Chapter 22 · Chapter 22 Viruses, Food and Environment Gary Grohmann and Alvin Lee 561 Introduction...

Chapter 22

Viruses, Food and Environment

Gary Grohmann and Alvin Lee

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Introduction

Characteristics of foodborne virusesNoroviruses

TaxonomyDiagnosis and detectionEpidemiology and pathogenesis

Enteric hepatitis virusesTaxonomyDiagnosis and detectionEpidemiology and pathogenesis

AstrovirusesTaxonomyDiagnosis and detectionEpidemiology and pathogenesis

RotavirusesParvo-like virusesEnterovirusesTick-borne encephalitis virusOther viruses

Detection in food and waterDetection of RNA viruses using cell culture techniquesDetection of RNA viruses using PCR Limitations of PCR for food and water samplesInterpretation of PCR resultsQuantitative RT-PCRImproving sensitivity and specificity


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ControlFood handlersEnvironmental controlShellfishFresh produce

Viral inactivation by non-thermal processing techniques

Challenges for the future

References

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Introduction

Enteric viruses such as hepatitis A virus,noroviruses (formerly Norwalk-like viruses),enteroviruses, astroviruses, adenoviruses,rotaviruses and hepatitis E virus have all beenimplicated in food and/or waterborne outbreaks ofillness. Data from the USA, Europe, Japan andthe UK shows that viruses are responsible for themajority of foodborne outbreaks with norovirusesbeing the most common cause of disease (38, 44,58, 59, 76, 77, 88). Moreover, there have beenmany anecdotal reports in ProMed during2001–2002 that have highlighted consecutiveoutbreaks of gastroenteritis due to noroviruses inhospital settings in the UK and Ireland, and invarious other settings elsewhere including day-care settings, homes for the elderly, cruise shipsand restaurants. Increased sporadic activity ofnoroviruses in the community has also beenobserved in most parts of the world. Hepatitis Ahas also commonly been reported with recentProMed reports highlighting outbreaks in NewZealand due to blueberries that were contaminatedby sewage affected groundwater.

The epidemiological evidence for thetransmission of viral disease via the food andwater route is best defined with hepatitis A virus,noroviruses and enteroviruses, since infectionsresult in a specific illness and outbreaks tend tobe linked to a point source or vehicle oftransmission. Outbreaks of food and waterbornehepatitis A still occur despite increasingstandards of hygiene and sanitation. In theUnited States the number of waterborne hepatitisA outbreaks has not declined since 1960 (11, 12,15). Since 1970, many outbreaks of food andwaterborne gastroenteritis have been identifiedas due to noroviruses, as techniques for theiridentification improve and awareness of theirpublic health importance increases. However,despite advances in detection techniques and thesubsequent identification of many new strainsand diverse genetic clusters, there are stilldifficulties in discerning viral outbreaks of illnessvia contaminated food and water. Some of thereasons for this include:1. The occurrence of subclinical infection.2. The presentation of a variety of clinical

symptoms associated with norovirus andenterovirus infections; so without virusidentification from environmental samplesand/or patient specimens it is impossible toclearly associate an outbreak of illness tothese viral agents.

3. The occurrence of secondary and tertiary

person-to-person transmission which maymask a food- or water-borne source oftransmission.Sometimes outbreaks due to food, shellfish

and contaminated water can be sudden anddramatic: e.g. at least 100 000 persons were affectedin waterborne outbreaks of hepatitis E virus inMexico, India and the former Soviet Union (28);over 100 000 persons contracted hepatitis A fromcontaminated clams in China (57), and over 4700persons in Japan contracted foodborne gastro-enteritis due to astrovirus (96).

Enteric viruses can survive for long periods infood and water and are often detected in theabsence of indicator bacteria. They are generallymore resistant to chemical and UV disinfection,filtration and pasteurisation than bacterialindicators but may be removed by ultrafiltrationmembranes or inactivated by prolonged heatingor optimal UV treatment. In general, theseviruses will survive reasonably well in adverseconditions, e.g. heating to 50°C, pH 2.7–9.6,microbial proteolysis and fermentation. Specificdata on virus survival and stability in foods havebeen previously reviewed and summarised (35).

The entry and survival of viruses in theenvironment is well documented. They aredifficult to eliminate in sewerage treatmentprocesses. They may survive post-treatment UVand chlorination, and be discharged fromtreatment plants into surface and ground waters.Viruses are protected in the environment by theirassociation with faecal matter and particulates.They move freely through the environmentsurviving in waterways, sludge, sediment, soil,shellfish and on crops which have been irrigatedwith recycled effluent thus potentially infectinghumans. In laboratory experiments, entericviruses have survived in tap water, seawater, soiland oysters for periods of three months or more.They can adsorb to sediment and particulates inraw and estuarine waters and soil and may bereleased under certain conditions (e.g. presence ofcations, soluble organics, pH and soil type). Aftera number of adsorption/elution events, virusescan migrate long distances in soil and waterways.Viruses can also survive on crops and in preparedor 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 foodborneoutbreaks is either from food handlers (involvingfaecal-oral and aerosol spread of faecal materialand vomitus) or from sewage contaminated food

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and water. In general, water used in the foodindustry is strictly controlled and only water ofpotable quality is permitted. However, anoutbreak of norovirus gastroenteritis on domesticair flights in Australia was due to orange juicewhich apparently used a contaminated ‘potable’water source (105).

Shellfish present a particular problem as theyare often affected by sewage discharges. Theyhave been shown to accumulate viruses and havebeen clearly associated with many outbreaks ofhepatitis and viral gastroenteritis. Some 2–3outbreaks of gastroenteritis per year have beenreported in Australia alone, often due tonoroviruses. Recently, the first documentedAustralian oyster associated hepatitis A outbreakoccurred and was traced to oysters harvested inWallis Lake in NSW (26).

Shellfish can filter some 10–20 L per hour ofwater under ideal conditions but also concentrateinfectious agents that are present in the marineenvironment. Being bivalve molluscs they feedand respire by inducing a current of water to flowover a series of complex gill structures, andcapture suspended particulate matter passing ittowards the mouth where it may be ingested orrejected as pseudo-faeces. Oysters are veryeffective filter feeders capable of concentratingviruses that may be present in water, resulting inviral concentrations within the oyster farexceeding those of the surrounding water. Inunpolluted estuarine environments there will belittle risk to consumers. However, if the environ-ment is polluted, oysters can act as a vehicle oftransmission for viral disease (26, 51, 93, 114).

The use of recycled effluent and sludge is alsoa matter of public health concern as infectiousviruses entering the sewerage system canultimately contaminate water and theenvironment affecting crops, soil, the water tableand shellfish. Recent hepatitis A outbreaksinvolving oysters in Australia and strawberries inthe USA, may have been partly due tocontamination by recycled effluent (16, 26, 94).

Until recently it has not been possible toroutinely detect viruses in food and water, butnucleic acid amplification techniques, such aspolymerase chain reaction (PCR), have now beendeveloped 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 environmentand on surfaces, and their poor correlation withindicator bacteria, assessment of water and foodfor these pathogens will become necessary inindustries involving potentially contaminatedwater or shellfish and also in investigations offood and water associated outbreaks of disease.

Characteristics of foodborne viruses

Over 150 types of enteric viruses falling into fivemajor viral families are potentially present infaecal material and sewage effluent, depending onthe season of the year and the viruses circulatingin the community. They may cause a variety ofdiseases in humans ranging from skin, eye andrespiratory infections to fever, meningitis,myalgia, hepatitis and gastroenteritis, but manyviral infections are silent or asymptomatic. Someof the characteristics of typical foodborne virusesare shown in Table 22.1.

Noroviruses, formerly Norwalk-like orsmall round structured viruses (SRSV)Taxonomy. These ssRNA viruses have beenclassified in the family Caliciviridae as theircapsid contains a single polypeptide of 59 000 MW.During 1993 the complete nucleotide sequences ofNorwalk virus and Southampton virus werepublished, leading to their final classificationwithin the Caliciviridae even though examinationby electron microscopy shows that they lackCaliciviridae’s typical cup-shaped morphology.Little is known of the replication strategy of theseviruses. A possible subgenomic mRNA has beenidentified in faeces (65), and studies using cell-free expression systems have been successful inexpressing capsids which have been useful forserological studies (64). The noroviruses are agroup of 27–35 nm particles that lack a distinctstructure when visualised by electron microscopy,hence they are were originally named small roundstructured viruses or SRSV. Norwalk virus is theprototype of this group. Three genotypes have nowbeen described which can be divided further intoat least 17 distinct genetic clusters. Genotypes G1and GII commonly infect humans and can co-circulate (39, 44) although GII types have predom-inated since the 1990s. Distinct, well characterisedserotypes in the norovirus genus include: Norwalkvirus, Desert Shield virus, Lordsdale virus,Mexico virus, Hawaii virus, Snow Mountain virusand Southampton virus. A second genus, theSapovirus genus, forms a third genogroup, G3,which is also now been identified within theCaliciviridae. Well-characterised and distinctviruses include: Saporovirus, Houston/86,Houston/90, London 29845, Manchester virus andParkville virus.Diagnosis and detection. Progress in thediagnosis of these viruses has been slow followingtheir detection by electron microscopy in 1972 forseveral reasons; they have not been isolated in cellculture, there are no readily available animalmodels and there are no serological reagents forthe detection of antigenically diverse strains.

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The diagnosis of a norovirus infection is basedon the clinical signs, and the detection of viralantigen or nucleic acid, or seroconversion by thepatient. Molecular tests, such as PCR, are nowcommonly used to detect noroviruses usingvarious primer sets (6–9, 39, 81, 82).Epidemiology and pathogenesis. Theseviruses cause acute gastroenteritis in humansworldwide and commonly cause epidemic viralgastroenteritis in all age groups. The onset ofvomiting and/or diarrhoea is sudden, accompaniedby anorexia, headache, abdominal discomfort,nausea, and low grade fever, often in severaldifferent combinations (50). The incubation periodis approximately 24 h and recovery from illness isusually complete within 72 h. Occasionallysymptoms can be very severe sometimes leadingto hospitalisation and often leading to workingdays being lost. The mortality rate is very low inindustrialised countries.

Outbreaks usually last about 7–10 d,although longer epidemics involving weeks ormonths have been recorded (54). Outbreaks areoften confined to families or closed communitiessuch as schools, restaurants, hospital wards,

nursing homes, caravan parks, cruise ships or themilitary (17, 18, 56, 59, 60, 61, 68, 72, 88, 90). Theviruses are spread via the faecal-oral route andaerosolised vomitus. Identified sources ofoutbreaks include contaminated water, ice,shellfish and food contaminated via food handlerssuch as salads, fruit, shrimp, school lunches andbakery products (5, 11, 31, 38, 48, 50, 68, 71-75,79). Outbreaks of illness in Australia attributableto these viruses have also been recorded, the firstfoodborne outbreak occurring in 1978, caused byconsumption of oysters from the Georges River inSydney (50, 51, 85, 93). Other outbreaks inAustralia due to enteric viruses have involvedcontaminated bore water (92), drinking water(90), orange juice (affecting over 4000 persons)(105), and septic tank contact (20). However, manymore outbreaks remain undocumented because ofthe difficulties in reporting and diagnosis.

Enteric hepatitis virusesTwo different viruses have been clearly associatedwith food and waterborne outbreaks; hepatitis Avirus and hepatitis E virus.

Table 22.1. Some characteristics of common foodborne viruses

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Taxonomy. Hepatitis A virus is 28–30 nm indiameter and a member of a separate genus,Hepatovirus, within the family Picornaviridae.Hepatitis E virus, is 32 nm in diameter with somesimilarity to the Caliciviridae in genomeorganisation and morphology however, it also hasamino acid similarity in its replicative enzymeswith the Togaviridae and its classification is stillunder review. Diagnosis and detection. Illness caused byhepatitis A or hepatitis E viruses is not clinicallydistinguishable (28). Illness may last severalweeks and usually includes malaise, nausea,jaundice, anorexia and vomiting with an abruptonset. Neither virus can be easily culturedalthough several laboratory strains of hepatitis Avirus have been cultured. Primer sets areavailable for PCR tests, which can be used todetect these viruses in food and water or clinicalspecimens. The detection of IgM in patients isdiagnostic. Hepatitis A virus is very much moreresistant to drying and heating than otherpicornaviruses and is also more resistant to pH 2,gamma rays, UV light and low levels of chlorineand ozone. Unlike other enteric viruses, hepatitisE virus is extremely labile and easily inactivatedby proteolysis, freeze-thawing, heating andultracentrifugation. Epidemiology and pathogenesis. Hepatitis Avirus enters the body by ingestion and multipliesin intestinal epithelial cells followed by aviraemia. The virus then infects the parenchymalcells in the liver and the host’s immune responsedestroys infected hepatocytes via cytotoxic T cells.Immunity is life long although relapses have beenrecorded and death is very rare. Only one serotypeof hepatitis A and E viruses exist and both arelikely to cause extensive subclinical infection inchildhood. The incubation period for hepatitis Aaverages 28–30 days and for hepatitis E, 40 days.Both viruses are predominately shed during theincubation period with hepatitis A virus beingshed in low numbers for up to two weeks afteronset of illness. Both viruses can cause a moresevere illness in pregnant women resulting in ahigh mortality rate – 17% and 33% for hepatitis Avirus and hepatitis E virus respectively. Thepathogenesis of hepatitis E is probably similar tothat of hepatitis A based on studies carried out inprimates (28).

Both viruses are spread via the faecal-oralroute. Direct person-to-person contact is the mostcommon method of transmission of these virusesin the community with contaminated food andwater playing a role, particularly in developingnations. Clinical cases are generally seen in youngadults or children.

Hepatitis A can be a particular problem inunsewered areas, in lower socioeconomic groupsand in persons with high-risk behaviour patternssuch as male homosexuals and intravenous drugusers. Persons travelling through developingcountries are also at greater risk. Outbreaks oftenoccur in communities having a poor or marginallevel of hygiene such as day-care centres, homesfor the mentally retarded, prisons, mentalhospitals, army camps, etc. Most outbreaksinvolve specific foods (15, 43, 94, 99) or shellfish(22–27).

Shellfish have been regularly involved inhepatitis A outbreaks in the USA and Europe andfrom January to March 1997, outbreaks ofhepatitis A occurred throughout Australia whichwere associated with oysters grown in the WallisLake Area in NSW (26). In this outbreak over 70%of cases were oyster associated and withsubsequent secondary spread affecting at least460 persons. Moreover, oysters met allbacteriological standards and had undergonedepuration (purification). This outbreak wasunusual in that hepatitis A from contaminatedoysters had not occurred in Australia previously.The fact that hepatitis A cases are increasing inboth the USA and Australia indicates that thisvirus may well be a re-emerging disease in thecommunity putting seronegative and immuno-compromised individuals at greatest risk.

Hepatitis E is now the most frequent cause ofhepatitis in Asia (28) and must be considered apotential emerging viral disease for Australia.Both hepatitis A and E tend to be most prevalentin autumn in Asia often following extensive rainor flood. Waterborne outbreaks of hepatitis E arecommon in the Asian/Indian area as well as inMexico (28). Surprisingly, no documentedoutbreaks of foodborne hepatitis E have beenreported, the virus being mainly transmitted bythe water route and person-to-person spread.

AstrovirusesAstroviruses affect a wide range of animals andcause gastroenteritis in humans. They appear tobe ubiquitous in young children and often affectthe elderly. They emerged as a potentiallysignificant foodborne pathogen when over 4700persons were infected by astrovirus in schools inOsaka, Japan (96). Taxonomy. Astroviruses are 28 nm sphericalparticles often exhibiting five- or six-pointed star-like patterns. They contain single stranded RNAand are resistant to pH 3 and heating to 50°C for30 min and 60°C for 5 min. They belong to a newfamily, the Astroviridae and eight serotypes ofhuman astrovirus are known.

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Diagnosis and detection. Astrovirus gastro-enteritis has an incubation period of 3–4 dayswith diarrhoea being more typical than vomiting,the clinical signs usually lasting 2–4 days. Viralshedding in faeces usually follows the duration ofsymptoms, however HIV positive patients withastrovirus gastroenteritis may shed the virusintermittently for months (53). The virus can beisolated using CaCo-2 cells in the presence oftrypsin. Enzyme immunoassays and PCR testscan also be used to detect the virus in faeces. PCRcan be used to detect the virus in food, water andeffluent samples.Epidemiology and pathogenesis. Astrovirusesare transmitted by the faecal-oral route viaperson-to-person spread and have a peakprevalence in the winter months, but theseviruses are also likely to be transmitted by foodand water. Most infections in the community aresubclinical with 4–6% of infantile diarrhoea beingattributed to astroviruses. Outbreaks are commonin families, day-care centres, hospital wards andnursing homes. These viruses replicate in thesmall intestine destroying mature enterocytes onthe villi, which are regenerated after a few days.Type specific antibody is produced with acquiredimmunity being monotypic.

RotavirusesRotaviruses are the most common cause of viralgastroenteritis in children, particularly indeveloping nations where they are responsible forhigh morbidity and mortality rates and are oftenassociated with waterborne disease. They aremainly transmitted by person-to-person spreadand sometimes by food handlers. Rotavirusesbelong to the Reoviridae and fall into three majorsubgroups. Group A has at least 13 differentserotypes and like astroviruses, they infectenterocytes in the small intestine causing cellulardamage, malabsorption and diarrhoea. Symptomsinclude diarrhoea, vomiting and fever that maylast for 4–6 days. Viral shedding in faeces ismaximal for 8 d. The virus can survive for weeksat 4°C but is less stable than other enteric virusesin food being almost completely inactivated at56°C for 30 min and unstable outside pH 3–10.

Parvo-like virusesSmall, 20–26 nm, featureless virus particles havebeen regularly associated with outbreaks ofgastroenteritis, sometimes associated withshellfish (5, 20). Because similar viruses are oftenseen in stool specimens in conjunction with knownagents of gastrointestinal illness, such asnoroviruses, parvo-like viruses could becommensals of humans or defective satellite

viruses requiring the genome of another entericvirus to replicate. The role of these agents as acause of gastroenteritis remains uncertain.

EnterovirusesPolioviruses, coxsackie viruses and echovirusesare members of the enterovirus group. Theseviruses are now rarely associated with foodborneoutbreaks despite the fact that they are oftencause subclinical infections in humans and areexcreted in faeces. Poliovirus was the first virus tobe associated with foodborne outbreaks of viraldisease but has now been almost eradicated byvaccination. Coxsackie B viruses and echoviruseshave been occasionally detected in foodborneoutbreaks in the USSR and in the USA due to foodhandlers contaminating foods (24). These virusesare however commonly detected in sewageeffluent and contaminated water (52,78) and havebeen detected in a variety of shellfish (26, 34). Forthis reason, enteric viruses such as enteroviruses,adenoviruses and reoviruses, all of which arecommonly detected in sewage effluent, may beuseful indicators of the presence of more commonviral pathogens such as hepatitis viruses andnoroviruses in shellfish or polluted waters. Forexample, these viruses have been detected incontaminated oysters and sediments wherenoroviruses have been implicated (34). In therecent Australian outbreak of oyster associatedhepatitis A at Wallis Lake, most oyster andsediment samples were positive for enterovirusesor adenoviruses rather than the aetiologicalagent, hepatitis A virus (26).

Tick-borne encephalitis virusSeveral viruses can in theory be transmitted viamilk. Dairy animals in Slovakia have beenreported to shed tick-borne encephalitis virusafter being bitten by infected ticks andsubsequently infecting humans (49). The problemhighlights the possibility of other zoonoticinfections from unpasteurised milk and milkproducts and affirms the need for properpasteurisation to occur to protect the communityfrom new and emerging viruses. Also ofimportance is the transmission of cytomegalovirus,human immunodeficiency viruses and somehuman T cell leukaemia viruses to childrenthrough breastfeeding.

Other virusesEnteric coronaviruses (4), adenoviruses, picobir-naviruses (53), herpesviruses and HIV are alsopotentially excreted in faecal material but theseagents are not generally associated withfoodborne outbreaks of disease.

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Detection in food and water

Despite the development of diagnostic methodsfor the detection of viral RNA or viruses fromwater and various food matrices, there are noroutine standard methods available for use bynon-research laboratories (6–9, 41, 42, 46, 76,81–84). Contaminated food products, in general,may contain very low viral numbers making themdifficult to detect even by nucleic acid amplificationtechnologies. Although the average infectiousdose of enteric viruses such as the noroviruses orhepatitis A virus is not known, it is believed to beless then 100 virions. Therefore, methods used forthe concentration, isolation and detection ofenteric viruses would require a high degree ofsensitivity and specificity (25).

In general, viruses need to be extracted fromfoods using blending techniques with appropriatebuffers and then concentrated by eitherpolyethylene glycol (PEG), AlCl3, iron oxide orFeCl3 precipitation, organic flocculation usingbeef extract or casein, or immobilisation byzirconium hydroxide (22, 25, 29, 46, 63, 80–84).The concentrate would then be resuspended in anappropriate buffer and examined for viruses usingcell culture techniques and/or various molecularbiology techniques including PCR (8, 9, 30, 38,39). Immunoassays are too insensitive for virusdetection in food (and water) but are useful indetecting serum antibodies and viral antigens inbody fluids (47, 64). A model scheme for virusesdetection in food is shown in Figure 22.1.

The detection of viruses in water is moreadvanced, with the primary concentration fromlarge volumes of 10–1000 L relying on flow-through sampling techniques based on either:1. Adsorption to filters, iron oxide,

polyelectrolytes etc. followed by elution, or

2. Hollow-fibre ultrafiltration (HFU) and otherfiltration methods where viruses are retainedbased on size or molecular weight.Using these techniques samples can be

reduced to 1 L or less. Samples are thenreconcentrated to 20–50 mL by precipitation asoutlined above, ultracentrifugation, organicflocculation using beef extract or casein, or theuse of iron oxide. The final concentrate can beexamined for viruses by inoculation of cellcultures and/or molecular biology techniques suchas the PCR (52, 103).

In all approaches it is important that the finalconcentrate � is not toxic to the cell cultures� does not interfere with virus-cell interactions� does not inhibit growth of viruses

� does not interfere with nucleic acid extractionfor PCR

� does not inhibit PCR reactions.Unfortunately, most concentration procedures

for viruses also concentrate organic materials,heavy metal complexes as well as humic andfulvic acids that are both cytotoxic and inhibitoryto PCR.

For the detection of viruses in water, filteradsorption/elution techniques are commonly used.However, they have low recovery rates and areoften used in conjunction with beef extract, highpH eluants and metal ions to enhance recovery.These conditions will inhibit both cell culture andPCR assays but over the last few years, thedevelopment of novel reagents such as zirconiumhydroxide (29), proprietary reagents Viraffinity™and Pro-Cipitate (62) have been used for betterrecovery of viruses without most of the inhibitingcompounds.

HFU is a very sensitive technique butrequires minimal turbidity in water samples.Both primary concentration systems referred togenerally need in-line prefilters to remove grossdebris that should also be analysed for viruses.The use of PEG is probably the best method forreconcentration and has the advantage ofremoving some inhibitors to cell culture and PCRassays. Studies using HFU, PEG and cell cultureon water samples in the environs of Sydney haveshown the presence of viruses in some 60–70% ofeffluent samples and in 15% of water samplesused for recreation (52, 78). Enteric viruses werealso occasionally detected in drinking waterstorages. A model detection scheme for thedetection of viruses in water is shown in Figure22.2.

Detection of RNA viruses using cellculture techniquesFor many culturable viruses, includingpolioviruses, enteroviruses, and certain strains ofhepatitis A virus and rotavirus, there have beennumerous reports on the development of virusextraction and assay procedures from foods andenvironmental samples. For example, cell culturetechniques for the propagation of polioviruses arereadily available. Due to the widespread use ofattenuated, oral poliovirus vaccines, the virus isshed in faeces and it may be possible to use thesevaccine strains of poliovirus as an indicator offaecal contamination and presence of otherpathogenic viruses for which there are no currentdetection procedures (97).

Cell culture techniques and procedures utiliseassays which develop plaques and cytopathiceffects to enumerate levels of viable viruses.However, such techniques and procedures are

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Figure 22.1. Model scheme for the detection of viruses in foods (based on 9)

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Figure 22.2. Schemes for the detection of viruses in water or treated effluent

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either ineffective or not ideal for the detection andenumeration of most hepatitis A viruses,rotaviruses, astroviruses and noroviruses.Furthermore, these methods commonly involvelaborious extraction and concentration procedures,removal of substances that may interfere with orbe cytotoxic to cell line growth, high assay andlabour costs, and the need for highly trainedpersonnel. The success of cell culture techniquesis also heavily dependent on the different growthrates of specific cell lines for particular viruses,presently laboratories with complex logisticalproblems.

Detection of RNA viruses using PCRAll enteric viruses, with the exception ofadenoviruses, have a small RNA genome. PCR isa process that allows the in vitro exponentialamplification of DNA that has been targeted byspecific oligonucleotides or primers. Amplificationand detection of viral RNA requires the use ofReverse Transcriptase (RT) to produce cDNAprior to PCR, a process known as RT-PCR. RT-PCR is inexpensive and offers a high degree ofsensitivity and specificity for the detection ofviruses, through the use of specific oligonucleotides,in food and water providing an additional test totraditional cell culture assays. The use of RT-PCRand other molecular biology techniques includingcloning and nucleic acid sequencing, the viralgenomes of a number of viruses such aspoliovirus, hepatitis A virus and noroviruses havebeen elucidated (7–9, 27, 45, 62, 63, 103, 115).With the development and implementation ofreal-time quantitative PCR, results that are bothqualitative and quantitative are possible in hoursrather than the weeks required for cell culturetechniques. Both traditional PCR and real-timePCR have provided various approaches for thereliable detection of non-culturable (e.g.noroviruses) and difficult to culture viruses (e.g.hepatitis A virus, astrovirus and rotavirus) aswell as the detection of culturable viruses (e.g.enteroviruses, adenoviruses).

Limitations of PCR for food and watersamplesThe presence of humic acids, lipopolysaccharides,glycogen, lipids and metal ions may limit the useof PCR techniques on food and water samples.These substances are commonly found in manyfood matrices and are usually concentrated insamples during virus extraction and concentrationprocedures. Humic acids bind strongly to proteinsand inhibit enzymes from functioning in PCRreactions, whilst metal ions will interfere withenzyme activity. The presence of these compoundsmay result in false negative results or non-specific

reactions. PCR is also inhibited by beef extractcommonly added in adsorption/elution concen-tration methods for viruses in water samples.

However, it is possible to remove inhibitorycompounds such as humic acid, beef extract andmetal ions from samples by using resins such asChelex-100 or hydrophilic gels such as Sephadex(112). When used in combination with spun-column chromatography, inhibitory substancesare removed from water samples. Sephadex G-200alone has also been shown to largely remove theinhibitory effects of beef extract on RT-PCR.

A number of other protocols have beendeveloped to extract viral RNA from variousfoods. Such protocols commonly require multiplesteps involving the use of various reagents suchas guanidinium thiocyanate, polyethylene glycol,phenol-chloroform and more recently Pro-Cipitateand Viraffinity (LigoChem, Fairfield, N.J.). Thesereagents have been previously reported to reducecontaminating polysaccharides from oysters andclam tissues (31, 62).

Many of the recently developed protocols forviral extraction, purification and concentrationand detection methods have not been tested onfood products, e.g. shellfish obtained from differentgeographical areas and varying seasons whereviral levels may vary. Other factors that mayinfluence viral titres in shellfish include the typesof algae and other substances consumed, glycogenlevels within shellfish tissues and the physiolo-gical state of the shellfish (98). Much of the currentwork utilising such protocols has been carried outon artificially inoculated shellfish with high titresof virus and has not been tested on shellfish thatare naturally contaminated with viruses.

Interpretation of PCR resultsPositive and negative controls must be includedwith every sample tested by PCR. If a positiveresult cannot be obtained after addition to thesample of an appropriate template, then thevalidity of negative results is in doubt. Negativecontrols ensure the samples are not crosscontaminated either by other samples oraerosolised PCR products.

Although PCR is an extremely useful tool fordetection of viruses, it will detect viable as well asnon-viable virus particles, and new or emergingviruses may not necessarily be detected. Only cellculture techniques will allow the detection ofviable and new viruses. Food processing, such aschemical or enzymatic treatment, high pressureprocessing, or UV irradiation, may result indamage to viral nucleic acids. These damagedviruses may not represent a public health threat,however the damaged nucleic acids could still beamplified by RT-PCR or PCR.

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The detection of viable and/or non-viableenteric viruses by PCR from food and watersamples is cause for concern, but does notnecessarily imply an actual risk of disease.However, in the case of non-culturable or difficultto culture viruses, a positive RT-PCR result wouldneed to be interpreted as the presumptivepresence of such viruses, since no cell cultureassays exist. Rather than a sole diagnostic tool,PCR can be used as a rapid screen for viruseswhile the more time consuming cell culture assaysare undertaken. PCR can also be used effectivelyto screen for viable viruses in cell culture. Ascheme for the detection of viruses by PCR isshown in Figure 22.3.

Quantitative RT-PCRThe first studies into quantitative methods forenumerating viruses used synthesised seeded orspiked oligonucleotides as ‘mimic fragments’(those having identical priming sites but a PCRproduct of increased molecular weight). Differentdilutions were used across several reactions andanalysed using mean probable number (MPN)methods. Such techniques would be accurate if alltarget sequences were amplified and wererandomly distributed in the sample.

The recent development of real-time PCRtechnology has brought the possibility ofenumerating viruses in foods a step closer toreality. Real-time PCR allows constantmonitoring of the generation of PCR productssimultaneously with the amplification process,allowing faster detection times and eliminatingthe need for amplicon detection by agarose gels.Quantification is achieved through the use offluorescence labelled probes, e.g. TaqMan probes,or DNA intercalating dyes such as SyberGreen,and the measurement of detected fluorescenceduring the amplification process. The use ofcomplex algorithms to establish a calibrationcurve using known viral titres enablesquantification of the viral target in a sample. Areal-time PCR method quantifying humanparvovirus using a duplex amplification with aninternal standard and two-colour fluorescencedetection, resulted in a detection sensitivity of 102 viruses/mL (55). Although the are few reportson the application of real-time PCR technology tofood and environmental samples, viral concen-tration and purification methods are currentlybeing modified for compatibility to real-time PCRtechnology (55, 91, 104).

Improving sensitivity and specificityImproved RT-PCR results may be obtained byoptimising different nucleic acid extractiontechniques. For example, most of the enteric

viruses have an RNA genome and the use of lowpH buffered phenol for nucleic acid extraction willpreferentially extract RNA, leaving proteins andDNA in the organic phase. This could lower theoverall nucleic acid ‘background’ in RT-PCRleading to more sensitive virus detection. Use ofthe cationic detergent cetyl-trimethylammoniumbromide (CTAB) and other commercially availablereagents, e.g. Pro-Cipitate, Viraffinity and TRIzolReagent (Gibco BRL, Rockville, MD), have alsoproved useful in purifying RNA from shellfishspecimens (8, 9, 31, 62). These reagents aredeveloped to precipitate nucleic acids while otherproteins and polysaccharides remain in solutionand inhibitors removed prior to analysis forenteric virus detection by RT-PCR.

Although PCR is a powerful technique, it canbe subjected to misuse and misinterpretation. Thepresence of PCR product or amplicons mayrepresent the presence of virus-specific products.However, such amplicons can also be associatedwith contaminating DNA present in the sample.Oligonucleotides used in PCR, specific to targetedviruses, can amplify contaminating DNA so longas the contaminating DNA contains a regionhomologous to the oligonucleotides. Although theoccurrence of such phenomena is rare, to rule outfalse positives, amplicons may be separated byagarose gels, and detected using digoxigenin-labelled (DIG) probes (30, 80), radiolabelledprobes (95), chemiluminescence (106, 109) orcolorimetric (19) detection systems that are suitablefor implementation in food testing laboratories.

Increased sensitivity and specificity of PCRcan also be achieved by using nested PCR or semi-nested PCR, i.e. a series of two PCR reactionswith different primer sets. The first reaction willamplify a portion of a viral genome while thesecond PCR is performed on products from thefirst reaction using oligonucleotides directedtowards an internal region of the target DNA (asshown in Figure 22.4) and is useful in assayingfood and water samples as inhibitors are dilutedfor the second PCR assay.

With the advancement of molecular biologicaltechniques, food and waterborne outbreaks can bemore adequately studied and early warningsystems could be implemented to protect publichealth and the food industry from viral disease.

Finally, despite the significant advancesmade in the detection of viruses, there are someareas of research which still need to beundertaken, including:� Determination of a practical viral indicator(s)

for the presence of enteric viruses in food andwater.

� The efficiency of concentration methods onsamples of differing type and quality.

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Figure 22.3. Scheme for nucleic acid amplification (PCR) for RNA viruses

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� The removal from food and water samples ofinhibitors of molecular and cell cultureassays.

� Methods for quantitative PCR for food andenvironmental samples.

� Methods for the extraction and detection ofviruses in food and shellfish.

Control

Sources of viruses in food include food handlers,contaminated surfaces in food handling areas,shellfish and contaminated water. Successfulprevention of foodborne illness relies on avoidingfaecal contamination of food and water. As anoverall strategy, vaccination of all food handlers

with hepatitis A virus and poliovirus vaccinesshould be encouraged to help stop the spread offoodborne hepatitis A and the (unlikely) potentialthreat of poliovirus. A rotavirus vaccine is likelyto be available within the next three years and foodhandlers should be encouraged to obtain it and anyother relevant vaccines as they become available.

Food handlersFood handlers need to be aware that entericviruses can be shed in faeces during asymptomaticphases of infection as well as during times of overtillness. Good personal hygiene practices are vitalin the prevention of foodborne viral infection.Liberal hand washing with soap, the use of iodinebased skin disinfectants (e.g. Betadine surgicalscrub) and the use of disposable gloves areessential. If vomiting occurs, noroviruses may be

Figure 22.4. Nested PCR for virus detection or confirmation of 1st round product

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spread over a large area in aerosol droplets (33,89) and therefore, uncovered food should bediscarded and the environment including worksurfaces should be thoroughly cleaned. Moreover,it is important that any food handler withdiarrhoea or vomiting must not return to work fora minimum period of 48 h after complete recovery.Training of personnel and food handlers isimportant in preventing outbreaks (66).

Environmental controlViruses can survive on surfaces and instrumentsused for food preparation for extended periods (1)and the use of carefully selected chemicaldisinfectants is important if viruses are to beinactivated and controlled in the environment.Enteric viruses are resistant to commonly usedantibacterial disinfectants (e.g. phenolics,ethanol, quaternary ammonium compounds) butare susceptible to free chlorine, iodine andaldehydes (formaldehyde and glutaraldehyde).These antiviral disinfectants need to be used withcaution as the aldehydes are potentialcarcinogens and may also damage certainsurfaces and instruments on prolonged exposure.

ShellfishIn many countries, commercial shellfish arecultured frequently in estuarine waters adjacentto populated areas. Often, these waters arecontaminated with sewage effluent and thepotential for transmission of hepatitis A virus,noroviruses and other enteric viruses exists.

In Australia, the cultivation and distributionof oysters and other shellfish is carefullyregulated. However, these regulations aredifficult and sometimes impossible to enforce.Moreover, the harvest from an individual leasemay be sent to a variety of suppliers and thendistributed in small quantities to a large numberof outlets (14). These factors, coupled with thelong incubation period of some diseases likehepatitis, make shellfish associated outbreaksvery difficult to document.

To reduce the risk of oyster borne infections,strict quality control must be observed in oystercultivation, including a period of depurationwhere oysters are held in tanks of disinfectedwater. Purification of oysters in NSW wasintroduced following the 1978 outbreaks of foodpoisoning (93) and remains a statutoryrequirement (14). For this reason, purificationtechniques are based on the immersion ofshellfish in seawater of good quality for at least 36 h, a process known as depuration.

Depuration may take place in closed loopcircuits where the seawater is continuouslydisinfected via UV lamps or in semi-open circuits

where the seawater of good quality is renewedevery 12 or 24 h. It has been shown that thesemethods, when carefully performed, yieldsatisfactory bacteriological results. However,virological results are not always satisfactory asshellfish may still contain enteric viruses afterpurification (26, 50). The fact that viral outbreaksstill occur via contaminated oysters, despiteoyster depuration, indicates that currentdepuration techniques are inadequate for theremoval of pathogenic viruses. They may have tobe combined with water disinfection techniquessuch as ozonation to be truly effective.

Since it is impossible to prevent thecontamination of coastal waters by sewageeffluent, the prevention of shellfish borne diseasesrequires bacteriological monitoring of the marineenvironment and shellfish flesh (14). Suchsurveillance allows the classification of growingareas’ suitability for harvesting and distributionof shellfish. However this surveillance is notalways sensitive enough as bacteria are a poorindicator of viral contamination of oysters (2, 14,26, 52). PCR testing for viruses in oysters and theenvironment is possible, although not a standardroutine test, and is 10–100 times more sensitivethan culture techniques (25).

Fresh produce Fresh fruits and vegetables can becomecontaminated by enteric viruses, possibly throughthe use of contaminated fertilisers or contaminatedirrigation water supplies. A range of entericviruses has been reported to survive long periodsof storage up to 60 days (1, 10, 73, 74, 101, 102,107). Chlorine has been widely used for freshproduce washing due to its availability, low costand its effectiveness on a wide range ofmicroorganisms. Chlorine based disinfectantshave been considered to be the most effectiveagainst many enteric viruses. However, chlorineresistant noroviruses, hepatitis A viruses andfeline calicivirus have been reported (32, 40, 67).It should be noted that chlorine based productsare susceptible to pH changes and requireconstant monitoring to maintain optimal efficacy.Ozone is another alternative that has been shownto be effective on a number of microorganismsincluding enteric viruses (36, 69). Ozone is apowerful oxidising agent and highly reactive, doesnot leave any residues on/in food and water andnaturally decomposes into oxygen. Disadvantagesof using ozone include its instability and that itcannot be stored and has therefore to begenerated on-site. A number of other alternativedisinfectants, including organic acids andsurfactants, have also been used with variablerates of success (3, 100, 101, 108, 116).

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Viral inactivation by non-thermalprocessing techniques

With the increasing global demand for freshproduce and seafood, there is added pressure onsuppliers to provide good quality and safe foods.Heat treatments have been used withconsiderable success in inactivating mostmicrobial pathogens including viruses. However,fresh produce and shellfish are consumed raw andhave a relatively short shelf life. Moreover, heattreatments are inappropriate for viralinactivation for these products.

Non-thermal processing techniques such asgamma irradiation and high hydrostatic pressureprocessing offer possibilities both for foodprocessing and microbial and viral inactivation.These processes have various advantagesincluding rapid application and minimalalterations to the food’s taste, odour and texture,resulting in the retention of freshness. Althoughsuch processes are currently expensive, they arean appealing alternative to thermal processing.Gamma irradiation offers a safe alternative forthe decontamination of food, especially for freshproduce (110, 111). There are currently nostandards on allowable gamma irradiation doses,based on the nutritional, toxicological andmicrobiological data, for use on various foods.Bidawid et al. (13) provides one of the few reportson viral inactivation from the use of gammairradiation on foods. These authors treated lettuceand strawberries that were inoculated withhepatitis A viruses using varying doses of gammairradiation. Their data indicated that gammairradiation doses between 2.7 and 3.0 kGy wererequired to achieve at least 90% (1 log10 reduction)inactivation of hepatitis A virus in lettuce andstrawberries respectively. Gamma irradiation iscurrently not used in Australia for processing offoods and more work is required to determine itsefficacy on fresh produce and shellfish.

High hydrostatic pressure processing (HPP)has been used to inactivate various problematicmicroorganisms, including viruses, in foods (70,86, A. Lee - unpublished results). HPP is rapid,and pressure is applied uniformly resulting inlittle damage to the product with minimalchanges to the physical properties of foods. HPPhas been used on shellfish to eliminate Vibrio spp.(87) providing shelf life extension for shuckedoysters. Treatment of shellfish at pressuresranging from 250 MPa to 400 MPa does not affectthe taste of raw shellfish and minimises handlingand wastage in oyster and clam shucking.

The effects of HPP in inactivating entericviruses is not well studied and preliminary results

have shown that feline calicivirus and hepatitis Avirus acting as model viruses for the (non-culturable) noroviruses, could be inactivated inbuffer suspensions at pressures between 300 MPaand 450 MPa (70, A. Lee - unpublished results).However, other studies on polioviruses showedresistance to pressures up to 600 MPa (70, 113). Itis clear that the effects of HPP on viruses invarious food matrices such as oyster homogenatesor whole oysters need further study.

Challenges for the future

Over the next ten years there will be manychallenges facing suppliers, retailers, regulatorsand researchers involved in food and watervirology. Complacency amongst food handlers willalways be an ongoing challenge and the need forcontinuing education and scrupulous personalhygiene will be paramount in the control of mostfoodborne disease of viral origin in thecommunity. Other needs likely to be addressed orrequiring vigilance include:� The promotion of relevant available viral

vaccines and proper hygiene training for foodhandlers.

� The development of guidelines forimmunocompromised persons, e.g. shouldraw foods, particularly shellfish, be avoided?

� The development of novel non-thermalprocessing techniques and processingparameters for the inactivation of viralpathogens from high risk foods to improve theshelf life of such foods.

� Research and monitoring of prion diseases toavoid their entry into the food chain.

� Monitoring of polluted water environmentsand any pollution sources identified and ifpossible neutralised. Sewage treatmentmethods should be upgraded to removeviruses from entering waterways. Shellfishshould not be harvested from polluted waters.

� Ensuring that recycled effluent for potableand non potable uses is free from known andunknown viruses and prions. Membrane andchemical disinfection processes will be thelikely key to remove these agents.

� The use of improved oyster depurationpractices possibly involving centraliseddepuration and the use of ozone or similar asa disinfectant.

� The development of risk assessment modelsfor viruses in food and water.

� The development and standardisation ofmolecular biology techniques for the rapiddetection of enteric viruses, particularly the

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development of PCR tests that indicate virusviability and that can be used quantitatively.

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Chapter 22 · Chapter 22 Viruses, Food and Environment Gary Grohmann and Alvin Lee 561 Introduction...

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