Small Round Structured Viruses Research Articles

Molecular epidemiology and clinical characteristics of norovirus gastroenteritis with seizures in children in Taiwan, 2006-2015.

This study examined the characteristics of norovirus (NoV) gastroenteritis associated with convulsions in children and its molecular epidemiology. From July 2006 through December 2015, NoV infection was confirmed by the genome detection using reverse transcriptase polymerase chain reaction. Viral genotyping with strain validation was achieved using sequence analyses with Basic Local Alignment Search Tool genome identification. The patients’ clinical features were assessed retrospectively, focusing on convulsive disorders. The diagnosis of encephalitis followed the International Encephalitis Consortium. Seizures occurred in 52 (20.9%) of 249 NoV infections. GII.4 Den_Haag_2006b (n = 22, 42.3%) and GII.4 Sydney 2012 (n = 10, 19.2%) were major variants correlated with convulsions. Patient with convulsions tend to have GII.4 genotype infection (P < .001), short vomiting (≤2 days) (P < .001), and no fever (P = .002). Compared to GII.4 Den_Haag_2006b, the GII.4 Sydney 2012-associated convulsions had similar manifestations except without significant winter preponderance (P = .049). The NoV infection with convulsions had less febrile course, specific genotype (GII.4) infections, and with shorter symptom of vomiting. Continuous surveillance is important for uncommon disease associated with emerging NoV strain infections. The prevention of NoV diseases requires the development of vaccines targeting highly virulent variants.

Norovirus Outbreak in Iran

Implication for health policy/practice/research/medical education: Norovirus is the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide, and a major cause of foodborne illnesses. Due to the recent outbreak of norovirus in the northern area of Tehran, this report reviews the recent advances and provides guidelines for outbreak management and disease prevention for medical practitioners Copyright © 2013, Shahid Beheshti University of Medical Sciencces;. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Due to the recent outbreak of norovirus in the northern area of Tehran, this report reviews the recent advances and provides guidelines for outbreak management and disease prevention for medical practitioners. Norovirus is the most common cause of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide, and a major cause of foodborne illnesses. In the United States, approximately 21 million illnesses attributable to norovirus are estimated to occur annually. Since 2001, when the most recent norovirus recommendations were published (1), substantial advances have been made in norovirus epidemiology, immunology, diagnostic methods, and infection control. As molecular diagnostic techniques have improved and become more widely available, detection and reporting of norovirus outbreaks have increased. Although the inability to culture human noroviruses in vitro has hampered the progression, assessment of the performance of disinfectants has been facilitated by the discovery of new, cultivable surrogates for human noroviruses. In addition, the periodic emergences of epidemic strains (from genogroup II type 4, GII.4) as well as outbreaks in specific populations (e.g. the elderly in nursing homes) have been characterized (1). The prototype norovirus was first identified as the cause of a gastroenteritis outbreak in Norwalk, Ohio, in 1968 (2). However, the epidemiology of noroviruses remained poorly characterized until relatively recently, because of the lack of widespread availability of sensitive diagnostic methods (3). Noroviruses are a group of nonenveloped, single-stranded RNA viruses, classified into the genus norovirus (previously referred to as Norwalk-like viruses (NLVs) or small round-structured viruses (SRSVs) of the caliciviridae family) (4). Noroviruses cause acute gastroenteritis in all ages. The illness typically begins after an incubation period of 12 48 hours and is characterized by acute onset, nonbloody diarrhea, vomiting, nausea, and abdominal cramps. Some patients might only experience vomiting or diarrhea. Low-grade fever and body aches might also be associated with the infection, and thus, the term stomach flu is often used to describe the illness, although there is no biological association with influenza. Although symptoms might be severe, they typically resolve without treatment after 1 3 days in otherwise healthy persons. However, more prolonged courses of illness lasting 4 6 days can occur, particularly among young children, elderly persons, and hospitalized patients (5, 6). Approximately 10% of cases with norovirus gastroenteritis seek medical attention, which might include hospitalization and treatment for dehydration with oral or intravenous fluid therapy. Norovirus-associated deaths have been reported among elderly and in the context of outbreaks in long-term-care facilities (7). Necrotizing enterocolitis in neonates, chronic diarrhea in immunosuppressed patients and post-infectious irritable bowel syndrome have also been reported in association with norovirus infections (8). Norovirus is extremely contagious, with an estimated infectious dose as low as 18 viral particles, suggesting that approximately 5 billion infectious doses might be contained in each gram of feces during peak shedding. Humans are the only known reservoirs for human norovirus infections and transmission occurs by three general routes: person-toperson, foodborne and waterborne. Person-to-person transmission might occur directly through the fecaloral route, by ingestion of aerosolized vomitus, or by indirect exposure to fomites or contaminated environmental surfaces. Foodborne transmission typically occurs via contamination by infected food handlers durAr ch ive f S ID

STUDY ON THE NOROVIRUS PRESENCE IN MYTILUS GALLOPROVINCIALIS SUBJECTED TO DEPURATION IN TWO C.D.Ms. IN THE SARDINIA REGION

Noroviruses (NoVs), known as Norwalk-Like Viruses (NLV) or Small-Round-Structured-Viruses (SRVS), are among the most frequent causes of acute viral gastroenteritis in human beings, often associated with food poisoning, if raw or poorly cooked bivalve molluscs (mussels, clams and oysters) are ingested. In compliance with EU regulations, the safety of these products is evaluated according to bacteriological paramenters (Salmonella and E.coli) as provided for by Regulation (EC) No. 2073/2005, biotoxicological parameters, as provided for by Regulation (EC) No. 853/2004, and chemical parameters, as provided for by Regulation (EC) No. 1881/06. This essay focuses on the evaluation of NoV concentration levels in Mytilus galloprovincialis populations, farmed in 2009 in two different Mussels Depuration Centers (CDMs) in Sardinia (Italy). During the assessment procedures, Noroviruses were detected for the first time on the regional territory with one-step TaqMan real-time RT-PCR.

Inactivation of feline calicivirus as a surrogate for norovirus on lettuce by electron beam irradiation.

Caliciviridae, including norovirus, are considered important sources of human gastroenteritis. As leafy green vegetables are commonly consumed without additional processing, it is important to evaluate interventions to reduce the presence of human pathogens in these products. Feline calicivirus was used as a model for small round structured viruses on lettuce. The lettuce was inoculated by immersion to simulate contamination from irrigation or wash water. The inoculated lettuce was then exposed to electron beam irradiation at various dose levels to determine survival. The D₁₀-value of the calicivirus on lettuce was determined to be 2.95 kGy. Irradiation to reduce bacterial pathogens on cut lettuce could also reduce the risk associated with small round structured viruses on lettuce.

Development of a Rapid High-Throughput Method for High-Resolution Melting Analysis for Routine Detection and Genotyping of Noroviruses

We developed a simple, rapid, high-throughput detection and genotyping method for noroviruses using real-time reverse transcription-PCR (RT-PCR) and high-resolution melting (HRM) analysis to create a difference plot. The capsid gene was amplified by real-time RT-PCR in the presence of ResoLight HRM dye, a saturating DNA dye. Following optimization of the HRM assay conditions, the major norovirus genotypes were selected. Because we had only small quantities of the patient stool samples used in this study, we constructed plasmids for each genotype and used these to optimize the HRM assay. We selected six stool samples, each positive for one of the six dominant subtypes of noroviruses that have been circulating in Japan, namely, genotypes 4, 8, and 9 from genogroup 1 and genotypes 3, 4, and 10 from genogroup 2. The specific high-resolution derivate plot of the HRM assay for each plasmid was constructed by subtracting the melting-curve shape of the plasmid from the reference or base curve. The RNAs extracted from 14 clinical samples positive for small round structured viruses were then directly analyzed using the HRM assay. The HRM data from the clinical RNA samples corresponded with the genotype results obtained by RT-PCR and sequencing of the clinical samples. In addition, the HRM data from the clinical RNA samples corresponded with the HRM data from the six reference plasmid DNAs, indicating that this assay is useful for the direct detection and genotyping of noroviruses in clinical samples. This assay requires no multiplexing or hybridization probes and provides a new approach to the genetic screening of noroviruses in clinical virology laboratories.

Noroviruses: Simple Detection for Complex Epidemiology

Nearly 25 years ago, Kaplan et al. [1] developed criteria for recognizing outbreaks of gastroenteritis due to norovirus. In those days, the state-of-the-art diagnostic test for these ubiquitous and ecologically clever RNA viruses was transmission electron microscopy. Preparing stool specimens for analysis under the microscope is not a particularly fulfilling exercise, given the frustratingly low sensitivity of visualizing the small round structured virus particles [2]. The criteria of Kaplan et al. responded to these limitations in diagnostic capability. Of course, in the intervening years, tremendous progress has been made in monoclonal antibody technology and nucleic acid detection (e.g., [3, 4]). Given all of the diagnostic advances of the past 2 decades, it is both remarkable and a little bit sad that the criteria of Kaplan et al. remain such a relevant tool for recognizing outbreaks caused by norovirus. Turcios et al. [5] have confirmed, on a much larger and more varied data set than Kaplan et al. [ 1] had at their disposal, what these earlier investigators originally suggested. These criteria are wonderfully specific-99%-and reasonably sensitive-

Enterically infecting viruses: pathogenicity, transmission and significance for food and waterborne infection

Enterically infecting viruses are ubiquitous agents, mostly inducing silent infections. Several are however associated with significant diseases in man from diarrhoea and vomiting to hepatitis and meningitis. These viruses arc drawn from a variety of virus families and have different structures and genetic material, yet all are suited to this means of transmission: Normally they are shed in high numbers (assisting environmental transit) and exhibit great particle stability (permitting survival both outside the body and on passage through the stomach). Human activities particularly associated with food and water processing and distribution have the capacity to influence the epidemiology of these viruses. This review provides a description of viruses spreading by these means, their significance as pathogens and considers their behavior in these human-assisted processes.

English

English

Virological control of groundwater quality using biomolecular tests

Deep groundwater, even if generally protected, could be contaminated by surface or rain water infiltration through soil fractures, septic tanks, cesspits, land irrigation, disposal of wastewater and disposal of muds from depuration systems. The sanitary importance of such possible contamination is related to the different uses of the water and it is at the maximum level when it is intended for human use. Routine microbiological analyses do not consider viruses, only bacterial parameters, as contamination indicators. However, it is known that enteric viruses can survive a long time in deep aquifers and that they may not always be associated with bacterial indicators. The virological analysis of waters intended for drinking use is provided only as an occasional control exercised at the discretion of the sanitary authority. Technological difficulties with obtaining data about groundwater viral contamination led to a study to devise rapid and efficient methods for their detection and the application of these methods to samples from different sources. Four acid nucleic extraction techniques have been tested (classic proteinase K- phenol/chloroform, QIAamp Viral RNA Kit (Qiagen), SV Total RNA Isolation System (Promega) and NucleoSpin Virus L (Macherey-Nagel). Sensitivity and specificity of RT-PCR protocols for entero- (EV), hepatitis A (HAV) and small round structured (SRSV) viruses have been verified. Deep groundwater samples (100 L) were concentrated (2-step tangential flow ultrafiltration) and the concentrate contaminated with serial 10-fold dilutions of a known titre of poliovirus type 3. Extracted RNA was concentrated (microcon-100) and analysed by RT-PCR using specific EV primers and visualising amplification products by agarose gel electrophoresis. In addition, two different methods of RT-PCR for non-cultivable viruses have been tested: (a) RT-PCR and nested RT-PCR for HAV and (b) RT-PCR with generic primers and RT-PCR with specific primers for SRSV. Different specificity tests have been carried out in the presence of some of the commoner microorganisms. The most efficient, sensitive and specific protocols were used to test 35 x 100L deep groundwater samples. Sample concentrates were split with one part treated with chloroform and analysed by cell culture (BGM and Frp/3, derived from FrHK/4, cells) and the other tested by RT-PCR for HAV, EV and SRSV. Results demonstrated the high efficiency of the classic and QIAamp methods. Microcon-100 did not increase the sensitivity of the technique used. The highest sensitivity was observed for RT-PCR with specific primers for SRSV and for nested RT-PCR for HAV. One sample showed a cytopathic effect, not confirmed at the third subculture, while the RT-PCR allowed the detection of echovirus 7. Cell culture did not allow detection of the majority of the enteric viruses while PCR gave sensitive, specific and rapid detection of a range of agents in the same samples. Even if it was impossible to fix a virological quality standard, it would be necessary to find a viral indicator in order to achieve a complete preventive check which would be particularly useful in some cases (e.g. water never used before, after pollution accidents, for seasonal checking).

Foodborne viruses and fresh produce.

Raw and minimally processed fruits and vegetables are typically sold to the consumer in a ready-to-use or ready-to-eat form. These products do not generally contain preservatives or antimicrobial agents and rarely undergo any heat processing prior to consumption. For many years raw fruits and vegetables have been implicated as vehicles for transmission of infectious micro-organisms. Although fresh produce can support the growth and/or survival of many pathogenic bacteria there is little published information on the stability of human pathogenic viruses on these food products. Viruses cannot grow in or on foods but may sometimes be present on fresh produce as a result of faecal contamination. This contamination can arise at source in the growth and harvesting area from contact with polluted water and inadequately or untreated sewage sludge used for irrigation and fertilization. Alternatively, fruits or vegetables handled by an infected person might become contaminated with virus and transmit infection. The most frequently reported foodborne viral infections are viral gastroenteritis and hepatitis A: both have been associated with the consumption of fresh fruit or vegetables.

The Role of Viruses in Gastrointestinal Disease in the Home

Edited transcript of an oral presentation Looking at the survival of viruses on environmental surfaces, most data have come from laboratory studies, from data obtained from outbreaks and from hygiene intervention studies, collected over several years. Consider what happens if we place an object contaminated by a virus in the environment, how quickly will that virus circulate? One recent study looked at this problem. 1 Viruses spread in childcare facilities were studied using modified cauliflower virus DNA as an environmental marker. The viral DNA, introduced through treated toy balls, spread within a few hours of handling. Although the marker treated balls were removed after 1 day, the viral DNA continued circulating in the facilities for up to 2 weeks. The markers could also be detected in the children’s homes, on the hands of family members, and on environmental surfaces within those homes, including toys – this shows the potential for the environment to spread viruses. Many studies have been carried out to determine the level of environmental contamination in the course of rotavirus infections, in childcare centres, paediatric wards and so on. 2–4 Up to 10 12 virus particles, per ml of faeces, are shed when someone has rotavirus diarrhoea, so if we consider splashing effects in toilets, changing nappies and handling young children who are not toilet trained, there is great potential for the virus to spread. In such studies rotavirus was found to be widely disseminated on refrigerator handles, door handles, toilet handles, telephone receivers, toys and in play areas, moist surfaces such as sinks, water play at tables, and so on. Work by Ward et al. 5 examined the role of the environment in the acquisition of rotavirus more critically. The authors looked at surfaces that had been contaminated with rotavirus seeded in non-fat milk and dried onto a surface. They worked with volunteers, males aged 18–45 years, and in a controlled experiment they had the volunteers drink a virus-seeded salt solution, after which increases in the level of viral shedding and antibodies were measured, and found that seven out of eight volunteers became infected. They also looked at how the virus could be transmitted by licking the surface – normally we would not lick surfaces but we do use knives and forks, cups and so on. Eight volunteers became infected from directly licking a contaminated surface. When a surface was wiped with a finger and transferred to the mouth five out of eight volunteers became infected. These adults had no clinical symptoms but became shedders of the virus. Adults do not normally show symptoms; it is only in children and the elderly that rotavirus can become a serious problem. These workers also showed that spraying rotavirus-contaminated surfaces with a disinfectant (79% ethyl alcohol and 0.1% phenylphenol) prevented infection. Volunteers touching the disinfected surfaces with their fingers and then their mouths did not acquire infection. Let us consider environmental contamination with the Norwalk-like viruses (NLVs – also known as small roundstructured viruses) and the use of the reverse-transcriptase polymerase chain reaction (RT-PCR) assay for detection. As NLV cannot be grown in tissue culture RT-PCR has become an important assay for detection. RT-PCR can be used to determine whether viral nucleic acid is present in the environment. However, the assay does not indicate whether an intact, infectious virus particle is present in the sample, it only detects the nucleic acid component. Nevertheless, RT-PCR is the only way we have of looking for NLV in the environment.

Viruses causing diarrhoea in AIDS.

Opportunistic viral enteritis is an important gastrointestinal manifestation of HIV related disease. Cytomegalovirus (CMV) is a well established aetiological agent of disease in the gastrointestinal tract in this group. CMV enteritis may affect any region of the bowel, most commonly the colon. Diagnosis and management of these infections may be difficult. The role of other viruses in so-called 'pathogen-negative' diarrhoea remains controversial. The clinical importance of HIV-specific enteropathy is probably limited. Several viruses including astrovirus, picobirnavirus, small round structured virus and rotavirus have been implicated HIV-related diarrhoea. In addition, adenovirus has been linked to persistent diarrhoea in patients with a characteristic adenovirus colitis. The spectrum of disease morbidity and mortality amongst HIV patients has altered dramatically since the wide spread use of highly active antiretroviral therapy (HAART). Opportunistic infections, including CMV infection of the gastrointestinal tract in patients with AIDS, have diminished greatly. AIDS patients with CMV are able successfully to discontinue anti-CMV treatment without disease reactivation and with a parallel reduction in CMV viraemia following the initiation of HAART.

Characterisation of the bovine enteric calici-like virus, Newbury agent 1.

The bovine enteric calici‐like virus, Newbury agent 1 (NA1) was characterised to determine if it is a member of the Caliciviridae and to establish its antigenic relationship to the established bovine enteric calicivirus Newbury agent 2 (NA2). Solid phase immune electron microscopy (SPIEM) allowed quantification of NA1 virions and identification of faecal samples with optimal virus levels. NA1 particles were 36.6 nm in diameter, had an indefinite surface structure resembling that of human small round structured viruses (SRSVs), and a buoyant density of 1.34 g ml−1. A single capsid protein of 49.4 kDa was detected by Western blotting in purified NA1 preparations prepared from post‐infection but not pre‐infection faecal samples and with post‐ but not pre‐infection sera. NA1 was antigenically unrelated to the bovine enteric calicivirus NA2 by SPIEM. These properties were consistent with classification of NA1 within the Caliciviridae but demonstrated heterogeneity in the capsid composition of bovine enteric caliciviruses.

Characterisation of the bovine enteric calici-like virus, Newbury agent 1

The bovine enteric calici-like virus, Newbury agent 1 (NA1) was characterised to determine if it is a member of the Caliciviridae and to establish its antigenic relationship to the established bovine enteric calicivirus Newbury agent 2 (NA2). Solid phase immune electron microscopy (SPIEM) allowed quantification of NA1 virions and identification of faecal samples with optimal virus levels. NA1 particles were 36.6 nm in diameter, had an indefinite surface structure resembling that of human small round structured viruses (SRSVs), and a buoyant density of 1.34 g ml −1. A single capsid protein of 49.4 kDa was detected by Western blotting in purified NA1 preparations prepared from post-infection but not pre-infection faecal samples and with post- but not pre-infection sera. NA1 was antigenically unrelated to the bovine enteric calicivirus NA2 by SPIEM. These properties were consistent with classification of NA1 within the Caliciviridae but demonstrated heterogeneity in the capsid composition of bovine enteric caliciviruses.

A waterborne outbreak of small round structured virus, campylobacter and shigella co-infections in La Neuveville, Switzerland, 1998.

An outbreak of gastro-enteritis occurred in La Neuveville, township with 3358 inhabitants. A retrospective cohort study of 1915 participants showed that 1607 (84%) had been ill. Campylobacter jejuni was isolated from 28 patient faecal samples, Shigella sonnei from 21 patients and small round structured viruses (SRSV) from 6 patients. More than one pathogen was identified in eight persons. The epidemic curve was characteristic of a point-source outbreak. The risk for illness was significantly higher among persons who had drank unboiled drinking water than among those who had not (1290 [80.3%] of 1607 vs. 86 [27.9%] of 308; RR = 2.87; 95% CI 2.40-3.45). Risk increased significantly with the quantity of water consumed (P < 0.00 x 10(-6)). An SRSV isolate from water and one human faeces had an identical DNA sequence. The outbreak was due to a pump failure producing a spill of sewage into the groundwater. We conclude that transmission was waterborne and that measures including early warning, basic hygiene and sanitation improvements controlled this epidemic.

Infantile convulsions with mild gastroenteritis.

The development of sensitive new molecular genetic techniques has led to the detection of rotavirus in cerebrospinal fluid, stools and throat swabs from patients with gastroenteritis with accompanying clinical symptoms similar to infantile benign convulsions. Small round structured virus (SRSV) has also been found in stools of patients with similar clinical symptoms by a new procedure. However, the mechanism by which these viral infections induce benign convulsions remains to be elucidated. The present paper reviews recent virological and clinical studies of seizures probably caused by gastroenteritis viruses including rotavirus, SRSV and other viruses.

The heteroduplex mobility assay (HMA) as a pre-sequencing screen for Norwalk-like viruses

Molecular epidemiological studies of Norwalk-like viruses (NLVs), previously known as small round structured viruses (SRSVs), are dependant currently on DNA sequencing of PCR amplicons, which is expensive and time consuming. The Heteroduplex Mobility assay (HMA) was evaluated as a method for identification of PCR amplicons from the commonly circulating NLV strains without DNA sequencing. The procedure was developed for use with two reference strains, a Mexico virus-like strain (MXV-like; Hu\\NLV\\RBH\\1993\\UK) and the Grimsby virus strain (Hu\\NLV\\Gimsby\\1995\\UK), and was optimised with regards to the annealing and electrophoresis conditions and the electrophoresis gel matrix. Using the optimised conditions, amplicons of less than 90% sequence identity formed visible heteroduplexes, allowing the strains to be placed into three categories; Mexico-like, Grimsby-like and non-Mexico virus/non-Grimsby virus strains. Outbreak strains ‘genotyped’ previously by DNA sequencing as Mexico virus or Grimsby virus were identified correctly by the heteroduplex mobility assay. The procedure was applied prospectively to strains from 130 outbreaks occurring in the UK between 1997 and 1998. Heteroduplex mobility assay was successful on 120 (92%) strains of which 68 (57%) were GRV-like strains, three (2.5%) were Mexico virus-like strains and 49 (41%) were categorised as non- Mexico/non-Grimsby virus strains. Amplicons from 50 of the 120 strains were sequenced and there was perfect correlation between the heteroduplex mobility assay categorisation and phylogenetic analysis. HMA offers a rapid, robust and far cheaper alternative to sequencing for the identification of prevalent Norwalk-like virus genotypes for molecular epidemiological studies.

The detection of small round-structured viruses in water and environmental materials

A procedure for concentrating small round-structured viruses (SRSVs) (Norwalk-like viruses) from water and other environmental materials is described. Primers based on the helicase region of the SRSV genome were confirmed as specific by reaction with typed specimens, and used to detect virus in concentrates of unseeded and seeded samples. Virus was detected in estuarine recreational water polluted by untreated sewage, although not in seawater samples taken some distance from outfall discharges. It was also detected in river water downstream of a sewage treatment plant. Virus could be detected in all matrices when they were seeded with a positive stool extract, including sewage seeded with as little as 2 μl stool extract, thus confirming the suitability of the method for environmental monitoring.

Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.

An outbreak of gastroenteritis followed a meal in a large hotel during which one of the diners vomited. The clinical features of the illness suggested Norwalk-like virus (NLV, small round structured virus) infection, and this was confirmed by electron microscopy and reverse transcriptase polymerase chain reaction (RT-PCR) of stool samples. Further characterization of the virus by nucleotide sequence analysis of the PCR amplicons revealed identical strains in all the affected individuals. The foods served at the meal could not be demonstrated to be the cause of the outbreak. Analysis of attack rates by dining table showed an inverse relationship with the distance from the person who vomited. No one eating in a separate restaurant reported illness. Transmission from person-to-person or direct contamination of food seems unlikely in this outbreak. However, the findings are consistent with airborne spread of NLV with infection by inhalation with subsequent ingestion of virus particles.

Multi-state outbreaks of acute gastroenteritis traced to fecal-contaminated oysters harvested in Louisiana.

Norwalk-like viruses (NLVs), or small round structured viruses, are known to cause acute gastroenteritis associated with eating contaminated shellfish. Between 1993 and 1996, three oyster-related gastroenteritis outbreaks attributed to NLV occurred in Louisiana. Intensive trace-back and environmental investigations revealed that the overboard disposal of sewage by oyster harvesters into oyster-bed waters was the most likely source of contamination in at least two of the outbreaks. The small infectious dose of NLV, the large quantity of virus particles in stool, and the ability of oysters to concentrate virus particles suggest that oyster-related outbreaks will continue unless strong control measures are established. Efforts to halt improper sewage disposal in oyster-harvesting waters, including overboard sewage discharge, must be undertaken if future outbreaks are to be prevented.