Norovirus and Other Human Enteric Viruses in Moroccan Shellfish

The aim of this study was to evaluate the presence of human enteric viruses in shellfish collected along the Mediterranean Sea and Atlantic Coast of Morocco. A total of 77 samples were collected from areas potentially contaminated by human sewage. Noroviruses were detected in 30 % of samples, with an equal representation of GI and GII strains, but were much more frequently found in cockles or clams than in oysters. The method used, including extraction efficiency controls, allowed the quantification of virus concentration. As in previous reports, results showed levels of contamination between 100 and 1,000 copies/g of digestive tissues. Sapoviruses were detected in 13 % of samples mainly in oyster and clam samples. Hepatitis A virus was detected in two samples, with concentrations around 100 RNA copies/g of digestive tissues. Only two samples were contaminated with enterovirus and none with norovirus GIV or Aichi virus. This study highlights the interest of studying shellfish samples from different countries and different production areas. A better knowledge of shellfish contamination helps us to understand virus levels in shellfish and to improve shellfish safety, thus protecting consumers.     

Human Enteric Viruses Occurrence in Shellfish from European Markets

Different sources were consulted to obtain information on the occurrence of viruses in bivalve molluscs on the European market. Twenty-six peer-reviewed articles were identified reporting on the molecular detection of viral RNA in 4,260 samples in total. The data obtained will be presented geographically on virus types detected, the origin and treatment of the shellfish, and the detection technique applied. The data demonstrate that viral RNA can be detected in shellfish from polluted areas, in depurated shellfish as well as those for human consumption. The European Rapid Alert System for Food and Feed (RASFF) database was consulted as another source. Twenty-eight notifications were identified on the presence of hepatitis A virus or norovirus in shellfish on the European market. The most recent report of the European laboratory network was referred to, to gain insight into the laboratory capability at present for the analyses of shellfish on the presence of viruses. Approximately 67% of 27 participating laboratories obtained intended results for all samples, consisting of lenticules loaded with 10³ copies norovirus (genogroup I (GGI) and/or genogroup II (GGII)) and/or 1 × 10⁵–8 × 10⁴ copies of hepatitis A virus. From 1993, there has been a continuous development of molecular detection techniques and tools have been described to ensure quality assurance. End product testing will, however, not be achievable. As depuration has been shown not to be effective for the complete elimination of viruses, shellfish should not be in contact with faecal contaminated water in order to minimise the risk of shellfish-transmittable viral diseases.     

Detection of Human Enteric Viruses in Freshwater from European Countries

The transmission of water-borne pathogens typically occurs by a faecal–oral route, through inhalation of aerosols, or by direct or indirect contact with contaminated water. Previous molecular-based studies have identified viral particles of zoonotic and human nature in surface waters. Contaminated water can lead to human health issues, and the development of rapid methods for the detection of pathogenic microorganisms is a valuable tool for the prevention of their spread. The aims of this work were to determine the presence and identity of representative human pathogenic enteric viruses in water samples from six European countries by quantitative polymerase chain reaction (q-PCR) and to develop two quantitative PCR methods for Adenovirus 41 and Mammalian Orthoreoviruses. A 2-year survey showed that Norovirus, Mammalian Orthoreovirus and Adenoviruses were the most frequently identified enteric viruses in the sampled surface waters. Although it was not possible to establish viability and infectivity of the viruses considered, the detectable presence of pathogenic viruses may represent a potential risk for human health. The methodology developed may aid in rapid detection of these pathogens for monitoring quality of surface waters.     

A 1-Year Study on the Detection of Human Enteric Viruses in New Caledonia

Human enteric viruses occur in high concentrations in wastewater and can contaminate receiving environmental waters. Due to the lack of data on the prevalence of enteric viruses in New Caledonia, the presence and the concentrations of enteric viruses in wastewater and seawater were determined. Untreated wastewater and seawater samples were collected monthly for 1 year from a wastewater treatment plant (WWTP) and from the WWTP’s outlet, located directly on a popular recreational beach. Samples were tested for norovirus genogroups I and II (NoV GI and GII), astroviruses (AsV), sapoviruses (SaV), enteroviruses (EV), hepatitis A viruses (HAV), rotaviruses (RoV), human adenoviruses (HAdV) and human polyomaviruses (HPyV). To support these data, faecal samples from cases of gastroenteritis were tested for the first time for NoV and detected in the population. NoV GI, NoV GII, EV, SaV, HAdV and HPyV were detected in all wastewaters, RoV in 75 % and AsV in 67 %. HAV were not detected in wastewater. Overall, 92 % of seawater samples were positive for at least one virus. HPyV were detected most frequently in 92 % of samples and at concentrations up to 7.7 × 10³ genome copies/L. NoV GI, NoV GII, EV, SaV, RoV and HAdV were found in 33, 66, 41, 33, 16 and 66 % of seawater samples, respectively. AsV were not detected in seawater. This study reports for the first time the presence of NoV and other enteric viruses in New Caledonia and highlights the year-round presence of enteric viruses in the seawater of a popular beach.     

Occurrence of Human Enteric Viruses in Commercial Mussels at Retail Level in Three European Countries

In this study, the prevalence of different enteric viruses in commercial mussels was evaluated at the retail level in three European countries (Finland, Greece and Spain). A total of 153 mussel samples from different origins were analysed for human norovirus (NoV) genogroups I and II, hepatitis A virus (HAV) and hepatitis E virus (HEV). Human adenovirus (HAdV) was also tested as an indicator of human faecal contamination. A full set of controls (such as sample process control, internal amplification controls, and positive and negative controls) were implemented during the process. The use of a sample process control allowed us to calculate the efficiencies of extraction, which ranged from 79 to 0.5?%, with an average value of 10?%. Samples were positive in 41?% of cases, with HAdV being the most prevalent virus detected (36?%), but no significant correlation was found between the presence of HAdV and human NoV, HAV and HEV. The prevalences of human norovirus genogroup II, HEV and human NoV genogroup I were 16, 3 and 0.7?%, respectively, and HAV was not detected. The estimated number of PCR detectable units varied between 24 and 1.4?×?10³?g^?1 of digestive tract. Interestingly, there appeared to be a significant association between the type of mussel species (M. galloprovincialis) and the positive result of samples, although a complete overlap between country and species examined required this finding to be confirmed including samples of both species from all possible countries of origin.     

Occurrence of Human Enteric Viruses in Water Sources and Shellfish: A Focus on Africa

Enteric viruses are a diverse group of human pathogens which are primarily transmitted by the faecal–oral route and are a major cause of non-bacterial diarrhoeal disease in both developed and developing countries. Because they are shed in high numbers by infected individuals and can persist for a long time in the environment, they pose a serious threat to human health globally. Enteric viruses end up in the environment mainly through discharge or leakage of raw or inadequately treated sewage into water sources such as springs, rivers, dams, or marine estuaries. Human exposure then follows when contaminated water is used for drinking, cooking, or recreation and, importantly, when filter-feeding bivalve shellfish are consumed. The human health hazard posed by enteric viruses is particularly serious in Africa where rapid urbanisation in a relatively short period of time has led to the expansion of informal settlements with poor sanitation and failing or non-existent wastewater treatment infrastructure, and where rural communities with limited or no access to municipal water are dependent on nearby open water sources for their subsistence. The role of sewage-contaminated water and bivalve shellfish as vehicles for transmission of enteric viruses is well documented but, to our knowledge, has not been comprehensively reviewed in the African context. Here we provide an overview of enteric viruses and then review the growing body of research where these viruses have been detected in association with sewage-contaminated water or food in several African countries. These studies highlight the need for more research into the prevalence, molecular epidemiology and circulation of these viruses in Africa, as well as for development and application of innovative wastewater treatment approaches to reduce environmental pollution and its impact on human health on the continent.

     

Identification of Enteric Viruses in Foods from Mexico City

Foodborne viruses are a common and, probably, the most under-recognized cause of outbreaks of gastroenteritis. Among the main foods involved in the transmission of human enteric viruses are mollusks, and fruits and vegetables irrigated with wastewater and/or washed with non-potable water or contaminated by contact with surfaces or hands of the infected personnel during its preparation. In this study, 134 food samples were analyzed for the detection of Norovirus, Rotavirus, and Hepatitis A virus (HAV) by amplification of conserved regions of these viruses. From the 134 analyzed samples, 14 were positive for HAV, 6 for Norovirus, and 11 for Rotavirus. This is the first report in Mexico where emphasis is given to the presence of HAV and Norovirus on perishable foods and food from fisheries, as well as Rotavirus on frozen vegetables, confirming the role of vegetables and bivalve mollusks as transmitting vehicles of enteric viruses.     

Seasonal Occurrence of Human Enteric Viruses in River Water Samples Collected from Rural Areas of South-East Poland

The study was carried out in 2007, and its main aim was 1 year monitoring of surface water of the River Wieprz in Poland for the presence of human adenoviruses group F and noroviruses (NoVs). In total, 60 water samples were collected from four sampling sites situated along the river. The viruses were concentrated from water samples using glass wool, followed by elution with a glycine buffer containing skimmed milk powder. Subsequently, the viral nucleic acids were extracted and purified from water concentrates using a NucliSENS^® kit and a QIAamp Viral RNA Mini Kit^®. The presence of viral nucleic acids was confirmed by applying traditional PCR-based methods with incorporated internal amplification controls. Human pathogenic viruses were detected in 35% of analysed water samples. Adenoviruses were detected in 28.3% of analysed samples, and were present at all seasons of the year. 11.6% of the samples were positive for NoVs; they were present only during summer, in contrast to conventional findings. Molecular identification of norovirus strains revealed that they belong to genogroup I and II.     

Reduction of Human Enteric and Indicator Viruses at a Wastewater Treatment Plant in Southern Louisiana,USA

This study assessed wastewater quality through the quantification of four human enteric viruses and the applicability of pepper mild mottle virus (PMMoV) a     

Enteric Viruses and Management of Shellfish Production in New Zealand

In New Zealand shellfish are a significant food resource and shellfish are harvested for both recreational and commercial use. Commercially harvested Greenshell mussels (Perna canaliculus) and Pacific oysters (Crassostrea gigas) from aquaculture farms dominate consumption in New Zealand. Other commercial species include cockles (Austrovenus stuchburyii) and surf clam species which are wild harvested. The consumption of shellfish has been associated with gastroenteritis outbreaks caused by noroviruses following faecal contamination of growing waters with human waste. In New Zealand, since 1994 over 50 norovirus outbreaks linked to consumption of either New Zealand commercially grown oysters or imported oysters have been reported. An IEC/ISO 17025 accredited method for detection of noroviruses in bivalve shellfish was established in 2007. This method has been used in outbreak investigations to analyse implicated shellfish, in virus prevalence surveys and monitoring programmes, and commercially for product clearances. Surveys have shown that enteric viruses occur frequently in non-commercial shellfish, especially near sewage outfalls and following sewage discharge events. Viral source tracking methods have assisted in identifying pollution sources. The commercial shellfish industry operates under the Bivalve Molluscan Shellfish Regulated Control Scheme (BMSRCS), administered by the New Zealand Food Safety Authority. Recently regulatory measures were introduced into the BMSRCS to manage viruses. These include the closure of harvest areas for at least 28 days after human sewage contamination events and norovirus outbreaks. These management strategies, coupled with new information on norovirus prevalence in shellfish, have helped to improve the quality and safety of New Zealand shellfish.     

Minimum Infective Dose of the Major Human Respiratory and Enteric Viruses Transmitted Through Food and the Environment

Viruses are a significant cause of morbidity and mortality around the world. Determining the minimum dose of virus particles that can initiate infection, termed the minimum infective dose (MID), is important for the development of risk assessment models in the fields of food and water treatment and the implementation of appropriate infection control strategies in healthcare settings. Both respiratory and enteric viruses can be shed at high titers from infected individuals even when the infection is asymptomatic. Presence of pre-existing antibodies has been shown to affect the infectious dose and to be protective against reinfection for many, but not all viruses. Most respiratory viruses appear to be as infective in humans as in tissue culture. Doses of <1 TCID₅₀ of influenza virus, rhinovirus, and adenovirus were reported to infect 50% of the tested population. Similarly, low doses of the enteric viruses, norovirus, rotavirus, echovirus, poliovirus, and hepatitis A virus, caused infection in at least some of the volunteers tested. A number of factors may influence viruses’ infectivity in experimentally infected human volunteers. These include host and pathogen factors as well as the experimental methodology. As a result, the reported infective doses of human viruses have to be interpreted with caution.     

Detection of Enteric Viruses from Wastewater and River Water in Botswana

Food and Environmental Virology - Waterborne diseases remain a public health concern in developing countries where many lack access to safe water. Water testing mainly uses bacterial indicators to...     

One-year Surveillance of Human Enteric Viruses in Raw and Treated Wastewaters,Downstream River Waters,and Drinking Waters

Human enteric viruses are a major cause of waterborne diseases, and can be transmitted by contaminated water of all kinds, including drinking and recreational water. The objectives of the present study were to assess the occurrence of enteric viruses (enterovirus, norovirus, adenovirus, hepatitis A and E virus) in raw and treated wastewaters, in rivers receiving wastewater discharges, and in drinking waters. Wastewater treatment plants’ (WWTP) pathogen removal efficiencies by adenovirus quantitative real-time PCR and the presence of infectious enterovirus, by cell culture assays, in treated wastewaters and in surface waters were also evaluated. A total of 90 water samples were collected: raw and treated wastewaters (treated effluents and ultrafiltered water reused for industrial purposes), water from two rivers receiving treated discharges, and drinking water. Nested PCR assays were used for the identification of viral DNA/RNA, followed by direct amplicon sequencing. All raw sewage samples (21/21), 61.9 % of treated wastewater samples (13/21), and 25 % of ultrafiltered water samples (3/12) were contaminated with at least one viral family. Multiple virus families and genera were frequently detected. Mean positive PCRs per sample decreased significantly from raw to treated sewage and to ultrafiltered waters. Moreover, quantitative adenovirus data showed a reduction in excess of 99 % in viral genome copies following wastewater treatment. In surface waters, 78.6 % (22/28) of samples tested positive for one or more viruses by molecular methods, but enterovirus-specific infectivity assays did not reveal infectious particles in these samples. All drinking water samples tested negative for all viruses, demonstrating the effectiveness of treatment in removing viral pathogens from drinking water. Integrated strategies to manage water from all sources are crucial to ensure water quality.     

Harmonised Investigation of the Occurrence of Human Enteric Viruses in the Leafy Green Vegetable Supply Chain in Three European Countries

Numerous outbreaks have been attributed to the consumption of raw or minimally processed leafy green vegetables contaminated with enteric viral pathogens. The aim of the present study was an integrated virological monitoring of the salad vegetables supply chain in Europe, from production, processing and point-of-sale. Samples were collected and analysed in Greece, Serbia and Poland, from ??general?? and ??ad hoc?? sampling points, which were perceived as critical points for virus contamination. General sampling points were identified through the analysis of background information questionnaires based on HACCP audit principles, and they were sampled during each sampling occasion where as-ad hoc sampling points were identified during food safety fact-finding visits and samples were only collected during the fact-finding visits. Human (hAdV) and porcine (pAdV) adenovirus, hepatitis A (HAV) and E (HEV) virus, norovirus GI and GII (NoV) and bovine polyomavirus (bPyV) were detected by means of real-time (RT-) PCR-based protocols. General samples were positive for hAdV, pAdV, HAV, HEV, NoV GI, NoV GII and bPyV at 20.09?% (134/667), 5.53?% (13/235), 1.32?% (4/304), 3.42?% (5/146), 2?% (6/299), 2.95?% (8/271) and 0.82?% (2/245), respectively. Ad hoc samples were positive for hAdV, pAdV, bPyV and NoV GI at 9?% (3/33), 9?% (2/22), 4.54?% (1/22) and 7.14?% (1/14), respectively. These results demonstrate the existence of viral contamination routes from human and animal sources to the salad vegetable supply chain and more specifically indicate the potential for public health risks due to the virus contamination of leafy green vegetables at primary production.     

Enteric Viruses in a Large Waterborne Outbreak of Acute Gastroenteritis in Finland

In Nokia city about 450,000 l of treated sewage water was for 2 days allowed to run into the drinking water supplies of the city due to a personal error of one employee. Within the next 5 weeks about 1,000 people sought care at the municipal health centre or regional hospital because of gastroenteritis. Here we report the results of viral analyses performed by gene amplification assays from the earliest water and sewage samples as well as from close to 300 patient samples. The contaminating treated sewage was shown to harbour several enteric viruses known to cause acute gastroenteritis. Likewise, the drinking water sample was positive for noro-, astro-, rota-, entero- and adenoviruses. Noroviruses were also found in 29.8% of stool samples from affected patients, while astro-, adeno-, rota- and enteroviruses were detected in 19.7, 18.2, 7.5 and 3.7% of the specimens, respectively. An erratum to this article can be found at     

Reduction of Enteric Viruses by Blueberry Juice and Blueberry Proanthocyanidins

Blueberry and blueberry extracts are known for their health benefits and antimicrobial properties. Natural therapeutic or preventive options to decrease the incidences of foodborne viral illnesses are becoming popular and being researched. This study aimed to determine the antiviral effects of blueberry juice (BJ) and blueberry proanthocyanidins (BB-PAC, B-type PAC structurally different from A-type PAC found in cranberries) against the infectivity of hepatitis A virus (HAV) and human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) at 37 °C over 24 h using standard plaque assays. Viruses at ~5 log PFU/ml were mixed with equal volumes of BJ (pH 2.8), neutralized BJ (pH 7.0), BB-PAC (1, 2, 4, and 10 mg/ml), malic acid (pH 3.0), or phosphate-buffered saline (pH 7.2) and incubated over 24 h at 37 °C. Each experiment was carried out in duplicate and replicated thrice. FCV-F9 titers were found to be reduced to undetectable levels with 1 and 2 mg/ml BB-PAC after 5 min, with 0.5 mg/ml BB-PAC after 1-h, and with BJ after 3-h. MNV-1 titers were reduced to undetectable levels after 3 h with 1, 2, and 5 mg/ml BB-PAC and after 6 h with BJ. HAV titers were reduced to undetectable levels after 30 min with 2 and 5 mg/ml BB-PAC, after 3 h with 1 mg/ml BB-PAC, and by ~2 log PFU/ml with BJ after 24-h. BB-PAC shows preventive potential against infection by the tested enteric viruses in a dose- and time-dependent manner, although further in vitro studies in model food systems and in vivo studies using animal models are warranted.     

Comparative Uptake of Enteric Viruses into Spinach and Green Onions

Root uptake of enteric pathogens and subsequent internalization has been a produce safety concern and is being investigated as a potential route of pre-harvest contamination. The objective of this study was to determine the ability of hepatitis A virus (HAV) and the human norovirus surrogate, murine norovirus (MNV), to internalize in spinach and green onions through root uptake in both soil and hydroponic systems. HAV or MNV was inoculated into soil matrices or into two hydroponic systems, floating and nutrient film technique systems. Viruses present within spinach and green onions were detected by RT-qPCR or infectivity assays after inactivating externally present viruses with Virkon^®. HAV and MNV were not detected in green onion plants grown up to 20 days and HAV was detected in only 1 of 64 spinach plants grown in contaminated soil substrate systems up to 20 days. Compared to soil systems, a drastic difference in virus internalization was observed in hydroponic systems; HAV or pressure-treated HAV and MNV were internalized up to 4 log RT-qPCR units and internalized MNV was shown to remain infectious. Understanding the interactions of human enteric viruses on produce can aid in the elucidation of the mechanisms of attachment and internalization, and aid in understanding risks associated with contamination events.