Deciphering the Diversities of Astroviruses and Noroviruses in Wastewater Treatment Plant Effluents by a High-Throughput Sequencing Method.

Although clinical epidemiology lists human enteric viruses to be among the primary causes of acute gastroenteritis in the human population, their circulation in the environment remains poorly investigated. These viruses are excreted by the human population into sewers and may be released into rivers through the effluents of wastewater treatment plants (WWTPs). In order to evaluate the viral diversity and loads in WWTP effluents of the Paris, France, urban area, which includes about 9 million inhabitants (approximately 15% of the French population), the seasonal occurrence of astroviruses and noroviruses in 100 WWTP effluent samples was investigated over 1 year. The coupling of these measurements with a high-throughput sequencing approach allowed the specific estimation of the diversity of human astroviruses (human astrovirus genotype 1 [HAstV-1], HAstV-2, HAstV-5, and HAstV-6), 7 genotypes of noroviruses (NoVs) of genogroup I (NoV GI.1 to NoV GI.6 and NoV GI.8), and 16 genotypes of NoVs of genogroup II (NoV GII.1 to NoV GII.7, NoV GII.9, NoV GII.12 to NoV GII.17, NoV GII.20, and NoV GII.21) in effluent samples. Comparison of the viral diversity in WWTP effluents to the viral diversity found by analysis of clinical data obtained throughout France underlined the consistency between the identified genotypes. However, some genotypes were locally present in effluents and were not found in the analysis of the clinical data. These findings could highlight an underestimation of the diversity of enteric viruses circulating in the human population. Consequently, analysis of WWTP effluents could allow the exploration of viral diversity not only in environmental waters but also in a human population linked to a sewerage network in order to better comprehend viral epidemiology and to forecast seasonal outbreaks.

A norovirus oyster-related outbreak in a nursing home in France, January 2012.

The presence of norovirus in shellfish is a public health concern in Europe. Here, we report the results of an investigation into a norovirus gastroenteritis outbreak following a festive lunch which affected 84 (57%) residents and staff members of a nursing home in January 2012 in France. Individuals who had eaten oysters had a significantly higher risk of developing symptoms in the following 2·5 days than those who had not, the risk increasing with the amount eaten [relative risk 2·2 (1·0-4·6) and 3·3 (1·6-6·6) for 3-4 and 5-12 oysters, respectively]. In healthy individuals during those days, 29 (32%) subsequently became ill, most of whom were staff members performing activities in close contact with residents. Genogroup II noroviruses were detected in faecal samples, in a sample of uneaten oysters and in oysters from the production area. Identifying a norovirus's infectious dose may facilitate the health-related management of contaminated shellfish.

Detection and genotyping of group A rotaviruses isolated from sewage samples in Monastir, Tunisia between April 2007 and April 2010.

AIMS: To ascertain the viral load, the distribution of G and P types of group A rotaviruses (RV-A) in sewage samples and to compare strains in clinical, animal and environmental samples. METHODS AND RESULTS: During our study from April 2007 to April 2010, 518 samples of raw and treated sewage were collected from two biological sewage treatment plants (STPs) located in the Monastir region, Tunisia. RV-A was detected by real-time RT-PCR in 375 (72·4%) sewage samples. According to the quantification results of RV-A, it appears that the viral load in raw and treated sewage of the two STPs was quite similar (P = 0·735). The genotyping of RV-A strains detected in sewage samples showed a great diversity with 10 G types and 8 P types. Most of them were described as common in humans, but we also detected genotypes commonly found in animals. All the genotypes detected in two previous studies performed in our laboratory on clinical and bovine samples were also found in environmental samples. However, some genotypes commonly found in animal were only found in sewage samples. CONCLUSION: The comparison of environmental, clinical and animal data suggests that STPs may convey not only human sewage but also animal wastes, both of them contaminated with numerous RV-A strains which are not efficiently eliminated by the sewage treatment process and may spread to surface waters. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates the potential release of human and animal RV-A into water sources, representing a public health risk, by inducing gastroenteritis in population, but also by increasing the risk of zoonotic transmission and formation of reassortant viruses which could get a higher infectious potential. Our findings also suggest that monitoring of sewage may provide an additional tool to determine the epidemiology of RV-A circulating in a given community.

Emergence of a novel GII.17 norovirus ­ End of the GII.4 era?

In the winter of 2014/15 a novel GII.P17-GII.17 norovirus strain (GII.17 Kawasaki 2014) emerged, as a major cause of gastroenteritis outbreaks in China and Japan. Since their emergence these novel GII.P17-GII.17 viruses have replaced the previously dominant GII.4 genotype Sydney 2012 variant in some areas in Asia but were only detected in a limited number of cases on other continents. This perspective provides an overview of the available information on GII.17 viruses in order to gain insight in the viral and host characteristics of this norovirus genotype. We further discuss the emergence of this novel GII.P17-GII.17 norovirus in context of current knowledge on the epidemiology of noroviruses. It remains to be seen if the currently dominant norovirus strain GII.4 Sydney 2012 will be replaced in other parts of the world. Nevertheless, the public health community and surveillance systems need to be prepared in case of a potential increase of norovirus activity in the next seasons caused by this novel GII.P17-GII.17 norovirus.

Distribution of G (VP7) and P (VP4) genotypes of group A bovine rotaviruses from Tunisian calves with diarrhoea.

AIMS: To investigate the incidence, viral load and genetic diversity of bovine rotaviruses strains in Tunisia. METHODS AND RESULTS: A total of 169 faecal specimens, collected from diarrhoeic calves from several farms located in the central eastern regions of Tunisia, between January 2006 and October 2010, were analysed by semi-nested multiplex RT-PCRs for P and G genotypes identification or were genotyped by DNA sequencing. Positive samples were tested by TaqMan real-time RT-PCR to quantify the viral load. Group A bovine rotaviruses were detected in 15·4% (26/169) of the total studied cases of diarrhoea. Overall, G10 was the predominant G type, detected in 12/26 samples (46·2%) and G6 accounted for 42·3% (11/26) while P[11] was the predominant P type, detected in 12/26 samples (46·2%). Two P[5] genotypes (7·7%) were found in the collection. Dual G or P combination and genotype G8 were not found. The most common VP7/VP4 combinations were G6P[11] (30·8%; n = 8) and G10P[11] (11·5%; n = 3). The combination G10P[14] was seen in one sample, and partial typing was assessed in 53·8% (n = 14) of the cases. The viral load determined by real-time RT-PCR showed an average of 1·68 × 10(9) genome copies/g of faeces. CONCLUSION: Knowledge of P and G types could help us understand the relatedness of animal rotaviruses to viruses causing disease in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the viral load and P types of bovine rotaviruses have been determined in Tunisia, and this study contributes to a better understanding of the epidemiology of such viruses circulating in Tunisia. Nevertheless, continuous surveillance is necessary to detect the emergence of new variants.

Surveillance for outbreaks of gastroenteritis in elderly long-term care facilities in France, November 2010 to May 2012.

This article describes outbreaks of gastroenteritis in elderly long-term care facilities (LTCF) in France from November 2010 to May 2012 reported through the surveillance system for gastroenteritis outbreaks in LTCF. A total of 1,072 outbreaks were reported, causing 26,551 episodes of illness and 60 deaths. The median attack rate (AR) among residents was 32%. Norovirus and person-to-person transmission were the most frequently reported aetiology and mode of transmission. Control measures were implemented in 1,054 (98%) outbreaks and for 928 outbreaks, the timing of such measures could be inferred. Of these, 799 (86%) had put control measures into effect within three days of the occurrence of the first case. Outbreaks of gastroenteritis in LTCF cause substantial morbidity and mortality among elderly people in France. LTCF are encouraged to develop infection prevention and control plans and to notify any gastroenteritis outbreak to health authorities to ensure rapid control.

A major outbreak of gastroenteritis in Réunion Island in 2012: first identification of G12 rotavirus on the Island.

Between August and November 2012 a severe outbreak of gastroenteritis occurred on Réunion Island, affecting more than 50,000 cases, particularly young children. Virological analyses showed that the virus responsible for this epidemic was rotavirus. Genotyping of stool samples indicated circulation of rotavirus type G3P[8] but also G12P[8], highlighting the risk of global emergence of this genotype in the coming years.

Indications for worldwide increased norovirus activity associated with emergence of a new variant of genotype II.4, late 2012.

Globally, surveillance systems showed an increasein norovirus activity in late 2012. Molecular datashared through the NoroNet network suggest thatthis increase is related to the emergence of a newnorovirus genotype II.4 variant, termed Sydney 2012.Healthcare institutions are advised to be prepared fora severe norovirus season.

Gastroenteritis outbreaks in elderly homes in the east of France during winter 2009/10: aetiology research for a series of 37 outbreaks.

Although acute gastroenteritis is a common cause of morbi-mortality in care homes, there is no national surveillance system in France except for food-borne gastroenteritis. Since 2008, a specific surveillance system has been operating in nursing homes in Alsace, a region in eastern French. In the winter season 2009/10 we had the opportunity to study 37 outbreaks, collecting data on attack rate, duration and aetiology as well as epidemic management in nursing homes. We noticed the responsiveness of the institutions, with a mean period of 1.6 days between the onset of first symptoms and the implementation of management measures (95% confidence interval (CI): 1.0­2.2). One or several stool samples were taken in 27 of the 37 described outbreaks. The only pathogen detected was norovirus, and the positive samples were from outbreaks with a very typical pattern: vomiting in 36 of the outbreaks, a high average attack rate of 36.8% (95% CI: 31.5­42.2) and a short average duration of 8.9 days (95% CI: 5.8­7.0). No severe cases, hospitalisations or deaths were reported. The high frequency of norovirus isolation indicated that systematic bacteriological analysis in local laboratories is not cost-effective. Consequently, Cire Est recommends to test for bacteria and viruses only in cases presenting with fever or atypical symptoms. Nevertheless, Cire Est also recommends to continue sending stool samples to the French National Centre for enteric viruses, more for the benefit of the virological surveillance programme than for diagnostic purposes.

Respiratory tract outbreak modeling with case definition criteria: A proposal for a standardized clinical approach in nursing homes.

OBJECTIVES: This study aimed to monitor respiratory tract outbreaks in nursing homes (NH) by applying standardized case definition criteria, pathogen identification and estimated mortality impact. PATIENTS AND METHODS: This longitudinal, observational study described NH outbreaks with temperature (T°), upper respiratory sign, lower respiratory sign (LRS), general status deterioration, general signs, and mortality. Viral examinations allowed for analysis of developing outbreaks based on positive, negative, or no tests (Flu+/Flu-/NT-Flu). RESULTS: Forty-six influenza identification episodes (Flu+, 1067 patients), 14 Flu- (409 patients), and 18 NT-Flu (381 patients) were analyzed. Viral examinations were conducted mainly among residents with T° (84.8% [302/356]). A specific temperature pattern was observed in Flu+ outbreaks: 35.1% of infected residents with T° without LRS, 15.6% in Flu- episodes, and 17.1% vs. 29.1% in LRS without T°. A median temperature (MT) of ≥38.3 °C was observed in Flu+ outbreaks. MT analysis of the 18 NT-Flu episodes identified five outbreaks with high temperatures (MT ≥38.2 °C) and high mortality. Conversely, the 13 NT-Flu outbreaks with lower MT (<38.0 °C) were associated with lower total mortality. Similar clinical pictures led to closely comparable all-cause mortality impacts, particularly in Flu+, Flu-, and NT-Flu with MT of ≥38.2 °C. CONCLUSIONS: Validated sign/symptom monitoring highlighted some specificities of respiratory NH outbreaks and could be a complementary approach, taking into account common and atypical clinical pictures, assessing mortality and initiating virological investigations and infection control measures.

Evidence of emergence of new GGII.4 norovirus variants from gastroenteritis outbreak survey in France during the 2007-to-2008 and 2008-to-2009 winter seasons.

Analysis of 316 outbreaks of gastroenteritis in France from September 2007 through March 2009 showed that genogroup II.4 (GGII.4) noroviruses were predominant and mostly belonged to the 2006b variant. However, the new GGII.4 variants, variant 2008 and the newly discovered Cairo variant from the Middle East, were also detected. The epidemiological survey suggests that these new variants might become the next predominant strains.

First molecular detection of Aichi virus in sewage and shellfish samples in the Monastir region of Tunisia.

The aims of our investigations were (1) to look for Aichi virus in environmental samples and (2) to compare the Aichi virus strains in both clinical and environmental samples in order to evaluate the role of environmental contamination as a possible vehicle for viral transmission. Aichi virus was detected in 15 (6%) sewage samples and in 4 (6.6%) shellfish samples. Aichi virus was identified for the first time in water samples. Phylogenetic analysis revealed several clusters that occurred sequentially in time, suggesting some parallelism in the evolution of environmental and human strains. Aichi virus present in sewage reflects the viruses circulating in the community.

Detection and molecular characterization of enteric viruses in environmental samples in Monastir, Tunisia between January 2003 and April 2007.

AIMS: A prospective study was performed to characterize the main human enteric viruses able to persist in sewage samples and in shellfish tissues, and to establish the correlation between environmental strains and viral infantile diarrhoea observed in the same area during the same period. METHODS AND RESULTS: A total of 250 sewage (raw and treated) and 60 shellfish samples were collected between January 2003 and April 2007 in Monastir region, Tunisia. Group A rotavirus (RVA) was detected in 80 (32%) sewage samples, norovirus (NoV) in 11 (4·4%) and enteric adenovirus (AdV) in 1 (0·4%). Among 60 shellfish samples collected near sewage effluents, one was contaminated by NoV (1·6%). CONCLUSION: Our data represent the first documentation in Tunisia, combining gastroenteritis viruses circulating in the environment and in clinical isolates. We observed a correlation between environmental strains and those found in children suffering from gastroenteritis during the same period study. This suggests the existence of a relationship between water contamination and paediatric diarrhoea. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results address the potential health risks associated with transmission of human enteric viruses through water-related environmental routes. The research findings will aid in elucidating the molecular epidemiology and circulation of enteric viruses in Tunisia and in Africa, where data are rare.

Prevalence and genetic diversity of Aichi virus strains in stool samples from community and hospitalized patients.

Aichi virus has been proposed as a causative agent of gastroenteritis. A total of 457 stool specimens from children hospitalized with acute diarrhea and 566 stool specimens from adults and children involved in 110 gastroenteritis outbreaks were screened for the presence of Aichi virus by reverse transcription-PCR (RT-PCR) amplification of the genomic region of the 3C and 3D (3CD) nonstructural proteins. Our results show a low incidence of Aichi virus in pediatric samples and the existence of mixed infections with other microbiological agents in some cases. From the outbreak survey, it appears that the presence of Aichi virus is an indicator of mixed infections causing gastroenteritis outbreaks and that it could be involved in half of the oyster-associated outbreaks. A second RT-PCR was developed to amplify a part of the VP1 gene. The phylogenetic analysis showed a good correlation between the two classifications based on 3CD and VP1 gene sequences and revealed the prevalence of genotype A in France. It also allowed us to partially describe an Aichi virus strain that could represent a new genotype, thus suggesting the existence of a certain diversity.

Import of norovirus infections in the Netherlands and Ireland following pilgrimages to Lourdes, 2008--preliminary report.

Between mid-September and 19 October 2008, nine clusters of norovirus infection involving around 90 primary cases and over a hundred secondary cases were identified in patients from the Netherlands, Ireland, Italy and France, linked to pilgrimage to Lourdes, France.

Viral prevalence and laboratory investigations of gastroenteritis in institutions for dependent people.

OBJECTIVES: Viruses are recognized as important agents of gastroenteritis outbreaks in institutions caring for dependent people. We aimed to define norovirus (NoV) and rotavirus (RV) immunochromatographic tests (ICT) and reverse transcription-polymerase chain reaction (RT-PCR) used in gastroenteritis investigations. METHODS: Fourteen sites were monitored from 2010 to 2015, with 360 laboratory investigations. Twenty-two outbreaks and 44 sporadic case patients were investigated with ICTs (114 NoVs and 80 RVs) and RT-PCRs (114 NoVs and 52 RVs). RESULTS: ICTs were useful during outbreaks (identification of NoVs and RVs in respectively 76.5% and 75.0% of episodes) despite the NoV sensitivity limit (55.1%) and the four RV false positive results observed for 10 samples. Given the NoV ICT performance and the observed variations of the NoV and RV prevalence (between 20.0% and 5.0%), ICTs are not appropriate to identify sporadic gastroenteritis case patients. Positive predictive values <60.0% were observed when the prevalence of RV and NoV was low (<5.0%). NoV and RV RT-PCR indications are sporadic gastroenteritis case patients, negative NoV and RV ICT during outbreaks, control of positive RV ICT in cases of suspected NoV and RV co-circulation, patients with long symptom duration, and NoV genogroup and genotype identifications (infection control and epidemiological surveillance). Inclusion of patients with specific clinical symptoms is recommended irrespective of the technique. CONCLUSION: On the basis of the ICT limits identified in this work, RT-PCR development seems essential to improve viral gastroenteritis investigations in institutions caring for dependent people.

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.

Relationship between GII.3 norovirus infections and blood group antigens in young children in Tunisia.

Noroviruses (NoVs) constitute a major cause of gastroenteritis in Tunisia. One hundred and fourteen matched saliva and stool samples were collected from children (n = 114) suffering from acute gastroenteritis at the hospital of Monastir during the winter season 2011-2012. For 98 of 114 children, blood samples were collected for secretor genotyping. NoVs were associated with 36.8% (n = 42/114) of the gastroenteritis cases. The GII.3 genotype was the most common (69% of all NoVs). For patients who were phenotyped (n = 114) for human blood group antigens (HBGAs), the secretor and non-secretor phenotypes represented 79% and 21%, respectively. Of the NoV infections, 83% were detected in all ABO groups. Five GII.3 isolates, one GII.1 isolate and one GII.7 isolate were detected in Lewis-positive non-secretors, confirmed by genotyping of the FUT2 gene. Even though our data showed that GII.3 NoVs could infect non-secretors, no binding was observed with saliva and GII.3 baculovirus-expressed virus-like particles from the same symptomatic non-secretor individual. This suggests that other factors might also participate in NoV attachment in children and newborns.

Burden of gastroenteritis outbreaks: specific epidemiology in a cohort of institutions caring for dependent people.

BACKGROUND: In institutions caring for dependent people, viral gastroenteritis occurs frequently and is highly contagious. In elderly persons, these episodes can lead to hospitalization and occasionally death. AIM: To study the impact of gastroenteritis outbreaks (GOs) in institutions caring for dependent people. METHODS: This study was conducted on 18 sites consisting of 35 units from four different disciplines (geriatric medicine and rehabilitation, psychogeriatrics, geriatric nursing homes, and specialized care homes for adults with physical and mental disabilities). Spatio-temporal analysis of GOs was performed during six winter seasons, and clinical and viral data were analysed with regard to structural parameters (size of the sites and dining-room organization), virus epidemiology and chronology of the outbreaks and type of activities. RESULTS: A total of 98 outbreaks were recorded in the 35 units. The risk of GO was high even outside national epidemic periods. Viruses were searched for in 86 outbreaks and were identified in 96.5% (83/86) of these outbreaks: norovirus genotype GII.4 (59.0%, 49/83), other viruses (41.0%, 34/83). There were variations between surveillance periods in terms of GO frequencies and attack rates and types of viruses. Dining-room organization could be a factor in cross-infection at a site. CONCLUSION: Specific surveillance that takes into account the precise epidemiology needs to be developed in institutions caring for dependent people in order to improve infectious disease control and information for healthcare workers.

The burden of norovirus gastroenteritis: an important foodborne and healthcare-related infection.

Human norovirus (NoV) is now recognized as one of the most important causative agents of gastroenteritis in all age groups worldwide. During the course of NoV infection, symptoms are usually mild and disappear within 48 h after onset. The incidence of NoV infection is high, with hundreds of cases per 10 000 of the population, although the number of infections is still underestimated. Epidemiological surveys conducted in Europe and North America have shown that NoV infections constitute a major disease burden, especially for young children and the elderly, in whom NoV infection leads to high rates of hospitalization and mortality. NoV infections are also of concern in hospitals, where viral infections can be persistent in immunocompromised patients. Although the cost of NoV infection in the hospital community has not yet been clearly established, it appears that NoV infections could cost hundreds of thousands of euros in terms of unit closure, and NoV-related sickness in patients and health workers. Besides their clinical burden, NoVs, as foodborne pathogens, also cause to millions of dollars of losses for the healthcare system and the food industry. Recent estimates in the USA showed that, annually, NoV illness cost $2 billion and led to a loss of approximately 5000 quality-adjusted life-years, making NoV one of the top five pathogens causing enteric illnesses. The highest cost among 14 foodborne pathogens is also attributed to human NoV in The Netherlands. This accumulation of evidence underlines the enormous impact of NoV on populations.