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Introduction and Review of literature 1 Introduction and review of literature 1.1 Water as a mode of transmission for viruses: Water has a profound influence on human health, since water is required for consumption on a daily basis for survival and therefore access to some form of water is essential for life. However, water has much broader influences on health and well-being and issues such as the quantity and quality of the water supplied are important in determining the health of individuals and whole communities. Waterborne and water-related diseases are associated with exposure to water environments in many ways. These include treated waters like those used for drinking and recreation in swimming pools and related facilities, in food processing and other industrial activities, as well as untreated waters used for recreation and agricultural purposes such as crop irrigation and animal husbandry. The microbiological quality of drinking water has been implicated in the spread of important infectious diseases such as cholera, typhoid, dysentery, hepatitis, giardiasis, guinea worm and schistosomiasis. Virtually all forms of life starting from simplest unicellular organisms like bacteria to organisms with highest degree of biological complexity like plants and animals including human beings are affected by one or other type of viruses. Transmission of viruses between hosts takes place by a variety of mechanisms including direct inoculation of contaminated body fluids from an infected host into the tissue or blood stream of a new host (Human Immunodeficiency Virus, Hepatitis B Virus, Hepatitis C Virus, rabies and haemorrhagic fever viruses), inhalation of air containing the viruses by a new host (influenza and measles) and transmission by the fecal-oral route often involving the ingestion of food and water contaminated with the viruses. These enteric viruses will be discussed in detail subsequently. Water-related disease places an excessive burden on the population and health services of many countries worldwide and in particular those in developing countries. Infectious diarrhoea or gastroenteritis is the most frequent, non-vector, water-related health outcome in both the developed and 1 developing world. The World Health Organization (WHO) estimated that every year there are 1.7 million deaths related to unsafe water, sanitation and hygiene, mainly through infectious diarrhoea. Approximately 4 billion cases of diarrhoea annually account for over 82 million disability adjusted life years (DALYs), representing 5.7% of the global burden of disease and placing diarrhoeal diseases as the third highest cause of morbidity and sixth highest cause of mortality (Pruss and Havelaar, 2001). Although the developing world is hardest hit by waterborne diseases, developed countries are also affected. For instance, the largest outbreak of a waterborne disease on record with some 403,000 cases of cryptosporidiosis occurred in 1993 in Milwaukee, a highly developed modern city in the USA (MacKenzie et al., 1994). 1.2 History of drinking water treatment: Storage and distribution of water for drinking purposes has been practiced by human beings for centuries. In ancient times when people lived as hunters/ collectors, river water was used for drinking water purposes. When people permanently stayed in one place for a long period of time, proximity to a river or lake was the most important criteria and this fact holds true even today. About 7000 years ago, Jericho (Israel) stored water in wells that were used as source of drinking water. People also started to develop drinking water transport systems. The transport took place through simple channels, dug in the sand or in rocks. Later on one also started using hollow tubes. Egypt used hollow palm trees and China and Japan used bamboo trunks. Eventually one started using clay, wood and even metal. In Persia people searched for underground rivers and lakes. The water went through holes in rocks into the wells on the plains. Around 3000 B.C., the city of Mohenjo-Daro (currently in Pakistan) used a very extensive water supply. In this city there were public bathing facilities with water boiler installations and bathrooms, demonstrating existence of very early technological developments. In ancient Greece, spring water, well water, and rainwater were used. Because of a fast increase in urban population, Greece was forced to store 2 water in wells and transport to the people through a distribution network. The water that was used was carried away through sewers, along with the rainwater. When valleys were reached, the water was led through hills under pressure. The Greeks were among the first to gain an interest in water quality. They used aeration basins for water purification. The Romans were the greatest architects and constructors of water distribution networks in history. They used river, spring or groundwater for provisioning. The Romans built dams on rivers, causing lakes to form. The lake water was aerated and then supplied. Mountain water was the most popular type of water because of its quality. For water transport the aquaducts where built. Through these aquaducts water was transported for tens of miles. Water winnings were protected from foreign pollutants. After the fall of the Roman Empire, the aquaducts were no longer used. From 500 to 1500 A.D. there was little development in the water treatment area. Wooden plumbing was used in the cities during the Middle Ages. The water was extracted from rivers or wells, or from outside the city. Soon, circumstances became highly unhygienic, because waste and excrements were discharged into the water. People who drank this water fell ill and often died. To solve this problem people started drinking water from outside the city, where rivers where unpolluted. This water was carried to the city by so-called water-bearers. John Gibb built the first drinking water supply that supplied an entire city in Paisley, Scotland in 1804, in order to supply his bleachery and the entire city with water. Within three years, filtered water was transported to Glasgow. In 1806 Paris operated a large water treatment plant. The water settled for 12 hours, before it was filtered. Filters consisted of sand and charcoal and were replaced every six hours. In 1827, the Englishman James Simpson built a sand filter for drinking water purification. Today, we still call this the number one tribute to public health. Public treatment facilities use a variety of different steps in their water purification process before the final product is tested and stored for consumer use. In general, the methods used include; 3 Physical processes (filtration and sedimentation). Biological processes (slow sand filters or activated sludge). Chemical processes including flocculation, chlorination and the use of electromagnetic radiation such as ultraviolet light. During the purification process the concentration of particulate matter including suspended particles, parasites, bacteria, algae, viruses, fungi; and a range of dissolved and particulate material may be reduced. The standards for drinking water quality are developed by Governments or by international agencies setting criteria for allowing maximum concentrations of contaminants in water intended for public use. 1.3 Viruses in water Viruses predominantly associated with waterborne transmission are referred to as enteric viruses that primarily infect cells of the gastrointestinal tract, and are excreted in the faeces of infected individuals. Enteric viruses can be transported in the water environment through groundwater, estuarine water, seawater, rivers, aerosols emitted from sewage treatment plants, insufficiently treated water, drinking water, and private wells that receive treated or untreated wastewater either directly or indirectly (Bitton and Gerba, 1984; Lipp et al., 2002; Rose et al., 1987; Sobsey et al., 1986; Yates et al., 1985). Over 100 virus species are present in contaminated waters causing a wide variety of illnesses in man (Table 1) including hepatitis, gastroenteritis, meningitis, fever, rash and conjunctivitis (Bosch, 1998). The viruses concerned are highly host specific. Their presence in water environments is a sound evidence of human faecal pollution. The extent of the host specificity of enteric viruses is such that it is used as a valuable tool to distinguish between faecal pollution of human and animal origin, or to identify the origin of faecal pollution (Maluquer de Motes et al.,2004; Hundesa et al., 2006). The hepatitis E virus may be the only meaningful exception to this rule, having strains which seem to infect both humans and certain animals, complying with the definition of a zoonosis (Reuter et al., 2009). The earliest record of diseases caused by enteric viruses may well be the report in the 4 Babylonian Talmud that hepatitis was common in the fifth century BC (Zuckerman, 1983). Table 1- Enteric viruses present in water environments. Genus Enterovirus Popular name Disease caused Poliovirus Paralysis, meningitis, fever Coxsackievirus, A, B Herpangina, meningitis, fever, hand-footand-mouth disease, myocarditis, rush, pleurodynia Echovirus Meningitis, fever, respiratory disease, rush, gastroenteritis Hepatovirus Hepatitis A Hepatitis Reovirus Human reovirus encephalitis and myocarditis Rotavirus Human rotavirus Gastroenteritis Mastadenovirus Human adenovirus Gastroenteritis, respiratory disease, conjunctivitis Calicivirus Human calicivirus Gastroenteritis Norovirus Norovirus Gastroenteritis, fever Astrovirus Human astrovirus Gastroenteritis Hepevirus Hepatitis E Hepatitis Parvovirus Human parvovirus Gastroenteritis Coronavirus Human coronavirus Gastroenteritis, respiratory disease Torovirus Human torovirus Gastroenteritis 1.4 Waterborne outbreak of viral diseases: Viruses are a major cause of waterborne and water-related diseases. Extreme examples include the outbreak of 300,000 cases of hepatitis A and 25,000 cases of viral gastroenteritis in 1988 in Shanghai caused by shellfish harvested from a sewage- polluted estuary (Halliday et al., 1991). 30 000 cases were reported in New Delhi, India, (1955 - 1956) after the flooding of the river Yamuna and contamination of the city's drinking water supply system (Viswanathan, 1957). In 1991, an outbreak of 79,000 cases of hepatitis E in Kanpur was ascribed to polluted drinking water (Aggarwal and Naik, 1994). 52 000 cases were reported in Kashmir, India, in 1978 (Khuroo et al., 1981). In 2003, 7,653 cases of hepatitis A were reported to CDC, which, when 5 corrected for underreporting and asymptomatic infections (Armstrong and Bell 2002), represents an estimated 36,700 cases and 79,600 infections. Although the mortality of many waterborne diseases is relatively low, the socioeconomic impact even of non-fatal infections is immense. There is reason to believe that the health impact of waterborne diseases, and particularly those caused by viruses, tends to be underestimated (Regli et al., 1991; Gerba et al., 1996a). For instance, mortality data do not reflect the large number of infected individuals who suffer from clinical manifestations that range from mild unreported discomfort to non-fatal severe illness, with far-reaching socio-economic implications (Pegram et al., 1998). 1.5 Waterborne viruses: Commonly studied groups of enteric viruses belong to the families Picornaviridae (polioviruses, enteroviruses, coxsakieviruses, hepatitis A virus, and echoviruses), Adenoviridae (adenoviruses), Caliciviridae (noroviruses, caliciviruses, astroviruses, and small round-structured viruses), and Reoviridae (reoviruses and rotaviruses). Enteric virus groups that are considered to be emerging waterborne pathogens include; Circoviruses consisting of torque tenovirus and torque tenovirus-like virus. These are nonenveloped viruses with single-stranded circular DNA and are resistant to heat inactivation. Picobirnaviridae, small nonenveloped viruses with bisegmented doublestranded RNA that are extremely resistant to UV light inactivation Parvoviruses, the smallest known enteric viruses, with single stranded RNA and high heat resistance Polyomaviruses including JC virus, BK virus, and simian virus 40. These are nonenveloped double-stranded DNA viruses that have been found to be very heat stable but are less resistant to chlorination than enteroviruses (Bofill-Mas et al., 2000, Brauniger et al., 2000, Engelbrecht et al., 1980, Wilhelmi et al., 2003). Enteric viruses are of immense public health concern due to their low infectious dose (Haas et al., 1993). For example, the probability of 6 infection from exposure to single rotavirus particle is 31%, and no more than 1 PFU is required to cause infection in 1% of healthy adults with no antibody against the virus (Schiff et al., 1984). Haas et al. (1993) concluded that the risk of infection when consuming viruses in drinking water is 10- to 10,000- fold greater than that for pathogenic bacteria at similar exposures. Because of the potential for contamination from a variety of sources, enteric viruses in water are of particular concern. Since the 1980s, with significant advancements in the area of environmental virology, enteric viruses have been recognized as the causative agents in many non-bacterial gastroenteritis cases and outbreaks (Bosch 1998). Enteric viruses have been isolated from and linked to outbreaks originating from contaminated drinking water sources, recreational waters (e.g., waters for swimming, canoeing, surfing, etc.), urban rivers, and shellfish harvested from contaminated waters (Cecuk et al., 1993; Dewailly et al., 1986; Jiang et al., 2001; Lee and Kim, 2002; Lipp and Rose, 1997; Muscillo et al., 1994; Patti et al., 1996). Between 1975 and 1979, water, followed by shellfish, was reported to be the main vehicle in outbreaks of vehicle-associated viral disease in the United States (Cliver, 1984). Several reports indicate that only a fraction of waterborne disease incidences are ever reported; Craun (1991) suggested that fewer than half of waterborne outbreaks occurring in the United States are investigated and reported. Nonpotable water, such as seawater, is also important; enteric viruses are able to persist for extended periods in the marine environment, which increases the probability of human exposure by recreational contact and accumulation in shellfish (Lipp and rose, 1997). Because shellfish are filter feeders, the concentration of viruses accumulated in their edible tissues may be much higher than that in the surrounding water (Abad et al., 1997). 1.5.1 Enteric hepatitis viruses: The term ‘‘jaundice’’ was used as early as in the ancient Greece when Hippocrates described an illness probably corresponding to a viral hepatitis. However, it was not until the beginning of the twentieth century when a form 7 of hepatitis was associated to an infectious disease occurring in epidemics and the term ‘‘infectious hepatitis’’ was established. In the early 1940s two separate entities were defined: ‘‘infectious’’ and ‘‘serum’’ hepatitis, and from 1965 to nowadays the different etiological agents of viral hepatitis have been identified. Although all viral hepatitis are infectious the term “infectious” refers to the fecal-oral mode of transmission and the ‘‘serum’’ hepatitis to those transmitted parenterally. 1.5.1.1 Hepatitis A The etiological agent of hepatitis A, hepatitis A virus (HAV) belongs to genus Hepatovirus within family Picornaviridae and consists of a nonenvoloped icosaedral capsid of around 30 nm in diameter containing a positive ssRNA genomic molecule of 7.5Kb (Fauquet et al., 2005). The genome contains a single open reading frame (ORF) encoding a polyprotein of around 2225 amino acids (aa) preceded by a 5’ non-coding region (5’NCR) that makes around 10% of the total genome, and followed by a much shorter 3’NCR that contains a poly(A) tract (Baroudy et al., 1985; Cohen et al., 1987). This genome is uncapped but covalently linked to a small viral protein (VPg) (Weitz et al., 1986). The singly translated polyprotein is subsequently cleaved into 11 proteins through a cascade of proteolytic events brought about mainly by the viral 3C protease (Schultheiss et al., 1994; Schultheiss et al., 1995). Different HAV isolates have been classified in to 7 genotypes (Robertson et al., 1992) Human isolates belong to genotypes I, II, III and VII while genotypes IV, V and VI represent isolates from Old World monkeys. Additional analyses indicated, however, that genotypes II and VII should be reclassified as subtypes A and B of genotype II (Costa-Mattioli et al., 2002). Importantly, a single serotype exists. In developing countries, hepatitis A is highly endemic and a large proportion of population acquires immunity through asymptomatic infection early in life (Gust, 1992). Improvement in hygienic and socioeconomic conditions has resulted in a decrease in the number of natural childhood infections. As a consequence, an increase in the susceptible adults with associated increased proportion of clinical disease is noted (Hadler, 1991) 8 HAV is shed in the feces of infected patients. The viral concentration in such stools is highest (up to 1011 genome copies/g of feces) after two weeks of the onset of symptoms and lasts at least four more weeks (Costafreda et al., 2006). HAV infection is mainly propagated via the fecal-oral route as the person-to-person contact is the most common mode of transmission (Mast and Alter, 1993). HAV survival in contaminated fomites, such as sanitary paper, sanitary tile and latex gloves, is very long (Abad et al., 1994a). In consequence, given the high excretion level of HAV, transmission of the infection is facilitated when poor sanitary conditions occur. Nevertheless, transmission through the parental route may occasionally occur (Noble et al., 1984; Sheretz et al., 2005). Viruses present in the stool of infected patients are discharged into sewage which ultimately may contaminate surface waters (Thornton et al., 1995), drinking water supply and seawater. The virus may be acquired and concentrated by shellfish growing in these waters or contaminate the vegetables irrigated with the polluted waters. Several waterborne (Bloch et al., 1990; Mahoney et al., 1992; Sutmoller et al., 1982, Arankalle et al., 2006) and foodborne outbreaks of the disease have been reported. Within this latter category, shellfish grown and harvested from waters receiving urban contaminants is a cause of large outbreaks of infectious hepatitis (Halliday et al., 1991; Sanchez et al., 2002). Additionally, large outbreaks associated with the consumption of berry fruits (Reid and Robinson, 1987) and vegetables (Rosemblum et al., 1990; Dentinger et al., 2001) irrigated with contaminated waters have been documented. Waterborne outbreaks are less common since the introduction of drinking water treatments. However, reports exist when these measures fail and outbreaks of hepatitis A occur (Bosch et al., 1991). HAV is stable in the environment, especially when associated with organic matter, and is resistant to low pH and heating (Hollinger and Emerson, 2001, Abad et al., 1994b; Bosch et al., 1994). These characteristics facilitate the likelihood of transmission by contaminated food and water and also improve the likelihood of detection in environmental samples, including water and sewage (Biziagos et al., 1988). Thus, although not compulsory, screening for HAV is advisable at least in specific samples when suspicion of contamination exists. 9 1.5.1.2 Hepatitis E HEV is the sole member of the genus Hepevirus in the family Hepeviridae (Emerson et al., 2004). It is a small, round non-enveloped particle of 27–34 nm with a genome approximately 7.2 kb in length that consists of a polydenylated, single-strand RNA molecule containing three discontinuous and partially overlapping ORFs (Tam et al., 1991), and 5’ and 3’ cis-acting elements which have important roles to play in HEV replication and transcription. HEV sequences have been classified into four genotypes, namely, genotypes 1, 2, 3, and 4 (Schlauder and Mushahwar, 2001). Genotype 1 of HEV consists of epidemic strains in developing countries in Asia and Africa; genotype 2 has been found in Mexico and Africa; genotype 3 is widely distributed throughout the world and has been isolated from sporadic cases of an acute HEV infection and/or domestic pigs in the United States, several European countries and Japan. Genotype 4 is found mainly in Asian countries and comprises strains from human and domestic pigs (Meng et al., 1998; Schlauder and Mushahwar, 2001; Lu et al., 2004; Lorenzo et al., 2007; Okamoto, 2007). A novel strain of HEV that was isolated from chicken with hepatitis splenomegaly syndrome was proposed to belong to either a new genotype 5 or to a separate genus (Haqshenas et al., 2001; Huang et al., 2002). A large number of hepatitis E epidemics have been reported from India, Nepal, Burma, the former USSR, Pakistan, Mexico, China, Somalia, Ivory Coast, Algeria and other African countries (Ramalingaswami and Purcell, 1988, Ticehurst, 1991). HEV is an important cause of sporadic acute viral hepatitis among Indian adults (Arankalle et al., 1993). During epidemics, the virus is associated with high mortality among pregnant women (Khuroo et al., 1981). HEV has also been shown to be responsible for a substantial proportion of sporadic fulminant cases (Arankalle et al., 1995). With the sequencing information available so far, genotype 1 appears to be the only genotype circulating in Indian patients, both in epidemic as well as sporadic situations (Arankalle et al., 2002, Arankalle et al., 2007). Many epidemics of water-borne hepatitis have occurred throughout India. These were thought to be epidemics of hepatitis A until 1980, when evidence for an enterically 10 transmitted non-A, non-B hepatitis was first reported. Subsequently, hepatitis E virus was discovered and most recent epidemics of enterically transmitted non-A, non-B hepatitis have been attributed to hepatitis E virus infection (Arankalle et al., 1994). 1.5.2 Enteroviruses Human enteroviruses (EVs) belong to the genus Enterovirus of the Picornaviridae family (Stanway et al., 2005). EVs are small RNA viruses with about 7.5 kilobase messenger-sense genome and an icosahedral nonenveloped capsid composed of 60 copies of each of the four different capsid proteins referred to as VP1 through VP4. Traditional division organizes this taxonomic group into the subgenera polioviruses, coxackieviruses (group A, B), echoviruses and a group of enteroviruses marked according to their serotype number (66–71 and newly identified 73–75, 77, 78) (Khetsuriani et al., 2006). The group of polioviruses includes 3 different serotypes; type 1 and 3 are recognized as epidemic while type 2 as endemic. Coxackieviruses are divided into 2 groups A and B. Group A includes 24 serological types, whereas B group comprises 6 serotypes. In 2003, the International Committee on Taxonomy of Viruses created a new taxonomy classification. Enteroviruses henceforth were divided into 5 groups of species based on their molecular properties (Khetsuriani and Parashar, 2003). The distribution of enteroviruses by species only partially corresponds to the groups in the traditional classification. Because molecular techniques of enterovirus typing are becoming increasingly available, new enteroviruses continue to be identified, and enteroviruses 79--101 have been recently described (Oberste et al., 1999; 2005; 2006; Norder et al., 2003). The spread of enteroviral infections occur mainly by the faecal-oral and oral-oral route, but also through direct contact with secretions from ophthalmic and dermal lesions. Infection is transmitted by contact with water or food contaminated with infected feaces (Pallansch and Roos, 2001). It poses a threat of transmission of infection and even an epidemic outbreak (Fong and Lipp 2005). Although most enterovirus (EV) infections are asymptomatic, estimates are that as many as 5 to 10 million symptomatic EV infections occur 11 each year in the United States (Strikas et al., 1986). An infected individual may shed enteroviruses in the stool for up to 16 weeks (Romero, 1999). This excreted virus in the faeces is translocated to the environment. The amounts excreted in the case of poliovirus are known to vary with maximal amounts reaching 106 infectious doses per gram (Melnick and Rennick, 1980). Laboratory methods used for enterovirus identification have changed substantially over time. Initially, enteroviruses were detected exclusively by viral culture (in vitro or in suckling mice) and identified by neutralization reaction using intersecting pools of type-specific antisera (Pallansch and Roos, 2001). Immunofluorescent assays using monoclonal antibodies became available for some enteroviruses in the 1980s (Khetsuriani and Parashar, 2003). In the 1990s, new molecular methods of enterovirus detection and identification were introduced. Beginning in the mid-1990s, the panenterovirus-polymerase chain reaction (PCR) assay which detects all enteroviruses but does not allow their differentiation became increasingly available and supplanted viral culture in many diagnostic laboratories (Rotbart, 1990; Byington et al., 1999; Hamilton et al., 1999). The molecular method for enterovirus typing based on sequence of the VP1 gene (which encodes important type-specific epitopes and, therefore, correlates with serotype) was developed in the late 1990s and is being used by increasing numbers of laboratories (Oberste et al., 1999, Caro et al., 2001). Enteroviruses are tolerant to residual chlorine from sewage treatment (Keswick et al., 1984) and a wide range of salinities (Skraber et al., 2004; Wetz et al., 2004), facilitating their survival in environmental waters. Enteroviruses are relatively thermostable, but less so than hepatitis A virus. Most enteroviruses are readily inactivated at 42°C, although some sulfhydral reducing agents and magnesium cations can stabilize viruses so that they are relatively stable at 50°C (Ackermann et al., 1970; Dorval et al., 1989). Due to the stability of the virion in the environment, multiple studies have been conducted in order to demonstrate the presence of enteroviruses in different kinds of aquatic environments such as seas, rivers, streams, drinking water, ground water, and sewage worldwide. (Lodder and de Roda Husman, 2005; 12 Abbaszadegan et al., 1999; Lodder-Verschoor et al., 2005; Amvrosieva et al., 2006). 1.5.3 Waterborne viruses causing gastroenteritis Acute gastroenteritis is one of the most common diseases in humans, and continues to be a significant cause of morbidity and mortality worldwide (Glass and Kilgore, 1997). Children under 5 years of age are particularly prone and it is calculated that in this group there are more than 700 million cases of acute diarrhea every year (Snyder and Merson, 1982). The mortality associated with gastroenteritis has been estimated to be 3–5 million cases per year, the majority of which occur in developing countries (Bern et al., 1994; Guerrant et al., 1990; Warren, 1990). In the developed world, the impact of the illness is seen in its high morbidity and in the high incidence of hospitalization (Christensen, 1999). Since the 1940s, viruses have been suspected of being important causes of gastroenteritis, as the etiology remained unknown in most cases (Kapikian and Chanock, 1996; Parashar et al., 1998). However, it was not until 1972 that Kapikian et al. (1972) first identified a virus (Norwalk virus) in feces after an outbreak of diarrhea as a cause of gastroenteritis. One year later, Bishop et al. (1973) observed the presence of rotavirus in the deodenal mucosa of children with gastroenteritis and in 1975, astroviruses (Madeley and Cosgrove, 1975) and enteric adenoviruses were identified in the feces of children with acute diarrhea (Flewett et al., 1975). Since then, the number of viruses associated with acute gastroenteritis has steadily increased. Thus, coronaviruses (Caul et al., 1975), picobirnaviruses Chandra, 1997; Grohmann et al., 1993; Ludert and Liprandi, 1993), pestiviruses (Yolken et al., 1989) and toroviruses (Beards et al., 1986) which produce diarrhea in animals are emerging as causes of viral gastroenteritis in humans (Jamieson et al., 1998; Kapikian, 1997; Kilgore and Glass, 1997). 1.5.3.1 Rotavirus Rotaviruses are members of the Reoviridae family (Matthews, 1979), and are characterized by their non-enveloped icosahedral structure and 70nm diameter. When observed under an electron microscope, they have a 13 'wheel' shape (Kapikian et al., 1996; Offit and Clark, 1995). The capsid consists of a double protein layer; the outer capsid is composed of the structural proteins VP7 and VP4, and the inner capsid mainly of VP6. The core is found inside the inner capsid, and encloses the rotavirus genome composed of 11 segments of double-stranded RNA. Given the segmented nature of the RNA genome, co-infection of cells with two different strains of rotavirus may result in reassortant virus, with RNA segments from each of the progenitors (Desselberger 1996). Each of the genomic segments encodes the structural VP proteins (VP1, VP2, VP3, VP4 (VP5 + VP8), VP6 and VP7) and the non-structural NSP proteins (NSP1, NSP2, NSP3, NSP4 and NSP5) (Kapikian and Chanock 1996). Rotaviruses are classified into groups, subgroups and serotypes according to the antigenic properties of the capsid proteins. Protein VP6 is the group reactivity determinant, with seven groups currently in existence, labeled A–G, and two subgroups, I and II (Estes and Cohen, 1989). Groups A, B and C are those which produce infection in humans. Classification into serotypes is based on the antigenic differences in the proteins of the outer capsid, VP7 and VP4. The first, a glycoprotein, determines the G-type specificity, and the second, the P-type specificity, owing to its protease sensitivity. At present, there are 15 G types (Rao et al., 2000), with G1, G2, G3 and G4 being the predominant ones throughout the world (Christensen, 1999). However, there have been reports of infections by unusual G types (Beards et al., 1992; Gouvea and Santos, 1999; Santos et al., 1998; Steele et al., 1999; Adah et al., 2001; Cunliffe et al., 2001). Rotaviruses are the single most important cause of severe diarrheal illness in infants and young children in both developed and developing countries worldwide (Bryce et al., 2005, WHO, 2005). Although rotavirus diarrhea occurs with high frequency in the developed countries, mortality is low. In the United States, rotaviruses cause about 5% to 10% of all diarrheal episodes in infants and children under 5 years of age; however, these viruses account for 30% to 50% of the severe diarrheal episodes (Parashar et al., 2006; Parashar et al., 2003). In developing countries, rotaviruses are documented consistently as the leading cause of life-threatening diarrhea 14 (Bern et al., 1994; Bresee et al., 2005; Glass et al., 2005). The global burden of rotavirus diarrheal disease in infants and young children under 5 years of age worldwide, but predominantly in developing countries, is estimated to be more than a 100 million episodes, over 20 million outpatient visits, and a staggering more than 600,000 deaths (range 454,000 to 705,000) (Parashar et al., 2006). Rotavirus genomes that were closely related to those of clinical strains were detected in raw sewage (11/29) and treated effluent (13/29) samples (Arraj et al., 2008). 1.5.3.2 Adenoviruses Adenoviruses were first isolated from humans and identified as the causative agent of epidemic febrile respiratory disease among military recruits in the 1950s (Hilleman and Werne,r 1954; Rowe et al., 1953). Adenoviruses are nonenveloped, range from 90 to 100 nm in diameter, and consist of double-stranded DNA (Kapikian and Wyatt, 1992). All adenoviruses with human or mammalian hosts are classified under genus Mastadenovirus (Ishibashi and Yasue, 1984). In 1998, adenoviruses were included in the “Candidate Contaminant List” as part of the Safe Drinking Water Act by the U.S. Environmental Protection Agency, and they are one of only four virus groups on the list (the three others are caliciviruses, coxsackieviruses, and echoviruses) (USEPA, 1998). Adenoviruses are included because of their public health implications and their frequent occurrence in many aquatic environments. In addition, adenoviruses have been shown to be up to 60 times more resistant to UV irradiation than RNA viruses, such as enteroviruses and hepatitis A virus. Because they are double stranded, an undamaged DNA strand in adenoviruses may serve as a template for repair by host enzymes; furthermore, these viruses have a high molecular weight that may also impart increased UV resistance (Gerba et al., 2002; Meng and Gerba, 1996; Roessler and Severin,.1996). Fifty-one serotypes of human adenoviruses (HAdV) have been identified (Gu et al., 2003). Human adenoviruses are the second most important viral pathogen of childhood gastroenteritis after rotavirus (Crabtree et al., 1997). They have been cited to cause symptomatic infections in several organ 15 systems, including the respiratory system (pharyngitis, acute respiratory disease, and pneumonia), eye (conjunctivitis), gastrointestinal tract (gastroenteritis), central nervous system (meningoencephalitis), and genitalia (urethritis and cervicitis) (Crabtree et al., 1997; Kapikian and Wyatt,. 1992). Human adenovirus types 40 and 41 have been associated with gastroenteritis in children, while human adenovirus type 4 is linked to persistent epidemics of acute respiratory disease in the United States (Cruz et al., 1990, McNeil et al., 1999). Transmission includes the fecal-oral route and inhalation of aerosols (Jiang et al., 2001). In India enteric adenoviruses were found in acute gastroenteritis patients. Sequence based analysis of the partial hexon and/or fiber genes showed the presence of adenovirus serotypes 40, 41, and 31 (Verma et al., 2009). The viruses are shed for extended periods in feces, urine, and respiratory secretions of infected persons (Crabtree et al., 1997). In contrast to the notion that only those adenoviruses that infect the intestinal tract of the host will be excreted in feces, adenoviruses type 5, the nonenteric adenovirus strain that accounts for 11% of clinical adenovirus cases reported to World Health Organization, is also frequently detected in aquatic environments (Tani et al., 1995). 1.5.3.3 Astroviruses In 1993, the Astroviridae family was established with a single genus, the astrovirus, which encompasses human and animal viruses (Matsui and Greenberg, 1996; Monroe et al., 1993). Astrovirus has been reported as small round viruses of 28 nm with an appearance like that of a five- or sixpointed star by direct visualization with electron microscopy. The name stems from the Greek astron, meaning star (Madeley and Cosgrove 1975). However, it has recently been verified that this virus has a different morphology, with an icosahedric appearance, a diameter of 41 nm, and well defined spikes. When these viruses are subjected to a high pH, they transform and present the typical morphology of the initially described star (Risco et al., 1995). The genome of human astroviruses is composed of single-stranded, positive-sense RNA which contains three 'open reading frames' (ORFs). ORF1a and ORF1b encode viral protease and polymerase, respectively. ORF2 encodes protein capsid precursor and is found at the 3'16 terminus of the genome (Strain et al., 2008). Astroviruses are classified into serotypes based on the reactivity of the capsid proteins with polyclonal sera and monoclonal antibodies (Matsui and Greenberg,1996). To date, there have been reports of four neutralizing monoclonal antibodies, developed by Sanchez-Fauquier et al. (1994) against serotype 2 human astrovirus and by Bass and Upadhyayula (1997) against serotype 1 astrovirus. They all react with the VP26 capsid protein, involved in the neutralization of human astroviruses. Astroviruses can also be classified into genotypes on the basis of the nucleotide sequence of a 348-bp region of the ORF2, and there is a good correlation with the serotypes (Noel et al., 1995). There are seven established genotypes, which correspond with seven serotypes. The existence of an eighth genotype has been suggested, due to the sequence of a putative serotype 8 (Mustafa et al., 2000). Serotype 1 is predominant in most studies, followed by 2, 3, 4 and 5. Serotypes 6, 7 and 8 are rarely detected (Lee and Kurtz, 1994; Gaggero et al., 1998; McIver et al., 2000; Palombo and Bishop, 1996; Unicomb et al., 1998). Astroviruses have been frequently detected in different water environments. Arraj et al (2008) detected genomes of human astrovirus in samples of raw sewage (11/29) and treated effluent samples (13/29). 1.5.3.4 Human caliciviruses Human caliciviruses are members of the Caliciviridae family, and two genera have been described, the Norwalk-like viruses (NLVs) and Sapporolike viruses (SLVs) (Berke et al., 1997). The virions are composed of a single structural capsid protein, with icosahedric symmetry (Prasad et al., 1999). The genome of the NLVs consists of positive-sense, single-stranded RNA organized into three ORFs. ORF1 encodes the non-structural proteins, such as RNA-dependent RNA polymerase and helicase (Seah et al., 1999), ORF2 encodes the structural protein of the capsid, and ORF3 encodes for a small protein whose function is unknown (Atmar and Estes, 2001). The genome of the SLVs differs from the NLV genome in that the ORF1 encodes the nonstructural proteins as well as the structural protein of the capsid (Liu et al., 17 1995a; 1995b). ORF2 encodes a small protein of unknown function, and the significance of ORF3 is still uncertain. Human calicivirus was first identified as a cause of outbreaks of nonbacterial diarrhea, initially using electron microscopic techniques (Kapikian et al., 1972). In recent studies, with the application of new assays such as EIA and reverse transcription–polymerase chain reaction (RT-PCR), knowledge of the epidemiology of human calicivirus has changed, especially for Norwalktype virus (Atmar and Estes, 2001). Thus, these viruses are recognized as the main agents responsible for outbreaks of non-bacterial diarrhea, and new estimates suggest that they represent the most common cause of illness with a food origin (Frankhauser et al., 1998; Koopmans et al., 2000; Mead et al., 1999; Nakata et al., 2000). Similarly, recent studies investigating calicivirus in sporadic cases of gastroenteritis in children have found that Norwalk-type viruses comprise the second cause of viral gastroenteritis after rotavirus (Bon et al., 1999; Lew et al., 1994; Pang et al., 2000; Pang et al., 1999). 1.5.3.5 Other gastroenteritis-producing viruses Torovirus Torovirus is a genus within the Coronaviridae family. Torovirus was detected for the first time in the feces of patients with gastroenteritis in 1984 (Beards et al., 1986). These viruses have an envelope of 100–140 nm, with a capsid of helicoidal symmetry and a single-stranded RNA genome of positive sense (Horzinek, 1999). They are associated with persistent and acute diarrhea in children, and may represent an important cause of nosocomial diarrhea (Jamieson et al., 1998; Koopmans et al., 1997). Coronavirus Included in the Coronaviridae family, these viruses are between 60 and 220 nm, with helicoidal symmetry and a spiculated envelope that gives them the appearance of a crown. The genome is composed of positive monocatenary RNA (McIntosh et al., 2000, Holmes and Lai,1996). Coronavirus was linked with diarrhea in humans for the first time in 1975, but 18 studies have not yet been able to establish a definite etiologic role (Glass et al., 1995). Picobirnaviruses These are small viruses, without an envelope, 30–40 nm in diameter, with a capsid of icosahedric symmetry, and a genome made up of two or three segments of bicatenary RNA. They were identified for the first time by Pereira et al. in 1988 (Pereira et al., 1988). Since then, they have been found in a wide variety of animal species (Chandra et al., 1997) and in both children and adults with diarrhea, including immuno supressed patients (Grohmann et al., 1993; Gallimore et al., 1995; Cascio et al., 1996). 1.6 Factors controlling virus survival 1.6.1 Temperature Temperature is the most significant factor controlling virus survival (Yates et al., 1985). The survival of viruses in most environments can be predicted by temperature. The lower the temperature, the longer viruses persist. Temperature affects the rate at which protein and nucleic acid denaturation occurs as well as chemical reactions in general that can degrade the viral capsid (e.g. enzymes). At freezing or near freezing temperatures, viruses may survive for many months (Goyal, 1984). Kutz and Gerba (1988) analyzed all the data on virus survival in surface waters and found that viruses survive longer in ground than surface waters held in the laboratory. At 810C in groundwater and 410C in surface waters, virus inactivation approached less than 0.01 log10 per day. Hepatitis A, adenoviruses, and parvoviruses are among the most thermal resistant of the enteric viruses (Crance et al., 1998; Bruniger et al., 2000). 1.6.2 pH In general, at the pH of most natural waters (pH 5–9), enteric viruses are very stable. Most enteric viruses are more stable at a pH between 3 and 5 than at pH 9 and 12. Enteroviruses can survive at a pH of 11.0–11.5 and 1.0– 2.0 for short periods of time; however, adenoviruses and rotaviruses are sensitive to inactivation at a pH of 10.0 or greater (Gerba and Goyal, 1982). 19 1.6.3 Light The ultraviolet (UV) light in sunlight can inactivate viruses by causing cross-linking among the nucleotides. Fujioka and Yoneyama (2002) found that several enteroviruses in seawater were inactivated much more rapidly in the presence of sunlight than in the dark. The rate of inactivation was much less in the winter than in the summer (Fattal et al., 1983). The doublestranded DNA viruses (e.g. adenoviruses) are significantly much more resistant to UV light inactivation than enteroviruses because they can use host cell repair enzymes to repair the UV light damage (Gerba et al., 2002). 1.6.4 Salts and metals Certain heavy metals such as copper and silver are known to have antiviral properties; their concentrations in water are usually too low to have an effect on viruses in natural waters (Thurman and Gerba, 1988). Viruses almost always survive longer in freshwater than in seawater. This appears to be largely due to the presence of antagonistic microorganisms rather than the increased salt concentration (Kapuscinski and Mitchell, 1980). 1.6.5 Interfaces Virus adsorption to solids plays a major role in their survival in natural waters. Enteric viruses readily associate with particulate matter in water and sediments. Thus, viruses are usually found in greater numbers in sediments than in the overlaying water (Goyal et al., 1978). Adsorption may act to prolong the survival of the virus, but such associations may also enhance virus inactivation (Murray and Laband, 1979). Studies have shown that viruses readily adsorb to sand, pure clays, bacterial cells, particulate organic matter, silts, etc. Adsorption of enteric viruses to sediments has been demonstrated to prolong the survival of enteroviruses (Smith et al., 1978). 1.6.6 Microflora Treating natural waters to eliminate or kill bacteria or fungi by autoclaving, filtration, addition of antibiotics, or treatment with UV light increases survival of viruses (Melnick and Gerba, 1980). The greater antiviral activity in seawater compared to freshwater appears to be related to the 20 presence of bacteria indigenous to seawater (Fujioka et al., 1980). Studies in freshwaters have suggested that enteroviruses are degraded by the indigenous microflora (Cliver and Hermann, 1972). The longer survival of enteric virus in water compared to other viruses may be due to their greater resistance to proteolytic enzymes (Chang, 1971). Polioviruses and coxsackie viruses have been shown to be resistant to a wide range of photolytic enzymes (Cliver and Hermann, 1972). However, this may vary with the serotype of enterovirus. 1.6.7 Other factors High hydrostatic pressures can inactivate enteric viruses. A 7 log10 drop in hepatitis A virus occurred when exposed to 450MPa for 5 min (Kingsley et al., 2002). However, poliovirus was unaffected by a 5 min exposure to 600 MPa. In the presence of seawater, the resistance of hepatitis A virus increased. Exposure of the virus to RNase indicated that inactivation was due to alteration of the viral capsid proteins. 1.7 Viruses versus bacteria as indicator of microbiological contamination of water Bacterial indicators such as enterococci and fecal coliform and total coliform bacteria are usually relied upon as an indicator of microbiological quality of water; however, these indicators do not always reflect the risk from many pathogenic viruses (Geldenhuys et. al. 1989). Infectious enteric viruses have been isolated from aquatic environments that are in compliance with bacterial indicator standards, and there have been several virus-related outbreaks linked to ingestion of waters that met fecal coliform standards (Craun. 1991, McAnulty et. al. 1993). One of the major drawbacks in using fecal coliform bacteria and other traditional indicators (e.g., enterococci) is that these indicators may be found in both human and animal feces and naturally in soils. Furthermore, they may regrow in the environment after being excreted from their host (Springthorpe et. al 1993). Viral pathogens, because of their host specificity, have been suggested as one of the most promising tools to determine the sources of fecal 21 contaminants in aquatic environments and may be used in conjunction with bacterial indicators to assess water quality and improve public health surveillance (Fong et. al 2005, McNeil et. al 1999). Pathogenic viruses are generally more resistant than bacterial indicators during conventional wastewater treatment such as chlorination and filtration and are able to withstand lipid solvents (Fujioka and Yoneyama, 2002, Jiang et. al 2001, Thurston-Enriquez et. al 2003). 1.8 Virus removal during drinking water treatment Understanding virus removal from water intended for drinking has assumed more importance because increased urbanization and rapidly surging human populations result in increased wastes levels, more reuse of virus-contaminated wastewater and increasing land disposal of sewage sludges (Sattar et al., 1999; Fane et al., 2002; White et al., 2003). When such wastes enter fluvial waters, the impact of contained viral pathogens can be felt far from the original contamination source. Another reason to understand virus reduction during water treatment comes from virus contamination during or subsequent to catastrophic events, including the potential risks to potable water from deliberate contamination with viral bioagents. The presence of human pathogenic viruses in drinking water with the amounted risk to human health depends on the quality of the water source, the multiple-barrier approach employed to treat it, and the nature of the pathogens themselves. Each of these factors has significant variability influenced by the presence, persistence and aggregation of the viruses in different water environments, and the types and efficiencies of the natural or engineered treatment processes applied. Viruses are extremely small and have a very high surface area to volume ratio. Traditionally, virus size is compared with bacterial size only in terms of particle diameters, with the former being 20–300 nm whereas the latter are usually from 0.5 mm to several mm. When considering virus removal, it is important to consider virus size in comparison to bacteria in three dimensions. For a typical small virus with a virion diameter of 30 nm, the volume is 22 approximately 1 X104nm3. For a typical bacterium, 1 mm in diameter and 2 mm in length, the volume is approximately 7 X109nm3. This approximately five to six order of magnitude size differences could lead to potentially very different particle behaviour between viruses and bacteria. (Wellings et al., 1976). Thus, during conventional water treatment processes the efficiency of coagulation, sedimentation and filtration may be significant for virus removal. However, some small but undefined fraction of virions may also behave as very small particles and potentially evade the removal process. In considering the disinfection step of water treatment, the small size and large surface area of the virions may make them vulnerable to rapid decontamination, but also assist in their escape from disinfection if they are able to ‘hide’ within organic matrices or be trapped in biofilms. In summary, the ability of viruses to be particle associated is likely to assist their removal but inhibit their disinfection, whereas the reverse is expected to be true for free virions. Virus removal relies on reduction of virus numbers by flocculation and sedimentation with other organic materials as well as during passage through filters of granular media or membranes. Although virus removal is focused on physical removal processes, concurrent chemical effects and biological decay are also confounded in the overall process measurements at any particular location. Holding of water supplies prior to treatment to permit biological decay to occur could be considered a removal mechanism. The focus on conventionally engineered water treatment processes can be justified due to the large populations who rely on conventional treatment for their drinking water. Flocculants may be based on aluminum or ferric compounds and may be simple or polymeric. Granulated media may be sand beds (e.g. slow sand filters, bank filtration) or the mixed bed filters used in many water treatment plants. Additional granular activated carbon columns are also present in some locations. Filtration processes are usually preceded by coagulation, flocculation and sedimentation of the particulates in drinking water so that the filters do not block too rapidly. Use of membrane filters as a final removal step for drinking water treatment is becoming more common, especially in smaller plants. Whether viruses are removed by coagulation and sedimentation, filtration through filter beds or by membranes, they end up in a 23 residue that must be properly disposed. Such residues can very well be a risk if viruses contained in them survive and are transported to another treatment facility or bathing area. 1.8.1 Experimental addition of viruses (or seeding studies) Relatively few studies have examined the reduction in viral pathogens employing conventional treatment process. These studies may be of two types: seeding studies with known viruses and/or phages, or removal of indigenous viruses and detection of survivors by a selection of standardized assays. Overall a wide variation is observed in the treatment protocols as well as evaluation methods used by different investigators. Gerba et al. (2003) used feline calicivirus (FCV) as a surrogate for human noroviruses, and for comparison, three bacteriophages were seeded into a pilot plant. The pilot plant received, in addition to the microbial seeds, alum and polymer. Coagulation occurred in three mixed and one static tank before the floc was allowed to settle in the sedimentation basin. The hydraulic retention time in the mixed tanks was 27 min each and in the static tank and sedimentation basin together was 99 min. The water was then filtered through a mixed bed consisting of granular activated carbon, sand and gravel. The mean range of inactivation measured for the phages was 0.78–1.97 log10 and 1.85–3.21 log10 after sedimentation and filtration, respectively. For the FCV, the corresponding figures were 2.49 log10 and 3.05 log10. In similar seeding experiments at another pilot plant (Hendricks et al., 2005) the phages used were reduced by approximately 3 log10 (MS-2) and 5 log10 (Phi-X 174). The viruses, attenuated poliovirus and echovirus 12, were each reduced by just more than 2 log10. In multiple experiments on the phages, they observed no difference between mono- or dual-filtration media but a marked effect of alum concentration, with greater removals at higher alum concentration. This effect was most marked for Phi-X 174; there was little or no phage removal in the absence of alum. A comparison of phage removal between the experimental pilot plant and a full-scale plant and pilot plant from a different location showed very different phage removal at the alternate locations (Hendricks et al, 2005); this difference was ascribed to 24 the lower alum concentration. Thus, there are differences in the ability of conventional coagulation, sedimentation and filtration processes to remove viruses that can depend on the efficiency of each of those steps as well as the nature of the virus. Since the hydraulic conditions differ somewhat between plants and between full scale and pilot plants, the real virus reductions in fullscale plants, which are difficult to measure by seeding experiments, are uncertain. Comparison of coagulants (ferric, alum and polymer) with two human viruses (poliovirus and echovirus) and two phages (MS-2 and PRD-1) showed relatively similar results among different coagulants; the greatest reduction was for poliovirus, which therefore appeared to show the most variation (Bell et al., 2000). MS-2 reduction was consistently higher than PRD-1; based on this data the latter appeared to be more suitable phage to represent the behaviour of the human viruses. In a laboratory study using flocculation and high-rate sedimentation with hepatitis A virus (HAV) and poliovirus, as well as F+ bacteriophage, Nasser et al. (1995) determined the optimum flocculent dose for virus removal. They showed that humic acid interfered with virus removal whereas addition of a small quantity (1 mg/l) of a cationic polyelectrolyte improved the performance of alum flocculation for HAV and bacteriophage removal, poliovirus removal was unaffected. Many smaller plants do not have access to full conventional treatment including flocculation, sedimentation and filtration, so virus removal can remain a challenge. Yahya et al. (1993) studied virus removal in a pilot plant with relevance to application in small plants using surface water. They used slow sand filtration followed by nanofiltration and studied the removal of bacteriophages MS-2 and PRD-1. The slow sand filter removed about 2 log10 of each of the phages and there was a 4–6 log10 reduction in phages in water from the nanofilters; PRD-1 was removed to a greater extent than MS-2 by both the sand filter and the nanofilter. No human viruses were included, but at least for the nanofilter, where removal is primarily size-dependant, human viruses of comparable size can be expected to be removed in a similar 25 magnitude to MS-2 (HAV, enteroviruses, caliciviruses) or PRD-1 (rotaviruses, reoviruses, adenoviruses). An alternative to following spiked microorganisms through a fulltreatment process can be assessment of individual steps following spiking of the known amount of virus. In their pilot-scale investigation of different treatment stages prior to a full plant upgrade, Huertas et al. (2003) used known titres of MS-2 coliphage and found virus removal: 0.86 log10 (85.1%) from coagulation-flocculation-sedimentation whereas more than 3 log10 were removed by microfiltration or ozonation. 1.8.2 Indigenous virus studies One of the early studies to examine indigenous virus removal during treatment was performed by Keswick et al. (1984) on a highly polluted water source. After an essentially complete treatment process (clarification, sand filtration and chlorination), virus was still found in 83% of the finished water by culture. Another study in Canada (Payment et al., 1985) used seven treatment plants in the Montreal region. Three plants used a treatment regime consisting of prechlorination, sedimentation, filtration, ozonation and chlorine, two more were similar but lacked the ozonation, one used only filtration and chlorination and one used chlorine alone. The first group showed about 97% of virus removal after chlorination and about 97.5% after sedimentation with further minor reduction after filtration and a final significant drop in virus content after ozonation; some virus was detectable in finished water. The second protocol that lacked ozonation resulted in similar reduction after sedimentation that increased slightly after filtration and in the finished water; these differences are unlikely to be significant. The last two plants had lower levels of virus contamination to contend with, but virus was found in the finished water of the one that had filtration and chlorination. 1.9 Methods for concentration of viruses from water samples. The prerequisite for the virological analysis of water is an efficient, inexpensive, and simple virus concentration method because the viruses may be present in very low numbers making it essential to start with a large sample volume and concentrate it to several orders of magnitude (Ehlers et 26 al., 2005; Lodder et al., 2005; Griffin et al., 1999; Lipp et al., 2001; Van Heerden et al., 2003). The concentration method should be applicable to wide spectrum of enteric viruses to facilitate simultaneous concentration. Wallis carried out pioneering work for concentration of viruses in 1960s (Wallis and melnick, 1967a; 1967b; Wallis et al., 1969). The basic principle involved was attractive force acting between opposite ionic charges and the procedure included the passage of the sample through a solid matrix under specific conditions of pH and ionic strength; the virus was adsorbed to the matrix and subsequently eluted in a suitable eluent. Over the span of fifty years, different types of matrix materials have been used for adsorption of enteric viruses. Choice of adsorbing matrix, eluting fluid and processing conditions are influenced by the nature of the sample, but elution is commonly done using a solution containing beef extract or skimmed milk, both at high pH, which displaces the virus from the adsorbing matrix into the eluant. Eluants comprising basic amino acids (glycine, lysine) are also used. 1.9.1 Adsorption to electronegative membranes and cartridges Electronegative membranes retain a net negative charge at pH values near neutrality, typical examples of electronegative membranes include cellulose acetate or cellulose nitrate membranes and Epoxy-Fiberglass filters. These membranes are available in various configurations and with different pore sizes for different applications. The basic principle involved in virus concentration by these membranes is adsorption of the virus particles to the filter by electrostatic attractive forces rather than size exclusion. It is possible to get good recoveries of the virus accompanied by good flow rates and a minimum of filter clogging even from turbid waters, and many solidsassociated viruses can be recovered. In the process of virus concentration by electronegative membranes, the virus-containing sample is passed through the membrane under positive pressure or vacuum. If the water sample to be concentrated contains high level of particulate impurities that may cause clogging of the membrane, then a pre-filtration step is added before actual concentration of the virus by the electronegative membrane. Since viruses 27 and the filter materials are both negatively charged at neutral pH the water sample must be conditioned to allow electrostatic binding of virus particles to the filter matrix. The water sample is adjusted to pH 3.5 and Al3+ or Mg2+ions may be added. Berg et al. (1971) used cellulose nitrate filters for recovery of small quantities of viruses from clean waters. The method consisted of adsorbing viruses onto cellulose nitrate membrane filters (0.45 m pore size) from water containing sufficient Na2HPO4 to produce a molarity of 0.05 and sufficient citric acid to produce a pH of 7, and eluting the adsorbed viruses in 3% beef extract under extended sonic treatment. Complete recovery of poliovirus 1, echovirus 7, and coxsackievirus B3 resulted when less than 100 plaqueforming units were added to 1-liter quantities of water. Recoveries of reovirus 1 were almost as good. Preliminary studies indicate that good recoveries can be made from 25-gal quantities of water. The method described was efficient in waters of high quality and may be useful for recovering viruses in renovated, and perhaps in tap waters, but not in waters containing certain organic matter unless that matter was first removed. In a study by Jakubowski et al., (1974), Epoxy-Fiberglass filters (Balston filters) were used for concentration of viruses from tap water. The authors described reliable concentration of poliovirus from 100 gal (about 380 liters) of drinking water at virus inputs of 1 infectious unit/gal (about 3.8 liters) or less. The filter unit used by them was 11.43 cm high and 6.67 cm in diameter (4.5 by 2.63 inches) and consisted of aluminum housing with a polycarbonate bowl (type 92, Balston, Inc., Lexington, Mass.). The virusadsorbing filter tube was 2.54 cm in diameter and 6.35 cm high (1 by 2.5 inches) and composed of glass microfibers bonded with epoxy resin. Three of the units were assembled in parallel by using stainless-steel, quick-disconnect couplings and fittings. Experimentally, virus adsorption was enhanced by adding HCl to adjust the pH to 3.5, and sodium thiosulfate was added to a final concentration of 50 mg/liter to neutralize chlorine. Attenuated poliovirus type 1 (Lsc 2ab strain) was continually added to the tap water with the thiosulfate solution. The flow rate was controlled at 1 gal/min by a limitingorifice valve (Dole Valve Co., Morton Grove, Ill.), and 100 gal was processed 28 in each run. At the end of each run, residual water was drained from the filter housings before eluting. Virus was eluted from the filters with 1-liter volumes of 0.05 M glycine buffer at pH 11.5. The eluate was then adjusted to pH 3.5 with 0.05 M glycine buffer (pH 1.1), and AlCI3 was added to a final concentration of 0.0005 M. This eluate was reconcentrated by filtering through a stacked series of epoxy-fiberglass disk filters (47 mm) Virus recovery ranged from 42 to 57% at virus inputs of approximately 1 PFU/5 gal to 1 PFU/gal. Fibreglass filters and cellulose nitrate membrane filters were evaluated by Morris and Waite (1980) for their suitability in recovering seeded poliovirus type 2 from tap and river water. They found that Fibreglass filters gave best recoveries in the evaluation when used with 0.0005 M AlCl36H2O, being up to 4 times more efficient than filters used without Al3+. No such Al3+ requirement was found for cellulose nitrate membrane filters. Fiberglass filters are less expensive and can be obtained in sterile cartridges in disposable form. However, they are prone to clogging, cannot be used with even moderately turbid water and at high flow rates (Gerba et al., 1987). Because of problems of clogging of membrane or tube filters, the processing of seawater samples in this way is limited to a maximum of 20 liter before filters have to be changed (Block and Schwartzbrod, 1989). Increasing the surface area available for filtration by the use of larger cartridge filters, where sheets of negatively charged pleated filter material are rolled and used in cm cartridge holders can overcome the problem of clogging. This also alleviates the need to change membranes or tubes frequently. Farrah et al (1976) carried out evaluation of various negatively charged filters. Nitrocellulose filters (type HA, Millipore Corp., Bedford, Mass.), epoxy-fiberglass filters (series AA, Cox Instrument Corp., Detroit, Mich.), acrylonitrile polyvinylchloride copolymer filters (Acropor series, Gelman Instrument Co., Ann Arbor, Mich.), 10-inch (ca. 25.4 cm) glass fiber, melamine-impregnated paper, epoxy filters (Duo- Fine series, Filterite Corp., Timonium, Md.), and 10-inch honeycomb, fiberglass depth filters (model K-27, Commercial Filters Division, Carborundum Co., Lebanon, Ind.) were used in their study. Seeded poliovirus was recovered from 378-liter volumes of water 29 with 53% efficiency. The authors reported that the filters could be regenerated up to five times by soaking for 5 min in 0.1M NaOH. Generally, virus recovery rates are variable with negatively charged filter media when applied for concentration of viruses from different types of water systems. Block and Schwartzbrod (1989) considered cellulose nitrate membranes relatively efficient as they gave 60% recovery of virus; the same authors recorded glass fibre filters giving a poor average yield from wastewater but 70% recovery with river water. Payment and Trudel (1979) using glass fibre filters, reported 38–58% recovery of 102–106 pfu seeded in 100 ml–1000 l volumes. Papaventsis et al. (2005) reported a modification of the use of negatively charged membranes wherein they were able to culture sewage-derived enteroviruses directly from the filter without elution, thus reducing the total time required for analysis. 1.9.2 Adsorption to electropositive membranes and cartridges Positively charged filters retain a net positive charge at near neutral pH values, hence they adsorb virus from water and other materials without the need for prior conditioning of the sample. Initial work for concentration of viruses using positively charged filters was done by Sobsey and Jones (1979) and by Hou et al. (1980). Sobsey reported that “Electropositive filters appear to offer distinct advantages over conventional negatively charged filters for concentrating enteric viruses from water, and their behavior tends to confirm the importance of electrostatic forces in virus recovery from water by microporous filter adsorption-elution methods”. Zeta Plus filters used by Sobsey were composed of diatomaceous earth-cellulose-"charge-modified" resin mixtures and having a net positive charge up to pH 5 to 6 efficiently adsorbed poliovirus from tap water at ambient pH levels 7.0 to 7.5 without added multivalent cation salts. The adsorbed viruses were eluted with glycineNaOH, pH 9.5 to 11.5. Electropositive asbestos-cellulose filters efficiently adsorbed poliovirus from tap water without added multivalent cation salts between pH 3.5 and 9.0, and the absorbed viruses could be eluted with 3% beef extract, pH 9, but not with pH 9.5 to 11.5 glycine-NaOH. Under water quality conditions in which poliovirus recoveries from large volumes of water 30 were less than 5% with conventional negatively charged filters and standard methods, recoveries with Zeta Plus filters averaged 64 and 22.5% for oneand two-stage concentration procedures, respectively. Hou et al. (1980) demonstrated that electropositive depth filters were capable of adsorbing viruses and endotoxins many times smaller than the average pore size of the filter. Electronegative filters of similar porosity or electropositive filters that had been treated to destroy the positive charge were almost ineffective under similar conditions for the removal of viruses and small latex spheres. The results of this study indicate that electropositive filters are highly effective in the removal of a wide range of contaminants over a wide range of pH values and ionic conditions. Generally electropositive filters adsorb virus in the pH range 3 - 6; at pH values above 7 the adsorption falls off rapidly, so the pH still needs to be carefully controlled. These properties make the use of positively charged filters attractive, not only for the convenience of not having to condition the sample but also because it makes possible the concentration of other viruses such as rotavirus and coliphages, which are sensitive to the low pH conditions needed for adsorption to negatively charged media. Keswick et al (1983) reported that type 1 poliovirus and rotavirus SA11 survived at least 5 weeks on electropositive filters at 410C, which makes them useful for on-site concentration. They are used in the same way as electronegative materials. The virus is eluted from the filter and secondary concentration is carried out as for the electronegative types. Sobsey & Glass (1980) compared the electronegative Filterite (fibreglass) pleated cartridge filter with two electropositive filters, Zeta-plus Series S filters made of a cellulose/diatomaceous earth/ion-exchange resin mixture and Virozorb 1MDS pleated-sheet cartridges filters containing two identical layers of a thin-sheet medium consisting of surface-modified fiber glass and cellulose mixtures for recovery of poliovirus from 1000 l tap water. These researchers observed that virus adsorption from tap water between pH 3.5 and 7.5 was more efficient with electropositive filters than with Filterite filters. Elution of adsorbed viruses was more efficient with beef extract in glycine, pH 9.5, than with glycine-NaOH, pH 11.0. In paired comparative 31 studies, electropositive filters, with adsorption at pH 7.5 and no added polyvalent cation salts, gave less variable virus concentration efficiencies than did Filterite filters with adsorption at pH 3.5 plus added MgCl2. Recovery of poliovirus from 1,000-liter tap water volumes was approximately 30% efficient with both Virozorb 1MDS and Filterite pleated cartridge filters, but the former were much simpler to use. The virus adsorption behavior of these filters appeared to be related to their surface charge properties, with more electropositive filters giving more efficient virus adsorption from tap water at higher pH levels. The advantages of these electropositive filters lie in the large volumes they can handle without the need for conditioning the sample however elution from the filter still needs to be carried out at pH 9 or above. The high cost of electropositive filters can be overcome by regenerating and re-using these filters. The study by Cashdollar and Dahling (2006) describes the development of a method which allows a filter to be used up to three times, achieving comparable recoveries to new filters. Zetapor 1MDS and N66 Posidyne electropositive filters were tested. The method was analyzed using tap water and Ohio River water that was spiked with poliovirus. Tap water recoveries averaged 32% for new filters, 30% for filters used twice, and 38% for filters used three times. River water recoveries averaged 68% for new filters, 83% for filters used twice, and 100% for filters used three times. Organic materials in the sample, especially fulvic acid, were reported to interfere with virus recovery from Virozorb cartridges and glass-fibre materials In a study by Sobsey and Hickey (1985) solutions of activated carbontreated tap water containing 3, 10, and 30-mg/liter concentrations of humic or fulvic acid were seeded with known amounts of poliovirus and processed with Virosorb 1MDS filters at pH 7.5 or Filterite filters at pH 3.5 (with and without 5 mM MgCl2). Organic acids caused appreciable reductions in virus adsorption and recovery efficiencies with both types of filter. Fulvic acid caused greater reductions in poliovirus recovery with Virosorb 1MDS filters than with Filterite filters. Fulvic acid interference with poliovirus recovery by Filterite filters was overcome by the presence of 5 mM MgCl2. Although humic acid reduced poliovirus recoveries by both types of filter, its greatest effect was on virus elution and recovery from Filterite 32 filters. Single-particle analyses demonstrated MgCl2 enhancement of poliovirus association with both organic acids at pH 3.5. Guttman-Bass and Catalano-Sherman (1986) tested two electropositively charged types of filter (Seitz S and Zeta Plus 60S) to concentrate poliovirus in the presence of humic materials. Humic acid inhibited virus adsorption, but even at the highest humic acid concentrations tested (200 mg/liter), 30 to 40% of the virus was recovered by the filters. Fulvic acid, tested with Zeta Plus filters, did not affect virus recovery. For comparison, two electronegatively charged filter types were tested (Cox and Balston). These two types of filter were more sensitive to interference at lower concentrations of humic acid than the more positively charged filters. With Balston filters, at humic acid concentrations above 10 mg/liter, most of the virus was recovered in the filtrate. Fulvic acid, tested with Balston filters, did not interfere with virus recovery. With the electropositively charged filters, the humic materials adsorbed efficiently, even at high input concentrations. Interference with virus adsorption occurred at humic acid concentrations which were below the level of saturation of the filters. In addition, in highvolume experiments, humic acid led to premature blockage of the filters. Such filters are used extensively in the USA for concentration of many types of viruses from treated drinking water to sewage effluent (Sedmak et al., 2005). A different electropositive MK filter that is cheaper then other electropositive filters was evaluated by Ma et al. (1994). Haramoto et al. (2004) reported improvements in poliovirus and norovirus recovery from tap water samples by coating of electronegative filters with cations through passage of AlCl3 prior to filtration. Poliovirus type 1 (LSc 2ab Sabin strain) inoculated into 40 ml of MilliQ (ultrapure) water was adsorbed effectively to a negatively charged filter (Millipore HA, 0.45- micro m pore size) coated with aluminum ions, 99% of which were recovered by elution with 1.0 mM NaOH (pH 10.8) following an acid rinse with 0.5 mM H2SO4 (pH 3.0). More than 80% poliovirus recovery yields were obtained from 500-ml, 1,000-ml, and 10-liter MilliQ water samples and from tap water samples. However the acid rinse step did not improve poliovirus recovery using Zetapor filters. 33 Advances in membrane technology have also resulted in chargemodified nylon membranes being available for concentration of viruses from water. Gilgen et al (1995; 1997) described the use of positively charged nylon membranes coupled with ultrafiltration for the concentration of a variety of enteric viruses prior to detection by RT-PCR. Other nylon membranes are also available which are made in various pore sizes, which would permit passage of the virus (0.45, 1.2 and 3 mm) and have a positive surface charge over the pH range 3–10, which would promote strong binding of negatively charged particles. Triple-layered polyvinylidene fluoride (PVDF) membranes and cartridges have been used in industry for the removal of polio and influenza viruses from pharmaceutical products (AranhaCreado et al., 1997). During the 2002/2003 outbreak of severe acute respiratory syndrome (SARS) attention was focused on possible transmission of the SARS-corona virus (SARSCoV) in sewage since SARS-CoV RNA was found in the stools of affected patients. Electropositive filters were used to concentrate the virus from sewage (Wang et al., 2005) and SARS-CoV RNA was recovered from sewage concentrates. 1.9.3 Adsorption to glass wool The technique of virus adsorption to glass wool was pioneered in France principally by Vilagine`s et al (1993), they applied it for the concentration of a range of viruses from surface, drinking and wastewaters. The authors reported a survey of two rivers over a 44-month period and concluded that the technique was robust enough to be used for routine monitoring of surface waters. Glass wool packed into holders at a density of 0.5 g/cm3 was washed through in sequence with HCl, water, NaOH and finally with water again to neutral pH before the sample is passed through the filter. Glass wool is an economic alternative to microporous filters. It is used in a column and if evenly packed to an adequate density, adsorption of viruses appears to be at least as efficient as with other filter types. An advantage of the method is that the virus will adsorb to the filter matrix at or near neutral pH, and without the addition of cations, which makes it suitable for viruses sensitive to acid. However, elution still has to be done at high pH. Different sizes of filter can be 34 prepared according to the type of water and flow rate. The only pre-treatment necessary is the dechlorination of drinking waters. Glass wool has been used in many other laboratories; Hugues et al. (1991) found it more sensitive than the glass powder method; they demonstrated that virus could be readily recovered from the first 25 ml of eluate of glass wool rather than from a reconcentration of the entire eluate, either by organic flocculation (83% of positivity vs 44% respectively) or double precipitation by PEG (85% of positivity vs 61% respectively). Concentration of viruses on glass wool was used by several investigators. Wolfaardt et al. (1995) used it to concentrate small round-structured viruses (SRSVs, now noroviruses) from spiked sewage and polluted water samples prior to detection by RT-PCR. Ehlers et al. (2005a) employed the same for concentration of enteroviruses from sewage and treated drinking water and for detection of adenoviruses in river and treated drinking water in South Africa (Ehlers et al., 2005b). Van Heerden et al. (2003; 2005) used glass wool to recover human adenoviruses from 200 liter treated drinking water samples and 25 liter river water samples. Adsorption to the filters was done on site and the filters were transported to the laboratory for elution. Grabow et al. (2001) used glass wool filtration for detection of enteroviruses in drinking water samples (17%), adenoviruses in 4% and hepatitis A virus in 3% of samples. In addition to these viruses, astro- and rotaviruses were detected in raw water samples. 1.9.4 Adsorption to glass powder Glass beads constitute a fluidised bed and so have the advantage that the filter matrix cannot get clogged as with glass-fibre systems. Sarrette et al. (1977) first developed this technique. The sample to be analyzed, was adjusted to pH = 3.5 with hydrochloric acid. It was then injected under low pressure (0.15 bar) by the lower end of a column containing glass powder (50 g). The calibration of the water's upward speed enabled the glass powder to remain in dynamic suspension. Under these conditions, poliovirus was quantitatively adsorbed on the glass powder. The virus was then eluted from the glass powder with 50 ml of a glycine buffer (pH = 11.5) and thus 35 concentrated 200 times in the final eluate. The total duration of operation did not exceeded 2 h 30 min. The recuperation efficiency was about 40–60% and did not depend on initial virus concentration in sample for virus concentrations ranging from 1 to 104PFU/L. Negatively and positively-charged glass powder were tested for the concentration of hepatitis A virus from different types of water samples (Gajardo et al. 1991). The efficiency of recovery of HAV with negatively charged glass powder was 100% for tap water, 80% for freshwater, 75% for sea water and 61% for sewage. The charge of glass powder was modified by polyethylenimine treatment to avoid the need to pretreat the sample. Concentration efficiencies of HAV through adsorption to and elution from positively-charged glass powder were 100% for tap water, 94% for sea water, and 61% for fresh water and sewage. Coronaviruses have also been concentrated by adsorption-elution on glass powder (Collomb et al. 1986). Virus adsorption was studied in relation to pH: optimal adsorption yield between 50 to 75% (mean value: 62%) was obtained at pH less than or equal to 3.5. Elution was compared using either a 0.05 M glycine solution or 3% beef extract solution. Virus elution occurred mostly at alkaline pH with a maximum yield between 38 and 55% for pH values between 9 and 11. Owing to the sensitivity of coronavirus to acid (pH 3) and alkaline pH (pH greater than or equal to 10), adsorption was optimal at pH 3.3 and elution at pH 9. Under such conditions, the overall efficiency of the method appears to stand between 24 and 28%. 1.9.5 Other adsorbents Apart from the adsorbent materials mentioned in the previous sections, several others have been used for concentration of enteric viruses from different types of water samples. Silicon dioxide (SiO2) was applied for detection of viruses in surface water, recreational waters and sewage samples (Baggi et al., 2000; Pallin et al., 1997). The procedure involved adsorption of the virus capsids to silica particles under acidic conditions, allowing their recovery by relatively gentle centrifugation. 36 Powdered coal has been used as an adsorbent with a view to transferring the virus concentration and water purification technology to developing countries (Dahling et al., 1985; Lahke and Parhad, 1988; Chaudhiri et al., 1986). This filter was effective over a pH range of 3.0 to 7.0. Poliovirus type 1 recoveries from 100-liter seeded samples of Cincinnati tap water did not vary significantly when compared with those of identical samples processed through Filterite and Millipore filters. In studies with raw domestic sewage, virus recoveries were nearly identical from comparable samples filtered through coal and Millipore disk filters. The same kind of matrix in a more refined state was used as granular activated carbon by Jothikumar et al. (1995) for the first stage concentration of enteroviruses, hepatitis E virus (HEV) and rotaviruses. Using RT-PCR as a detection method, these authors reported 74% recovery of poliovirus 1. A range of viruses can be concentrated from different waters using talc (magnesium silicate) mixed with celite (diatomaceous earth) (Sattar and Westwood, 1978; Ramia and Sattar, 1979; Sattar and Ramia, 1979). The efficiency of the talc-Celite technique was tested by using potable water samples experimentally-contaminated with (a) strains of enteric viruses recovered from field samples of wastewaters (b) indigenous enteric viruses contaminated, in raw sewage. An average of 88.0% of input virus could be recovered when potable water samples contaminated, in separate experiments, with five different isolates of enteric viruses (a vaccine strain of polio 1, non-vaccine strain of polio 1, echo I, coxsackie B3, and reovirus) were concentrated by this technique. When potable water samples containing either 0.05% or 0.1% of raw sewage were processed by this method, about 86.0% of the indigenous enteric virus plaque forming units could be recovered. 1.9.6 Ultrafiltration Ultrafiltration technique is based on the principle of size exclusion in which the virus in a sample is bound to a filter matrix principally by virtue of its size rather than by any charges on the particle, though in practice electrostatic effects can also exert an effect. Divizia et al (1989a; 1989b) evaluated the 37 efficiency of polysulfonate membrane of 10,000 molecular weight limit for recovery of seeded hepatitis A virus and poliovirus from 1 liter of dechlorinated tap water or different buffer. Under standard conditions hepatitis A virus recovery was 100% of the input, but the percentage was reduced dramatically when the inflow pressure was increased. In contrast, poliovirus recovery was low under standard conditions, but it improved when the membranes were pretreated with different buffers. The best recovery was obtained using beef extract at neutral pH. Rotem et al (1979) used a capillary membrane ultrafiltration unit for the concentration of poliovirus I from a volume of 100 liter tap water. Backwashing the units with either high pH glycine buffer or beef extract resulted in an average virus recovery of about 67%. In several cases where ultrafiltration and organic flocculation methods were combined and initial virus input was relatively low (between 39 pfu/100L and 200 pfu/100L of water), average virus recovery of about 80% was obtained. Average filtration time was 86 min. for all experiments. Virus concentration by ultrafiltration using hollow fibre membrane filter was also reported (Belfort et al., 1982). Most laboratories use membranes or fibre systems with cut-off levels of 30–100 kDa. Systems in which the fluid passes directly through the filter, non-filterable components quickly clog the filter or precipitate at the membrane surface, thus this type of filter is only useful for small volumes (<1000 ml). Some ultrafilters employ tangential flow or vortex flow (VFF), which reduces clogging. Tsai et al. (1993) used VFF for processing inshore water samples in Southern California. Fifteen litres of each sample were concentrated to 100 ml using a 100 kDa cut-off membrane and the samples were further concentrated to 100 l using Centriprep and Centricon units at 1000 X g. In a study Poliovirus 1 concentration tests were carried out in artificially contaminated water by tangential flow ultrafiltration with Polysulfone filters 100000 Molecular Weight Cut Off (Bigliardi et al., 2004). The tests were performed in 1 and in 20 liters of waters. The filters were conditioned and eluted respectively with Beef extract 3% and with glicina 1% at pH 7 and pH 9. The recovery mean using Beef extract was about 83%. The viral recovery was low using the glycine for conditioning and eluting the filters. 38 The use of ultrafiltration as a concentration method to recover viruses from environmental waters was investigated by Olszewski et al. (Olszewski et al., 2005). Two ultrafiltration systems (hollow fiber and tangential flow) in a large (100 L) and small-scale (2 L) configuration were able to recover greater than 50% of multiple viruses (bacteriophage PP7 and T1 and poliovirus type 2) from varying water turbidities (10-157 nephelometric turbidity units (NTU)) simultaneously. Mean recoveries (n = 3) in ground and surface water by the large-scale hollow fiber ultrafiltration system (100 L) were comparable to recoveries observed in the small-scale system (2 L). Recovery of seeded viruses in highly turbid waters from small-scale tangential flow (2 L) (screen and open channel) and hollow fiber ultrafilters (2 L) (small pilot) were greater than 70%. Clogging occurred in the hollow fiber pencil module and when particulate concentrations exceeded 1.6 g/L and 5.5 g/L (dry mass) in the screen and open channel filters, respectively. The small pilot module was able to filter all concentrates without clogging. The small pilot hollow fiber ultrafilter was used to test recovery of seeded viruses from surface waters from different geographical regions in 10-L volumes. Recoveries >70% were observed from all locations. Hill et al. (2005) showed that it was possible to concentrate viruses and other microorganisms simultaneously using hollow fibre technology, and used sodium polyphosphate to minimize adhesion of organisms to the filter. Rutjes et al. (2005) used a membrane ultrafilter of 10 kDa cut off for secondary concentration of enteroviruses following primary concentration by adsorption/elution; the starting primary concentrate volumes were approximately 650 ml (raw sewage) and 1800 ml (river water). Some workers have experienced binding of the virus to the membrane rather than just retention. In these cases the virus was eluted by backwashing with glycine buffer or beef extract and the eluate was reconcentrated by organic flocculation. The advantages of ultrafiltration are principally that the sample requires no pre-conditioning and that a wide range of viruses can therefore be recovered, including those sensitive to the pH changes necessary in most adsorption/elution procedures, and also bacteriophages (Nupen et al., 1981; 39 Urase et al., 1994). Efficiency of recovery is usually good, though as with all methods it is variable. Surface water samples may take a long time to process if they are turbid; Nupen et al. were able to filter 50 l volumes but this took about 40–72 h depending on the sample. Systems have high capital cost, though disposable cartridges have recently become available. The technique is sometimes seen as an advance on the adsorption/elution technique (Grabow et al., 1984; Muscillo et al., 1997). 1.9.7 Ultracentrifugation Ultracentrifugation is a useful method and it is capable of concentrating all viruses in a sample if optimum centrifugal-force and time are used. Differential ultracentrifugation allows separation of different virus types. In a study by Mack et al (1972), 5 gallons of water from the well used by a restaurant was concentrated by flocculation and ultracentrifugation and the sediment was tested for virus in cell cultures. Type II poliovirus was recovered. In another study, viral numbers in natural waters were reported to be as high as 2.5 X 108/ml, 103–107 times higher than the plaque assay (Bergh et al., 1989). However the limited volumes that can be processed even using continuous flow systems together with the high capital costs and lack of portability of the equipment limit its usefulness in concentrating viruses directly from natural waters. During investigation of gastroenteritis outbreak associated with polluted drinking water Murphy et al. (1983), concentrated 5 L samples of borehole water to 50 ml using an ultrafiltration hollow fibre system followed by ultracentrifugation to pellet the virus for electron microscopical examination. They were able to detect rapidly rotaviruses, adenoviruses and SRSVs (noroviruses) as well as enteroviruses, which were confirmed by cell culture. In an investigation to detect HEV in sewage, Pina et al. (1998a) concentrated viruses and removed suspended solids from 40 ml samples by differential ultracentrifugation; Vaidya et al. (2002) used the same protocol to detect HEV and HAV in sewage samples. Le Cann et al. (2004) concentrated astroviruses from sewage samples by ultracentrifugation and extracted the RNA from the pellets. 40 1.9.8 Other methods Several other methods have been applied for concentration of viruses from a variety of environmental samples, but none has been extensively tested for simultaneous recovery of enteric viruses, cost effectiveness and ability to handle large amount of water samples. These include hydroextraction with hygroscopic solids (Wellings et al., 1976; Ramia and Sattar, 1979), iron oxide flocculation (Rao et al., 1968; Bitton et al., 1976), two-phase separation (Lund and Hedstrom. 1966) and freezedrying (Bosch et al., 1988; Kittigul et al., 2001). Affinity columns were used by Schwab et al. (1996) in a broad-based antibody capture technique for a variety of viruses and Myrmel et al. (2000) described the separation of noroviruses in this way. An important attribute of this method is that it acts as a clean-up stage to remove RT-PCR inhibitors. Cromeans et al. (2004) reported the preparation and use of a soluble Coxsackie virus-adenovirus (sCAR) receptor immobilised to magnetic beads for the concentration of Coxsackie and adenoviruses from water sample concentrates. The receptor, which neutralized Coxsackie virus B3, also reacted with other Coxsackie B types. The group also reported the use of a neutralising monoclonal antibody for immunocapture of the same viruses. 1.9.9 Secondary concentration Gilgen et al. (1997) developed a protocol for analysis of bathing waters and drinking water using filtration through positively charged membranes followed by ultrafiltration as a secondary concentration step. Huang et al. (2000) used positively charged membranes followed by beef extract elution and PEG precipitation for the concentration of caliciviruses in water. Secondary concentration can also be accomplished using two-phase separation, usually with polyethylene glycol (PEG)/NaCl, or PEG and dextran T40. Rutjes et al. (2006) compared two-phase separation (PEG and dextran T40) with ultrafiltration for secondary concentration of noroviruses from water following primary concentration by adsorption/elution and observed ultrafiltration to be better. If molecular biological analysis is to be done, the volume may be reduced to about 1ml by dialysis, in spin-columns or 41 microconcentrators with a Membrane cut-off of 100,000 KDa. Where proteinaceous eluent fluids are used, the most commonly employed secondary concentration technique is that of Katzenelson et al. (1976); the pH of the primary eluate is reduced to 3.5–4.5 leading to isoelectric coagulation (flocculation) of the protein. The virus adsorbs to the flock which is deposited by centrifugation and dissolved in 5–10 ml neutral phosphate buffer. If the concentrate is to be inoculated into cell cultures it is common to filter it through a 0.22 mm pore diameter filter to remove contaminating bacteria. 1.10 Methods for Detection of viruses: 1.10.1 Cell culture assays Concentrated samples can be either extracted for viral nucleic acid analysis (PCR amplification) or inoculated onto common cell lines, such as buffalo green monkey kidney (BGM) cells, MA104 cells, RD cells, A549 cells, FRhK-4 cells, CaCo-2 cells, Madin-Darby bovine kidney (MDBK) cells, and pig kidney (PK-15 A) cells, specific to each virus type for isolation and quantification of infectious viruses (Doherty et al., 1999; Ley et al., 2002; Pina et al., 1998b). The cell culture technique was the most widely used technique to determine the occurrence of infectious enteroviruses in environmental samples before the development of molecular methods and is still the best method to isolate and determine infectivity of viruses from environmental samples. After inoculation of a chosen cell line, flasks are evaluated for the presence of damaged cells or rounding of cells and sloughing of the monolayer (cytopathogenic effects [CPE]) as evidence for viral infection. The major drawback to the cell culture assay is that it is laborious and time-consuming; it requires days to weeks of incubation and several passages to confirm both positive and negative results. In addition, some samples may be cytotoxic and produce CPE on cells. A universal cell line that can be used for culturing all enteric viruses has not been established, and there are many viruses that cannot be detected through cell culture assay because they either do not produce CPE, are extremely slow growing, or do not grow on 42 established cell lines (Chapron et al., 2000; Lipp et al., 2001; Pommepuy and Guyader, 1998). For example, adenoviruses, which are one of the most important human pathogens and are often detected in greater numbers than enteroviruses in wastewater, are slow growing, often do not produce CPE, and are consistently underestimated when fast-growing enteroviruses are present (Irving et al., 1981; Tani et al., 1995). Likewise, noroviruses, one of the major causative agents for viral gastroenteritis and food-borne outbreaks, cannot be propagated in cell culture (Haramoto 2004). 1.10.2 Molecular assays. The use of molecular biological detection techniques has permitted faster detection times and, in many cases, increases in sensitivity. It is particularly useful in the detection of viruses which do not multiply in cell culture and, since most of the gastroenteritis viruses, Hepatitis A virus and Hepatitis E Virus fall into this category, this is an important development. With both concentrated samples and infected cultured cells, viral nucleic acids can be extracted and purified to remove cell debris and inhibitors before being amplified and detected by PCR (Griffin et al., 2003; Lipp et al., 2001). One of the most widely used methods for viral nucleic acid extraction and purification was developed by Boom et al. (1990) and is based on guanidium thiocyanate extraction and use of silica columns to bind and wash nucleic acids. This method is rapid, easy to use, and efficient in removing inhibitors (Jiang et al., 2001, Pommepuy et al., 1998). Casas et al. (Casas et al., 1995) developed an extraction method with the use of guanidium thiocyanate and an inorganic solvent to purify both viral RNA and DNA in a single extraction step. Other methods for viral nucleic acid extraction and purification include proteinase K treatment followed by phenol- chloroform extraction and ethanol precipitation. (Albert and Schwartzbrod, 1991; Bosch et al., 1997, Chapron et al., 2000; Green et al., 1995; Le Guyader et al., 1994; Monpoeho et al., 2001). 1.10.2.1 Polymerase Chain Reaction: Molecular techniques have been used extensively to detect enteric viruses from environmental samples since the early 1990s. Molecular 43 detection assays, such as PCR and hybridization, usually are based on the detection of a part of the viral genome that is highly conserved with broad homology within a specific group of viruses (Allard et al., 1992; De Leon et al., 1990). PCR-based assays offer several advantages over cell culture assays in detecting viral pathogens from environmental samples. PCR is rapid, highly sensitive, and specific if a well-designed assay is developed. PCR viral detection is less laborious and time-consuming and also more specific and sensitive than cell culture (chung et al., 1996; Hafliger et al., 1997). Results from PCR assays can be obtained within 24 h of sampling, compared to days or weeks of incubation for cell culture assay (Griffin et al., 2001; Noble et al., 2003). PCR is also capable of differentiating specific viruses (Katayama et al., 2002). For example, PCR primers can be designed to target whole virus orders (e.g., enteroviruses or adenoviruses) or may be specific to a single type of virus (e.g., poliovirus) or tailored for virus serotypes within a host group (e.g., humans, cattle, and pigs) (Gu et al., 2003, Xu et al., 2000). PCR assay can be automated allowing handling of a large number of samples in a short time. PCR is also highly sensitive and is capable of detecting viruses present in low numbers in environmental samples or those which are difficult to grow in cultured cells or replicate without producing CPE (Chapron et al.,2000; Pommepuy et al., 1998). On account of sensitivity of PCR it is realized that use of cell culture alone can grossly underestimate viral contamination. Pina et al. (1998b) suggested that PCR has led to higher rates of detection of adenoviruses in environmental samples. Borchardt et al. (2003) detected enteric viruses (enteroviruses, rotavirus, Norwalk-like virus [norovirus], and hepatitis A virus) from 4 (8%) of 50 household wells by PCR, while no virus was detected by cell culture. In contrast to cell culture the infectivity of viruses detected by molecular methods is often questionable. The other side of the PCR coin is possibility of contamination leading to false positive results. In order to reduce false positive rates, stringent quality control measures, such as use of aerosol-resistant pipette tips or positive-displacement pipettors, decontamination of instruments between experiments, physical separation of pre- and post-PCR procedures and adequate controls are 44 mandatory to ensure the quality of PCR results. Likewise, false-negative results may also be a problem when inhibitors in environmental samples are present. Humic and fulvic acids, heavy metals, and phenolic compounds may inhibit the activity of polymerase enzyme (Straub et al., 1995; Wilson 1997; Young et al., 1993). Additional manipulations, including resin treatments, polyethylene glycol precipitation- resuspension, immunomagnetic capture, and glass purification are sometimes required to remove inhibitors. Additives may also be used in PCR, directly, to reduce the effects of inhibitory compounds. One of the limitations of traditional PCR has been the inability to enumerate viruses. Recently, conventional PCR has been modified to improve specificity, sensitivity and efficiency but also to quantify the number of viruses detected. Some variations of conventional PCR include nested PCR, multiplex PCR, and real-time PCR (for quantification). Seminested PCR and nested PCR assays increase the sensitivity and specificity of PCR with the use of an internal primer or primer set and are sometimes used as a confirmation step. 1.10.2.2 Multiplex PCR. Multiplex PCR (where several sets of primers against several targets are included in a single PCR) is a novel and useful tool saving time and cost, because several types of viruses can be detected in a single PCR assay (Fout et al., 2003). The development of a multiplex PCR assay, however, is not easy and requires careful optimization of reaction mixtures and PCR conditions (Green and Lewis, 1999; Tsai et al., 1993). 1.10.2.3 Real-time PCR. Real-time PCR provides quantitative data for the presence of enteric viral genomes in environmental samples with the use of a fluorescent dye, such as SYBR Green that will bind to amplified cDNA or with fluorochrometagged probes that fluoresce when bound to complementary sequences in the amplified region. The procedure is less time-consuming because a confirmation step such as agarose gel electrophoresis and additional hybridization are generally not required. The entire analysis can be done in a 45 closed system, reducing the potential contamination. Real-time PCR assays have shown detection sensitivities comparable to or greater than those of conventional PCR in several studies. Beuret (2004) reported that real-time reverse transcription-PCR (RT-PCR) detection of norovirus and enteroviruses in seeded samples showed an increased sensitivity of 1 and 2 orders of magnitude, respectively, compared to the conventional RT-PCR protocol. The real-time RT-PCR assay developed by Donaldson et al. (2002) for detection of enteroviruses showed a detection limit of 9.3 viral particles ml-1 for seawater and 155 viral particles g-1 for sponge. However, the cost of a realtime PCR instrument is still substantially more than that of a conventional PCR instrument, and in some cases, real-time PCR has been shown to be less sensitive than conventional RT-PCR and nested PCR. Noble et al. (2003) reported that human adenovirus 40 was detected by real-time PCR in only two of the four samples positive for adenoviruses by conventional nested PCR; none of the samples that were positive for enteroviruses by conventional RT-PCR was detected by real-time RT-PCR. 1.10.2.4 Integrated cell culture PCR. While PCR-based methods offer many advantages in sensitivity, specificity, and efficiency over cell culture, they still cannot provide information on the infectivity of viruses detected with the reliability of cell culture. To address this, several studies have combined cell culture and PCR and have reported that this method improves the specific detection of infectious enteric virus from environmental samples. The hypothesis behind this method is that after inoculation of a cell line, only infectious viruses, if present, will propagate; the cells can then be extracted and tested for viruses by PCR before CPE is noted. This is also appropriate for viruses that do not produce CPE but still infect and grow in a cell line. Chapron et al. (2000) noted that an integrated cell culture-RT-nested PCR (ICC-RT-PCR) procedure provided increased sensitivity compared to the conventional cell culture method (CPE only). By ICC-RT-PCR, 68.9% of samples were positive for an infectious virus, compared to 17.2% determined by traditional cell culture. Detection of infectious adenoviruses also showed significant improvement with this method; the percentage of positive environmental samples (including sewage, 46 sludge, river, and shellfish samples) increased from 28.6% by conventional cell culture to 50% by ICC-PCR (Greening et al., 2002). ICC-(RT)-PCR also increase the frequency at which viruses are detected from environmental samples that normally have very low levels of infectious enteric viruses, including potable water (Lee and Kim, 2002). ICCPCR can also produce results in a shorter period than traditional cell culture (i.e., <3 days) (Greening et al., 2002). However, recent work by Ko et al. (2003) suggests that carryover of nucleic acids of inactivated viruses inoculated onto cultured cells might result in a false-positive result from samples containing no infectious viruses. To address this, the authors developed an ICC-RTPCR- based assay to detect viral mRNA rather than DNA in the case of adenoviruses; mRNA is only transcribed by infectious adenoviruses during replication. After exposure to different doses of UV radiation, adenovirus DNA was detected consistently in inoculated cell culture lysate by PCR even when adenovirus mRNA could no longer be detected. 1.11 Assessing viral infectivity. While information derived from direct molecular detection assays for viruses can indicate contamination in an area, data on viral infectivity is extremely important to determine health risks. Therefore, understanding the relationship between viral detection by molecular methods and infectivity as determined by in-vitro assays is critical to ultimately use molecularly derived data for risk assessment. Several recent studies have provided evidence that degradation of viral nucleic acids (particularly RNA) is well correlated with loss of infectivity (based on loss of CPE in cell culture), even when the viral genome is more persistent than infectious viruses (Dubois et al., 1997; Skraber 2004; Tsai et al., 1995; Wetz et al., 2004). However, because RNA degrades relatively rapidly in the environment (in a few minutes) compared to DNA, viruses that are no longer infectious because of damage to the capsid also experience damage to the RNA on the same time scale, thus becoming undetectable by both cell culture and RT-PCR (Limsawat and Ohgaki, 1997). Wetz et al. (2004) showed that the detection rate for polioviruses varied little between cell culture and RT-PCR in unfiltered (natural) seawater. 47 Tsai et al. (1995) showed that naked enteroviral RNA could not be detected by RT-PCR and dot blot hybridization after 2 days of incubation at both 4°C and 23°C in unfiltered seawater. Skraber et al. (2004) observed that although the poliovirus genome has a higher persistence than an infectious poliovirus, the loss in detection of the viral genome is directly correlated to the disappearance of infectious virus, suggesting that viral nucleic acids may indeed serve as an efficient indicator for infectious viruses in aquatic environments. 1.12 Detection of enteric viruses in environmental samples: The Indian scenario. The presence of Hepatitis A, Hepatitis E and other enteric viruses in environmental samples has been demonstrated in India (Jothikumar et al., 1995; 2000; Vaidya et al., 2002) and these viruses are posing a considerable threat to public health especially in rural and under-developed areas (Arankalle et al., 2006), but no systematic and large scale initiative has been taken for the surveillance of these viruses in the water bodies. Some smallscale studies have been done but a lot of work needs to be done. 48