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STORAGE OF WATER FOR ENTERIC VIRUS DETECTION
By:
OGBONNNA, EMMANUEL OKEZIE
Registration Number: 2011/177638
Department of Microbiology
University of Nigeria
Nsukka
March, 2015
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University of Nigeria, Nsukka
Department of Microbiology
Course Code: MCB 481
Course Title: Seminar in Microbiology
SEMINAR TOPIC
By:
Ogbonna, Emmanuel Okezie
Registration Number: 2011/177638
Supervisor:
Vincent N. Chigor, PhD
March, 2015
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This Seminar has been approved for the Department of Microbiology, Faculty of Biological Sciences,
University of Nigeria, Nsukka
By:
Vincent N. Chigor, PhD ____________________________ _______________________
(Supervisor) Signature Date
Professor Anene N. Moneke ____________________________ _______________________
(Supervisor) Signature Date
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DEDICATION
This seminar work is dedicated to the Almighty God, the author of life and master designer of all
human destinies, whose understanding and wisdom is far beyond the cumulative knowledge of
all earth, to whom all things is ascribed to and would always be; And also to my parents Mr. and
Mrs. Moses Ogbonna who bore me into this world.
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ACKNOWLEDGEMENT
To God be the glory and honour to him will I give all the thanks for keeping me all through my
tender age until this time of my life. I would like to thank Vincent N. Chigor, PhD for allowing
me the honour of being one of his seminar students and, to double the thanks for his patience
with me as I worked on this Seminar topic. I also wish to thank my lecturers at the University of
Nigeria, Nsukka, my (HOD) Professor Anene N. Moneke and not forgetting one of the wonders
of the twenty first century being the internet which has succeeded in bridging the gap between
researchers and reducing the distance to information to just a click.
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Summary
Human enteric viruses are water-borne infectious agents that can be detected
appropriately only if an effective virus concentration method is applied often the adsorption-
elution principle is used, reasons for concentration prior to analysis could range from the
unavailability of the required facility or the needed technical skills, in addition to this, is the
recognition that water samples collected sometimes need to be stored for a longer duration before
analysis is performed of which temperature plays a crucial role in the retention of virus viability,
infectivity and survival, most of the enteric viruses are known to survive better at lower
temperatures as opposed to higher temperatures even though some of the enteric viruses have or
must have developed the ability to survive at a higher temperature by the production of stressor
proteins, storage of water samples for enteric virus detection could range from days to months.
Human enteric viruses are known to be transmitted by various means which could be faecal-oral
route, aerosol, even food borne transmission has been recorded, they are known to cause a
variety of illnesses such as gastroenteritis, hepatitis, myocarditis etc. Human enteric viruses have
been known to be a major cause of water-related diseases, various methods have been developed
for the detection of enteric viruses with the various limitations of each necessitating the invention
of a more promising method, example of these methods include cell culture, molecular technique
and integrated cell culture polymerase chase reaction.
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TABLE OF CONTENTS
i---------------------------------------------------------------------------------------------------TITLE PAGE
ii-------------------------------------------------------------------------------------------APPROVAL PAGE
iii------------------------------------------------------------------------------------------------DEDICATION
iv----------------------------------------------------------------------------------ACKNOWLEDGEMENT
v----------------------------------------------------------------------------------------------------SUMMARY
vi------------------------------------------------------------------------------------TABLE OF CONTENTS
CHAPTER ONE: INTRODUCTION
1--------------------------------------------------------------------------------------------INTRODUCTION
4----------------------------------------------------------------------------------TRANSMISSION MODES
CHAPTER TWO: METHODS OF DETECTING ENTERIC VIRUSES
8--------------------------------------------------------------------------CONCENTRATION METHODS
13------------------------------------------------------------------------------CELL CULTURE METHOD
19---------------------------------------------------------------------------------MOLECULAR METHOD
26-------------------------INTEGRATED CELL CULTURE POLYMERASE CHAIN REACTION
CHAPTER THREE: STORAGE OF WATER SAMPLE
31-------------------------------------------------------------------------NEED FOR WATER STORAGE
CHAPTER FOUR: CONCLUSION
35----------------------------------------------------------------------------------------------CONCLUSION
37-----------------------------------------------------------------------------------------------REFERENCES
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INTRODUCTION
Viruses that primarily infect and replicate in the gastrointestinal tract are known as enteric
viruses. These include enteroviruses, noroviruses, rotaviruses, hepatitis A virus, adenoviruses,
and reoviruses, amongst others. Enteric viruses can be present in human and animal faeces,
which can contaminate recreational and drinking water sources. Enteric viruses are extremely
small particles ranging from 20 nanometres (mu) to 85 mu in diameter.
Enteric viruses can be transmitted by the water route. The most common mode of Enteric
Virus transmission is by person-to-person contact. Small children are the most susceptible
because of their close contact with other children and their less than optimal hygienic habits.
Adults are generally less subject to infection because of immunity acquired by previous exposure
to the virus. Enteric viruses including polioviruses, hepatitis A virus (HAV) and rotaviruses are
found in faecally contaminated water and food and are an important public health concern
(Metcalf et al, 1995). Water borne outbreaks of viral hepatitis and gastroenteritis caused by
rotaviruses have been reported (Hopkins et al, 1984; Wen et al, 1992; De Serres et al, 1999). The
outbreaks were attributed to the consumption of drinking water and food that was considered
safe by bacteriological standard methods (Hejkal et al, 1982). Rotaviruses can survive well
enough in treated drinking water (Sattar et al, 1984), raw and treated river water (Raphael et al,
1985).
The inability to remove or inactivate enteric viruses from contaminated foodstuffs leads
inevitably to human disease in some susceptible consumers. The diseases caused by enteric
viruses fall into three main types: gastroenteritis, enterically transmitted hepatitis, and illnesses
that can affect other parts of the body such as the eye, the respiratory system, and the central
nervous system including conjunctivitis, poliomyelitis.
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The determination of enteric viruses in environmental water samples is, however, rather
difficult because their densities are so low that an efficient virus concentration is necessary.
Several methods for concentration of enteric viruses from water have been proposed and the
most promising method is the virus adsorption-elution technique (Toranzos and Gerba, 1989; Li
et al, 1998; Abbaszadegan et al, 1999) Human enteric viruses are water-borne infectious agents
that can be detected appropriately only if an effective virus concentration method is applied.
Often the adsorption-elution principle is used. As a result of the low concentration of human
pathogenic viruses in drinking water it is essential to concentrate big sample volumes before
detection is possible. This is done using filtration techniques, flocculation or affinity
chromatography and is usually associated with virus loss and sometimes inactivation due to the
treatment (Auckenthaler, 2003).
There are three broad methods of detecting virus in the environment and they are cell
culture, molecular method and the integrated cell culture polymerase chain reaction. Many of the
enteric viruses such as astroviruses, enteric adenoviruses, HAV, and rotaviruses are fastidious in
their in vitro growth requirements but can still be grown in cell cultures. Noroviruses, on the
other hand, do not grow in vitro, and no animal model exists for the human noroviruses yet
Human noroviruses are nonculturable, and until recently no animal model had been identified.
More than several hundreds of enteric microbial pathogens are known to infect man as human
enteric viruses, because they preferably multiply in the intestine (except hepatitis and poliovirus).
The transmission route is faecal-oral. Viruses transmitted via drinking water are usually of
human origin, but for rotavirus (Cook et al., 2004) and for hepatitis E virus (Clemente-Casares et
al., 2003) animal reservoirs have also been identified. The most common technique for detection
after concentration is to grow viruses on cell cultures and subsequent analysis of the plaques,
which are formed on the cell monolayer. Unfortunately not all viruses are able to grow in culture.
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Furthermore, the technique takes a lot of time (one week), is very expensive and needs skilled
laboratory workers. New molecular methods like reverse transcriptase (real-time) PCR seem to
be a good alternative and circumvent problems connected with the cell culture approach (Beuret,
2004). PCR is very sensitive, but also associated with several disadvantages. Detected viruses are
not essentially infective and substances present in the water matrix, like humic acids, can inhibit
the amplification reaction (Walter, 2000). Furthermore, RNA is very unstable and the capsid is
often very well structured and therefore not easy to break up.
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TRANSMISSION MODES
Human pathogenic viruses commonly referred to as enteric viruses can be transmitted by
a variety of routes, including direct and indirect contact, vector transmission, and vehicle
transmission. The enteric viruses like the enteroviruses, rotaviruses, Hepatitis A & E,
Noroviruses, adenoviruses, reoviruses, and others are transmitted by the faecal-oral route, infect
the gastrointestinal tract and are capable of causing a wide range of illnesses including diarrhoea,
fever, hepatitis, paralysis, meningitis and heart disease. Evidence for faecal contamination of
surface and ground waters is provided by the detection of enteric viruses in both surface and
groundwater and the continued occurrence of outbreaks of waterborne disease. The methods for
determination of enteric viruses contaminated water samples are required for monitoring of food
and water quality in the presence of viral pollution in order to enable effective management of
public water supplies, implementation of appropriate preventive control and curative measures.
River waters has been frequently and inevitably contaminated with human enteric viruses via the
discharge of untreated domestic and industrial waste water action. The human enteric viruses are
resistant in the environment. They are capable to remain infective in the aquatic environment for
several months. These agents are responsible for a wide spectrum of symptoms ranging from
gastroenteritis to neurological infections. Foods play an important role in the transmission of
enteric viruses. Enteric viruses can cause surface contamination of fruits and vegetables by
adsorption following exposure to faecally contaminated soil or groundwater. All enteric viruses
except the adenoviruses contain RNA rather than DNA, have a protein capsid protecting the
nucleic acid, and are non-enveloped. The enteric viruses are inert particles and do not replicate or
metabolize because, like all viruses, they are obligate pathogens and require living cells to
multiply. Enteric viruses are excreted in the faeces of infected individuals and may directly or
indirectly contaminate water intended for drinking. These microbes are excreted in high
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numbers, 105-1011 per gram of faeces of infected individuals and are commonly isolated in
domestic wastewater, even after disinfection. Once in the environment they can survive for long
period of time, even months under the right conditions. Ground and surface water may be
subjected to faecal contamination from a variety of sources, including sewage treatment plant
effluents, on-site septic waste treatment discharges, land runoff from urban, agricultural and
natural areas, and leachates from sanitary landfills. The enteroviruses (poliovirus, coxsackie A
and B viruses, echovirus) can cause a variety of illnesses ranging from gastroenteritis to
myocarditis and aseptic meningitis (Melnick, 1990). Numerous studies have documented the
presence of enteroviruses in raw and treated drinking water (Keswick et al., 1984, wastewater
Payment 1981) and sludge (Craun, 1984). Enteroviruses in the environment pose a public health
risk because these viruses can be transmitted via the faecal-oral route through contaminated
water (Craun, 1984) and low numbers are able to initiate infection in humans. One of the enteric
viruses e.g. Hepatitis A virus (HAV) is an important waterborne virus because of the severity of
the disease it may cause in susceptible individuals. HAV is the cause of acute infectious hepatitis
and was the first enteric viruses for which a waterborne outbreak was documented in the United
States. This virus survives more than four months at 5o
and 25o
C in water, wastewater and
sediments (Sobsey et al., 1988). As with the enteroviruses, the full genome of various strains of
HAV has been sequenced. Rotaviruses are a significant cause of acute diarrheal illness,
especially in young children. Rotaviruses group A has been documented as causes of waterborne
outbreaks in humans (Gerba and Rose, 1990). These viruses have a segmented genome
consisting of 11 segments of double stranded RNA. Segments designating subgroup and serotype
specificity have been sequenced for several strains and Serotypes. Enteric viruses are shed in
extremely high numbers in the faeces of infected individuals; patients suffering from diarrhoea
or hepatitis may excrete from 105 to 1011 virus particles per gram of stool (Farthing, 1989).
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Furthermore, a single episode of vomit of a patient with norovirus gastroenteritis may contain
around 107 particles (Cheesbrough et al., 1997). Ingestion of sewage contaminated water or food
is the main route of infection with enteric viruses, although the role of inanimate surfaces serving
as vehicles for virus infection must not be underestimated. Viruses with a viremic phase, such as
the hepatitis viruses, may also be parentally transmitted, although these days it is considered to
be a much less frequent mode of transmission .Enteric virus infections through shellfish grown in
contaminated waters, contaminated drinking water, food crops grown in land irrigated with
wastewater and/or fertilized with sewage, and, to a lesser extent, sewage-polluted recreational
water Pathogenic viruses are routinely introduced into the environment through the discharge of
treated and untreated wastes, as current treatment practices are unable to provide virus-free
wastewater effluents. Enteric viruses are generally resistant to environmental stressors, including
heat and acid. Most resist freezing and drying and are stable in the presence of lipid solvents.
The resistance of enteric viruses to environmental stressors allows them to resist both the acidic
environment of the mammalian gut and also the proteolytic and alkaline activity of the
duodenum so that they are able to pass through these regions and colonize the lower digestive
tract. These properties also allow survival of enteric viruses in acidic, marinated, and pickled
foods; frozen foods; and lightly cooked foods such as shellfish. Most enteric viruses are believed
to have a low infectious dose of 10–100 particles or possibly even less. Hence, although they do
not multiply in food, enough infectious virions may survive in food, and when consumed can
cause disease.
Projectile vomiting is a characteristic symptom that can contribute to secondary spread
through droplet infection, where droplets containing virus may contaminate surfaces or be
swallowed. Evidence that norovirus transmission occurs through aerosolization of vomit was
clearly demonstrated at a United Kingdom hotel. During a meal, a guest vomited at the table, and
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norovirus infection spread in a radial pattern through the restaurant, progressively decreasing
from 91% attack rate among those seated at the same table to an attack rate of 25% in those
patrons who were seated the farthest distance away from the guest who vomited (Marks et al.,
2000). Noroviruses are the main cause of food-borne viral gastroenteritis worldwide with food-
borne transmission accounting for a large proportion of norovirus outbreaks in many countries.
Determination of the original source of the virus is often problematic because several modes of
transmission frequently operate during norovirus gastroenteritis outbreaks. Although the initial
transmission route may be through consumption of contaminated foods, secondary transmission
via direct contamination of the environment or person-to-person contact also often occurs. This
results in wide dissemination where infection quickly spreads through institutions, schools,
camps, resorts, and cruise ships and causes large-scale epidemics with more than 50% attack
rates.
CONCENTRATION METHODS
The determination of enteric viruses in environmental water samples is however rather
difficult because their densities are so low that an efficient virus concentration is necessary.
Several methods for concentration of enteric viruses from water have been proposed and the
most promising method is the virus adsorption-elution technique (Toranzos and Gerba, 1989; Li
et al, 1998; Abbaszadegan et al, 1999). A good concentration method should fulfil several
requirements: it should be technically simple, fast, provide high virus recoveries, be adequate for
a wide range of enteric viruses, provide a small volume of concentrate, and be inexpensive
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(Bosch et al., 2008), but with a large volume of water the second step becomes necessary
(Katzenelson et al., 1976). The ideal method for virus concentration from water should be
capable of processing large volumes of a variety of waters in the least possible time, sensitive
enough to concentrate most types of viruses known to be present in water and wastewater, easy
to perform and economical to use, and able to detect viral aggregates and viruses adsorbed to
suspended solids (Gerba and Goyal, 1982). Adsorption-elution of viruses with an electropositive
filter is one of the most commonly used techniques and is the method for recovery of enteric
viruses from water. Under ambient conditions, enteric viruses are negatively charged and will
adsorb to a positively charged membrane under acidic conditions. In addition, as a second
concentration method an organic flocculation method which commonly uses 3% beef extract
with high pH (pH 9.5) is the most widely used to elute absorbed viruses from filters and gives a
high viral recovery. In organic flocculation, buffered beef extract is used to precipitate viruses
from concentrated samples by lowering pH of a protein solution to 3.5 (Fong and Lipp, 2005;
Lakhe and Paunikar, 2002 and Katzenelson et al., 1976).A study carried out by making a
comparative study of some primary concentration methods as well as two secondary virus
concentration methods. To evaluate the efficacy of the methods The results showed that the
adsorption / elution on cellulose acetate filter is more effective than on silica gel or floated
gauze. For secondary concentration the use of PEG 8000 gave better results than organic
flocculation. The secondary concentration step is an improvement of the virus detection protocol.
The PEG concentration and organic flocculation are normally used leading to a better detection
than single primary concentration. The same data showed that most amplification were better
with PEG concentration than organic flocculation. For the analysis of water and food virus
concentration step is crucial. This step is described as critical as a result of an analysis of water
depends largely on it. The role of concentration methods can be confirmed when it is observed
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that the tests made on samples without any virus concentration, had given a negative results.
Despite the importance of the concentration some authors were able to obtain amplification of
human enteric viruses’ genome from raw sewage without concentration steps. However, it
remains a negligible approach. Various techniques have been used for virus concentration from
water samples. The easiest ones are adsorption/elution based since others used more
technological approach such as ultrafiltration The interest of a successful concentration method
is to improve detection in water and wastewater that may contain various viral pathogen load,
depending on the health status of people whose homes are drained, Researchers have variously
described protocols for concentrating viruses form large water samples. Virus detection methods
share a common step of concentrating a large water sample (up to 2000 litres) to as little as a few
millilitres. One of these methods involves binding viruses to membrane filters and eluting viruses
from the membrane with a beef extract solution (Stetler, 1984). Concentration is required
because virus concentrations are low and samples must be of a manageable size. Sample
concentration is a particularly critical step because the viruses may be present in such low
numbers that concentration of the water samples is indispensable to reduce the volume to be
assayed to a few millilitres or even microlitres. In relatively non polluted waters, the virus levels
are likely to be so low that optimally hundreds, or even thousands, of litres should be sampled to
increase the probability of virus detection. A good concentration method should fulfill several
criteria: it should be technically simple, fast, provide high virus recoveries, be adequate for a
wide range of enteric viruses, provide a small volume of concentrate, and be inexpensive.
Basically, all available procedures have been evaluated using samples spiked with known
viruses. It is known that the recovery efficiency recorded with experimentally contaminated
water dramatically decrease when the method is applied in actual field trials. Additionally, none
of the existing concentration procedures has been tested with all of the medically important virus
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groups; normally, a few specific enteric viruses have been employed to conduct the evaluation
trials. However, several virus concentration methods have been used successfully to recover
naturally occurring enteric viruses in water (Finance et al., 1982; Gerba and Goyal, 1982; Goyal)
the methods include Adsorption-elution methods, Precipitation methods, Ultracentrifugation,
Lyophilization, Ultrafiltration.
Most of the procedures for concentrating and extracting viruses make use of the properties of the
viral proteinaceous macromolecules. Certain protein structures confer on viruses in an aquatic
environment the properties of a hydrophilic colloid of an amphoteric nature whose electric
charge varies according to the pH and the ionic force of the environment. Viruses can therefore
be adsorbed onto and then detach themselves from different substrates that are positively or
negatively charged depending on their pH. Methods based on the adsorption of viruses from the
sampled water onto a suitable solid surface from which they may subsequently be eluted into a
much smaller volume are preferred for use with large-volume samples. Different types of filters
have been evaluated for the recuperation of aquatic viruses, in the form of flat membranes or
cartridges. Cartridge-type filters have the advantage to allow filtration of large volumes of
moderately turbid water within a relatively short time. Their chemical composition, Diameter,
and porosity vary enormously. A whole range of “negatively” or “positively “charged filters now
exist. Their efficiency depends on the type of water being tested and the presence of interfering
substances such as detergents, suspended solid matter, or organic matter, which can affect the
adsorption of viruses on these filters (Sobsey and Glass, 1984; Sobsey and Hickey, 1985, Gilgen
et al., 1997). The disadvantage of the negatively charged membranes or cartridges (Farrah et al.,
1976) is that the water sample must be pre-treated prior to concentration. This includes
acidification of water and addition of salts to the water sample to facilitate virus adsorption
because electronegative filters do not adsorb viruses well under ambient water conditions (Rao
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and Melnick, 1986). The necessity of this pre-treatment step limits the on-location use of this
method to a certain extent, although automatic injection systems do exist for treating several
hundred litres of water. Virus concentration with electropositive filters may be performed on
location at ambient conditions and without any prior amendment of the sample, which make this
procedure most suited for in-field studies, provided that the sample pH is lower than 8.5 (Sobsey
and Jones, 1979). Glass powder (Sarrette et al., 1977; Schwartzbrod and Lucena, 1978; Gajardo
et al., 1991) or glass fiber (Vilaginès et al., 1997) have also been satisfactorily used in different
laboratories as adsorbent materials for virus concentration. Viruses in eluate volumes too large to
be conveniently and economically assayed directly for viruses, such as those obtained from
processing large volumes of water through cartridge or large disk filters, can be reconcentrated
by several methods. Obviously, the recovery of small quantities of viruses from natural waters is
dependent not only on the efficacy of primary concentration from the original large volume but
also on the reconcentration of the primary eluate to a smaller volume. Methods such as
aluminium hydroxide adsorption-precipitation (American Public Health Association, 1998),
polyethylene glycol hydro extraction (Farrah et al., 1978; Lewis and Metcalf, 1988), organic
flocculation (Katzenel- son et al., 1976), and ammonium sulphate precipitation (Shields and
Farrah, 1986; Bosch et al., 1988b) that are impractical for processing large fluid volumes are
suitable for second-step concentration procedures. Alternatively, viruses can be sedimented
depending on their molecular weight using ultracentrifugation (Steinman, 1981; Mehnert et al.,
1997). Freeze-drying of samples (Gajardo et al., 1995; Pintó et al., 2001) and rehydration in a
smaller volume provides a procedure for both virus concentration and removal of PCR inhibitors.
Ultrafiltration (Divizia et al., 1989b) can utilize size exclusion rather than adsorption and (or)
elution to concentrate viruses and can provide consistent recoveries of different viruses under
widely varying water conditions.
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CELL CULTURE
Traditionally, detection of enteric viruses in environmental samples is done by cell
culture approach involving propagation of viruses in a susceptible cell line producing cytopathic
effects (CPE) (Zurbriggen et al., 2008). However, it has been discovered that a single cell line
can not suffice for propagation and detection of all or even some of enteric viruses, because each
virus behaves differentially in a particular cell line (Rodríguez et al., 2008; Lee et al., 2004;
Chonmaitree et al., 1988). Therefore, use of multiple cell lines becomes essential, making the
process time-consuming, laborious and expensive. Also some enteric viruses having significant
epidemiological potential, such as hepatitis A virus (HAV), hepatitis E virus (HEV), norovirus
and adenovirus 40 and 41 do not grow efficiently in cell culture (Emerson et al., 1991; Divizia et
al., 1999; Cromeans et al. 2008; Duizer et al., 2004). Cell culture assays on FRhk-4 (Fetal
Rhesus monkey Kidney), A549 (Human Lung Carcinoma) and BGM (Buffalo Green Monkey
kidney) were performed to test for the infectivity of viruses. Normally viruses can be detected by
cell culture techniques but the technique also has some shortcomings, e.g. cell culture needs high
technical demand, great effort, is time consuming appears to take more than 3 days or some time
6-8 weeks for pathological effects Presently, the common methods for the detection of poliovirus
in drinking water samples involve cell culture assay which is expensive and time consuming, it
require at least 2 weeks. PCR is an attractive method for the regular monitoring of poliovirus in
water samples because of it is more rapid, simpler and less expensive than conventional cell
culture method. Noroviruses, on the other hand, do not grow in vitro, and no animal model exists
for the human noroviruses yet. For many years, the lack of a culture system limited
investigations focusing on the role of noroviruses in food-borne disease, although progress is
now being made after the in vitro culture of a mouse norovirus (Wobus et al., 2004). Cell
cultures are generally used for the analysis of culturable viruses. Using culture methods,
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infectious viruses can be identified through their ability to produce changes in inoculated cells
(cytopathic effects or CPE) or through expression of viral antigens that may be detected
serologically. The advantages of culture-based methodology is that it can be either quantitative
or qualitative and produces unambiguous results with respect to virus presence and infectivity.
HAV can be cultured in several different primate cell lines including African green monkey
kidney cells (BSC-1), fetal rhesus monkey kidney cells (FRhK-4 and FRhK-6), and human
fibroblasts (HF), but wild-type strains are difficult to culture and generally do not produce CPE
in cell cultures. Human noroviruses are nonculturable, and until recently no animal model had
been identified. Inoculation of chimpanzees with Norwalk virus elicited immune responses but
no symptoms developed and no virus was shed in faeces (Wyatt et al., 1978). Sustained attempts
have been made to culture human noroviruses over the past 10–15 years but without success.
More than 26 different cell lines combined with many varied cell culture supplements and
growth conditions have been evaluated, but no norovirus-induced CPE or replicating norovirus
was obtained (Duizer et al., 2004). Noroviruses have now been identified that infect animals,
including pigs, cattle, and mice, and progress in this field is now being made with the growth of a
mouse norovirus in artificial culture (Wobus et al., 2004). This culture system will help to
discover more about human noroviruses and their mechanisms of pathogenicity. The infected
mice develop gastroenteritis, and so this discovery holds potential as a future model for human
norovirus disease. Although many rotaviruses can be grown in cell cultures, they have proved
difficult to cultivate in vitro, and growth is restricted to a few cell lines derived mainly from
monkey kidneys. Addition of trypsin to the culture medium is required to enhance viral growth
in cell cultures. Serotypes 40 and 41 of enteric adenoviruses are difficult to grow in cell cultures,
whereas most of the non-faecal types are culturable. Adenoviruses are slow-growing compared
with a majority of enteroviruses and can be quickly overgrown in some cell lines. The A549 and
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293 cell lines have been successfully used for the isolation of adenoviruses from food and
environmental samples. Conventional methodology for the detection of enteric viruses from the
environment relies on a few established cell lines. The Buffalo Green Monkey (BGM) kidney
cell line is the most commonly used for the detection of enteroviruses in the environment
(Dahling et al., 1984). This cell is preferred over others, including primary cells, because it
provides high sensitivity to natural isolates of enteroviruses (Dahling and Wright, 1986). Its
sensitivity can be further enhanced by pre-treatment of the cells with enzymes or other
substances (Benton and Hurst, 1986). Unfortunately, the use of other cell lines is required to
detect other groups of enteric viruses (Smith and Gerba, 1982). This can greatly increase the
cost and time of the assay. While the cell culture assay can detect infectious viruses in
environmental samples, without additional tests, no determination can be made as to the
particular strain of virus present in a sample. Additionally, the length of time needed to detect
infection in the cell culture can vary greatly, from a few days to several weeks, depending on the
type and number of viruses present. The use of cell culture for virus detection and the PCR assay
for viruses differ significantly in several ways. For cell culture, the minimum detection level of
viruses in a sample is, by definition, one PFU per unit volume - a quantity of virus particles that
may range from just a few or many more at least some of which must be infectious. In addition,
when a sample tests positive for viral infectivity using cell culture, the infectious agent is not
necessarily known. The BGM cell line, routinely used for enterovirus assays, is susceptible to
infection by many viruses, including reoviruses such as rotavirus, a pathogen often present in
environmental samples in numbers greater than enterovirus (Puig et al., 1994). Cell culture
protocols do not detect all human viruses present in the environment. Norwalk virus, for
instance, has yet to be successfully grown in cell cultures, and therefore environmental samples
cannot be assayed for this pathogen. Finally, since each environmental sample is unique, little is
22. 15_Chigor
known regarding possible sample components that may inhibit the viral infectivity in culture.
Cell culture, however, does offers the advantages of isolating an infectious viral pathogen, and is
widely accepted as the standard method for viral detection in water. Most cell culture protocols
call for a 14-day initial passage and for a 14-day secondary passage of the sample on cells,
followed by a seven-day confirmation passage of putative positive samples. To test for different
viruses, multiple cell lines must be maintained, different growth media must be purchased and
stored, and different protocols followed. The cost of one sample by cell culture assay in a lab
may be approximately $650.00 Traditional methods for detecting enteric viruses in water rely on
tissue culture with the Buffalo Green Monkey cell culture line and take several days to yield
results (Havelaar, 1993; Kott et al., 1974). Primary tissue culture methods require a high degree
of technical ability, and are too expensive to be used practically in many areas of the world (Kott
et al., 1974; Havelaar, 1993). The cell culture procedure detects enterovirus and orthoreovirus
species that are capable of infecting and producing cytopathic effects (CPE) in the Buffalo Green
Monkey kidney (BGM) cell line. Although this cell line is considered a “gold standard” for
detection of infectious waterborne viruses, noroviruses and a number of enteroviruses do not
replicate in BGM cells. There is no established cell line for detection of infectious human
noroviruses, but a prototype research method is under development .Cytopathic effect (CPE) –
The degeneration of cells caused by virus replication. It often involves the complete
disintegration of cells but also may be identified through changes in cell morphology. However,
care must be taken in using changes in cell morphology as evidence of CPE, because uninfected
23. 16_Chigor
BGM cells change morphology during mitosis. True CPE is always progressive.
Figure 1. Uninfected BGM cells
Figure 2. BGM cells showing early cytopathic effect from poliovirus
Buffalo Green Monkey kidney (BGM) cells – This is a stable cell line of monkey kidney cells
that were originally developed at the University of Buffalo for clinical isolation of enteroviruses
and later adapted for use in detecting infectious viruses in environmental samples. BGM cells
form a monolayer of cells when propagated in tissue culture vessels. Figure 1 is a micrograph of
24. 17_Chigor
uninfected BGM cells growing as a monolayer. Cell culture is considered by some experts to be
the best way to isolate and determine infectious virus from an environmental sample (Fong and
Lipp, 2005). Plaques assays utilizing cell culture are typically used with wastewater and bio solid
samples Thus, the detection of viral mRNA in cell culture indicates the presence of infectious
viral particles a major problem with cell culture procedures is that they do not detect viruses that
fail to cause cytopathic effects (CPE).
MOLECULAR TECHNIQUES
Owing to the limitations with cell culture method nucleic acid-based methods such as
PCR and hybridization have proved to be important tools. PCR was shown to be more
effective than cell culture technique for detection of enteric viruses (Abbaszadegan et al.,
1999). However, the use of PCR for detection of multiple viral targets in environmental
samples is limited due to high cost and sometimes the unavailability of adequate test sample
volume for several individual reactions. Unlike single PCR, multiplex PCR (Chamberlain et
al., 1988) with different pairs of specific primers for amplifying different viral genomes in
one reaction tube enables detection of two or more targets in a single test, hence making the
later more cost-effective and less time consuming. Multiplex PCR has been used for
simultaneous detection of enteric viruses from environmental samples and food samples (Fout
et al., 2003; Tsai et al., 1993) The advent of molecular techniques, and particularly
procedures based on nucleic acid amplification through the polymerase chain reaction (PCR)
25. 18_Chigor
provided tools for the specific and sensitive monitoring of health significant enteric viruses in
water, thus enabling a safer evaluation of water virological quality. Today, RT-PCR
technique has become the standard for diagnosis of norovirus infection worldwide; among the
problems with traditional RT-PCR has been the inability to enumerate viruses (Jothikumar et
al., 2005). Recently, most reported conventional RT-PCR assays have been modified, because
it is unable to detect all norovirus, to increase specificity, sensitivity and efficiency (Fong and
Lipp, 2005 and O'Neill et al., 2002). NRT-PCR assays, with the use of an internal primer or
primer set, have been successfully employed for amplification of low levels of enteric viruses
found in naturally contaminated food and water (O'Neill et al., 2001; Oh et al., 2003 and
Schreier et al., 2000). This method is one of highly specific and sensitive assays. More
sensitive technology and faster methods is now available for the detection of viruses from
both environmental and clinical samples. More reliable and efficient method such as PCR and
nucleic acid hybridization for the detection of different enteric viruses from water samples are
well documented. The advantage of PCR over cell culture is due to decreased time and cost
and increased sensitivity of RT- PCR which facilitate the detection of low numbers of target
RNAs usually found in environmental samples. Although PCR become the attractive
alternative method over cell culture but in environmental samples, organic matter or metal ion
causes inhibition of enzymatic amplification. In recent years, PCR has been adopted
extensively in detecting poliovirus in environment owing to its high specificity and simpler
operations. Although PCR can pick out even there is only one virus but its sensitivity still
does not come up to the standards for testing virus present in water because the concentrated
sample say 10ml is even still too large for PCR reaction. Testing capability is inconsistent
even positive sample can’t decide the infectivity of the virus until the introduction of
molecular methods, enteric viruses were mainly identified by electron microscopy (EM)
26. 19_Chigor
including solid-phase immune electron microscopy (SPIEM). The SPIEM is more sensitive
than direct EM because, in the presence of specific antibodies, the virus particles are coated
with specific antibody and aggregated together, making them more easily distinguishable
from the background matrix. Many of the “small round viruses,” which include astroviruses,
noroviruses, sapoviruses, and parvoviruses, were first discovered through the use of EM.
Molecular methods are now the most commonly used techniques for the identification of
enteric viruses in foods, but other methods are also available for virus detection in human
specimens. Identification of enteric viruses can also be carried out by enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (RIA), and, for the culturable viruses,
culture-PCR, which is a combination of cell culture and polymerase chain reaction (PCR)
methods. The development of new molecular methods, including real-time PCR–based
methods, for the detection of non-culturable or difficult to culture viruses has shown their
frequent presence in the environment prior to the development of molecular methods, there
was limited knowledge about these viruses because their identification was difficult. The
inability to culture noroviruses coupled with the problems associated with identification of the
virus by EM restricted their detection for many years. Noroviruses are difficult to identify by
direct EM in faecal samples and foods because of their small size and the nature of the
background matrices. Immune electron microscopy (IEM) is frequently used to improve the
sensitivity of detection, but the antibody coating can mask the appearance of the virus. The
development of assays such as reverse transcription- polymerase chain reaction (RT-PCR)
has facilitated the detection and identification of these viruses, and consequently the role of
noroviruses in gastroenteritis outbreaks has been clarified. Noroviruses show great genetic
diversity, which has complicated their identification by molecular assays. To date, none of the
numerous norovirus primer sets designed have been able to detect 100% of known norovirus
27. 20_Chigor
strains, but some sets have been found to be more sensitive and have a broader detection
range than others (Vinje et al., 2003). Since its invention, PCR has become one of the most
widely used biochemical assays. The speed, specificity and low cost of the procedure has led
to its use in such fields as criminal and pathological forensics, genetic mapping, disease
diagnosis, systematics and evolutionary studies, and environmental science. PCR can be used
to amplify, to detectable levels, nucleic acids associated with pathogens that may be present
in low numbers in water samples. PCR assays must be able to detect viruses after
concentration from large volumes (100 to 1,500 litres) of water. This is usually accomplished
by a filter-adsorption and elution method, resulting in a concentrate containing microbes, and
organic and dissolved solids. Compounds, such as humic substances, once concentrated, can
interfere with the activity of the enzymes used in PCR assay. PCR is a process in which target
DNA, polymerase enzyme and the DNA subunits are combined in a test tube and subjected to
the temperature changes needed for the DNA duplication to occur. By repeating this process
many times, a large amount of DNA is generated. This reaction, termed the Polymerase
Chain Reaction (Mullis et al., 1987, Saiki et al., 1988), or PCR, can, under ideal conditions,
generate millions of copies of a single DNA molecule in just 20 to 30 repetitions of the
temperature cycle - each cycle requiring only a few minutes. The PCR assay can selectively
amplify only a portion of the target DNA for diagnostic applications. PCR, however, did
reveal a greater level of viral contamination than did the cell culture assay. This could be due
to the greater sensitivity of the PCR method for the detection of viruses in water samples, the
ability of PCR to detect a wider variety of viruses than the cell culture method, and the
possibility that PCR detected non-infectious viral nucleic acids. Conversely, RT-PCR is
potentially much more sensitive assay for virus detection, in that it is possible to detect as
little as a single molecule of RNA. The technique can detect less than one PFU of a virus
28. 21_Chigor
(since some virus particles may not be infectious), and PCR can detect both infectious and
non-infectious viruses. A single RT-PCR virus assay can easily be accomplished in two to
three days, including confirmation, and costs less than $200.00. This price includes the cost
of a large volume 1MDS filter, elution, and concentration. A single RT-PCR reaction costs
less than $20.00. Given its increased sensitivity and ability to detect an intact virus particle
(Abbaszadegan et al. 1997), PCR analysis would be expected to reveal more positive results
than cell culture analysis. Since either cell culture analysis or PCR can only reveal a
“snapshot” of the quality of the groundwater being sampled, PCR would be a desirable rapid
initial screening tool, in that the presence of even non-infectious or non-intact viruses would
suggest that a groundwater supply may be subject to contamination. While the detection of
viral RNA does not show an infectious level of contamination, the presence of viral RNA
does suggest a source of viral contamination and thus the potential for health risk. The most
sensitive method of detection would be the most desirable, even without the ability to confirm
infectivity of the sample contamination. The latest methods to be used in detecting human
enteric viruses in water are based on the polymerase chain reaction (PCR) (Schwab et al.,
1993), nucleic acid hybridization (Margolin et al., 1989) and immunological methods (Kfir
and Genthe, 1993). The use of nucleic acid probes and PCR to detect viruses in water has
several limitations (Alvarez, et al., 1993). The use of these methods requires high levels of
technical skill, knowledge and expensive reagents which are prohibitive in most laboratories
(Alvarez et al., 1993). Molecular detection approaches such as PCR or RT-PCR are normally
employed for fastidious virus analysis. However, they are unable to differentiate between
infectious and non-infectious particles (Kopecka et al., 1993; American Public Health
Association, 1998) and are, therefore, unsuitable for virus persistence studies, even when
quantitative real-time procedures are employed. Although reports on the presence of
29. 22_Chigor
norovirus sequences in bottled mineral water raised a lot of concern (Beuret et al., 2002),
many authors have shown the lack of correlation between virus persistence and molecular
detection of virus genomes. It now seems obvious that infectious particles are degraded more
rapidly than virus genomes. Reverse transcriptase PCR (RT-PCR) involves a step in which
the viral RNA genome is reverse transcribed to a complementary DNA strand (cDNA) prior
to the PCR and has been successfully used to monitor water for enteric RNA viral
contamination. Over conventional (qualitative) RT-PCR, real-time RT-PCR has the
advantage of enabling sensitive and rapid determination of concentrations of viral pathogens
in environmental samples. A major limitation of the PCR assays is their inability to determine
the viability and infectivity of viruses detected, as the presence of viral nucleic acid does not
necessarily indicate the presence of infectious viruses. However, a recent study has
demonstrated a statistical correlation between genome copy numbers and infectious enteric
viral particles in wastewater samples and proposed that a cut off value of 200 genome copies
could be used to indicate viral survival in environmental monitoring. The use of propidium
monoazide in RT-PCR (PMA-RT-PCR) has also been shown to be effective for
distinguishing between infectious and non-infectious enteric RNA viruses in water samples
despite the advantages, molecular techniques are subject to three main limitations. First, PCR
methods assay smaller volumes than culture methods, resulting in lower detection limits.
Second, these methods are sensitive to inhibitors that are present in some environmental
samples; to address this problem, controls are used to determine whether negative results are
true negative or false negative values. Finally, molecular methods do not distinguish between
infectious and non-infectious viruses; therefore, a positive PCR assay for a particular
pathogen in drinking water indicates the presence of viral nucleic acid, and does not directly
address issues of public health. PCR detection can be sensitive and specific. The efficiency of
30. 23_Chigor
viral amplification from environmental samples by PCR is influenced by the ability to recover
the virus from the environmental matrix and the purity of the recovered nucleic acid (Metcalf
et al., 1995). Traditionally, PCR gives a positive or negative result, however real-time PCR
can quantify the amount of virus in the sample. Other advantages include a smaller time
frame to obtain results because an agarose gel is not necessary and a closed system which is
less likely to be contaminated (Fong and Lipp, 54 2005). Like traditional PCR, real-time PCR
does not indicate infectivity. PCR may detect non-infectious virus particles and thus is
currently unable to provide information on the public health significance of such analysis.
Molecular techniques, such as PCR-based methods, are commonly used to detect and identify
viral contamination in water, particularly those viruses that do not multiply easily in cell
culture. PCR alone does not allow the discrimination between infectious and non-infectious
viral particles.
31. 24_Chigor
INTEGRATED CELL CULTURE POLYMERASE CHAIN REACTION
The Integrated Cell Culture with PCR or qPCR (ICC-PCR) is a method that combines the
high sensitivity of cell culture with the high specificity of PCR together and avoids the
shortcomings of low specificity and long testing period of cell culture (Li et al., 2002). ICC-PCR
method has still the possibility to detect nucleic acids of inactivated viruses from environmental
samples simply adsorbed onto cell receptors without cell infection resulting in false positives
infectious data (Sobsey et al., 1988). Therefore, other strategies are required to confirm
infectious viruses by assaying infection of the permissive cells; this can be based on the use of
viral mRNA transcribed into infected cells as RT-PCR templates (ICC-RT-PCR). Thus, the
detection of viral mRNA in cell culture indicates the presence of infectious viral particles;
specificity and sensitivity are also important aspects to consider, as the ICC-RT-qPCR relies on
mRNA and thus avoids false negatives or positives (Ko, Cro- means & Sobsey et al., 2003;
Rigotto et al., 2010). Studies emphasize the importance of using ICC-RT-PCR when is
necessary to measure infectious pathogens, explaining that this technique is safe and accurate
(Ko, Cromeans & Sobsey, 2003). However, the majority of studies using ICC-RT-qPCR attempt
to estimate the viral infectivity of artificially contaminated samples, but rarely employ such a
technique to evaluate virus from environmental samples (Gal- lagher & Margolin, 2007;
Lambertini et al., 2010). Compared with ICC-RT-PCR used for virus detection, PCR has several
advantages: the time required for this test can be reduced from days or weeks to hours, costs for
implementing this technique are substantially smaller, besides being a methodology easy to
perform and has high specificity and sensitivity (Nu- anualsuwan & Cliver, 2002; Carducci et al.,
2003). In addition, this method facilitates the identification of fastidious pathogenous viruses that
do not grow well in cell culture assays, such as rotavirus, calicivirus, adenoviruses and some
HAV, and extends information previously available for enterovirus, which shows good growth in
32. 25_Chigor
culture cells (Wynn-Jones & Sellwwod, 2001; Carducci et al. 2003, Girones, et al. 2010). The
use of the assay of ICC-RT-PCR or qPCR was reported as a rapid and accurate for detection of
HAV in environmental monitoring (Gallagher & Margolin 2007; Rigotto et al., 2010; Fongaro et
al., 2013).
Culture-PCR detects both infectious and non-infectious viruses. PCR only detects the infectious
virus. Molecular techniques, including culture-PCR, have become the method of choice for
detection of virus in nonhuman samples, PCR-based methods currently detect both infectious
and non-infectious viruses and are not able to determine viral infectivity, which is the key factor
when assessing human health risks from food-borne pathogens. It is important that data
generated solely from molecular-based assays is judiciously interpreted when studying these
viruses. Use of cell culture combined with PCR methods (culture-PCR) can overcome some of
these problems for those viruses that are difficult to grow. Unfortunately, the infectivity status of
the main food-borne viral pathogen, norovirus, still cannot be determined by in vitro methods.
This has limited our knowledge of the natural history and biological properties of this pathogen
and has also slowed progress in the development of effective control and intervention strategies.
The advantages of PCR are numerous. When compared with techniques such as cell culture for
the detection of viruses, the time required for the assay can be reduced from days or weeks to
hours. Both the initial and recurring costs for PCR are much less than cell culture techniques and
the technique is easily performed. Additionally, PCR can be used to identify a specific pathogen
found in water. Standard PCR cannot, however, be used to detect the infectious state of an
organism only the presence or absence of pathogen-specific DNA or RNA. PCR assays have
been applied to the detection of enteroviruses and other pathogens in clinical (Rotbart, 1990) and
environmental samples (Abbaszadegan et al., 1993 & 1999; Pillai et al., 1991). Some health
significant enteric viruses, such as rotavirus, astrovirus, and enteric adenovirus, replicate poorly
33. 26_Chigor
in cell cultures; yet their persistence may be evaluated by integrated cell culture RT-PCR assays
(Pintó et al., 1995; Reynolds et al., 1996; Abad et al., 1997; Reynolds et al., 2001). For this
purpose, cells supporting the propagation of a wide variety of enteric viruses, such as CaCo-2
(colonic carcinoma) or PLC/PRF/5 cells (human liver hepatoma), are used for an in vivo
amplification step prior to molecular amplification (Grabow et al., 1993; Pintó et al., 1994). A
new method of virus detection utilizing cultural and molecular techniques, known as the
integrated cell culture- polymerase chain reaction (ICC-PCR), provides a more rapid and
sensitive means for isolating low levels of infective virus, including elusive strains that do not
produce CPE. ICC/PCR has recently been used to detect viruses after exposure to chlorine
disinfectant. This method detected viruses after eight minutes of chlorine treatment, four times
longer than the recommended exposure time of two minutes based on cell culture analysis alone.
This has serious implications since the determination of inactivation rates of waterborne virus is
crucial to the drinking water industry. This phenomenon could help to explain why infectious
viruses have been detected in drinking water receiving what was believed to be adequate
disinfecting. Currently, the University of Arizona and County Sanitation Districts of Los Angeles
County are conducting a collaborative study to assess potential virus contamination in: 1) water
reclamation plant disinfected tertiary effluents used for recharge, 2) groundwater monitoring
wells, 3) sites impacted by reclaimed water, and 4) sites not exposed to reclaimed water to
determine the safety of groundwater recharge practices using the ICC/PCR methodology
Alarmingly, conventional methods of virus growth and isolation were not effective in detecting
these certain virus populations. Instead, ICC/PCR was needed to determine the presence of these
infectious agents. The fact that these viruses survive extensive disinfecting practices causes
concern over whether these organisms are more resistant to disinfectants or perhaps altered by
the disinfecting process. Certainly, evidence suggests that researchers may be overestimating the
34. 27_Chigor
effectiveness of their disinfecting procedures while underestimating the survival and transport
capabilities of the elusive virus populations. Development of an integrated cell culture-
polymerase chain reaction (ICC-PCR) assay has allowed detection of viruses that are under
detected and undetected by the plaque assay. Integrated cell culture-PCR (ICC-PCR) overcomes
the individual disadvantages of cell culture and PCR. Additionally, ICC-PCR permits evaluation
of a much larger percentage of the original sample as compared to traditional PCR. Several
studies have considered the sensitivity, efficiency, and ease of ICC-PCR and found it be better
than either traditional PCR or cell culture methods alone (Blackmer et al., 2000; Chapron et 71
al., 2000; Greening et al., 2002; Jiang et al., 2004; Ko et al., 72 2003; Lee and Jeong, 2004; Lee
et al., 2005; Reynolds et al., 73 1997, 1996, 2001). A combined cell culture/PCR method enables
rapid and specific detection of viable viruses with greater sensitivity than either method alone.
The integrated approach was more sensitive than direct PCR for the detection of enteroviruses in
highly inhibitory water concentrates. The integrated cell culture/RT-PCR approach was up to
five times more rapid at detecting enteroviruses in water samples. The integrated technology
eliminated common problems of PCR inhibition and enabled rapid examination of larger
equivalent sample volumes. ICC/PCR was capable of detecting elusive virus populations
following chlorine disinfection that first passage cell culture falsely determines absent.
Molecular sequencing methodology was effective and important for definitive identification of
specific human virus strains. A combination of cell culture and PCR has allowed detection of
infectious viruses that grow slowly or fail to produce cytopathic effects (CPE) in cell culture.
Integrated cell culture PCR (ICC-PCR) has the benefits of cell culture coupled to PCR and
attempts to compensate for several cell culture disadvantages, such as time-consuming and
limited detection sensitivity. The integrated cell culture-PCR (ICC-PCR) method has been
suggested as an improved method for detection of viruses in water environments. A study carried
35. 28_Chigor
out by testing 57 source waters including finished water samples in Gyeonggido for enteric viral
contamination using total cultural virus assay (TCVA) using BGMK cells and ICC-PCR.
Nineteen of the 57 source water samples (33.3%) exhibited the cytopathic effect (CPE) on
BGMK cells and no finished water did exhibited CPE. Nineteen samples (33.3%) of the 57 were
positive for reoviruses. For the enteroviruses, only 3 samples (5.3%) of the 57 samples showed
positive results. By using ICC-PCR method, 202 flasks from source water samples were positive
for enteroviruses and reoviruses. Three samples from source water were positive for both
viruses. However, any flasks tested was not co-infected with two types of viruses. While the
enteric viral frequencies in TCVA and ICC-PCR were similar, the viral frequency for reoviruses
at first passage in two type of method was higher in ICC-PCR (94.7%) than TCVA (56.9%).
NEED FOR STORAGE
Storage of water at refrigerated temperatures (4o
C) is a common approach to prolong the
viability of an enteric viruses in water, also, it has been discovered that the prolong viability and
survival of enteric virus in water at 4o
c is relative to storage at room temperature. For example,
Bidawid et al found approximately a two-fold reduction in hepatitis A virus titre on lettuce stored
36. 29_Chigor
at 4o
C for 12 days compared to a 10,000-fold reduction when the lettuce was stored at room
temperature. In a field survey of enteric viruses, water samples collected sometimes need to be
stored for a long duration before analysis is performed. Three types of sample storage methods
were evaluated using MilliQ water, pond water, and treated sewage inoculated with poliovirus
and norovirus: (i) storage followed by the full concentration procedure, (ii) filtration and storage
followed by the remaining concentration procedure, and (iii) the full concentration procedure
before storage. Among the three methods tested, the method of storing the eluted samples was
judged to be most appropriate for detection of viruses from water samples. This method does not
require any special equipment and can be easily adopted in field surveys, especially in
developing countries. In a field survey of enteric viruses, the water samples are supposed to be
transported to a laboratory as soon as possible after collection, followed by procedures including
concentration, detection, and identification of viruses. However, because of the limited
availability of laboratories, water samples sometimes need to be stored for several days or even
longer. This is particularly true for developing countries. Currently, no storage method has been
established for detecting viruses in water samples at remote locations on the other hand, no clear
difference in Poliovirus recovery was found between the two storage methods when the samples
from the treated sewage were stored at 4o
C. It is therefore suggested that the temperature of
sample storage is an important factor in degradation of Poliovirus recovery, and that the method
of storing the eluted samples can usually be the most appropriate method of retaining higher
virus recovery. One advantage of the method of storing the eluted samples is that it could remove
coexisting substances in the original water sample during the concentration procedures; thus, the
eluted sample can be subjected to long-term storage. If the sample storage was performed at a
cool temperature, an increase in virus Recovery is usually recorded, as observed in the
experiments using treated sewage. Considering that the samples were not always stored at a cool
37. 30_Chigor
temperature during transportation, the method of storing the eluted samples after filtration and
elution was judged to be most appropriate for detecting enteric viruses from water samples. The
sample storage method developed in this study of Poliovirus does not require any special
equipment and can be easily adopted in field surveys conducted away from laboratories. This
method has been applied to a field survey in Jakarta, Indonesia, where Noroviruses (GI and GII),
enteroviruses, adenoviruses, and the hepatitis A virus were successfully detected in
environmental water samples concentrated on-site and analysed in a laboratory in Japan. The
occurrence of viruses has also been investigated in other countries such as Cambodia, China, and
Vietnam. The samples were concentrated followed by storage at -20o
C until use. Norovirus in
the environmental condition are stable and can survive under power of hydrogen (pH, 3-10) for
prolong time at low temperatures. There was a case were 135 tap water samples were collected
from different areas of Lahore and Islamabad in two litres autoclaved sterilized bottles, pH and
temperature were calculated at the time of sample collection, and samples were stored at room
temperature before the final detection process. Temperature is one of the physical factors best
recognized to play a role in virus stability in water. Virus survival can be prolonged on foods
stored at 4o
C, as indicated by the detection of infectious poliovirus on celery that had been
irrigated with virus seeded wastewater for more than 2 months. The level of persistence varies
with the vegetable matrix. No decline in infectious poliovirus titre was observed after two weeks
at 4°C on green onion or fresh raspberries, but a ten-fold reduction was observed after 11.6 days
on lettuce, 14.2 days on white cabbage and 8.4 days on frozen strawberries. Higher temperatures,
such as those achieved in cooking or pasteurization, increase the rate of virus inactivation.
However, certain constituents or additives in foods may stabilize the virus, protecting it from
inactivation. For example, heat inactivation of HAV is less efficient in dairy products containing
a greater fractional content of fat (e.g., cream) compared to products with less fat (e.g., skim
38. 31_Chigor
milk), and a higher fat content in ground beef decreases the thermal inactivation of poliovirus.
Higher sucrose concentrations, such as those used as stabilizers in some fruit-based products,
increase HAV resistance to heat inactivation in strawberries. On the other hand, higher acidity
can increase viral susceptibility to heat inactivation. Rotavirus infectivity was stable over 3 days
at 4°C in a stored water sample. A number of physical, chemical and biological factors influence
the rate of virus inactivation in the environment which subsequently reduces the number of virus
gotten from a stored water sample. Exposure to higher temperatures (heat), ultraviolet light,
lower relative humidity, high pressure and radiation are physical factors that all contribute to loss
of virus infectivity. Viral infectivity declined more rapidly when the water were stored at a higher
temperature (18°C).The length of virus survival appears to be temperature dependent and is
inversely related to increased temperature. The enteric viruses may survive longer if attached to
particulate matter or sediments, where they can present a greater potential risk to human health
(Jaykus et al., 1994). Several enteroviruses have been reported to survive during storage for
periods of up to 5 weeks in water storage, temperature is usually at −80 °C until further analysis.
Water for the detection of hepatitis A virus is stored for 90 days at -20 °C, this is due to the
prolonged virus survival and viability as well as its retention of infectivity in the water samples.
A study carried out on Rotavirus showed that Rotaviruses do not show the same tolerance to
extreme conditions as other enteric viruses, although they are stable in the environment and can
be stored for several months at 4°C or even 20°C. They are resistant to drying and may survive
on fomites and surfaces. Heating at 50°C for 30min reduces their infectivity by 99%,and
infectivity is rapidly lost at pH <3.0 and >10.0.
39. 32_Chigor
CONCLUSION
Various research journals reviewed in the process of writing on this seminar topic has
shown that in other to detect enteric viruses contamination of water in developing countries,
more water samples must be collected from developing countries. The water samples are
supposed to be transported to a laboratory as soon as possible after collection, followed by
procedures including concentration, detection, and identification of enteric viruses. However,
because of the limited availability of laboratories, expensive equipment, and sometimes the
unavailable technical skills, water samples sometimes need to be stored for several days or even
longer and subsequent transportation to place with the required equipment, this is particularly
true for developing countries. This has undoubtedly led to few studies available on detection of
enteric viruses in water samples in developing countries as opposed to many studies conducted
mainly in developed countries. Enteric viruses can be transmitted by various routes, including
direct and indirect contact, vector transmission, and vehicle transmission. Adsorption-elution
technique has been the method of choice when it comes to enteric viruses concentration from
water samples this is because it is simple, fast, cheap and comes with less virus specificity. The
detection of enteric viruses in water samples requires various methods such as the cell culture,
the molecular techniques and the integrated cell culture polymerase chain reaction. The cell
culture has the advantage of detecting if the enteric viruses is infectious or not, its disadvantages
are wastage of time which could be days to weeks, expensive. The molecular techniques is
cheap, faster but can’t detect if the enteric viruses is infectious or not. The two methods above
still do not resolve the problem associated with enteric viruses detection completely as not all
enteric viruses can be cultivated using the cell culture, also those detected by the molecular
methods still do not tell us if they are infectious or not hence, the need for the integrated cell
culture polymerase chain reaction which is known to combine the advantages of both the cell
41. 34_Chigor
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