Risk From Viral Pathogens in Seafood

CHAPTE R 15

Risk From Viral Pathogens in Seafood Samanta S. Khora VIT University, Vellore, Tamil Nadu, India

1 Introduction It is well known throughout the world that seafood (marine fish and shellfish) plays a very important role in human nutrition. In the recent decades, a strong move to healthier eating habits and the substitution of meat with seafood in the diet has resulted in greater demands for marine fish and shellfish makes seafood one of the major foods consumed worldwide (Wild and Lehrer, 2005). Consequently, the international trade of seafood has been growing rapidly, which reflects on the popularity and frequency of ingestion. Expansion of the seafood market has led to an increasing number of workers engaged in the capture and culture marine fisheries. This increased production and consumption of seafood has been accompanied by more frequent reports of adverse health problems among consumers, as well as processors of seafood. Although the safety of seafood has increased globally in recent decades, there are still a number of environmental chemical contaminants, naturally occurring marine toxins, and microbiological hazards that are present in seafood (Schmid and Wuthrich, 1997). The valuation and seriousness of the hazard to a population caused by the consumption of food is labeled risk. Seafoodborne illnesses can be broadly divided into allergenic, intoxication, and infection categories. In the first two cases, the causative agent is a toxic compound that contaminates the seafood or is produced by a biological agent in the marine products. Infection, the third seafood-associated illness, is caused by microbes (mainly bacteria, viruses, and parasites). All of these pathogenic varieties must be ingested alive, resulting in invasion of the intestinal mucous membrane or other organs to produce endotoxins (Venkitanarayanan and Doyle, 2002). Spread of the illness is mainly through personal contact and infected food and water. Ingestion of seafood contaminated with causative microbial organisms continues to pose a large-scale health threat. The reported number of illnesses from seafoodborne microbes has remained steady over the past several decades. Exposure to Vibrio, a bacterium that contaminates raw oysters and causes illness, and norovirus infection are still a concern. Viruses are stable in the environment, and transmission occurs through ingestion of contaminated water or food or contact with a contaminated surfaces. The viruses cause a wide range of diseases in different individuals (from aseptic meningitis

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440  Chapter 15 which may be very mild, to chronic diseases, such as myocarditis) and may not be easily related to a common source outbreak. Outbreaks associated with shellfish have not been readily documented. Viral disease transmission to human via consumption of seafood has been known since the 1950s (Roos, 1956), and norovirus is a leading cause of foodborne illness and outbreaks. Bivalve molluscan shellfish (oysters, clams, and mussels) may become contaminated by bioaccumulating human pathogens from surrounding polluted waters, thus becoming vehicles of disease transmission to humans. The severity of this risk depends upon the nature of contamination, and may range from mild diarrhea even death. The variation in the occurrence of viral disease estimates is high. For instance, viral disease associated with seafood for hepatitis A (HAV) and for NoV are ∼5% and 12%–47%, respectively. By various means of data collection and estimation, it has been shown that seafoodborne viral illness occurs globally. In this chapter, the author reviews viral pathogens most commonly associated with seafood. General characteristics, viral type, pathogenicity, mode of transmission, and association with seafood of each viral species are discussed, as well as the detection and diagnosis, risk factors, and management—whether preventive measures have been effective in decreasing or controlling risks.

2  Risks From Pathogenic Microbes in Seafood Seafood, like any food item, has the potential to cause disease from a variety of pathogenic bacterial, viral, and parasitic microorganisms under certain circumstances. Risks associated with more acute seafoodborne illness are microbiological hazards (FAO/WHO, 2008a,b). The greatest risk to human health is from pathogens in seafood. Some of the pathogenic microbes that occur naturally in seafood or in the marine environments are associated with sewage contamination of harvesting areas, or can be acquired during seafood harvest or processing. The microbiological risk associated with seafood other than raw molluscan shellfish is much lower and appears to result from recontamination or cross-contamination of cooked by raw product, or from contamination during preparation followed by time/temperature abuse. Many of these microorganisms pose only a slight risk to normal human populations, but all are pathogens and some pose serious risk to specific population groups, such as persons with defective immune systems (Archer and Young, 1988). Epidemiologic investigations have associated Vibrio cholera O1 illness with eating crabs, shrimp, and raw oysters harvested along the Gulf Coast (Blake et al., 1980). V. parahaemolyticus is a common marine isolate, with isolation reported from water, sediment, suspended particulates, plankton, fish, and shellfish (Joseph, 1988). However, it is likely that only a small fraction of marine isolates are potentially pathogenous. The CDC valuation of these microbiological hazards during the years prior to 1980 (with less informative reporting) suggested approximately 11% of all foodborne outbreaks (an outbreak involves two or more cases from a common source) implicated fish, molluscs, or crustaceans as the food

Risk From Viral Pathogens in Seafood  441 vehicle (Bryan, 1986). Compilations of CDC data from 1978–87 indicate fish and shellfish constituted only 10.5% of foodborne outbreaks and 3.6% of total cases (IOM, 1991). FAO’s compilation of the CDC data indicates that the number of cases remains higher for shellfish, but that outbreaks for fish, of which 90% can be linked to the cause, are more common (Huss et al., 2004). The association between exposure and illness is considered higher for seafood than for other foods due to the early onset of symptoms and the particular symptoms per types of seafood. Seafood is responsible for an important proportions of infectious illnesses, which range from mild gastroenteritis to life-threatening syndromes, each with its own epidemiology. These are prevalent and result in outbreaks everywhere in the world. This diverse group of pathogens results in a wide variety of clinical syndromes. At low levels, pathogenic microorganisms cause no problems. At illness thresholds, however, they can make people ill and cause death. Most bacterial and viral food poisoning appears within 8 h of ingesting food. The signs and symptoms of poisoning include nausea, vomiting, diarrhea, muscle aches, and lowgrade fever. Food poisoning can also occur from the ingestion of parasites. These agents are acquired from three sources: (1) mainly fecal pollution of coastal environs, (2) general aquatic environs, and (3) industrial, retail, restaurant, or home processing and preparation. Consumption of seafood contaminated with fecal organisms continues to pose a large-scale health threat.

3  Risks From Pathogenic Viruses in Seafood Viruses are small acellular structures with diameters of 15–400 nm, and have a simple protein coat surrounding structure, having RNA or DNA as genetic material. Viruses are abundant in nature and most are not pathogenic to humans. There are millions of virus-like particles in a milliliter of seawater, and they are a major cause of natural mortality in bacteria and protista. These are pathologically active in all biota including humans and cause various illnesses, such as smallpox, chicken pox, herpes, Ebola, rabies, and HIV/AIDS. Viruses may also play a role in autoimmune diseases, such as multiple sclerosis and diabetes and pose public health risks. Viruses produce an immense range of symptoms in humans, for example, diarrhea, aseptic meningitis, paralysis, conjunctivitis, myocarditis, and hepatitis. Viral replication occurs initially within the intestinal tract and leads to the excretion of large numbers of virus particles in the feces, after which survival in the environment can be prolonged due to the structure of viruses. There are over 120 enteric viruses, which may be found in human sewage. The viruses include the enteroviruses (poliovirus, echoviruses, coxsackie A and B), rotavirus, adenoviruses, hepatitis A, non-A, hepatitis E, and small round viruses (calicivirus, astrovirus, Snow Mountain agent, Norwalk virus). In raw sewage, levels as high as 492,000 viral units per liter have been detected, and in a secondary effluent following disinfection,

442  Chapter 15 levels may pull down between 2 and 7,150 viral units per liter (Rose, 1986). There is a significant body of information linking hepatitis and viral gastrointestinal disorders by the ingestion of infected shellfish in the USA and worldwide (Appleton, 1987; De Leon and Gerba, 1990; Gerba, 1988; Jaykus et al., 1993; Richards, 1985). Over 100 incidents have been reported in the USA, increasing from fewer than 10 in the years 1966–70 to more than 50 in the years 1981–85, after more routine use was made of the electron microscope for detection (Richards, 1985). The pathogenic viruses may be accumulated at a higher degree than that of surrounding seawater in some filter-feeding shellfish. Some movable shellfish, such as lobsters and crabs can act as accumulators of viruses in polluted seawaters and cause a problem by changing their positions, moving toward cleaner waters and subsequently acting as carriers of pathogenic viruses. Ingestion of pathogenic viruses can cause polio, gastroenteritis, and hepatitis (Lees, 2000; Svensson, 2000). Families of viral pathogens associated with pollution of harvesting waters include picornaviruses, reoviruses, adenoviruses, caliciviruses, astroviruses, and noroviruses. Of these enteric viruses, only HAV, caliciviruses, astroviruses, and norovirus enteral hepatitis virus have been documented to cause seafood-associated illness (Table 15.1) (Bryan, 1986; CDC, 1989; Cliver, 1988; Gerba, 1988; Richards, 1985, 1987; Rippey and Verber, 1988).

4  Historical Perspectives Poliovirus (PV) is an ancient virus that has caused crippling and death globally. The polioviruses were the first viruses shown to be foodborne, but virulent strains are now rare, and the vaccine strains are potential indicators of the possible presence of other, virulent viruses in food and water (Alhajjar et al., 1988). Although poliomyelitis was first recognized in 1789, outbreaks have continued to occur in the USA and Europe with increasing severity (CDC, 2013). Poliovirus was first identified in 1909 through inoculation of specimens into monkeys. Poliomyelitis has been a notable disease in Australia since 1922. The peak of poliomyelitis infections occurred in 1952, with more than 21,000 paralytic cases. The virus was first grown in 1949 for cell culture, which became the basis for vaccines. This is a virus that could be eliminated permanently, as its reservoir is the human being (Dowdle and Birmingham, 1997). Viral disease transmission to humans via consumption of seafood has been known since the 1950s (Roos, 1956). The enteric viruses appear to be the major cause of shellfish-associated disease. The first seafood-associated infection with hepatitis A was recorded in Sweden by the ingestion of oysters, which resulted in 629 cases in the year 1956 (Mason and McLean, 1962; Wanke and Guerrant, 1987). In 1961, the first seafood-associated outbreak in the USA was reported and was traced to oysters and clams (Goh et al., 1987). In the United

Table 15.1: Characteristics of seafoodborne viruses. Genome Viruses Sizes (kb)

Associated Seafoods

Infective Transmissions Doses

Incubation Periods

Durations of Illnesses

Illnesses (Major Signs)

HAV

Single stranded RNA (7.5)

Fecal–oral; person-toperson

3–6 Weeks

4–6 Weeks

Fever, malaise, and Winter and Supportive abdominal epigas- early spring tric pain

Bialek et al. (2007)

HEV

Single stranded RNA (7.2)

Both raw and steamed clams, oysters, cockles, and mussels Shrimp or small prawns, salmon, cod, mussels, hake, and squid

Fecal–oral >106 Viral route; person- particles to-person spread is not common

3–8 Weeks

2 Weeks

ET-NANBH

Rainy

Supportive therapy

NoV

Single stranded RNA (7.4–7.7) Single stranded RNA (7.4–7.7)

Oysters, mussels, cockles, clams

Fecal–oral, person-toperson

<100 Viruses

18–48 h

24–48 h

Year round

Supportive

Detected from shellfish (oysters, cockles, and smooth clams) Detected in shellfish

Human fecal origin

1,015– 2,800 Viral particles

<1–4 Days

1–4 Weeks

Nausea, vomiting, diarrhea, abdominal cramps, and occasionally fever Diarrhea and vomiting.

Crossan et al. (2012); Said et al. (2009); USPHS (1995) Porter and Sarkin (1987)

Cold season

Supportive therapy

Oka et al. (2015)

Fecal–oral route

1,000 to 1,000,000 Viruses

6–20 Days

3–35 Days

Summer

Supportive therapy and OPV and IPV

Larkin and Hunt (1982)

Detection in shellfish reported

primarily via the fecal–oral route and respiratory aerosols

—

3–8 Eeeks

10 Days

Asymptomatic infection, viral meningitis, paraalytic disease, poliomyelitis Group A: widespread myositis. flaccid paralysis; group B: heart CNS focal myositis myocarditis, hepatitis, and encephalitis

Summer and fall months

Supportive care

Stewart et al. (2013)

SaV

PV

Single stranded RNA (7.2–8.4)

CVs

Single stranded RNA (7.2–8.4)

10–100 Viruses

Seasons of Illnesses

Treatments References

(Continued)

Table 15.1: Characteristics of seafoodborne viruses. (cont.) Genome Viruses Sizes (kb) EchoV Singlestranded positivesense RNA (7.2–8.4) AiV-1 Singlestranded positivesense RNA (7.2–8.4) AstV Single stranded RNA (7–8)

Associated Seafoods Detection in shellfish reported

Infective Transmissions Doses Fecal–oral 919 pfu

Durations of Illnesses 5–6 Weeks

Illnesses (Major Signs) Acute onset of fever, headache, photophobia, nausea, and vomiting

Seasons of Illnesses Summer and fall months

Treatments References Supportive Pallansch care and Roos (2007)

Oysters, Fecal–oral clams, cockles

—

—

—

Diarrhea, abdomi- Winter nal pain, nausea, vomiting, and fever

Supportive care

Le Guyader et al. (2008)

Musseles, oysters

Fecal–oral route

—

1–3 Days

2–3 Days

Diarrhea

Winter

Maunula et al. (2004)

10 to 100 Infectious viral particles 1014 Viral particles

1–2 Days

3–8 Days

Fever, vomiting, watery diarrhea in children

Late autumn, winter

Symptomatic treatment, and rehydration Supportiveoral, IV rehydration

Year round

Supportive care

Winter

Supportive care

Viguier et al. (2001) MuniainMujika et al. (2003)

RV

DoubleIsolated from stranded sewage RNA (16–27)

Fecal–oral route

ParV

Single— stranded DNA (5) DoubleDetected in stranded shellfish DNA (28–45)

Close contact and respiratory route Fecal–oral route

AdV

Incubation Periods 2–10 Days

>1011 Viruses

10–12 Days 2–3 Months Erythema infectiosum or “fifth disease” 3–10 Days 10–14 Days Respiratory, eye, and gastrointestinal infections

Wilhelmi (2003)

AdV, Adenovirus; AiV, aichivirus; AstV, astrovirus; CVs, coxackieviruses; ET-NANBH, enterically transmitted non-A non-B hepatitis; HAV, hepatitis A; HEV, hepatitis E; NoV, norovirus; ParV, parvovirus; PV, poliovirus; RV, rotavirus; SaV, sapovirus.

Risk From Viral Pathogens in Seafood  445 Kingdom during the winter of 1976–77, the connection of viruses with shellfish-associated gastroenteritis was established, as cooked cockles were epidemiologically linked to 33 incidents affecting almost 800 persons (Appleton and Pereira, 1977). Eventually, more outbreaks were reported globally, linking to HAV. The largest one was in Shanghai, China, involving around 300,000 individuals who consumed raw/undercooked clams (Halliday et al., 1991). In 1997 an outbreak in Australia was reported in which 467 patients were infected: again, oysters were implicated as the source of infection (Conaty et al., 2000). In the largest outbreak in Italy in 1996–97, 11,000 people were infected. In 2005, hepatitis A virus was confirmed as causing illness in four states among restaurant patrons who ate Gulf Coast oysters. Coxsackie virus was first isolated from human feces in the town of Coxsackie, New York, by G. Dalldorf in 1948. Also in 1948, a new group of agents was identified by inoculation of newborn mice from two children with paralytic disease. These agents were named coxsackieviruses after the town in New York state. Coxsackieviruses A and B were identified on the basis of the histopathological changes they produced in newborn mice and their capacity to grow in cell cultures. The Norwalk virus was the first gastroenteritis virus reported to be foodborne (Greenberg et al., 1979). Its name came from the location of the first gastroenteritis outbreak, which was in Norwalk, Ohio, in 1968 (Gerba et al., 1985). It is now considered a major cause of nonbacterial gastroenteritis. Since then norovirus has become recognized as the most frequent cause of nonbacterial acute gastroenteritis in the USA, accounting for 52% or more of such cases. It is mainly due to the ingestion of oysters in particular and raw shellfish in general that created gastroenteritis. Hepatis E virus (HEV) has been recognized as a prominent cause of human disease only as recently as 1980. HEV is a known cause of epidemic and intermittent (sporadic) cases of enterically transmitted acute hepatitis. Astroviruses (AstVs) are viruses that are small, round, and star-shaped in appearance. At present, only seven serotypes of AstVs have been implicated in gastroenteritis, principally in children. These pathogens have a minor impact in an adult. A few reports have connected these to shellfish ingestion (Yamashita et al., 1991; Caul, 1996). Adenoviruses (AdV) were first isolated in the 1950s in adenoid tissue. Rotavirus was first described in 1973 by electron microscopy from duodenal biopsy specimens from children with diarrhea. It is one of the most common causes of infantile diarrhea worldwide. Sapoviruses were first discovered in 1976 by electron microscopy in human diarrheic samples. To date, sapoviruses have also been detected in several animals: pigs, mink, dogs, sea lions (Oka et al., 2015). The first echoviruses were accidentally discovered in human feces, unassociated with human disease, during epidemiological studies of polioviruses. The name echovirus comes from

446  Chapter 15 “enteric, cytopathic, human, orphan viruses.” Echoviruses were later identified as producing cytopathic changes in cell culture and being nonpathogenic for newborn mice and subhuman primates.

5  Seafood as Vehicles for Viruses Foodborne viruses are derived from the human gut, water, and foods due to contamination with sewage and infection transmitted by food handlers with poor hygiene. Seafood includes molluscs (e.g., oysters, clams, and mussels), finfish (e.g., salmon and tuna), marine mammals (e.g., seal and whale), fish eggs (roe), and crustaceans (e.g., shrimp, crab, and lobster) (Table 15.2). Some seafood commodities are inherently more risky than others owing to many factors, including the nature of the environment from which they come, their Table 15.2: Classification of seafood assigned to viral risks. Seafood Groups

Phylums

Invertebrates (without backbone)

Arthropoda Crustacean

Shellfish

Mollusca

Vertebrates (with backbone)

Finfish

Mammals

Chordata

Classes

Common Names Shrimp, lobsters, crabs

Gastropoda

Viruses

HAV, enteroviruses Desenclos et al. (1991); Morse et al. (1986); Botero et al. (1996) NoV, SaV Ozawa et al. (2015) NoV, HAV, AiV Iizuka et al. (2010); Xia Ming et al. (2013)

Conch, sea snails Bivalvia Hard clams, soft clams, Manila clams, razor clams, blood clams, clams, cockles, scallops, mussels, and oysters Cephalopoda Squids HEV

Osteichthyes

Mammalia

Salmon (salm- HAV, HEV on eggs), cod, tuna, whitefish, hake Seal meat, NoV, HAV, SaV whale, sea lions

AiV, Aichivirus; HAV, hepatitis A; HEV, hepatitis E; NoV, norovirus; SaV, sapovirus.

References

Crossan et al. (2012); Said et al. (2009) Desenclos et al. (1991); Morse et al. (1986) Iwamoto et al. (2010); Li et al. (2011)

Risk From Viral Pathogens in Seafood  447 mode of feeding, the season during which they are harvested, and how they are prepared and served. Filter-feeding shellfish are notorious as a source of seafoodborne viral infections because they actively concentrate viruses from contaminated water. Infectious viruses can be detected for up to 6 weeks without any loss in quality of the shellfish. Depuration, a practice that may reduce bacterial contamination, is not as effective in reducing the viral load. Viruses are believed to live up to 10 weeks in live shellfish gut. The persistence of viruses after depuration in the case of NoV has also been reported (Le Guyader et al., 2006). Additional research findings have shown some risk of human infection by ingestion of contaminated shellfish in raw or undercooked state (Croci et al., 2005; Hewitt and Greening, 2004). Fish, molluscs, and crustaceans can acquire pathogens from various sources. All seafood can be susceptible to surface or tissue contamination originating from the marine environment. Bivalve molluscs feed by filtering large volumes of seawater. During this process, they can accumulate and concentrate pathogenic microorganisms that are naturally present in harvest waters, such as vibrios. Contamination of seafood by pathogens with a human reservoir can occur when growing areas are contaminated with human sewage (Koopmans, 2009). Species of fish and shellfish harvested from inshore waters that are subject to contamination by human or terrestrial animal feces, or by other industrial/agricultural pollutants, may contain bacteria and viruses that are pathogenic for humans (Liston, 1980). This is especially true for filter feeders that concentrate bacteria and viruses present in polluted waters. Oysters harvested at growth from waters contaminated with human sewage have been associated with many outbreaks of enteric disease (Son and Fleet, 1980). The shellfish collect viruses in the course of their filter-feeding activity. Human viruses do not infect these species, but they are harbored for days or weeks in the shellfish digestive tract and are apparently more difficult to remove than bacteria during processes intended to cleanse the shellfish (Grohmann et al., 1981; Power and Collins, 1989). Unlike many other seafoods, shellfish are usually eaten with their digestive tracts in place. They are often eaten raw or lightly cooked: shellfish, unlike other foods, may also protect viruses from thermal inactivation during cooking (DiGirolamo et al., 1970). Bivalve molluscs, such as clams, cockles, mussels, and oysters, are especially prone to transmit viruses.

6  Outbreaks and Prevalence Foodborne viral illnesses are understudied and underreported. However, NoV gastroenteritis and Hepatitis A virus infections by shellfish ingestion have drawn attention because of the need for control. Globally, viruses, such as NoV, enteroviruses, and hepatitis A viruses are commonly present in contaminated seafood. Hepatitis A, primarily associated with bivalve molluscs from polluted waters, causes more than 4% of all seafood-related outbreaks and illnesses. The largest reported outbreak has been from China, where around 300,000

448  Chapter 15 individuals were infected by the ingestion of clams from a sewage-contaminated area. Common-source foodborne outbreaks occur. For example, Hepatitis A infections with clams and oysters have been reported in the USA since the 1960s (Portnoy et al., 1975; Desenclos et al., 1991). In the last few decades the occurrence has been reduced due to strategic control measures. However, some outbreaks of HAV infections (7 outbreaks: 77.7%) were associated with molluscs. Tuna and crab were each implicated in one outbreak. Most outbreaks occurred during the 1980s (Desenclos et al., 1991; Morse et al., 1986), none was reported during the 1990s, and only two were reported after 2000 (Bialek et al., 2007). Norovirus (NoV) is a leading cause of foodborne illness and outbreaks (Widdowson et al., 2005), although the number of reported outbreaks is likely grossly underestimated because of a lack of routine availability or use of diagnostic testing. Seafood harvested from sewage-contaminated waters caused outbreaks of norovirus gastroenteritis. From 1976 to 1980, the CDC reported that 42% of the outbreaks of nonbacterial gastroenteritis were caused by norovirus (Gerba et al., 1985). Major incidents have occurred by the ingestion of undercooked/raw shellfish, such as a series of oyster- and clam-associated outbreaks in which northeastern US coastal waters were implicated. Molluscan shellfish, particularly oysters, have been commonly identified in NoV-related gastroenteritis outbreaks, worldwide. Noroviruses from Genogroup II, genotype 4 (abbreviated as GII.4) account for the majority of adult outbreaks of gastroenteritis and often sweep across the globe. Confirmed outbreaks of norovirus infection have been reported increasingly during the past decade: 26 of 31 (83.8%) outbreaks were reported during the period of 1998–2006. These have been followed by a decrease in reported outbreaks of viral illness that lasted until 2000, when reports again increased, likely due to better diagnostic testing, an improved ability to confirm etiology, and improved public health surveillance for outbreaks. Overall, norovirus is the third most commonly reported pathogen associated with seafood and the most common viral agent, causing 31 (77.5%) of viral illness outbreaks. Unlike outbreaks of bacterial illness, outbreaks of norovirus illness are more common during cooler months, with more than three-fourths (77.4%) of outbreaks occurring from October to March. An estimated 527,000 children under the age of 5 die from rotavirus diarrhea each year, with over 85% of the deaths occurring in low income countries in Africa and Asia (Parashar et al., 2006). AdVs account for 10% of the gastroenteritis in children. In Japan, it is the second most common cause of hospitalization due to diarrhea in children. In Finland, enteric adenoviruses were found in 6% of the cases of children between 2 months and 2 years (Pang et al., 2000). Shellfish (prawns, lobsters, crabs, mussels, and scallops) and mixed seafood (a mixture of shrimp or small prawns, salmon, cod, mussels, hake, and squid) caused acute HEV infections in 33 participants on a world cruise in 2008 (Said et al., 2009). Only one outbreak of foodborne coxsackievirus illness has been reported (Osherovich and Chasovnikova, 1967). Although the reported numbers of outbreaks are less for sapoviruses than for noroviruses (Iritani et al., 2014), sapovirus gastroenteritis outbreaks occur throughout

Risk From Viral Pathogens in Seafood  449 the year in all ages of people in various settings, such as child day care centers, kindergartens, schools, colleges, hospitals, nursing homes, restaurants, hotels, wedding halls, and ships (Lee et al., 2012). An AstV epidemiological survey revealed that at least 242 persons were affected (Maunula et al., 2004). These viruses are often hard to recognize under electron microscopy and thus they are most likely considerably underreported. Since the launch of the Global Polio Eradication Initiative in 1988, the incidence of wild poliovirus has reduced by 99%: from 350,000 children paralyzed or killed annually in 125 endemic countries in 1988 to 620 cases reported in 16 countries in 2011 (as of January 3, 2012). In 2006, the number of polio-endemic countries (countries that have never stopped indigenous wild poliovirus transmission) was confined to Afghanistan, Pakistan, India, and Nigeria. Molluscs were implicated in 85 (45.2%) of outbreaks, followed by fish in 73 (38.8%) outbreaks, and crustaceans in 30 (16.0%) outbreaks (Table 15.3). All of the molluscconnected outbreaks were associated with bivalve molluscs, including oysters, clams, and mussels. In fish-associated outbreaks, the most commonly implicated fish was salmon, including fermented fish parts and fermented salmon eggs, which accounted for 10 of these outbreaks. Other commonly reported fish vehicles were tuna, seal meat, whale, and whitefish. Among crustacean-associated outbreaks, the most commonly implicated items were crab and shrimp. Comparing seafood categories within different periods, the number and proportion of mollusc-associated outbreaks increased greatly.

7  Viral Pathogens in Seafood Viruses are more abundant than any other organism in the sea. They are diverse and participate in food webs. They can infect bacteria, shellfish, finfish, turtles, and mammals of the seas. Humans are also vulnerable if they eat infected seafood. Diseases caused by viruses include mild common flu to more severe AIDS. They cause common diarrhea and serious liver ailments, leading to bedridden patients, even death. Human enteric virus causes two types of seafoodborne diseases: gastroenteritis and hepatitis (Caul, 2000). These are mainly due to ingestion of uncooked molluscan shellfish. Viruses are categorized on the basis of structure and function of genomes (Table 15.4). It is either DNA or RNA, as well as double- or single-stranded, segmented or nonsegmented, linear or circular. Asingle-stranded RNA virus is further categorized by whether it can function as messenger RNA (mRNA) and, in addition, its structure (symmetry, enveloped or not, number of capsomeres). Presently, molecular technology has helped to classify and take taxonomic account of viruses, which was not so easy by electronic microscopy. Viral disease transmission to humans via consumption of seafood has been known since the 1950s (Roos, 1956). Human enteric viruses appear to be the major cause of shellfish-associated

450  Chapter 15 Table 15.3: Outbreaks and prevalence of viral pathogens from seafood. Locations

No. of Cases

Years

Remarks

Viruses (%)

Seafood Groups

Sweden

629 Cases

1956

HAV

Oyster

East coast, India

50 Samples

2006–07

First documented outbreak Detected in shellfish

West coast, India Japan

194 Samples

2007

AdV

53

1984–87

Detected in shellfish —

Japan

27 Samples

2012

—

Sea snails

Japan

5 Outbreaks

1989

Sydney, Australia Australia

2000 Cases

1978

NoV

Oysters

13 Outbreaks

2001–06

First recognition First NoV outbreak —

NoV (55.6%) SaV (44.4%) AiV-1

NoV (50%)

Oysters

Australia

467 Patients

1997

—

HAV

Oysters

Globally

368 Outbreaks

1980–2012 —

Shanghai, China

288,000 People 1988

The largest outbreak

NoV (83.7%) HAV (12.8%) HAV

Oysters (58.4%) Raw clams

United Kingdom

33 Participants 2008

In world cruise HEV ship

England

41 Cases

1978

HAV

England

800 People

1994

Boiled and steamed —

Shrimp, salmon, cod, mussels, hake, and squid Mussels

Parvoviruses

Cockles

France

111 Cases

2007

—

HAV

Oysters

France

8 Samples

2006

—

AiV

Oysters

Czech Republic Norway

285 Cases were 2003 recorded 100 Samples 2000

—

AdV

Shellfish

—

HAV (47%)

Shellfish

Norwegian coast

681 Sample

—

AdV (18.6%)

Shellfish

2000–03

NoV 12 (24%) Shellfish

NoV

Oysters, clams Oysters

Oysters

References Mason and McLean (1962) Anbazhagi and Kamatchiammal (2010) Umesha et al. (2008) Sekine et al. (1989) Ozawa et al. (2015) Yamashita et al. (2000) Murphy et al. (1979) Huppatz et al. (2008) Conaty et al. (2000) Bellou et al. (2013) Lees (2000); Butt et al. (2004) Crossan et al. (2012); Said et al. (2009)

Bostock et al. (1979) Appleton (1994) Guillois-Bécel et al. (2009) Le Guyader et al. (2008) Drapal et al. (2003) MuniainMujika et al. (2003) Myrmel et al. (2004)

Risk From Viral Pathogens in Seafood  451 Table 15.3: Outbreaks and prevalence of viral pathogens from seafood. (cont.) Viruses (%) HAV

Seafood Groups Mussels/ clams

1996–97

Remarks Identified as: annual incidence rate doubled —

HAV

Raw seafood

882 People

2004

—

HAV

Shellfish

Mississippi/ Alabama, USA USA

84 Cases

1961

—

HAV

Oysters and clams

368 Outbreaks

1980–2012 —

Louisiana, USA

263 Cases

1973

NoV (83.7%) HAV (12.8%) HAV

Shellfish Oys- Bellou et al. ters (58.4%). (2013) Oysters Portnoy et al. (1975)

Louisiana, USA

180 Cases

1993

NoV (small round structured virus)

Raw/steamed CDC (1993) oysters

Massachusetts, USA

83 Cases

1977

Clams

Truman et al. (1987)

California, USA

171 Cases

1998

NoV (Snow Mountain agent) NoV

Oysters

CDHS (1998)

Locations Livorno, Italy

No. of Cases 75 Cases

Years 1984

Italy

11,000 People

Italy

Harvested from approved oyster-growing areas Raw or steamed oysters affected; 14 states received the product First outbreak, clams were baked Raw or undercooked oysters

References Mele et al. (1989)

Butt et al. (2004) Pontrelli et al. (2008) Goh et al. (1987)

AdV, Adenoviruses; AiV, aichivirus; HAV, hepatitis A; HEV, hepatitis E; NoV, norovirus; SaV, sapovirus.

disease. Man is the only known reservoir for the major viruses causing foodborne diseases: calicivirus and hepatitis A virus (Svensson, 2000). Molluscan in general and bivalves in particular are well-defined agents for pathogenic viral transmission (Lees, 2000). The high risk results from two factors: shellfish are filter feeders and many shellfish are ingested either raw, lightly cooked, or whole. The risk is increased because many species are cultivated in near-coastal waters, where contamination with human sewage may easily be found, which may contain high levels of viral particles. Ingestion of pathogenic viruses can cause polio, gastroenteritis, and hepatitis (Lees, 2000; Svensson, 2000). Presently there are more than 100 known enteric viruses, which are excreted in human feces and find their way into domestic sewage. Caliciviruses, such as norovirus (Norwalk virus), are recognized as the

Table 15.4: Classification of viruses assigned to seafood risks.

Phylum

Class

Orders

Families

Genera

Species

Vira

Ribohelica

Picornavirales

Picornaviridae

Hepatovirus/ HAV Enterovirus

Shapes and Natures of Sizes (in nm) Genomes Featureless spheres (27)

PV

Spherical shape (27)

Coxsackieviruses (A and B)

Round or oval shape (20–30)

Echoviruses

Spherical shape (30)

(+) ssRNA, unenveloped with icosahedral symmetry (+) ssRNA, unenveloped with icosahedral symmetry

Illnesses/ Clinical Features Inflammation of liver, hepatitis A

Associated Seafoods Bivalve molluscs

Poliomyelitis, Detection meningitis, in shellfish encephalitis reported; no seafood associated outbreak reported (+) ssRNA, Poliomyelitis, Detection unenvelmeningitis, in shellfish oped with encephalitis reported; icosahedral no seafood symmetry associated outbreak reported (+) ssRNA, Aseptic men- No outbreaks unenveloped ingitis, febrile associwith an illnesses with ated with the icosahedral or without consumption configuration rash, comof shellfish mon colds, have been and acute reported hemorrhagic conjunctivitis

Calicivirales

Caliciviridae

Kobuvirus

AiV

Norovirus

NoV

Sapovirus

SaV

Hepevirales

Hepeviridae

Hepevirus

HEV

Astrovirales

Astroviridae

Mamastrovirus

AstV

Round-struc- (+) ssRNA, tured (30) unenveloped with icosahedral symmetry Cup-shaped (+) ssRNA, (27–32) nonenveloped with icosahedral symmetry Cup-shaped (+) ssRNA, (30–38) unenveloped with icosahedral symmetry Cup-like (+) ssRNA, (32–34) unenveloped, linear, with icosahedral symmetry

Gastroenteritis

Mussels, clams, cockle, oysters

Epidemic gastroenteritis

Bivalve molluscs

Diarihea (infants and children)

Detected from shellfish (oysters and clams)

Inflammation of liver, hepatitis E

Star-shaped (28)

Gastroenteritis

Shrimp or small prawns, salmon, cod, mussels, hake, and squid Epidemiological evidence limited, but shellfish associated outbreak reported

AiV, Aichivirus; AstV, astrovirus; HAV, hepatitis A; HEV, hepatitis E; NoV, norovirus; PV, polioviruses; SaV, sapovirus.

(+) ssRNA, unenveloped with icosahedral symmetry

454  Chapter 15 major cause of seafood-associated gastroenteritis. Over 80 per cent of the outbreaks of nonbacterial gastroenteritis in the USA and Europe are currently attributed to caliciviruses (Svensson, 2000).

7.1  Hepatitis A Virus (HAV) 7.1.1  Systematics and morphology HAV comes under Hepotovirus genus of the Picornaviridae family and order Picornavirrales and class Ribohelica (D’Souza and Jaykus, 2002). HAV is a small (27–32 nm) nonenveloped virus with icosahedral symmetry, single-stranded positive-sense RNA of 7.5 kb in size, and a buoyant density in cesium chloride of 1.33–1.34 g/mL (Gerba et al., 1985). 7.1.2  Virus types There is one species, HAV, with two strains or biotypes: human HAV and simian HAV. These two distinct strains are phylogenetically distinct and have different preferred hosts. Now, at least five serological types of hepatitis viruses are recognized, belonging to various taxonomic groups, but only hepatitis A is documented as being foodborne (Cromeans et al., 1994). Human HAV infects all species of primates including humans, chimpanzees, owl monkeys, and marmosets, whereas simian HAV infects green monkeys and cynomolgus monkeys. Seven genotypes have been recognized, of which four infect humans and the remaining three infect nonhuman primates. Notwithstanding the genetic variation, human HAV comprises a single serotype (D’Souza and Jaykus, 2002). 7.1.3 Pathogenicity Most commonly, HAV is an acute, self-limiting illness associated with fever, malaise, jaundice, anorexia, and nausea; symptoms may last from several weeks to several months. Deaths from fulminant hepatitis can occur. Severity increases with age, and in rare cases, there is a prolonged, relapsing hepatitis. In infants and young children, the infection is often asymptomatic. Symptoms begin after4 weeks of exposure. Initial symptoms generally include fever, weakness, epigastric abdominal pain, and malaise. As it progresses, individuals are affected by jaundice and produce dark urine. The illness progresses from mild to severe, leading to hospitalization. Fatalities are only 0.1%, primarily among the elderly and people with an underlying disease (Bryan, 1986). The vaccine is produced from a mutant strain of the virus that replicates in cell culture. The wild type virus either does not replicate in cell culture or replicates very slowly, often without cytopathic effects (Cromeans et al., 1994). Diagnosis is made using serologic tests that measure immunoglobulin M antibody for hepatitis A. 7.1.4  Mode of transmission Humans are the only reservoir and transmission is generally by the fecal–oral route. Food becomes contaminated via fecally soiled hands of infected persons or via fecally

Risk From Viral Pathogens in Seafood  455 contaminated water, as is usual with shellfish. Hepatitis A is one of the more severe of foodborne diseases, especially among those caused by viruses. Although HAV cannot grow in the environment, it is considered to be extremely stable under a wide range of environmental conditions, including freezing, heat, chemicals, and desiccation. 7.1.5  Associated with seafood The first HAV-linked seafoodborne outbreak implicating oysters was recorded in Sweden in 1955 (Lindberg-Braman, 1956). Subsequent outbreaks of HAV implicating oysters from certified areas have been reported (Mackowiak et al., 1976; Portnoy et al., 1975). Soft clams, hard clams, mussels, and oysters have been linked to outbreaks of HAV (Dienstag et al., 1976; Feingold, 1973; Gerba and Goyal, 1978; Mackowiak et al., 1976; Portnoy et al., 1975). There is a higher risk of infection for coastal populations as compared to the inhabitants of inland states (Goyal, 1984; Goyal et al., 1979). Although clinical manifestations vary in severity, hepatitis A virus infection is the most serious viral infection associated with seafood consumption.

7.2  Poliovirus (PV) 7.2.1  Systematics and morphology PV is the species under the genus Enterovirus of Picornaviridae in the order Picornavirales under the class Ribohelica. It is monopartite, linear ssRNA(+) with icosahedral symmetry of 7.2–8.5 kb, polyadenylated, composed of a single open-reading frame (ORF) encoding a polyprotein. Humans are the only susceptible hosts. Polioviruses are distributed globally. Before the availability of immunization, almost 100% of children under the age of 5 from developing countries were at risk. 7.2.2  Virus types There are three distinct serotypes of poliovirus (P1, P2, and P3). Immunity to one serotype does not produce immunity to the other serotypes. 7.2.3 Pathogenicity Poliovirus principally affects motor neurons and autonomic neurons. Neuronal destruction is accompanied by an inflammatory infiltrate of polymorphonuclear leukocytes, lymphocytes, and macrophages. The lesions are characteristically distributed throughout the gray matter of the anterior horn of the spinal column and the motor nuclei of the pons and medulla. Clinical symptoms depend on the severity of lesions rather than on their distribution. Almost 95% of infections are asymptomatic. One in 200 infections leads to irreversible paralysis (usually in the legs). Among those paralyzed, 5% to 10% die when their breathing muscles become immobilized.

456  Chapter 15 7.2.4  Mode of transmission Polio is usually transmitted by contact, as well as via fecal–oral routes. This virus is transmitted from the stool of an infected person to the mouth of another person via contaminated hands or such objects as eating utensils. Some cases may be spread directly through the oral–oral route. 7.2.5  Associated with seafood Some of the most frequently recovered viruses from shellfish are the polioviruses because of the common practice of immunizing American children against polio (Larkin and Hunt, 1982). The vaccine consists of live attenuated viruses that replicate in the intestine but produce few or no clinical symptoms. Children who have been immunized excrete viruses (from 1,000 to 1,000,000 viruses/g feces) for several days after the vaccine is administered. An examination of 20% of the polioviruses isolated from the Texas Gulf showed that all were of vaccinal origin. Since the viruses in the vaccine are modified, they present no health hazard if consumed by humans.

7.3  Coxackieviruses (CVs) 7.3.1  Systematics and morphology Coxsackieviruses (CVs) are a member of the genus Enterovirus under the family Picornaviridae of the order Picornavirales under the class Ribohelica. CV is nonenveloped with a linear single strand of ribonucleic acid (RNA) for genetic material. CV and PV have a common nature. After success with the control of PV infection, global attentions have fallen on nonpolio-enteroviruses, such as CV. 7.3.2  Virus types CV is separable into two groups, A (CVA) and B (CVB), which are based on their effects on newborn mice and their capacity to grow in cell cultures (van Regenmortel et al., 2000). There are 23 serotypes (1–22, 24) in CVA but in CVB 6 serotypes are reported. CVB have a type-specific antigen. In addition, all from group B and one from group A (A9) share a group Ag. Cross-reactivities have also been demonstrated between several groups of A viruses but no common group antigen has been found. 7.3.3 Pathogenicity Usually CVA produces herpangia, acute-hemorrhagic-conjunctivitis, and hand-foot-mouth diseases (HFMD). CVB produces pleurodynia, myocarditis, pericarditis, and hepatitis, infecting the heart, pleura, pancreas, as well as liver. Moreover, these two groups produce rashes, aseptic meningitis, febrile illnesses, and upper respiratory tract disease. Numerous CVA are responsible for causing CNS disease similar to poliomyelitis. Systemic neonatal

Risk From Viral Pathogens in Seafood  457 disease is often associated with group CVB. Coxsackieviruses are RNA viruses that may be causal for HFMD. HFMD usually occurs in children but can occur in adults as well (Stewart et al., 2013). The majority of HFMD infections are self-limiting, so no treatment is required. HFMD usually resolves in about 10 days with no scarring, but the person may shed CV for several weeks. More than 90% of CV infections are asymptomatic or cause nonspecific febrile illnesses. In neonates, they are the most common cause of febrile illnesses during the summer and fall. The CDC has established that CV infections accounted for approximately 25% of all neonatal enterovirus infections (26,737) from 1983 to 2003.Those due to coxsackievirus B4 were associated with a higher mortality rate than any other serotype. The viruses initially replicate in the upper respiratory tract and the distal small bowel. They have been found in the respiratory tract up to 3 weeks after initial infection and in feces up to 8 weeks after initial infection. The viruses have been found to replicate in the submucosal lymph tissue and spread to reticulo-endothelial organs. More propagation to other organs occurs following a secondary viremia. Immunity is thought to be chiefly humoral. 7.3.4  Mode of transmission CV is transmitted primarily via the fecal-oral route and respiratory aerosols, although transmission via fomites is possible. Infection is usually spread by fecal–oral contamination, although occasionally the virus is spread by droplets expelled by infected individuals. 7.3.5  Associated with seafood Although the coxsackieviruses have sometimes been reported in association with food and water, a single foodborne coxsackievirus illness outbreak has been confirmed (Osherovich and Chasovnikova, 1967). Moreover, foodborne illness outbreaks associated with coxsackieviruses and echoviruses have also been registered (Cliver, 1997; Sattar, 2001).

7.4 Echoviruses 7.4.1  Systematics and morphology Echoviruses are under the genus Enterovirus of the family Picornaviridae to order Picornavirales under the class Ribohelica. They are small (24–30 nm) linear single strand ribonucleic acid (RNA), nonenveloped with an icosahedral symmetry. Four proteins VP1VP4 form a capsid having 60 subunits. Proteins of these play important roles in terms of determining host range and tropism and in delivering the RNA genome into the cytoplasm of the host’s cells. Echoviruses (ECHO: enteric cytopathogenic human orphan viruses) are grouped together because they infect the human enteric tract and because they can be recovered from humans only by inoculation of certain tissue cultures. Most echoviruses are no longer considered orphans.

458  Chapter 15 7.4.2  Virus types Echoviruses make up the largest enterovirus subgroup, consisting of 32 serotypes (types 1–34; echovirus 10 and 28 were found to be other viruses and thus the numbers are unused), but not all cause human illness. Echoviruses 10 and 28 have been reclassified as reoviruses, and echovirus 9 is now recognized as the same as coxsackievirus A23. 7.4.3 Pathogenicity Echoviruses are common human pathogens that cause a range of illnesses, from minor febrile illness to severe, potentially fatal conditions (e.g., aseptic meningitis, encephalitis, paralysis, myocarditis). Individual serotypes have different temporal patterns of circulation and cause different clinical manifestations. Aseptic meningitis, febrile illnesses with or without rash, common colds, and acute hemorrhagic conjunctivitis are among the diseases caused by echoviruses. 7.4.4  Mode of transmission Echoviruses are infective over a wide range of pH (3–10) and are resistant to ether and alcohol. 7.4.5  Associated with seafood Two foodborne outbreaks of echovirus illness have been recorded (NYDH, 1989; USDHEW, 1979). Changes in circulating serotypes can be associated with large-scale outbreaks.

7.5  Aichivirus A (AiV-1) 7.5.1  Systematics and morphology Aichivirus belongs to genus Kobuvirus under Picornaviridae family in the order Picornavirales under the class Ribohelica (Sasaki et al., 2001). It is a small, unenveloped with single stranded positive sense ribonucleic acid virus with 8.2 kb genome. The single, large ORF encodes a polyprotein of 2,432 amino acids that is cleaved into the typical picornavirus structural proteins (VP0, VP3, VP1) and nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, 3D). Based on the phylogenetic analysis of 519-bp sequences at the 3C-3D (3 CD) junction. A morphological study on purified Aichivirus virions indicated that the surface structure features a small round structured virus (Yamashita et al., 1991). 7.5.2  Virus types Aichivirus A consists of three genetically distinct members with different host species, namely, AiV-1 (Aichivirus in humans), canine kobuvirus 1, and murine kobuvirus 1 (Kitajima and Gerba, 2015).

Risk From Viral Pathogens in Seafood  459 7.5.3 Pathogenicity The clinical signs and symptoms of AiV-1 infection typically include diarrhea, abdominal pain, nausea, vomiting, and fever. It produces diarrhea in patients in the 15- to 34-year-old age group, with 50%–76% possessing neutralizing antibodies. It has been isolated in studies of Finnish children, Pakistani children, and Japanese travelers. A number of clinical investigations demonstrated a low incidence of AiV-1 infection in patients with either sporadic or epidemic gastroenteritis. However, recent clinical studies have shown that AiV-1 is usually present together with other enteric viruses in fecal samples of gastroenteritis patients (Alcalá et al., 2010). These findings suggest: AiV-1 might be circulating without causing any symptoms, AiV-1 could be responsible for a portion of subclinical gastroenteritis infections requiring no medical attention, and/or AiV-1 may contribute to mixed viral infections leading to enteric disease. 7.5.4  Mode of transmission One indication that Aichi viruses may be transmitted by the fecal–oral route is the detection of these viruses in sewage samples in Tunisia (Sdiri-Loulizi et al., 2010), in surface waters in Venezuela, and in sewage and river waters in Japan. 7.5.5  Associated with seafood AiV was first recognized in 1989 as the cause of oyster-associated nonbacterial gastroenteritis in Japan (Yamashita et al., 2000). The detection of AiV in shellfish from the contaminated harvesting area and the identification of this virus as the sole pathogen found in at least one person’s stool sample suggest that AiV contributed to the illness burden seen in outbreaks in France. Le Guyader et al. (2008) cited that 8% of oyster samples from France were implicated with AiV-1 genotype A. Similarly, it has been reported in 6.6% of shellfish in Tunisia (Sdiri-Loulizi et al., 2010). The virus is found to be mostly foodborne.

7.6  Norovirus (NoV) 7.6.1  Systematics and morphology NoV are a member of the genus Norovirus (previously referred to as Norwalk-like viruses [NLVs] or small round-structured viruses [SRSVs]) belonging to the family Calciviridae of the order Calcivirales under the class Ribohelica. The noroviruses are 28- to 35-nm, nonenveloped, single strand of RNA virus with a linear genome of ∼7.6 kb in icosahedral symmetry having buoyant density in cesium chloride gradient of 1.36–1.41 g/mL. 7.6.2  Virus types NoVs are categorized in 5 genogroups, designated GI–GV, based on amino acid identity in the major structural protein (VP1). The strains that infect humans (referred to collectively as “human noroviruses”) are in G1, G2, and G4 while G3 infects cows and mice. These strains are causal for outbreaks of NoV globally (Siebenga et al., 2009).

460  Chapter 15 7.6.3 Pathogenicity NoV infection produces a self-limiting disease of abdominal cramps nausea, vomiting, myalgia, low fever, and sometimes headache. Symptoms of gastroenteritis usually begin within 40 h (mostly 12–72 h) after consumption of contaminated food. Gastroenteritis caused by NoV is a self-limiting illness which usually persists 48 h, but can last a long as 1 week (Bryan, 1986; Grohmann et al., 1981; Gunn et al., 1982; Morse et al., 1986; Porter and Sarkin, 1987). Infectious viruses can be transmitted not only at the time of illness but also during the incubation period and after recovery, with 30% of cases shedding virus for up to 3 weeks after infection. NoV can persist in the environment and can infect large numbers of people irrespective of ages and seasons. Although it generally causes a mild illness, the severity of risk—even death—increases for elderly and underlying sick people. 7.6.4  Mode of transmission Humans are the only known reservoir, and NoV is passed on individual to individual by infected food, water, or through the fecal–oral way, as well as the environment. Many foodborne NoV outbreaks have been described, often caused by infected food handlers. NoVs can survive outside the host, are resistant to common disinfectants and extreme pH fluctuations, and are highly infectious. As a result, transmission of virus via fomites is likely. 7.6.5  Associated with seafood NoV is associated with both bivalve molluscs and finfish, causing 16% of all seafood-related outbreaks and almost 30% of the illnesses reported from 1973 to 2006. Illness from Norwalk virus has been associated with eating clams (both raw and steamed) (Morse et al., 1986; Porter and Sarkin, 1987), oysters (Gunn et al., 1982), and cockles (Gunn et al., 1982). It is now considered a major cause of nonbacterial gastroenteritis. Shellfish, particularly bivalve molluscs that are filter feeders, can accumulate large numbers of viruses. Contamination of harvest areas with human sewage, which has occurred following overboard sewage dumping by oyster harvest boats and from sewage runoff into harvest areas following heavy rains, has been an important source of outbreaks. Additionally, insufficient cooking, such as steaming clams only until they open rather than to higher temperatures that kill NoV, has contributed to illness and outbreaks. Control measures have focused on monitoring harvest waters for fecal coliforms and encouraging adequate cooking of seafood; however, continued outbreaks of illness have brought renewed attention to the risks associated with oyster consumption.

7.7  Sapoviruses (SaV) 7.7.1  Systematics and morphology Sapoviruses (SaV) were previously called “typical human caliciviruses” or “Sapporo-like viruses.” They belong to the genus Sapovirus within the family Caliciviridae of the order Calcivirales under the class Ribohelica. SaV are 28- to 35-nm, nonenveloped, positive-

Risk From Viral Pathogens in Seafood  461 sense, single-stranded RNA viruses with a genome of approximately 7.6 kb and icosahedral capsid symmetry and exhibit the properties of the Caliciviridae. SaV are morphologically distinguishable from other gastroenteritis pathogens (e.g., norovirus, rotavirus, astrovirus, or adenovirus) by their typical “star of David” surface morphology under the electron microscope. 7.7.2  Virus types SaV are divided into multiple genogroups based on complete VP1 sequences. At present, four genogroups of human SaV (G1, G2, G4, as well as G5) out of five are categorized (Farkas et al., 2004; Oka et al., 2012). 7.7.3 Pathogenicity Major clinical symptoms include diarrhea and vomiting; however, additional constitutional symptoms (i.e., nausea, stomach/abdominal cramps, chills, headache, myalgia, or malaise) are also frequently reported. Similar to the case for NoV illness, fever is a rare clinical symptom. Diarrhea usually resolves within 1 week, however, individuals showing symptoms for a longer time (i.e., from over a week to up to 20 days) were also reported. In general, the severity of sapovirus gastroenteritis is milder than that for rotavirus and norovirus. Based on the epidemiological data from patients with sapovirus gastroenteritis, the incubation period ranges from less than 1–4 days (Oka et al., 2015). Gastroenteritis symptoms are usually self-limiting, and patients usually recover within a couple of days; however, the symptoms, severity, and duration of disease are dependent on the individual, and sapovirus infection sometimes leads to hospitalization (Monica et al., 2007; Zintz et al., 2005). Mortality is rare, but it has been reported from outbreaks that occurred in a long-term-care facility for the elderly (Lee et al., 2012). SaV infects and causes disease in humans of all ages, in both sporadic cases and outbreaks. Infectious dose and stability in the environment remain unknown. 7.7.4  Mode of transmission SaV were likely viruses of human fecal origin that were discharged into environmental waters and accumulated in shellfish (i.e., oysters or clams) (Oka et al., 2015). SaV contamination levels were ∼1.6 × 104 copies/g of digestive tissue in various types of shellfish (oyster, cockle, and smooth clam) (Benabbes et al., 2013). Transmission of SaV passes the fecal–oral way, as well as in infected food, water, materials, and individual-to-individual contacts. The foodborne transmission is rare, while seafood has so far been not implicated (Greening, 2006). 7.7.5  Associated with seafood SaV are genetically indistinguishable (i.e., similar or identical based on partial virus genome sequences) between those detected in human clinical specimens and those detected in

462  Chapter 15 shellfish (oysters and clams) (Hansman et al., 2007; Iizuka et al., 2010, 2013; Oka et al., 2015; Ueki et al., 2010).

7.8  Hepatitis E Virus (HEV) 7.8.1  Systematics and morphology HEV belongs to Hepevirus genus of Hepeviridae family in the order Hepevirales under the class Ribohelica. HEV is a small (32–34 nm) single (+) stranded RNA coated with protein with a linear genome of 7.2 kb that shows characteristic cup-like depressions (Cromeans et al., 1994). The capsid symmetry is icosahedral, and the buoyant density in potassium tartrate-glycerol gradient is 1.29 g/mL. HEV and caliciviruses have more common and distinctive features but genomically they are quite dissimilar. 7.8.2  Virus types Two HEV serotypes and four major HEV genotypes have been identified based on nucleotide and protein sequencing. Genotype 1 includes Asian and African strains whereas genotype 2 is Mexican, while genotype 3 is from the USA—strains for swine and humans—and genotype 4 comprises strains from China, Japan, and Taiwan (Emerson and Purcell, 2003). 7.8.3 Pathogenicity The disease caused by HEV is called hepatitis E/enterically transmitted non-A and non-B hepatitis (ET-NANBH)/fecal–oral non-A and non-B hepatitis/A-like non-A non-B hepatitis. Clinical manifestations of HEV and HAV have many similarities like acute and self-limiting live disorder, but HEV has a higher mortality rate for infected pregnant women. Moreover, HEV is prevalent in Asian and other developing countries. 7.8.4  Mode of transmission It is a non-A and non-B hepatitis virus that is known to be transmitted by the fecal–oral route. Waterborne outbreaks are common; but for some reason, foodborne outbreaks have not yet been reported. However, HEV waterborne infections include the risk of shellfish mediated transmission. 7.8.5  Associated with seafood Environmental contact of HEV with possible transmission by eating shellfish in the United Kingdom warranted investigation (Crossan et al., 2012). Shellfish (prawns, lobster, crab, mussels, and scallops) and mixed seafood (a mixture of shrimp or small prawns, salmon, cod, mussels, hake, and squid) caused acute infections of HEV in 33 participants in a world cruise in 2008 (Said et al., 2009). Many infections by non-A and non-B hepatitis viruses are associated with shellfish ingestion (Jaykus et al., 1993; Torne et al., 1988). Presently one cannot show proof but it is a clear suggestion of shellfish-associated HEV transmission.

Risk From Viral Pathogens in Seafood  463

7.9  Rotavirus (RV) 7.9.1  Systematics and morphology Rotaviruses (RVs) belong to genus Rotavirus in the family Reoviridae of order Reovirales under the class Ribohelica. Rotaviruses are 60–80 nm, nonenveloped, linear segmented double-stranded RNA viruses with icosahedral capsid symmetry. The 16–27 kb genome is enclosed by a triple-layered capsid surrounded by a double protein coat (Sattar et al., 1994). Electron micrographs of RVs show a characteristic wheel-like appearance, hence the name rotavirus, derived from the Latin meaning “wheel.” 7.9.2  Virus types There are five species of RV, designated rotavirus A (simian rotavirus) through rotavirus E (porcine rotavirus). Two possible species, rotavirus F (avian) and rotavirus G (avian) are also listed but they differ in their ability to reassort the genome segments. Most human infections are caused by rotavirus A, B, and C, but all rotaviral species can infect a range of vertebrates (van Regenmortel et al., 2000; Sattar, 2001). 7.9.3 Pathogenicity Rotavirus is the main causal for severe acute diarrhea among young children worldwide (Parashar et al., 2006). The disease, which affects all age groups, is generally considered a mild infection in adults. The incubation period for RV infection is 1–2 days. Typical symptoms are vomiting and watery diarrhea, which develop quickly and persist for 3–8 days. Dehydration is a key factor that contributes to the high infant death rate, especially in developing countries where there is no availability of good treatment. Globally, human rotaviruses (HRV) are causal for high dehydrating viral gastroenteritis among infants, as well as children. These lead to the main mortality rate of children in developing countries. The WHO surveillance networks have revealed that between 2001 and 2008, approximately 40% of the hospitalizations for diarrhea among children under the age of 5 were attributed to HRV. 7.9.4  Mode of transmission Globally, principal mode of HRV transmission is by individual-to-individual contact. However, under poor hygienic conditions, water and foodborne transmission is possible. 7.9.5  Associated with seafood HRV are passed on through the fecal–oral way and infection is not generally looked upon as foodborne. There have been some reports of outbreaks that were associated with food and water in a number of countries (Sattar, 2001). Italy has witnessed a major viral gastroenteritis through drinking water, which was attributed to contamination of both RoVs and NoVs. The origin of contamination could not be found, but extra chlorination of the water solved the problem (Martinelli et al., 2007). In the Netherlands, poor food hygiene was identified

464  Chapter 15 as one of the major risk factors for RoV infection (De Wit et al., 2001). RoV are found in wastewater and can also be concentrated by shellfish, however, HRV have not been linked with infectious disease following seafood consumption (Cook et al., 2004; Lees, 2000). Food contaminated after being cooked may also be the source of viral infection (Svensson, 2000; Cook et al., 2004).

7.10  Astroviruses (AstVs) 7.10.1  Systematics and morphology AstV belong to the genus Mamastrovirus of the family Astroviridae in the order Astrovirales under the class Ribohelica. Human astroviruses (HAstVs) contain a single positive strand of RNA of about 7.5 kb surrounded by a protein capsid of 28–30 nm diameter. A five- or sixpointed star shape can be observed on the particles under the electron microscope. Mature virions contain two major coat proteins of about 33 kDa each. They are so called because their surface has a star-shaped configuration, which is not seen on all particles. Its structure is unknown but is unlikely to be based on an icosahedron, with which the six-pointed stars are incompatible. 7.10.2  Virus types There are eight serotypes, HAstVs-1 to HAstVs-8, with HAstVs-1 being most frequently associated with gastroenteritis. 7 serotypes of HAstVs are found by neutralization tests, which mainly cause gastroenteritis in children. There is no group antigen. 7.10.3 Pathogenicity HAstVs are the principal agents for acute diarrhea among children resulting in many outbreaks and leading to hospitalization. AstV disease is generally milder than that caused by RoVs. The frequent coinfections of AstV, RoV, and CVs in diarrhea in children makes it difficult to understand its epidemiology. Infections are more common in winter. The usual features of the disease vary slightly from those of the Norwalk-like viruses: the incubation period is somewhat longer, vomiting is less common, and very young children (1 year) are more often affected. 7.10.4  Mode of transmission Epidemiologic evidence of transmission via foods is limited. Infection is most common in the winter and is spread by fecal–oral transmission. 7.10.5  Associated with seafood Astroviruses, adenoviruses, and parvoviruses are enteric viruses that have been implicated in recreational water and foodborne (seafood) outbreaks. Even though some reports of shellfishassociated AstV infections are available, their role seems of minor magnitude as for the immunity of adults.

Risk From Viral Pathogens in Seafood  465

7.11  Parvovirus (ParV) 7.11.1  Systematics and morphology Parvoviruses (ParV) belong to the genus Parvovirus of Parvoviridae family in order Parvovirales under the class Deoxyhelica. Parvoviruses are linear, nonpermuted singlestranded DNA viruses and are among the smallest known viruses at 20–30 nm in diameter, unenveloped, icosahedral symmetry, and genome of ∼5 kb. They have a smooth surface with no discernable features and were included with “small round viruses” before definitive classification of these viruses was completed. They have been proposed as causal agents of human gastroenteritis but their role in viral gastroenteritis of some animal species has also been well documented. 7.11.2  Virus types Parvoviridae family has three genera: Depedovirus, Densovirus and the Parvovirus which members inflect vertebrates including humans. Moreover, human parvoviruses 4 and 5 have recently been isolated from humans but until now have not been classified. Human parvovirus B19V genotype 1 is a global and common infectious pathogen in humans. 7.11.3 Pathogenicity Human parvovirus B19 (B19) has been linked with a broad spectrum of clinical syndromes, including erythema infectiosum, transient aplastic crisis, persistent infection manifesting as pure red cell aplasia in immunocompromised individuals, nonimmune hydrops fetalis, and arthritis (Barah et al., 2014). Human parvovirus infection has since been associated with arthritis, erythema infectiosum (fifth disease), fetal death, and hydrops fetalis (Joseph, 1988). The infection can occur in any month of the year in sporadic or epidemic form. In temperate climates, however, infections usually occur in the spring with epidemic peaks every 2–4 years. 7.11.4  Mode of transmission B19V transmission generally occurs by the respiratory route. Transmission takes place frequently among household and school contacts during outbreaks of B19V infection, with higher risks being associated with close contact between infected and susceptible individuals for a prolonged time. 7.11.5  Associated with seafood There is limited evidence of parvovirus association with foodborne disease, but it has been linked to the ingestion of infected shellfish (Appleton, 2001; Appleton and Pereira, 1977). ParV was linked to a large outbreak in the United Kingdom by the ingestion of infected cockles (Appleton, 2001). More than 800 confirmed cases of gastroenteritis occurred, and parvovirus was identified in all stools examined from this large gastroenteritis outbreak.

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7.12  Adenoviruses (AdVs) 7.12.1  Systematics and morphology AdVs belong to the Adenoviridae family of order Adenovirales under the class Deoxyhelica. They are classified into two genera: the Mastadenovirus, which infects mammals, and the Aviadenovirus, which infects birds. More than 100 members of the Adenoviridae have been isolated from humans and animals, including birds and amphibians. AdVs have spherical, middle-sized (68–85 nm) double-stranded DNA, unenveloped, linear with icosahedral symmetry under electron microscopy (Votava et al., 2003). They are described as a nonenveloped virus, which makes them extremely stable in the environment. 7.12.2  Virus types Six species of human adenoviruses (HAdV-A to HAdV-F) have been identified according to DNA homology (van Regenmortel et al., 2000). Between 50% and 90% DNA homology exists within these species, but only 5%–20% homology exists between the species. To date, there are at least 52 immunologically distinct types that can cause human infections. The most common one used in research is Adenovirus-type 5 (Ad-5, Ad5, Adeno5). 7.12.3 Pathogenicity A series of infections in humans are produced by this group of viruses, which include gastroenteritis, conjunctivitis, and several respiratory conditions. Together with adenoviruses causing respiratory diseases they can be isolated from feces of affected children. Incidence of AdV infections in the Czech Republic is low; 285 cases were recorded in the year 2003 (Drapal et al., 2003). AdVs are a group of viruses that cause respiratory infection, which may have a range of symptoms from the common cold to pneumonia (infection of the lungs). Adenoviruses may also produce infections in the eyes, in the brain, and spinal cord, in the urinary tract of children, and present as diarrhea in infants. Patients with poorly functioning immune systems are especially prone to severe and life-threatening infections. AdVs are a frequent cause of acute upper respiratory tract infections, such as the common cold, but have also been associated with other forms of illness, such as conjunctivitis (pink eye), gastrointestinal illness, and urinary tract infections. 7.12.4  Mode of transmission AdVs can spread not only via droplet infection, but also via the fecal–oral route. 7.12.5  Associated with seafood AdVs may be detected in sewage, seawater, and shellfish (Pina et al., 1998; Vantarakis and Papapetropoulou, 1999). AdVs were detected in 18.6% of the shellfish samples from the Norwegian coast with more positive samples during winter (Myrmel et al., 2004). MuniainMujika et al. (2002) have also reported the presence of pathogenic viruses from shellfish

Risk From Viral Pathogens in Seafood  467 where human adenoviruses were found in 47% of the samples. It was determined that all the samples positive for enterovirus and HAV were also positive for human AdV. The human adenoviruses detected by polymerase chain reaction (PCR) correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish (Formiga-Cruz et al., 2002).

8  Risk Factors The viruses and the nucleic acid signature survive for an extended period in the marine environment. One of the primary concerns of public health officials to relate presence of pathogens and the recreational as well seafood risks to human health in polluted marine environments. Virus survival and transmission in seafood (Rodríguez-Lázaro et al., 2012) can be affected by diverse factors: 1. We are infected through food contaminated with human sewage carrying viral pathogens. These types of infections have been well recognized by the detection of HAV from sewage treatment workers (Cadilhac and Roudot-Thoraval, 1996). Studies from the USA, Europe, and Japan have resulted positive for enteric viruses (da Silva et al., 2007; Gregory et al., 2006; La Rosa et al., 2007; Laverick et al., 2004; Myrmel et al., 2006; Ueki et al., 2005; van den Berg et al., 2005; Villar et al., 2007). 2. Infected food handlers are capable of seafood contamination. Carrying handlers can shed viruses 12 h after infection of NoV and for several weeks for other viruses (Rockx et al., 2002). 3. The other main factor responsible for seafoodborne viral transmission is the stability in food processing. Enteric viruses are recalcitrant to common food processing and preservative methods. Though refrigeration and freezing have little effect, freezing provides a more effective viral preservation method (Papafragkou et al., 2006). Because of concerns about virus persistence in food processing, effective control strategies need to focus on prevention of contamination. Such prevention will have to occur at the preharvest level for some products (bivalve molluscs, fresh produce for raw consumption), and at the postharvest phase for others (prepared and ready-to-eat foods). The persistence of viruses in foods contaminated at either phase is well documented in epidemiological publications. Present findings envisage the binding of NoV in receptor sites of shellfish tissues, as well as continuing for 8–10 weeks, persisting even after depuration (FAO/WHO, 2008a,b; Le Guyader et al., 2006). 4. The stability of seafoodborne viruses in marine environs affects overall viral diseases. 5. The most important risk factor in the primary cases (HAV) was consumption of raw seafood, while person-to-person contact, perhaps between children, played a major part in secondary cases (Germinario et al., 2000). 6. The risk is increased because many species are cultivated in near-coastal waters, where contamination with human sewage-which may contain high levels of viral particles may

468  Chapter 15 easily occur. Ingestion of shellfish that have not been sufficiently heated poses a high risk for norovirus infection. 7. Persons with underlying medical conditions, such as liver disease, diabetes, or immunosuppressing conditions and those on organ transplant or cancer medications are at higher risk of acquiring severe infection also particularly vulnerable. Some studies have reported advanced age and a lower socioeconomic status to be risk factors for NoV infection. Pregnant women and their unborn babies, young children and older people are at particular risk.

9  Detection and Diagnosis There are a large number of seafoodborne illnesses of unknown etiology generally classified as viral (e.g., norovirusal). Evidence is lacking due to limitations in the methodology for culturing and enumerating viruses; further identification is difficult. Detection of virus in contaminated seafood will be useful for studying related issues and developing interventions that will result in improved outbreak prevention and control. Detection of viruses in seafood is most likely to be undertaken when an outbreak has occurred. Outbreak investigation and routine monitoring present very different sets of priorities. It would be helpful, however, if some routine testing method was available to apply to seafood, such as shellfish, that often serve as vehicles for viruses. Viruses are obligate parasites on living cells of specific hosts. There is no specific proof that seafood (finfish and shellfish) hosts as replicating vectors but they have some role in a passive transfer of viruses to humans (Bosch et al., 2008). Moreover, meager viral presence in shellfish could pose a health risk (Bosch et al., 1994; Le Guyader et al., 2008, 2006; Sánchez et al., 2002). RT-PCR assay is being used to find out entericviruses from shellfish (Costafreda et al., 2006; Jothikumar et al., 2005; Loisy et al., 2005). This application can also quantify viruses from a sample (Bosch et al., 2008; Torok, 2013). The clinical diagnosis of viral infection can be achieved by performing analytical tests on serum, stool, and, in some instances, vomitus. Diagnosis also can be achieved by examining blood serum samples for a rise in virus-specific serum antibody titers, measured by enzyme immunoassay (i.e., ELISA or EIA). Electron microscopy and polyacrylamide gel electrophoresis are used in some laboratories. There is no test available in routine hospital laboratories but specialist virology laboratories can use a molecular test to detect the virus. With the recent application of seminested or nested PCR (second-round PCR with an internal set of primers) (Green et al., 1998; Hafliger et al., 1997), subsequent characterization of PCR amplicons derived from shellfish has been improved significantly. Ample PCR products from the second-round amplification facilitate the success of cloning and sequencing of an amplicon. Nucleotide sequence information from the amplified products can be used to confirm the result of Southern hybridization and to genotype the virus strains derived from

Risk From Viral Pathogens in Seafood  469 contaminated shellfish (Butt et al., 2004). Successful PCR detection of viruses in shellfish depends upon the efficient recovery of viruses and the effective removal of PCR inhibitors naturally occurring in shellfish. These two criteria are important for PCR detection of low levels of viruses (Carol Shieh et al., 2000). Despite the increased sensitivity of quantitative RT-PCR methods for detecting viruses, the risk associated with illness from consuming contaminated food is not clear for two reasons: (1) the limit of detection is not sensitive enough to detect a theoretical infectious dose, and (2) viral copies detected by PCR are not directly related to infective viral particles as nonencapsidated RNA and degraded viral RNA (EFSA, 2011). Hence, the RT-PCR is the most advanced and sensitive methodology currently available (Ozawa et al., 2015). However, research studies in several countries suggest that virus is commonly detected in commercially produced bivalves using PCR (Bosch et al., 2009). A major factor is the current absence of any standardized and validated methods with the demonstrable performance necessary to both protect public health and avoid trade disputes. Within the European Union (member organization), a network of specialist reference laboratories, participating in the European Committee for Standardizationm, is working toward the development of a standard method for detection of human pathogenic viruses in foods (including bivalve molluscs), which may help resolve some of these issues (FAO, 2014). Further, there is a need for easier availability in improved methods to detect infectious and pathogenic viruses in seafood.

10  Risk Management Prevention of seafood-associated viral infections requires an understanding of not only the etiologic agents and seafood commodities associated with illness but also the mechanisms of contamination that are amenable to control. Risk management and communication are often performed by public health organizations including risk evaluation, option assessment, option implementation, monitoring and review, and dissemination of information (FAO/WHO, 1997; FAO/WHO, 2008a,b). Extensive environmental monitoring and seasonal quarantine of a harvest are employed to reduce the risk of diseases by safety measures, such as good handling practices, good manufacturing practices, and the hazard analysis critical control point (HACCP). The criteria for viral risk management are as follows: 1. Strict implementation of hygienic rules is currently considered the most important preventive measure. Foodhandlers with gastroenteritis should immediately be removed from the seafood chain. More problematic are outbreaks linked to asymptomatic, presymptomatic, and postsymptomatic shedders. The kinetics of viral shedding has been studied in only a few infected volunteers and may not reflect real-life situations in which people may have been infected with a low dose of infectious virus. Given the highly infectious nature of NoV and the documented risk of virus transmission to seafood during the incubation period, it is suggested that guidelines be developed that include

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2.

3.

4.

5. 6.

the occurrence of gastroenteritis in contacts (e.g., children) of people working at critical points in the food chain. This should be based on data on the kinetics of viral shedding after natural infection. The globalization of the food market has hampered the implementation of control measures to assure safe seafood, as it is often difficult to exactly trace the food. Routine monitoring is not yet feasible: first, because there are no good methods, and, second, because end-product testing is not reliable to assure food safety on statistical grounds. Documented outbreaks of foodborne infections could be reported faster using a system, such as the “rapid alert system for seafood” of the European Union. However, this would be much more informative if typing information of virus strains were included. Seafoodborne infections by human enteric viruses in raw and improperly cooked molluscan shellfish could be decreased significantly by the development of valid growing water indicator(s) and of direct detection methodologies for entericviruses. The FDA plays an important role in establishing guidelines and providing oversight to ensure safety. Prevention strategies developed by the FDA and the seafood industry to minimize the risk of microbial contamination and decrease the risk of seafoodassociated infection include good manufacturing practices, which address sanitation conditions and practices, and the HACCP program. The purpose of a HACCP is to identify sources and points in processes at which the risk of contamination is high, from harvest to consumption, so that processes to decrease these risks can be implemented and monitored. During the mid-1990s, the FDA issued regulations for the seafood industry, relying on HACCP-based principles. Every seafood harvester and processor is now required to use a HACCP-based method. The national shellfish sanitation program constituted by FDA and ISSC guidelines for storage times and temperatures for specific seafood products. Current methods for molecular detection of viruses in oysters cannot be readily adapted for routine monitoring by local regulatory laboratories. Until means for routine monitoring become available (e.g., viral indicators), the most effective preventative measures are to educate the public regarding appropriate disposal of human waste, especially near the vicinity of shellfish-growing waters. Local authorities are encouraged to establish means to minimize or eliminate human fecal contamination in shellfish-growing areas, such as portable toilets in remote areas used for recreation, routine inspection of sewage-treatment systems, mandatory requirements for boats to be equipped with marine sanitation devices, and making waste pump-out facilities available. Many postharvest controls are not effective in completely eliminating viruses from contaminated seafood.

11 Conclusions The occurrence of several human enteric viruses: NoV, SaV, RV, PV, CVs, EchoV, AiV, ParV, AdV, AstV, HAV, and HEV have been identified in seafood, but not all have been clearly linked with disease outbreaks. These viruses, that is, AdV, AstV, NoV, RV, and also SaV

Risk From Viral Pathogens in Seafood  471 produce gastroenteritis, HAV, as well as HEV cause enterically transmitted hepatitis; PV, CVs, and EchoV induce illness migrating to CNS after replicating in the human intestine. Both HAV and NoV have emerged as broadly associated with seafood. Moreover, HEV has been partnered with infected oyster ingestion.

Acknowledgments The author is grateful to the authorities of VIT University, Vellore 632014, Tamil Nadu, for the facilities and support. He also thanks one of his research scholars (Ms. Kirti) for her assistance with preparation of the References.

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Further Reading Ambert-Balay, K., Lorrot, M., Bon, F., Giraudon, H., Kaplon, J., Wolfer, M., Lebon, P., Gendrel, D., Pothier, P., 2008. Prevalence and genetic diversity of Aichi virus in community and hospitalized patients. J. Clin. Microbiol. 46, 1252–1258. Atmar, R.L., Estes, M.K., 2001. Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin. Microbiol. Rev. 14, 15–37. Bobowick, A., Brody, J., Mathews, M., Roos, R., Gajdusek, D., 1973. Creutzfeld-Jacob disease: a case-control study. Am. J. Epidemiol. 98, 381–394. Bosch, A., 2007. Human Viruses in Water. Perspectives in Medical Virology Series, vol. 17. Elsevier, Amsterdam, The Netherlands. Bryan, F.L., 1980. Epidemiology of foodborne diseases transmitted by fish, shellfish and marine crustaceans in the United States 1970-1978. J. Food. Prot. 43, 859–876.

Risk From Viral Pathogens in Seafood  479 Centers for Disease Control and Prevention (CDC), 1998. Outbreak of Vibrio parahaemolyticus infections associated with eating raw oysters—Pacific Northwest, 1997. Morb. Mortal.Weekly Rep. 47, 457–461. Centers for Disease Control and Prevention (CDC), 1994. Viral hepatitis surveillance program, 1990–1992. Hepatitis Surveillance Report No. 55. US Department of Health and Human Services, Atlanta, GA, pp. 19–34. Centers for Disease Control and Prevention (CDC), 1982. Foodborne disease outbreaks, annual summary 1982. Issued September 1985. USDHHS Publication No. (CDC) 85-8185. Centers for Disease Control and Prevention (CDC), 1980. Foodborne disease outbreaks, annual summary 1980. Issued February 1983. USDHHS Publication No. (CDC) 83-8185. Cole, M.T., Kilgen, M.B., Reily, L.A., Hackney, C.R., 1986. Detection of enteroviruses and bacterial indicators and pathogens in Louisiana oysters and their overlying waters. J. Food Protect. 49, 596–601. Croci, L., De Medici, D., Scalfar, C., Fior, A., Toti, L., 2002. The survival of hepatitis: a virus in fresh produce. Int. J. Food Microbiol. 73, 29–34. Davanipour, Z., Alter, M., Sobel, E., Asher, D., Gajdusek, D., 1985. A case-control study of Ceutzfeld-Jacob disease: dietary risk factors. Am. J. Epidemiol. 122, 443–451. De Grazia, S., Medici, M.C., Pinto, P., Moschidou, P., Tummolo, F., Calderaro, A., Bonura, F., Banyai, K., Giammanco, G.M., Martella, V., 2012. Genetic heterogeneity and recombination in human type 2 astroviruses. J. Clin. Microbiol. 50, 3760–3764. De Grazia, S., Platia, M.A., Rotolo, V., Colomba, C., Martella, V., Giammanco, G.M., 2011. Surveillance of human astrovirus circulation in Italy 2002-2005: emergence of lineage 2c strains. Clin. Microbiol. Infect. 17, 97–101. De Leon, R., Matsui, S.M., Baric, R.S., et al., 1992. Detection of Norwalk virus in stool specimens by reverse transcriptase–polymerase chain reaction and nonradioactive oligoprobes. J. Clin. Microbiol. 30, 3151–3157. DePaola, A., Jones, J.L., Woods, J., Burkhardt, W., Calci, K.R., Krantz, J.A., Bowers, J.C., Kasturi, K., Byars, R.H., Jacobs, E., Williams-Hill, D., Nabe, K., 2010. Bacterial and Viral Pathogens in Live Oysters: 2007 United States Market Survey. Appl. Environ. Microbiol. 76, 2754–2768. Dismukes, W., Bisno, A., Katz, S., Johnson, R., 1969. An outbreak of infectious hepatitis attributed to raw clams. Am. J. Epidemiol. 89, 555–561. European Food Safety Authority, 2012. Scientific opinion on norovirus (NoV) in oysters: methods, limits and controloptions. EFSA Journal 10, 2500. Elliot, E.L., Colwell, R.R., 1985. Indicator organisms for estuarine and marine waters. FEMS Microbiol. Rev. 32, 61–79. EFSA/ECDPC (European Food Safety Authority/European Center for DiseasePrevention and Control), 2012. Report on trends and sources of zoonoses, zoonoticagents and food-borne outbreaks in 2010. EFSA Journal 10, 2597. Greening, G.E., Hewitt, J., Hay, B.E., Grant, C.M., 2003. Persistence of Norwalklike viruses over time in Pacific oysters grown in the natural environment. In: Villalba, A., Reguera, B., Romalde, J.L., and Beiras, R. (Eds.), Molluscan Shellfish Safety: Proceedings of the 4th International Conference on Molluscan Shellfish Safety. Consellería de Pesca e Asuntos Marítimos da Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, Santiago de Compostela, Spain, pp. 367–377. Hafliger, D., Hubner, P., Luthy, J., 2000. Outbreak of viral gastroenteritis due to sewage-contaminated drinking water. Int. J. Food Microbiol. 54, 123–126. Hiroki, O., Kumazaki, M., Ueki, S., Morita, M., Usuku, S., 2015. Detection and genetic analysis of noroviruses and sapoviruses in sea snail. Food Environ. Virol. 7, 325–332. Jaykus, L., D’Souza, D., Moe, C., 2013. Foodborne viral pathogens. In: Doyle, M., Buchanan, R. (Eds.), Food Microbiology. ASM Press, Washington, DC, pp. 619–649. Jiang, X., Wilton, N., Zhong, W.M., et al., 2000. Diagnosis of human caliciviruses by use of enzyme immunoassays. J. Infect. Dis. 181, 349–359. Kapikian, A.Z., Wyatt, R.G., Dolin, R., Thornhill, T.S., Kalica, A.R., Chanock, R.M., 1972. Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis. J. Virol. 10, 1075–1081.

480  Chapter 15 Kilgen, M.B., Cole, M.T., Hackney, C.R., 1988. Shellfish sanitation studies in Louisiana. J. Shellfish Res. 7, 527–530. Koff, R.S., Grady, G.F., Chalmers, T.C., Mosley, J.W., Swartz, B.L., and the Boston Inter-Hospital Liver Group, 1967. Viral hepatitis in a group of Boston hospitals. III. Importance of exposure to shellfish in a nonepidemic period. N. Engl. J. Med. 276, 703–710. Koopmans, M., Duizer, E., 2004. Foodborne viruses: an emerging problem. Int. J. Food. Microbiol. 90, 23–41. Koopmans, M., von Bonsdorff, C.H., Vinjé, J., de Medici, D., Monroe, S., 2002. Foodborne viruses. FEMS Microbiol. 26, 187–205. Le Guyader, F., Neill, F.H., Estes, M.K., Monroe, S.S., Ando, T., Atmar, R.L., 1996. Detection and analysis of a small round-structured virus strain in oysters implicated in an outbreak of acute gastroenteritis. Appl. Environ. Microbiol. 62, 4268–4272. Leoni, E., Bevini, C., Degli, Esposti, S., Graziano, A., 1998. An outbreak of intrafamiliar hepatitis A associated with clam consumption: epidemic transmission to a school community. Eur. J. Epidemiol. 14, 187–192. Martella, V., Moschidou, P., Catella, C., Larocca, V., Pinto, P., Losurdo, M., Corrente, M., Lorusso, E., Bànyai, K., Decaro, N., Lavazza, A., Buonavoglia, C., 2012. Enteric disease in dogs naturally infected by a novel canine astrovirus. J. Clin. Microbiol. 50, 1066–1069. Martella, V., Moschidou, P., Pinto, P., Catella, C., Desario, C., Larocca, V., Circella, E., Bànyai, K., Lavazza, A., Magistrali, C., Decaro, N., Buonavoglia, C., 2011. Astroviruses in rabbits. Emerg. Infect. Dis. 17, 2287–2293. Martelli, F., Caprioli, A., Zengarini, M., Marata, A., Fiegna, C., Di Bartolo, I., Ruggeri, F.M., Delogu, M., Ostanello, F., 2008. Detection of hepatits E virus (HEV) in a demographic managed wild boar (Sus scrofa scrofa) population in Italy. Vet. Microbiol. 126, 74–81. Matches, J.R., Abeyta, C., 1983. Indicator organisms in fish and shellfish. J. Food Protect. 37, 114–117. Medici, M.C., Tummolo, F., Albonetti, V., Abelli, L.A., Chezzi, C., Calderaro, A., 2012. Molecular detection and epidemiology of astrovirus, bocavirus, and sapovirus in Italian children admitted to hospital with acute gastroenteritis, 2008–2009. J. Med. Virol. 84, 643–650. Melnick, J.L., Gerba, C.P., Wallis, C., 1978. Viruses in water. Bull. World. Health Org. 56, 499–504. Meng, X.J., 2011. From barnyard to food table: the omnipresence of hepatitis E virus and risk for zoonotic infection and food safety. Virus Res. 161, 23–30. Mirazo, S., Ramos, N., Mainardi, V., Gerona, S., Arbiza, J., 2014. Transmission, diagnosis, and management of hepatitis E: an update. Hepat. Med. 3, 45–59. Moschidou, P., Martella, V., Lorusso, E., Desario, C., Pinto, P., Losurdo, M., Catella, C., Parisi, A., Bányai, K., Buonavoglia, C., 2011. Mixed infection by feline astrovirus and feline panleukopenia virus in a domestic cat with gastroenteritis and panleukopenia. J. Vet. Diagn. Invest. 23, 581–584. Nishida, T., Nishio, O., Kato, M., Chuma, T., Kato, H., Iwata, H., Kimura, H., 2007. Genotyping and quantitation of noroviruses in oysters from two distinct sea areas in Japan. Microbiol. Immunol. 51, 177–184. Overby, L.R., Deinhardt, F., Deinhardt, J. (Eds.), 1983. Viral Hepatitis: Second International Max von Pettenkofer Symposium. Marcel Dekker, New York, NY. Potasman, I., Paz, A., Odeh, M., 2002. Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective. Clin. Infect. Dis. 35, 921–928. Purcell, R.H., Emerson, S.U., 2000. Hepatitis E virus. Mandel, G., Bennett, J., Dolin, R. (Eds.), Principles and Practice of Infectious Diseases, 2, Churchill Livingstone, Philadelphia, PA,, pp. 1958–1970. Rabenau, H.F., Sturmer, M., Buxbaum, S., Walczok, A., Preiser, W., Doerr, H.W., 2003. Laboratory diagnosis of norovirus: which method is the best? Intervirology 46, 232–238. Richards, A.F., Lopman, B., Gunn, A., et al., 2003. Evaluation of a commercial ELISA for detecting Norwalk-like virus antigen in faeces. J. Clin. Virol. 26, 109–115. Rutjes, S.A., Lodder, W.J., Bouwknegt, M., de Roda Husman, A.M., 2007. Increased hepatitis E virus prevalence on Dutch pig farms from 33 to 55% by using appropriate internal quality controls for RTPCR. J. Virol. Methods 143, 112–116. Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., Widdowson, M.A., Roy, S.L., Jones, J.L., Griffin, P.M., 2011. Foodborne illness acquired in the United States: major pathogens. Emerg. Infect. Dis. 17, 7–15.

Risk From Viral Pathogens in Seafood  481 Sobsey, M., Shields, P., Hauchman, F., Davis, A., Rullman, V., Bosch, A., 1988. Survival and persistence of hepatitis A virus in environmental samples. In: Zuckermann, A. (Ed.), Viral Hepatitis and Liver Disease. Alan Liss, New York, NY, pp. 121–124. Sobsey, M.D., Hackney, C.R., Carrick, R.J., Ray, B., Speck, M.G., 1980. Occurrence of enteric bacteria and viruses in oysters. J. Food Protect. 43, 111–128. Storelli, M.M., 2008. Potential human health risks from metals (Hg, Cd, and Pb) and polychlorinated biphenyls (PCBs) via seafood consumption: estimation of target hazard quotients (THQs) and toxic equivalents (TEQs). Food Chem. Toxicol. 46, 2782–2788. Stroffolini, T., Biagini, W., Lorenzoni, L., Palazzesi, G.P., Divizia, M., Frongillo, R., 1990. An outbreak of hepatitis A in young adults in central Italy. Eur. J. Epidemiol. 6, 156–159. Tang, Y.W., Wang, J.X., Xu, Z.Y., Guo, Y.F., Qian, W.H., Xu, J.X., 1991. A serologically confirmed, case-control study, of a large outbreak of hepatitis A in China, associated with consumption of clams. Epidemiol. Infect. 107, 651–657. Thurn, J., 1988. Human parvovirus B19: historical and clinical review. Rev. Infect. Dis. 10, 1005–1011. Tomoichiro, O., Wang, Q., Katayama, K., Linda, J., 2015. Saif comprehensive review of human sapoviruses. Clin. Microbiol. Rev. 28, 32–53. US Food and Drug Administration (USFDA), 1984. Bacteriological analytical methods, sixth ed. Association of Official Analytical Chemists, Arlington, VA. Valeria, T., 2013. Review of foodborne viruses in shellfish and current detection methodologies. South Australian Research and Development Institute. White, D.O., Fenner, F., 1986. Medical Virology, third ed. Academic Press, Orlando, FL, USA. Yugo, D.M., Meng, X.J., 2013. Hepatitis E virus: foodborne, waterborne and zoonotic transmission. Int. J. Environ. Res. Public Health 25, 4507–4533.