Environmental factors associated with childhood norovirus diarrhoea in León, Nicaragua.

Norovirus is detected in one in five diarrhoea episodes in children, yet little is known about environmental risk factors associated with this disease, especially in low-income settings. The objective of this study was to examine environmental risk factors, and spatial and seasonal patterns of norovirus diarrhoea episodes in children in León, Nicaragua. We followed a population-based cohort of children under age 5 years for norovirus diarrhoea over a 1-year period. At baseline, characteristics of each household were recorded. Households were geocoded and spatial locations of garbage dumps, rivers, and markets were collected. In bivariate analysis we observed younger children and those with animals in their households were more likely to have experienced norovirus episodes. In adjusted models, younger children remained at higher risk for norovirus episodes, but only modest associations were observed with family and environmental characteristics. We next identified symptomatic children living in the same household and within 500 m buffer zones around the household of another child infected with the same genotype. Norovirus diarrhoea episodes peaked early in the rainy season. These findings contribute to our understanding of environmental factors and norovirus infection.

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.

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.

Epidemiological and genetic characteristics of norovirus outbreaks in long-term care facilities, 2003-2006.

To identify the epidemiological and genetic characteristics of norovirus (NoV) outbreaks and estimate the impact of NoV infections in an older population, we analysed epidemiological and laboratory data collected using standardized methods from long-term care facilities (LTCFs) during 2003-2006. Faecal specimens were tested for NoV by real-time reverse transcriptase-polymerase chain reaction. NoV strains were genotyped by sequencing. Of the 234 acute gastroenteritis (AGE) outbreaks reported, 163 (70%) were caused by NoV. The annual attack rate of outbreak-associated NoV infection in LTCF residents was 4%, with a case-hospitalization rate of 3·1% and a case-fatality rate of 0·5%. GII.4 strains accounted for 84% of NoV outbreaks. Median duration of illness was longer for GII.4 infections than non-GII.4 infections (33 vs. 24 h, P<0·001). Emerging GII.4 strains (Hunter/2004, Minerva/2006b, Terneuzen/2006a) gradually replaced the previously dominant strain (Farmington Hills/2002) during 2004-2006. NoV GII.4 strains are now associated with the majority of AGE outbreaks in LTCFs and prolonged illness in Oregon.

Hospital-based surveillance of rotavirus and other viral agents of diarrhea in children and adults in Russia, 2005-2007.

During a 2-year period in 2005-2007, we conducted surveillance of group A rotaviruses and other enteric agents among patients hospitalized with acute gastroenteritis in 8 different cities of the Russian Federation. Fecal specimens were gathered from 3208 children (including 2848 children aged <5 years) and 1354 adults who were admitted to hospitals in Moscow, St. Petersburg, Chelyabinsk, Nizhnii Novgorod, Tyumen, Khabarovsk, Makhachkala, and Yakutsk. Polymerase chain reaction was performed to detect rotaviruses of groups A and C, noroviruses of genogroups I and II, astrovirus, sapovirus, and enteric adenoviruses (group F). Group A rotavirus was the most common viral pathogen detected among children aged <5 years (43.6%), followed by norovirus (12.5%), whereas norovirus was the pathogen most commonly detected in adults (11.9%). P and G genotypes were determined for 515 rotavirus specimens, and the most prevalent genotypes were G1P[8] (44.9%), G4P[8] (40.0%), G2P[4] (8.5%), and G3P[8] (6.6%). This study is the first multicenter study of rotaviruses in the Russian Federation and documents the important burden of disease caused by this pathogen, which soon may be preventable by vaccination.

PATRIC: the VBI PathoSystems Resource Integration Center.

The PathoSystems Resource Integration Center (PATRIC) is one of eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infection Diseases (NIAID) to create a data and analysis resource for selected NIAID priority pathogens, specifically proteobacteria of the genera Brucella, Rickettsia and Coxiella, and corona-, calici- and lyssaviruses and viruses associated with hepatitis A and E. The goal of the project is to provide a comprehensive bioinformatics resource for these pathogens, including consistently annotated genome, proteome and metabolic pathway data to facilitate research into counter-measures, including drugs, vaccines and diagnostics. The project's curation strategy has three prongs: 'breadth first' beginning with whole-genome and proteome curation using standardized protocols, a 'targeted' approach addressing the specific needs of researchers and an integrative strategy to leverage high-throughput experimental data (e.g. microarrays, proteomics) and literature. The PATRIC infrastructure consists of a relational database, analytical pipelines and a website which supports browsing, querying, data visualization and the ability to download raw and curated data in standard formats. At present, the site warehouses complete sequences for 17 bacterial and 332 viral genomes. The PATRIC website (https://patric.vbi.vt.edu) will continually grow with the addition of data, analysis and functionality over the course of the project.

Evaluation of RT-PCR and reverse line blot hybridization for detection and genotyping F+ RNA coliphages from estuarine waters and molluscan shellfish.

AIMS: To evaluate a PCR-based detection and typing method for faecal indicator viruses (F+ RNA coliphages) in water and shellfish, and apply the method for better understanding of the ecology and microbial source tracking potential of these viruses. METHODS AND RESULTS: Water and shellfish samples were collected over 3 years at nine estuaries in the East, West and Gulf Coasts of the USA, providing 1033 F+ RNA coliphage isolates. F+ RNA coliphage genotyping rates by reverse transcriptase-PCR-reverse line blot (RLB) hybridization ranged from 94.7% to 100% among estuaries, and were not significantly different in oysters, clams, mussels or water (P = 0.8427). Twenty samples negative by RLB were nucleotide sequenced for confirmation, and to refine RLB probes. More F+ RNA coliphages were genotyped from colder water than warmer waters, while the water salinity did not affect F+ RNA coliphage levels. CONCLUSIONS: RT-PCR-RLB was a robust method for detecting and genotyping F+ RNA coliphages from diverse coastal areas, which provided new information on the ecology of F+ RNA coliphages. SIGNIFICANCE AND IMPACT OF THE STUDY: This performance-validated F+ RNA coliphage method can be used for faecal indicator monitoring and microbial source tracking, to protect recreational bathers and shellfish consumers from exposure to pathogenic virus and their disease risks.

Animal noroviruses.

Among enteric caliciviruses, noroviruses belong to the genus Norovirus, one of the four accepted genera in the family Caliciviridae. These single-stranded, positive-sense RNA viruses are highly variable both genetically and antigenically. Several animal enteric caliciviruses that are morphologically indistinguishable and genetically closely related to human noroviruses have been identified. The first bovine enteric noroviruses were described in Great Britain and are known as Newbury Agent 2. At least three genetic clusters of porcine noroviruses join together within genogroup II noroviruses. Human noroviruses are the most important cause of acute gastroenteritis illness in people of all ages. In the USA, they are associated with approximately 30-50% of all food-borne outbreaks. Until now, noroviruses have not been associated with gastroenteritis outbreaks in immunocompetent animals. Neither bovine nor porcine noroviruses can replicate in cell culture, although human norovirus can grow in a complex 3D culture system. However, the recently discovered murine noroviruses can replicate in cell culture and are therefore used as model viruses to study human noroviruses. This review focusses on virus classification, virion structure, pathogenesis, epidemiology, immune response and diagnosis of animal noroviruses in comparison with human noroviruses. The classification of animal enteric caliciviruses within the Norovirus genus raises the question of whether transmission from an animal reservoir to humans could occur. Answering this question is important in determining the risk of cross-species infections affecting the epidemiology and evolution of these viruses and so complicating the control of human norovirus infections.

Evaluation of RIDA®GENE norovirus GI/GII real time RT-PCR using stool specimens collected from children and adults with acute gastroenteritis.

BACKGROUND: Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. OBJECTIVE: To evaluate RIDA®GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. STUDY DESIGN: Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014-2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5'end of the norovirus ORF2 gene. RESULTS: A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5-93.5) and 99.1% (98.0-99.6) and for GII was 94.8% (90.8-97.2) and 98.6% (96.9-99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. CONCLUSIONS: The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE.

Molecular epidemiology of human enteric caliciviruses in The Netherlands.

Caliciviruses are among the most common causes of gastroenteritis in people of all age groups. These antigenetically and genetically diverse viruses have been grouped into two genera within the family Caliciviridae, designated Norwalk-like viruses (NLV) and Sapporo-like viruses (SLV). To gain more insight in their epidemiology, we have developed a tentative genotyping scheme, which was used to differentiate the viruses detected in a set of epidemiological studies. NLVs and SLVs were detected by generic RT-PCR in stool specimens from 5.1% and 2.4% of cases with acute gastroenteritis for which a general practitioner was consulted, and in 16.5% and 6.3% of community cases of gastroenteritis. In addition, NLVs were associated with more than 80% of reported outbreaks of gastroenteritis from 1994-1999. Typically, several genotypes of NLV co-circulate in the community. Occasionally, however, several consecutive outbreaks were caused by essentially the same virus, although an epidemiological link had not previously been noted. This was most pronounced in 1995/1996, when a Lordsdale-like variant was detected that subsequently was found worldwide. This epidemic spread suggests differences in virulence or mode of transmission. In addition, we found that related NLVs are highly prevalent in calves in The Netherlands, raising questions about their potential for zoonotic transmission.

Infectious gastro-enteritis: an uncommon cause of diarrhoea in adult allogeneic and autologous stem cell transplant recipients.

The incidence and aetiology of acute diarrhoea in 60 adult allogeneic or autologous stem cell transplant (SCT) recipients was determined in a prospective study. Stool specimens were obtained prior to SCT and on days +20, +40, +60 and +100 post transplant. Microbiological evaluation was performed for pathogenic bacteria, fungi, parasites and viruses. Forty-seven patients were evaluable of whom 31 had a total of 48 acute diarrhoeal episodes. Diarrhoea occurred in 79% of allogeneic and 47% of autologous SCT recipients (P < 0.05). Intestinal infections were found in three of 48 (6%) diarrhoeal episodes. Clostridium difficile with positive toxin was cultured twice and one stool specimen was positive for cryptosporidium. Intestinal pathogens were identified in 13 out of 172 stool specimens from asymptomatic patients and included: rotavirus (4), adenovirus (3), C. difficile, toxin positive (2), and others (4). Graft-versus-host disease was confirmed by biopsy in two of 36 episodes of diarrhoea in allogeneic patients, and in three patients a relationship between reactivation of cytomegalovirus and diarrhoea was suspected. In 40 of 48 (83%) episodes of diarrhoea no clear aetiology could be found.

Epidemiology of Norwalk-like human caliciviruses in hospital outbreaks of acute gastroenteritis in the Stockholm area in 1996.

OBJECTIVES: Outbreaks of acute gastroenteritis associated with 'Norwalk-like viruses' (NLVs) cause significant health problems in hospitals. Hospital outbreaks in the Stockholm area in 1996 were investigated, in order to identify the magnitude of the problem, the mode of transmission, the effect of control measures and the genetic variability of outbreak strains. Determining the epidemiological and clinical significance involves a broad range of possibilities. METHODS: Ten hospitals, representing 66% of the hospitals in the Stockholm area, participated in the study, which included 211 wards. Of these, 18 were selected as control. A standardized protocol that included personal contacts was administered. Outbreak wards were visited between 5 and 10 times. Wards that had reported outbreaks in 1996 were prospectively followed through 1999 by personal contacts, and the available data from 1991 on outbreak reports were collected. A total of 253 stool samples from outbreaks in 1996 were analyzed by electron microscopy (EM) for the presence of NLVs. Positive samples were confirmed by the reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In total, 4 326 patients and 1 119 staff were exposed on the 43 wards that reported 54 outbreaks. The mean attack rate was 13% for patients and 21% for staff. The number of outbreaks in 1996 outnumbered the reported outbreaks in the preceding years (4-70%) and later years (35-40%). Admission to 24 (56%) of the outbreak wards was stopped. The mean duration of illness for patients was 35 hours and for staff, 30 hours. The main symptoms were diarrhoea (80%) and vomiting (68%). Genotyping revealed that the majority of the hospital outbreaks in the Stockholm area in 1996 were caused by a single NLV strain. CONCLUSIONS: The study confirmed that outbreaks of NLV are an increasing public-health problem in hospitals. The risk of being affected by an outbreak was significantly greater on wards that had reported outbreaks in the previous year. It was not obvious which measures had helped to shorten the outbreaks to any appreciable extent. Different managements must therefore be carefully interpreted and adapted to the prevailing circumstances. Genotyping of strains is an important tool of getting a better insight into transmission routes and the mechanism behind the appearance of epidemic strains.

An outbreak of Norwalk like virus gastroenteritis in a nursing home in Rotterdam.

Sixty-two per cent of elderly and disabled residents of a Dutch nursing home (74/120) and 32% of staff (33/102) became ill in an outbreak of Norwalk-like viral gastroenteritis. The outbreak spread from person to person was supported by temporal clustering

[A gastroenteritis epidemic caused by a Norwalk-like virus after 2 weddings in a restaurant; a plea for integral microbiological investigation]. / Een epidemie van gastro-enteritis door een Norwalk-achtig virus na twee bruiloften in een restaurant; een pleidooi voor integraal microbiologisch onderzoek.

OBJECTIVE: To elucidate the cause of an epidemic of gastroenteritis amongst the guests and waiting staff of two weddings celebrated on the same day in the same restaurant. DESIGN: Retrospective and descriptive. METHOD: Following an outbreak of gastroenteritis amongst 215 wedding guests and restaurant staff in 1999, the Public Health Institute Midden-Limburg, the Netherlands, inventoried the demographic and clinical variables and consumed foods by means of a structured written interview. Faecal samples and remaining food products were bacteriologically examined, and later on faeces were virologically examined for the presence of the Norwalk-like virus (NLV). The attack rates and the incidence rates of NLV-positive faecal specimens amongst consumers and non-consumers of specific food products were calculated, as well as the corresponding relative risks with 95% confidence intervals. RESULTS: The overall attack rate was 66%. An NLV with the same genotype was found statistically significantly more frequently in faecal specimens of ill persons compared with non-ill individuals. Of the 61 different dishes served, two showed a statistically significant relation with being ill. These food products were eaten by 26% of the ill persons. No statistically significant association was found between NLV-positive faeces and consumption of a specific dish. The index case began having symptoms of gastroenteritis at the morning of the wedding party, before food was served. The guests of the two wedding parties used the same entrance and toilets. CONCLUSION: The epidemic was caused by a single NLV strain. No association could be found between the consumption of certain food products and being ill. The NLV epidemic was probably caused through direct or indirect contact between wedding guests and restaurant personnel. In the case of such reports of gastroenteritis epidemics, it is strongly recommended to test faecal, food product and environment samples for both bacteria and viruses, with an emphasis on NLV, to ensure early diagnosis.

Infection control for norovirus.

Norovirus infections are notoriously difficult to prevent and control, owing to their low infectious dose, high shedding titre, and environmental stability. The virus can spread through multiple transmission routes, of which person-to-person and foodborne are the most important. Recent advances in molecular diagnostics have helped to establish norovirus as the most common cause of sporadic gastroenteritis and the most common cause of outbreaks of acute gastroenteritis across all ages. In this article, we review the epidemiology and virology of noroviruses, and prevention and control guidelines, with a focus on the principles of disinfection and decontamination. Outbreak management relies on sound infection control principles, including hand hygiene, limiting exposure to infectious individuals, and thorough environmental decontamination. Ideally, all infection control recommendations would rely on empirical evidence, but a number of challenges, including the inability to culture noroviruses in the laboratory and the challenges of outbreak management in complex environments, has made it difficult to garner clear evidence of efficacy in certain areas of infection control. New experimental data on cultivable surrogates for human norovirus and on environmental survivability and relative resistance to commonly used disinfectants are providing new insights for further refinining disinfection practices. Finally, clinical trials are underway to evaluate the efficacy of vaccines, which may shift the current infection control principles to more targeted interventions.

An automated genotyping tool for enteroviruses and noroviruses.

BACKGROUND: Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. OBJECTIVE: To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. STUDY DESIGN: An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. RESULTS: Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. DISCUSSION AND CONCLUSIONS: The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons.

Fate and transport of zoonotic, bacterial, viral, and parasitic pathogens during swine manure treatment, storage, and land application.

Members of the public are always somewhat aware of foodborne and other zoonotic pathogens; however, recent illnesses traced to produce and the emergence of pandemic H1N1 influenza virus have increased the scrutiny on all areas of food production. The Council for Agricultural Science and Technology has recently published a comprehensive review of the fate and transport of zoonotic pathogens that can be associated with swine manure. The majority of microbes in swine manure are not zoonotic, but several bacterial, viral, and parasitic pathogens have been detected. Awareness of the potential zoonotic pathogens in swine manure and how treatment, storage, and handling affect their survival and their potential to persist in the environment is critical to ensure that producers and consumers are not at risk. This review discusses the primary zoonotic pathogens associated with swine manure, including bacteria, viruses, and parasites, as well as their fate and transport. Because the ecology of microbes in swine waste is still poorly described, several recommendations for future research are made to better understand and reduce human health risks. These recommendations include examination of environmental and ecological conditions that contribute to off-farm transport and development of quantitative risk assessments.

Failure to detect infection by oral polio vaccine virus following natural exposure among inactivated polio vaccine recipients.

While oral polio vaccine (OPV) has been shown to be safe and effective, it has been observed that it can circulate within a susceptible population and revert to a virulent form. Inactivated polio vaccine (IPV) confers protection from paralytic disease, but provides limited protection against infection. It is possible, then, that an IPV-immunized population, when exposed to OPV, could sustain undetected circulation of vaccine-derived poliovirus. This study examines the possibility of polio vaccine virus circulating within the United States (highly IPV-immunized) population that borders Mexico (OPV-immunized). A total of 653 stool and 20 sewage samples collected on the US side of the border were tested for the presence of poliovirus. All samples were found to be negative. These results suggest that the risk of circulating vaccine-derived poliovirus is low in fully immunized IPV-using populations in developed countries that border OPV-using populations.

Molecular detection and epidemiology of small round-structured viruses in outbreaks of gastroenteritis in the Netherlands.

To study the epidemiology of small round-structured viruses (SRSV) in the Netherlands, all outbreaks of gastroenteritis that were reported to the Research Laboratory for Infectious Diseases, Department of Virology, National Institute of Public Health and the Environment (RIVM) in 1994 and 1995 were examined using electron microscopy (EM), single-round reverse transcription-polymerase chain reaction (RT-PCR), and sequencing. To enable this, a generic SRSV-specific primer pair was developed that could detect 85% of a panel of antigenically diverse SRSV. By EM, SRSV could be detected in 86% and by RT-PCR in 91% of the reported gastroenteritis outbreaks. Partial sequence analysis of the polymerase region of these viruses revealed that two different clusters of viruses were responsible for the majority of the outbreaks. This strongly suggests epidemic spread of SRSV in the Netherlands.

Simultaneous detection and genotyping of "Norwalk-like viruses" by oligonucleotide array in a reverse line blot hybridization format.

"Norwalk-like viruses" (NLVs) are the most common cause of outbreaks of nonbacterial gastroenteritis worldwide. To date, the method most widely used for typing of NLV strains is sequencing and subsequent phylogenetic analysis of reverse transcription (RT)-PCR products, which has revealed the existence of stable distinct lineages (genotypes). This typing method is rather costly, not routinely used in clinical laboratories, and not very suitable for the analysis of large numbers of samples. Therefore, we have developed a rapid and simple method for genotyping of NLVs. The method, designated reverse line blot hybridization, is based on the nucleotide divergence of a region of the gene for RNA polymerase which can be used to classify NLVs into genotypes. NLV RNA was amplified by RT-PCR and then hybridized to 18 different membrane-bound oligonucleotides that were able to discriminate among 13 NLV genotypes. Application of the method to a panel of 132 positive stool samples from 34 outbreaks and 20 sporadic cases of gastroenteritis collected in a 6-year period (1994 to 1999) resulted in successful genotyping of 124 samples (94%), as confirmed by phylogenetic analysis. The nucleotide sequences of the remaning eight strains (6%) from three outbreaks did not cluster with the known NLV genotypes. Phylogenetic analysis of the complete and partial open reading frame 2 (capsid gene) sequences of these strains revealed the existence of one novel genotype (Alphatron) and one potentially novel genotype (Amsterdam). This novel method, which allows simultaneous detection and genotyping of NLVs, is useful in the diagnosis and typing of NLVs obtained from outbreaks and in large-scale epidemiological studies.