Download Introduction and Review of literature

Introduction
and
Review of literature
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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
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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
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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;
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
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
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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
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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
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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
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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)
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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.
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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
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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.
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