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
                                  FINAL DRAFT

-------
            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
                                  FINAL DRAFT

-------
                            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
 EPA OW/OST/HECD                         iii                  Enterovirus Criteria Document

                                    FINAL DRAFT

-------
                   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
EPA OW/OST/HECD                         v                   Enterovirus Criteria Document

                                    FINAL DRAFT

-------
                               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
 EPA OW/OST/HECD
                                         Vll
                                     FINAL DRAFT
Enterovirus Criteria Document

-------
                                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.








 EPA OW/OST/HECD                          M                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
       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








 EPA OW/OST/HECD                      i    1-2Enterovirus Criteria Document



                                      FINAL DRAFT

-------
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








 EPA OW/OSf /HECD1^3Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          1^4Enteroviras Criteria Document



                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          T5                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          1^6                   Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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,








 EPA OW/OST/HECD                           K7                    Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          Tl                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








EPA OW/OST/HECD                           N9                    Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                          1-10                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                        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,
 EPA OW/OST/HECD                          2-1                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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).
  EPA OW/OST/HECD                          2-2                   Enterovirus Criteria Document




                                        FINAL DRAFT

-------
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).








EPA OW/OST/HECD                          2^3                   Enterovirus Criteria Document



                                      FINAL DRAFT

-------
                                                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.
EPA OW/OST/HECD
    2-4

FINAL DRAFT
                                      Enterovirus Criteria Document

-------
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).
 EPA OW/OST/HECD                          2-5                    Enteroviras Criteria Document




                                      FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          2^6                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          2-7                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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).
 EPA OW/OST/HECD                         2-8                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                         2^9                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








EPA OW/OST/HECD                   :       2-10                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
                                             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.
EPA OW/OST/HECD
   2-11

FINAL DRAFT
Enterovirus Criteria Document

-------
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
 EPA OW/OST/HECD                         2-12                   Enteroviras Criteria Document




                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD            •             2-13                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                                       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.
EPA OW/OST/HECD
   2-14



FINAL DRAFT
Enterovirus Criteria Document

-------
                                          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.
EPA OW/OST/HECD
   2-15

FINAL DRAFT
Enterovirus Criteria Document

-------
                                             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
EPA OW/OST/HECD
   2-16

FINAL DRAFT
Enteroviras Criteria Document

-------
                              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.
EPA OW/OST/HECD
  2-17

FINAL DRAFT
Enterovirus Criteria Document

-------
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








 EPA OW/OST/HECD            :              2^18                 Enterovirus Criteria Document



                                      FINAL DRAFT

-------
                                               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
EPA OW/OST/HECD
   2-19

FINAL DRAFT
                                                                         Enterovirus Criteria Document

-------
                                            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
EPA OW/OST/HECD
   2-20

FINAL DRAFT
Enterovirus Criteria Document

-------
                                           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.
EPA OW/OST/HECD
   2-21

FINAL DRAFT
Enterovirus Criteria Document

-------
                                       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,
EPA OW/OST/HECD
   2-22

FINAL DRAFT
Enterovirus Criteria Document

-------
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
 EPA OW/OST/HECD                        2-23                   Enterovirus Criteria Document




                                      FINAL DRAFT

-------
                                           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.
EPA OW/OST/HECD
   2-24

FINAL DRAFT
Enterovirus Criteria Document

-------
                                             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).
EPA OW/OST/HECD
                                                   2-25

                                               FINAL DRAFT
            Enterovirus Criteria Document

-------
                             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.
EPA OW/OST/HECD
   2-26

FINAL DRAFT
Enterovirus Criteria Document

-------
(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),
 EPA OW/OST/HECD                         2-27                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                                  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).
EPA OW/OST/HECD
    2-28

FINAL DRAFT
Enterovirus Criteria Document

-------
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








 EPA OW/OST/HECD                        2X29                   Enterovirus Criteria Document



                                      FINAL DRAFT

-------
                                 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.
 EPA OW/OST/HECD
    2-30

FINAL DRAFT
Enteroviras Criteria Document

-------
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








 EPA OW/OST/HECD                         2^31                  Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                         2-32                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                               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








 EPA OW/OST/HECD                          ~l                   Enterovirus Criteria Document



                                      FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          3^2                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








EPA OW/OST/HECD                         3^3                  Enterovirus Criteria Document



                                      FINAL DRAFT

-------
       •  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.
 EPA OW/OST/HECD                         3-4                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                          3-5                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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








EPA OW/OST/HECD                          3^6                   Enteroviras Criteria Document



                                      FINAL DRAFT

-------
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)








 EPA OW/OST/HECD                          3^7                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
       •  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).








 EPA OW/OST/HECD                          3^1                   Enterovirus Criteria Document



                                      FINAL DRAFT

-------
       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








 EPA OW/OST/HECD                           3^9                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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.








 EPA  OW/OST/HECD                          MO                   Enterovirus Criteria Document



                                        FINAL DRAFT

-------
 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.




 EPA OW/OST/HECD                         JTi                   Enterovirus Criteria Document


                                        FINAL DRAFT

-------
      •   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








EPA OW/OST/HECD                          3^12  '                 Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                         3-13                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
        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
  EPA OW/OST/HEGD                          3-14                   Enterovirus Criteria Document

                                        FINAL DRAFT

-------
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








 EPA OW/OST/HECD                         3-15Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                          3-16                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
       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








 EPA OW/OST/HECD                          M7                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                         3^18                  Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                          3-19                    Enterovirus Criteria Document




                                        FINAL DRAFT

-------
                             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



 EPA OW/OST/HECD                        4^1                     Enterovirus Criteria Document

                                      FINAL DRAFT

-------
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


 EPA OW/OST/HECD                       4^2                     Enterovirus Criteria Document
                                       FINAL DRAFT

-------
exist a few reports on the detection of enteroviruses in domestic pets.  However, the impact of




this on public health is unknown.
 EPA OW/OST/HECD                       4-3                     Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                              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.








EPA OW/OST/HECD                        5^1                     Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD        '            '    5-2                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








EPA OW/OST/HECD                       5^3                    Enterovirus Criteria Document



                                      FINAL DRAFT

-------
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;








 EPA OW/OST/HECD                       5-4                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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).
 EPA OW/OST/HECD                        5-5                     Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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.








 EPAOW/OST/HECD                         fP>                     Enterovirus Criteria Document



                                        FINAL DRAFT

-------
                               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
EPA OW/OST/HECD
   5-7

FINAL DRAFT
Enterovirus Criteria Document

-------
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
 EPA OW/OST/HECD                         5-8                     Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                               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
EPA OW/OST/HECD
   5-9

FINAL DRAFT
Enterovirus Criteria Document

-------
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
 EPA OW/OST/HECD                        5-10                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                               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
EPA OW/OST/HECD
  5-11

FINAL DRAFT
Enterovirus Criteria Document

-------
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.
  EPA OW/OST/HECD                        5-12                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                               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
EPA OW/OST/HECD
  5-13

FINAL DRAFT
Enterovirus Criteria Document

-------
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)








 EPA OW/OST/HECD                        5-14                     Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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).








 EPA OW/OST/HECD                       5-15                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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).








 EPA OW/OST/HECD                       5-16Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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).
  EPA OW/OST/HECD                        5-17                    Entero.viras Criteria Document




                                       FINAL DRAFT

-------
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








EPA OW/OST/HECD                        S48                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                       5-19                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                       5-20                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                               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








 EPA OW/OST/HECD                         HTl                      Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                        6-2                    Enteroviras Criteria Document




                                       FINAL DRAFT

-------
                                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
EPA OW/OST/HECD
   6-3

FINAL DRAFT
Enterovirus Criteria Document

-------
       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








 EPA OW/OST/HECD                         fr4                     Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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.








 EPA OW/OST/HECD                         6^5                     Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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








EPA OW/OST/HECD                        6^6                     Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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)
 EPA OW/OST/HECD                         6-7                     Enterovirus Criteria Document




                                        FINAL DRAFT

-------
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).








 EPA OW/OST/HECD                         W5                     Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                         6-9                     Enterovirus Criteria Document




                                        FINAL DRAFT

-------
                                 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
 EPA OW/OST/HECD                         7-1                   Enterovirus Criteria Document




                                      FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          7-2                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
                         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.
EPA OW/OST/HECD
   7-3

FINAL DRAFT
Enterovirus Criteria Document

-------
                                    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.
EPA OW/OST/HECD
                    7-4

               FINAL DRAFT
Enterovirus Criteria Document

-------
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








 EPA OW/OST/HECD                           7^5                    Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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








 EPA OW/OST7HECD                           7-6                   Enteroviras Criteria Document



                                        FINAL DRAFT

-------
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.








EPA OW/OST/HECD                          7^7                    Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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.








 EPA OW/OST/HECD                           7^8                    Enterovirus Criteria Document



                                        FINAL DRAFT

-------
       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.
 EPA OW/OST/HECD                           7-9                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                                  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.
EPA OW/OST/HECD
    7-10

FINAL DRAFT
Enterovirus Criteria Document

-------
                                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.
EPA OW/OST/HECD
    7-11

FINAL DRAFT
Enterovirus Criteria Document

-------
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.
 EPA OW/OST/HECD                         7-12                  Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                          7-13                   Enterovirus Criteria Document
                                       FINAL DRAFT

-------
       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.
 EPA OW/OST/HECD                         7-14                   Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                                   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
 EPA OW/OST/HECD                        8-1                     Enterovirus Criteria Document




                                      FINAL DRAFT

-------
                                                  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.
EPA OW/OST/HECD
   8-2

FINAL DRAFT
                                         Enterovirus Criteria Document

-------
                                             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.
EPA OW/OST/HECD
   8-3




FINAL DRAFT
Enterovirus Criteria Document

-------
                                             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.
EPA OW/OST/HECD
   8-4




FINAL DRAFT
Enterovirus Criteria Document

-------
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
 EPA OW/OST/HECD                        8-5                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                                 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.
EPA OW/OST/HECD
                            8-6
                         FINAL DRAFT
 Enterovirus Criteria Document

-------
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








 EPA OW/OST/HECD                         8^7                     Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                         8^8                     Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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
 EPA OW/OST/HECD                         8-9                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                        iTlOEnterovirus Criteria Document



                                      FINAL DRAFT

-------
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
 EPA OW/OST/HECD                        8-11                     Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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).
 EPA OW/OST/HECD                        8-12                   Enterovirus Criteria Document




                                      FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                        8-13                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                         8-14                     Enterovirus Criteria Document




                                        FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                        8-15                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                     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,








EPA OW/OST/HECD                          
-------
      •   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).
EPA OW/OST/HECD                          9-2                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
       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.
 EPA OW/OST/HECD                          9-3                     Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                          
-------
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








 EPA OW/OST/HECD                          9^5                     Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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
 EPA OW/OST/HECD                         9-6                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                          9-7                    Enteroviras Criteria Document




                                       FINAL DRAFT

-------
       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-








EPA OW/OST/HECD                          9^8                   Enterovirus Criteria Document



                                      FINAL DRAFT

-------
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
 EPA OW/OST/HECD                          9-9                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                          9-10                    Enteroviras Criteria Document




                                        FINAL DRAFT

-------
                                    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,








EPA OW/OST/HECD                        KM                    Enterovirus Criteria Document



                                      FINAL DRAFT

-------
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








 EPA OW/OST/HECD                        ia5Enterovirus Criteria Document



                                       FINAL DRAFT

-------
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).
 EPA OW/OST/HECD                        10-3                    Enterovirus Criteria Document




                                      FINAL DRAFT

-------
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








 EPA OW/OST/HECD        •                 KM                     Enterovirus Criteria Document



                                        FINAL DRAFT

-------
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








 EPA OW/OST/HECD                        10^5                    Enteroviras Criteria Document



                                       FINAL DRAFT

-------
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,
 EPA OW/OST/HECD                        10-6                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
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








 EPA OW/OST/HECD                        W^JEnterovirus Criteria Document



                                       FINAL DRAFT

-------
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








EPA OW/OST/HECD                         HWJEnterovirus Criteria Document



                                      FINAL DRAFT

-------
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.
 EPA OW/OST/HECD                        10-9                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
       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).
 EPA OW/OST/HECD                       10-10                    Enterovirus Criteria Document




                                       FINAL DRAFT

-------
                                              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
EPA OW/OST/HECD
   10-11

FINAL DRAFT
Enterovirus Criteria Document

-------
                                          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
EPA OW/OST/HECD
   10-12




FINAL DRAFT
Enterovirus Criteria Document

-------
                                                    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).
EPA OW/OST/HECD
    10-13

FINAL DRAFT
Enterovirus Criteria Document

-------
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.
 EPA OW/OST/HECD                        10-14                   Enterovirus Criteria Document



                                       FINAL DRAFT

-------
                                    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

-------