5.1.3 Viruses (Lone Gram)

Viruses are very small microorganisms (typically 25-70 nm) which consist of genetic material (RNA or DNA) and a protein cover. Viruses are obligatory intracellular pathogens and cannot, as bacteria, yeasts and filamentous fungi, multiply outside host cells. Thus virus particles per se are totally inert. The marine environment is full of viruses which represent the most abundant life form in the sea, typically numbering ten billion per litre, however, none of these are pathogenic to man (Lees, 2000). Viruses being implicated in seafood-borne diseases all have their niche in the human gastro-intestinal (GI) tract and their presence in water and seafood is a consequence of poor hygiene; either water being contaminated with sewage or products being contaminated by food handlers.

The diseases caused by human enteric viruses fall into two major categories: viral gastroenteritis and viral hepatitis (Caul, 2000). As is evident from data in section 4.2, viruses are responsible for the largest number of cases of seafood-borne diseases and is in particular associated with raw (under-cooked) molluscan shellfish. Notably, the largest outbreak of food-borne disease ever to be recorded was an outbreak of Hepatitis A in Shanghai, China, in 1988 where more than 290 000 people were infected by eating clams harvested in a sewage polluted area (Lees, 2000; Halliday et al., 1991; Tang et al., 1991).

Viruses may cause a range of diseases in humans and are the cause of mild diseases as flu and cold as well as more serious diseases as AIDS. Viruses traced to seafood-borne diseases are primarily so-called Norwalk-like virus and Hepatitis A virus.

Viruses are divided into groups depending on the organization and transcription of the genetic material (Table 5.18). The genetic material is DNA or RNA and they carry a single or a double stranded strand of genetic information. Many viral orders and families exist. Norwalk virus belongs to the viral family, Caliciviridae, which also includes three other genera, including the Sapporo-like virus. Norwalk-like virus are sometimes also called small-round-structured virus (SRSV). Hepatitis A belongs to the Picornaviridae family. Taxonomy of virus was for a long time dependent on electron microscopically classification (Caul, 2000) but has been greatly facilitated by molecular techniques allowing sequencing and molecular phylogenetic studies.

Studies of viral (seafood-borne) diseases have been and are greatly hampered by lack of methods for culturing and enumeration. Several viruses, including the Norwalk-like virus cannot be cultured on cell lines and enumeration relies on molecular (e.g. PCR-based) detection. Some laboratory adapted strains of hepatitis A can be cultured but most wild type strains escape culturing.

Table 5.18 Groups of viruses causing gastrointestinal diseases from seafood. Based on Lees (2000) and Caul (2000).

1. SS = Single Stranded
2. DS = Double Stranded

a) The disease and some epidemiological aspects

Norwalk-like virus (NLV) forms a distinct group of viruses which includes the "classical" Norwalk virus as well as Snow mountain virus, the Hawaii agent and the Montgomery agent. Disease is caused by ingesting viruses and symptoms appear after approximately 24 hours. These are sudden in onset and typically include nausea, vomiting, low-grade fever and diarrhoea. In general, NLV infections are mild and self-limiting and cease after 1-4 days. Due to the short duration and the self limiting disease, the number of NLV cases (from all sources) is probably underreported (EC, 2002). The infective dose of NLV - and most other viruses - is not known but several studies with human volunteers ingesting enteric virus point to low MIDs; probably less than 50 plaque forming units (PFU) (Gerba and Haas, 1988).

NLV is highly transmissible and the attack rate, i.e. the number of people becoming ill following ingestion, is high, typically between 50 and 90%. NLV is transmitted by perosn-to-person contact, by contaminated environments and by water and food (EC, 2002). Food-borne NLV gastroenteritis is especially caused by consuming contaminated molluscan shellfish. The link between molluscan shellfish and NLV was made in the UK where electron microscopy of faecal material from "winter vomiting disease" patients revealed virus particles.

Hepatitis A virus (HAV) causes a food- and water-borne infectious viral disease which lasts for several weeks. The liver is typically infected and jaundice, anorexia, vomiting and profound malaise are characteristic symptoms. The incubation period range from 15 to 50 days. The patient develops immunity but relapses and sequela may appear. Vaccines are available in both Europe and the United States and it has been suggested that food handlers should be immunized (Cliver, 1997).

b) The niche and prevalence in fish and fishery products

Like all viral diseases transmitted by seafood, NLV and HAV are associated with the gastrointestinal tract of humans and are shed in large quantities on faeces of infected persons. NLV are shedded from infected people, and food handlers must not work with foods for at least two days after symptoms have disappeared. In contrast, HAV is often shed in faeces from infected people 10-14 days before onset of disease and continues 1-2 weeks after onset.

The most common cause of viral gastroenteritis is live molluscan shellfish in which viral particles from the surrounding (contaminated) water are filtered and accumulated in the animals. However, a range of other foods have been implicated in viral diseases. Hepatitis A has been caused by orange juice, salads, bakery goods and lettuce. NLV has cause outbreaks involving butter cream, cool drinks and fresh cut fruits.

c) Growth and survival in fish and fishery products

Viruses do not multiply outside the host, and thus their numbers will not increase after the initial contamination event. Subsequent processing will affect the survival of the viruses, although little is known about the effect of food processing parameters on NLV and HAV. In general, viruses are more resistant to preservation parameters and processing steps than vegetative bacteria. Virus particles are stable at refrigeration temperatures when they are not de-stabilised by other factors, and frozen storage will only cause a slight increase in rate of inactivation (ICMSF, 1996). Heat inactivates viruses and D-values are typically measured in seconds at temperatures > 60°C but in minutes at temperatures in the range of 50 to 60°C. This means that household cooking / steaming often is not sufficient to inactivate viruses.

HAV is more resistant to heat and drying than other enterovirus but heating to 85-90°C caused a 4 log reduction in PFUs (Millard et al., 1987). HAV is resistant to short exposures to acid (pH 2). Due to the lack of culture methods for NLV, studies on the influence of food relevant parameters on virus survival are almost impossible to conduct. Based on studies of food-borne outbreaks it can be concluded that infectivity persists for 3 h at pH 2.7 at room temperature and for 60 min at neutral pH at 60°C.

Temperature has a major impact on survival of virus in seawater. At 4°C, it took 671 days to reduce HAV with 90% whereas the same reduction was obtained in 25 days at 25°C (Gantzer et al., 1998). UV-light inactivates virus and HAV was reduced with 90% in 2.6 minutes at 42 mW s /cm (Gantzer et al., 1998).

d) Prevention and control

Control of seafood-borne viral disease is, in principle, simple since the source of disease is indirect or direct faecal contamination. Thus, measures that prevent this contamination control the disease. Bivalve shellfish are suited for human consumption if harvested from waters free from sewage and pollution. Alternatively, the processing can include a virucidal treatment such as heat treatment at high temperatures (e.g. canning), or the viruses can be removed from the shellfish before consumption.

Depuration. Molluscan shellfish are filter feeding animals and the viruses - and other pathogenic agents accumulated - may be removed by depuration. This involves the transfer of the animals to clean water enabling them to shed the virus and other agents. The process is very difficult to control and there is no simple test to indicate that a shellfish has been depurated effectively. Several studies have shown that viruses are retained in the animals longer than bacteria. Epidemiological evidence indicate strongly that depuration may fail to eliminate enteric viruses from contaminated shellfish and that compliance with bacterial standards do not guarantee absence of viruses (Lees, 2000) (Figure 5.8). Several studies have suggested the use of a viral indicator such as the F+ RNA bacteriophage. This enteric virus is culturable and numbers correlates with the presence of NLV and the outbreaks of diseases (Doré et al., 2000).

However, since such viral indicators are not widely accepted and most viruses like NLV cannot be cultured, waters in which bivalves are harvested are monitored using bacterial counts. The EU and the US both have several guidelines and standards relating to bacteriological quality of live bivalves or shellfish growing waters. Due to the lack of correlation between water quality and presence of pathogens in the animals, EU has set standards for the animals (EC, 1991) while US standards refer to the quality of the water in harvesting areas (see section 11.2).

Hygienic practices. Contamination by food handlers can be prevented by good personal hygiene and education. As mentioned, food handlers must not handle foods for 2 days following an outbreak of NLV. Disposable gloves may be worn since viruses are difficult to remove by hand washing. Viruses are relatively resistant to disinfectants (e.g. phenolics, quaternary ammonium compounds, ethanol) while halogens (chlorine, iodine) inactivate viruses in water and on clean surfaces. The sensitivity to halogens is, however, lower than that of vegetative bacterial cells. Levels of > 10 mg chlorine / litre for 30 min are sufficient to inactivate the viruses.