Norovirus Distribution within an Estuarine Environment

Abstract

Human norovirus (NoV) is the leading cause of nonbacterial gastroenteritis worldwide (3). The Centers for Disease Control and Prevention (CDC) estimate that 23 million cases of acute gastroenteritis due to NoV occur each year, with symptoms including acute-onset vomiting, watery nonbloody diarrhea with abdominal cramps, and nausea (35). NoV outbreaks are pervasive for many reasons, but particularly because the virus is highly contagious and environmentally hardy (7). Additionally, infected individuals can excrete millions of viral particles in feces, leading to large numbers in sewage (16). Without proper removal or inactivation during wastewater treatment, the viruses can be released into recreational and shellfishharvesting water bodies. Complete inactivation of NoV during sewage treatment is rare, and even in areas with proper wastewater treatment, contamination of oyster beds has been reported (5,16,17,32,38). Because bivalve molluscan shellfish are believed to act as filters for viruses and other microbes and because NoV is extremely infectious (as little as one viral particle is required for disease), the disease risk for consumption of raw oysters is high (27,33,40).Human NoV genogroup I (GI) and GII have been detected in oyster samples harvested from bays and estuaries worldwide (5,10,20). Ueki et al. (42) detected NoV in both shellfish and the surrounding river water in Japan and concluded that NoV contamination was most likely due to sewage and treated wastewater input into the river; however, no study has yet been able to characterize how NoV may be naturally distributed in an estuarine system, including in water, adhered to particles (including plankton), and in shellfish. The limitations are due in part to a lack of adequate detection methods specifically adapted to different environmental-sample types (8). Using a newly developed detection and quantification protocol (21), this study aimed to examine the distribution of NoV genogroups across a range of sample types within an estuarine system with the goal of better characterizing possible circulation of viruses between water, plankton, and oysters. MATERIALS AND METHODS Controls. (i) NoV-positive controls.Three NoV-positive fecal samples, representing genotypes GI.4, GI.3b, and GII.4 Minerva, were provided as controls for this study by the CDC. Stool samples were diluted to obtain a 20% suspension in phosphate-buffered saline (PBS), vortexed, and centrifuged at 15,700 ϫ g for 2 min.(ii) Viral-RNA extraction from stool. For stool samples, viral RNA was extracted from the clarified PBS extracts using the MagMAX-96 Viral Isolation Kit (Ambion, Austin, TX) and the KingFisher Instrument (Thermo Electron Corporation, Waltham, MA), which automatically purifies viral RNA. The purified RNA was eluted into 55 l elution buffer, provided in the kit.(iii) RNA transcript standards. To enable quantification by real-time reverse transcription (RT)-PCR, we used GI and GII plasmids (1) to generate RNA runoff transcripts. Briefly, the norovirus 3-kb plasmids were pu...

Histo-Blood Group Antigen-Like Substances of Human Enteric Bacteria as Specific Adsorbents for Human Noroviruses

“…Sano et al demonstrated that indigenous noroviruses were not present in the permeates of mixed liquor and treated wastewater filtered using a microfiltration membrane (pore size, 0.1 m) (46). Gentry et al showed that an unexpectedly large number of HuNoVs were present in the fraction collected using 63-and 200-m-mesh plankton nets, which suggested the binding of HuNoV particles to phytoplankton and zooplankton (47). Viral particles associated with environmental materials are physically more stable than freely moving viral particles (48).…”

“…This value is comparable with that obtained for the interactions between ß-galactose-specific lectins and lactose-carrying glycopolystyrenes, which have binding constants of 10 6 to 10 7 M Ϫ1 (54). The interactions between enteric viruses and environmental adsorbents were previously shown to have no binding specificity (41)(42)(43)(44)(45)47), whereas HuNoV binding to bacterial HBGAs is regarded as specific, as illustrated by human HBGAs and HuNoVs (12). In addition to enthalpy-driven adsorption via an array of hydrogen binding routes (28), the entropy effect caused by multiple binding sites also contributes to the increased binding avidity although structural analyses of bacterial EPS are essential to obtain an insight into the true value of the binding constant.…”

Environmental Transmission of Human Noroviruses in Shellfish Waters

“…Surveillance of NoV in commercial oyster production areas in the United Kingdom, Ireland, and France detected total levels of NoV (GI plus GII) ranging from Ͻ100 genome copies/g to 10 4 genome copies/g (33). Levels of NoV GI in oysters collected from commercial beds in Georgia in the United States ranged from 10 3 to 10 8 copies/g (88). Monitoring of NoV in mussels grown in floating rafts and wild shellfish (mussels, clams, and cockles) in Galicia (Spain) detected concentrations of the virus ranging from 10 2 to 10 3 copies/g for GI and from 10 1 to 10 4 copies/g for GII, with GII levels being generally higher in cultivated areas than in wild shellfish (120).…”

Real-time PCR quantitative assessment of hepatitis A virus, rotaviruses and enteroviruses in the Tyume River located in the Eastern Cape Province, South Africa

“…Aw and Gin (2010) reported the detection of both NoV genogroups GI and GII in 100% of sewage and secondary effluent samples in Singapore while Lee and Kim (2008) reported genetic diversity of HuNoVs detected in river water in Korea. Numerous studies have also detected NoVs in environmental waters in Europe (Lysen et al, 2009;Lodder and de Roda Husman, 2005), in the United States of America (Gentry et al, 2009) and in South America (Victoria et al, 2010). A recent report on the detection of enteric viruses in selected urban and rural river water and sewage in Kenya (Kiulia et al, 2010) revealed that HuNoVs GI and GII were detected in 90% of samples collected from urban rivers and streams.…”

“…They comprise of non-enveloped, single-stranded RNA viruses which have been found in humans, pigs, cattle, sheep and mice (Wolf et al, 2010). Human NoVs (HuNoVs) are the most common etiological agent for gastroenteritis outbreaks as well as the leading cause of non-bacterial gastroenteritis in all age groups (Gentry et al, 2009;Siebenga et al, 2009). Human noroviruses GI and GII have been detected in both freshwaters and estuarine waters worldwide.…”