Prevalence of Norovirus Infection in Children and Adults with Acute Gastroenteritis,Tehran, Iran, 2008–2009

Noroviruses are one of important agents that cause acute viral gastroenteritis worldwide. These viruses are belonging to Caliciviridae family and are genetically diverse. To date, there is no valuable data about prevalence of norovirus infection and the dominant genogroup/genotype among Iranian population. The objective of this study was to determine the frequency of norovirus infection in Iranian patients with gastroenteritis referred to three hospitals of Tehran and to specify the dominant genogroup/genotype of this virus among our study population. A total of 293 patients with acute gastroenteritis were included in the study. Detection of norovirus was performed using RT-PCR method and confirmed by direct sequencing with specific designed primers for capsid region of norovirus genome. Phylogenetic analysis was performed using the neighbor-joining method. Norovirus strains identified in our study were subsequently categorized according to previously defined genogroup/genotypes. Of these, norovirus GII was dominant genogroup. Sixty-five percent (17 of 26) of positive samples were determined as GII and 35% (9 of 26) were determined as GI, respectively, in 2008–2009. And among 8 sequenced strains of genogroup II the most frequent genotype was GII.3. The results of this study indicated that norovirus must be considered as one of the infectious causes of acute gastroenteritis among Iranian population. We also found that GII.3 is more prevalent in our study population. To the best of our knowledge there is limited data about the role of noroviruses in children and adults’ acute gastroenteritis among Iranian patients and this prevalence and genotyping report of norovirus infection could be remarkable for further studies.     

Challenging Investigation of a Norovirus Foodborne Disease Outbreak During a Military Deployment in Central African Republic

In January 2016, a large-scale outbreak of acute gastroenteritis was reported among French armed forces deployed in the Central African Republic. Challenging investigations, conducted from France, made it possible to identify a norovirus genogroup II in both stool and food samples, confirming a norovirus foodborne disease outbreak. Infected food handler management is discussed.     

Detection and Sequencing of Multiple Human Norovirus Genotypes from Imported Frozen Raspberries Linked to Outbreaks in the Province of Quebec,Canada, in 2017

Food and Environmental Virology - Human noroviruses are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several norovirus food-related outbreaks....     

Virucidal Efficacy of Olanexidine Gluconate as a Hand Antiseptic Against Human Norovirus

Food and Environmental Virology - Human noroviruses are the major cause of non-bacterial acute gastroenteritis worldwide. Since no therapeutic agent has been proven to prevent human norovirus...     

Occurrence of Norovirus GIV in Environmental Water Samples from Belém City,Amazon Region,Brazil

Noroviruses are the major cause of non-bacterial acute gastroenteritis outbreaks in humans, with few reports about the occurrence of the norovirus GIV strain. We investigated the presence of norovirus GIV in surface water (river, bay, and stream) and untreated sewage, and we determined a positivity rate of 9.4 % (9/96). The strains genotyped were GIV.1. To our knowledge, this is the first report of GIV in Brazil.     

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n?=?6), GII.4 (n?=?1), GII.17 (n?=?3), and GII.unclassified (n?=?3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83–1.85?×?10⁴ genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00?×?10¹ genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

     

Noroviruses: State of the Art

Noroviruses are a common cause of both endemic and epidemic gastroenteritis. These highly infectious viruses usually cause self-limited disease, but chronic infections occur in highly immunocompromised patients and unusual manifestations are also being described in some populations. Histoblood-group antigen expression is now recognized as an important susceptibility factor for many norovirus strains, but a correlate of acquired immunity to infection or illness has not yet been identified. Currently, treatment and prevention strategies rely on non-specific measures. However, virus-like particles containing capsid antigens are undergoing evaluation as a vaccine candidate for illness prevention. This article reviews the biologic properties, epidemiology, clinical features, host susceptibility, diagnosis, and treatment and prevention of norovirus infection.     

Application of a Swab Sampling Method for the Detection of Norovirus and Rotavirus on Artificially Contaminated Food and Environmental Surfaces

Noroviruses and rotaviruses are the leading causes of non-bacterial gastroenteritis in humans worldwide. Virus-contaminated food and surfaces represent an important risk to public health. However, established detection methods for the viruses in food products are laborious and time-consuming. Here, we describe a detailed swabbing protocol combined with real-time RT-PCR for norovirus and rotavirus detection on artificially contaminated food and environmental surfaces. Recovery rates between 2 and 78% for norovirus and between 8 and 42% for rotavirus were determined for contaminated food surfaces of apple, pepper, cooked ham and salami. From contaminated environmental surfaces (stainless steel, ceramic plate, polyethylene, wood), recovery rates between 26 and 52% (norovirus) and between 10 and 58% (rotavirus) were determined. The results demonstrate the suitability of the swab sample method for virus detection on food and environmental surfaces. Compared to other methods, it is easy to perform and significantly time-saving, predestining it for routine testing.     

Detection and Typing of Norovirus from Frozen Strawberries Involved in a Large-Scale Gastroenteritis Outbreak in Germany

During September/October 2012, a norovirus gastroenteritis outbreak affecting about 11,000 people occurred in Germany. Epidemiological studies suggested that frozen strawberries represented the vehicle of infection. We describe here the analysis of frozen strawberries for the presence of norovirus. Samples were taken by applying a stratified subsampling scheme. Two different methods for virus extraction from strawberries were compared. First, viruses were eluted from strawberries under alkaline conditions and concentrated using a polyethylene glycol precipitation. Second, ultrafiltration was applied for concentration of viruses rinsed off of the berries. In both cases, RNA was extracted and analyzed by real-time RT-PCR. Application of the ultrafiltration method generally resulted in a lower detection rate. Noroviruses were detected in 7/11 samples derived from the lot of strawberries implicated in the outbreak using the precipitation method. Typing of norovirus revealed three different genotypes including a combination of norovirus genotype II.16 (viral polymerase) and II.13 (viral capsid). This genotype combination was also found in some of the patients that were involved in the outbreak, but that had not been reported in Germany so far. In conclusion, heterogeneously distributed noroviruses in frozen strawberries can be detected by applying an optimized combination of sampling procedures, virus extraction methods, and real-time RT-PCR protocols. The detection of several different genotypes in the strawberries may suggest contamination from sewage rather than from a single infected food handler.     

A One-Year Survey of Norovirus in UK Oysters Collected at the Point of Sale

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a food safety risk and a potential contributor to the overall burden of gastroenteritis in the community. The United Kingdom (UK) has comprehensive national baseline data on the prevalence, levels, and seasonality of norovirus in oysters in production areas resulting from a previous two-year study (2009–2011). However, previously, data on final product as sold to the consumer have been lacking. As part of a wider project to establish the overall burden of foodborne norovirus in the UK, this study aimed to address this data gap. A one-year survey of oysters collected from the point-of-sale to the consumer was carried out from March 2015 to March 2016. A total of 630 samples, originating in five different European Union Member States, were collected from 21 regions across the UK using a randomised sampling plan, and tested for norovirus using a method compliant with ISO 15216-1, in addition to Escherichia coli as the statutory indicator of hygiene status. As in the previous production area study, norovirus RNA was detected in a high proportion of samples (68.7%), with a strong winter seasonality noted. Some statistically significant differences in prevalences and levels in oysters from different countries were noted, with samples originating in the Netherlands showing lower prevalences and levels than those from either the UK or Ireland. Overall, levels detected in positive samples were considerably lower than seen previously. Investigation of potential contributing factors to this pattern of results was carried out. Application of normalisation factors to the data from the two studies based on both the numbers of norovirus illness reports received by national surveillance systems, and the national average environmental temperatures during the two study periods resulted in a much closer agreement between the two data sets, with the notably different numbers of illness reports making the major contribution to the differences observed in norovirus levels in oysters. The large majority of samples (76.5%) contained no detectable E. coli; however, in a small number of samples (2.4%) levels above the statutory end product standard (230 MPN/100 g) were detected. This study both revealed the high prevalence of norovirus RNA in oysters directly available to the UK consumer, despite the high level of compliance with the existing E. coli-based health standards, while also highlighting the difficulty in comparing the results of surveys carried out in different time periods, due to variability in risk factors.     

Human Enteric Viruses in Groundwater

Waterborne outbreaks of enteric viruses are a major public health concern. The present study has been carried out to assess the presence of enteric viruses responsible for human acute gastroenteritis (AGE) in groundwater intended for drinking and produce washing. In total, 62 samples from groundwater for drinking and produce washing collected from Dec 2007 to Dec 2008 in Seoul were tested for enteric viruses using conventional RT–PCR, ELISA, and real-time RT–PCR. Our results showed that enteric viruses were detected in 7 (8.8%) groundwater samples. Rotaviruses were detected in 3 (4.8%) of the samples by ELISA; human adenoviruses were detected in 2 (3.2%) of the samples by ELISA; and nested RT–PCR detected noroviruses in 2 (3.2%) of the samples. In one of the groundwater sample, the norovirus RNA was detected by conventional RT–PCR which was confirmed positive by real-time RT–PCR. Additionally, real-time RT–PCR successfully detected norovirus RNA in five out of 62 water samples (8.1%). The data demonstrate that real-time RT–PCR will be useful as a rapid and sensitive method for detecting norovirus in water samples. Phylogenetic analysis revealed that the noroviruses detected in two of the groundwater samples belonged to GII-4. These studies can provide important information for the prevalence of enteric viruses in Korean groundwater.     

Re-emergence of a GII.4 Norovirus Sydney 2012 Variant Equipped with GII.P16 RdRp and Its Predominance over Novel Variants of GII.17 in South Korea in 2016

Noroviruses are major causative pathogen of nonbacterial acute gastroenteritis worldwide. Of the seven genogroups of noroviruses suggested recently, genogroup II genotype 4 (GII.4) had been the most common genotype identified in hospitalized patients in the last few decades. However, since the latter half of 2014, new variants of GII.17 have been reported as the main causes of outbreaks over GII.4 in East Asia and have also occurred in America and Europe. In this study, we monitored norovirus GII in coastal streams at South Gyeongsang province and South Jeolla province of South Korea from March 2015 to May 2016. Norovirus GII.17 capsid sequences were predominantly detected until September 2015 in water samples. However, we found that the number of positive cases of the norovirus GII.4 Sydney 2012 capsid sequence has been increasing since December 2015, overtaking that of GII.17 in 2016. The RdRp genotype of this predominant GII.4 variant in 2016 was identified as GII.P16. The emergence and predominance of the GII.4 pandemic capsid sequence harboring a different RdRp genotype suggested the potential for a future pandemic.     

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.     

An Outbreak of Norovirus Infections Among Lunch Customers at a Restaurant,Tampere, Finland, 2015

On January 29, 2015, the city of Tampere environmental health officers were informed of a possible foodborne outbreak among customers who had eaten lunch in restaurant X. Employees of electric companies A and B had a sudden onset of gastrointestinal symptoms. We conducted a retrospective cohort study to identify the vehicle, source, and causative agent of the outbreak. A case was defined as an employee of companies A or B with diarrhea and/or vomiting who ate lunch at Restaurant X on January 26, 2015. All employees of the companies attending the implicated lunch were invited to participate in the cohort study. Environmental investigation was conducted. Twenty-one responders were included in statistical analysis, of which 11 met with the case definition. Of the 15 food items consumed by participants, four food items were associated with gastroenteritis. Of four kitchen staff, three tested positive for norovirus GIP7, the strain was found earlier in the community. No patient samples were obtained. Level of hygiene in the kitchen was inadequate. Infected kitchen staff probably transmitted norovirus by inadequate hygiene practices. No new cases associated with Restaurant X were reported after the hygiene practices were improved.     

Prevalence and Molecular Genotyping of Noroviruses in Market Oysters,Mussels, and Cockles in Bangkok,Thailand

Noroviruses are the most common cause of acute gastroenteritis associated with bivalve shellfish consumption. This study aimed to detect and characterize noroviruses in three bivalve shellfish species: oysters (Saccostrea forskali), cockles (Anadara nodifera), and mussels (Perna viridis). The virus concentration procedure (adsorption-twice elution-extraction) and a molecular method were employed to identify noroviruses in shellfish. RT-nested PCR was able to detect known norovirus GII.4 of 8.8 × 10^?2 genome copies/g of digestive tissues from oyster and cockle concentrates, whereas in mussel concentrates, the positive result was seen at 8.8 × 10² copies/g of digestive tissues. From August 2011 to July 2012, a total of 300 shellfish samples, including each of 100 samples from oysters, cockles, and mussels were collected and tested for noroviruses. Norovirus RNA was detected in 12.3 % of shellfish samples. Of the noroviruses, 7.7 % were of the genogroup (G) I, 2.6 % GII, and 2.0 % were mixed GI and GII. The detection rate of norovirus GI was 2.1 times higher than GII. With regards to the different shellfish species, 17 % of the oyster samples were positive, while 14.0 and 6.0 % were positive for noroviruses found in mussels and cockles, respectively. Norovirus contamination in the shellfish occurred throughout the year with the highest peak in September. Seventeen norovirus-positive PCR products were characterized upon a partial sequence analysis of the capsid gene. Based on phylogenetic analysis, five different genotypes of norovirus GI (GI.2, GI.3, GI.4, GI.5, and GI.9) and four different genotypes of GII (GII.1, GII.2, GII.3, and GII.4) were identified. These findings indicate the prevalence and distribution of noroviruses in three shellfish species. The high prevalence of noroviruses in oysters contributes to the optimization of monitoring plans to improve the preventive strategies of acute gastroenteritis.     

Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels

Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic polymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log₁₀ between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

     

International Standardisation of a Method for Detection of Human Pathogenic Viruses in Molluscan Shellfish

The viruses primarily associated with shellfish-borne illness are norovirus, causing gastroenteritis and hepatitis A virus (HAV). Recent years have seen a proliferation of publications on methods for detection of these viruses in shellfish using polymerase chain reaction (PCR). However, currently no standard harmonised procedures have been published. Standardisation is necessary before virus methods can be considered for adoption within a regulatory framework. A European standardisation working group is developing a two-part (quantitative and qualitative) standard method for virus detection in foodstuffs, including shellfish, which has the potential to be incorporated into EU legislation as a reference method. This article describes the development of the standard method and outlines the key methodology principles adopted, the controls and other quality assurance measures supporting the method and future necessary developments in the area.