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1.  Mechanisms of GII.4 Norovirus Persistence in Human Populations  
PLoS Medicine  2008;5(2):e31.
Background
Noroviruses are the leading cause of viral acute gastroenteritis in humans, noted for causing epidemic outbreaks in communities, the military, cruise ships, hospitals, and assisted living communities. The evolutionary mechanisms governing the persistence and emergence of new norovirus strains in human populations are unknown. Primarily organized by sequence homology into two major human genogroups defined by multiple genoclusters, the majority of norovirus outbreaks are caused by viruses from the GII.4 genocluster, which was first recognized as the major epidemic strain in the mid-1990s. Previous studies by our laboratory and others indicate that some noroviruses readily infect individuals who carry a gene encoding a functional alpha-1,2-fucosyltransferase (FUT2) and are designated “secretor-positive” to indicate that they express ABH histo-blood group antigens (HBGAs), a highly heterogeneous group of related carbohydrates on mucosal surfaces. Individuals with defects in the FUT2 gene are termed secretor-negative, do not express the appropriate HBGA necessary for docking, and are resistant to Norwalk infection. These data argue that FUT2 and other genes encoding enzymes that regulate processing of the HBGA carbohydrates function as susceptibility alleles. However, secretor-negative individuals can be infected with other norovirus strains, and reinfection with the GII.4 strains is common in human populations. In this article, we analyze molecular mechanisms governing GII.4 epidemiology, susceptibility, and persistence in human populations.
Methods and Findings
Phylogenetic analyses of the GII.4 capsid sequences suggested an epochal evolution over the last 20 y with periods of stasis followed by rapid evolution of novel epidemic strains. The epidemic strains show a linear relationship in time, whereby serial replacements emerge from the previous cluster. Five major evolutionary clusters were identified, and representative ORF2 capsid genes for each cluster were expressed as virus-like particles (VLPs). Using salivary and carbohydrate-binding assays, we showed that GII.4 VLP-carbohydrate ligand binding patterns have changed over time and include carbohydrates regulated by the human FUT2 and FUT3 pathways, suggesting that strain sensitivity to human susceptibility alleles will vary. Variation in surface-exposed residues and in residues that surround the fucose ligand interaction domain suggests that antigenic drift may promote GII.4 persistence in human populations. Evidence supporting antigenic drift was obtained by measuring the antigenic relatedness of GII.4 VLPs using murine and human sera and demonstrating strain-specific serologic and carbohydrate-binding blockade responses. These data suggest that the GII.4 noroviruses persist by altering their HBGA carbohydrate-binding targets over time, which not only allows for escape from highly penetrant host susceptibility alleles, but simultaneously allows for immune-driven selection in the receptor-binding region to facilitate escape from protective herd immunity.
Conclusions
Our data suggest that the surface-exposed carbohydrate ligand binding domain in the norovirus capsid is under heavy immune selection and likely evolves by antigenic drift in the face of human herd immunity. Variation in the capsid carbohydrate-binding domain is tolerated because of the large repertoire of similar, yet distinct HBGA carbohydrate receptors available on mucosal surfaces that could interface with the remodeled architecture of the capsid ligand-binding pocket. The continuing evolution of new replacement strains suggests that, as with influenza viruses, vaccines could be targeted that protect against norovirus infections, and that continued epidemiologic surveillance and reformulations of norovirus vaccines will be essential in the control of future outbreaks.
Through phylogenetic analysis of norovirus isolates, Ralph Baric and colleagues show that new epidemic strains arise as the variety of available cellular receptors permits antigenic drift in the viral capsid.
Editors' Summary
Background.
Noroviruses are the leading cause of viral gastroenteritis (stomach flu), the symptoms of which include nausea, vomiting, and diarrhea. There is no treatment for infection with these highly contagious viruses. While most people recover within a few days, the very young and old may experience severe disease. Like influenza, large outbreaks (epidemics) of norovirus infection occur periodically (often in closed communities such as cruise ships), and most people have several norovirus infections during their lifetime. Currently, 100,000–200,000 people are being infected each week in England with a new GII.4 variant. There are several reasons for this pattern of infection and reinfection. First, the immune response induced by a norovirus infection is short-lived in some people, but not all. Second, there are many different noroviruses. Based on their genomes (genetic blueprints), noroviruses belong to five “genogroups,” which are further subdivided into “genotypes.” An immune response to one norovirus provides little protection against noroviruses of other genogroups or genotypes. Third, like influenza viruses, noroviruses frequently acquire small changes in their genome. This process is called antigenic drift (antigens are the molecules on the surface of infectious agents that stimulate the production of antibodies, proteins that help the immune system recognize and deal with foreign invaders). Norovirus epidemics occur when virus variants emerge to which the human population has no immunity.
Why Was This Study Done?
It is unknown exactly how noroviruses change over time or how they persist in human populations. In addition, little is known about susceptibility to norovirus infections except that secretor-positive individuals—people who express “histoblood group antigens” (HBGAs, a heterogeneous group of sugar molecules by which noroviruses attach themselves to human cells) on the cells that line their mouths and guts—are more susceptible than secretor-negative people, who express these antigens only on red blood cells. Information of this sort is needed to devise effective intervention strategies, therapies, and vaccines to reduce the illness and economic costs associated with norovirus outbreaks. In this study, the researchers investigate the molecular mechanisms governing the emergence and persistence of epidemic norovirus strains in human populations by analyzing how GII.4 norovirus strains (the genotype usually associated with epidemics) have changed over time.
What Did the Researchers Do and Find?
The researchers analyzed the relationships among the sequences of the gene encoding the capsid protein of GII.4 norovirus strains isolated over the past 20 years. The capsid protein forms a shell around noroviruses and is involved in their binding to HBGAs and their recognition by the human immune system. The researchers found that the virus evolved in fits and starts. That is, for several years, one cluster of strains was predominant but then new epidemic strains emerged rapidly from the cluster. In all, the researchers identified five major evolutionary clusters. They then created “virus-like particles” (VLPs) using representative capsid genes from each cluster and showed that these VLPs bound to different HBGAs. Finally they measured the antigenic relatedness of the different VLPs using human blood collected during a 1988 GII.4 outbreak. Antibodies in these samples recognized the VLPs representing early GII.4 strains better than VLPs representing recent GII.4 strains. The ability of the blood samples to block the interaction of VLPs with their matching HBGAs showed a similar pattern.
What Do These Findings Mean?
These findings suggest that the part of the norovirus capsid protein that binds to sugars on host cells is under heavy immune selection and evolves over time by antigenic drift. They show that, like influenza viruses, GII.4 viruses evolve through serial changes in the capsid sequence that occur sporadically after periods of stability, probably to evade the build up of immunity within the human population. Variation in this region of the viral genome is possible because human populations express a great variety of HBGA molecules so there is always likely to be a subpopulation of people that is susceptible to the altered virus. Overall, these findings suggest that it should be possible to develop vaccines to protect against norovirus infections but, just as with influenza virus, surveillance systems will have to monitor how the virus is changing and vaccines will need to be reformulated frequently to provide effective protection against norovirus outbreaks.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050031.
See a related PLoS Medicine Perspective article
The MedlinePlus encyclopedia has a page on viral gastroenteritis (in English and Spanish)
The US Centers for Disease Control and Prevention provides information on viral gastroenteritis (in English and Spanish) and on noroviruses
The UK National Health Service's health website (NHS Direct) provides information about noroviruses
The UK Health Protection Agency and the US Food & Drug Administration also provide information about noroviruses
doi:10.1371/journal.pmed.0050031
PMCID: PMC2235898  PMID: 18271619
2.  Broad Blockade Antibody Responses in Human Volunteers after Immunization with a Multivalent Norovirus VLP Candidate Vaccine: Immunological Analyses from a Phase I Clinical Trial 
PLoS Medicine  2015;12(3):e1001807.
Background
Human noroviruses (NoVs) are the primary cause of acute gastroenteritis and are characterized by antigenic variation between genogroups and genotypes and antigenic drift of strains within the predominant GII.4 genotype. In the context of this diversity, an effective NoV vaccine must elicit broadly protective immunity. We used an antibody (Ab) binding blockade assay to measure the potential cross-strain protection provided by a multivalent NoV virus-like particle (VLP) candidate vaccine in human volunteers.
Methods and Findings
Sera from ten human volunteers immunized with a multivalent NoV VLP vaccine (genotypes GI.1/GII.4) were analyzed for IgG and Ab blockade of VLP interaction with carbohydrate ligand, a potential correlate of protective immunity to NoV infection and illness. Immunization resulted in rapid rises in IgG and blockade Ab titers against both vaccine components and additional VLPs representing diverse strains and genotypes not represented in the vaccine. Importantly, vaccination induced blockade Ab to two novel GII.4 strains not in circulation at the time of vaccination or sample collection. GII.4 cross-reactive blockade Ab titers were more potent than responses against non-GII.4 VLPs, suggesting that previous exposure history to this dominant circulating genotype may impact the vaccine Ab response. Further, antigenic cartography indicated that vaccination preferentially activated preexisting Ab responses to epitopes associated with GII.4.1997. Study interpretations may be limited by the relevance of the surrogate neutralization assay and the number of immunized participants evaluated.
Conclusions
Vaccination with a multivalent NoV VLP vaccine induces a broadly blocking Ab response to multiple epitopes within vaccine and non-vaccine NoV strains and to novel antigenic variants not yet circulating at the time of vaccination. These data reveal new information about complex NoV immune responses to both natural exposure and to vaccination, and support the potential feasibility of an efficacious multivalent NoV VLP vaccine for future use in human populations.
Trial Registration
ClinicalTrials.gov NCT01168401
Lisa Lindesmith and colleagues assess the potential of a candidate virus-like particle (VLP) vaccine to induce antibody responses to antigenically divergent norovirus strains.
Editors' Summary
Background
Worldwide, noroviruses cause one in five cases of viral gastroenteritis (often called stomach flu or winter vomiting disease), the symptoms of which include nausea, vomiting, and diarrhea. There is no specific treatment for infection with these highly contagious viruses, and no established approach to vaccine development. While most people recover from the symptoms of norovirus infection within a few days, young children and the elderly may become severely ill or die. An estimated annual 300 million cases of norovirus infection contribute to roughly 260,000 deaths, mostly among this vulnerable demographic and mostly in low-income countries. Like influenza viruses, many noroviruses are evolving via a process known as antigenic drift. Antigens are components of infectious agents (including viruses) that are recognized by antibodies, proteins that bind to and neutralize foreign invaders. Over time, noroviruses develop small changes in their antigens that allow them to escape from antibodies produced in response to earlier infections. Every two to four years, because of accumulated antigenic drift, a new strain of norovirus emerges to which the human population has no direct antibody immunity, and an outbreak occurs. Because vaccines usually contain a component of the infectious agent that stimulates immunity, antigenic drift complicates the process of vaccine development. To be worth the cost and effort, a norovirus vaccine must confer immunity against a diverse range of norovirus strains, ideally including strains beyond those represented within the vaccine itself.
Partly because there is not a reliable method for growing noroviruses in the laboratory, recent efforts have focused on developing candidate vaccines using virus-like particles (VLPs). VLPs are constructed from laboratory-generated molecules of the virus’s capsid (outer shell). These capsid proteins self-assemble into icosahedral VLPs, which resemble the viral shell. VLPs cannot infect people or cause illness, but because they contain viral antigens, they can induce the immune system to produce antibodies that may neutralize actual viruses. VLPs can also be used to study the antibodies that people produce in response to vaccination or infection.
Why Was This Study Done?
VLP-based vaccines are relatively new, and their capacity to elicit a broad immune response conferring protection to an evolving range of norovirus strains is not established. One VLP vaccine based on a single strain that circulates primarily in children conferred immunity to that strain. Another, multivalent (containing a mix of VLPs from more than one strain) VLP vaccine elicited antibody generation, but in a phase I clinical trial did not confer immunity to infection by a strain that had previously circulated globally. In the current study, the researchers explored two key questions using laboratory analysis of blood samples drawn from participants in that trial. First, they tested whether the vaccine elicits antibody responses to a broad range of norovirus strains, as antibody responses can provide clues to the potential for this type of vaccine to confer broad immunity in the future. Second, they investigated how preexisting exposure to noroviruses affects the immune system’s response to a vaccine—strategic information that could aid in future vaccine development.
What Did the Researchers Do and Find?
The researchers tested serum (blood without cells or clotting proteins; serum contains the antibodies generated by the immune system) collected from ten participants receiving one injection of the VLP vaccine followed by a second injection 28 days later. They analyzed the serum specimens for antibodies to vaccine VLPs and also to VLPs representing viruses that were not contained in the vaccine. They used two methods, both utilizing VLPs generated from 11 norovirus strains: a traditional method that assesses binding of serum antibodies to each of these VLPs, and a more recent method that assays the ability of antibodies to block the interaction of each VLP with a molecule on intestinal cells that binds to the virus (the gut epithelial ligand), enabling norovirus to enter and infect cells. Prior studies suggest that this latter assay may be a better proxy for actual immunity.
The researchers’ major finding is that a multivalent VLP vaccine (two VLPs representing four strains of norovirus: one from a subgroup called genotype GI.1 and another consensus VLP of three strains from the subgroup GII.4) can rapidly elicit serum antibodies that bind a range of vaccine and non-vaccine VLPs, and that block binding of these VLPs to the gut epithelial ligand. Notably, vaccine recipients also generated antibodies reactive to two novel VLPs representing human noroviruses that they could not have previously encountered, indicating that prior exposure to each norovirus strain was not required for the full antibody response following vaccination. However, based on an analysis of which specific epitopes (small regions on an antigen) the population of antibodies binds, the authors report that antibody responses to the vaccine prominently target epitopes of a 1997 strain of human GII.4 norovirus, and propose that exposure history does influence the antibody response.
What Do these Findings Mean?
These findings raise the possibility that the VLP vaccine may induce immunity not only to norovirus strains that have caused past outbreaks, but also to variants that have yet to enter the population—a necessary attribute given the antigenic drift observed among noroviruses. The study also indicates that VLP-induced antibody responses to norovirus are consistent with the “antigenic seniority” model, in which strains to which an individual was previously exposed influence the binding properties of a vaccine-induced antibody population. This latter finding may influence the design of future norovirus vaccines.
These results must be interpreted cautiously, particularly as they pertain to the potential for a norovirus vaccine to protect against natural infection. The study is small, and antibody binding and blocking assays may not replicate how the immune system of a vaccine recipient will respond to true norovirus infection. Additionally, the study participants were all adults aged 18 to 49 years, while a vaccine is most needed for young children (who account for the majority of severe infections) and the elderly (who are most likely to die from infection). Unlike the study participants, young children lack preexisting antibodies to norovirus. Older people are more likely to have been previously exposed to norovirus, but may show attenuated immune responses to vaccination. Adapting to the different immune responses of these two groups remains a central challenge to norovirus vaccine development.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001807.
The World Health Organization provides a comprehensive description of the disease burden from diarrheal disease
The MedlinePlus encyclopedia has a page on viral gastroenteritis (in English and Spanish)
The US Centers for Disease Control and Prevention provides information on disease trends and outbreaks
The US Department of Health and Human Services offers guidance for prevention based on food safety
A 2014 interview with Academic Editor Benjamin Lopman explores the difficulty of developing a norovirus vaccine
The authors have previously published findings on the evolution of norovirus strains in PLOS Medicine and have discussed the challenges of norovirus therapeutic design in PLOS Pathogens
doi:10.1371/journal.pmed.1001807
PMCID: PMC4371888  PMID: 25803642
3.  Etiological study of enteric viruses and the genetic diversity of norovirus, sapovirus, adenovirus, and astrovirus in children with diarrhea in Chongqing, China 
BMC Infectious Diseases  2013;13:412.
Background
Enteric viruses are a major cause of diarrhea in children, especially those <5 years old. Identifying the viral agents is critical to the development of effective preventive measures. This study aimed to determine the prevalence of common enteric viruses in children <5 years old presented with diarrhea to the Children’s Hospital of Chongqing Medical University.
Methods
Five hundred fecal samples were collected between August and November 2010 from children <5 years of age who presented with acute diarrhea at the Children’s Hospital of Chongqing Medical University. All samples were tested for rotaviruses A, B, and C, noroviruses GI and GII, adenovirus, sapovirus, and astrovirus using enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction (RT-PCR), or PCR. Partial sequences of norovirus, sapovirus, adenovirus, and astrovirus were phylogenetically analyzed to determine the genotype.
Results
Enteric viruses were detected in 302 of the 500 children who presented with acute diarrhea (277/477; 58.07%) and persistent diarrhea (5/23; 21.74%). In 277 samples from children with acute diarrhea in whom at least one viral agent was found, rotavirus A was the most frequent virus identified (132 cases; 27.67%), followed by norovirus GII in 130 cases (27.25%), adenovirus in 30 cases (6.29%), sapovirus in 9 cases (1.89%) and astrovirus in one case (0.21%). Twenty-two of the norovirus GII-positive cases were randomly selected for genotyping. GII/4 was the predominant strain, followed by GII/6, GII/2, GII/3, and GII/7. Sapovirus was classified into four genotypes: GI/1 was predominant, followed by GI/2, GII/1, and GIV. The predominant adenovirus was type 41. Mixed infections were found in 25 cases, all of which presented with acute diarrhea (25/477; 5.24%). Viruses were positive in 5/23 (21.74%) cases with persistent diarrhea. Neither rotavirus B, rotavirus C, nor norovirus GI were found in any of the samples.
Conclusions
Enteric viruses are a major cause of diarrhea in children <5 years old in Chongqing. Rotavirus A is the most common etiological agent, follow by norovirus.
doi:10.1186/1471-2334-13-412
PMCID: PMC3766652  PMID: 24004442
4.  Rotavirus and Norovirus infections among acute gastroenteritis children in Morocco 
BMC Infectious Diseases  2014;14:300.
Background
Acute gastroenteritis is a serious cause of child mortality and morbidity in resource-limited countries. A viral etiology is most common, and rotavirus and norovirus are reported to be the leading causative agents. There are still few epidemiological data on the simultaneous occurrence of these viruses in Morocco. The aim of this study was to provide useful epidemiological data on the gastroenteritis associated with rotavirus and norovirus among children aged less than 5 years.
Methods
From January to December 2011, 335 samples were tested for rotavirus and norovirus using enzyme-linked immunosorbent assay, reverse-transcription-polymerase chain reaction (RT-multiplex PCR) and real-time RT-PCR. Partial sequences of the norovirus were phylogenetically analyzed to determine the genotype.
Results
The overall rates of rotavirus and norovirus infections were 26.6% and 16.1%, respectively. Mixed viral infections were detected in 9 of 335 stool specimens (2.7%).
The most common genotype combination in the rotavirus strains was G1[P8] (51.7%), followed by G2[P4] (10.1%), G2[P8] (4.5%), G9[P8] (3.4%), G4[P8] (3.4%), and G1[P6] (2.3%). Among patients positive for norovirus, 42 (77.8%) tested positive for GII and 12 (22.2%) for GI. Thirty-three (78.6%) of the norovirus GII-positive cases were successfully characterized. Genotype GII.4 was the most prevalent (n = 27; 81.8%), followed by GII.3 (n = 2; 6.1%), GII.13 (n = 2; 6.1%), GII.16 (n = 1; 3%), and GII.17 (n = 1; 3%).
Conclusion
This study suggests that in Morocco, norovirus is the most frequent cause of acute gastroenteritis after rotavirus, but further enteric viruses need to be integrated in the surveillance system so that a conclusion could be drawn.
doi:10.1186/1471-2334-14-300
PMCID: PMC4057912  PMID: 24894194
Norovirus; Rotavirus; Acute gastroenteritis; Morocco
5.  Aetiology of childhood viral gastroenteritis in Lucknow, north India 
Background & objectives:
Due to limited availability of data on viral aetiology of acute gastroenteritis in north India, the present study was planned to detect rotavirus, norovirus, sapovirus and astrovirus in stool samples of both in hospitalized and non-hospitalized children less than five years of age presenting with acute gastroenteritis.
Methods:
A total of 278 stool samples from equal number of children were tested for rotavirus antigen using ELISA and for norovirus, sapovirus and astroviruses by reverse transcription (RT)-PCR.
Results:
Of the 169 samples from hospitalized patients, rotavirus, norovirus, sapovirus and astrovirus were detected in 19.5, 2.3, 3.5 and 2.9 per cent samples, respectively. Of the 109 samples collected from the non-hospitalized patients, frequency of rotavirus and sapovirus detection was 9.1 and 1.8 per cent, respectively while norovirus and astrovirus were not detected.
Interpretation & conclusions:
Rotavirus was the most frequent cause of viral gastroenteritis in both hospitalized and non-hospitalized children. Maximum positivity of the viruses was seen in children less than two years of age.
doi:10.4103/0971-5916.159298
PMCID: PMC4510728  PMID: 26112849
Astrovirus; diarrhoea; norovirus; rotavirus; sapovirus; stool samples; viral gastroenteritis
6.  Epidemiology of norovirus infections among diarrhea outpatients in a diarrhea surveillance system in Shanghai, China: a cross-sectional study 
BMC Infectious Diseases  2015;15:183.
Background
Norovirus is an important cause of gastroenteritis both in children and adults. In China, few studies have been conducted on adult populations. This study aimed to determine the contribution of norovirus to gastroenteritis, characterize the features of norovirus infections, compare them with other pathogens, and test the effectiveness of the surveillance system.
Methods
A citywide surveillance network on diarrhea patients was established. Samples were collected with intervals from both children and adults among diarrhea outpatients in hospitals and tested for viruses using rRT-PCR and for bacteria in CDCs. Patient information was acquired through interviews and recorded into a dedicated online system. The Pearsonχ2 test, multivariate logistic regression models and discriminant models were fitted into its comparisons with the non-norovirus group and other pathogens.
Results
Norovirus was detected in 22.91% of sampled diarrhea patients. The seasonal distribution of norovirus infections was different from non-norovirus patients (p < 0.001), with a half-year peak. Higher proportions of males (p = 0.001, OR = 1.303, 95% CI = 1.110-1.529), local citizens (p < 0.001) and officials/clerks (p = 0.001, OR = 1.348, 95% CI = 1.124-1.618) were affected with norovirus when compared with non-norovirus patients. Diarrhea patients affected with norovirus featured nausea (p < 0.001, OR = 1.418, 95% CI = 1.176-1.709) and vomiting (p < 0.001, OR = 1.969, 95% CI = 1.618-2.398), while fewer manifested fever (p = 0.046, OR = 0.758, 95% CI = 0.577-0.996) and abdominal pain (p = 0.018, OR = 0.815, 95% CI = 0.689-0.965). Children were more vulnerable to rotavirus (p = 0.008, OR = 1.637, 95% CI = 1.136-2.358) and bacteria (p = 0.027, OR = 1.511, 95% CI = 1.053-2.169) than norovirus. There was a seasonal difference between the GI and GII genotypes (p < 0.001). Officials or clerks were more easily affected with GI than GII (p = 0.006, OR = 1.888, 95% CI = 1.205-2.958).
Conclusions
This study was based on a citywide hospital-sentinel surveillance system with multiple enteric pathogens included. Norovirus was recognized as the most prevalent enteric pathogen in Shanghai. The seasonal peak was from October to April. Males had a higher prevalence than females. Local citizens and officials/clerks were more vulnerable to norovirus than other pathogens. Compared with rotavirus and bacteria, children were less frequently affected by norovirus. Nausea and vomiting were typical of norovirus, whereas fever and abdominal pain were uncommon symptoms of this pathogen. GI and GII infections were centered in different seasons. Officials and clerks were more easily affected by GI than GII.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-015-0922-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12879-015-0922-z
PMCID: PMC4438334  PMID: 25884557
Human norovirus; Diarrhea; Surveillance; Epidemiology; Sporadic; All age groups; rRT-PCR
7.  Norovirus Diversity in Diarrheic Children from an African-Descendant Settlement in Belém, Northern Brazil 
PLoS ONE  2013;8(2):e56608.
Norovirus (NoV), sapovirus (SaV) and human astrovirus (HAstV) are viral pathogens that are associated with outbreaks and sporadic cases of gastroenteritis. However, little is known about the occurrence of these pathogens in relatively isolated communities, such as the remnants of African-descendant villages (“Quilombola”). The objective of this study was the frequency determination of these viruses in children under 10 years, with and without gastroenteritis, from a “Quilombola” Community, Northern Brazil. A total of 159 stool samples were obtained from April/2008 to July/2010 and tested by an enzyme immunoassay (EIA) and reverse transcription-polymerase chain reaction (RT-PCR) to detect NoV, SaV and HAstV, and further molecular characterization was performed. These viruses were detected only in the diarrheic group. NoV was the most frequent viral agent detected (19.7%-16/81), followed by SaV (2.5%-2/81) and HAstV (1.2%-1/81). Of the 16 NoV-positive samples, 14 were sequenced with primers targeting the B region of the polymerase (ORF1) and the D region of the capsid (ORF2). The results showed a broad genetic diversity of NoV, with 12 strains being classified as GII-4 (5–41.7%), GII-6 (3–25%), GII-7 (2–16.7%), GII-17 (1–8.3%) and GI-2 (1–8.3%), as based on the polymerase region; 12 samples were classified, based on the capsid region, as GII-4 (6–50%, being 3–2006b variant and 3–2010 variant), GII-6 (3–25%), GII-17 (2–16.7%) and GII-20 (1–8.3%). One NoV-strain showed dual genotype specificity, based on the polymerase and capsid region (GII-7/GII-20). This study provides, for the first time, epidemiological and molecular information on the circulation of NoV, SaV and HAstV in African-descendant communities in Northern Brazil and identifies NoV genotypes that were different from those detected previously in studies conducted in the urban area of Belém. It remains to be determined why a broader NoV diversity was observed in such a semi-isolated community.
doi:10.1371/journal.pone.0056608
PMCID: PMC3574080  PMID: 23457593
8.  Evaluation and Verification of the Seeplex Diarrhea-V ACE Assay for Simultaneous Detection of Adenovirus, Rotavirus, and Norovirus Genogroups I and II in Clinical Stool Specimens ▿  
Journal of Clinical Microbiology  2011;49(9):3154-3162.
Acute viral gastroenteritis is an intestinal infection that can be caused by several different viruses. Here we describe the evaluation and verification of Seeplex Diarrhea-V ACE (Seeplex DV), a novel commercial multiplex reverse transcription-PCR (RT-PCR) assay that detects 5 diarrheal pathogens, including adenovirus, rotavirus, norovirus genogroup I (GI) and GII, and astrovirus. We describe a retrospective study of 200 clinical specimens of which 177 were stool specimens previously tested for the presence of gastrointestinal viruses by electron microscopy (EM) and/or real-time RT-PCR (rRT-PCR). The remaining 23 specimens comprised other human pathogens of viral or bacterial origin. Discordant norovirus GI and GII results were resolved using a commercial kit; discordant adenovirus and rotavirus results were resolved using a home brew multiplex rRT-PCR assay. Diagnostic sensitivities and specificities were calculated before and after discordant analysis. After discordant analysis, estimated diagnostic sensitivities were 100% for adenovirus, rotavirus, and norovirus GI and 97% for norovirus GII. Diagnostic specificities after discordant analysis were 100% for adenovirus, rotavirus, and norovirus GI and 99.4% for norovirus GII. The 95% limits of detection were 31, 10, 2, and 1 genome equivalent per reaction for adenovirus, rotavirus, and norovirus GI and GII, respectively. The results demonstrate that the Seeplex DV assay is sensitive, specific, convenient, and reliable for the simultaneous detection of several viral pathogens directly in specimens from patients with gastroenteritis. Importantly, this novel multiplex PCR assay enabled the identification of viral coinfections in 12 (6.8%) stool specimens.
doi:10.1128/JCM.00599-11
PMCID: PMC3165607  PMID: 21775550
9.  Norovirus Infections in Symptomatic and Asymptomatic Food Handlers in Japan▿  
Journal of Clinical Microbiology  2007;45(12):3996-4005.
Noroviruses are the leading cause of outbreaks of gastroenteritis in the world. At present, norovirus genogroup II, genotype 4 (GII/4), strains are the most prevalent in many countries. In this study we investigated 55 outbreaks and 35 sporadic cases of norovirus-associated gastroenteritis in food handlers in food-catering settings between 10 November 2005 and 9 December 2006 in Japan. Stool specimens were collected from both symptomatic and asymptomatic individuals and were examined for norovirus by real-time reverse transcription-PCR; the results were then confirmed by sequence analysis. Norovirus was detected in 449 of 2,376 (19%) specimens. Four genogroup I (GI) genotypes and 12 GII genotypes, including one new GII genotype, were detected. The GII/4 sequences were predominant, accounting for 19 of 55 (35%) outbreaks and 16 of 35 (46%) sporadic cases. Our results also showed that a large number of asymptomatic food handlers were infected with norovirus GII/4 strains. Norovirus GII had a slightly higher mean viral load (1 log unit higher) than norovirus GI, i.e., 3.81 × 108 versus 2.79 × 107 copies/g of stool. Among norovirus GI strains, GI/4 had the highest mean viral load, whereas among GII strains, GII/4 had the highest mean viral load (2.02 × 108 and 7.96 × 109 copies/g of stool, respectively). Importantly, we found that asymptomatic individuals had mean viral loads similar to those of symptomatic individuals, which may account for the increased number of infections and the predominance of an asymptomatic transmission route.
doi:10.1128/JCM.01516-07
PMCID: PMC2168587  PMID: 17928420
10.  Evaluation and Validation of Real-Time Reverse Transcription-PCR Assay Using the LightCycler System for Detection and Quantitation of Norovirus 
Journal of Clinical Microbiology  2004;42(10):4679-4685.
We developed an assay for the detection and quantitation of norovirus with the LightCycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR) and previously published primers in the capsid and the polymerase gene. One hundred thirty-two stool specimens from the Provincial Laboratory for Public Health (Microbiology), Alberta, Canada, and the Centers for Disease Control and Prevention, Atlanta, Ga., were used to validate the new assay. The samples were collected from patients involved in outbreaks of acute gastroenteritis or children who presented with sporadic gastroenteritis. The real-time LC RT-PCR assay detected norovirus strains from three genogroup I (G-I) clusters (G-I/1, G-I/2, and G-I/3) and 10 genogroup II (G-II) clusters (G-II/1, G-II/2, G-II/3, G-II/4, G-II/6, G-II/7, G-II/10, G-II/12, G-II/15, and G-II/16). There was 100% concordance with the results from 58 stool specimens which tested positive by conventional RT-PCR assays. By dilution analysis, the real-time LC RT-PCR was 10,000 times more sensitive than the conventional RT-PCR. The new assay increased the number of samples in which noroviruses were detected by 19%. The real-time LC RT-PCR had a wide dynamic range, detecting from 5 to 5 × 106 copies of RNA per reaction, resulting in a theoretical lower limit of detection of 25,000 copies of RNA per g of stool. No cross-reactions were found with specimens containing sapovirus, rotavirus, astrovirus, and adenovirus. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of norovirus infection.
doi:10.1128/JCM.42.10.4679-4685.2004
PMCID: PMC522381  PMID: 15472327
11.  Clinical Features and Role of Viral Isolates from Stool Samples of Intussuception in Children 
Purpose
To detect major acute gastroenteritis virus (rotavirus, norovirus, astrovirus, and enteric adenovirus) and non-enteric type of adenovirus (AdV) in the stools of intussusception patients and to investigate the clinical role of detected viruses.
Methods
From March 2012 to February 2013, major acute gastroenteritis virus and non-enteric type of AdV were isolated from stool samples that collected from 44 patients treated for intussusception in Chungnam National University Hospital. Patients were divided according to age and isolated virus.
Results
Virus was detected in 28 (63%) stool specimens. The virus detection rate was significantly lower in patients aged under 12 months (p = 0.04). Twenty-two patients (78.6%) had non-enteric adenovirus, 4 (14.3%) had norovirus, 1 (3.6%) had sapovirus, and 1 (3.6%) had astrovirus. AdV subgroup C (AdV 1, 2, 5, and 6) comprised the majority with 20 cases (90.9%). A monthly increment-and-decrement pattern of intussusception was similar to that of viral detection in the stool samples. Enema reductions were successful in 39 patients and surgical manual reductions were performed in 5 patients. Virus was detected in 24 patients (61.5%) of enema reduction group and 4 patients (80.0%) of surgical manual reduction group. All of the detected viruses were non-enteric adenovirus subgroup C (AdV 1, 5, and 6) in surgical reduction patients.
Conclusions
The virus detection rate was high in the stools of intussusception patients. The pattern of seasonal intussusception occurrence rate was parallel with seasonal these viral detection rate in the stool samples. These findings suggest that viral infection plays an important role in the development of intussusception and further research is warranted.
doi:10.5223/pghn.2013.16.3.162
PMCID: PMC3819690  PMID: 24224149
Intussusception; Non-enteric type of adenovirus; Norovirus
12.  Etiological Role of Viruses in Outbreaks of Acute Gastroenteritis in The Netherlands from 1994 through 2005▿  
Journal of Clinical Microbiology  2007;45(5):1389-1394.
Acute gastroenteritis is one of the most common diseases worldwide. In developed countries, viruses, particularly noroviruses, are recognized as the leading cause. In The Netherlands, the surveillance of gastroenteritis outbreaks with suspected viral etiologies (as determined by Kaplan criteria) was established by the National Institute for Public Health and the Environment in 1994. This paper presents an overview of viral gastroenteritis outbreaks reported from 1994 through 2005. A minimum epidemiological data set consisting of the associated setting(s), the probable transmission mode, the date of the first illness and the date of sampling, the number of persons affected, and the number of hospitalizations was requested for each reported outbreak. Stool samples were tested for the presence of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, and Aichi virus by electron microscopy, enzyme-linked immunosorbent assay, and/or reverse transcription-PCR. A total of 6,707 stool samples from 941 gastroenteritis outbreaks were investigated. Noroviruses were detected as the causative agent in 735 (78.1%) of the outbreaks, and rotaviruses, adenoviruses, and astroviruses were found to be responsible for 46 (4.9%), 9 (1.0%), and 5 (0.5%) outbreaks, respectively. Among the gastroenteritis outbreaks in which a mode of transmission was identified, most outbreaks (38.1%) were associated with person-to-person transmission, and the majority (54.9%) of the outbreaks investigated were reported by residential institutions. Since 2002, the total number of outbreaks reported and the number of unexplained outbreaks have increased. Furthermore, the number of rotavirus-associated outbreaks has increased, especially in nursing homes. Despite thorough testing, 115 (12.2%) outbreaks suspected of having viral etiologies remain unexplained. Increases in numbers of reported outbreaks may indicate undefined changes in the criteria for reporting or the emergence of new pathogens.
doi:10.1128/JCM.02305-06
PMCID: PMC1865895  PMID: 17360839
13.  Influence of Novel Norovirus GII.4 Variants on Gastroenteritis Outbreak Dynamics in Alberta and the Northern Territories, Canada between 2000 and 2008 
PLoS ONE  2010;5(7):e11599.
Background
Norovirus GII.4 is the predominant genotype circulating worldwide over the last decade causing 80% of all norovirus outbreaks with new GII.4 variants reported in parallel with periodic epidemic waves of norovirus outbreaks. The circulating new GII.4 variants and the epidemiology of norovirus outbreaks in Alberta, Canada have not been described. Our hypothesis is that the periodic epidemic norovirus outbreak activity in Alberta was driven by new GII.4 variants evolving by genetic drift.
Methodology/Principal Findings
The Alberta Provincial Public Health Laboratory performed norovirus testing using RT-PCR for suspected norovirus outbreaks in the province and the northern Territories between 2000 and 2008. At least one norovirus strain from 707 out of 1,057 (66.9%) confirmed norovirus outbreaks were successfully sequenced. Phylogenetic analysis was performed using BioNumerics and 617 (91.1%) outbreaks were characterized as caused by GII.4 with 598 assigned as novel variants including: GII.4-1996, GII.4-2002, GII.4-2004, GII.4-2006a, GII.4-2006b, GII.4-2008a and GII.4-2008b. Defining July to June of the following year as the yearly observation period, there was clear biannual pattern of low and high outbreak activity in Alberta. Within this biannual pattern, high outbreak activity followed the emergence of novel GII.4 variants. The two variants that emerged in 2006 had wider geographic distribution and resulted in higher outbreak activity compared to other variants. The outbreak settings were analyzed. Community-based group residence was the most common for both GII.4 variants and non-GII.4 variants. GII.4 variants were more commonly associated with outbreaks in acute care hospitals while outbreaks associated non-GII.4 variants were more commonly seen in school and community social events settings (p<0.01).
Conclusions/Significance
The emergence of new norovirus GII.4 variants resulted in an increased norovirus outbreak activity in the following season in a unique biannual pattern in Alberta over an eight year period. The association between antigenic drift of GII.4 strains and epidemic norovirus outbreak activity could be due to changes in host immunity, viral receptor binding efficiency or virulence factors in the new variants. Early detection of novel GII.4 variants provides vital information that could be used to forecast the norovirus outbreak burden, enhance public health preparedness and allocate appropriate resources for outbreak management.
doi:10.1371/journal.pone.0011599
PMCID: PMC2905434  PMID: 20661286
14.  Personalized genetic testing and norovirus susceptibility 
BACKGROUND:
The availability of direct-to-consumer personalized genetic testing has enabled the public to access and interpret their own genetic information. Various genetic traits can be determined including resistance to norovirus through a nonsense mutation (G428A) in the FUT2 gene. Although this trait is believed to confer resistance to the most dominant norovirus genotype (GII.4), the spectrum of resistance to other norovirus strains is unknown. The present report describes a cluster of symptomatic norovirus GI.6 infection in a family identified to have norovirus resistance through personalized genetic testing.
CASE PRESENTATION:
In January 2013, four members of a family determined by a direct-to-consumer genetic test to be homozygous for the norovirus resistance trait (A/A genotype for single nucleotide polymorphism rs601338) developed symptoms consistent with acute viral gastroenteritis. Stool and vomitus samples were submitted for enteric viral pathogen testing. Samples were positive for norovirus GI.6 in three of the four cases.
CONCLUSIONS:
The present report is the first to describe norovirus GI.6 infection in patients with the G428A nonsense mutation in FUT2; this cluster of cases suggests that the G428A mutation in FUT2 may not confer resistance to norovirus GI.6. Direct-to-consumer genetic testing is empowering members of the public to identify novel associations with their genetic traits. Expert consultation is important for the interpretation of personalized genetic test results, and follow-up laboratory testing can confirm any potentially novel associations.
PMCID: PMC4173944  PMID: 25285128
Direct-to-consumer genetic testing; Norovirus; Norovirus resistance
15.  Assessment of Gastroenteric Viruses Frequency in a Children's Day Care Center in Rio De Janeiro, Brazil: A Fifteen Year Study (1994–2008) 
PLoS ONE  2012;7(3):e33754.
This 15-year study aimed to determine the role of the main viruses responsible for acute infantile gastroenteritis cases in a day care center in the city of Rio de Janeiro, Brazil. From 1994 to 2008, 539 fecal samples were obtained from 23 outbreaks as well as sporadic cases that occurred in this period. The detection of Rotavirus group A (RVA), norovirus (NoV) and astrovirus (AstV) was investigated both by classical and molecular methods of viral detection. RVA was detected by enzymatic immune assay and/or polyacrylamide gel electrophoresis and genotyped by using semi-nested multiplex PCR. NoV and AstV were subsequently tested by real time PCR in all RVA-negative samples and genotyped throughout genome sequencing. Three protocols for molecular characterization of NoV nucleotide sequencing were performed with the partial nucleotide sequencing of genomic regions known as region B (polymerase gen), C and D (capsid gen).Viruses were identified in 47.7% (257/539) of the cases, and the detection rates of RVA, NoV and AstV in16.1% (87/539), 33.4% (151/452), and 6.3% (19/301), respectively. Most gastroenteritis cases were reported in autumn and winter, although NoV presented a broader monthly distribution. Viruses' detection rates were significantly higher among children aged less than 24 months old, although NoV cases were detected in all age groups. RVA genotypes as G1P[8], G9P[8], G2P[4], G3P[8] and G1+G3P[8] and RVA was no longer detected after 2005. NoV characterization revealed genotypes variability circulating in the period as GI.2, GI.3, GI.8 GII.2, GII.3, GII.4, GII.4 variants 2001 and 2006b, GII.6, GII.7, GII.12 and GII.17. AstV genotypes 1, 2, 4 and 5 were also characterized. Those data demonstrate the impact of NoV infection in cases of infantile gastroenteritis, surpassing RVA infection responsible for high morbidity rate in children under five years old.
doi:10.1371/journal.pone.0033754
PMCID: PMC3309004  PMID: 22448271
16.  Epidemiology and clinical features of rotavirus and norovirus infection among children in Ji’nan, China 
Virology Journal  2013;10:302.
Background
Acute gastroenteritis caused by bacteria, virus and parasite is an important cause of childhood morbidity and mortality in developing countries. Rotavirus and norovirus have been recognized as the most common pathogens causing acute gastroenteritis among children. However, there is still no valuable data about infections of rotavirus and norovirus in children in Ji’nan, an eastern city in China. The aims of the present study are to determine the incidence of rotavirus and norovirus associated acute gastroenteritis in Ji’nan among children, to characterize rotavirus and norovirus strains circulating during this period; and to provide useful epidemiological and clinical data.
Methods
Fecal specimens and clinical data were collected from 767 children (502 outpatients and 265 inpatients) under 5 years of age with acute diarrhea at Shandong University Qilu Hospital and Qilu children’s Hospital in Ji’nan, China between February 2011 and January 2012. Virus RNA was extracted, amplified, electrophoresed, sequenced and phylogenetically analyzed to determine the prevalent genotypes. Chi-square and U test were used to compare characteristics of clinical manifestation in each group.
Results
Of the 767 specimens 263 (34.3%) were positive for rotavirus and 80 (10.4%) were positive for norovirus. Among 263 rotavirus positive cases, G3 (40.7%) was the most prevalent serotype, P[8] (46.8%) was the dominant genotype and G3P[8] (31.9%) was the most common combination. All of the norovirus strains belonged to GII genogroup including GII.3, GII.4 and GII.6, of which GII.4 (61.2%) was the predominant genotype. Phylogenetic analysis of the GII.4 sequences showed that 18 GII.4 strains belonged to GII.4 2004–2006 cluster and 31 GII.4 strains were divided into GII.4 2006b cluster. A peak number of rotavirus infections was observed during the cold season from November to next January. Higher rates of norovirus infections were detected from September to November. Most patients with rotavirus and norovirus associated diarrhea experienced vomiting (88.2% and 67.5%, respectively) and fever (79.1% and 46.3%, respectively).
Conclusions
The present study showed that rotavirus and norovirus were still the important causative agents of pediatric diarrhea in Ji’nan during this period.
doi:10.1186/1743-422X-10-302
PMCID: PMC3851746  PMID: 24099150
Rotavirus; Norovirus; Acute gastroenteritis; Epidemiology; Clinical symptom
17.  Rapid Evolution of Pandemic Noroviruses of the GII.4 Lineage 
PLoS Pathogens  2010;6(3):e1000831.
Over the last fifteen years there have been five pandemics of norovirus (NoV) associated gastroenteritis, and the period of stasis between each pandemic has been progressively shortening. NoV is classified into five genogroups, which can be further classified into 25 or more different human NoV genotypes; however, only one, genogroup II genotype 4 (GII.4), is associated with pandemics. Hence, GII.4 viruses have both a higher frequency in the host population and greater epidemiological fitness. The aim of this study was to investigate if the accuracy and rate of replication are contributing to the increased epidemiological fitness of the GII.4 strains. The replication and mutation rates were determined using in vitro RNA dependent RNA polymerase (RdRp) assays, and rates of evolution were determined by bioinformatics. GII.4 strains were compared to the second most reported genotype, recombinant GII.b/GII.3, the rarely detected GII.3 and GII.7 and as a control, hepatitis C virus (HCV). The predominant GII.4 strains had a higher mutation rate and rate of evolution compared to the less frequently detected GII.b, GII.3 and GII.7 strains. Furthermore, the GII.4 lineage had on average a 1.7-fold higher rate of evolution within the capsid sequence and a greater number of non-synonymous changes compared to other NoVs, supporting the theory that it is undergoing antigenic drift at a faster rate. Interestingly, the non-synonymous mutations for all three NoV genotypes were localised to common structural residues in the capsid, indicating that these sites are likely to be under immune selection. This study supports the hypothesis that the ability of the virus to generate genetic diversity is vital for viral fitness.
Author Summary
Since 1995, norovirus has caused five pandemics of acute gastroenteritis. These pandemics spread across the globe within a few months, causing great economic burden on society due to medical and social expenses. Norovirus, like influenza virus, has over 40 genotypes circulating within the population at the same time. However, it is only a single genotype, known as genogroup II genotype 4 (GII.4), that causes mass outbreaks and pandemics. Very little research has been conducted to determine why GII.4 viruses can cause pandemics. Consequently, we compared the evolution properties of several pandemic GII.4 strains to non-pandemic strains and found that the GII.4 viruses were undergoing evolution at a much higher rate than the non-pandemic norovirus strains. This phenomenon is similar to influenza virus, where an increase in antigenic drift has been associated with increased outbreaks. This discovery has important implications in understanding norovirus incidence and also the development of a vaccine and treatment for norovirus.
doi:10.1371/journal.ppat.1000831
PMCID: PMC2847951  PMID: 20360972
18.  RNA Populations in Immunocompromised Patients as Reservoirs for Novel Norovirus Variants 
Journal of Virology  2014;88(24):14184-14196.
ABSTRACT
Noroviruses are the leading cause of acute gastroenteritis outbreaks worldwide. The majority of norovirus outbreaks are caused by genogroup II.4 (GII.4). Novel GII.4 strains emerge every 2 to 4 years and replace older variants as the dominant norovirus. Novel variants emerge through a combination of recombination, genetic drift, and selection driven by population immunity, but the exact mechanism of how or where is not known. We detected two previously unknown novel GII.4 variants, termed GII.4 UNK1 and GII.4 UNK2, and a diverse norovirus population in fecal specimens from immunocompromised individuals with diarrhea after they had undergone bone marrow transplantation. We hypothesized that immunocompromised individuals can serve as reservoirs for novel norovirus variants. To test our hypothesis, metagenomic analysis of viral RNA populations was combined with a full-genome bioinformatic analysis of publicly available GII.4 norovirus sequences from 1974 to 2014 to identify converging sites. Variable sites were proportionally more likely to be within two amino acids (P < 0.05) of positively selected sites. Further analysis using a hypergeometric distribution indicated that polymorphic site distribution was random and its proximity to positively selected sites was dependent on the size of the norovirus genome and the number of positively selected sites.In conclusion, random mutations may have a positive impact on driving norovirus evolution, and immunocompromised individuals could serve as potential reservoirs for novel GII.4 strains.
IMPORTANCE Norovirus is the most common cause of viral gastroenteritis in the United States. Every 2 to 3 years novel norovirus variants emerge and replace dominant strains. The continual emergence of novel noroviruses is believed to be caused by a combination of genetic drift, population immunity, and recombination, but exactly how this emergence occurs remains unknown. In this study, we identified two novel GII.4 variants in immunocompromised bone marrow transplant patients. Using metagenomic and bioinformatic analysis, we showed that most genetic polymorphisms in the novel variants occur near 0 to 2 amino acids of positively selected sites, but the distribution of mutations was random; clustering of polymorphisms with positively selected sites was a result of genome size and number of mutations and positively selected sites. This study shows that immunocompromised patients can harbor infectious novel norovirus variants, and although mutations in viruses are random, they can have a positive effect on viral evolution.
doi:10.1128/JVI.02494-14
PMCID: PMC4249157  PMID: 25275120
19.  Replacing Traditional Diagnostics of Fecal Viral Pathogens by a Comprehensive Panel of Real-Time PCRs▿ 
Journal of Clinical Microbiology  2011;49(5):1926-1931.
Molecular DNA-based diagnostics are increasingly being used for diagnosis of viral infections. For enteric viruses, PCR assays have also been developed. The aims of this study were to compile and evaluate a comprehensive panel of PCR assays for diagnosis of viruses causing diarrheal disease and to evaluate its use in a largely pediatric population in a 750-bed university medical center. The PCR panel was designed to include assays for detection of adenovirus, astrovirus, enterovirus, norovirus, parechovirus, rotavirus, and sapovirus. The results of the PCR panel were evaluated in relation to conventional viral diagnostics consisting of viral culture and/or rotavirus and adenovirus rapid antigen tests on samples that were taken for routine diagnostics. Comparing conventional with PCR-based testing, the number of viruses detected increased dramatically from 25 to 106 when PCR assays were used. This increase was due mainly to detection of previously undetected viruses, i.e., astrovirus, norovirus, and sapovirus. In 24% of the samples, norovirus was detected. Also, the lower detection limit of PCR-based adenovirus, enterovirus, parechovirus, and rotavirus diagnostics further increased the detection rate. By focusing on samples from patients with complaints of gastroenteritis, detection of a causative agent was increased from 49% by conventional tests to 97% by molecular diagnostics. However, many samples containing low viral loads were found in patients with complaints other than intestinal complaints. In conclusion, the proposed comprehensive PCR panel with appropriate cutoff values can be used for sensitive, rapid, and clinically relevant diagnosis of gastrointestinal viruses.
doi:10.1128/JCM.01925-10
PMCID: PMC3122640  PMID: 21430103
20.  Evaluation of a New Immunochromatographic Assay Kit for the Rapid Detection of Norovirus in Fecal Specimens 
Annals of Laboratory Medicine  2011;32(1):79-81.
Rapid and accurate detection of norovirus is essential for the prevention and control of norovirus outbreaks. This study compared the effectiveness of a new immunochromatographic assay kit (SD BIOLINE Norovirus; Standard Diagnostics, Korea) and real-time reverse transcription-PCR (RT-PCR) for detecting norovirus in fecal specimens. Compared with real-time RT-PCR, the new assay had sensitivity, specificity, positive predictive value, and negative predictive value of 76.5% (52/68), 99.7% (342/343), 98.1% (52/53), and 95.5% (342/358), respectively. The sensitivity of the assay was 81.8% (18/22) for GII.3 and 75.7% (28/37) for GII.4. None of the 38 enteric virus-positive specimens (3 for astrovirus, 5 for enteric adenovirus, and 30 for rotavirus) tested positive in the cross-reactivity test performed by using this assay. The new immunochromatographic assay may be a useful screening tool for the rapid detection of norovirus in sporadic and outbreak cases; however, negative results may require confirmatory assays of greater sensitivity.
doi:10.3343/alm.2012.32.1.79
PMCID: PMC3255496  PMID: 22259783
Immunochromatographic assay; Norovirus; Sensitivity; Specificity
21.  Etiology of Sporadic Cases of Pediatric Acute Gastroenteritis in Asturias, Spain, and Genotyping and Characterization of Norovirus Strains Involved 
Journal of Clinical Microbiology  2004;42(6):2668-2674.
From November 2000 to October 2001, a reverse transcription-PCR using primers directed to the norovirus RNA polymerase coding region was included in a viral and bacterial routine screening to diagnose sporadic cases of acute gastroenteritis among children in Asturias, Spain. The role of noroviruses (8.6% of the positively diagnosed cases) as the cause of sporadic pediatric gastroenteritis was evaluated with respect to the detection rates of other gastroenteritis-associated viruses and bacteria. The results indicated that noroviruses were less common than rotaviruses (36.9%), Campylobacter spp. (28.8%), and Salmonella spp. (18.4%) but more frequent than astroviruses (4.3%), adenoviruses (3.8%), and Yersinia spp. (2.2%). Mixed infections involving noroviruses were rarely observed (0.5%). The presence of a norovirus-associated pediatric gastroenteritis peak in summer, as well as the complete absence of norovirus-associated cases in colder months, challenges the view that norovirus infections exclusively have wintertime seasonality. On the other hand, phylogenetic analysis of the amplified fragments showed that the norovirus strains responsible were closely related. A further study using the full-length capsid region showed that these strains could be included into genogroup II, Bristol/Lorsdale cluster, and were closely related to the 1995 and 1996 U.S. subset of strains associated with outbreaks recorded worldwide between 1995 and 1996.
doi:10.1128/JCM.42.6.2668-2674.2004
PMCID: PMC427848  PMID: 15184450
22.  Inactivation of a Foodborne Norovirus Outbreak Strain with Nonthermal Atmospheric Pressure Plasma 
mBio  2015;6(1):e02300-14.
ABSTRACT 
Human norovirus (NoV) is the most frequent cause of epidemic nonbacterial acute gastroenteritis worldwide. We investigated the impact of nonthermal or cold atmospheric pressure plasma (CAPP) on the inactivation of a clinical human outbreak NoV, GII.4. Three different dilutions of a NoV-positive stool sample were prepared and subsequently treated with CAPP for various lengths of time, up to 15 min. NoV viral loads were quantified by quantitative real-time reverse transcription PCR (RT-qPCR). Increased CAPP treatment time led to increased NoV reduction; samples treated for the longest time had the lowest viral load. From the initial starting quantity of 2.36 × 104 genomic equivalents/ml, sample exposure to CAPP reduced this value by 1.23 log10 and 1.69 log10 genomic equivalents/ml after 10 and 15 min, respectively (P < 0.01). CAPP treatment of surfaces carrying a lower viral load reduced NoV by at least 1 log10 after CAPP exposure for 2 min (P < 0.05) and 1 min (P < 0.05), respectively. Our results suggest that NoV can be inactivated by CAPP treatment. The lack of cell culture assays prevents our ability to estimate infectivity. It is possible that some detectable, intact virus particles were rendered noninfectious. We conclude that CAPP treatment of surfaces may be a useful strategy to reduce the risk of NoV transmission in crowded environments.
Importance  Human gastroenteritis is most frequently caused by noroviruses, which are spread person to person and via surfaces, often in facilities with crowds of people. Disinfection of surfaces that come into contact with infected humans is critical for the prevention of cross-contamination and further transmission of the virus. However, effective disinfection cannot be done easily in mass catering environments or health care facilities. We evaluated the efficacy of cold atmospheric pressure plasma, an innovative airborne disinfection method, on surfaces inoculated with norovirus. We used a clinically relevant strain of norovirus from an outbreak in Germany. Cold plasma was able to inactivate the virus on the tested surfaces, suggesting that this method could be used for continuous disinfection of contaminated surfaces. The use of a clinical strain of norovirus strengthens the reliability of our results as it is a strain relevant to outbreaks in humans.
Importance 
Human gastroenteritis is most frequently caused by noroviruses, which are spread person to person and via surfaces, often in facilities with crowds of people. Disinfection of surfaces that come into contact with infected humans is critical for the prevention of cross-contamination and further transmission of the virus. However, effective disinfection cannot be done easily in mass catering environments or health care facilities. We evaluated the efficacy of cold atmospheric pressure plasma, an innovative airborne disinfection method, on surfaces inoculated with norovirus. We used a clinically relevant strain of norovirus from an outbreak in Germany. Cold plasma was able to inactivate the virus on the tested surfaces, suggesting that this method could be used for continuous disinfection of contaminated surfaces. The use of a clinical strain of norovirus strengthens the reliability of our results as it is a strain relevant to outbreaks in humans.
doi:10.1128/mBio.02300-14
PMCID: PMC4311907  PMID: 25587014
23.  Phylodynamic Reconstruction Reveals Norovirus GII.4 Epidemic Expansions and their Molecular Determinants 
PLoS Pathogens  2010;6(5):e1000884.
Noroviruses are the most common cause of viral gastroenteritis. An increase in the number of globally reported norovirus outbreaks was seen the past decade, especially for outbreaks caused by successive genogroup II genotype 4 (GII.4) variants. Whether this observed increase was due to an upswing in the number of infections, or to a surveillance artifact caused by heightened awareness and concomitant improved reporting, remained unclear. Therefore, we set out to study the population structure and changes thereof of GII.4 strains detected through systematic outbreak surveillance since the early 1990s. We collected 1383 partial polymerase and 194 full capsid GII.4 sequences. A Bayesian MCMC coalescent analysis revealed an increase in the number of GII.4 infections during the last decade. The GII.4 strains included in our analyses evolved at a rate of 4.3–9.0×10−3 mutations per site per year, and share a most recent common ancestor in the early 1980s. Determinants of adaptation in the capsid protein were studied using different maximum likelihood approaches to identify sites subject to diversifying or directional selection and sites that co-evolved. While a number of the computationally determined adaptively evolving sites were on the surface of the capsid and possible subject to immune selection, we also detected sites that were subject to constrained or compensatory evolution due to secondary RNA structures, relevant in virus-replication. We highlight codons that may prove useful in identifying emerging novel variants, and, using these, indicate that the novel 2008 variant is more likely to cause a future epidemic than the 2007 variant. While norovirus infections are generally mild and self-limiting, more severe outcomes of infection frequently occur in elderly and immunocompromized people, and no treatment is available. The observed pattern of continually emerging novel variants of GII.4, causing elevated numbers of infections, is therefore a cause for concern.
Author Summary
Noroviruses, known as the viruses that cause the ‘stomach flu’ or as the ‘cruise ship virus’, cause sporadic cases and large outbreaks of gastrointestinal illness in humans. An increase in norovirus outbreaks was reported globally around 2002. Doubts remained as to whether this increase was real, or caused by improved detection-techniques and increased awareness. This study was performed to address this ambiguity, and to determine the possible virological causes for such changes. Using a population genetic approach, we studied sequences of epidemic norovirus strains collected through time and we indeed demonstrated expanding epidemic dynamics. Global epidemics were caused by subsequent variants of norovirus, observed in 2002, 2004 and 2006 and at a smaller scale in 1996, whereas no evidence for such epidemic evolutionary patterns occurring previous to these peaks. Based on the sequences analyzed the strains of the genotype under study here were shown to have circulated at least since the early 1980s, and likely earlier. We showed that not only surface exposed sites on the outside of the virus shell were under selective pressure, involved in avoiding host immune responses, but also codons that are apparently conserved for the purpose of virus replication.
doi:10.1371/journal.ppat.1000884
PMCID: PMC2865530  PMID: 20463813
24.  Sensitive Detection of Multiple Rotavirus Genotypes with a Single Reverse Transcription-Real-Time Quantitative PCR Assay▿  
Journal of Clinical Microbiology  2008;46(8):2547-2554.
Rotaviruses are one of the major causes of diarrhea in infants and children under 5 years old, especially affecting developing countries. In natural disasters, fecal matter and potable waters can mix, allowing low, yet infective, concentrations of rotavirus to be present in water supplies, constituting a risk for the population. Any of the most commonly detected rotavirus genotypes could originate an outbreak. The development of a fast and sensitive method that could detect the broadest possible range of rotavirus genotypes would help with efficient diagnosis and prevention. We have designed a reverse transcription (RT)-real-time quantitative PCR approach targeted to the rotaviral VP2 gene, based on a multiple-sequence alignment of different human rotaviral strains. To overcome the high nucleotide sequence diversity, multiple forward and reverse primers were used, in addition to a degenerate probe. The performance of the assay was tested on isolates representing the most prevalent human genotypes: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8]. The developed method improved classical rotavirus detection by enzyme-linked immunosorbent assay and nested RT-PCR by 5 and at least 1 order of magnitude, respectively. A survey of 159 stool samples indicated that the method can efficiently detect a broad range of rotavirus strains, including different G-P genotype combinations of human, porcine, and bovine origin. No cross-reactivity was observed with other enteric viruses, such as astrovirus, sapovirus, and norovirus.
doi:10.1128/JCM.02428-07
PMCID: PMC2519481  PMID: 18524966
25.  European Multicenter Evaluation of Commercial Enzyme Immunoassays for Detecting Norovirus Antigen in Fecal Samples▿  
Clinical and Vaccine Immunology : CVI  2007;14(10):1349-1355.
A total of 2,254 fecal samples were tested in a European multicenter evaluation of commercially available norovirus antigen detection assays. Two commercial enzyme immunoassays, IDEIA Norovirus (Oxoid; Thermo Fisher Scientific, Ely, United Kingdom) and RIDASCREEN Norovirus (R-Biopharm, Darmstadt, Germany), were included in the evaluation, and their performance was compared with the results of reverse transcription-PCR (RT-PCR). Included in the evaluation were samples collected in sporadic cases of gastroenteritis, samples from outbreaks in which two or more samples were collected, well-characterized samples representing genotypes currently cocirculating within Europe, and samples collected from patients with gastroenteritis caused by a pathogen other than norovirus. The sensitivities and specificities of the IDEIA Norovirus and RIDASCREEN Norovirus assays were 58.93 and 43.81% and 93.91 and 96.37%, respectively, compared with RT-PCR. The sensitivities of both assays for outbreak investigations improved when six or more samples from an outbreak were examined. The IDEIA Norovirus assay exhibited reactivity to a broader range of norovirus genotypes than the RIDASCREEN Norovirus assay, which showed genotype-dependent sensitivities. The results indicate that, if used, these assays should serve as screening assays and the results should be confirmed by RT-PCR.
doi:10.1128/CVI.00214-07
PMCID: PMC2168115  PMID: 17715333

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