Chapter 22
Viruses, Food and Environment
Gary Grohmann and Alvin Lee
Introduction
Characteristics of foodborne viruses
Noroviruses
Taxonomy
Diagnosis and detection
Epidemiology and pathogenesis
Enteric hepatitis viruses
Taxonomy
Diagnosis and detection
Epidemiology and pathogenesis
Astroviruses
Taxonomy
Diagnosis and detection
Epidemiology and pathogenesis
Rotaviruses
Parvo-like viruses
Enteroviruses
Tick-borne encephalitis virus
Other viruses
Detection in food and water
Detection of RNA viruses using cell culture techniques
Detection of RNA viruses using PCR
Limitations of PCR for food and water samples
Interpretation of PCR results
Quantitative RT-PCR
Improving sensitivity and specificity
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Gary Grohmann and Alvin Lee
Control
Food handlers
Environmental control
Shellfish
Fresh produce
Viral inactivation by non-thermal processing techniques
Challenges for the future
References
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Viruses, Food and Environment
Introduction
Enteric viruses such as hepatitis A virus,
noroviruses (formerly Norwalk-like viruses),
enteroviruses,
astroviruses,
adenoviruses,
rotaviruses and hepatitis E virus have all been
implicated in food and/or waterborne outbreaks of
illness. Data from the USA, Europe, Japan and
the UK shows that viruses are responsible for the
majority of foodborne outbreaks with noroviruses
being the most common cause of disease (38, 44,
58, 59, 76, 77, 88). Moreover, there have been
many anecdotal reports in ProMed during
2001–2002 that have highlighted consecutive
outbreaks of gastroenteritis due to noroviruses in
hospital settings in the UK and Ireland, and in
various other settings elsewhere including daycare settings, homes for the elderly, cruise ships
and restaurants. Increased sporadic activity of
noroviruses in the community has also been
observed in most parts of the world. Hepatitis A
has also commonly been reported with recent
ProMed reports highlighting outbreaks in New
Zealand due to blueberries that were contaminated
by sewage affected groundwater.
The epidemiological evidence for the
transmission of viral disease via the food and
water route is best defined with hepatitis A virus,
noroviruses and enteroviruses, since infections
result in a specific illness and outbreaks tend to
be linked to a point source or vehicle of
transmission. Outbreaks of food and waterborne
hepatitis A still occur despite increasing
standards of hygiene and sanitation. In the
United States the number of waterborne hepatitis
A outbreaks has not declined since 1960 (11, 12,
15). Since 1970, many outbreaks of food and
waterborne gastroenteritis have been identified
as due to noroviruses, as techniques for their
identification improve and awareness of their
public health importance increases. However,
despite advances in detection techniques and the
subsequent identification of many new strains
and diverse genetic clusters, there are still
difficulties in discerning viral outbreaks of illness
via contaminated food and water. Some of the
reasons for this include:
1. The occurrence of subclinical infection.
2. The presentation of a variety of clinical
symptoms associated with norovirus and
enterovirus infections; so without virus
identification from environmental samples
and/or patient specimens it is impossible to
clearly associate an outbreak of illness to
these viral agents.
3. The occurrence of secondary and tertiary
person-to-person transmission which may
mask a food- or water-borne source of
transmission.
Sometimes outbreaks due to food, shellfish
and contaminated water can be sudden and
dramatic: e.g. at least 100 000 persons were affected
in waterborne outbreaks of hepatitis E virus in
Mexico, India and the former Soviet Union (28);
over 100 000 persons contracted hepatitis A from
contaminated clams in China (57), and over 4700
persons in Japan contracted foodborne gastroenteritis due to astrovirus (96).
Enteric viruses can survive for long periods in
food and water and are often detected in the
absence of indicator bacteria. They are generally
more resistant to chemical and UV disinfection,
filtration and pasteurisation than bacterial
indicators but may be removed by ultrafiltration
membranes or inactivated by prolonged heating
or optimal UV treatment. In general, these
viruses will survive reasonably well in adverse
conditions, e.g. heating to 50°C, pH 2.7–9.6,
microbial proteolysis and fermentation. Specific
data on virus survival and stability in foods have
been previously reviewed and summarised (35).
The entry and survival of viruses in the
environment is well documented. They are
difficult to eliminate in sewerage treatment
processes. They may survive post-treatment UV
and chlorination, and be discharged from
treatment plants into surface and ground waters.
Viruses are protected in the environment by their
association with faecal matter and particulates.
They move freely through the environment
surviving in waterways, sludge, sediment, soil,
shellfish and on crops which have been irrigated
with recycled effluent thus potentially infecting
humans. In laboratory experiments, enteric
viruses have survived in tap water, seawater, soil
and oysters for periods of three months or more.
They can adsorb to sediment and particulates in
raw and estuarine waters and soil and may be
released under certain conditions (e.g. presence of
cations, soluble organics, pH and soil type). After
a number of adsorption/elution events, viruses
can migrate long distances in soil and waterways.
Viruses can also survive on crops and in prepared
or processed foods but are susceptible to heating,
drying out and the effect of natural UV light
(1, 10, 21, 24, 28).
The origin of viral pathogens in foodborne
outbreaks is either from food handlers (involving
faecal-oral and aerosol spread of faecal material
and vomitus) or from sewage contaminated food
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Gary Grohmann and Alvin Lee
and water. In general, water used in the food
industry is strictly controlled and only water of
potable quality is permitted. However, an
outbreak of norovirus gastroenteritis on domestic
air flights in Australia was due to orange juice
which apparently used a contaminated ‘potable’
water source (105).
Shellfish present a particular problem as they
are often affected by sewage discharges. They
have been shown to accumulate viruses and have
been clearly associated with many outbreaks of
hepatitis and viral gastroenteritis. Some 2–3
outbreaks of gastroenteritis per year have been
reported in Australia alone, often due to
noroviruses. Recently, the first documented
Australian oyster associated hepatitis A outbreak
occurred and was traced to oysters harvested in
Wallis Lake in NSW (26).
Shellfish can filter some 10–20 L per hour of
water under ideal conditions but also concentrate
infectious agents that are present in the marine
environment. Being bivalve molluscs they feed
and respire by inducing a current of water to flow
over a series of complex gill structures, and
capture suspended particulate matter passing it
towards the mouth where it may be ingested or
rejected as pseudo-faeces. Oysters are very
effective filter feeders capable of concentrating
viruses that may be present in water, resulting in
viral concentrations within the oyster far
exceeding those of the surrounding water. In
unpolluted estuarine environments there will be
little risk to consumers. However, if the environment is polluted, oysters can act as a vehicle of
transmission for viral disease (26, 51, 93, 114).
The use of recycled effluent and sludge is also
a matter of public health concern as infectious
viruses entering the sewerage system can
ultimately contaminate water and the
environment affecting crops, soil, the water table
and shellfish. Recent hepatitis A outbreaks
involving oysters in Australia and strawberries in
the USA, may have been partly due to
contamination by recycled effluent (16, 26, 94).
Until recently it has not been possible to
routinely detect viruses in food and water, but
nucleic acid amplification techniques, such as
polymerase chain reaction (PCR), have now been
developed to detect all the enteric viruses (2, 8, 9,
37, 81, 82, 112). Given their low infectious dose,
the fact that they survive well in the environment
and on surfaces, and their poor correlation with
indicator bacteria, assessment of water and food
for these pathogens will become necessary in
industries involving potentially contaminated
water or shellfish and also in investigations of
food and water associated outbreaks of disease.
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Characteristics of foodborne viruses
Over 150 types of enteric viruses falling into five
major viral families are potentially present in
faecal material and sewage effluent, depending on
the season of the year and the viruses circulating
in the community. They may cause a variety of
diseases in humans ranging from skin, eye and
respiratory infections to fever, meningitis,
myalgia, hepatitis and gastroenteritis, but many
viral infections are silent or asymptomatic. Some
of the characteristics of typical foodborne viruses
are shown in Table 22.1.
Noroviruses, formerly Norwalk-like or
small round structured viruses (SRSV)
Taxonomy. These ssRNA viruses have been
classified in the family Caliciviridae as their
capsid contains a single polypeptide of 59 000 MW.
During 1993 the complete nucleotide sequences of
Norwalk virus and Southampton virus were
published, leading to their final classification
within the Caliciviridae even though examination
by electron microscopy shows that they lack
Caliciviridae’s typical cup-shaped morphology.
Little is known of the replication strategy of these
viruses. A possible subgenomic mRNA has been
identified in faeces (65), and studies using cellfree expression systems have been successful in
expressing capsids which have been useful for
serological studies (64). The noroviruses are a
group of 27–35 nm particles that lack a distinct
structure when visualised by electron microscopy,
hence they are were originally named small round
structured viruses or SRSV. Norwalk virus is the
prototype of this group. Three genotypes have now
been described which can be divided further into
at least 17 distinct genetic clusters. Genotypes G1
and GII commonly infect humans and can cocirculate (39, 44) although GII types have predominated since the 1990s. Distinct, well characterised
serotypes in the norovirus genus include: Norwalk
virus, Desert Shield virus, Lordsdale virus,
Mexico virus, Hawaii virus, Snow Mountain virus
and Southampton virus. A second genus, the
Sapovirus genus, forms a third genogroup, G3,
which is also now been identified within the
Caliciviridae. Well-characterised and distinct
viruses include: Saporovirus, Houston/86,
Houston/90, London 29845, Manchester virus and
Parkville virus.
Diagnosis and detection. Progress in the
diagnosis of these viruses has been slow following
their detection by electron microscopy in 1972 for
several reasons; they have not been isolated in cell
culture, there are no readily available animal
models and there are no serological reagents for
the detection of antigenically diverse strains.
Viruses, Food and Environment
The diagnosis of a norovirus infection is based
on the clinical signs, and the detection of viral
antigen or nucleic acid, or seroconversion by the
patient. Molecular tests, such as PCR, are now
commonly used to detect noroviruses using
various primer sets (6–9, 39, 81, 82).
Epidemiology and pathogenesis. These
viruses cause acute gastroenteritis in humans
worldwide and commonly cause epidemic viral
gastroenteritis in all age groups. The onset of
vomiting and/or diarrhoea is sudden, accompanied
by anorexia, headache, abdominal discomfort,
nausea, and low grade fever, often in several
different combinations (50). The incubation period
is approximately 24 h and recovery from illness is
usually complete within 72 h. Occasionally
symptoms can be very severe sometimes leading
to hospitalisation and often leading to working
days being lost. The mortality rate is very low in
industrialised countries.
Outbreaks usually last about 7–10 d,
although longer epidemics involving weeks or
months have been recorded (54). Outbreaks are
often confined to families or closed communities
such as schools, restaurants, hospital wards,
nursing homes, caravan parks, cruise ships or the
military (17, 18, 56, 59, 60, 61, 68, 72, 88, 90). The
viruses are spread via the faecal-oral route and
aerosolised vomitus. Identified sources of
outbreaks include contaminated water, ice,
shellfish and food contaminated via food handlers
such as salads, fruit, shrimp, school lunches and
bakery products (5, 11, 31, 38, 48, 50, 68, 71-75,
79). Outbreaks of illness in Australia attributable
to these viruses have also been recorded, the first
foodborne outbreak occurring in 1978, caused by
consumption of oysters from the Georges River in
Sydney (50, 51, 85, 93). Other outbreaks in
Australia due to enteric viruses have involved
contaminated bore water (92), drinking water
(90), orange juice (affecting over 4000 persons)
(105), and septic tank contact (20). However, many
more outbreaks remain undocumented because of
the difficulties in reporting and diagnosis.
Enteric hepatitis viruses
Two different viruses have been clearly associated
with food and waterborne outbreaks; hepatitis A
virus and hepatitis E virus.
Table 22.1. Some characteristics of common foodborne viruses
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Gary Grohmann and Alvin Lee
Taxonomy. Hepatitis A virus is 28–30 nm in
diameter and a member of a separate genus,
Hepatovirus, within the family Picornaviridae.
Hepatitis E virus, is 32 nm in diameter with some
similarity to the Caliciviridae in genome
organisation and morphology however, it also has
amino acid similarity in its replicative enzymes
with the Togaviridae and its classification is still
under review.
Diagnosis and detection. Illness caused by
hepatitis A or hepatitis E viruses is not clinically
distinguishable (28). Illness may last several
weeks and usually includes malaise, nausea,
jaundice, anorexia and vomiting with an abrupt
onset. Neither virus can be easily cultured
although several laboratory strains of hepatitis A
virus have been cultured. Primer sets are
available for PCR tests, which can be used to
detect these viruses in food and water or clinical
specimens. The detection of IgM in patients is
diagnostic. Hepatitis A virus is very much more
resistant to drying and heating than other
picornaviruses and is also more resistant to pH 2,
gamma rays, UV light and low levels of chlorine
and ozone. Unlike other enteric viruses, hepatitis
E virus is extremely labile and easily inactivated
by proteolysis, freeze-thawing, heating and
ultracentrifugation.
Epidemiology and pathogenesis. Hepatitis A
virus enters the body by ingestion and multiplies
in intestinal epithelial cells followed by a
viraemia. The virus then infects the parenchymal
cells in the liver and the host’s immune response
destroys infected hepatocytes via cytotoxic T cells.
Immunity is life long although relapses have been
recorded and death is very rare. Only one serotype
of hepatitis A and E viruses exist and both are
likely to cause extensive subclinical infection in
childhood. The incubation period for hepatitis A
averages 28–30 days and for hepatitis E, 40 days.
Both viruses are predominately shed during the
incubation period with hepatitis A virus being
shed in low numbers for up to two weeks after
onset of illness. Both viruses can cause a more
severe illness in pregnant women resulting in a
high mortality rate – 17% and 33% for hepatitis A
virus and hepatitis E virus respectively. The
pathogenesis of hepatitis E is probably similar to
that of hepatitis A based on studies carried out in
primates (28).
Both viruses are spread via the faecal-oral
route. Direct person-to-person contact is the most
common method of transmission of these viruses
in the community with contaminated food and
water playing a role, particularly in developing
nations. Clinical cases are generally seen in young
adults or children.
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Hepatitis A can be a particular problem in
unsewered areas, in lower socioeconomic groups
and in persons with high-risk behaviour patterns
such as male homosexuals and intravenous drug
users. Persons travelling through developing
countries are also at greater risk. Outbreaks often
occur in communities having a poor or marginal
level of hygiene such as day-care centres, homes
for the mentally retarded, prisons, mental
hospitals, army camps, etc. Most outbreaks
involve specific foods (15, 43, 94, 99) or shellfish
(22–27).
Shellfish have been regularly involved in
hepatitis A outbreaks in the USA and Europe and
from January to March 1997, outbreaks of
hepatitis A occurred throughout Australia which
were associated with oysters grown in the Wallis
Lake Area in NSW (26). In this outbreak over 70%
of cases were oyster associated and with
subsequent secondary spread affecting at least
460 persons. Moreover, oysters met all
bacteriological standards and had undergone
depuration (purification). This outbreak was
unusual in that hepatitis A from contaminated
oysters had not occurred in Australia previously.
The fact that hepatitis A cases are increasing in
both the USA and Australia indicates that this
virus may well be a re-emerging disease in the
community putting seronegative and immunocompromised individuals at greatest risk.
Hepatitis E is now the most frequent cause of
hepatitis in Asia (28) and must be considered a
potential emerging viral disease for Australia.
Both hepatitis A and E tend to be most prevalent
in autumn in Asia often following extensive rain
or flood. Waterborne outbreaks of hepatitis E are
common in the Asian/Indian area as well as in
Mexico (28). Surprisingly, no documented
outbreaks of foodborne hepatitis E have been
reported, the virus being mainly transmitted by
the water route and person-to-person spread.
Astroviruses
Astroviruses affect a wide range of animals and
cause gastroenteritis in humans. They appear to
be ubiquitous in young children and often affect
the elderly. They emerged as a potentially
significant foodborne pathogen when over 4700
persons were infected by astrovirus in schools in
Osaka, Japan (96).
Taxonomy. Astroviruses are 28 nm spherical
particles often exhibiting five- or six-pointed starlike patterns. They contain single stranded RNA
and are resistant to pH 3 and heating to 50°C for
30 min and 60°C for 5 min. They belong to a new
family, the Astroviridae and eight serotypes of
human astrovirus are known.
Viruses, Food and Environment
Diagnosis and detection. Astrovirus gastroenteritis has an incubation period of 3–4 days
with diarrhoea being more typical than vomiting,
the clinical signs usually lasting 2–4 days. Viral
shedding in faeces usually follows the duration of
symptoms, however HIV positive patients with
astrovirus gastroenteritis may shed the virus
intermittently for months (53). The virus can be
isolated using CaCo-2 cells in the presence of
trypsin. Enzyme immunoassays and PCR tests
can also be used to detect the virus in faeces. PCR
can be used to detect the virus in food, water and
effluent samples.
Epidemiology and pathogenesis. Astroviruses
are transmitted by the faecal-oral route via
person-to-person spread and have a peak
prevalence in the winter months, but these
viruses are also likely to be transmitted by food
and water. Most infections in the community are
subclinical with 4–6% of infantile diarrhoea being
attributed to astroviruses. Outbreaks are common
in families, day-care centres, hospital wards and
nursing homes. These viruses replicate in the
small intestine destroying mature enterocytes on
the villi, which are regenerated after a few days.
Type specific antibody is produced with acquired
immunity being monotypic.
Rotaviruses
Rotaviruses are the most common cause of viral
gastroenteritis in children, particularly in
developing nations where they are responsible for
high morbidity and mortality rates and are often
associated with waterborne disease. They are
mainly transmitted by person-to-person spread
and sometimes by food handlers. Rotaviruses
belong to the Reoviridae and fall into three major
subgroups. Group A has at least 13 different
serotypes and like astroviruses, they infect
enterocytes in the small intestine causing cellular
damage, malabsorption and diarrhoea. Symptoms
include diarrhoea, vomiting and fever that may
last for 4–6 days. Viral shedding in faeces is
maximal for 8 d. The virus can survive for weeks
at 4°C but is less stable than other enteric viruses
in food being almost completely inactivated at
56°C for 30 min and unstable outside pH 3–10.
Parvo-like viruses
Small, 20–26 nm, featureless virus particles have
been regularly associated with outbreaks of
gastroenteritis, sometimes associated with
shellfish (5, 20). Because similar viruses are often
seen in stool specimens in conjunction with known
agents of gastrointestinal illness, such as
noroviruses, parvo-like viruses could be
commensals of humans or defective satellite
viruses requiring the genome of another enteric
virus to replicate. The role of these agents as a
cause of gastroenteritis remains uncertain.
Enteroviruses
Polioviruses, coxsackie viruses and echoviruses
are members of the enterovirus group. These
viruses are now rarely associated with foodborne
outbreaks despite the fact that they are often
cause subclinical infections in humans and are
excreted in faeces. Poliovirus was the first virus to
be associated with foodborne outbreaks of viral
disease but has now been almost eradicated by
vaccination. Coxsackie B viruses and echoviruses
have been occasionally detected in foodborne
outbreaks in the USSR and in the USA due to food
handlers contaminating foods (24). These viruses
are however commonly detected in sewage
effluent and contaminated water (52,78) and have
been detected in a variety of shellfish (26, 34). For
this reason, enteric viruses such as enteroviruses,
adenoviruses and reoviruses, all of which are
commonly detected in sewage effluent, may be
useful indicators of the presence of more common
viral pathogens such as hepatitis viruses and
noroviruses in shellfish or polluted waters. For
example, these viruses have been detected in
contaminated oysters and sediments where
noroviruses have been implicated (34). In the
recent Australian outbreak of oyster associated
hepatitis A at Wallis Lake, most oyster and
sediment samples were positive for enteroviruses
or adenoviruses rather than the aetiological
agent, hepatitis A virus (26).
Tick-borne encephalitis virus
Several viruses can in theory be transmitted via
milk. Dairy animals in Slovakia have been
reported to shed tick-borne encephalitis virus
after being bitten by infected ticks and
subsequently infecting humans (49). The problem
highlights the possibility of other zoonotic
infections from unpasteurised milk and milk
products and affirms the need for proper
pasteurisation to occur to protect the community
from new and emerging viruses. Also of
importance is the transmission of cytomegalovirus,
human immunodeficiency viruses and some
human T cell leukaemia viruses to children
through breastfeeding.
Other viruses
Enteric coronaviruses (4), adenoviruses, picobirnaviruses (53), herpesviruses and HIV are also
potentially excreted in faecal material but these
agents are not generally associated with
foodborne outbreaks of disease.
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Detection in food and water
Despite the development of diagnostic methods
for the detection of viral RNA or viruses from
water and various food matrices, there are no
routine standard methods available for use by
non-research laboratories (6–9, 41, 42, 46, 76,
81–84). Contaminated food products, in general,
may contain very low viral numbers making them
difficult to detect even by nucleic acid amplification
technologies. Although the average infectious
dose of enteric viruses such as the noroviruses or
hepatitis A virus is not known, it is believed to be
less then 100 virions. Therefore, methods used for
the concentration, isolation and detection of
enteric viruses would require a high degree of
sensitivity and specificity (25).
In general, viruses need to be extracted from
foods using blending techniques with appropriate
buffers and then concentrated by either
polyethylene glycol (PEG), AlCl3, iron oxide or
FeCl3 precipitation, organic flocculation using
beef extract or casein, or immobilisation by
zirconium hydroxide (22, 25, 29, 46, 63, 80–84).
The concentrate would then be resuspended in an
appropriate buffer and examined for viruses using
cell culture techniques and/or various molecular
biology techniques including PCR (8, 9, 30, 38,
39). Immunoassays are too insensitive for virus
detection in food (and water) but are useful in
detecting serum antibodies and viral antigens in
body fluids (47, 64). A model scheme for viruses
detection in food is shown in Figure 22.1.
The detection of viruses in water is more
advanced, with the primary concentration from
large volumes of 10–1000 L relying on flowthrough sampling techniques based on either:
1. Adsorption
to
filters,
iron
oxide,
polyelectrolytes etc. followed by elution,
or
2. Hollow-fibre ultrafiltration (HFU) and other
filtration methods where viruses are retained
based on size or molecular weight.
Using these techniques samples can be
reduced to 1 L or less. Samples are then
reconcentrated to 20–50 mL by precipitation as
outlined above, ultracentrifugation, organic
flocculation using beef extract or casein, or the
use of iron oxide. The final concentrate can be
examined for viruses by inoculation of cell
cultures and/or molecular biology techniques such
as the PCR (52, 103).
In all approaches it is important that the final
concentrate
䊉
is not toxic to the cell cultures
䊉
does not interfere with virus-cell interactions
䊉
does not inhibit growth of viruses
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does not interfere with nucleic acid extraction
for PCR
䊉
does not inhibit PCR reactions.
Unfortunately, most concentration procedures
for viruses also concentrate organic materials,
heavy metal complexes as well as humic and
fulvic acids that are both cytotoxic and inhibitory
to PCR.
For the detection of viruses in water, filter
adsorption/elution techniques are commonly used.
However, they have low recovery rates and are
often used in conjunction with beef extract, high
pH eluants and metal ions to enhance recovery.
These conditions will inhibit both cell culture and
PCR assays but over the last few years, the
development of novel reagents such as zirconium
hydroxide (29), proprietary reagents Viraffinity™
and Pro-Cipitate (62) have been used for better
recovery of viruses without most of the inhibiting
compounds.
HFU is a very sensitive technique but
requires minimal turbidity in water samples.
Both primary concentration systems referred to
generally need in-line prefilters to remove gross
debris that should also be analysed for viruses.
The use of PEG is probably the best method for
reconcentration and has the advantage of
removing some inhibitors to cell culture and PCR
assays. Studies using HFU, PEG and cell culture
on water samples in the environs of Sydney have
shown the presence of viruses in some 60–70% of
effluent samples and in 15% of water samples
used for recreation (52, 78). Enteric viruses were
also occasionally detected in drinking water
storages. A model detection scheme for the
detection of viruses in water is shown in Figure
22.2.
䊉
Detection of RNA viruses using cell
culture techniques
For many culturable viruses, including
polioviruses, enteroviruses, and certain strains of
hepatitis A virus and rotavirus, there have been
numerous reports on the development of virus
extraction and assay procedures from foods and
environmental samples. For example, cell culture
techniques for the propagation of polioviruses are
readily available. Due to the widespread use of
attenuated, oral poliovirus vaccines, the virus is
shed in faeces and it may be possible to use these
vaccine strains of poliovirus as an indicator of
faecal contamination and presence of other
pathogenic viruses for which there are no current
detection procedures (97).
Cell culture techniques and procedures utilise
assays which develop plaques and cytopathic
effects to enumerate levels of viable viruses.
However, such techniques and procedures are
Viruses, Food and Environment
Figure 22.1. Model scheme for the detection of viruses in foods (based on 9)
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Gary Grohmann and Alvin Lee
Figure 22.2. Schemes for the detection of viruses in water or treated effluent
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Viruses, Food and Environment
either ineffective or not ideal for the detection and
enumeration of most hepatitis A viruses,
rotaviruses, astroviruses and noroviruses.
Furthermore, these methods commonly involve
laborious extraction and concentration procedures,
removal of substances that may interfere with or
be cytotoxic to cell line growth, high assay and
labour costs, and the need for highly trained
personnel. The success of cell culture techniques
is also heavily dependent on the different growth
rates of specific cell lines for particular viruses,
presently laboratories with complex logistical
problems.
Detection of RNA viruses using PCR
All enteric viruses, with the exception of
adenoviruses, have a small RNA genome. PCR is
a process that allows the in vitro exponential
amplification of DNA that has been targeted by
specific oligonucleotides or primers. Amplification
and detection of viral RNA requires the use of
Reverse Transcriptase (RT) to produce cDNA
prior to PCR, a process known as RT-PCR. RTPCR is inexpensive and offers a high degree of
sensitivity and specificity for the detection of
viruses, through the use of specific oligonucleotides,
in food and water providing an additional test to
traditional cell culture assays. The use of RT-PCR
and other molecular biology techniques including
cloning and nucleic acid sequencing, the viral
genomes of a number of viruses such as
poliovirus, hepatitis A virus and noroviruses have
been elucidated (7–9, 27, 45, 62, 63, 103, 115).
With the development and implementation of
real-time quantitative PCR, results that are both
qualitative and quantitative are possible in hours
rather than the weeks required for cell culture
techniques. Both traditional PCR and real-time
PCR have provided various approaches for the
reliable detection of non-culturable (e.g.
noroviruses) and difficult to culture viruses (e.g.
hepatitis A virus, astrovirus and rotavirus) as
well as the detection of culturable viruses (e.g.
enteroviruses, adenoviruses).
Limitations of PCR for food and water
samples
The presence of humic acids, lipopolysaccharides,
glycogen, lipids and metal ions may limit the use
of PCR techniques on food and water samples.
These substances are commonly found in many
food matrices and are usually concentrated in
samples during virus extraction and concentration
procedures. Humic acids bind strongly to proteins
and inhibit enzymes from functioning in PCR
reactions, whilst metal ions will interfere with
enzyme activity. The presence of these compounds
may result in false negative results or non-specific
reactions. PCR is also inhibited by beef extract
commonly added in adsorption/elution concentration methods for viruses in water samples.
However, it is possible to remove inhibitory
compounds such as humic acid, beef extract and
metal ions from samples by using resins such as
Chelex-100 or hydrophilic gels such as Sephadex
(112). When used in combination with spuncolumn chromatography, inhibitory substances
are removed from water samples. Sephadex G-200
alone has also been shown to largely remove the
inhibitory effects of beef extract on RT-PCR.
A number of other protocols have been
developed to extract viral RNA from various
foods. Such protocols commonly require multiple
steps involving the use of various reagents such
as guanidinium thiocyanate, polyethylene glycol,
phenol-chloroform and more recently Pro-Cipitate
and Viraffinity (LigoChem, Fairfield, N.J.). These
reagents have been previously reported to reduce
contaminating polysaccharides from oysters and
clam tissues (31, 62).
Many of the recently developed protocols for
viral extraction, purification and concentration
and detection methods have not been tested on
food products, e.g. shellfish obtained from different
geographical areas and varying seasons where
viral levels may vary. Other factors that may
influence viral titres in shellfish include the types
of algae and other substances consumed, glycogen
levels within shellfish tissues and the physiological state of the shellfish (98). Much of the current
work utilising such protocols has been carried out
on artificially inoculated shellfish with high titres
of virus and has not been tested on shellfish that
are naturally contaminated with viruses.
Interpretation of PCR results
Positive and negative controls must be included
with every sample tested by PCR. If a positive
result cannot be obtained after addition to the
sample of an appropriate template, then the
validity of negative results is in doubt. Negative
controls ensure the samples are not cross
contaminated either by other samples or
aerosolised PCR products.
Although PCR is an extremely useful tool for
detection of viruses, it will detect viable as well as
non-viable virus particles, and new or emerging
viruses may not necessarily be detected. Only cell
culture techniques will allow the detection of
viable and new viruses. Food processing, such as
chemical or enzymatic treatment, high pressure
processing, or UV irradiation, may result in
damage to viral nucleic acids. These damaged
viruses may not represent a public health threat,
however the damaged nucleic acids could still be
amplified by RT-PCR or PCR.
571
Gary Grohmann and Alvin Lee
The detection of viable and/or non-viable
enteric viruses by PCR from food and water
samples is cause for concern, but does not
necessarily imply an actual risk of disease.
However, in the case of non-culturable or difficult
to culture viruses, a positive RT-PCR result would
need to be interpreted as the presumptive
presence of such viruses, since no cell culture
assays exist. Rather than a sole diagnostic tool,
PCR can be used as a rapid screen for viruses
while the more time consuming cell culture assays
are undertaken. PCR can also be used effectively
to screen for viable viruses in cell culture. A
scheme for the detection of viruses by PCR is
shown in Figure 22.3.
Quantitative RT-PCR
The first studies into quantitative methods for
enumerating viruses used synthesised seeded or
spiked oligonucleotides as ‘mimic fragments’
(those having identical priming sites but a PCR
product of increased molecular weight). Different
dilutions were used across several reactions and
analysed using mean probable number (MPN)
methods. Such techniques would be accurate if all
target sequences were amplified and were
randomly distributed in the sample.
The recent development of real-time PCR
technology has brought the possibility of
enumerating viruses in foods a step closer to
reality. Real-time PCR allows constant
monitoring of the generation of PCR products
simultaneously with the amplification process,
allowing faster detection times and eliminating
the need for amplicon detection by agarose gels.
Quantification is achieved through the use of
fluorescence labelled probes, e.g. TaqMan probes,
or DNA intercalating dyes such as SyberGreen,
and the measurement of detected fluorescence
during the amplification process. The use of
complex algorithms to establish a calibration
curve using known viral titres enables
quantification of the viral target in a sample. A
real-time PCR method quantifying human
parvovirus using a duplex amplification with an
internal standard and two-colour fluorescence
detection, resulted in a detection sensitivity of
102 viruses/mL (55). Although the are few reports
on the application of real-time PCR technology to
food and environmental samples, viral concentration and purification methods are currently
being modified for compatibility to real-time PCR
technology (55, 91, 104).
Improving sensitivity and specificity
Improved RT-PCR results may be obtained by
optimising different nucleic acid extraction
techniques. For example, most of the enteric
572
viruses have an RNA genome and the use of low
pH buffered phenol for nucleic acid extraction will
preferentially extract RNA, leaving proteins and
DNA in the organic phase. This could lower the
overall nucleic acid ‘background’ in RT-PCR
leading to more sensitive virus detection. Use of
the cationic detergent cetyl-trimethylammonium
bromide (CTAB) and other commercially available
reagents, e.g. Pro-Cipitate, Viraffinity and TRIzol
Reagent (Gibco BRL, Rockville, MD), have also
proved useful in purifying RNA from shellfish
specimens (8, 9, 31, 62). These reagents are
developed to precipitate nucleic acids while other
proteins and polysaccharides remain in solution
and inhibitors removed prior to analysis for
enteric virus detection by RT-PCR.
Although PCR is a powerful technique, it can
be subjected to misuse and misinterpretation. The
presence of PCR product or amplicons may
represent the presence of virus-specific products.
However, such amplicons can also be associated
with contaminating DNA present in the sample.
Oligonucleotides used in PCR, specific to targeted
viruses, can amplify contaminating DNA so long
as the contaminating DNA contains a region
homologous to the oligonucleotides. Although the
occurrence of such phenomena is rare, to rule out
false positives, amplicons may be separated by
agarose gels, and detected using digoxigeninlabelled (DIG) probes (30, 80), radiolabelled
probes (95), chemiluminescence (106, 109) or
colorimetric (19) detection systems that are suitable
for implementation in food testing laboratories.
Increased sensitivity and specificity of PCR
can also be achieved by using nested PCR or seminested PCR, i.e. a series of two PCR reactions
with different primer sets. The first reaction will
amplify a portion of a viral genome while the
second PCR is performed on products from the
first reaction using oligonucleotides directed
towards an internal region of the target DNA (as
shown in Figure 22.4) and is useful in assaying
food and water samples as inhibitors are diluted
for the second PCR assay.
With the advancement of molecular biological
techniques, food and waterborne outbreaks can be
more adequately studied and early warning
systems could be implemented to protect public
health and the food industry from viral disease.
Finally, despite the significant advances
made in the detection of viruses, there are some
areas of research which still need to be
undertaken, including:
䊉
Determination of a practical viral indicator(s)
for the presence of enteric viruses in food and
water.
䊉
The efficiency of concentration methods on
samples of differing type and quality.
Viruses, Food and Environment
Figure 22.3. Scheme for nucleic acid amplification (PCR) for RNA viruses
573
Gary Grohmann and Alvin Lee
䊉
䊉
䊉
The removal from food and water samples of
inhibitors of molecular and cell culture
assays.
Methods for quantitative PCR for food and
environmental samples.
Methods for the extraction and detection of
viruses in food and shellfish.
with hepatitis A virus and poliovirus vaccines
should be encouraged to help stop the spread of
foodborne hepatitis A and the (unlikely) potential
threat of poliovirus. A rotavirus vaccine is likely
to be available within the next three years and food
handlers should be encouraged to obtain it and any
other relevant vaccines as they become available.
Food handlers
Control
Sources of viruses in food include food handlers,
contaminated surfaces in food handling areas,
shellfish and contaminated water. Successful
prevention of foodborne illness relies on avoiding
faecal contamination of food and water. As an
overall strategy, vaccination of all food handlers
Food handlers need to be aware that enteric
viruses can be shed in faeces during asymptomatic
phases of infection as well as during times of overt
illness. Good personal hygiene practices are vital
in the prevention of foodborne viral infection.
Liberal hand washing with soap, the use of iodine
based skin disinfectants (e.g. Betadine surgical
scrub) and the use of disposable gloves are
essential. If vomiting occurs, noroviruses may be
Figure 22.4. Nested PCR for virus detection or confirmation of 1st round product
574
Viruses, Food and Environment
spread over a large area in aerosol droplets (33,
89) and therefore, uncovered food should be
discarded and the environment including work
surfaces should be thoroughly cleaned. Moreover,
it is important that any food handler with
diarrhoea or vomiting must not return to work for
a minimum period of 48 h after complete recovery.
Training of personnel and food handlers is
important in preventing outbreaks (66).
Environmental control
Viruses can survive on surfaces and instruments
used for food preparation for extended periods (1)
and the use of carefully selected chemical
disinfectants is important if viruses are to be
inactivated and controlled in the environment.
Enteric viruses are resistant to commonly used
antibacterial disinfectants (e.g. phenolics,
ethanol, quaternary ammonium compounds) but
are susceptible to free chlorine, iodine and
aldehydes (formaldehyde and glutaraldehyde).
These antiviral disinfectants need to be used with
caution as the aldehydes are potential
carcinogens and may also damage certain
surfaces and instruments on prolonged exposure.
Shellfish
In many countries, commercial shellfish are
cultured frequently in estuarine waters adjacent
to populated areas. Often, these waters are
contaminated with sewage effluent and the
potential for transmission of hepatitis A virus,
noroviruses and other enteric viruses exists.
In Australia, the cultivation and distribution
of oysters and other shellfish is carefully
regulated. However, these regulations are
difficult and sometimes impossible to enforce.
Moreover, the harvest from an individual lease
may be sent to a variety of suppliers and then
distributed in small quantities to a large number
of outlets (14). These factors, coupled with the
long incubation period of some diseases like
hepatitis, make shellfish associated outbreaks
very difficult to document.
To reduce the risk of oyster borne infections,
strict quality control must be observed in oyster
cultivation, including a period of depuration
where oysters are held in tanks of disinfected
water. Purification of oysters in NSW was
introduced following the 1978 outbreaks of food
poisoning (93) and remains a statutory
requirement (14). For this reason, purification
techniques are based on the immersion of
shellfish in seawater of good quality for at least
36 h, a process known as depuration.
Depuration may take place in closed loop
circuits where the seawater is continuously
disinfected via UV lamps or in semi-open circuits
where the seawater of good quality is renewed
every 12 or 24 h. It has been shown that these
methods, when carefully performed, yield
satisfactory bacteriological results. However,
virological results are not always satisfactory as
shellfish may still contain enteric viruses after
purification (26, 50). The fact that viral outbreaks
still occur via contaminated oysters, despite
oyster depuration, indicates that current
depuration techniques are inadequate for the
removal of pathogenic viruses. They may have to
be combined with water disinfection techniques
such as ozonation to be truly effective.
Since it is impossible to prevent the
contamination of coastal waters by sewage
effluent, the prevention of shellfish borne diseases
requires bacteriological monitoring of the marine
environment and shellfish flesh (14). Such
surveillance allows the classification of growing
areas’ suitability for harvesting and distribution
of shellfish. However this surveillance is not
always sensitive enough as bacteria are a poor
indicator of viral contamination of oysters (2, 14,
26, 52). PCR testing for viruses in oysters and the
environment is possible, although not a standard
routine test, and is 10–100 times more sensitive
than culture techniques (25).
Fresh produce
Fresh fruits and vegetables can become
contaminated by enteric viruses, possibly through
the use of contaminated fertilisers or contaminated
irrigation water supplies. A range of enteric
viruses has been reported to survive long periods
of storage up to 60 days (1, 10, 73, 74, 101, 102,
107). Chlorine has been widely used for fresh
produce washing due to its availability, low cost
and its effectiveness on a wide range of
microorganisms. Chlorine based disinfectants
have been considered to be the most effective
against many enteric viruses. However, chlorine
resistant noroviruses, hepatitis A viruses and
feline calicivirus have been reported (32, 40, 67).
It should be noted that chlorine based products
are susceptible to pH changes and require
constant monitoring to maintain optimal efficacy.
Ozone is another alternative that has been shown
to be effective on a number of microorganisms
including enteric viruses (36, 69). Ozone is a
powerful oxidising agent and highly reactive, does
not leave any residues on/in food and water and
naturally decomposes into oxygen. Disadvantages
of using ozone include its instability and that it
cannot be stored and has therefore to be
generated on-site. A number of other alternative
disinfectants, including organic acids and
surfactants, have also been used with variable
rates of success (3, 100, 101, 108, 116).
575
Gary Grohmann and Alvin Lee
Viral inactivation by non-thermal
processing techniques
With the increasing global demand for fresh
produce and seafood, there is added pressure on
suppliers to provide good quality and safe foods.
Heat treatments have been used with
considerable success in inactivating most
microbial pathogens including viruses. However,
fresh produce and shellfish are consumed raw and
have a relatively short shelf life. Moreover, heat
treatments are inappropriate for viral
inactivation for these products.
Non-thermal processing techniques such as
gamma irradiation and high hydrostatic pressure
processing offer possibilities both for food
processing and microbial and viral inactivation.
These processes have various advantages
including rapid application and minimal
alterations to the food’s taste, odour and texture,
resulting in the retention of freshness. Although
such processes are currently expensive, they are
an appealing alternative to thermal processing.
Gamma irradiation offers a safe alternative for
the decontamination of food, especially for fresh
produce (110, 111). There are currently no
standards on allowable gamma irradiation doses,
based on the nutritional, toxicological and
microbiological data, for use on various foods.
Bidawid et al. (13) provides one of the few reports
on viral inactivation from the use of gamma
irradiation on foods. These authors treated lettuce
and strawberries that were inoculated with
hepatitis A viruses using varying doses of gamma
irradiation. Their data indicated that gamma
irradiation doses between 2.7 and 3.0 kGy were
required to achieve at least 90% (1 log10 reduction)
inactivation of hepatitis A virus in lettuce and
strawberries respectively. Gamma irradiation is
currently not used in Australia for processing of
foods and more work is required to determine its
efficacy on fresh produce and shellfish.
High hydrostatic pressure processing (HPP)
has been used to inactivate various problematic
microorganisms, including viruses, in foods (70,
86, A. Lee - unpublished results). HPP is rapid,
and pressure is applied uniformly resulting in
little damage to the product with minimal
changes to the physical properties of foods. HPP
has been used on shellfish to eliminate Vibrio spp.
(87) providing shelf life extension for shucked
oysters. Treatment of shellfish at pressures
ranging from 250 MPa to 400 MPa does not affect
the taste of raw shellfish and minimises handling
and wastage in oyster and clam shucking.
The effects of HPP in inactivating enteric
viruses is not well studied and preliminary results
576
have shown that feline calicivirus and hepatitis A
virus acting as model viruses for the (nonculturable) noroviruses, could be inactivated in
buffer suspensions at pressures between 300 MPa
and 450 MPa (70, A. Lee - unpublished results).
However, other studies on polioviruses showed
resistance to pressures up to 600 MPa (70, 113). It
is clear that the effects of HPP on viruses in
various food matrices such as oyster homogenates
or whole oysters need further study.
Challenges for the future
Over the next ten years there will be many
challenges facing suppliers, retailers, regulators
and researchers involved in food and water
virology. Complacency amongst food handlers will
always be an ongoing challenge and the need for
continuing education and scrupulous personal
hygiene will be paramount in the control of most
foodborne disease of viral origin in the
community. Other needs likely to be addressed or
requiring vigilance include:
䊉
The promotion of relevant available viral
vaccines and proper hygiene training for food
handlers.
䊉
The
development
of
guidelines
for
immunocompromised persons, e.g. should
raw foods, particularly shellfish, be avoided?
䊉
The development of novel non-thermal
processing techniques and processing
parameters for the inactivation of viral
pathogens from high risk foods to improve the
shelf life of such foods.
䊉
Research and monitoring of prion diseases to
avoid their entry into the food chain.
䊉
Monitoring of polluted water environments
and any pollution sources identified and if
possible neutralised. Sewage treatment
methods should be upgraded to remove
viruses from entering waterways. Shellfish
should not be harvested from polluted waters.
䊉
Ensuring that recycled effluent for potable
and non potable uses is free from known and
unknown viruses and prions. Membrane and
chemical disinfection processes will be the
likely key to remove these agents.
䊉
The use of improved oyster depuration
practices possibly involving centralised
depuration and the use of ozone or similar as
a disinfectant.
䊉
The development of risk assessment models
for viruses in food and water.
䊉
The development and standardisation of
molecular biology techniques for the rapid
detection of enteric viruses, particularly the
Viruses, Food and Environment
development of PCR tests that indicate virus
viability and that can be used quantitatively.
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