC, 1998a; Metcalf et al., 1995;
Lederberg, 1992
Metcalf etal., 1995; Lederberg, 1992
MetcalfetaL, 1995; Lederberg, 1992
Metcalf etal., 1995; Lederberg, 1992
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5.3.6 Hepatitis A Virus Health Effects
HAV is one of the most common causes of infectious jaundice in the world today. The
name hepatitis is derived from the Greek word "Hepar" for liver because the human liver is the
primary attack site of hepatitis A and all the other hepatitis viruses (B, C, D, E, and F). Only A
and E are waterborne viruses (Grabow, 1997).
Different terminologies are used in various publications to describe the disease caused by
hepatitis A virus, and consequently, health effects publications use these terms interchangeably.
The equivalent terminologies for hepatitis A are "epidemic jaundice" as opposed to homologous
jaundice caused by hepatitis B, "short incubation hepatitis" as opposed to long-incubation
jaundice caused by hepatitis B, and "infectious hepatitis" as opposed to serum or transfusion
hepatitis caused by hepatitis B. The preferred and consistent terminology that has replaced all
previous designations mentioned above is viral hepatitis type A or hepatitis A (U.S. FDA, 1992;
LennetteetaL, 1985).
Hepatitis A is a nationally notifiable disease in the United States. Data for nationally
notifiable diseases are reported by the 50 states, the District of Columbia, and the U.S. territories
and are published weekly in the Morbidity and Mortality Weekly Report (mmwr/about.htm 1998)
Since 1991, the number of reported cases of hepatitis A has increased nationwide (CDC, 1998).
In 1996, the rate of hepatitis A in the western United States was threefold the average rate in
other regions (CDC, 1998). The Hepatitis Foundation estimates that 10 million cases of hepatitis
A infection occur worldwide each year. The foundation also reports that 152,000 of those cases
occur in the United States with 100 deaths attributed to hepatitis A each year. The annual cost
associated with hepatitis A in the United States has been estimated at $200 million (1991 dollars)
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by the Hepatitis Foundation (1998). The worldwide incidence of hepatitis A is estimated to cost
$1.5 to 3.0 billion dollars in health care annually (Hollinger and Ticehurst, 1996). The total
number of all viral hepatitis cases reported to the National Morbidity Reporting System of the
CDC in the United States in 1993 was 43,012. Of these cases, 56% were reported to be HAV,
31% as HBV, and 11% as HCV (Hollinger and Ticehurst, 1996).
Viral hepatitis surveillance in 1993 revealed an association between HAV and race
(ethnicity), with international travel as a risk factor. Hispanic patients accounted for 47%, non-
Hispanic whites 43%, Asian Pacific Islanders 8%, and non-Hispanic blacks accounted for less
than 2% of travel-related cases. There was also an association between race and location visited.
Of the Hispanic patients, 92% had visited South/Central America, and 75% of non-Hispanics had
visited the same region (Hepatitis Surveillance, 1996).
Hepatitis infection is more prevalent in males than females with an attack ratio of 2:1. In
a Baltimore hepatitis epidemic that occurred from November 1988 to December 1989, of 607
reported cases, 57% occurred in males (Stone et al., 1993).
5.3.6.1 Clinical Symptoms
HAV is known clinically for its association with a short and acute onset of highly
infectious and catarrhal jaundice. Hepatitis A symptoms are classified into 3 groups with 3
phases; severely symptomatic, symptomatic, and asymptomatic. The disease can be an
inapparent hepatitis (asymptomatic), anicteric hepatitis (symptomatic without jaundice), and
icteric hepatitis (symptomatic with jaundice) (Shapiro, 1997). Approximately 75% of adults
infected with hepatitis A are symptomatic (anicteric and icteric) (Koff and Galambos, 1987).
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Most cases of HAV are mild and anicteric, and often go unreported, leading to an inaccurate
determination of the incidence in the population (Levinthal and Ray, 1996).
The most common source of transmission of HAV is person to person. Household spread
has been reported by Shapiro (1997) to be 25-30%. Day care centers are reported to be the
second most common source of secondary person-to-person spread (15%). A 1998 HAV
epidemic in Baltimore, Maryland, resulting in 607 reported cases consisting of 57% males,
showed that 56% of children and 21% of adults had contact with confirmed and suspected cases
of HAV (Stone et al., 1993).
The average incubation perjod for hepatitis A is 30 days, with a range of 15 to 50 days
(Alter and Mast, 1994; Koff, 1992; Shapiro, 1997; Shapiro et al., 1997). The basic function of
the liver is the removal of bilirubin from the blood system. Infection with hepatitis virus causes
liver failure and an accumulation of bilirubin, which is yellow. When bilirubin becomes visible
in the eyes and palms of an infected person, it is called jaundice.
Gerba et al. (1996a) indicate that there is no increased risk to the sensitive subpopulation
from infection due to HAV. There is also no report of increased risk of pregnancy complications
for infected pregnant women (Dinsmoor, 1997; Koff, 1992).
According to Shapiro (1997), the fatality rate from reported cases of HAV is 0.4%, This
ratio can be higher in older persons (>40 years) (Shapiro et al., 1997). Alter and Mast (1994)
indicate that the overall reported case fatality rate is low (less than 1/1000). Tolsa and Bryant
(1976) report a 0.2% case fatality rate, whereas Levinthal and Ray (1966) gave a figure of as
much as 35%, but this was given 32 years ago in 1966. An 80% or greater fatality rate has been
reported for infants with neonatal hepatitis within 1-3 weeks of infection (Modlin, 1997).
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5.3.6.2 Seasonality
The highest incidence of HAV is reported to be in the autumn months and generally in
young children from age 1 to 15 years. Levinthal and Ray (1966) suggest that there is a
worldwide shift from children to adults in age-specific attacks of HAV, which has resulted in
decreased immunity in the adult population.
5.3.6.3 Immunity
Infection and recovery from hepatitis A infection confer lifetime immunity. Once a
person is infected, the immunoglobulin M (IgM) anti-HAV antibodies appear in the blood and
are usually a confirmation of acute hepatitis A infection. After several months following
infection, the IgM anti-HAV antibody titer decreases, giving rise to an IgG anti-HAV antibody
that persists indefinitely. Blood from donors positive for anti-HAV IgG is usually rejected by
blood banks (Hepatitis Foundation., 1998).
Nasser (1994) reports that HAV is the most commonly reported agent, and its prevalence
is related to the socioeconomic level of the population.
5.3.6.4 Vaccine
Two hepatitis A vaccines are currently licensed in the United States, HAVRIX and
VAQTA. Both are vaccines made from inactivated viruses (CDC, 1996b), The two vaccines are
licensed for persons older than 2 years of age and are administered intramuscularly (Shapiro et
al, 1997).
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5.4 Minimal Infective Dose
A minimal infective dose (MID) is the minimum concentration of a pathogenic organism
required to produce infection in a human or animal. A virus can enter the body through several
routes: ingestion, eyes, skin, genitourinary tract, inhalation, and even bathing (Dufour, 1986).
But MID studies are generally conducted by the oral and nasal entry sites because the respiratory
and GI tract are essentially the most important modes of entry for viruses (Ward and Akin, 1984).
Some enteric viruses such as coxsackievirus A21 have been shown to infect the respiratory tract
more frequently than the intestinal tract. Since the primary site of enterovirus replication is the
intestine, infectious dose studies in humans have been conducted by the oral route only with
poliovirus and echovirus type 12 (Ward and Akin, 1984). In theory, however, the minimum
infective dose is one single infectious virus particle.
In a search and review of available information, it became clear that only a few
publications were available on this subject. Since human volunteers are necessary for such
testing, and feeding them strains of pathogenic viruses that may produce myocarditis or other
diseases known to be caused by enteroviruses is not an option, other means of determining
infectivity must be sought. The few available publications have all referred to the human
volunteer study of Schiff et al. (1984), who conducted a study with 149 healthy human volunteers
with no detectable antibody to determine the minimum infective dose of echovirus 12. The
volunteers were given various doses of echovirus 12, ranging from 0 to 330,000 PFU of virus
suspended in nonchlorinated water. A fourfold or greater rise in antibody titer or fecal shedding
of virus was considered an infection. An HID50 (dose required for the infection of 50% of
volunteers) was given as 919 PFU. Using a probit statistical transformation, the investigators
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showed the average minimum infective dose to be 17 PFU. Schiff s study also showed that a
dose of 1,500 PFU of echoviras 12 was required to infect 60% of healthy volunteers with no
detectable levels of neutralizing antibody, while the same dosage was required to reinfect 72% of
previously infected volunteers.
Studies on minimum infective dosage on human volunteers have often used vaccine
strains rather than wild-type strains. Koprowski et al. (1956) conducted a study on human
volunteers with poliovirus type 1 in gelatin capsules. Data indicated a minimum infective dose
of 2 PFU. The minimum infective dose for most enteric viruses studied is considered to be very
low (Gerba and Rose, 1993; Payment, 1993; EPA, 1985; Ward and Akin, 1984). The minimum
dose required to initiate an infection varies from organism to organism and from host to host. A
number of factors are involved in the interactions that lead to an infection. These factors include
immune status, age, and underlying health effects. The most important factor, however, is the
immune status of the host.
Duncan and Edberg (1995) indicate that an initiation of an infection requires the
interaction of three factors: (a) the number of microorganisms, (b) virulence characteristics, and
(c) immune status of host. The authors explain the relationship among the key factors in
infection with an equation, as follows:
(Infection = # of microbes x virulence characteristics/immune status of the host).
For an infection to occur, however, a target organ must first come into contact with a sufficient
number of virulent microbes. The target organ, at the same time, should be susceptible and be
overwhelmed. The most commonly used units for describing infectious dose are the PFU and the
(ID50) or HID50 (the infectious dose/human infectious dose that will infect 50% of individuals) or
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LD50 (dosage that is lethal to 50% of challenged individuals). Although a single virion can infect
the target cell, the infectious or lethal dose can vary from one to many thousands (EPA, 1985).
An infectious dose value comparison by Duncan and Edberg (1995) for the various
common GI pathogens is presented in Table 5-5. The infectious dose value for enteroviruses is
shown to be from 1 to 4. However, it should be noted that with viruses, there can be both
infective and noninfective virions present, creating competition for the host's cell receptors and
thus preventing infection. In addition, the initial exposure dose may be low enough to be
overcome by the individual's immune system and yet high enough to cause infection in another
individual, further confounding the application of infectious dose patterns in real-life situations.
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6.0 Mechanisms of Disease
6.1 Factors That Affect Disease Occurrence
The initiation of a disease condition in a healthy individual is dependent upon the
virulence of the microorganism and the overall susceptibility of the individual to infection.
6.1.1 Virulence
Virulence is the ability of a pathogenic organism to cause disease. The virulence of a
virus strain involves numerous genetically controlled factors. The primary factor is the ability of
the virus to multiply and destroy the infected host cells. A virulent virus strain can multiply at
human fever temperatures. Dodet et al. (1997) reported that the severity of the clinical
symptoms, such as diarrhea, vomiting, and dehydration,, is dependent on the virulence of a strain,
and its ability to destroy host cells.
6.1.2 Susceptibility of Host Cells
6.1.2.1 Cell Receptors
The receptor sites on a host are very important in viral susceptibility. Receptor site
specificity makes it possible for a virus to attack one cell type but not another. Some other form
of activation may still be required before a given virus can attach and begin the infection process.
Viruses display a high degree of specificity with regard to their host range and to the specific cell
type and organ that can be invaded. Coxsackie B viruses have been shown to use a 100-KDa
specific surface antigen on host cells for entry (Verdugo et al., 1995). Echo virus 7 utilizes the
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decay-accelerating factor as its binding site on cell surfaces and Clarkson et al. (1995) have
shown that antibody against the receptor interferes with echoviras 7 binding, Echovirus 1 and 8
appear to use a specific antigen receptor found on the surface of human cells that differs from
that used by echovirus 22 (Bergelson et al., 1993).
6.1,3 Secondary Spread
A primary enteroviras infection usually occurs through the ingestion of contaminated
water. Enteroviras infection can also be spread through person-to-person contact, by food, or by
contact with contaminated fomites (inanimate objects). Secondary spread attack rates for
enteroviruses have been reported by Gerba and Rose (1993) to be 90% for poliovirus, 75% for
coxsackievirus, 45% for echovirus, and 76% for HAV.
6-1-4 Sensitive Subpopulations .
A sensitive subpopulation is that segment of the population that is at a higher risk for
infection than the rest of the population. It has been estimated that the sensitive subpopulation
constitutes 20% of the entire population in the United States (Gerba et al., 1996a). Included in
this group are infants; the elderly; burn victims; the immunocompromised, such as those with
acquired immune deficiency syndrome (AIDS); radiation and chemotherapy patients; diabetics;
and pregnant women. The number of persons at higher risk in the United States has substantially
increased since 1985. Individuals that comprise the sensitive subpopulation group are presented
in Table 6-1.
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TABLE 6-1
Sensitive Subpopulations in the United States
Subpopulation
AIDS patients
Pregnant women
Organ transplant patients
Cancer patients
Neonates
Elderly (over 65)
Nursing home residents
Number of
individuals
64,966
6,272,000
17,095
1,853,795
4,002,000
29,400,000
1,553,000
Year
1997
1996
1994
1992
1989
1989
1986
Reference
EPA, 1997a
EPA, 1997a
EPA, 1997a
EPA, 1997a
GerbaetaL, 1996a
Gerbaetal., 1996a
Gerbaetal., 1996a
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The severity of a disease cannot be assumed equivalent between the normal population
and the population at higher risk. Diseases that may not be serious for a normal person could
cause devastating effects in persons with depressed immune systems. Immune suppression is
defined by Ades et al. (1992) as the dampening of the natural occurrence of the process of
nonself recognition known by a,repertoire of negative responses that occur during a normal
immune response. Immune suppression can be caused by a variety of factors such as viral
infections, chronic illnesses such as cancer, diabetes, chemical and radiation therapy, physical
trauma such as burns and surgery, and natural causes such as age and pregnancy that are also
associated with a suppressed immune system. Exposure to many viruses causes a transient
immune suppression since many viruses undergo replication in the cells of the immune system
such as the T cells, macrophages, and bone marrow cells. Some viruses such as polio can
suppress the immune system by activating prostaglandin syntheses and thereby leading to the
impairment of the ability of T cells to respond to cytokines (Ades et al., 1992). According to
Dodet et al. (1997), enteric viruses can cause more severe and persistent diarrhea in patients with
reduced immunity. Modlin (1997) indicates that 80% of infants infected with neonatal hepatitis
die within 1-3 weeks.
6.2 Chronic Sequelae
Human exposure to coxsackievirus can in extreme cases result in serious illness such as
myocarditis and insulin-dependent diabetes mellitus (Melnick, 1996b; Yoon et al., 1995).
Melnick (1996b) indicates that about 5% of all symptomatic coxsackievirus infections result in
heart disease. A mortality rate of 50-60% from coxsackievirus-related myocarditis in infants in
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two nursery outbreaks was reported by Gerba et al. (1996a). Modlin (1995) reported that most
patients infected with coxsackievirus and eehovirus encephalitis beyond the neonatal period
eventually recover fully. However, chronic neurologic sequelae following encephalitis have been
associated with eehovirus type 9 and other enteroviruses (Kibrick, 1964). Static neurologic
sequelae and rare deaths have also been reported (Modlin, 1997).
Patients with aseptic meningitis due to enterovirus infection generally recover
uneventfully, but fatigue can persist for months after the acute illness (Modlin, 1995).
In a case study conducted in a hospital serving a predominantly black population, 6 out of
7 children from 2 months to 12 years hospitalized for paralysis due to coxsackievirus B3
infection, died of multiple limb paralysis (Yui and Gledhill, 1991).
Enterovirus hepatitis has been linked to chronic hepatic insufficiency. Survivors of
neonatal hepatitis may develop cirrhosis and chronic hepatic insufficiency (Modlin, 1997).
6.3 States of Disease
Two states of disease, apparent and inapparent, can occur in response to a viral infection.
In an apparent disease state, there are clinical manifestations. In an inapparent disease state, the
infection is subclmical and there is no obvious manifestation of disease. Both states result in the
production of an immune response to infection.
A viral infection may be either localized or disseminated. When the infection is
disseminated throughout the body, it can lead to viremia (virus in blood), and the lymphatic and
blood system will carry the virus to other target organs, resulting in an amplification of the viral
load.
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6.3.1 Apparent Infections
Apparent infections for enteric viruses are usually accompanied by GI illnesses of varying
severity. In a HAV infection, jaundice would result. Coxsackie infections are frequently
accompanied by severe diarrhea, vomiting, dehydration, and even death (especially in small
children). Clinical manifestations of virus infections include nausea, vomiting, diarrhea,
abdominal cramps, headache, fever, chills, myalgia, and sore throat. These may last from 2 hours
to several days, but more than likely 12-60 hours (EPA, 1996).
6.3.2 Inapparent Infections
Inapparent infections are those which result in no apparent symptoms or very mild
symptoms, such as a mild and short-lived diarrhea, loose stools, or no apparent disturbances in
bowel movement pattern. However, individuals with inapparent infections will still produce an
antibody response that may confer long-term immunity. It was reported by Schiff et al. (1984)
that antibodies to echovirus 12 do not appear to protect individuals from reinfection. It also has
been shown that 50-75% of adults in the United States and more in other countries test positive
for enterovirus antibodies even though most remain asymptomatic (EPA, 1996).
6.4 Host Defense Systems
The immune system is made up of two major groups of components (EPA, 1997). The
first group is specific and requires activation by a foreign substance (antigen or immunogen). It
consists of antigen-presenting cells, B-lymphocytes, T lymphocytes, and cytokines which up-
regulate or down-regulate either a humoral or cell-mediated response. The second group of
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defenses consists of a nonspecific component that does not require activation by an antigen. This
group includes natural killer (NK) cells, phagocytic cells, and chemokines that inhibit foreign
antigens or prevent the replication of viruses in uninfected cells. The two groups of responses
are important in eradicating viral infections or in the establishment of immunity after exposure to
viral antigens.
6,4.1 Antibodies
The progenitors of antibodies are derived from the bone marrow and the thymus. The B-
cells (from bone marrow) differentiate into cells that are endowed with specific immunoglobulin
(antibody)-producing capabilities. These cells will mature and be present in the spleen, lymph
nodes, and mucosal sites. Upon encountering a given virus, these mature cells will secrete
specific antibody molecules capable of reacting specifically to the virus strain that induced them.
T-cell progenitors, which originate in the thymus, give rise to mature T-cells that can recognize
each antigen present in the vast universe of antigens (more than 108 molecular arrangements).
After clonal selection of T-cells has been completed, these thymocytes are exported to the
peripheral lymphoid tissues (spleen, lymph nodes, and mucosal sites) where the unique T-cell
receptors are capable of binding with specific viral antigens. Certain T-cells are capable of
lysing virus-infected cells. The mucosal sites are especially important for protection against
enteroviruses because of the intimate contact with viruses that occurs upon invasion of the GI
mucosa. Mucosa-associated lymphoid tissue (MALT) represents a major line of immunological
defense in the GI tract (EPA, 1997). B-cells, T-cells, and antigen-presenting cells (APC)
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comprise the MALT and interact cooperatively to initiate and sustain IgA antibody formation.
The MALT protects humans against viral invasion of the GI system.
About 50-75% of adults in the United States have antibodies to enterovirus even though
most of these adults may remain asymptomatic (EPA, 1996; Modlin, 1997). Antibodies to
echovirus 12 are not protective (Schiff et al,, 1984). Antibodies to poliovirus infection are
developed as early as 7 days following infection and before onset of paralysis (Melnick, 1996).
Antibodies to HAV infection confer lifetime immunity (Hepatitis Foundation, 1998).
A cross-sectional survey of the seroprevalence of HAV antibodies was conducted in a
healthy population in Nicaragua by, Perez et al. (1996), The results indicate that the overall
prevalence of antibodies to HAV was 94.6%. The authors concluded that HAV is a childhood
disease in Nicaragua, and that the spread of the infection is facilitated by poor socioeconomic
conditions.
6.4.2 Cell-Mediated Immunity
In addition to the specific T-cell receptors (TCRs), mature T-cells express other important
surface molecules, such as T-helper cells and the T-cytotoxic (Tc) cells, which are capable of
recognizing viral antigens on the infected target cells, reacting with them, and destroying the
cells. T-helper (TH) cells stimulate the expression of numerous cytokines which serve to enhance
the immune response mechanism. It also results in the stimulation of antiviral factors such as
interferon that inhibit the intracellular replication of viruses in uninfected cells. The Tc cells are
responsible for defense against viruses and other intracellular pathogenic microorganisms (EPA,
1997).
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6.4.3 Nonspecific Factors in Immunity
Nonspecific factors that protect against infection include NK cells that are specialized in
killing aberrant target cells such as certain virus-infected cells. Unlike Tc cells, the NK cells are
nonspecific with regard to the virus-infected target cells they destroy. They are activated by
cytokines, and once activated will produce enzymes that are toxic to target cells, such as
enterovirus-infected cells (EPA, 1997).
6.5 Summary
The ability of a virus to produce disease and the severity of the disease depend on the
virulence of the virus and the susceptibility of the host. A primary infection occurs by the
ingestion of contaminated water. But infection can occur by person-to-person contact.
Secondary spread of enterovirus infections ranges from 45% for echovirus to 76% for HAV and
90% for Poliovirus. The diseases produced by enteroviruses are mostly asymptomatic.
Poliovirus can suppress the immune system by interfering with the ability of T cells to respond to
cytokines. Chronic sequelae due to enterovirus include myocarditis and diabetes mellitus.
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7.0 Risk Assessment
7,1 Introduction
A pathogen risk assessment is defined by the International Life Science Institute (ILSI)
Risk Science Institute Pathogenic Assessment Working Group, as a process that evaluates the
likelihood of adverse human health effects following exposure to pathogenic microorganisms in
a medium such as water (ILSI, 1996). The assessment of risk to human health is currently
patterned after a widely accepted paradigm on chemical risk assessment developed by the
National Academy of Sciences (NAS) in 1983. This paradigm was reiterated by NAS in 1994
(Science and Judgement in Risk Assessment, NAS, 1994). *
7.2 NAS Risk Assessment Framework Document
The NAS risk assessment paradigm contains four elements in its framework: hazard
identification, dose response assessment, exposure assessment, and risk characterization.
7.3 Ecological Risk Assessment Framework
Another framework for risk assessment is the ecological risk assessment, which is similar
to the NAS human health approach. It differs in three areas, however. First, the ecological
framework can examine effects on a population, ecosystem, or community instead of individuals
of a single species. Second, ecological risk assessment considers nonchemical, as well as
chemical stressors, such as loss of habitat, instead of toxic stresses induced by individual or
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groups of chemicals. Third, no single set of ecological values to be protected can be applied in
general (NRG, 1993; EPA, 1992).
7.4 Microbial Risk Assessment Framework
The microbial risk assessment framework was developed because the framework for
chemical exposures is generally regarded as inadequate for microbial risk assessment. This is
because several issues unique to pathogenic microorganisms are not accounted for in the
chemical risk assessment approach. They include:
• pathogen-host interactions,
» secondary spread of microorganisms,
» short- and long-term immunity,
* the carrier state,
• host animal reservoirs,
» animal-to-human transmission,
» human-to-human transmission, and
» conditions that lead to multiplication of microorganisms.
An alternative framework, recently developed by the ILSI Pathogen Assessment Working
Group, is presented in Figures 7-1 and 7-2. It consists of three phases: problem formulation
phase, analysis phase (human exposure characterization and human health effects), and risk
characterization phase. Pathogen properties that affect the ability of the organisms to be
transmitted, to infect, and to cause disease in the host must be considered in microbial risk
assessment. In addition, intrinsic genotypic and phenotypic characteristics that influence host
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Figure 7-1.
Generalized framework for microbial risk assessment of
\\atcrborne contaminants
MICROBIAL RISK ASSESSMENT
Problem Formulation
A 1 I
N
A
L
Y
S
i
characterization
of
exposure
characterization
of
human health
effects
s 1 I
V
Risk Characterization
Adapted from; ILSI Risk Science Institute Pathogen Risk Assessment Working
Group, 1996.
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Figure 7-2.
Analysis phase of microbial risk assessment
Characterization of exposure
Pathogen
characterization
Exposure
analysis
Hazard
occurrence
Exposure
profile
Characterization of
human hea|th effects
Host
characterization
Host
microbe
profl|e
Adapted from: ILSI Risk Science Institute Pathogen Risk Assessment Working Group, 1996.
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specificity, pathogenicity, virulence, multiplication, and survival in the environment uniquely
distinguish a living, replicating pathogen from chemicals. Transmission pathways can be
important in determining the entry portal as well as the potential for secondary spread and
multiplication. The transmission of diseases via fecal/oral route and secondary spread for
enterovirus was discussed in the health effects section (Chapter 5).
Understanding the pathogen's niche, as well as its seasonally of occurrence, plays a very
important role in exposure characterization. In addition, establishing the host-pathogen profile
involves many more factors than does the dose-response analysis used in chemical risk analysis.
Eisenberg et al. (1996) recently developed a new approach to quantify waterborne
pathogen risk based on an epidemiological framework. Their approach uses Monte Carlo
simulation with input on identified conditions that occur in either outbreak or nonoutbreak
conditions. The approach also accounts for the uncertainties associated with the risk prediction.
Traditionally, microbial risk assessors use point estimates to evaluate the probability that
an individual will be infected by a waterborne pathogen. This approach shifts the risk
characterization to a distributional estimate, and from a single individual to the population as a
whole. The model tracks the traditional epidemiological variables such as the number of
individuals that are susceptible, infected, diseased, or immune. It also takes into account virus
shedding and concentration.
The Monte Carlo simulation acknowledges the uncertainty and variability of the data and
assigns probability distributions to each of the parameters. Case studies can be analyzed by
sampling from these distributions for Monte Carlo simulations, using a binary classification to
assess the output of each simulation. Eisenberg et al. (1996) used literature-based information to
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assign parameter ranges and discovered that pathogenic microorganism shedding was an
important contributor to the uncertainty of the risk.
The two risk assessment approaches discussed above, the ILSI approach and the
Rosenberg approach, focus on those parameters that make risk assessment for waterborne
pathogens uniquely different from toxic chemicals that may be present in our drinking water
sources.
The normal population and sensitive subpopulation in the United States are at risk for
waterborne virus infections. There is substantial evidence of gastrointestinal illness due to
enteric viruses. These have also occurred worldwide (Corwin et al., 1996; Nasser, 1994).
Modeling the risk from microbes in drinking water has not had much practical application
because of limitations placed on the accurate enumeration of pathogens, uncertainties associated
with infectivity and virulence, diversity in organism occurrence, and the large water sample
volumes required to demonstrate negligible risk (Haas, 1993; Regli et al., 1991). Haas et al.
(1993,1983) have developed various models for predicting risks from viruses in drinking water.
Methods for modeling waterborne risk from Giardia and viruses have been suggested
(Regli et al., 1991). Using conservative assumptions, including the dose response for rotavirus
(which is the most infectious virus for which data are available), Regli et al. (1991) calculated
that the virus concentration in drinking water should not be more than 2.22 x 10~7/L
(2 virus particles per 10 million liters of water), to achieve less than one infection per 10,000
people per year.
Gale (1996) reviewed many of the available microbial risk assessment models for
drinking water. He believes that pathogen exposure calculations in microbial risk assessment are
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of limited value because pathogen density data for drinking water supplies are available only for
large-volume samples. He indicates that such volumes are much larger than the actual amount of
water ingested by a normal consumer. Gale (1996) also points out that pathogen dispersion data
in large sample volumes were lacking. There is evidence that pathogens are not evenly
distributed, but rather clustered in water sample volumes. This means that some individuals are
exposed to larger numbers of microbes than others. A microbial assessment without such data
will overestimate the risk for some pathogens while underestimating the risk for others.
Asano et al. (1992) developed a quantitative risk assessment for several exposure
scenarios using reclaimed waste water, which was either disinfected or not disinfected. Exposure
scenarios were presented for ground water recharge, golf course irrigation, irrigation of food
crops, and unrestricted recreational use of reclaimed water. The virus concentration in the waste
water was assumed to be either 1 virus unit/100 L (limit of detection) or 111 virus units per 100
L (the highest virus concentration detected in monitored waste water). The highest annual risk
was associated with recreational use of the waste water and was as high as 10"2 for waste water
containing an assumed level of 1 virus unit/100 L. All other exposure scenarios resulted in risks
of 10"4 or lower. Thus, if a disinfected waste water containing less than 1 virus unit/100 L is
reclaimed, the associated risks of infection are very low.
\
7.5. Enterovirus Risk Assessment
7.5.1 Current Limitations to Microbial Risk Assessment
The lack of occurrence data, dose-response data, and exposure data of pathogens of
interest including enteroviruses has limited microbial risk assessments of many microorganisms.
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Waterborne outbreak reports are usually about gastrointestinal diseases (discussed in Chapter 2),
The severe chronic diseases associated with microorganisms associated with those outbreaks are
usually ignored. This leads to incomplete information for hazard identification in risk
assessment.
7.5.2 Risk Assessment of Coxsackievirus
There is no available risk assessment information on enteric viruses except that developed
on the risks associated with exposure to waterborne adenovirus by Crabtree et al. (1997) and for
waterborne rotavirus by Gerba et al., (1996b).
Mena et al. (1998) recently assessed the risk associated with exposure to waterborne
coxsackievirus type B4. The specific serotype was used because it is frequently isolated from
water and more virulent than group A coxsackievirus. In the risk assessment, the authors
considered the severity and frequency of occurrence of different diseases caused by
coxsackievirus (many of which have been discussed in Chapter 5) and the waterborne outbreaks
associated with the virus.
7.5.3 Pose-Response Model and Risk Characterization
Mena et al. (1998) evaluated the dose-response data of coxsackievirus and developed the
best-fit model for determining the probability of infection. To address the exposure scenario of
coxsackievirus, the authors summarized data on the concentration of coxsackievirus found in
sewage, treated sewage, fresh water, and drinking water, and on susceptibility to treatment
inactivation, resistance to inactivation, and persistence in the environment.
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The exponential risk assessment model was used to assess the exposure risk to
eoxsaekievirus and the point estimate developed for comparison used the following formula:
p. = 1 _ e[-(l/k)N]
Pj = probability of becoming infected
N = number of virus ingested (cell culture PFU) where N was calculated with 2
liter/person/day of drinking water for the general population, 4 liters for the elderly,
and 100 ml for daily exposure for recreational swimming in fresh water.
K = (129) was estimated from the dose-response experiments on coxsaekievirus B4 by
Suptel (1963).
The probability of clinical illness was calculated by multiplying the probability of
infection, Pi by the morbidity ratio of 0.75 (Cherry, 1981). The probability of death from an
infection was calculated by multiplying morbidity ratio (P,X) of 0.75 by case fatality ratio. Mena
et al. (1998) used a mortality ratio of 0.0059 even though the authors indicate that a higher
mortality ratio of 0.0094 had been reported for coxsaekievirus by Assad and Borecka (1977).
The risks of infection, illness, and death associated with coxsaekievirus in drinking water
(surface water and ground water) and contaminated fresh water are presented in Tables 7-1 and
7-2. The authors conclude that coxsaekievirus poses a very significant risk in water even though
epidemiological evidence for eoxsaekievirus-associated diseases transmitted by water is very
limited.
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TABLE 7-1.
Risk Associated with Coxsackievirus in Drinking Water3
Concentration/Exposure
5x10 3 MPNCU/Lb Surface Water 0.13 PFU/LC Ground Water
Day Year Day Year
Riskoflnfectiond
Riskoflllnessd
RiskofDeathd
Risk of Death6
7.75xlO'5
5.81xlO'5
3. 43x1 0-7
6.86x1 0'7
2.79x1 0-2
2.09x1 0'2
1.23xlO-4
2.43x1 0'4
2.01xlO'3
l.SlxlO'3
8.91xlO-6
1.78xlO-5
5.21x10-'
3.91x10-'
2.30xlO-3
3.41xlO'3
aMena et al. (submitted 1998).
bPayment et al., 1985 (MPNCU = most probable number cytopathic units).
cHejkal et al., 1982 (PFU = plaque-forming units).
dGeneral population at 2 L/person-day exposure.
eElderly at 4 L/person-day exposure.
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TABLE 7-2.
Risks Associated With Swimming in
Coxsaekievirus-Contaminated Fresh Water3
Enterovirus Concentration
0.67 MPNCU/L" 5.44 MPNCU/I/
IDay 5 Days 10 Days 1 Day 5 Days 10 Days
Risk of Infection6
Risk of Illness0
Risk of Death0
5.19xlO'4
3. 89x1 0-4
2.30x1 0-6
2.59x1 0'3
1.94xlO-3
1.15xlO-s
5.18xlO-3
3. 89x1 0-3
2.29x1 Q-5
4.21xlO-3
3.16xlO-3
1.86xlO-5
2.09x1 0-2
1.56xlO-2
9.23x1 0-5
4.13xlO-2
3.10xlO'2
1.83xlO'4
aMena et al. (submitted 1998).
bLucena et al., 1985 (MPNCU = most probable number of cytopathic units).
°General population at 100-mL single exposure.
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7.6 Transmission of Viruses by Drinking Water
The transmission of enteroviruses and HAV is mostly person-to-person through fecal-oral
route or oral-oral route. Several reports have shown the transmission of viruses by drinking
water. Some of the information indicates that the drinking water implicated in the outbreaks met
microbiological standards for safety (Payment, 1989; Bitton et al., 1986; Gerba et al., 1984;
Marzouk et al., 1980). Some of these reports were discussed in Chapter 2. Enteroviruses can
also be transmitted by food.
7.6.1 Endpoints
Some of the effects of infection due to enteroviruses and HAV include diarrhea, fever,
vomiting, dehydration, headaches, and jaundice. HAV infection is self-limiting and does not
result in chronic liver disease, and treatment is gradually supportive (Shapiro, 1997). The
symptoms manifested may last for several weeks and usually not longer than 2 months, although
some cases show relapsing signs and symptoms for up to 6 months (Shapiro, 1997).
Aseptic meningitis in infants due to coxsackievirus and echovirus may last for 4-6 days
(Kaplan et al., 1983), but most infants with fever will recover in 2-10 days without
complications (Modlin, 1997). Coxsackievirus B2-produced paralysis has been shown to last for
2 weeks with full recovery (Cherry, 1995). Kibriek (1964) reported that infection with
coxsackievirus B during the first month of life may result in severe and frequently fatal disease
characterized by myocarditis.
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7.6.2 Epidemiological Evidence for Viral Transmission in Water
Several studies have described the transmission of viruses in drinking water (Payment,
1993,1989; Gerba et aL, 1993). Some reports show that drinking water that had met
bacteriological standards of safely potentially could be responsible for outbreaks caused by viral
contamination. In all reported outbreaks since the surveillance collaboration began between
CDC and EPA, AGI has been associated with the greatest number of outbreaks. It does not
appear that entero viruses are responsible for many outbreaks of the identified etiologic agents;
however, HAV appears to be one of the pathogenic viruses frequently reported.
t '
7.7 Summary
The chemical risk assessment approach does not account for several unique issues
associated with pathogenic microorganisms such as pathogen-host interactions, secondary spread
of microorganisms, and multiplication of microorganisms. The ILSI microbial risk assessment
framework incorporates these issues in its framework with three phases. Risk assessment of
microorganisms is limited by the lack of information on occurrence, dose-response data, and
exposure data. The data available on outbreaks are usually on gastrointestinal illness and do not
include the chronic diseases associated with the microorganisms of interest.
Risk assessment has not been developed for the enteric viruses except for rotavirus,
adenovirus, and recently for coxsackievirus. Risks of exposure to coxsackievirus in drinking
water have been calculated based on 2 liters for the general population and 4 liters for the elderly.
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The transmission of enteroviruses and HAV is mostly person-to-person through the fecal-
oral route or oral-oral route. The epidemiological evidence for enteroviras transmission through
water is limited.
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8,0 Methodology
8.1 Introduction
Enteric viruses are found in environmental waters at very low concentrations. This
concentration is further reduced by dilution through the mixing of treated wastewater effluent
and untreated sewage with natural waters. One of the major problems in virus recovery in
environmental waters is the large volume of water that must be concentrated to obtain virus
numbers that are within the detection limits of existing technologies. Without adequate recovery
methods for the detection of viral contaminants, appropriate monitoring of water cannot be
accomplished and increased risks of infection become a very serious hazard.
Virus detection methods have been greatly improved over the last 15 years. Of all the
detection methods available today, PCR, RT-PCR is considered the most promising in water
virology.
8.1.1 Virus Concentration and Recovery
Virus sampling and enumeration in raw and finished water are important for assessing the
safety of drinking water for public consumption. The main problem in virus recovery from water
has been and still is the large volumes of sample necessary. Because of their small size, low
numbers, and difficulty in cultivation, it has been difficult to obtain an accurate assessment of the
presence of waterborne viruses. Various methods for the concentration of viruses in water have
been summarized in Table 8-1. In the past, methods developed for waterborne virus recovery
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TABLE 8-1
Methods for Concentrating Viruses from Water*
Method
Filter adsorption-elution
Negatively charged filters
Filter adsorption-elution
Positively charged filters
Adsorption to metal salt
precipitate, aluminum
hydroxide, ferric hydroxide
Polyelectrolytes - PE60
Ammonium sulfate
flocculation
Bentonite
Iron oxide
Talcum powder
Gauze pad
Volume of
Water Processed
large
large
small
large
medium
small
small
large
large
Applications
all but very turbid
waters
all but very turbid
waters; tap water
tap water, sewage
tap water, lake water,
sewage
tap water, sewage,
reconcentration
tap water, sewage
tap water, sewage
tap water, sewage
—
Remarks
Efficient method for concentrating viruses from large
volumes of tap water, sewage, seawater and other natural
waters. Cationic salt concentration and pH must be
adjusted before processing.
Efficient method for concentrating viruses from large
volumes of tap water, sewage, seawater and other natural
waters. Samples can be processed over a wide pH range
(3-9). No preconditioning of waters is generally necessary.
Have been useful as reconcentration methods.
Because of its unstable nature and lot-to-lot variations in
efficiency for concentrating viruses, the method has not
been used in recent years.
Useful when fluctuations in pH are undesirable.
Can be used in sandwich between filter paper supports to
process up to 100 L volumes.
First method developed for detecting viruses in water, but
not quantitative.
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TABLE 8-1. Continued
Method
Glass powder
Glass wool
Organic flocculation
Polyethylene glycol
precipitation
Protamine sulfate
Polymer two-phase
Hydroextraction
Ultracentrifugation
Ultrafiltration: Soluble filters
Volume of
Water Processed
large
large
small
medium
small
small
small
small
small
Applications
tap water
all but very turbid
waters
reconcentration
tap water,
reconcentration
sewage
sewage
sewage
reconcentration
clean waters
Remarks
Columns containing glass powder have been made that are
capable of processing 400 L volumes.
Works well within a wide pH range (3-9). Has shown
higher recovery efficiencies for several viruses.
Widely used method for reconcentrating viruses from
primary filter eluates.
Sample volumes were reduced 250-300 fold and levels of
viruses recovered were > 50%. May require several
precipitation steps followed by ultrafiltration.
Efficient method for concentrating Reoviruses and
Adenoviruses from small volumes of sewage.
Processing is slow; method has been used to reconcentrate
viruses from primary eluates.
Often used as a method for reconcentrating viruses from
primary eluates.
Reduced sample volume by 8,000 fold.
Clog rapidly even with low turbidity.
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TABLE 8-1. Continued
Method
Ultrafiltration: Flat membranes
Ultrafiltration: Hollow fiber or
capillary
Reverse osmosis
Volume of
Water Processed
small
large
large
Applications
clean waters
tap water, lake water
clean waters
Remarks
Clog rapidly even with low turbidity.
Up to 100 L have been processed, but water must often be
prefiltered.
Also concentrates cytotoxic compounds that adversely
affect assay methods.
*Sources: EPA, 1985; APHA, 1989; EPA, 1984.
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from raw and finished water samples have yielded mixed results. More methods continue to be
developed. The methods are discussed in greater detail in the 1985 document.
In the last 15 years, improved methods have been developed and compared for virus
recoveiy efficiency. These methods include continuous flow centrifugation, continuous
immunomagnetic capture, cross-flow filtration, and vortex flow filtration. Hock (1996) indicates
that the recovery efficiency of these methods is not quite definitive because the recovery range
may vary from about 20%-80%, even when high concentrations of virus particles are present.
8.2 Detection Methods for Viruses in Water
8.2.1 Cell Culture Assays
The conventional method for viral detection is mammalian cell culture assay. Cell
culture techniques have been available for several decades, and represent a definitive method for
determining viral infectivity in a sample. There are a few established cell lines used for viral
detection in environmental samples. These cell lines are summarized in Table 8-2. The most
commonly used cell line for culturing enteroviruses is Buffalo green monkey (BGM) kidney cell
line. The BGM cells are grown to confluent monolayers in 25 cm2 plastic flasks. The
monolayers are washed with buffered saline solution. A 1-rnL volume of concentrated virus
samples are inoculated into the flasks, which are incubated at 37 °C. The incubated flasks are
examined daily for viral cytopathic effect (CPE). Observed viral CPE is confirmed by a passage
into a fresh BGM monolayer, and a resultant CPE. Samples that show negative CPE on first
passage are passed a second time on BGM monolayers. The time period required for each
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TABLE 8-2
Commonly Used Cell Cultures for Propagating Human Enteric Viruses8
Virus
Cell Culture
Cox A Cox B
Echo
Polio
Primary
RhMk
AGMK
HEK
Diploid or
continuous
MFK
BGM
BSC-1
Vero
MA-104
HFDK
Hep-2 (HeLa)
RD
Graham 293
++
++
++
++
aSource: EPA, 1985.
bCoxsackieviruses A9 and A16 replicate in primary monkey kidney cells.
Cell cultures: RhMK, rhesus monkey kidney; AGMK, African green monkey kidney;
HEK, human embryonic kidney; MFK, MA-104, fetal rhesus monkey kidney; BGM,
Buffalo green monkey kidney; HFDK, human fetal diploid kidney; HEp-2, HeLa,
human malignant epithelial cells; RD, human rhabdomyosarcoma.
(-), Virus replicates poorly or not at all in this cell culture; (+) virus replicates in this
cell culture; (++), optimal cell culture for virus replication.
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sample assay is 30 to 45 days (Abbaszadegan and DeLeon, 1997). Although the cell culture
method is very useful for detecting cultivable viruses, it is time- consuming and expensive.
Some enteric viruses such as HAV grow poorly in cell culture and some such as Norwalk viruses
do not grow at all (Nasser et al., 1995).
Margolin et al. (1993) tested 233 water samples for the presence of polio viruses using
tissue culture assay and a poliovirus cDNA probe labeled with radioactive phosphorus. Twenty-
eight samples were positive for viruses by the tissue culture technique, and 36 samples were
positive by the cDNA probe technique while 22 samples were positive by both techniques. The
gene probe assay does not strictly correlate with infectivity, but provides a quick and reliable
screening for viral presence.
Enriquez et al. (1993) detected poliovirus by tissue culture and gene probe assays, in
order to determine whether there was a correlation between the molecular assay method and the
assay for infectious virus. In all water samples, the number of viable viruses declined over time.
There was a similar decline in virus detectable by the gene probe, except in autoclaved well
water and phosphate-buffered water samples. Although environmental nucleases could account
for the decline in nucleic acid available for gene probe analysis, the authors concluded from their
results that the gene probe would detect, mostly, viable virus in water samples.
8.3 Molecular Methods
8.3.1 PCR Assays
Newer methods for viral detection include PCR and RT-PCR assays that can specifically
identify viruses by targeting specific sequences in the target nucleic acid (DNA and RNA). Since
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the development of the PCR technique for the detection of sickle cell hemoglobin by Saiki et al,
(1985), the technique has gained wide use and acceptance in the environmental industry and has
become an important detection and diagnostic tool in clinical and environmental samples
(Abbaszadegan et al., 1993; Puig et al., 1994; Shieh et al., 1997; Straub et al., 1994). PCR assays
have been used in the detection of enteroviruses in environmental samples (Abbaszadegan et al.,
1993; DeLeon et al., 1990; Muscillo et al., 1995).
The genome of the enteroviruses and HAV is single-stranded RNA. In PCR assays, the
RNA will be converted into DNA before amplification by the polymerase chain reaction. This
conversion is through a reverse transcriptase (RT) action, hence an RT-PCR. In the use of the
PCR method for enterovirus detection, Muscillo et al. (1995) indicate that most studies use
universal primers that are selected in a highly conserved area of the 5'-noncoding region of the
enteroviral genome. Identification of the amplified fragment is based on molecular weight.
Amplification is followed by hybridization with oligonucleotide probes or by RFLP. The
hybridization, in essence, is what differentiates the enteroviruses (Muscillo et al., 1995).
The drawback of the PCR method is its inability to distinguish between infective and
noninfective viral particles. It is also difficult to use with environmental samples because of
inhibitory substances and false positive results. The PCR method requires specialized training
for result interpretation.
8.3.2 PCR Method Studies
Abbaszadegan et al. (1993) described a sensitive and specific detection method for
viruses using PCR. This method does not require the use of tissue cultures, but rather uses the
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genetic "fingerprint" of the viral RNA to determine the presence of viruses in environmental
samples. The method is specific for a number of human enteroviruses because they share a
conserved region on their genome. The authors suggest that this method is faster, simpler, and
less expensive than the standard cell culture detection methods. However, with the PCR method,
there is a problem with organic substance interference in the water samples. These organic
substances as well as metal ions that interfere with enzymatic reactions can be removed by
treatments with Sephadex and Chelex resins. The sensitivity of the PCR method is reported to be
similar and possibly superior to conventional cell culture methods.
As stated above, the disadvantage of PCR is its inability to distinguish between amplified
viral sequence and noninfectious viral sequence. Reynolds et al. (1996), however, have used a
combination of cell cultural assay and the molecular PCR technique to detect infeetivity and
increase detection of poliovirus type 1. The combined technique used the advantageous features
of both methods and together, the methods eliminated the disadvantage encountered when each
method was used separately. In the study of Reynolds et al. (1996), concentrated samples were
inoculated into the BGM kidney continuous cell lines, incubated overnight and lysate-analyzed
by PCR. The combined technique decreased time associated with cell culturing for the
production of cytopathic effects. Incubation in cell culture increased the infectious virus
concentration. PCR allowed detection of small numbers of target RNAs and DMAs and a rapid
detection of the infectious viruses.
Another often cited disadvantage of PCR is its inability to provide quantitative
information. Tsai and Parker (1998) recently described a quantitative method for poliovirus in
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sewage and seawater by competitive RT-PCR. The method uses a cloned internal standard and
specific primers.
Jothikumar et al. (1995) developed a simple device for concentration and detection of
enterovirus, hepatitis E virus, and rotaviras from drinking water samples by RT-PCR. The
drinking water samples were run through a filtration column filled with granular activated carbon
(GAG). Viruses from GAC were eluted with urea-arginine phosphate buffer (UAPB) at pH 9.0
and further concentrated with magnesium chloride. This concentration procedure facilitated
nucleic acid extraction, cDNA synthesis, and amplification with a specific set of primers for
enterovirus, hepatitis E virus, and rotavirus. The PCR products were confirmed by southern
transfer and hybridization with the relevant probes. The authors reported that the efficiency of
the protocol was 74% for virus recovery in GAC-based UAPB-RT-PCR. The Jothikumar
method uses positively charged 1-MDS membrane filters for concentration (Jothikumar et al.,
1998).
Regan and Margolin (1997) describe an RT-PCR assay capable of monitoring for the
recovery of small amounts of polio virus RNA from environmental samples. In order to avoid the
interference from organic and other contaminations, the poliovirus RNA was isolated and
captured using magnetic bead technology, which allows the RT-PCR to take place on the
magnetic bead surface. This method is highly sensitive and can detect one PFU of virus in a
seeded sample and can be applied to other viruses. The authors suggest that the vaccine strain of
poliovirus may be the most suitable indicator of viral contamination of drinking water.
Inhibitors in environmental samples are a major problem in the use of PCR methodology
because these inhibitors obscure the detection of nucleic acids (Metcalf et al., 1995). Ijzerman et
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al. (1997) described a purification method in conjunction with RT-PCR that can detect
waterborne human enteric viruses in the presence of environmental inhibitors. The method
included concentration of the inhibitors along with viruses during water sample processing, and
then removal of the inhibitors by dialysis, solvent extraction, ultrafiltration, or glass purification.
The investigators evaluated the method by spiking sodium phosphate buffer with poliovirus type
1 with or without inhibitors (humic or fulvic acids), and then assessing virus recovery by plaque
assay and RT-PCR. This study showed 90% of the spiked virus recovery from samples at the
end of the ultrafiltration step.
A broad-spectrum immunoeapture method for concentration and purification of enteric
viruses developed by Schwab et al. (1996) is an indirect antibody capture (AbCAP) of intact
viruses followed by a release of virion genomic RNA and RT-PCR for amplification and
oligoprobe hybridization for detection. This technique involves concentrating enteric viruses
from large volumes of water by standard filtration-elution techniques with Sobsey filters (1-
MDS) and using 1 L of 1% beef extract-0.05 M glycine (BE/G) as an eluate. Using the AbCAP
method, the authors reported 9 positive samples for enteric viruses from 11 field samples of
fecal-contaminated surface water. Four of the 11 samples were positive for enteric viruses by
direct RNA extraction of a small portion of the second PEG concentrate; and 4 of 11 samples
were positive for enteric viruses when assessed for cell culture infectivity.
Ma et al. (1995) report a PCR method for increasing the sensitivity of enteric virus
detection in tap water concentrates. The substances inhibitory of PCR were first removed, then a
GAC-based method for concentration of viruses from water samples was used and viruses were
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subsequently detected effectively in reduced volumes of virus-containing water concentrate.
Poliovirus 1 and coxsackievirus B3 were seeded into 378 L of tap water, concentrated with
1-MDS filters, and reconcentrated by organic flocculation. Phenol-chloroform-isoamyl (PCI)
alcohol extraction was compared with Sephadex G-100 and Chelex-100 column for PCR
inhibitor removal. Using PCI, the authors could remove sufficient inhibitory substances to
perform RT-seminested PCR with a sensitivity of 0.2 PFU/10 ^L of tap water. Sephadex G-50
plus a Chelex-100 column also were capable of removing inhibitory substances. The Chelex
column was able to remove 99% of the viruses. Sephadex G-100 in combination with Chelex-
100 has also been shown to be very'effective in the removal of inhibitory factors for the detection
of enteroviruses by PCR in ground water (Abbaszadegan et al., 1993).
An RT-PCR assay for detecting human enteroviruses in water samples was described by
Tougianidou and Botzenhart (1993), The PCR method can be subject to contamination by the
nucleic acid of other microbial species, whereas the method described by the investigators
overcomes this problem by using confirmative hybridization with an oligomeric probe.
Tougianidou and Botzenhart (1993) indicate that this method is faster and cheaper than the
conventional detection methods used for viruses, such as cell culture, and also could be done
with smaller sample volumes.
The inhibitors of PCR reaction during sample concentration are well documented.
Several studies have also been conducted to solve the problem of PCR inhibition (Sobsey, 1994;
Straub et al., 1994; Abbaszadegan et al., 1993).
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8.4 Imnmnoassay Technique
Many of the recent advances in viral detection are based on immunoassay technology,
which does not require cross-linking of antigens by antibodies (Hock, 1996). Assays with high
detection sensitivity have been developed using labels, such as fluorescent dyes or enzymes, to
assess specific viral antigen-binding by antibody. Other techniques have been developed such as
the dipstick or dot blot tests for shorter analysis time. Some of the immunological techniques
include enzyme-linked immunosorbent assay (ELISA), immuno-fluorescent techniques (IF), and
radio-immunoassays (RIA).
The immunological assays involve the use of specific viral antibodies and antigens.
Immunological assays are subject to interference due to cross-reaction of similar antibodies
(Metcalfetal., 1995).
8-5 Other Methods
Margolin et al. (1991) described a sensitive and specific recombinant DMA method for
the detection of small numbers of poliovirus in tissue culture and environmental samples. The
viral cDNA, inserted into a plasmid vector was highly radiolabeled with 32P. The sensitivity of
the method was about one viral unit and the test was completed in 48 hours. The possibility for
false positive results was investigated and it was determined that, with the use of appropriate
techniques, false positive results were not likely to occur.
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8.5.1 Electron Microscopy (EM)
High resolution EM with negative staining and immune EM can be used to visualize viral
particles. The technique requires a highly skilled electron microscopist and viral particles that
are >106 concentration (Hurst et aL, 1989). Proctor (1997) discussed the use of microscopy to
study free viruses and to compare the ultrastracture of free viruses with bacteriophages and
viruses cultured from marine hosts. The author concluded that gross virus ultrastructure cannot
be used as the only criterion for determining marine virus diversity, because many viruses have
similar morphological characters. The morphology of all the enteroviruses has been shown to be
indistinguishable and termed structureless (Williams, 1989).
8.5.2 ELISA Method
Nasser et al. (1995) conducted a method comparative study in Israel to determine the
detection of viable poliovirus in ground water and waste water at different temperatures using the
plaque assay (BGM cell line) and the ELISA (nylon filter) serological techniques. The results
obtained by the investigators showed that at 4°C, no die-off of virus was detected by either
technique in 20 days. At 20°C and 30°C, a die-off of virus occurred. Poliovirus was no longer
detectable by ELISA after 2 days at 20 °C, whereas the plaque assay still detected virus (2 log
loss). The investigators concluded that the plaque assay was more sensitive for detecting virus
than the ELISA serological assay, while the ELISA method was faster.
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8.6 Summary
The small size of viruses and their low concentration pose a problem in virus recovery.
Large volumes of water must be concentrated to obtain virus numbers within detection limits of
available methods. Cell culture is the traditional method for virus detection and can differentiate
between infectious and noninfectious particles. However, some viruses cannot grow or grow
poorly on cell culture. PCR and RT-PCR methods are the newest and most sensitive detection
methods. However, the PCR method cannot differentiate between infectious and noninfectious
virus particles. Another drawback to this method is the inhibitory factors in environmental
samples.
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9.0 Potential Indicators for Waterborne Viruses
An indicator organism is a microorganism that indicates by its presence in water, possible
or actual contamination by fecal material (CDC, 1996c). Indicator microorganisms are used in
drinking water to assess contamination. They are usually not pathogenic and they are found in
larger numbers in contaminated waters than pathogens.
Detection of indicator organisms in drinking water may point to fecal contamination,
inadequate water treatment efficiency, or a distribution system problem. Fecal coliform bacteria
are universally used as an indicator-of fecal contamination in water and sewage. Coliforms are
present in large numbers in the GI tract and feces of humans. It is estimated that billions of
coliform bacteria are excreted daily by an average person (Pelzar et al., 1986). Although fecal
coliforms have been useful indicators for microbial pathogens, the detection of pathogenic
viruses from drinking water that met microbiological standards for safety based on fecal
coliforms as indicators has raised important questions as to the usefulness of such coliforms as a
standard for the virological quality of water (Marzouk et al., 1986; Rose et al., 1986; Rivera et
al., 1988).
An ideal drinking water indicator organism should have the following attributes (Berger
etal., 1992):
• Be suitable for all types of drinking water,
• Be nonpathogenic,
• Be capable of surviving for an extended period of time,
• Be poorly adsorbed to soils,
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• Be present when pathogens are present and absent when pathogens are absent,
• Not multiply in the environment,
• Be as resistant to disinfection and adverse environmental factors as the pathogens,
• Have a consistent presence ratio with the pathogen,
• Be easy to identify and quantify, and
• Be present in greater number than the pathogen.
9.1 Specific Indicators
9.1.1 Total/Fecal Coliforms
Total coliforms are used worldwide as an indication of water quality. Monitoring for
coliforms in drinking water provides a way of evaluating the overall efficiency of the treatment
system. Total coliforms are not generally regarded as a good indicator of fecal contamination.
The number of viral-infected individuals within a given community bears no relationship to the
ubiquitous presence of total coliforms in the receiving waters, and the absence or presence of
coliforms does not always correlate with the presence of enteric viruses (EPA, 1985). The
absence of coliforms, on the other hand, does not preclude the usefulness of coliforms in
indirectly detecting viral presence in water.
While the absence of fecal coliform does not indicate an effective removal of enteric
viruses by treatment, the presence of fecal coliforms in marine sediments where waste water
sludge had been disposed of has been interpreted as an indication of improperly disinfected waste
water sludge (Baker, 1995).
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Marzouk et al. (1980) conducted a study to determine the correlation between the
presence of enteroviruses and fecal coliforms or fecal streptococci using standard methods. A
total of 155 samples were collected from surface water, ground water, and potable water. The
investigators isolated viruses (echovirus, poliovirus, and coxsackievirus group B) in water with
no detectable fecal and total coliform bacteria, and which met bacteriological treatment
standards. Further, the investigators did not find a significant correlation between the occurrence
of bacterial indicators and the presence of viruses. The authors question the validity of using the
current indicator method for the prediction of virus contamination, especially in countries with a
high incidence of enteric viral disease.
9.1.2 Escherichia coli
E. coli is a good indicator for evaluating the presence of fecal pollution. It is not,
however, a good indicator for viral contamination. Nasser et al. (1993) conducted a comparative
study of the survival patterns of E. coli, poliovirus, HAV, and F+ coliphage. These authors
studied the die-off of viruses and E. coli in several types of water and at several temperatures. E.
coli was subject to die-off in all waters and was most susceptible to cooler temperatures (10°C).
Viruses, on the other hand, survived well at the lower temperature but were affected by a
temperature of 30°C. The authors concluded that E. coli was not an appropriate indicator
organism for potential viral contamination.
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9.1.3 Heterotrophic Bacteria
Heterotrophic bacteria need organic nutrients for multiplication and therefore their
presence will indicate the presence of organic matter (including feces) contamination in intake or
finished drinking water. However, interpreting the data beyond this level could be risky because
viruses can be present in treated water, even in the absence of heterotrophic bacteria. In addition,
the counting results of heterotrophic plate counts can be variable and temperature dependent
(Coallier et al.s 1994).
9.1.3.1 Fecal Streptococci/Enterococci
Monitoring for the presence of fecal streptococci may be a good alternative indicator of
fecal pollution. Enterococci persist longer in water than do the fecal coliforms. They may also
survive chlorination that has effectively inactivated fecal and total coliforms. In a study designed
to compare several alternative approaches to determining fecal contamination, Lucena et al.
(1994) assayed for total coliforms, fecal coliforms, fecal streptococcus (enterocpccus),
Clostridium perfringem, bacteriophages (of B. fragilis and E. coif), and enteroviruses. They
concluded that the fate of bacteriophages of B. fragilis released into a marine environment could
more closely approximate the fate of human viruses than any of the other microorganisms
examined.
9.1.4 Clostridium perfringens
C. perfringens is highly resistant to chlorination and could be present in water that has
been adequately treated for drinking (EPA, 1985). However, this bacterium is more difficult and
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expensive to monitor than other available alternatives. Researchers at EPA Cincinnati indicate
problems with the culture method for C. perfringens, particularly with the rapid fading of the
chromogenic confirmation assays.
9.1.4.1 Bacteriophages
A bacteriophage is a virus that infects bacteria. Bacteriophages are proposed as
alternative indicators of viral contamination of drinking water and whose presence can be used to
evaluate treatment efficiency. A coliphage is a bacteriophage which specifically attacks E. coli.
Somatic bacteriophage will attach itself to the bacterial cell wall, while F-specific or male-
specific bacteriophage will attach itself to bacterial F pili (hairlike projections). Therefore, a
bacterium without a pilus cannot be infected by an F-specific or male-specific bacteriophage.
Several studies have examined the usefulness of bacteriophages as surrogates for
monitoring the presence of human enteric viruses. The study of Sobsey et al. (1990) indicates
that male-specific bacteriophages can be useful surrogates for monitoring the presence of human
enteroviruses in drinking water. The investigators showed that male-specific bacteriophages
were similar to enteric viruses in shape, size, survival rate, and soil transport behavior.
Havelaar et al. (1993) have also shown a correlation between the presence of male-
specific RNA bacteriophages and enteric viruses in fresh water. The coagulation removal pattern
of enteroviruses has been reported to be similar to that of male-specific phages (Abbaszadegan et
al., 1997).
Armon (1993) conducted a 9-month survey of drinking water from various locations in
Israel to evaluate the relationship between the presence of drinking water indicators and
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bacteriophages. A total of 2,187 drinking water samples were collected and analyzed for the
presence of somatic coliphage, F-specific coliphages, and Bacteroides fragilis phage. The study
simultaneously monitored the presence of total coliforms and fecal coliforms, standard indicators
for drinking water in Israel. Results obtained showed a poor correlation between the
bacteriophages and the standard indicators. A detection frequency of 11.5% was registered for
the somatic eoliphage, 6.46% for F-specific coliphage, and 5.48% for B. fragilis phages. No
correlation was found between the presence of the three phages evaluated and the presence of
total coliforms and fecal coliforms. Armon (1993) suggest that the poor correlation could be due
to chlorination because the study found bacteria inactivation while the bacteriophages remained
viable in the absence of coliforms. Another explanation given by the investigator for poor
correlation was the dilution factor created by distance.
An ongoing comprehensive study is being conducted by Abbaszadegan et al. (1998). The
study is designed to analyze the occurrence of enteric viruses in 550 ground water samples and
also determine the association of enterovirus presence with several potential biological and
physical indicators. The interim report on 250 samples (Abbaszadegan et al., 1998) indicates that
by using either the cell culture method or the RT-PCR method, their results show no consistent
relationship between indicator bacteria and bacteriophages and the presence of enteroviruses.
Keswick et al. (1982) studied the survival of several viruses and bacteria in ground water.
The decay rate was greatest for the bacteriophage £2, followed by E. coli, echovirus 1, fecal
streptococcus, poliovirus 1, and coxsackievirus B3. From these results the authors concluded
that the phage £2 is least suited as an indicator organism for viral contamination; on the other
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hand, poliovirus type 1 (vaccine strain) appeared to be a good indicator for determining viral
contamination of ground water.
Because coliphages such as F-specific are found in high numbers in human feces and in
sewage, and are known to persist but not multiply in the environment, they are considered
potential indicators for fecal pollution. Vaughn and Metcalf (1975) evaluated estuarine waters
and found a disparity in the ratio of enteric viruses and phages. Results showed that 63% of the
samples were positive for enteric viruses but were negative for coliphages. Delgado and
Toranzos (1995) studied phage survival and replication in a tropical pristine river in Puerto Rico.
The authors showed that coliphages, did not replicate under tropical environmental conditions in
all the host strains and survived from 5 to 15 days.
Snowdon and Oliver (1989) have suggested that coliphages could be useful indicators of
viral contamination of ground water. Coliphages are technically easier to detect than pathogenic
human viruses. However, for the coliphages to be useful and reliable predictors of enteroviras
contamination, their presence, persistence, and transport must be correlated with the actual
presence of pathogenic viruses.
Dutka et al. (1990) conducted a study in the province of Ontario in which raw and
chlorine-treated well waters were collected and analyzed for the presence of indicator organisms
of fecal contamination (total coliforms, fecal coliforms, or fecal streptococci). All of the samples
contained less than one organism/100 mL, the criterion for drinking water safety. However, the
raw and treated water samples contained significant numbers of coliphages and other
bacteriophages. Chlorination did not reduce the presence of these bacterial viruses.
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The coliphage QP was suggested by Woody and Oliver (1995) to be useful as an indicator
organism and as a virus model, for determining the contamination of ground water with
enteroviruses. The QP coliphage does not reproduce in E. coli at temperatures below 25 °C, thus,
its presence in cool ground water would indicate prior fecal contamination rather than replication
of the phage.
Good survival in the environment and no possibility for additional replication are
desirable features of an indicator organism. In a more recent article, Woody and Oliver (1997)
reported that F-specific RNA (fRNA) coliphages, might serve as indicators of human enteric
viruses in ground water, provided these phages do not replicate in ground water and replicate
only to a limited extent in waste water (because of the low amount of nutrient available in ground
and waste water).
Toranzos et al. (1988) found somatic coliphages in samples collected from Puerto Rican
source waters that were contaminated with sewage. None was found in the surface waters that
were considered pristine. However, this phenomenon has not been observed in temperate areas
of the world.
Williams and Stetler (1994) analyzed ground water samples from two sites in Alabama
that were 70 km from each other for FRNA coliphages using Salmonella typhimurium WG49 as
the host. Samples were assayed for FRNA coliphage plaques and the morphologies of the phage
isolates were examined by EM. Their results did not show a positive correlation.
Governal and Gerba (1997) evaluated the persistence of bacteriophages MS-2 and PRD-1
in tap water, in reverse osmosis permeate, and in three locations within an ultrapure water
system. Ultrapure samples that were studied included pre- and post-UV sterilization and post-
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mixed bed ion exchange tank. The inactivation rates for MS-2 were calculated as log 10
reduction per hour/per day: K= - (log 10 Ct/CO)/t. PRD-1 phage was found to persist with no
loss of infectivity in all three water purity environments evaluated, whereas MS-2 did not.
Beekwilder et al. (1996) reported that F-specific RNA phages can be used as indicator
organisms for enteric viruses to monitor the effectiveness of sewage treatment, and to assess the
potential contamination of surface water with these viruses. A method was described by this
group that identifies RNA phages quantitatively by a plaque hybridization assay.
Oligonucleotide probes were developed that can assign phages to their phylogenetic subgroups.
9.2 Other Indicators
The potential of other bacteria as indicators has also been studied. McFeters et al. (1974)
studied the survival of several bacteria in well water under laboratory conditions. The water
temperature ranged from 9.5 to 12.5°C. Under these conditions, Aeromonas sp. survived best,
followed by Shigella, fecal streptococci, coliforms including some Salmonella species,
Streptococcus equinus, Vibrio cholerae, Salmonella typhi, and Streptococcus bovis.
9.2.1 Bifidobacterium
The use of Bifidobacterium has in the past been suggested as an alternative indicator for
water pollution because it is present in high numbers in human feces. Evison and James (1975)
conducted a comparative study of the distribution of Bifidobacterium, coliforms, and E. coli and
fecal streptococcus in a variety of water in United Kingdom as an alternative indicator of water
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pollution. Their results showed a similar distribution of E. coli and bifidobacteria. The
drawback is that Bifidobacterium has rigorous growth requirements and is anaerobic.
9.3 Summary
The ideal indicator of fecal contamination and the presence of waterborne pathogenic
virus would be the pathogen itself. But that is not feasible because of related costs and because
there are many different pathogens, and some of the pathogens cannot be grown in available cell
lines or grow poorly. The next best indicator is one that survives well under the most hostile
environmental conditions, including exposure to disinfectants that do not kill the most hardy
virus. Several bacterial species have been investigated as suitable indicators of fecal
contamination, including E. coli, fecal streptococci, C. perfringens, and B. fragilis. However,
viruses can still be present in intake or finished drinking water or treated waste water that do not
have any of the bacterial species present. Coliphages, which would be introduced into water
along with their host bacteria, however, appear to survive as well as pathogenic human viruses.
There is no agreement as to the correlation between bacteriophage presence and virus presence.
E. coli and fecal coliforms still remain the most adequate of all suggested indicators, but further
research is needed.
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10,0 Treatment
10.1 Introduction
This chapter will briefly discuss the requirements of the Safe Drinking Water Act
(SDWA) as it relates to EPA regulations for drinking water treatment. It will also discuss the
disinfection methods in use in most water treatment systems and the effects of those methods on
enteroviruses and HAV. The chapter does not, however, present the chemistry of disinfectants
and their mechanisms of action.
Under the SDWA, EPA is required to publish National Primary Drinking Water
Regulations (NPDWRs) that will set a maximum contaminant level (MCL) or a water treatment
technique requirement for adverse health effects contaminants. The SDWA, in addition,
mandates EPA to specify monitoring and reporting requirements for each regulated contaminant
(Berger and Regli, 1990).
Under the same mandate, EPA will set an MCL for a contaminant if it is technologically
and financially feasible to determine the contaminant level. However, if this is not possible, then
EPA is required to set a water treatment technique. EPA will make this judgement based on
treatment method reliability, method detection limits, laboratory experience with available
methods, ability to relate the measurement to the determination of health risk significance, and
cost analysis (Berger and Regli, 1990).
Among the contaminants of concern are the enteric viruses. EPA's current regulations
use two indicators of microbiologically safe drinking water quality. These are total coliforms and
turbidity. The SDWA amendment of 1996 requires EPA to regulate viruses, Giardia lamblia,
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and Legionella, EPA is also required to publish regulations that will specify the criteria for a
filtration requirement as a treatment technique for those public water systems that have surface
water as their supply source. There is a requirement for EPA to publish regulations that require
disinfection as a treatment technique for all public water systems and as necessary for ground
water systems (Berger and Regli, 1990).
EPA will comply with all the above requirements under the 1996 SDWA amendments
through three rules. These EPA Rules are The Ground Water Rule (GWR), the Surface Water
Treatment Rule (SWTR), and the Revised Total Coliform Rule (TCR). EPA believes that these
three rules will reduce waterborne disease occurrence in the United States (Berger and Regli,
1990),
Enteric viruses including enteroviruses are affected by two of the rules discussed above,
the GWR and the SWTR.
10.1.1 Surface Water Treatment Rule as It Relates to Viruses
The SWTR will regulate viruses in surface waters. This is accomplished under SWTR by
establishing treatment technique requirements rather than MCLs. All water systems using
surface water as source must achieve at least a 99.99% (4-log) removal or inactivation of viruses
(Federal Register (FR), 1989).
10.1.2 Ground Water Rule
Section 1412 (b) (1) (A) of the SDWA requires EPA to establish NPDWR for a
contaminant if 1) the contaminant may have an adverse health effect, 2) it is known or likely to
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occur in public water systems with a frequency and at levels of public health concern, and 3) if
"regulation of such contaminant presents a meaningful opportunity for health risk reduction.
There is also a supplemental provision for an additional requirement under section 1412 (b) (8)
that EPA develop regulations specifying the use of disinfection for ground water systems "as
necessary," The GWR will specify appropriate use of disinfection and encourage the use of
alternative approaches which include best management practices and control of contamination at
its source (OW/OGWDW, 1998).
10.2 Conventional Water Treatment Method
Water treatment plants use various processes to treat drinking water. The most common
of these processes for surface water supplies is conventional treatment. Conventional treatment
consists of disinfection, coagulation, flocculation, sedimentation, and filtration. This may be
followed by a second disinfection step. Other additional steps may also be included and these are
preoxidation, preaeration, adsorption, and presedimentation (Montgomery, 1985; Hudson, 1981).
Coagulation, flocculation, sedimentation, and filtration are physical and chemical processes that
remove suspended solids in water.
10.2.1 Coagulation
In the coagulation step, a coagulant such as alum [A12(SO4)314H2O], ferric sulfate
[Fe2(SO4)3], and ferric chloride (FeCl3) may be added to alter the physical state of dissolved and
suspended solids to enhance their removal by sedimentation (Hudson, 1981; Montgomery, 1985).
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10.2.2 Flocculation
The flocculation process involves the gentle stirring of treated water to increase particle
collisions and large particle formation. An adequate flocculation can settle out most aggregates
in 1 to 2 hours of sedimentation.
10.2.3 Sedimentation
The sedimentation process involves the separation of suspended particles from water by
gravitational settling. Berger and Argaman (1983) indicate that coagulation and sedimentation
can effectively remove 88-95% of poliovirus and coxsackievirus.
10.2.4 Filtration
Sand and anthracite coal are used for filtration. The effluent obtained from sedimentation
process is subjected to rapid filtration to separate suspended solids. Rapid filters are usually 24
to 36 inches of 0.5 to 1 mm-diameter sand or anthracite. Suspended solids are removed through
filtration at rates of 1 to 6 gallons/minutes/square foot.
10.3 Disinfection
Disinfection is a process that kills or inactivates pathogenic microorganisms. Inactivation
is the removal of a pathogenic microorganism's ability to infect. Disinfection does not eliminate
all microorganisms, but sterilization does. While treatment of water intended for drinking is
important for public health protection against pathogenic microorganisms, drinking water
treatment does not always produce an absolute rnicrobiologically safe water because disinfection
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does not mean sterilization. No water supply can be treated to the point of elimination of all
pathogens (Haas et al., 1993). Numerous documented evidence exists of outbreaks due to
contamination of treated drinking water.
Adequate disinfection of water is defined as the application of chlorine to achieve a free
residual of at least 0.5 mg/L after a minimum contact time of 30 minutes and a turbidity level of
1 NTU and pH of <8.0. However, it depends on temperature and type of virus (see Table 10-1).
In terms of virus inactivation, the disinfection must achieve a 99.99% reduction of enteric viruses
(WHO, 1996).
There are four chemical disinfectants most commonly in use for treatment of water in the
United States. These are chlorine, chloramine, chlorine dioxide, and ozone. The effectiveness of
a chosen treatment system depends on the quality of the water before treatment. The more
polluted a water system, the more treatment is required.
10.3.1 Chlorination
The inactivation ability of chlorine is not absolute. This is because viruses have been
recovered from chlorinated drinking waters that were negative for total coliform, exposed to 0.5
mg/L of free chlorine for 30 minutes and also with turbidity of less than 1 NTU (EPA, 1985).
Chlorine, however, is the disinfectant of choice and has been for the past several decades.
The recommended chlorine dosage is generally adequate for chlorine demand and excess residual
to protect systems from recontamination. Some microbiai contaminants have been recovered
from systems providing a residual of chlorine as high as 5 mg/L. But it is now known that
chlorine can produce cancer-causing byproducts when certain precursor material is present in the
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source water prior to chlorination. The growing concern over the problem of chlorine byproducts
such as trihalomethanes (THMs) and health risks associated with these byproducts has created a
need for alternative disinfectants.
Microbial resistance to disinfectants varies. Studies have shown that viruses are more
resistant to inactivation than bacteria. Peterson et al. (1983) conducted a study to evaluate the
effect of chlorine treatment on the infectivity of HAV. The infectivity of HAV was tested
intramuscularly in marmoset monkeys. Chlorine residuals used for the study ranged from 0.5 to
2.5 mg/L with contact times of 15, 30, and 60 min. at 5°C. Results showed that untreated
inoculum induced seroconversion in 100% of the marmosets. A 0.5-1.5 mg of chlorine residual
induced hepatitis in 14% of the marmosets and induced seroconversion in 10% of the marmoset
monkeys while those solutions containing HAV treated with 2.0 and 2.5 mg of free residual
chlorine were not infectious. From these results, the investigators concluded that HAV was more
resistant to chlorine than other enterovirases.
Keswick et al. (1985) conducted tests to determine the inactivation of Norwalk virus,
polio virus, human rotavirus, and simian rotavirus, as well as bacteriophage £2. At residual
chlorine doses of up to 3.75 mg/L in the drinking water, some of the human volunteers became ill
from Norwalk vims. According to the authors, this dosage is similar to that in most municipal
water treatment systems. The authors concluded that chlorine treatment alone cannot be relied
upon to inactivate Norwalk virus, and that doses up to 10 mg/L might be required to inactivate
the virus. However, these authors found that polioviras and both types of rotaviruses could be
inactivated with 3.75 mg/L or somewhat higher doses of chlorine. The bacteriophage £2,
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however, was only partially inactivated at doses of 5.0 and 10 mg/L. Sobsey (1989) reported that
the most chlorine-resistant pathogens were enteric viruses, protozoan cysts, and mycobacteria.
Payment (1989) reported on a virus sampling program at various locations in seven
drinking water treatment plants and indicated that all of these plants delivered finished water that
was essentially free of indicator bacteria, and that the average cumulative reduction of viruses
was 95.15% after sedimentation and 99.7% after filtration, but that there were no significant
reductions in viruses after disinfection with chlorine or ozone.
The inactivation of polioviras and human rotaviros strain Wa was studied in several
waters: lake water, ground water, tap water, creek water, and secondary effluent (Pancorbo et at,
1987), Poliovirus, in general, survived longer than the rotavirus. Virus survival was
significantly affected by water type. Virus survival was greatest in lake and ground water and
lowest in tap water. The viruses survived 2-3 weeks in lake water, but only 2—3 days in tap
water.
Melnick (1996) reported that a free residual chlorine treatment of 0.3-0.5 ppm chlorine
can cause rapid inactivation of enteroviruses, but that the viruses are protected from such
inactivation by organic substances. Various chlorine-based studies that reflect a 99.99% (4-log)
removal of enteroviruses and HAV are summarized in Table 10-1.
10.3.2 Chloramine
Chloramine is used as a primary or secondary disinfectant. It is formed as a byproduct of
chlorination in the presence of ammonia in source waters, Chloramine has been shown to reduce
the formation of THMs. It has been estimated that 29% of community surface water systems and
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11% of community ground water systems in the United States that serve 10,000 people use
chloramine for disinfection (EPA, 1997b). Several studies have demonstrated the effectiveness
of chloramine. Table 10-1 presents the results of some of these studies.
10.3.3 Chlorine Dioxide
Chlorine dioxide is used as a disinfectant by some water treatment plants, either alone or
in combination with chlorine to control odor, taste, and color problems in drinking water. Only
10% of surface water plants and 1.0% of ground water plants in the United States use chlorine
dioxide as a disinfectant (EPA, 199,7b). Several studies have demonstrated the effectiveness of
chlorine dioxide; they are presented in Table 10-1.
10.3.4 Ozone
The use of ozone as a disinfectant was rarely practiced during the past 15 years in the
United States but this has changed and ozone use as a disinfectant has increased because of
concern over cancer-causing chlorine byproducts. Ozone is used frequently in Europe for the
disinfection of drinking water and was first used as a disinfectant in the Netherlands in 1893.
Forty water treatment plants in the United States serving more than 10,000 people used ozone in
1991. That number has increased to 201 in 1997 (EPA, 1997b). Ozone is the most oxidizing
agent of all disinfectants that are available for water treatment and can be generated with oxygen
or with air. It has been shown to be more effective than chlorine, chloramine, and chlorine
dioxide against viruses and requires a short contact time (EPA, 1997b). Unlike chlorine,
however, ozone does not leave a residual to protect against recontamination of treated water in
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the distribution system (EPA, 1991), The effects of ozone in enterovirus inactivation are
presented in Table 10-1.
10.3.5 Ultraviolet Light (UV)
Ultraviolet (UV) light produced by UV lamps has been shown to be an effective
disinfectant. UV disinfection involves exposure of a film of water to one or more quartz mercury
vapor arc lamps emitting UV radiation. The maximum absorption spectrum of DNA is between
a wavelength of 250-270 nm. Therefore, the UV process involves the transfer of
electromagnetic energy from a UV Jamp source to an organism's genetic material. The UV light
interferes with the genetic material of a microorganism at 254 nm. The sensitivity of viruses to
UV radiation is comparable with that of bacteria (Meulemans, 1987).
Viruses in water and on exposed surfaces can be inactivated with UV light (Cliver, 1997).
The factors that determine the amount of UV radiation needed to disinfect a body of water are
turbidity, color, and dissolved iron salts, which prevent the UV energy from penetrating the
water. UV light is generally not used for disinfecting turbid water because of interference, but
rather used for disinfecting ground water (EPA, 1991). Unlike chlorine, UV light does not have
a residual disinfecting capability and cannot prevent recontamination.
Battigelli et al. (1993) conducted a study to determine the effectiveness of UV radiation
on HAV, coxsackievirus B5, rotaviras, and bacteriophage MS-2. Their results showed a
(99.99%) 4-log removal for HAV after 20 mW sec/cm2. The bacteriophage MS-2 showed the
greatest resistance, with less than 1 log removal observed after exposure to 25 mW sec/cm2.
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Table 10-1 presents various studies that have demonstrated the effectiveness of UV
against enteroviruses and other microorganisms. All the studies show a 4-log removal (99,99%)
efficiency. The studies also identify the disinfectants that leave residuals and those that produce
byproducts.
10.4 Factors That Influence Treatment Efficiency
The temperature and pH of the water being disinfected are important factors that
influence most treatment efficacy. The higher the temperature, the greater and faster the
inactivation. Hoff (1990) indicates a 2 to 3 log,0 inactivation rate for every 10°C increase in
temperature. The hydrogen ion concentration (pH), on the other hand, is disinfectant specific.
An increase in pH decreases the effectiveness of free chlorine and chloramine but increases the
effectiveness of chlorine dioxide. The ability of ozone to inactivate a microorganism is not
affected by pH. Thorough mixing is an important factor in disinfectant effectiveness. A C-T
determination is important in the effectiveness of disinfection. C-T is the concentration (in
mg/L) of disinfectant multiplied by contact time (in minutes). A C-T determination is required
for unfiltered surface waters in the SWTR and is recommended by EPA for plants that filter
surface water. An accurate estimation of contact time is as important as the accurate
measurement of residual disinfectant (EPA, 1990).
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TABLE 10-1
Studies on Treatment Technologies Capable of 99.99% (4 Log) Virus Inactivation
Studies conducted3
Disinfectant
Chlorine
Chloramine
Chlorine
dioxide
Calcium
hypochlorite
Sodium
bypochlorite
Ozone
Virus(es)
studied
HAV
Coxsackie B5
Poliovirus 1
HAV
HAV
HAV
HAV
Poliovirus
Poliovirus 1
Effectiveness
Max.
l°g
removal
4
4
4
4
4
4
4
4
4
4-6
CT at max.
removal
4C
30C
-1.07
-7.8
994C
16.7C
4C
4C
0.6C
.008
Additional notes
Residual
Y
Y
Y
Y
Y
Y
Y
Y
N
N
DBPb
Y
Y
Y
Y
N
N
Y
Y
Y
Y
Comments
pH=10
pH = 6,T = 28°C
4-logat5°C
Add ammonia after
chlorine
Chlorite and chlorate
may be formed
CT based on chlorine
dosage
CT based on chlorine
dosage
NoTHM
T=10°C
Reference
SobseyetaL, 1988b
Sobseyetal., 1988b
Kelly & Sanderson,
1958
Kelly & Sanderson,
1958
Sobseyetal., 1988b
SobseyetaL, 1988b
Sobseyetal,, 1988b
Sobseyetal., 1988b
Roy etal., 1982
Herboldetal, 1989
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TABLE 10-1. Continued
Studies conducted3
Disinfectant
Ozone
Chlorinating
tablets
Anodic
oxidation
Reverse
osmosis
Ultraviolet6
Virus(es)
studied
Enterics
HAV
MS2
HAV
f-2
bacteriophage
<0.5nm
HAV
Effectiveness
Max.
log
removal
4
4
3.9-6.0
4-6
4
2.7-7
4
4
4
100%
removal
4
4
4
CT at max,
removal
5
3
-0.167
0.22
0.40
7.2
.013
6.2C
4C
50-70%
recovery
87.4-93
18.5
16
Additional notes
Residual
N
N
N
N
N
N
N
Y
Y
N
N
N
N
DBF"
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
N
N
Comments
Also MS2
T=10°C
T = 4°C
T = 22 °C, initial
T = 22 °C, residual
See calcium hypoclorite
Same CT as chlorine
suggested by EPA
MWCO <0.5 nm
Ground water pilot scale
Reference
Kaneko, 1989
Finch etal., 1992
Hall &Sobsey, 1993
Herbold etal., 1989
Vaughn etal., 1990
Finch etal., 1992
Finch etal., 1992
EPA, 1985
Bradford and Baker,
1994
Jacangelo et al.,
1995
SniceretaL, 1996
Wiedenmann et al.,
1993
Lobe, 1993
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TABLE 10-1. Continued
Studies conducted*
Disinfectant
Ultraviolet
Virus(es)
studied
Poliovirus
Coxsackie B5
Effectiveness
Max.
log
removal
4
4
4
4
3-4
4
CT at max.
removal
120
16
39.4
120
-30
29
Additional notes
Residual
N
N
N
N
N
N
DBP"
N
N
N
N
N
N
Comments
Safety factor = 3
Also Rotavirus SA1 1,
Poliovirus 1
Safety factor = 3, IT = 30
(1987)
Approximately 4-log
Approximately 4-log
Reference
Sobseyetal., 1988b
Battigelli et al.,
1993
Wilson et al., 1992
Harris etal., 1987
Chang etal., 1985
Battigelli et al.,
1993
"These studies reflect only those that give a 99,99% (4-log removal of viruses).
hDBP=disinfectant byproduct.
'Value for T=15°C.
•"Removal based on pore size,
"Inactivation is the product of the light intensity (I) and the contact time (T).
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10.5 Summary
Conventional water treatment methods include coagulation, flocculation, sedimentation,
and filtration. These methods can remove solids and clarity source waters. Sedimentation can
remove about 95% of polio virus and coxsackievirus.
Chlorination is the disinfectant of choice in the United States. The recommended
chlorine dosage is generally adequate for the chlorine demand needed for disinfection and for the
excess residual needed to protect the distribution system from recontamination. But it is now
known that chlorine can produce cancer-causing byproducts. The growing concern over the
problem of chlorine byproducts such as trmalomethanes and health risks associated with these
t '
byproducts has created a need for alternative disinfectants.
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11.0 References
Abad, F.X., Pinto, R.M., Diez, J.M., and Basket, J, 1994. Disinfection of human enteric viruses
in water by copper and silver in combination with low levels of chlorine. Appl. Environ.
Microbiol. 60 (7):2377-2383.
Abbaszadegan, M., and DeLeon, R. 1997. Detection of viruses in water samples by nucleic acid
amplification. In: Toranzos, G.A., ed., Environmental applications of nucleic acid amplification
techniques, Technemic Publishing Company, Lancaster, PA. pp. 113-127.
Abbaszadegan, M., Huber, M.S., Gerba, C.P., and Pepper, LL. 1993. Detection of enteroviruses
in ground water with the polyrnerase chain reaction. Appl. Environ. Microbiol. 59(5):1318-1324.
Abbaszadegan, M., Stewart, P.W., LeChevallier, M.W., Rosen, J.S., and Gerba, C.P. 1998.
Occurrence of viruses in ground water in the United States: Interim Report. March 1998, pp.
1-156.
Abbaszadegan, M., Stewart, P., LeChevallier, M., Yates, M., and Gerba, C. 1995. Occurrence of
enteroviruses in ground water and correlation with water quality parameters. Proceedings 1995
Water Quality Technology Conference, American Water Works Association, Nov. 12-16, New
Orleans, LA.
Ades, E.W., Bosse, D.C., and Parker, J.T. 1992. Immune suppression. In: Encyclopedia of
Microbiology, vol. 2. Orlando, FL: Academic Press, pp. 467-472.
Alexander, J.P., Chapman, L.E., Pallanseh, M.A., Stephensen, W.T., Torok, T.J., and Anderson,
L.J. 1993. Coxsackievirus B2 infection and aseptic meningitis: A focal outbreak among members
of a high school football team. J. Infect. Dis. 167:1201-1205.
Alhajjar, B.J., Steamer, S.L., Cliver, D.O., and Harkin, J.M. 1988. Transport modelling of
biological tracers from septic systems. Wat. Res. 22:907-915.
Alter, M.J., and Mast, E.E. 1994. The epidemiology of viral hepatitis in the United States.
Gastroenterol. Clin. North Am. 23 (3):437-455.
Amundson, D., Lindholm, C., Goyal, S.M., and Robinson R.A. 1988. Microbial pollution of well
water in southeastern Minnesota. J. Environ. Sci. Health A23 (5):453^t68.
APHA (American Public Health Association) 1989. Standard methods for the examination of
water and wastewater, 17th edition. Washington, DC: American Public Health Association.
Armon, R. 1993. Bacteriophage monitoring in drinking water: Do they fulfil the index or
indicator function? Wat. Sci. Tech. 27 (3-4):463^70.
EPA OW/OST/HECD 11-1 Enterovirus Criteria Document
FINAL DRAFT
-------
Asano, T., Leong, L.Y.C., Rigby, M.G., and Sakaji, R.H. 1992. Evaluation of the California
waste water reclamation criteria using enteric virus monitoring data, Wat. Sci. Tech.
26:1513-1524. •
Assaad, F., arid Borecka, I. 1977. Nine-year study of WHO virus reports on fatal virus infections.
Bull. Wld. Hlth. Org. 55:445.
Backlow,N.R., and Cukor, G. 1985. Viral gastroenteritis agents. In: Linnette, E.H., Balows, A.,
Hausler, W.J., and Shadomy, H.J., eds. Manual of Clinical Microbiology, 4th ed. Ch. 80.
Washington, D.C.: American Society for Microbiology, pp. 805-812.
Baker, K.H. 1995. Detection arid occurrence of indicator organisms and pathogens. Wat.
Environ. Res. 67 (4):406^10.
Bales, R.C., Li, S., Maguire, K.M., Yahya, M.Y., Gerba, C.P., and Harvey, R.W. 1995. Virus and
bacteria transport in a sandy aquifer, Cape Cod, MA. Ground Wat. 33 (4): 65 3-661.
Battigelli, D.A,, Sobsey, M.D., and Lobe, D.C. 1993. Inactivation of hepatitis A virus and other
model viruses by UV irradiation. Wat. Sci. Tech. 27 (3-4):339-342.
Beekwilder, J., Nieuwenhuizen, R., Havelaar, A.H., and van Duin, J, 1996. An oligonucleotide
hybridization assay for the identification and enumeration of F-specific RNA phages in surface
water. J. Appl. Bacteriol. 80 (2): 179-186.
Berg, G. 1978, Viruses in the environment: Criteria for risk. In: Sagik, B.P., and Sorber, C.A.,
eds. Risk Assessment and Health Effects of Land Application of Municipal Waste water and
Sludges. San Antonio, TX: Center for Applied Research and Technology, The University of
Texas at San Antonio.
Bergelson, J.M., St. John, N., Kawaguchi, S., Chan, M., Modlin, J., and Finberg, R.W. 1993.
Infection by echoviruses 1 and 8 depends on the alpha 2 subunit of human VLA-2. J. Virol.
67(ll):6847-6852.
Berger, P.S,, and Argaman, Y. 1983. Assessment of microbiology and turbidity standards for
drinking water. EPA Report #570-9-83-001. U.S. EPA Office of Drinking Water, Washington,
DC.
Berger, P.S., Clark, R.M., and Reasoner, D.J. 1992. Water, drinking. In: Encyclopedia of
Microbiology, Vol. 4. Orlando, FL: Academic Press, pp. 385-398,
Berger, P.S., and Regli, S. 1990. The safe drinking water act and the regulation of
microorganisms in drinking water. In: Craun, G.F., ed. Methods for the Investigation and
Prevention of Waterborne Disease Outbreaks. EPA/600/1-90/005a. U.S. Environmental
Protection Agency.
EPA OW/OST/HECD 11 -2 Enterovirus Criteria Document
FINAL DRAFT
-------
Berlin, L.E., and Rorabaugh, M.L. 1993. Aseptic meningitis in infants <2 years of age; Diagnosis
and etiology. J. Infect. Dis. 168:888-892.
Biggs, D.D., Toorkey, B.C., Carrigan, D.C., Hanson, G.A., and Ash, R.C. 1990. Disseminated
echovirus infection complicating bone marrow transplantation. Am. J. Med. 88:421-424.
Bitton, O.5 Davidson, J.M., and Farrah, S.R. 1979. On the value of soil columns for assessing the
transport pattern of viruses through soils: A critical outlook. Wat. Air Soil Pollut. 12:449^57.
Bitton, G., Farrah, S.R., Montague, C.L., and Elmer, E.W. 1986. Viruses in drinking water.
Environ. Sci. Tech. 20 (3):216-222.
Black, E.K., and Finch, O.K. 1993. Detection and occurrence of waterborne bacterial and viral
pathogens. Wat. Environ. Res. 65:295-300.
Bloeh, A.B., Stranier, S,L., Smith, D.J., Margolis, H.S., Fields, H.A., McKinley, T.W., Gerba,
C.P., Maynard, I.E., and Sikes, R.K, 1990. Recovery of hepatitis A virus from a water supply
responsible for a common source outbreak of hepatitis A. Am. J. Public Health 80 (4):428^30.
Bosch, A., Lucena, F., Girones, R., and Jofre, J. 1986. Survey of viral pollution in Besos River
(Barcelona). J. Wat Pollut. Control Fed. 58:87-91.
Bosch, A., Pinto, R.M., Blanch, A.R., and Jofre, J.T. 1988. Detection of human rotavirus in
sewage through two concentration procedures. Wat. Res. 22(3):343-348.
Bothner, M.H., et al. 1994. Sewage contamination in sediments beneath a deep-ocean dump site
off New York. Marine Environ. Res. 38:43.
Bowen, S.G., and McCarthy, M.A. 1983. Hepatitis A associated with a hardware store water
fountain and a contaminated well in Lancaster County, Pennsylvania, 1980. Am. J. Epidemiol.
117(6):695~-704.
Bradford, W.L., and Baker, F.A. 1994. Design, fabrication and testing of a laboratory test
electrolytic water disinfection unit (EWDU): Addendum Number 2. Los Alamos Technical
Report LATA/MX-94/OQ09. Los Alamos Technical Associates, Inc. Los Alamos, NM.
Brown, K.W., Wolf, H.W., Donnelly, K.C., and Slowey, J.F. 1979. The movement of fecal
coliforms and coliphages below septic lines. J. Environ. Qual. 8 (1): 121-125.
Bulkow, L.R., Wamwright, R.B., McMahon, B.J., Middaugh, J.P., Jenkerson, S.A., and
Margolis, H.S. 1993. Secular trends in hepatitis A virus infection among Alaskan Natives.
J. Infect. Dis. 168 (4):1017-1020.
Calderon, R.L. and Craun, G.R. 1998. Epidemiology of waterborne outbreaks, 1971-1996.
EPA OW/OST/HECD FTs Enterovirus Criteria Document
FINAL DRAFT
-------
CDC. 1998a. "Hepatitis A- reported cases per 100,000, population United States and territories,
1996." (http://www.cdc.gov/epo/dphsi/annsum/gph22.htm),
CDC. 1998b. "Poliomyelitis (paralytic) - by year, United States, 1966-1996."
(http://www.cdc.gov/epo/dphsi/annsum/gph33.htm).
CDC. 1998c. Enterovirus outbreak in Taiwan. (http://www.cdc.gov.od.oc.media/
pressrel/r980608.htm).
CDC. 1998d. Taiwan outbreak, (http://www.cde.gov/travel/taiwan.h1m).
CDC. 1997. Case definitions for infectious conditions under public health surveillance. Morbid.
Mortal. Weekly Rep. 46 (RR10).
CDC. 1996a. Surveillance for waterborne disease outbreaks-United States, 1993-1994. 45(ss-
CDC. 1996b. Hepatitis A vaccine and immune globulin disease and vaccine information.
(http://www.cdc.gov/travel/hepa_ig.htm).
CDC. 1996c. Hepatitis Surveillance: Viral Hepatitis Surveillance Program. Hepatitis
Surveillance Report 56. (http/www.cdc.gov/ncidod/diseases/hepatitis/h96surve.htm).
CDC. 1991. Waterborne disease outbreaks-United States, 1989-1990. 40(ss-3):l-22.
CDC. 1990. Waterborne disease outbreaks-United States, 1986-1988. 30(ss-l):l-13.
Chang, J.C.H., Ossoff, S.F., Lobe, D.C., Dorfrnan, M.H., Dumais, CM., Quails, R.G., and
Johnson, J.D. 1985. UV Inactivation of pathogenic and indicator microorganisms. Appl.
Environ. Microbiol 49:1361-1365.
Cherry, J.D. 1995. Enteroviruses. In: Remington and Klein, eds. Infectious Diseases of the Fetus
and Newborn Infant. Philadelphia: W.B. Saunders, pp. 404-446.
Cherry, J.D. 1981. Textbook of Pediatric Infectious Disease. Feigin, R.D., and Cherry, J.D., eds.
Philadelphia: W.B. Saunders Co.
Clapper, W.E. 1970. Comments on viruses recovered from dogs. J. Am. Vet. Med. Assoc.
156:1678-1680.
Clarkson, N.A., Kaufman, R., Lublin, D.M., Ward, T., Pipkin, P.A., Minor, P.O., Evans, D.J.,
and Almond, J.W. 1995. Characterization of the echovirus 7 receptor: domains of CD 55 critical
for virus binding. J. Virol. 69(9):5497-5501.
Cliver, D.O. 1997. Virus transmission via food. World Health Stat. 50 (1-2):90-101 .
EPA OW/OST/HECD 1M Enterovirus Criteria Document
FINAL DRAFT
-------
Oliver, D,O. 1987. Fate of viruses during sludge processing. In: Rao, V.C., ed. Human Viruses in
Sediments, Sludges, and Soils. Boca Raton, FL: CRC Press, pp. 111-127.
Corwin, A.L., Dai, T.C., Due, D.D., Suu, Van, N.I., and Ha, L.D. 1996. Acute viral hepatitis in
Hanoi, Viet Nam. Trans. R. Soc. Trop. Med. Hyg. 90 (6):647-648.
Crabtree, K.D., Gerba, C.P., Rose, J.B., and Haas, C.N. 1997. Waterborne adenovirus: A risk
assessment Wat. Sci. Tech. 35(11-12): 1-6,
Craun, G.F. 1990. Methods for the investigation and prevention of waterborne disease outbreaks.
EPA/600/1 -90/005a. Washington D.C.: U.S. Environmental Protection Agency.
Dagan, R. 1996. Nonpolio enteroviruses and the febrile young infant: epidemiologic, clinical,
and diagnostic aspects. Pediat. Infect. Dis. J. 15(1)67-71.
Dahling, D.R., and Safferman, R.S. 1979. Survival of enteric viruses under natural conditions in
a subarctic river. Appl. Environ. Microbiol. 38:1103-1110.
Dahling, D.R., Safferman, R.S., and Wright, B.A. 1989. Isolation of enterovirus and reovirus
from sewage and treated effluents in selected Puerto Rican communities. Appl. Environ.
Microbiol. 55:503-506.
Dalldorf, G., and Melnick, J.L. 1965. Coxsackieviruses. In: Horsefall, F.L., and Tamms, L., eds.
Viral and Rickettsial Infections of Man, 4th ed. Philadelphia: J.B. Lippincott, pp. 474—511.
Dalldorf, G. 1957. Neuropathogenicity of certain group A coxsaekie viruses. J. Exp. Med.
106:69.
DeLeon, R., and Gerba, C.P. 1991. Detection of rotaviruses in water by gene probes. Wat. Sci.
Tech. 24:281-284.
DeLeon, R., Shieh, C., Baric, R.S., and Sobsey, M.D. 1990. Detection of enteroviruses and
hepatitis A virus in environmental samples by gene probes and polymerase chain reaction. Proc.
1990 AWWA WQTC, San Diego, CA. AWWA, Denver, CO.
Dewilde, A., Pellieux, C., Hajjam, S., Wattre, P., Pierlot, C., Hober, D., and Aubry, J.M. 1996.
Virucidal activity of pure singlet oxygen generated by thermolysis of a water-soluble naphthalene
endoperoxide. J. Photochem. Photobiol. B. 36 (1):23—29.
Divizia, M., Gnesivo, C., Bonapaste, R.A., Morace, G., Pisani, G., and Pana, A. 1993. Hepatitis
A virus identification in an outbreak by enzymatic amplification. Eur. J. Epidemiol. 9
(2):203-208.
EPA OW/OST/HECD 11-5 Enterovirus Criteria Document
FINAL DRAFT
-------
Dodet, B,, Heseltine, E., Mary, C., and Saliou, P. 1997. Rotaviruses in human and veterinary
medicine, Sante 7 (3): 195-199.
Dufour, A.P. 1986. Diseases caused by water contact. In: Craun G.F., ed. Waterborne Diseases in
the United States. Boca Raton, FL: CRC Press, pp. 23-41.
Duncan, H.E., and Edberg, S.C. 1995. Host microbe interaction in the gastrointestinal tract.
Crit. Rev. Microbiol. 21(2):85-100.
Dubois, E., Le Guyader, F., Haugarreau, L., Kopecka, H., Cormier, M., and Pommepuy, M.
1997. Molecular epidemiological survey of rotaviruses in sewage by reverse transcriptase
seminested PCR and restriction fragment length polymorphism assay. Appl. Env. Microbiol.
63(5): 1794-1800.
Dutka, B.J. 1990. The presence of bacterial virus in ground water treated drinking water.
Environ. Pollut 63:293-298.
Eisenberg, J.N., Seto, E.Y.W., Olivieri, A.W., and Spear, R.C. 1996. Quantifying water pathogen
risk in an epidemiological framework. Risk Anal. 16 (4):549-563.
Enriquez, C., and Gerba C. 1995. Concentration of enteric adenovirus 40 from tap, sea and waste
water. Wat. Res. 29(11):2554-2560.
Enriquez, C.E., Abbaszadegan, M., Pepper, I.L., Richardson, K.J., Margolin, A.B., and Gerba,
C.P. 1993. Comparison of polio virus detection in water by cell culture and nucleic acid
hybridization. Wat. Sci. Tech. 27:315-319.
EPA. 1998. Demographic distribution of sensitive population groups. USEPA/OST/HECD.
Contract #68-06-0036 (WA-B-11/22).
EPA. 1997a. Draft final report on immune system impairment from chemical exposure and
mechanism of action. Office of Science and Technology, Human and Ecological Health Effects
Division, U.S. Environmental Protection Agency.
EPA. 1997b. Occurrence assessment for disinfectants and disinfectant byproducts in public
drinking water supplies. Office of Ground water and Drinking Water, U.S. Environmental
Protection Agency. EPA contract #68-06-0059.
EPA. 1996, ICRMicrobial Laboratory Manual. EPA/600/R-95/178. U.S. Environmental
Protection Agency.
EPA. 1994. Drinking water treatment for small communities: A focus on EPA's research.
EPA/640/K-94/003. Office of Research and Development, U.S. Environmental Protection
Agency.
EPA OW/OST/HECD 11 -6 Enterovirus Criteria Document
FINAL DRAFT
-------
EPA. 1990a. Citizens guide to ground water protection. EPA 440/6-90-004. U.S. Environmental
Protection Agency.
EPA. 1990b. Methods for the investigation and prevention of waterborne disease outbreaks.
EPA/600/1 -90/005a. U.S. Environmental Protection Agency.
EPA. 54FR 27486, Federal Register, June 29,1989.
EPA. 1985. Final draft drinking water criteria document for viruses. ECAO-CIN-451.
Environmental Criteria and Assessment Office, U.S. Environmental Protection Agency.
EPA. 1984. U.S. EPA Manual of methods for virology. EPA/600/4-84/013.
EPA. 1978. Report to Congress. Human viruses in the aquatic environment: a status report with
emphasis on the EPA research program. EPA-57Q/9-78-OQ6. U.S. Environmental Protection
Agency.
Every, L.V., and Dawson, S.D. 1995. Ground water as a vehicle for disease transmission in
southeastern Idaho: A case study.
Evison, L.M., and James, A, 1975. Bifodobacterium as an indicator of fecal pollution in water.
Prog. Wat. Technol. 7 (2):57-66.
Finch, G.R., Labatiuk, C.W., Helmer, R.D., and Belosevic, M. 1992. Ozone and ozone-peroxide
disinfection of Giardia and viruses. AWWA Research Foundation. Denver, CO.
Funderburg, S.W., Moore, B.E., Sagik, B.P., and Sorber, C.A. 1981. Viral transport through soil
columns under conditions of saturated flow. Wat. Res. 15:703-711.
Gale, P. 1996. Developments in microbial risk assessment methods for drinking water-a short
review. J. Appl. Bacteriol. 81(4):403-410.
Garthright, W.E., Archer, D.L., and Kvenberg, J.E. 1988. Estimates of incidence and costs of
intestinal infectious diseases. Public Health Rep. 103:107—116.
Geldreich, E.E. 1989. Drinking water microbiology—new directions toward water quality
enhancement. Int. J. Food Microbiol. 9:295-312.
Geldreich, E.E., Fox, K.R., Goodrich, J.A., Rice, E.W., Clark, R.M., and Swerdlow, D.L. 1992.
Searching for a water supply connection in the Cabool, Missouri disease outbreak of Escherichia
coli 0157:H7. Wat. Res. 26 (8): 1127-1137.
Gerba, C.P. 1983. Methods for recovering viruses from the water environment. In: Berg, G., ed.
Viral Pollution of the Environment. Boca Raton, FL: CRC Press, Inc.
EPA OW/OST/HECD 11 -7 Enterovirus Criteria Document
FINAL DRAFT
-------
Gerba, C.P., Hou, K., and Sobsey, M.D. 1985. Microbial removal and inactivation from water by
filters containing magnesium peroxide. J. Environ. Sci. Health 23:41-58.
Gerba, C.P., Keswick, B.H., Dupont, H.L., and Fields, H.A. 1984. Isolation of Rotavirus and
hepatitis A virus from drinking water. Monogr. Virol. 15:119-125.
Gerba C.P., and Rose, J.B. 1993. Estimating viral disease risk from drinking water. In: Cothern,
C.R., ed. Comparative Environmental Risk Assessment. Ch. 9. Ann Arbor, MI: Lewis
Publishers, pp.117-135.
Gerba, C.P., Rose, J.B., and Haas, C.N. 1996a. Sensitive populations: Who is at greatest risk?
Int. J. Food Microbiol. 30:113-123.
Gerba, C.P., Rose, J.B., and Haas, C.N. 1996b. Waterborne rotavirus: Risk assessment. Wat.
Res. 30:2929-2940.
Girones, R., Puig, M., Allard, A., L,ucena, F., Wadell, G., Jofre, J., Morris, R., Grabow, W.,
Botzenhart, K., and Wyn-Jones, A. 1995. Detection of adenovirus and enterovirus by PCR
amplification in polluted waters. Health Related Wat. Microbiol. 31(5-6):351-357.
Governal, R.A., and Gerba, C.P. 1997. Persistence of MS-2 and PRD-1 bacteriophages in an
ultrapure water system. J. Ind. Microbiol. BioTech. 18 (5):297-301.
Goyal, S.M., Keswick, B.H., and Gerba, C.P. 1984. Viruses in ground water beneath sewage
irrigated cropland. Wat. Res. 18 (3):299-302.
Grabow, W. 1997. Hepatitis viruses in water: Update on risk and control. Wat. SA 23
(4):379-385.
Grabow, W.O.K., Gauss-Muller, V., Prozesky, O.W., and Deinhardt, F. 1983. Inactivation of
hepatitis A virus and indicator organisms in water by free chlorine residuals. Appl. Environ.
Microbiol. 46:619-624.
Grew, N., Gohd, R.S., Arguedas, J., and Kato, J.I. 1970. Enteroviruses in rural families and their
domestic animals. Am. J. Epidemiol. 91:518-526.
Grinde, B., Jonassen, T.O., and Ushijima, H. 1995. Sensitive detection of group A Rotaviruses
by immunomagnetic separation and reverse transcription-polymerase chain reaction. J. Virol.
Meth. 55 (3):327-338.
Haas, C.N., Rose, J.B., Gerba, C,, and Regli, S. 1993. Risk assessment of virus in drinking water.
Risk Anal. 13 (5):545-552.
EPA OW/OST/HECD 11 -8 Enterovirus Criteria Document
FINAL DRAFT
-------
Hain, K.E., and O'Brien, R.T. 1979. The survival of enteric viruses in septic tanks and septic
tank drain fields. WRRI Report No. 108, New Mexico Wat. Res. Inst.
Hall, R.M., and Sobsey, M.D. 1993. Inactivation of hepatitis A virus and MS2 by ozone and
ozone-hydrogen peroxide in buffered water. Wat. Sci. Tech. 27(3/4):371-378.
Harris, G.D., Adams, V.D., Sorenson, D.L., and Curtis, M.S. 1987. Ultraviolet inactivation of
selected bacteria and viruses with photoreactivation of the bacteria. Wat. Res. 21:687-692.
Havelaar, A.H., and Pot-Hogeboom, W.M. 1988. F-specific RNA-bacteriophages as model
viruses in water hygiene: Ecological aspects. Wat. Sci. Tech. 20 (11/12):399-407.
Hawley, B.H., Morin, D.P., Geraghty, ME., Tomkow, J., and Phillips, C.A. 1973. Coxsackie B
epidemic at a boys' summer camp. JAMA 226 (1):33—37.
Hedberg, C.W., and Osterholm, M.T. 1993. Outbreaks of food-borne and waterborne viral
gastroenteritis. Clin. Microbiol. Rev. 6 (3):199-210.
Hejkal, T.W., Keswick, B., LaBelle, R.L., Gerba, C.P., Sanchez, Y., Dreesman, G., Hafkin, B.,
and Melnick, J.L. 1982. Viruses in a community water supply associated with an outbreak of
gastroenteritis and infectious hepatitis. J.A.W.W.A. 74:318—321.
Hepatitis Foundation International. 1998. Hepatitis statistics, pp. 1- 4.
(http ://www.hepfi ,org/stats.htm).
Hepatitis Foundation International. 1997. Hepatitis statistics/diagnosis and treatment.
(http://www.hepfi.org/stats.htm and http://www.hepfi.org/diagnosis.htm).
Herbold, K., Flehmig, B., and Botzenhart, K. 1989. Comparison of ozone inactivation, in flowing
water, of hepatitis A virus, polioviras 1, and indicator organisms. Appl. Environ. Microbiol.
55(ll):2949-2953.
Herbold-Paschke, K., Straub, U., Hahn, T., Teutsch, G., and Botzenhart, K. 1991. Behavior of
pathogenic bacteria, phages and viruses in ground water during transport and adsorption. Wat.
Sci. Tech. 24 (2):301-304.
Hemandez-Delgado and Toranzos, G.A. 1995. In situ replication studies of somatic and male-
specific coliphages in a tropical pristine river. Wat. Sci. Tech. 31(5-6):247-250.
Herwaldt, B.L., Craun, G.F., Strokes, S.L., and Juranek, D.D. 1992. Outbreaks of waterborne
disease in the United States: 1989-90. J.A.W.W.A. April 1991:129-135.
Highsmith, A.K., and Crow, S.A. 1992. Waterborne diseases. In: Encyclopedia of Microbiology,
vol. 4. Orlando, FL: Academic Press, pp. 377-384.
EPA OW/OST/HECD 1H? Enterovirus Criteria Document
FINAL DRAFT
-------
Hock, B. 1996. Advances in immunochemical detection of microorganisms. Ann. Biol. Clin. 54
(6):243-252.
Hoff, J.C. 1990, Principles of drinking water disinfection for pathogen control. In: Craun, G.F.,
ed. Methods for the Investigation and Prevention of Waterborne Disease Outbreaks. EP A/600/1 -
90/005a. U.S. Environmental Protection Agency.
Bellinger, B.F., and Ticehurst, J.R. 1996. In: Fields, B.N., Knipe, D.M., and Howley, P.M., eds.
Virology. Philadelphia: Lippincott-Raven Publishers, pp. 735-782.
Hovi, T., Stenvik, M., and Rosenlew, M. 1996. Relative abundance of enteroviras serotypes in
sewage differs from that in patients: Clinical and epidemiological implications. Epidemiol.
Infect. 116:91-97.
Hudson, H.E. 1981. Water clarification processes: Practical design and evaluation. New York,
NY: Van Nostrand Reinhold.
Hurst, C.J., Benton, W.H., and Stetler, R.E. 1989. Detecting viruses in water. J.A.W.W.A.
Ijzerman, M.M., Dahling, D.R., and Fout, G.S, 1997. A method to remove environmental
inhibitors prior to the detection of waterborne enteric viruses by reverse transcription-polymerase
chain reaction. J. Virol. Meth. 63 (1-2): 145-153.
Ijzerman, M.M., Hagedorn, C., and Reneau, R.B., Jr. 1992. Fecal indicator organisms below an
on-site waste water system with low pressure distribution. Wat. Air Soil Pollut. 63:201-210.
ILSI Risk Science Institute. 1996. A conceptual framework to assess the risks of human disease
following exposure to pathogens. Pathogen Risk Assessment Working Group. Risk Anal. 16
(6):841-847.
International Committee on Taxonomy of Viruses (ICTV). 1995. In: Murphy, F.A., Fauquet,
C.M., Bishop, D.H.L., Ghabrial, S.A., Jarvis, A.W., Martelli, M.A. Mayo, M.A., and Summers,
M.D., eds. Sixth Report of the ICTV. New York: Springer Verlag.
Jacangelo, J.G., Adham, S., and Laine, J-M. 1995. Application of membrane filtration techniques
for compliance with the surface water and ground water treatment rules. AWWA. Denver, CO.
Jansons, J., and Bucens, M.R, 1986. Concentration of Rotaviras by ultrafiltration. Wat. Res.
20:79-83.
Jorgensen, P.H., and Lund, E. 1995. Detection and stability of enteric viruses in sludge, soil, and
ground water. Wat. Sci. Tech. 17:185-195,
EPA OW/OST/HECD 11-10 Enteroviras Criteria Document
FINAL DRAFT
-------
Jothikumar, N., Cliver, D.O., and Mariam, T.W. 1998. Immunomagnetic capture PCR for rapid
concentration and detection of hepatitis A vims from environmental samples. Appl. Environ.
Mierobiol. 64:504-508.
Jothikumar, N., Khanna, P., Paulmurugan, R., Kamatchiammal, S., and Padmanabhan, P. 1995.
A simple device for the concentration and detection of enterovirus,, hepatitis E virus and
Rotavirus from water samples by reverse transcription-polymerase chain reaction. J. Virol. Meth.
55(3):401-415.
Kalijot, K.T., Ling, J.P., Gold, J.N.M., Laughon, B.E., Bartlett, J.G., et al. 1989. Prevalence of
acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea.
Gastroenterology 97:1031-1032.
Kaneko, M. 1989. Effect of suspended solids on inactivation of poliovirus and T-2 phage by
ozone. Wat. Sci. Tech. 21(3):215-219.
Kaplan, G.J. 1970. Echovims type 30 meningitis and related febrile illness: Epidemiologic study
of an outbreak in an Eskimo community. Am. J. Epidemiol. 92(4):257-265.
Kaplan, M.H., Klein, S.W., McPhee, J., and Harper, R.G. 1983. Group B coxsackievirus
infections in infants younger than three months of age: A serious childhood illness. Rev. Inf. Dis.
5 (6): 1019-1032.
Kelly, S., and Sanderson, W.W. 1958. The effect of chlorine in water on enteric viruses. Am. J,
Pub. Health 48(10): 1323-1334.
Keswick, B.H., and Gerba, C.P. 1980. Viruses in ground water. Env. Sci. Tech. 1290-1297.
Keswick, B.H., Gerba, C.P., DuPont, H.L., and Rose, J.B. 1984. Detection of enteric viruses in
treated drinking water. Appl. Environ. Mierobiol. 47 (6): 1290-1294.
Keswick, B.H., Gerba, C.P., Secor, S.L., and Cech, I. 1982. Survival of enteric viruses and
indicator bacteria in ground water. J. Environ. Health Sci. Al7:903-912.
Keswick, B.H., Satterwhite, T.K., Johnson, P.C., DuPont, H.L., Secor, S.L., Bitsura, J.A., Gary,
G.W., and Hoff, J.C. 1985. Inactivation of Norwalk virus in drinking water by chlorine. Appl.
Environ. Mierobiol. 50:261-264.
Kibrick, K. 1964. Current status of coxsaekie and echo viruses in human disease. Prog. Med.
Virol. 6:27-70.
Koff, R.S. 1992. Clinical manifestations and diagnosis of hepatitis A virus infection. Vaccine
(suppl. 1):S15-S17.
EPA OW/OST/HECD 11-11 Enterovirus Criteria Document
FINAL DRAFT
-------
Koff, R.S., and Galambos, J.T. 1987. Viral hepatitis. In: Schiff, L., and Schiff, E.R., eds.
Diseases of the Liver, 6th. ed. Philadelphia: J.B. Lippincott, pp. 457-582.
Kollar, J. 1975. Coliform contamination in rural water supplies in Aurora and Brule counties,
South Dakota. Proc. S. D. Acad. Sci. 54:223-228.
Koprowski, H., Norton, T.W., Jervis, G.A., Nelson, T.L., Chadwick, D., Nelsen, J.N., and
Meyer, C.F. 1956. Clinical investigations of attenuated strains of poliomyelitis virus: Use as a
method of immunization of children with living virus. JAMA 160:954-966.
Kott, Y., Roze, N., Sperber, S., and Betzer, N. 1974. Bacteriophages as viral pollution indicators.
Wat. Res. 8:165-171.
Kukkula, M., Arstila, P., Klossner, M., Maunula, L., Bonsdorff, C.V., and Jaatinen, P. 1997.
Waterborne outbreak of viral gastroenteritis. Scand. J. Infect. Dis. 29:415-419.
Kutz, S.M., and Gerba, C.P. 1988. Comparison of virus survival in freshwater sources. Wat. Sci.
Tech. 20(11/12):467-471.
Lawson, H.W., Braun, M.M., Glass, R.I.M., Stine, S.E., Monroe, S.S., Atrash, H.K., Lee, L.E.,
and Englender, S.J. 1991. Waterborne outbreak of Norwalk virus gastroenteritis at a southwest
U.S. resort: Role of geological formations in contamination of well water. Lancet
337:1200-1204.
Lennette, E.H., Balows, A., Hansler, W.J., and Shadomy, H.J. eds. Manual of Clinical
Microbiology, 4th ed. American Society for Microbiology, Washington, DC.
Leonardi, G.P., Greenberg, A.J., Costello, P., and Szabo, K. 1993. Echovirus type 30 infection
associated with aseptic meningitis in Nassau county, New York, USA. Intervirology 36:53-56.
Levinthal, G., and Ray, M. 1966. Hepatitis A: From epidemic jaundice to a vaccine-preventable
disease. The Gastroenterologist 4 (2): 107-115.
Lippy, E.G., and Waltrip, S.C. 1984. Waterborne disease outbreaks-1946-1980: A thirty-five
year perspective. J.A.W.W.A. 76:60-67.
Lopez-Pila, J.M., Dizer, H., and Dorau, W. 1996. Waste water treatment and elimination of
pathogens: New prospects for an old problem. Microbioldgia 12 (4):525-536.
Lucena, R., et al. 1994. Effect of distance from the polluting focus on the relative concentrations
of Bacteriodes fragilis phages and coliphages in mussels. Appl. Environ. Microbiol. 60:2272.
Lucena, F., Bosch, A., Jofre, J., and Schwartzbrod, L. 1985. Identification of viruses isolated
from sewage, riverwater, and coastal seawater in Barcelona. Wat. Res. 19:1237.
EPA OW/OST/HECD 11-12 Enterovirus Criteria Document
FINAL DRAFT
-------
Ma, J.F., Gerba, C.P., and Pepper, LL. 1995. Increased sensitivity of polioviras detection in tap
water concentrates by reverse transcriptase-polymerase chain reaction. J. Virol. Meth. 55
(3):295-302.
Margolin, A.B., Gerba, C.P., Richardson, K.J., andNaranjo, I.E. 1993. Comparison of cell
culture and a polio virus gene probe assay for the detection of enterovirases in environmental
water samples. Wat. Sci. Tech. 27 (3-4):311-314.
Margolin, A.B., Hewlett, M.J., and Gerba, C.P. 1991. The application of a polio virus cDNA
probe for the detection of enterovirases in water. Wat. Sci. Tech. 24:277-280.
Marzouk, Y., Goyal, S.M., and Gerba, C.P. 1980. Relationship of viruses and indicator bacteria
in water and waste water of Israel. Wat. Res. 14:1585-1590.
McDonnell, S., Kirkland, K.B., Hlady, W.G., Aristeguieta, C., Hopkins, R.S., Monroe, S.S., and
Glass, R.I. 1997. Failure of cooking to prevent shellfish-associated viral gastroenteritis. Arch.
Intern. Med. 157(1):111-116.
McFeters, G.A., Bissonnette, O.K., Jezeski, J.J., Thompson, C.A., and Stuart, D.G. 1974.
Comparative survival of indicator bacteria and enteric pathogens in well water. Appl. Microbiol.
27 (5):823-829.
Mehnert, D.U., Stewien, K.E., Harsi, C.M., Queiroz, A.P.S., Candeis, J.M.G., and Candeial,
J.A.N. 1997. Detection of rotavirus in sewage and creek water: efficiency of the concentration
method. Memorias do Instituto Oswaldo Cruz 92(1 ):97-100.
Melnick, J.L. 1996a. My role in the discovery and classification of the enterovimses. Annu. Rev.
Microbiol. 50:1-24.
Melnick, J.L. 1996b. Enteroviruses: Polioviras, coxsackieviruses, echovimses, and newer
enterovirases. In: Fields, B.N., Knipe, D.M., and Howley, P.M., eds. Fields Virology.
Philadelphia: Lippincott Raven Publishers.
Melnick, J.L. 1992. Enteroviruses. In: Encyclopedia of Microbiology, Vol. 2. Orlando, FL:
Academic Press, pp. 69-80.
Melnick, J.L. 1985. Taxonomy of Viruses. In: Lennette, E.H., Balows, A., Hausler, W.J., and
Shadomy, H.J., eds. Manual of Clinical Microbiology, 4th ed. Ch. 62. Washington, D.C.:
American Society for Microbiology, pp. 694-700.
Melnick, J.L., and Gerba, C.P. 1982. Viruses in surface, and drinking waters. Environ. Intern.
7:3.
EPA OW/OST/HECD 11-13 Enterovirus Criteria Document
FINAL DRAFT
-------
Melnick, J.L. 1965. Echoviruses. In: Horsefall, F.L., and Tamm, L., eds. Viral and Rickettsial
Infections of Man, 4th ed. Philadelphia: J.B. Lippincott, pp. 513-545.
Mena, K.D., Gerba, C.P., Haas, C.N., and Rose, J.B. Risk assessment of waterborne
coxsaekievirus (submitted 1998).
Metcalf, T.G., Melnick, J.L., and Estes, M.K. 1995. Environmental virology: From detection of
vims in sewage and water by isolation to identification by molecular biology—A trip of over 50
years. Annu. Rev. Microbiol. 49:461^187.
Modlin, J.F. 1997. Enteroviruses: Coxsackie, echoviruses, and newer enteroviruses. In: Long,
S.S., et al, eds. Principles and Practice of Pediatric Infectious Diseases. New York: Churchill
Livingstone.
Modlin, J.F. 1995. Coxsackieviruses, echoviruses, and newer enteroviruses. In: Mandell, G.L., et
al, eds. Principles and Practice of Pediatric Infectious Diseases. New York: Churchill
Livingstone.
Modlin, J.F., and Kinney, J.S. 1987. Perinatal enteroviras infections. Adv. Pediatr, Infect. Dis.
2:57-78.
Montgomery, J.M. 1985. Water Treatment Principles and Design. New York, NY: Wiley
Interscience.
Mullis, K.B., and Faloona, F.A. 1987. Specific synthesis of DNA in vitro via a polymerase-
catalyzed chain reaction. Meth. Enzymol. 155:335-350.
Muscillo, M., La Rosa, G., Aulicino, F.A., Orsini, P., Bellucci, C., and Miearelli, R. 1995.
Comparison of cDNA probe hybridizations and RT-PCR detection methods for the identification
and differentiation of enteroviruses isolated from seawater samples. Wat. Res. 29 (5): 1309—1316.
Nasser, A.M. 1994. Prevalence and fate of HAV in water. Crit. Rev. Environ. Sci. Tech.
24:281-323.
Nasser, A.M., Tchorch, Y., and Fattal, B. 1995. Validity of serological methods (ELISA) for
detecting infectious viruses in water. Wat. Sci. Tech. 31:307-310.
Nasser, A.M., Tchorch, Y., and Fattal, B. 1993. Comparative survival of E. Coli, F+
baeteriophages, HAV and Poliovirus 1 in waste water and ground water. Wat. Sci. Tech. 27 (3-
4):401-407.
National Academy of Sciences (NAS). 1994. Science and Judgement in Risk Assessment.
Washington, D.C.: National Academy Press.
EPA OW/OST/HECD 11-14 Enteroviras Criteria Document
FINAL DRAFT
-------
National Academy of Sciences (NAS). 1983. Risk Assessment in me Federal Government:
Managing the Process. Washington, B.C.; National Academy Press.
OW/OGWDW. 1998. Drinking water priority rulemaking: Ground Water Rule.
(http://www.epa.gov/OGWDW/standard/gwr.html).
Pancorbo, O.C., Evanshen, E.G., Campbell, W.F., Lambert, S., Curtis S.K., and Woolley, T.W.
1987. Infectivity and antigenicity reduction rates of human Rotavirus strain Wa in fresh waters.
Appl. Environ. Microbiol. 53 (8):1803-1811.
Paul, J.H., Rose, J.B., Jiang, S.C., London, P., Xhou, X., and Kellogg, C. 1997. Coliphage and
indigenous phage in Mamala Bay, Oahu, Hawaii. Appl. Environ. Microbiol. 63 (1):133-138.
Payment, P. 1993. Viruses: Prevalence of diseases, levels, and sources. In: Craun, G.F., ed.
Safety of Water Disinfection: Balancing Chemical and Microbial Risks. Washington, DC: ILSI
Press, pp. 99-113.
Payment, P. 1991. Fate of human enteric viruses, coliphages, and Clostridium perfringes during
drinking water treatment. Can. J. Microbiol. 37 (2): 154-157.
Payment, P. 1989. Presence of human and animal viruses in surface and ground water. Wat. Sci.
Tech. 21 (3):283-285.
Payment, P. and Armon, R. 1989. Virus removal by drinking water treatment process. Crit. Rev.
Env. Control 19(1): 15-32.
Payment, P.,*Tremblay, M., and Trudel, M. 1985. Relative resistance to chlorine of poliovirus
and coxsackievirus isolates from environmental sources and drinking water. Appl. Environ.
Microbiol. 49:981.
Payment P., Trudel, M., and Plante, R. 1985. Elimination of viruses and indicator bacteria at
each step of treatment during preparation of drinking water at seven water treatment plants.
Appl. Environ. Microbiol. 49:1418.
Pelzar, M.J., Chan, B.C., and Krieg, N.R. 1986. Microbiology. McGraw-Hill, New York.
Perez, O.M., Morales, W., Paniagua, M., and Strannegard, O. 1996. Prevalence of antibodies to
hepatitis A, B, C, and E viruses in a healthy population in Leon, Nicaragua. Am. J. Trop. Med.
Hyg. 55(1): 17-21.
Peterson, D.A., Hurley, T.R., Hoff, J.C., and Wolfe, L.G. 1983. Effect of chlorine treatment on
infectivity of hepatitis A virus. Appl. Env. Microbiol. 45 (l):223-227.
EPA OW/OST/HECD 11-15 Enteroviras Criteria Document
FINAL DRAFT
-------
Pfeil, R.M., Venkat, J.A., Plimmer, J.R., Sham, S., Davis, K., and Nair, P.P. 1994. Quantitative
assessment of ground water quality using a biological indicator: Some preliminary observations.
Arch. Environ. Contam. Toxicol. 26:201-207.
Powelson, D.K., and Gerba, C.P. 1994. Virus removal from sewage effluents during saturated
and unsaturated flow through soil columns. Wat. Res. 28 (10):2175-2181.
Powelson, D.K., Gerba, C.P., and Yahya, M.T. 1993. Virus transport and removal in waste water
during aquifer recharge. Wat. Res. 27 (4):583-590.
Prabhakar, B.S., Haspel, M.V., McClintock, P.R., andNotkins, A.L. 1982. High frequency of
antigenic variants among naturally occurring human coxsackie B4 virus isolates identified by
monoclonal antibodies. Nature 300:374-376.
Proctor, L.M. 1997. Advances in the study of marine viruses. Microsc. Res. Tech. 37
Puig, M., Jofre, G., Lucena, F., Allard, A., Wadell, G., and Girones, R. 1994. Detection of
adenoviruses and enteroviruses in polluted waters by nested PCR amplification. Appl. Environ.
Micro. 60:2963-2970.
Rao, V.C., Metcalf, T.G., and Melnick, J.L. 1987. Removal of indigenous rotavirus during
primary settling and activated-sludge treatment of raw sewage. Wat. Res. 2:171-177.
Rao, V.C., Metcalf, T.G., and Melnick, J.L. 1986. Human viruses in sediments, sludges and
soils. Bull. WHO 64(1): 1-1 4.
Regan, P.M., and Margolin, A.B. 1997. Development of a nucleic acid capture probe with
reverse transcriptase-polymerase chain reaction to detect poliovirus in ground water. J. Virol.
Meth. 64:65-72.
Regli, S., Rose, J.B., Haas, C.N., and Gerba, C.P. 1991. Modeling the risk from Giardia and
viruses in drinking water. J.A.W.W.A. 83:76-84.
Reynolds, K.A., Gerba, C.P., and Pepper, I.I. 1996. Detection of infectious enteroviruses by an
integrated cell culture-PCR procedure. Appl. Environ. Microbiol. 62 (4): 1424- 1427.
Rivera, S.C., Hazen, T.C., and Toranzos, G.A. 1988. Isolation of fecal coliforms from pristine
sites in a tropical rain forest. Appl. Environ. Microbiol. 54(2):513-517.
Robertson, J.B., and Edberg, S.C. 1997. Natural protection of spring and well drinking water
against surface microbial contamination. I. Hydrogeological parameters. Crit. Rev. Microbiol. 23
(2):143-178.
EPA OW/OST/HECD 11-16 Enterovirus Criteria Document
FINAL DRAFT
-------
Rose, J.B., Gerba, C.P., Singh, S.N., Toranzos, G.A. and Keswick, B.H. 1986. Isolating enteric
viruses from finished waters. J. Am. Wat. Works Assn. 78:51-61.
Roy, D., Engelbrecht, R.S., and Chian, E.S.K. 1982. Comparative interaction of six enteroviruses
by ozone. J. Am. Wat. Works Assoc. 74:660-664.
Roy, D., and Tittlebaum, M.E. 1982. Microbiology: Detection, occurrence and removal of
viruses. J.W.P.C.F. 54 (6):984-986.
Rusin, P.A., Sinclair, N.A., Gerba, C.P., and Gershman, M. 1992. Application of phage typing to
the identification of sources of ground water contamination. J. Contam. Hydrol. 11:173-188.
Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Erlich, H.A., and Arnheim, N. 1985. Enzymatic
amplification of B-globin genomic sequences and restriction site analysis for diagnosis of sickle
cell anemia. Science 230:1350-1354.
Salo, R.J. and Oliver, D.O. 1976. Effect of acid pH, salts, and temperature on the infectivity and
physical integrity of enteroviruses. Arch. Virol. 52:269-282.
Schaub, S.A., and Sorber, C.A. 1977. Virus and bacteria removal from waste water by rapid
infiltration through soil. Appl. Environ. Microbiol. 33 (3):609-619.
Schiff, G.M., Stefanovic, G.M., Young, E.G., Sander, D.S., Permekamp, J.K., and Ward, R.L.
1984. Studies of echo virus-12 in volunteers: determination of minimal infectious dose and the
effect of previous infection on infectious dose. J. Infect. Dis. 150 (6):858-866.
Schwab, K.J., De Leon, R., and Sobsey, M.D. 1996. Immunoaffinity concentration and
purification of waterborne enteric viruses for detection by reverse transcriptase PCR. Appl.
Environ. Microbiol. 62 (6):2086-2094.
Schwab, K., De Leon, R., and Sobsey, M.D. 1995. Concentration and purification of beef extract
mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and
Norwalk virus by reverse transcription-PCR. Appl. Environ. Microbiol. 61 (2):531-537.
Shapiro, C.N. 1997. Hepatitis A virus. In: Long, S.S., et al., eds. Principles and Practice of
Pediatric Infectious Diseases. New York: Livingstone, pp. 1295-1300.
Shapiro, C.N., Mahoney, F.J., and Mast, E.E. 1997. Hepatitis A. Ch. 3. pp. 1-8.
(http://www.cdc.gov/nip/manual/hep/hepa.htm).
Shieh, Y., Baric, R., and Sobsey, M. 1997. Detection of low levels of enteric viruses in
metropolitan and airplane sewage. Appl. Environ. Microbiol. 63:4401-4407.
EPA OW/OST/HECD 11-17 Enterovirus Criteria Document
FINAL DRAFT
-------
Sim, Y., and Chrysikopoulos, C.V. 1996, One-dimensional virus transport in porous media with
time-dependent inactivation rate coefficients. Wat. Res. 32 (8):2607-2611.
Smith, W.G. 1970. Coxsackie B myopericarditis in adults. Am. Heart J. 80 (l):34-46.
Snicer. 1996. Evaluation of ultraviolet (UV) technology for ground water disinfection.
AWWARF Draft Report.
Snowdon, J.A., and Oliver, C.O. 1989. Coliphages as indicators of human enteric viruses in
groundwater. Crit. Rev. Environ. Contr. 19:231—246.
Sobsey., M.D. 1989a. Simple membrane filter method to concentrate and enumerate male-specific
RNA coliphages. J.A.W.W.A. 82:52-59.
Sobsey, M.D. 1989b. Inactivation of health-related microorganisms in water by disinfection
processes. Wat. Sci. Tech. 21:179-195.
Sobsey, M.D., Fuji, T., and Shields, P.A. 1990. Inactivation of cell-associated and dispersed
hepatitis A virus by free and combined chlorine and chlorine dioxide. Proceedings of 1989 Water
Quality Technology Conference. American Water Works Association, Denver, CO. pp. 167-179.
Sobsey, M.D., Shields, P.A., Hauchman, F.S., Davis, A.L., Rullman, V.A., and Bosch, A. 1988a.
Survival and persistence of hepatitis A virus in environmental samples. In: Zuckerman, A.J,, ed.
Viral Hepatitis and Liver Disease. Alan Liss Inc., New York, pp. 121-124.
Sobsey, M.D., Fuji, T., and Shields, P.A. 1988b. Inactivation of hepatitis A virus and model
viruses in water by free chlorine and monochloramine. In: Proceedings of the International
Conference for Water and Wastewater Microbiology. IAWPRC. New York: Pergamon Press.
Sobsey, M.D., Shields, P.A., Hauchman, F.H., Hazard, R.L., and Caton, L.W. 1986. Survival and
transport of hepatitis A virus in soils, ground water and waste water. Wat. Sci. Tech. 18
(10):97-106.
Stewart, L.W., and Reneau, R.B., Jr. 1981. Spatial and temporal variation of fecal coliform
movement surrounding septic tank-soil absorption systems in two Atlantic Coastal Plain soils. J.
Environ. Qual. 10 (4):528-531.
Stone, S., Erickson, B., Alexander, M., Dunning, R.? Ebenezer, I. and Dwyer, D.M. 1993.
Characteristics of epidemic hepatitis A in Baltimore City: Implications for control measures.
Maryland Med. J. 42 (10):995-1000.
Storch, G., McFarland, L.M., Kelso, K., Heilman, J.C., and Caraway, C.T. 1979. Viral hepatitis
associated with day-care centers. JAMA 242 (14):1514-1518.
EPA OW/OST/HECD 11-18 Enterovirus Criteria Document
FINAL DRAFT
-------
Straub, M.T., Pepper, I.L., and Gerba, C.P. 1995. Comparison of PCR and cell culture for
detection of enterovirases in sludge-amended field soils and determination of their transport.
Appl. Env, Microbiol. 61 (5):2Q66-2068,
Straub, M.T., Pepper, I.L., and Gerba, C.P. 1994. Detection of naturally occurring enterovirus
and hepatitis A virus in undigested and anaerobically digested sludge using the polymerase chain
method. Can. J. Microbiol. 40:884-888.
Suptel, E.A. 1963. Pathogenesis of experimental coxsackievirus infection. Arch. Virol. 7:61.
Tani, N., Dohi, Y., Kuramatani, N., and Yonemasu, K. 1995. Seasonal distribution of
adenovirus, enteroviruses and reoviruses in urban river water, Microbiol. Immunol. 39
(8):577-580.
Thurman, R.B., and Gerba, C.R. 1987. Protecting ground water from viral contamination by soil
modification. J. Environ. Sci. Health A22 (4):396-388.
Tolsa, D.D., and Bryant, J.A. 1976. The economic impact of viral hepatitis in the United States.
Public Reports. 91(4): 349-353.
Toranzos, G.A., Gerba, C.P., and Hanssen, H. 1988. Enteric viruses and coliphages in Latin
America. Toxicol. Assess. Int. J. 3:491-510.
Toranzos, G.A., Gerba, C.P., and Hanssen, H. 1986. Occurrence of enteroviruses and rotaviruses
in drinking water in Colombia. Wat. Sci. Tech. 18:109-114.
Tougianidou, D., and Botzenhart, K. 1993. Detection of enteroviral RNA sequences in different
water samples. Wat. Sci. Tech. 27:219-222.
Townsend, T.R., Bolyard, E.A., Yolken, R.H., Bishop, C.A., Santos, G.W., Berschoner, W.E.,
Burns, W.H., and Saral, R. 1982. Outbreak of coxsackie Al gastroenteritis: A complication of
bone-marrow transplantation. Lancet 1:821-822.
Tsai, Y., and Parker, S. 1998. Quantification of poliovirus in seawater and sewage by
competitive reverse transcriptase-polymerase chain reaction. Can. J. Microbiol. 44:35-41.
U.S. Food and Drag Administration (FDA). 1992. Hepatitis A Virus. Foodborne Pathogenic
Microorganisms and Natural Toxins 1992 (Bad Bug Book). Center for Food Safety & Applied
Nutrition, (http://vm.cfsan.fda.gov/~mow/).
Vaughn, J.M., Chein, Y.S., Novotny, J.F., and Strout, D. 1990. Effects of ozone treatment on the
infectivity of hepatitis A virus. Can. J. Microbiol. 36(8):557-560.
EPA OW/OST/HECD 11-19 Enterovirus Criteria Document
FINAL DRAFT
-------
Vaughn, J.M., Landry, E.F., Baranosky, L.J., Beckwith, C.A., Dahl, M.C., and Delihas, N.C.
1978. Survey of human virus occurrence in waste water-recharged ground water on Long Island.
Appl. Environ. Microbiol. 36 (1):47-51.
Vaughn, J.M., Landry, E.F., Beckwith, C.A., and Thomas, M.Z. 1981. Virus removal during
ground water recharge: Effects of infiltration rate on adsorption of poliovirus to soil. Appl.
Environ. Microbiol. 41 (1):139-147.
Vaughn, J.M., Landry, E.F., and Thomas, M. 1983. Entrainment of human viruses through a
shallow, sandy soil aquifer. Appl. Environ. Microbiol. 45:1474-1480.
Vaughn, J.M., and Metcalf, T.G. 1975. Factors influencing use of coliphage as indicators of
enteric viruses in estuarine waters. Wat Res. 9:613-616.
Vaughn, J.M., and Novotny, J.F. 1991. Virus inactivation by disinfectants. In Hurst, C.J., ed.
"Modeling the Environmental Fate of Microorganisms." ASM Press, Washington, DC. pp. 217-
241.
Verdugo, U.R., Selinka, H.C., Huber, M., Kramer, B,, Kellermann, J., Hofschneider, P.H., and
Kandolf, R. 1995. Characterization of a 100-kilodalton binding protein for the six serotypes of
coxsackie B viruses. J. Virol. 69(11):6751-6757.
Vernon, A.A., Schable, C., and Francis, D. 1982. A large outbreak of hepatitis A in day-care
center. Am. J. Epidemiol. 115 (3):325—331.
Vilagines, P., Sarrette, B., Husson, G., and Vilagines, R. 1993. Glass wool for virus
concentration at ambient water pH level. Wat. Sci. Tech. 27 (3-4):299-306.
Wang, D., and Gerba, C.P. 1981. Evaluation of f2 coliphage for tracing movement of viruses in
ground water. (Invited paper: abstract).
Wang, D., Gerba, C.P., and Lance, C. 1981. Effect of soil permeability on virus removal through
soil columns. Appl. Environ. Microbiol. 42 (1):83—88.
Ward, R.L., and Akin, E.W. 1984. Minimum infectious dose of animal viruses. Crit. Rev.
Environ. Control 14 (4):297-310.
Wellings, P.M., Lewis, A.L., Mountain, C.W., and Pierce, L.V. 1975. Demonstration of virus in
ground water after effluent discharge onto soil. Appl. Microbiol. 29 (6):751-757.
Wiedenmann et al. 1993. Disinfection of hepatitis A virus and MS2 coliphage in water by
ultraviolet irradiation: Comparison of UV-susceptibility. Wat. Sci. Tech. 27(3-4):335-338.
Williams, P.P., and Akin, E.W. 1986. Waterborne viral gastroenteritis. J.A.W.W.A. 78 (l):34-39.
EPA OW/OST/HECD 11-20 Enterovirus Criteria Document
FINAL DRAFT
-------
Williams, P.P., and Stetler, R.E. 1994. Detection of FRNA coliphages in ground water:
Interference with the assay by somatic salmonella bacteriophages. Lett. Appl. Microbiol.
19:79-82.
Williams, S.V., Huff, J.C., and Bryan, J.A. 1975. "News from the Center for Disease Control"
hepatitis A and facilities for pre-school children. J. Infect. Dis. 131 (4):491-495.
Wilson, B.R., Roessler, P.P., VanDellen, E., Abbaszadegan, M, and Gerba, C.P, 1992.
Coliphage MS2 as a UV water disinfection efficacy test surrogate for bacterial and viral
pathogens. Proceedings of the Water Quality Technology Conference. May 1992. American
Water Works Association. Toronto, Ontario, Canada.
Woody, M.A., and Cliver, D.O. 1997. Replication of coliphage Q beta as affected by host cell
number, nutrition, competition from insusceptible cells and non-FRNA coliphages. J. Appl.
Microbiol. 82 (4):431-440.
Woody, M.A., and Cliver, D.O. 1995. Effects of temperature and host cell growth phase on
replication of F-specific RNA coliphage QB. Appl. Environ. Microbiol. 61 (4): 1520-1526.
World Health Organization (WHO). 1996. Microbial indicators of water quality. In: Guidelines
for Drinking Water Quality, vol. 2. pp. 82-99.
Yahya, M.T., Galsomies, L., Gerba, C.P., and Bales, R.C. 1993. Survival of bacteriophages MS-
2, and PRD-1 in ground water. Wat. Sci. Tech. 27 (3-4):409-412.
Yates, M.V. 1985. Septic tank density and ground water contamination. Ground Wat.
23:586-591r
Yates, M.V., and Gerba, C.P. 1985. Factors controlling the survival of viruses in ground water.
Wat. Sci. Tech. 17:681-687.
Yates, M.V., Gerba, C.P., and Kelley, L.M. 1985. Virus persistence in ground water. Appl.
Environ. Microbiol. 49 (4):778-781.
Yates, M.V., and Yates, S.R. 1988a. Septic tank setback distances: A way to minimize virus
contamination of drinking water. Ground Wat. 27 (2):202-208.
Yates, M.V., and Yates, S.R. 1988b. Virus survival and transport in ground water. Wat. Sci.
Tech.20(ll/12):301-306.
Yates, M.V., Yates, S.R., Warrick, A.W., and Gerba, C.P. 1986. Use of geostatistics to predict
virus decay rates for determination of septic tank setback distances. Appl. Environ. Microbiol. 52
(3):479-483.
EPA OW/OST/HECD 11-21 Enterovims Criteria Document
FINAL DRAFT
-------
Yeager, J,G.» and O'Brien, R.T. 1977. Enterovirus and bacteriophage inactivation in subsurface
waters and translocation in soil. WRRI Report No. 083. New Mexico Wat. Res. Inst,
Yoon, J.W., Austin, M., Onodera, T., and Notkins, A.L. 1979. Virus-induced diabetes mellitus.
N. Engl. J. Med. 200 (21):1173-1179.
Yui, L.A., and Gledhill, R.F. 1991. Limb paralysis as a manifestation of coxsackie B virus
infection. Dev. Med. Child Neurol. 33:427-438.
Zaoutis, T., and Klein, J.D. 1998. Enterovirus infections. Pediatr. Rev. 19(6):183-191.
EPA OW/OST/HECD 11 -22 Enterovirus Criteria Document
FINAL DRAFT
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