Detection Of Norovirus Research Articles

Affinity Peptide-guided Plasmonic Biosensor for Detection of Noroviral Protein and Human Norovirus

In this study, we developed an affinity peptide-guided plasmonic biosensor that is capable of detection for noroviral capsid proteins and human norovirus. Construction of plasmonic biosensor was achieved by immobilization of affinity peptides (named norovirus binding peptides) on the localized surface plasmonic sensor (LSPR) layer for detection of noroviral capsid proteins and human norovirus. The performance of the plasmonic biosensor in detection of their targets was monitored using LSPR techniques. This specific interaction is proportional to the absorbance of LSPR signals. The lowest detection value for noroviral capsid protein was 0.1 ng/mL in the presence of complex tissue culture media (MEM and FBS), and limit of detection (LOD) for human norovirus was found to be 9.9 copies/mL by the 3-σ rule. Interestingly, no dynamic binding response with norovirus binding peptides as affinity reagent was observed against rotavirus, suggesting that norovirus binding peptides have high selectivity for human norovirus. Thus, norovirus binding peptide-guided plasmonic biosensor could be used for the detection of norovirus-related foodborne pathogens.

Molecular epidemiology and host genetics of norovirus and rotavirus infections in Portuguese elderly living in aged care homes.

Norovirus (NoV) and rotavirus group A (RVA) are major agents of acute gastroenteritis worldwide. This study aimed to investigate their epidemiological profile in Portuguese elderly living in long-term care facilities and to assess the host genetic factors mediating infection susceptibility. From November 2013 to June 2015, 636 faecal specimens from 169 elderly, mainly asymptomatic, living in nursing homes in Greater Lisbon and Faro district, Portugal, were collected. NoV and RVA were detected by real-time polymerase chain reaction and NoV genotyped by phylogenetic analysis. NoV detection rate was 7.1% (12 of 169). Three GI.3 and one GII.6 strains were genotyped. RVA detection rate was 3.6% (6 of 169), exclusively in asymptomatic individuals. Host genetic factors associated with infection susceptibility were described on 250 samples by saliva-based enzyme-linked immunosorbent assays. The Lewis-negative phenotype was 8.8% (22 of 250) and the rate of nonsecretors was 16.8% (42 of 250). Association to NoV and RVA infection was performed in the subgroup of individuals (n = 147) who delivered both faecal and saliva samples. The majority of NoV- and RVA-positive individuals (90.9% and 83.3%, respectively) were secretor-positive, with Lewis B phenotype. In a subset of individuals, FUT2 and FUT3 genes were genotyped to assess mutations and validate the secretor and Lewis phenotypes. All sequenced nonsecretors were homozygous for FUT2 nonsense mutation G428A. In this study, low detection rates of NoV and RVA infections were found during two winter seasons. However, even in the absence of any outbreak, the importance of finding these infections in a nonepidemic situation in long-term care facilities may have important implications for infection control.

Comparative evaluation of sensitivity and specificity of immunochromatography kit for the rapid detection of norovirus and rotavirus in Bangladesh

We report a comprehensive analysis of sensitivity and specificity of immunochromatography kit (IC Kit) for the rapid detection of norovirus and rotavirus in Bangladesh. The IC kit (IP-Noro/Rota) provides highest sensitivity (100%) to both viruses compared to the reference method reverse transcription- polymerase chain reaction (RT- PCR) for diagnosis. Furthermore, the test provides a high specificity of 98.9% and 96.1% to diagnose norovirus and rotavirus, respectively, as well as good agreement with the reference method. We also found high prevalence of rotavirus infection (74%) among Bangladeshi pediatric population, of which most of the patients were less than five years old, suffering from severe dehydration, abdominal pain and vomiting. This study is the first to report the ease and rapid detection of norovirus and rotavirus by IC kits in Bangladesh. Therefore, IP-Noro/Rota kit is recommended for the rapid detection of these viruses in routine diagnosis as well as during outbreaks.

Utility of Droplet Digital PCR Assay for Quantitative Detection of Norovirus in Shellfish, from Production to Consumption in Guangxi, China

ObjectiveShellfish are recognized as important vehicles of norovirus-associated gastroenteritis. The present study aimed to monitor norovirus contamination in oysters along the farm-to-fork continuum in Guangxi, a major oyster production area in Southwestern China. MethodsOyster samples were collected monthly from farms, markets, and restaurants, from January to December 2016. Norovirus was detected and quantified by one-step reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR). ResultsA total of 480 oyster samples were collected and tested for norovirus genogroups I and II. Norovirus was detected in 20.7% of samples, with genogroup II predominating. No significant difference was observed in norovirus prevalence among different sampling sites. The norovirus levels varied widely, with a geometric mean of 19,300 copies/g in digestive glands. Both norovirus prevalence and viral loads showed obvious seasonality, with a strong winter bias. ConclusionThis study provides a systematic analysis of norovirus contamination ‘from the farm to the fork’ in Guangxi. RT-ddPCR can be a useful tool for detection and quantification of low amounts of norovirus in the presence of inhibitors found particularly in foodstuffs. This approach will contribute to the development of strategies for controlling and reducing the risk of human illness resulting from shellfish consumption.

Impact of long-term storage of clinical samples collected from 1996 to 2017 on RT-PCR detection of norovirus

Noroviruses are recognized as the leading cause of acute gastroenteritis globally. With improved molecular diagnostics developed over the last two decades, archived clinical specimens are increasingly used to investigate the historic prevalence and molecular epidemiology of human norovirus. Yet the impact of long-term storage on viral integrity in clinical specimens has not been evaluated. In this study, we retested 994 stool specimens collected between 1996 and 2017 that originally tested norovirus-positive to quantify the loss of norovirus RT-PCR positivity with increasing sample storage time at 4 °C. In all, 79% of samples tested positive after retesting, but there was an approximate 3% decline in the positivity ratio and 4% decline in the percentage of samples that could be genotyped with each additional year of sample storage. For samples that were originally quantified by real-time RT-PCR (collected between 2003 and 2017), there was an estimated 1-log loss of viral titer occurring every 7 years of sample storage. Few samples contained PCR inhibitors, assessed using a MS2 extraction control, indicating that loss of RT-PCR signal was due primarily to loss of viral RNA integrity after long-term storage of stool samples at 4 °C. Our results indicate that norovirus positive stool samples can be stored with minimal loss in RT-PCR positivity when stored less than a decade. Longer periods of storage may impair norovirus detection, potentially impacting historic estimates of norovirus prevalence and molecular epidemiology if derived by testing archival clinical specimens.

Ultrasensitive Colorimetric Detection of Murine Norovirus Using NanoZyme Aptasensor.

Human norovirus (NoV) remains the most common cause of viral gastroenteritis and the leading cause of viral foodborne outbreaks globally. NoV is highly pathogenic with an estimated median viral infective dose (ID50) ranging from 18 to 1015 genome copies. For NoV detection, the only reliable and sensitive method available for detection and quantification is reverse transcription quantitative polymerase chain reaction (RTqPCR). NoV detection in food is particularly challenging, requiring matrix specific concentration of the virus and removal of inhibitory compounds to detection assays. Hence, the RTqPCR method poses some challenges for rapid in-field or point-of-care diagnostic applications. We propose a new colorimetric NanoZyme aptasensor strategy for rapid (10 min) and ultrasensitive (calculated Limit of Detection (LoD) of 3 viruses per assay equivalent to 30 viruses/mL of sample and experimentally demonstrated LoD of 20 viruses per assay equivalent to 200 viruses/mL) detection of the infective murine norovirus (MNV), a readily cultivable surrogate for NoV. Our approach combines the enzyme-mimic catalytic activity of gold nanoparticles with high target specificity of an MNV aptamer to create sensor probes that produce a blue color in the presence of this norovirus, such that the color intensity provides the virus concentrations. Overall, our strategy offers the most sensitive detection of norovirus or a norovirus surrogate achieved to date using a biosensor approach, enabling for the first time, the detection of MNV virion corresponding to the lower end of the ID50 for NoV. We further demonstrate the robustness of the norovirus NanoZyme aptasensor by testing its performance in the presence of other nontarget microorganisms, human serum and shellfish homogenate, supporting the potential of detecting norovirus in complex matrices. This new assay format can, therefore, be of significant importance as it allows ultrasensitive norovirus detection rapidly within minutes, while also offering the simplicity of use and need for nonspecialized laboratory infrastructure.

Environmental monitoring of astrovirus and norovirus in the Rosetta branch of the River Nile and the El-Rahawy drain, Egypt

AbstractSewage discharge is considered to be the primary source of viral contamination in aquatic environments. This study was conducted to evaluate the impact of El-Rahawy wastewater on the water quality of the Rosetta branch of the River Nile (Rosetta River Nile) through detection of astrovirus (AstV) and norovirus (NoV) in the water and sediments of both sites. For this purpose, we collected 72 wastewater and 12 sediment samples from El-Rahawy drain, and 12 river water and 12 sediment samples from Rosetta River Nile before and after mixing with El-Rahawy wastewater between April 2017 and March 2018. AstVs and NoVs were identified in wastewater (40.2% versus 25%), El-Rahawy sediment (41.6% versus 20.8%), river water after mixing with wastewater (25% versus 16.6%), river water before mixing with wastewater (8.3% versus 0%), river sediment after mixing with wastewater (16.6% versus 8.3%), and no viruses were found in river sediments before mixing with wastewater. AstV genogroup B and NoV genogroup GI were the most frequently detected genotypes in the analyzed samples, with a peak incidence in the winter months. Increasing detection rates of both viruses in El-Rahawy drain samples and river water taken from the Rosetta branch after receiving El-Rahawy wastewater reflect the impact of this drain on the water quality of this stretch of the River Nile.

Molecular detection and characterisation of sapoviruses and noroviruses in outpatient children with diarrhoea in Northwest Ethiopia.

Childhood morbidity and mortality of diarrhoeal diseases are high, particularly in low-income countries and noroviruses and sapoviruses are among the most frequent causes worldwide. Their epidemiology and diversity remain not well studied in many African countries. To assess the positivity rate and the diversity of sapoviruses and noroviruses in Northwest Ethiopia, during November 2015 and April 2016, a total of 450 faecal samples were collected from outpatient children aged <5 years who presented with diarrhoea. Samples were screened for noroviruses and sapoviruses by real-time RT-PCR. Partial VP1 genes were sequenced, genotyped and phylogenetically analysed. Norovirus and sapovirus stool positivity rate was 13.3% and 10.0%, respectively. Noroviruses included GII.4 (35%), GII.6 (20%), GII.17 (13.3%), GII.10 (10%), GII.2 (6.7%), GII.16 (5%), GII.7 (3.3%), GII.9, GII.13, GII.20 and GI.3 (1.7% each) strains. For sapoviruses, GI.1, GII.1 (20.0% each), GII.6 (13.3%), GI.2 (8.9%), GII.2 (11.1%), GV.1 (8.9%), GIV.1 (6.7%), GI.3 and GII.4 (2.2% each) genotypes were detected. This study demonstrates a high genetic diversity of noroviruses and sapoviruses in Northwest Ethiopia. The positivity rate in stool samples from young children with diarrhoea was high for both caliciviruses. Continued monitoring is recommended to identify trends in genetic diversity and seasonal variations.

Ultrasensitive norovirus nanoimmunosensor based on concurrent axial super-localization of ellipsoidal point spread function by 3D light sheet microscopy

A fluorescence-free ultrasensitive norovirus nanoimmunosensor was investigated based on gradient-fitting super-localization by three-dimensional (3D) dual-view light sheet microscopy (DV-LSM). A highly contagious norovirus was detected by immunoreaction with 100-nm gold nanospots (AuNSs) and 40-nm silver nanoparticles (AgNPs) as imaging probes. Dual-view setup was added to the optical path to simultaneously image AgNP-labelled norovirus and AuNSs in real time. A cylindrical lens and dual-view identified axial distance differences between AuNS and AgNP images as a function of an elliptically distorted point spread function (PSF). Differences between PSFs in each plane at 10 nm slicing intervals were distinguished by gradient-fitting algorithm-based super-localization. This approach showed highly enhanced detection sensitivity and super-localization in the axial direction for norovirus. The axial distance between AuNS and AgNP in norovirus immnosensor was 23 ± 3 nm for 7.8 zM, and the distance increased with increased concentration. The method had an excellent limit of detection (LOD) of 7.8 zM with a wide linear dynamic range of 7.8 zM–240 aM. The LOD was 106 to 2,300,000 times lower than previous methods. Norovirus was detected in lettuce leaf extraction at 14.3 zM with 99.87% recovery. The 3D DV-LSM method with gradient-fitting super-localization was an effective method for ultra-trace norovirus detection at the single-molecule level with highly precise axial resolution.

Detection of Norovirus and Rotavirus Present in Suspended and Dissolved Forms in Drinking Water Sources.

We investigated the present forms of genogroup II norovirus and group A rotavirus in surface water used for drinking water production. River water samples (N = 15) collected at a drinking water treatment plant (DWTP) monthly from June 2017 to August 2018 were fractioned by filtration through 10- and 0.45-μm-pore-size membranes, and viruses present in suspended and dissolved forms were quantitatively detected. Norovirus GII was present in > 10-μm- and 0.45-10-μm-suspended and dissolved forms with detection rates of 33%, 60%, and 87%, respectively. Rotavirus A was detected more frequently than norovirus GII in each form (> 10 μm suspended, 73%; 0.45-10 μm suspended, 93%; dissolved, 100%). We also analyzed surface water samples from 21 DWTPs all over Japan in non-epidemic and epidemic seasons of gastroenteritis. Norovirus GII was detected in 48% and 81% of samples with the concentrations of up to 4.1 and 5.3 log10 copies/L in dissolved form in non-epidemic and epidemic seasons, respectively, and GII.4 Sydney 2012 was predominant genotype followed by GII.2. Rotavirus A was detected in 95% and 86% of samples with the maximum concentrations of 5.5 and 6.3 log10 copies/L in dissolved form in respective seasons. Concentration of norovirus GII was similar in 0.45-10-μm suspended and dissolved forms, while there was a significant difference for rotavirus A (P < 0.01, pared t test), indicating that rotavirus A was less associated with suspended solids in the surface water samples compared to norovirus GII. Our observations provide important implications for understanding of viral behavior in environmental waters.

A semi-nested RT-PCR assay for detection of norovirus in rat fecal samples.

Norovirus is a highly prevalent pathogen that can infect a wide range of host species. Thus far, there have only been two reports of norovirus infection in rats. Diagnostic assays for the detection of norovirus are well established, but a specific molecular assay for the diagnosis of norovirus infection in laboratory rats has not yet been reported. In this study, we describe the development of a sensitive, semi-nested RT-PCR assay for detection of norovirus in fecal samples from Rattus norvegicus, reared in animal facilities under different sanitary barrier conditions. Additionally, we describe the first report of the presence of norovirus in rat colonies from Brazilian animal facilities.

Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus

BackgroundMurine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV.ResultsThe detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1 × 102 copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA.ConclusionsA broadly reactive RT-RPA assay was successfully established for MNV detection.

Genetic Diversity of Norovirus Infections, Coinfections, and Undernutrition in Children From Brazilian Semiarid Region.

Norovirus (NoV) infections are known to have high-morbidity and mortality rates and are a major health problem globally. The impact of NoV on child development is, however, poorly understood. We evaluated the distribution of NoV genotypes in children from a low-income Brazilian semiarid region, in relation with their clinical symptoms, nutritional status, and co-pathogens. The test population included children aged 2 to 36 months from 6 cities of the Brazilian semiarid region. Fecal samples were collected from each child, along with the information regarding their socioeconomic/clinical conditions using a standardized questionnaire. Detection and quantification of NoV were performed by reverse-transcription quantitative polymerase chain reaction, followed by molecular and phylogenetic analyses. The NoV detection rate was 45.2%. Presence of NoV was associated with lower z scores for weight-for-age (P = 0.03), weight-for-height (P = 0.03), and body mass index-for-age (P = 0.03). NoV infection was associated with more frequent respiratory illnesses (P < 0.01). GII.P7 (polymerase) and GII.3 (capsid) were the most frequent NoV genotypes. Analysis of the open reading frame (ORF)1-2 junction identified recombinant NoV strains in 80% of the sequenced samples. Enteroaggregative Escherichia coli coinfection was the major predictor for diarrhea in NoV-positive samples (P < 0.02). Moreover, Shigella spp was also associated with NoV-positive diagnosis (P = 0.02). This study highlights the genetic variability of NoV and, associated co-infections and undernutrition in infants from low-income Brazilian semiarid region.

Influence of environmental and seasonal factors on microbial contamination levels in clam production areas in southern Tunisia: Escherichia coli, Salmonella spp., hepatitis A virus and norovirus.

To study the influence of environmental and biological factors on levels of contamination by Escherichia coli, Salmonella spp., hepatitis A virus (HAV) and norovirus in clam production areas, an epidemiological study was conducted on 791 samples of live clams (Ruditapes decussatus), 539 of which were sent for bacteriological analysis and 252 for detection of norovirus and HAV. These samples were collected in different production areas in the Sfax region of southern Tunisia over four consecutive years, from March 2013 to December 2016. The prevalence of positive samples was 36% for E. coli, 11% for Salmonella spp., 19% for norovirus and 3% for HAV. There was a significant correlation between contamination by E. coli and by Salmonella spp., as well as between contamination by noroviruses and by HAV and between contamination by noroviruses and by Salmonella spp. Temperature, the presence of migratory birds and tourism are the main factors affecting microbial contamination levels in bivalve molluscs.

Detection of norovirus in children with acute gastroenteritis in a hospital of Italy

Detection of norovirus in children with acute gastroenteritis in a hospital of Italy

Enhanced colorimetric detection of norovirus using in-situ growth of Ag shell on Au NPs

Norovirus (NoV) is a leading cause of acute gastroenteritis. The low infectious dose and environmental stability of NoV facilitate its effective transmission through a variety of modes such as food, water and person-to-person. The available enzyme-linked immunosorbent assay (ELISA) for NoV detection has low sensitivity due to the low catalytic activity of the peroxidase used, and thus, a reliable ultrasensitive bioassay is needed. In this study, we apply the enhanced peroxidase-like activity of silver ion-incorporated gold nanoparticles (Au/Ag NPs) in a colorimetric bioassay for NoV detection. NoV was captured by anti-NoV genogroup II antibodies, which were immobilized on the surface of a 96-well microtiter plate and formed a sandwich structure among anti-NoV Ab, NoV and Ab-Au NP. Then, Ag ion-containing hydroquinone was added to form Au/Ag core/shell NPs. When H2O2/3,3′,5,5′-tetramethylbenzidine (TMB) solution was added to the wells, Ag ions were liberated from the surface of Au/Ag NPs and enhanced the oxidation of TMB. These reactions enhanced the oxidation of TMB and developed an intense blue color. The Au/Ag NPs were shown to exhibit higher affinity and catalytic efficiency for H2O2 and higher catalytic velocity based on the kcat of up to 7-fold compared with Au NPs. The bioassay was then optimized to detect clinically isolated NoV using NoV-like particles (NoV-LPs). NoV-LPs were detected with a limit of detection of 10.8 pg/mL, corresponding to 1000- and 100-fold higher sensitivity compared to the gold-immunoassay and horseradish peroxidase-based ELISA, respectively. Clinically isolated NoV GII.4 and NoV GII.3 were detected in the range of 102–106 copies of viral RNA/mL fecal solution with a detection limit of 13.2 copies/mL fecal solution for NoV GII.4, equivalent to 132 copies of viral RNA/g feces and indicating significantly higher sensitivity compared to commercial immunoassay kits. This bioassay represents a workable detection assay for low concentrations of NoV that is applicable for early-stage diagnosis for public hygiene.

Development of a homogeneous time-resolved fluorescence assay for detection of viral double-stranded RNA

The group of positive-sense single-stranded RNA ((+) ssRNA) viruses includes many important human pathogens. However, specific antiviral agents are not currently available for many RNA viruses. For screening of antiviral agents, methods that are simple, rapid, and compatible with high-throughput are required. Here, we describe a novel method for measurement of double-stranded RNA using a homogeneous time-resolved fluorescence assay. This method allowed detection of human rhinovirus (HRV), enterovirus, coxsackievirus, and murine norovirus. Furthermore, this method detected antiviral activity of a HRV 3C protease inhibitor. The assay may be useful for discovery of antiviral agents against (+) ssRNA viruses.

Single-step detection of norovirus tuning localized surface plasmon resonance-induced optical signal between gold nanoparticles and quantum dots

A new method of label free sensing approach with superior selectivity and sensitivity towards virlabel-freeon is presented here, employing the localized surface plasmon resonance (LSPR) behavior of gold nanoparticles (AuNPs) and fluorescent CdSeTeS quantum dots (QDs). Inorganic quaternary alloyed CdSeTeS QDs were capped with L-cysteine via a ligand exchange reaction. Alternatively, citrate stabilized AuNPs were functionalized with 11-mercaptoundecanoic acid to generate carboxylic group on the gold surface. The carboxylic group on the AuNPs was subjected to bind covalently with the amine group of L-cysteine capped CdSeTeS QDs to form CdSeTeS QDs/AuNPs nanocomposites. The fluorescence of CdSeTeS QDs/AuNPs nanocomposite shows quenched spectrum of CdSeTeS QDs at 640 nm due to the close interaction with AuNPs. However, after successive addition of norovirus-like particles (NoV-LPs), steric hindrance-induced LSPR signal from the adjacent AuNPs triggered the fluorescence enhancement of QDs in proportion to the concentration of the target NoV-LPs. A linear range of 10−14 to 10−9 g mL−1 NoV-LPs with a detection limit of 12.1 × 10−15 g mL−1 was obtained. This method was further applied on clinically isolated norovirus detection, in the range of 102–105 copies mL−1 with a detection limit of 95.0 copies mL−1, which is 100-fold higher than commercial ELISA kit. The superiority of the proposed sensor over other conventional sensors is found in its ultrasensitive detectability at low virus concentration even in clinically isolated samples. This proposed detection method can pave an avenue for the development of high performance and robust sensing probes for detection of virus in biomedical applications.

Development of a rapid and sensitive electrochemical biosensor for detection of human norovirus via novel specific binding peptides

Human noroviruses cause acute foodborne gastroenteritis outbreaks worldwide. In this study, a highly sensitive and selective electrochemical biosensor was fabricated for the detection of human norovirus using novel peptides as recognition elements. The electrochemical biosensor was fabricated by assembling of eight novel peptides separately on the gold electrode and investigated their efficiencies for sensing human noroviruses. Among eight peptides, NoroBP peptide coated onto the gold electrode exhibited a high binding affinity towards human noroviruses, resulting a progressive decrease in current signals with increasing concentration of human norovirus (0–105 copies/mL). As a result, NoroBP-nonFoul(FlexL)2-coated gold electrode acts an efficient electrochemical biosensor for highly selective detection of human norovirus with a detection limit of 1.7 copies/mL, which is 3-fold lower than the reported methods. The developed electrochemical biosensor was successfully applied to detect human norovirus prepared by standard procedure from oyster, which suggests that the developed biosensor can be used as a very sensitive and selective point-of-care bioanalytical platform for the detection of human norovirus in various food samples.

Phylogenetic characterization of norovirus strains detected from sporadic gastroenteritis in Seoul during 2014\u20132016

BackgroundPhylogenetic analysis of norovirus (NoV) is efficient for tracking NoV transmission. To determine the widespread NoV strains in Seoul, we conducted an extensive phylogenetic characterization of NoV-positives from 1659 diarrheal specimens collected in 2014–2016 for the Seoul NoV-surveillance.ResultsWhen the large numbers of NoV partial VP1 genome sequences were analyzed in acute gastroenteritis patients along with the phylogenetic characterization, we could identify molecular epidemiologic patterns based on the genetic characteristics of sporadic NoV strains circulating in Seoul, which could provide a detailed description of the genome-wide and community-wide NoV evolution in each genotype. The average NoV detection rate in our study period was 16.34% that was increased by 7.44% from 13.17% in 2014 to 20.61% in 2016. Prevalence of NoV GI and GII was 4.43% and 93.36%, respectively, and the GII.4, GII.17, and GII.3 were found to be the major type among 17 genotypes of NoV. The most prevalent one was GII.4 (50.92%) that was followed by GII.17 (18.08%) and GII.3 (9.96%). According to an extensive phylogenetic analysis based on partial VP1 sequences of 1008 NoV (276 sporadic, 518 outbreak and 214 reference), pandemic strains of GII.17, GII.4 and GII.3 have emerged in succession during the 2014-2016 Seoul NoV-surveillance. GII.17 emerged as GII.17|Kawasaki323 in 2014, and became the predominant genotype in 2015 with GII.17|2014_Kawasaki lineages (CUHK-NS-616/Kawasaki308). The formerly predominant GII.4 remained high-level with GII.4|2012_Sydney in 2014 and internally replaced to GII.4|2016_Kawasaki194 lineage (NOR-2565/NOR-2558/OH16002) that caused the sporadic NoV explosion since December 2015. Sporadically prevalent GII.3|Hu/Aichio334-13/2013 failed to develop any outbreaks, whereas sporadic GII.3|Hu/3-28/2015/HNZZ/CHN caused heavy outbreaks in Seoul without preparation time since November 2016.ConclusionsThis is the first extensive phylogenetic study revealing the important events of NoV strains circulating in Seoul. Particularly, our study period from 2014 to 2016 was very dynamic with the emergences of the three main NoV strains (GII.17|2014_Kawasaki, GII.4|2016_Kawasaki194 and GII.3|Hu/3-28/2015/HNZZ/CHN) every year. We are sure that it is hard to detect above findings by simple conventional analysis. Our present study reports a future paradigm of the NoV molecular epidemiology, which might be highly valuable to track new strains and predict oncoming outbreaks.