key: cord-0040676-6nzsu5jf
authors: Bosch, A.; Pintó, R.M.; Lees, D.H.; von Bonsdorff, C.-H.; Croci, L.; De Medici, D.; Le Guyader, F.S.
title: Detecting virus contamination in seafood
date: 2014-03-27
journal: Improving Seafood Products for the Consumer
DOI: 10.1533/9781845694586.3.194
sha: 172d847e8d6812853296dfc5f732dd0a0671f9b1
doc_id: 40676
cord_uid: 6nzsu5jf

nan

Viruses are obligate intracellular parasites depending on living cells for replication and usually infecting only a restricted range of hosts. Thus viruses cannot multiply in dead or processed food and therefore are not responsible for food spoilage. Human viruses may contaminate seafood but since the commercially exploited species (fish and shellfish) are widely divergent from humans, there is no evidence that they can act as a replication vector. Viral problems are thus limited to the role of seafood in passive transfer of viruses to humans. The viruses most adapted and likely to be carried in this way are those transmitted by the fecal-oral route. These include viral agents causing gastrointestinal disease in humans but also agents such as hepatitis A virus and polio virus which although being transmitted by the fecal-oral route, and often having a growth phase in the gut, exhibit their classical clinical symptoms elsewhere in the body. Such viruses can contaminate seafood at source through fecal pollution of the aquatic environment, or through poor hygiene during seafood processing ( Fig. 12.1 ). Many viruses transmitted by the fecal-oral route are widely prevalent in the community and infected individuals can shed many millions of virus particles in their feces. Consequently viruses of many types occur in large numbers in municipal sewage and may also occur in other sources of human fecal contamination (Bosch, 1998) . Sewage treatment processes are generally only partially effective at virus removal (depending on the treatment level) and may also be bypassed during periods of heavy rain or during emergencies (see Chapter 13). Other sources of fecal pollution, for example septic tank overflows or boat discharges, may also contribute to marine contamination. Following marine discharge from such sources, viruses are capable of survival for long periods (Callahan et al., 1995; Gantzer et al., 1998; Nasser, 1994) . Thus, seafood harvested from coastal locations is vulnerable to contamination with enteric viruses of potential health significance for man (Fig. 12.1) . However, of the many harvested seafood species, only the filter-feeding bivalve molluscan shellfish (bivalve molluscs) have consistently proven to be an effective vehicle for the transmission of human viral diseases (Lees, 2000) .

Bivalve molluscs are a type of shellfish that have two shell halves which hinge together. Species commonly commercially exploited in Europe include the native or flat oyster (Ostrea edulis), pacific oyster (Crassostrea gigas), common blue mussel (Mytilus edulis) and Mediterranean blue mussel (Mytilus galloprovincialis), cockles (Cerastoderma edule), king scallops (Pecten maximus) and queen scallops (Chlamys opercularis), and various clams including the native clam or palourde (Tapes descussatus), the hard shell clam (Mercenaria mercenaria), the manila clam (Tapes philippinarum), and the razor shell clam (Ensis spp.). The bivalve molluscs are an effective vehicle for transmission of enteric disease agents for several reasons. A principal factor is that these animals obtain their food by filtering small particles from their surrounding water. In the process of filter-feeding, bivalve molluscs may also concentrate and retain human pathogens derived from sewage contamination of growing waters. Many bivalve molluscs are harvested from sheltered in-shore coastal locations, such as river estuaries, which are often also susceptible to fecal pollution (see Chapter 13). Important additional compounding factors are that a number of species of bivalve mollusc are traditionally consumed raw, or only lightly cooked, and are also consumed whole, i.e. including the viscera, which contains the bulk of contaminants. Disease incidents are most commonly associated with species consumed raw (or lightly cooked) and whole, such as oysters and clams and infrequently, or not at all, with species that are well cooked and where the viscera is not consumed, such as scallops. Disease incidents associated with bivalve molluscs have been extensively reported and have been reviewed by several authors (Richards, 1985; Rippey, 1994; Jaykus et al., 1994 , Lees, 2000 . The potential of bivalve molluscs to transmit enteric pathogens acquired through sewage pollution of growing areas first became recognised in the late 19th and early 20th century with numerous outbreaks of typhoid fever in several European countries, the US and elsewhere (Allen, 1899). Since this time there has been increasing recognition of the importance of human enteric viruses as the predominant aetiological agent in human illness incidents associated with consumption of bivalve molluscs. It is now well recognised that the most common illness associated with bivalve mollusc consumption is gastroenteritis caused by Norovirus (NoV). Other gastro enteric viruses, such as astroviruses and parvoviruses, have also occasionally been implicated in shellfish-related outbreaks, although their true epidemiological significance is not clear. NoV causes a relatively`mild' gastroenteritis, often including nausea, diarrhoea, vomiting, fever and abdominal pain. The incubation period is 1 to 4 days with a duration of about 2 days and generally followed by complete recovery. NoV has previously been known as Norwalk-like virus or as small round structured virus (SRSV).

Human NoV cannot be propagated using cell culture (Duizer et al., 2004) therefore characterisation and classification has been achieved largely using molecular techniques. It is now known that the Norovirus genus belongs to the Caliciviridae family and comprise a genetically diverse group of viruses which can be separated beneath this level into genogroups, clusters or genotypes, and individual strains (Zheng et al., 2006; Hansman et al., 2006) . NoVs infecting humans group into genogroup one (up to 8 clusters), and genogroup two (up to 17 clusters) (Zheng et al., 2006) . Genetically related animal NoV strains have also been described (Oliver et al., 2006) ; however, currently there is no evidence that they are capable of directly infecting man. The genetic diversity of NoV strains presents a difficult challenge for the design of molecular diagnostics capable of detecting all strains of health significance. It is now generally accepted that NoV is one of the most common causes of infectious intestinal disease in both outbreaks and in the community (Evans et al., 1998; Tompkins et al., 1999) . Infections occur in all age groups including older children and adults. NoV is highly transmissible and often becomes noticeable through epidemic spread of diarrhoea and vomiting in closed communities such as hospitals, cruise and military ships and old people's homes. NoV appears to be prevalent throughout the world. Infected individuals shed large amounts of virus in their faeces thus domestic sewage and other polluted waters can normally be expected to be heavily contaminated with this virus , with the obvious risks for impacted bivalve molluscs. There are numerous reports in the scientific literature documenting the occurrence of NoV gastro enteric illness outbreaks associated with consumption of bivalve mollusc. These have been reviewed by several authors (Jaykus et al.,1994; Lees, 2000) and continue to occur (Doyle et al., 2004) . The need for measures to more adequately protect the consumer against viral infection are widely noted in such outbreak reports. US FDA risk assessments estimate cases of NoV mediated gastroenteritis related to seafood consumption at some 100,000 per year (Williams and Zorn, 1997) . In addition to these direct health consequences, bivalve molluscs may also present a potent vector for emergence of recombinant NoV strains of enhanced virulence following contamination with multiple strains from human (and potentially animal) sources. Mixed human infections following consumption of bivalve molluscs with multiple contaminating NoV strains has been commonly reported (Gallimore et al., 2005; Prato et al., 2004; Kageyama et al., 2004) .

The other fecal-oral transmitted virus of major significance in bivalve molluscs related outbreaks is hepatitis A virus (HAV). HAV is a positive-strand RNA virus classified in its own genus of Hepatovirus within the Picornaviridae. There is only a single major serotype of HAV with three human antigenic variants and a number of genotypes identified by sequence analysis (Costa-Mattioli et al., 2003) . Compared to other enteric viruses HAV has an extended incubation period of about 4 weeks (range 2 to 6 weeks) and causes a serious debilitating disease progressing from a non-specific illness with fever, headache, nausea and malaise to vomiting, diarrhoea, abdominal pain and jaundice. HAV is self-limiting and rarely causes death but patients may be incapacitated for several months. Age has an important bearing on the severity of the infection with young children frequently experiencing only mild illness whereas overt hepatitis develops in the majority of infected adults. Recovery is complete and leads to long-term immunity from reinfection. HAV is a common endemic infection in developing countries with most children being seropositive by six years of age. However improving sanitary conditions in developed countries have lead to declining prevalence and resulted in large sectors of the population being susceptible to infection. HAV can be readily demonstrated in stools by molecular techniques (Yotsuyanagi et al., 1996) and has also been demonstrated in sewage effluents and polluted receiving waters (Tsai et al., 1994) . Thus bivalve molluscs have frequently been implicated as food vehicles in outbreaks of hepatitis A (Klontz and Rippey 1991; Conaty et al., 2000; Bosch et al., 2001) .

With the advent of molecular diagnostic methods in more recent years the polymerase chain reaction (PCR) has been used to study the contamination of molluscan shellfish with NoV and HAV at the low concentrations found in field samples. Various studies have shown rather high rates of viral contamination of commercially produced bivalve shellfish placed on the market in a number of different countries (Costantini et al., 2006; Cheng et al., 2005; Chironna et al., 2002; Formiga-Cruz et al., 2002; Nishida et al., 2003; Boxman et al., 2006) illustrating the potential health hazards and the urgent need for diagnostic methods capable of identifying the risk and better protecting the consumer. The REFHEPA project within the SEAFOODplus consortium is aimed at the development of standardised procedures for the detection of HAV and NoV in bivalve molluscan shellfish to the point at which they could be used successfully in a routine diagnostic context to increase the safety of shellfish delivered to the consumer. The methodologies developed in the REFHEPA project have been successfully applied in another project, REDRISK, to detect viruses in naturally contaminated shellfish from France and Spain.

Viruses are present in shellfish in very low numbers; however, in sufficient amounts to pose a health risk (Bosch et al., 1994; Sa Ânchez et al., 2002 , Le Guyader et al., 2003 , 2006a . This low contamination makes necessary the development of highly sensitive viral extraction methods ensuring the virus recovery from shellfish tissues. The hypothesis made in the 1980s, that viruses are concentrated in digestive diverticulum tissues (Metcalf et al., 1980) , represented a major step for the progress of the extraction methodologies. This hypothesis was later confirmed by detection of HAV using an in situ system in oysters artificially contaminated following virus bioaccumulation (Romalde et al., 1994) as well as through the tissue-specific quantification of infectious enteric adenoviruses and rotaviruses in mussels previously contaminated by bioaccumulation of such viruses (Abad et al., 1997a) . Additionally, a very interesting result has recently been described: the occurrence of a specific binding of Norwalk virus to oyster digestive tissues through the interaction with a N-acetylgalactosamine-containing receptor (Le Guyader et al., 2006b) . Analysis of digestive tissues provides several advantages, including increased sensitivity, decreased processing time and decreased interference with RT-PCR (Atmar et al., 1995) . Focusing the analysis of shellfish on the digestive tissues, where many of the viruses are concentrated, enhances assay performance by eliminating tissues (i.e. adductor muscle) that are rich in inhibitors but contain relatively little virus (Abad et al., 1997a) . As a matter of fact, the digestive tissues represent about one tenth of the total animal weight for oysters and mussels. Except for small species, such as clams or cockles, because dissection may be technically difficult, most of recent methods are based on dissected tissues and thus will be discussed here. Methods cited in the literature are diverse, complex, poorly standardised and restricted to a few specialist laboratories. It seems obvious that quality control and quality assurance issues must be solved, as well as simplification and automatation, of molecular procedures before they could be adopted by routine monitoring laboratories. All these latter issues have been addressed in the REFHEPA project of SEAFOODplus.

Extraction of enteric viruses from shellfish is based on several steps: virus elution from shellfish tissues, recovery of viral particles, and then virus concentration. The weight analysed generally ranges from 1.5 to 2 g of digestives tissues. Some recent methods propose larger weights for the first step but thereafter analysing only part of them (Boxman et al., 2006) . Viruses are eluted from shellfish digestive tissues using various buffers (i.e. chloroformbutanol or glycine) before being concentrated either by polyethylene glycol precipitation or ultracentrifugation (Atmar et al., 1995; Nishida et al., 2003; Myrmel et al. 2004) . Those approaches used in the analysis of whole shellfish meat, such as acidic adsorption prior to virus elution, are not applied to dissected tissues (Shieh et al., 1999; Mullendore et al., 2001) . When working on dissected tissues, and applying molecular techniques, direct lysis of virus particles can also be used. For example, proteinase K, or Trizol and lysis of shellfish tissues using Zirconia beads and a denaturing buffer have all been used for virus and/or nucleic acid elution (Lodder-Verschoor et al., 2005; Jothikumar et al., 2005) . A disadvantage of this direct approach is that a lower quantity of shellfish tissue is analysed in the RT-PCR assay.

Since the most relevant shellfish-borne viral pathogens, enteric hepatitis viruses A and E and noroviruses, are non-culturable RNA viruses, RT-PCR and now real-time RT-PCR are the methods of choice to set up sensitive protocol for their detection. The methods used for nucleic acid extraction are dependent on those used for virus elution and concentration. Most methods are based on guanidium extraction either using the methods described by Boom et al. (1990) or using a kit, based on similar chemistry (QIAamp or RNeasy kit by Qiagen Õ ) (Shieh et al., 1999; Loisy et al., 2000; Schwab et al., 2000) . Capsid lysis by proteinase K and then purification of nucleic acid using phenol-chloroform and CTAB precipitation is a more labour-intensive but was one of the first successful methods described (Atmar et al., 1993) . One of the goals of the extraction methods is to remove inhibitors of the RT and PCR reactions sufficiently to allow detection of viral nucleic acids. Polysaccharides present in shellfish tissue are at least one substance that can inhibit the PCR reaction (Atmar et al., 1993) . Several reported methods eliminate inhibitors to varying degrees, although no systematic evaluation of the efficiency of inhibitor removal has been performed, and only a few of them have been applied on naturally contaminated shellfish. Inhibitor elimination is difficult to evaluate and depending on the time of the year and shellfish life, different compounds may be present (Di Giralimo et al., 1977; Burkhardt and Calci, 2000) . Internal control standards have been used to detect the presence of significant sample inhibition, and the amount of sample inhibition has varied depending upon the shellfish tissue being analysed (Atmar et al., 1995; Schwab et al., 1998; Le Guyader et al., 2000) . Dilution of the extracted sample is the approach often used to overcome the inhibitor problem, leading to a smaller quantity of shellfish tissues being analysed. For most methods in the literature, the weight of digestive tissues analysed in each RT reaction varies between 0.01 g and 2.5 g. The method analysing the smallest shellfish tissue weight (0.01 g) is based on direct lysis of virus without a concentration step (Jothikumar et al., 2005) , while the method analysing the largest tissue weight (2.5 g) is based upon direct extraction of all nucleic acids followed by purification of nucleic acid using a poly A capture (Goswami et al., 2002) . It is important to promote methods allowing the biggest tissue analysis as it helps to improve the detection sensitivity.

Beside the inhibitor problem, molecular analysis of viruses in shellfish samples includes other frequent difficulties such as low virus concentrations, and sequence variation. As the extraction-concentration procedure is not virus specific, the nucleic acid of several viruses can be extracted at the same time. RT-PCR must be performed under stringent conditions and confirmed by hybridisation. The first important step for sensitivity and specificity is the synthesis of the complementary DNA (cDNA) by reverse transcription (RT). Most assays utilise a virus-specific primer in the RT reaction (Atmar et al., 1995; Le Guyader et al., 2000; Kingsley et al., 2002; Formiga-Cruz et al., 2002; Sa Ânchez et al., 2002; de Medici et al., 2004; Myrmel et al., 2004; Boxman et al., 2006) but random hexamers are also used in some assays (Chung et al., 1996; Green et al., 1998; Cheng et al., 2005) . PCR amplification is usually performed for at least 40 cycles; some methods use nested PCR formats with fewer than 40 cycles in the first amplification reaction. Probe hybridisation is then performed as a confirmation step and enhances both assay sensitivity and specificity (Atmar et al., 1995; Chung et al., 1996; Shieh et al., 1999; Le Guyader et al., 2000; Sa Ânchez et al., 2002; Costantini et al., 2006) .

Sometimes it is necessary to analyse the amplified sequence in order to characterise the viral strains, and virus-specific amplicons must be sequenced to obtain additional information about the virus(es) present in the sample. This is particularly important for NoV detection, due to its wide strain diversity. However, sequence analysis is hampered by the scarce product sometimes obtained after PCR amplification from shellfish tissues. One of the limitations in developing RT-PCR assays for the detection of NoV has been the selection of proper primer and probe combinations that allow the detection of most or all strains of concern. The high genetic diversity of NoV makes it necessary to use broadly reactive primers. Despite several improvements in the methodology, up to now no single primer set is able to amplify all strains (Atmar and Estes, 2001; Vinje et al., 2003) . In the absence of such a universal primer set, multiple sets increase the chance to detect a greater number of strains, and the homology of the primers with the NoV strain is important in terms of sensitivity (Le Guyader et al., 1996a , 2000 , 2006a . No single assay stands out as the best by all criteria, such as evaluation of sensitivity, detection limit and assay format, not even for the stool analysis being clearly more difficult in the case of shellfish samples with such very low contamination (Atmar and Estes, 2001; Vinje et al., 2003) . For example, in three outbreak reports, primer sets targeting different regions of the NoV genome were needed to be able to amplify the strain both in clinical or environmental samples Le Guyader et al., 1996b , 2006a .

For HAV, primer selection is easier since the degree of variation, particularly in the 5 H non-coding region, is significantly lower (Sa Ânchez et al., 2004; Costafreda et al., 2006) . However, when genotyping is required other regions must be analysed such as the VP1X2A junction (Robertson et al., 1992; Sa Ânchez et al., 2004) or even larger fragments. However, the low virus concentration very often hampers the amplification of such large fragments.

Real-time RT-PCR assays, in which the RT, PCR and hybridisation assays are combined in a single well, are being developed and used successfully to detect enteric viruses in shellfish (Nishida et al., 2003; Loisy et al., 2005; Jothikumar et al., 2005; Costafreda et al., 2006) . This technology takes advantage of not merely detecting but also quantifying the viruses present in the sample. However, for this last purpose it is necessary not only to develop but also to standardise the methodologies by including several controls at those most critical steps, let's say the nucleic acids extraction and the RT reaction (Costafreda et al., 2006) . The efficiency of the virus nucleic acids extraction must be controlled by means of a model virus while the efficiency of the RT-PCR reaction must be traced by means of a RNA molecule amplifiable and detectable with the same combination of primers and probes as those used for the actual virus. When these two reagents are added at known concentrations their recovery can be measured. Costafreda and colleagues (2006) proposed the use of the Mengo virus to evaluate the nucleic acid extraction efficiency, in general for any enteric virus and in particular for HAV, while the RNA molecule should be specific for each assay. The use of such an internal RNA control for the evaluation of the molecular reactions inhibition has been extensively used even in qualitative assays (Schwab et al., 1998; Le Guyader et al., 2003) .

Regarding other viral pathogens, such as rotaviruses and astroviruses, an interesting alternative exists based on their capability of replication in some tissue culture systems, such as the CaCo-2 cells, which represents a universal in vivo amplification system for the enteric viruses (Pinto  et al., 1994) combined with either molecular (Abad et al., 1997b; Pinto  et al., 1994 Pinto  et al., , 1996 Pinto  et al., , 1999 or immunological (Abad et al., 1998; Bosch et al., 2004) detection methods. Other cell culture molecular integrated systems have been proposed for enteroviruses (Reynolds et al., 1996) . Interestingly, these combinations allow the quantification of infectious viruses (Abad et al., 1997a) . However, although these techniques have been satisfactory evaluated and used in water samples, their application in shellfish is not common due to the infrequent shellfishborn viral outbreaks other than enteric hepatitis and norovirus gastroenteritis.

In summary, the quantitative assays open a new view in terms of analysis of the sanitary risks associated to the consumption of virus contaminated shellfish.

The gut as a`factory' of viruses is strongly selective. On one side, the alimentary tract with strong salivary enzymatic activity together with large pH shifts followed by bile acids and pancreatic enzymes aiming at breaking down foodstuff into its smallest components sets harsh conditions for a virus to survive. Most of the enteric viruses are small, non-enveloped RNA-viruses possessing an icosahedral capsid. However, an increasing number of enveloped viruses have also emerged that are capable of surviving the enteric route.

The abundance of viruses in the gut offers exceptionally favorable conditions for genetic modifications of enteric viruses. Not only mutations, but recombinations and reassortments may facilitate the appearance of new variants of the already recognised viruses. An example of this is the recent appearance of a new variant of the well-known GII.4 type NoV. Within weeks it was able to spread via a variety of epidemiological routes throughout the world causing extensive outbreaks. This pandemic also demonstrates how fast an enteric pathogen may spread. It emphasises the threat posed by a pathogen of high medical impact, too.

There is only a semantic difference between a virus called`new' and just a genetically modified old one. For practical reasons, a virus is`new' when the population immunity is missing totally or to a considerable part. An example would be the above mentioned GII.4 new variant NoV. Owing to their error prone polymerase, viruses that possess a ssRNA genome are constantly modified by mutations and may lead to strains or variants of high virulence. An example of this was the poliovirus type 3 that caused an outbreak in a vaccinated population in Finland (Hovi et al., 1986) . Human rotaviruses, having a segmented genome, can undergo genetic changes through interchange of RNA segments, i.e. give rise to reassortants. This has been demonstrated clearly among group A rotaviruses (Maunula and von Bonsdorff, 2002) . Waterborne outbreaks caused by group A rotaviruses have been detected (Villena et al., 2003; Divizia et al., 2004) but it is not known to which extent that virus possibly was modified. Rotaviruses of group B cause extensive outbreaks among adults, which appear to be restricted almost exclusively to China (Hung et al., 1984) . Also in China new unclassified rotaviruses have emerged causing outbreaks that are still only poorly defined (Yang et al., 2004) .

Rotaviruses of group C have been involved in cases of gastroenteritis throughout the world, both in sporadic cases and in outbreaks (Jiang et al., 1996; Brown et al., 1989) . However, in general, the rotavirus C infections seem to be rare (Abid et al., 2007) . Group C viruses are also found in animals, preferentially in pigs. The porcine strains are, however, not identical to the human ones. For both rotaviruses belonging to groups B and C there is the potential that they may undergo changes that could increase their pathogenicity. Some zoonotical agents have caught a lot of attention due to their potential to spread emerging infections. One of these agents is the severe acute respiratory syndrome (SARS, Peiris et al., 2005) . The causative virus belongs to the family Coronaviridae and is able to overcome the harsh alimentary tract conditions and is excreted in stools. However, whether this observation indicates an effective infection route for SARS remains to be determined, and thus a risk for seafood safety seems presently rather remote.

Another group of emerging viruses that has evoked a lot of attention are the highly pathogenic avian influenza viruses (HPAI), preferentially the ones classified as H5N1 and H7N3, reviewed by Horimoto and co-workers (2005).

These viruses have evolved from viruses of low pathogenicity by a mutation in the cleavage site of the hemagglutinin protein. This site is affected both by the local protein and the carbohydrate moieties (Stieneke-Gruber et al., 1992; Kawaoka and Webster, 1988) . The trypsin specific cleavage is changed to a less specific protease cleavage (Li et al., 2004; Glaser et al., 2005) . In seabird colonies, among which all known influenza A viruses reside, they seem to cause very little harm. Such pathogenic strains emerge occasionally, as is the case at the time of the writing of this chapter. In birds the viral infection is enteric, i.e. the virus is secreted in the chloaca. Especially waterfowl, such as ducks and other dabblers, that reside and excrete the virus in shallow waters are of importance (Markwell and Shortridge, 1982) . The inactivation of the viruses in water is rather slow lasting from weeks to months, depending on the conditions (Stallknecht et al., 1990) . Thus the viruses in water pose an infection risk for humans, too. The HPAI viruses show a varying pathogenecity among bird species. In general they cause mass death among cultured fowl like chicken, geese and turkey. Among wild bird species the pathogenicity varies. The reason for additional concern is the fact that they may infect humans and that the infections concur with a high mortality, up to 50%. The infections in man are, however, rare due to the receptor distribution in the respiratory pathway. It appears, that the`right' sialic acid construction is found only in alveolar cells, not in nasopharynx (2,3-vs 2,6-sialic acid bond) (Matrosovich et al., 2004) . Thus only directly inhaled viruses that reach the susceptible cells will lead to an infection.

Although an infected duck may excrete as much as 1,010 infectious doses per day, and the virus is able to survive in contaminated waters as long as 4 days at 22 ëC and 30 days at a 0 ëC, the risk to acquire the infection through bathing in contaminated waters has been estimated to be negligible (WHO, 2006) . Like HAV, hepatitis E virus (HEV) replicates in the gut epithelium. It evolves, however, into a systemic infection mostly affecting the liver. The disease is similar to that of HAV-infection with the exception of its devastating effect on pregnant women. Up to 20±30% of them succumb as a consequence of the infection (Khuroo, 1980) . It is still unclear what causes this high mortality. Large waterborne outbreaks caused by HEV have been reported originally from India, but the virus circulates widely in tropical and subtropical areas. HEV underwent several classification steps before it was placed into its present own family Hepeviridae (Reyes et al., 1990; Tam et al., 1991) . Most of the human hepeviruses belong to one serogroup, although a genome-based division into four genotypes has been defined (Schlauder and Mushahwar, 2001) . Apart from the human HEVs, they have also been broadly found among animals, most commonly among swine (Meng et al., 1997; Tei et al., 2003) . The swine HEV appear in three clusters, in two of these, human cases have been identified. The swine farms when contaminated provide a rich source of HEV with direct close contact to man but will also enter the circulation via water. The detection of porcine HEV in cases of human disease (van der Pool et al., 2001; Meng et al., 2002; Tamada et al., 2004; Li et al., 2006) indicates that this threat is real.

Nipah virus is a newly recognised zoonotic virus. The virus was 'discovered' in 1999 'discovered' in (Chua et al., 2000 . It has caused disease in animals and in humans, through contact with infectious animals. The virus is named after the location where it was first detected in Malaysia. Nipah is closely related to another newly recognised zoonotic virus called Hendra virus, named after the town where it first appeared in Australia. Both Nipah and Hendra are members of the virus family Paramyxoviridae (Eaton, 2001) . Although members of this group of viruses have only caused a few focal outbreaks, the biologic property of these viruses to infect a wide range of hosts and to produce a disease causing significant mortality in humans has made this emerging viral infection a public heath concern. In symptomatic cases, the onset is usually with`influenza-like' symptoms, with high fever and muscle pains (myalgia). The disease may progress to inflammation of the brain (encephalitis) with drowsiness, disorientation, convulsions and coma. Fifty percent of clinically apparent cases die. It is unlikely that Nipah virus is easily transmitted to man, although previous outbreak reports suggest that Nipah virus is transmitted from animals to humans more readily than Hendra virus. Pigs were the apparent source of infection among most human cases in the Malaysian outbreak of Nipah, but other sources, such as infected dogs and cats, cannot be excluded. Human-to-human transmission of Nipah virus has not been reported. The low stability of the paramyxovirus virions makes the shellfishborne transmission of Nipah virus an unrealistic possibility.

Advances in the detection tools for the`classic' enteric virus pathogens (rotavirus, astrovirus, adenovirus and norovirus) also evidenced the occurrence of a variety of other agents such as Aichi virus, belonging to genus Kobuvirus within the Picornavirus family, and picobirnavirus in the Birnaviridae family. Their apparent rather limited circulation or low pathogenicity for man may be just temporary. With the increasing spread and efficiency by which especially food-and waterborne viruses are propagated all over the world, one can foresee the emergence of some of them as pathogens with more serious impact on the disease burden.

Another important issue in the emergence and re-emergence of viruses is their potential implication in bioterrorism. Apart from the airborne route of infection, the most damaging spread of a pathogen is achieved if (drinking) water can be contaminated. This also applies for possible contamination of molluscs. For this purpose, viruses normally not transmitted through water or food may be employed, smallpox being an obvious candidate. However the potential of poliovirus as a bioterrorism weapon in a future immunologically naõ Ève population if poliomyelitis is finally eradicated should not be underestimated.

One key element in reducing foodborne spread of viruses is the implementation of surveillance, controls on the products before the commercialisation and awareness. Additionally, consumer-information campaigns must be strengthened, including the promotion of suitable procedures of food preparation and consumption.

The REFHEPA project (SEAFOODplus) has produced methods for the detection of HAV and NoV in bivalve molluscan shellfish to the point at which they could be included in regulatory standards for viruses in molluscan bivalves which would greatly increase the safety of these products for public consumption.

School outbreak of gastroenteritis due to atypical rotavirus

Selective accumulation may account for shellfishassociated viral illness

Comparative survival of hepatitis A virus, poliovirus and indicator viruses in geographically diverse seawaters

Norovirus contamination found in oysters worldwide

Detection of hepatitis A virus in mussels from different sources marketed in Puglia region

Detection of human enteric viruses in oysters by in vivo and in vitro amplification of nucleic acids

Hepatitis A in New South Wales, Australia from consumption of oysters: the first reported outbreak

Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples

Human and animal enteric caliciviruses in oysters from different coastal regions of the United States

Reverse transcription booster PCR for detection of noroviruses in shellfish

Ionic binding, the mechanism of viral uptake by shellfish mucu

Waterborne gastroenteritis outbreak in Albania

Norovirus foodborne outbreaks associated with the consumption of oysters from the Etang de

Laboratory efforts to cultivate noroviruses

Introduction to current focus on Hendra and Nipah viruses

General outbreaks of infectious intestinal disease in England and Wales

Distribution of human virus contamination in shellfish from different growing areas in Greece

Multiple norovirus genotypes characterised from an oyster-associated outbreak of gastroenteritis

Influence of environmental factors on the survival of enteric viruses in seawater

A single amino acid substitution in 1918 influenza virus hemagglutinin changes receptor binding specificity

A polymerase chain reaction based method for the detection of hepatitis A virus in produce and shellfish

A nested reverse transcriptase PCR assay for detection of small round structured viruses in environmentally contaminated molluscan shellfish

Genetic and antigenic diversity among noroviruses

Influenza: lessons from the past pandemics, warnings from current incidents

Outbreak of paralytic poliomyeleitis in Finland: widespread circulation of antigenically altered poliovirus type 3 in a vaccinated population

Waterborne outbreak of rotavirus diarrhea in adults in China caused by a novel rotavirus

Human Enteric Pathogenic Viruses

Outbreaks of gastroenteritis in elderly nursing homes and retirement facilities associated with human caliciviruses

Rapid and sensitive detection of noroviruses by using TaqMan based one-step reverse transcription-PCR assays and application to naturally contaminated shellfish samples

Coexistence of multiple genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to Norovirus in Japan

Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells

Study of an epidemic non-A, non-B hepatitis. Possibility of another human hepatitis virus distinct from post-transfusion non-A, non-B hepatitis

Detection of both hepatitis A virus and norwalk-like virus in imported clams associated with food-borne illness

Epidemiology of molluscan-borne illnesses

Viruses and bivalve shellfish

Evaluation of a degenerate primer for the PCR detection of human caliciviruses

Detection and analysis of a Small Round-Structured virus strain in oysters implicated in an outbreak of acute gastroenteritis

Three-year study to assess human enteric viruses in shellfish

A semi-quantitative approach to estimate Norwalk-like virus contamination of oysters implicated in an outbreak

Detection of noroviruses in an international gastroenteritis outbreak linked to oyster consumption

Norwalk virus specific binding to oyster digestive tissues

Genesis of highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia

Seroprevalence of hepatitis E virus infection, rural Southern Peoples Republic of China

Presence of noroviruses and other enteric viruses in sewage and surface waters in the Netherlands

Yearround screening of non commercial and commercial oysters for the presence of human pathogenic viruses

An improved method for the detection of Norwalk-like caliciviruses in environmental samples

Realtime RT-PCR for norovirus screening in shellfish

Possible waterborne transmission of influenza viruses in domestic ducks

Human and avian influenza viruses target different cell types in cultures of human airway epithelium

Frequent reassortments may explain the genetic heterogeneity of rotaviruses: analysis of Finnish rotavirus strains

A novel virus in swine is closely related to human hepatitis E virus

Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries

Improved method and test strategy for recovery of enteric viruses from shellfish

Improved method for the recovery of hepatitis A virus from oysters

Detection of enteric viruses in shellfish from the Norwegian coast

Detection, quantitation, and phylogenic analysis of noroviruses in japanese oysters

Genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses

Aetiology of SARS

Use of the colonic carcinoma cell line CaCo-2 for in vivo amplification and detection of enteric viruses

Detection of infectious astroviruses in water

Detection of fastidious infectious enteric viruses in water

Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy

Isolation of a cDNA from a virus responsible for enterically transmitted non-A, non-B hepatitis

Detection of infectious enteroviruses by an integrated cell culture-PCR procedure

Outbreaks of shellfish-associated enteric virus illness in the United States: requisite for development of viral guidelines

Infectious diseases associated with molluscan shellfish consumption

Genetic relatedness of hepatitis A virus strains recovered from different geographical regions

In situ detection of hepatitis A virus in cell cultures and shellfish tissues

Molecular characterization of hepatitis A virus isolates from a transcontinental shellfish-borne outbreak

Molecular detection and typing of hepatitis A virus

Genetic heterogeneity of hepatitis E virus

Improvements in the RT-PCR detection of enteric viruses in environmental samples

Development of methods to detect``Norwalk-Like Viruses'' (NLVs) and Hepatitis A virus in delicatessen foods: application to a food-Borne NLV outbreak

A method to detect low levels of enteric viruses in contaminated oysters

Detection of Norwalk-like virus in shellfish implicated in illness

Persistence of avian influenza viruses in water

Influenza virus hemagglutinin with multiple cleavage sites is activated by furin, a subtilisin-like endoprotease

Hepatitis E virus (HEV): molecular cloniong and sequencing of the full-length viral genome', Virology, 185, 120±131

Zoonotic transmission of hepatitis E virus from deer to human beings

A study of infectious intestinal disease in England: microbiological findings in cases and controls

Detection of poliovirus, hepatitis A virus, and rotavirus from sewage and ocean water by triplex reverse transcriptase PCR

Hepatitis E virus sequences in swine related to sequences in humans

A large infantile gastroenteritis outbreak in Albania caused by multiple emerging rotavirus genotypes

International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of Noroviruses

Review of latest available evidence on risks to human health through potential transmission of avian influenza (H5N1) through water and sewage

Hazard analysis and critical control point systems applied to public health risks: the example of seafood

Cloning and sequence analysis of dsRNA segments 5, 6 and 7 of novel non-group A, B, C adult rotavirus that caused an outbreak of gastroenteritis in China

Prolonged fecal excretion of hepatitis A virus in adult patients with hepatitis A as determined by polymerase chain reaction

Norovirus classification and proposed strain nomenclature