Norovirus Genotypes Research Articles

Complete Genome Sequence of a Human Norovirus Strain from the United States Classified as Genotype GII.P6_GII.6.

ABSTRACTWe report here the complete genome sequence of a GII.6 norovirus strain detected in a clinical fecal specimen from the United States. The virus genome has a length of 7,547 bp and a GC content of 50.1%. Complete norovirus genotyping of the full-genome sequence identified the virus genotype as GII.P6_GII.6.

Genetic and epidemiological analysis of norovirus from children with gastroenteritis in Botswana, 2013\u20132015

BackgroundNorovirus is a leading cause of viral gastroenteritis worldwide with a peak of disease seen in children. The epidemiological analysis regarding the virus strains in Africa is limited. The first report of norovirus in Botswana was in 2010 and currently, the prevalence and circulating genotypes of norovirus are unknown, as the country has no systems to report the norovirus cases. This study investigated the prevalence, patterns and molecular characteristics of norovirus infections among children ≤5 years of age admitted with acute gastroenteritis at four hospitals in Botswana.MethodsA total of 484 faecal samples were collected from children who were admitted with acute gastroenteritis during the rotavirus vaccine impact survey between July 2013 and December 2015. Norovirus was detected using real-time RT-PCR. Positive samples were genotyped using conventional RT-PCR followed by partial sequencing of the capsid and RdRp genes. Norovirus strains were determined by nucleotide sequence analysis using the online Norovirus Genotyping Tool Version 1.0, and confirmed using maximum likelihood tree construction as implemented in MEGA 6.0.ResultsThe prevalence of norovirus was 9.3% (95% CI 6.7–11.9). The genotype diversity was dominated by the GII.4 strain at 69.7%. This was followed by GII.2, GII.12 each at 9.1%, GI.9 at 6.6% and GII.6, GII.10 each at 3.0%. The most common combined RdRp/Capsid genotype was the GII.Pe/GII.4 Sydney 2012. Norovirus was detected during most part of the year; however, there was a preponderance of cases in the wet season (December to March).ConclusionThe study showed a possible decline of norovirus infections in the last 10 years since the first report. An upward trend seen between 2013 and 2015 may be attributable to the success of rotavirus vaccine introductions in 2012. Knowledge of circulating genotypes, seasonal trends and overall prevalence is critical for prevention programming and possible future vaccine design implications.

OI-165 - Norovirus Genotype II Outbreak in a Homeless Veterans' Residential Facility

OI-165 - Norovirus Genotype II Outbreak in a Homeless Veterans' Residential Facility

Research on the contamination levels of norovirus in food facilities using groundwater in South Korea, 2015–2016

Norovirus (NoV) is a major pathogenic virus that is responsible for foodborne and waterborne gastroenteritis outbreaks. Groundwater is an important source of drinking water and is used in agriculture and food manufacturing processes. This study investigated norovirus contamination of groundwater treatment systems at 1360 sites in seven metropolitan areas and nine provinces in 2015–2016. Temperature, pH, residual chlorine, and turbidity content were assessed to analyze the water quality. In 2015, six sites were positive for the presence of NoV (0.88%) and in 2016, two sites were positive (0.29%); in total, NoV was detected in 8 of the 1360 sample sites (0.59%) investigated. Identified genotypes of NoV in groundwater included GI.5, 9 and GII.4, 6, 13, 17, and 21. GII.17 was the most prevalent genotype in treated groundwater used in the food industry. This dominance of GII.17 was corroborated by NoV infection outbreak cases and the results of a survey of coastal waters in South Korea in 2014–2015. Although a low detection rate was observed in this study, NoV is a pathogen that can spread extensively. Therefore, it is necessary to periodically monitor levels of norovirus which is responsible for food poisoning in groundwater. This is a first report to reveal epidemic genotype shift of norovirus in groundwater treatment system of food facilities in South Korea. Our results may contribute to the enhancement of public health and sanitary conditions by providing molecular epidemiological information on groundwater NoV.

Waterborne Norovirus outbreak at a seaside resort likely originating from municipal water distribution system failure.

In May 2016 a Norovirus (NoV) gastroenteritis outbreak involved a high school class visiting a seaside resort near Taormina (Mascali, Sicily). Twenty-four students and a teacher were affected and 17 of them showed symptoms on the second day of the journey, while the others got ill within the following 2 days. Symptoms included vomiting, diarrhoea and fever, and 12 students required hospitalisation. Stool samples tested positive for NoV genome by Real-Time polymerase chain reaction assay in all 25 symptomatic subjects. The GII.P2/GII.2 NoV genotype was linked to the outbreak by ORF1/ORF2 sequence analysis. The epidemiological features of the outbreak were consistent with food/waterborne followed by person-to-person and/or vomit transmission. Food consumed at a shared lunch on the first day of the trip was associated to illness and drinking un-bottled tap water was also considered as a risk factor. The analysis of water samples revealed the presence of bacterial indicators of faecal contamination in the water used in the resort as well as in other areas of the municipal water network, linking the NoV gastroenteritis outbreak to tap water pollution from sewage leakage. From a single water sample, an amplicon whose sequence corresponded to the capsid genotype recovered from patients could be obtained.

Prevalence and genetic diversity of norovirus genogroup II in children less than 5 years of age with acute gastroenteritis in Tehran, Iran

Viral gastroenteritis is a major public health problem worldwide. In Iran, very limited studies have been performed with regard to the epidemiology of noroviruses. This study aimed to evaluate the prevalence and molecular epidemiology of GII noroviruses in hospitalized children less than 5years of age with acute gastroenteritis (AGE). A total of 210 stool specimens were collected from Ali Asghar Children's Hospital and Bahrami Children's Hospital in Tehran, from June 2015 to June 2016. The samples were screened by real-time RT-PCR for genogroup II (GII). Positive samples were genotyped by semi-nested PCR followed by Sanger sequencing and phylogenetic analysis. Norovirus was identified in 36 (17.1%) of 210 specimens. Based on genetic analysis of RdRp and capsid sequences, the strains were clustered into eight RdRp-capsid genotypes: GII.P4-GII.4 Sydney_2012 (41.7%), GII.Pe-GII.4 Sydney_2012 (30.6%), GII.P21-GII.3 (13.9%), GII.P16-GII.4 Sydney_2012 (2.8%), GII.P16-GII.12 (2.8%), GII.P2-GII.4 Sydney_2012 (2.8%), GII.P7-GII.7 (2.8%) and GII.P2-GII.2 (2.8%). We determined several different co-circulating norovirus genotypes in children < 5years of age with AGE in our hospital in Tehran, Iran. Continued molecular surveillance of noroviruses, including typing of both RdRp and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.

Molecular epidemiology and temporal evolution of norovirus associated with acute gastroenteritis in Amazonas state, Brazil

BackgroundGlobally, Norovirus (NoV) is considered the most common cause of diarrheal episodes across all age groups. Despite its wide genetic diversity, the GII.4 strain is the most predominant and has been associated with epidemics worldwide. In this study, we characterized sporadic cases of diarrhea from NoV-positive children, during a five-year period (2010–2014).MethodsA total of 250 NoV-positive samples identified by an enzyme immunoassay (EIA) were subjected to RT-PCR and partial nucleotide sequencing for polymerase and capsid genes. Phylogenetic analysis was performed to identify NoV genotypes using the binary classification. In addition, sequences from the P2 subdomain (capsid) gene of GII-4 variants were characterized by evolutionary analyses, using the MCMC method implemented in the BEAST package. A 3D structure was built using protein modeling.ResultsPhylogenetic analysis demonstrated a predominance of genotype GII.4 (52.4% - 99/189), variants New Orleans_2009 and Sydney_2012 followed by GII.P7/GII.6 with 6.3% (12/189). Amino acid analyses of the GII.4 strains showed several important amino acid changes. A higher evolutionary rate was found, 7.7 × 10− 3 in the Sydney variant and 6.3 × 10− 3 in the New Orleans. Based in evolutionary analysis the time to the most recent common ancestor (TMRCA) has been calculated as estimates of the population divergence time. Thus, TMRCA for New Orleans and Sydney variant were 2008.7 and 2010.7, respectively. Also, we observed a lineage of transition between New Orleans and Sydney.ConclusionThis study describes the different strains of norovirus isolated from Amazonas state in Brazil during a five-year period. Considering that NoV are capable of changing their antigenic epitopes rapidly, a continuous surveillance is important to monitor the occurrence and changes of the NoV in the community through epidemiological studies. These results contribute to the understanding of NoV molecular epidemiology and its evolutionary dynamics in Amazonas state, Brazil.

Synthesis and Evaluation of Biotinylated Bivalent HistoBlood Group Antigens for Capturing Human Noroviruses

Human noroviruses are the leading cause of epidemic and sporadic gastroenteritis in the US,1 and cause approximately 18% of all diarrheal infections globally.2 Noroviruses are a genetically and antigenically diverse group of nonenveloped single-stranded RNA viruses with a diameter of 27–40 nm and a genome of 7.5–7.7 kilobases in length. At least 7 different genogroups have been recognized of which viruses belonging to GI and GII cause the vast majority of infections in humans.3 The most commonly used diagnostic method is RT-qPCR assay. However, it is expensive and requires well-trained personnel. Marionneau and coworkers reported that recombinant Norwalk virus-like particles (VLPs) were able to recognize two types of HBGA, H type 1 and H type 3 antigens in 2002.4 Since then, the HBGA binding profiles of a wide variety of different norovirus genotypes have been studied that showed multiple different binding patterns.5 Herein, we report the synthesis of biotinylated bivalent H-type glycans using a modular synthetic strategy. The biotin provides a good handle for facile conjugation to high capacity streptavidin coated magnetic beads and the captured norovirus can be subjected to PCR analysis. These glycoconjugates were used to recover human norovirus from fecal samples using a magnetic bead-based assay, which exhibited similar or better capturing ability compared to commercial biotinylated glycopolymers.6 Support or Funding Information NIAID (R33-AI100246) Substrates and Workflow of the Assay This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elucidation of a 2.3 Å Resolution Norovirus GII.4 Protease Structure by X‐Ray Crystallography

According to a current report from the Centers for Disease Control (CDC), it is estimated that noroviruses are responsible for 685 million cases of acute gastroenteritis each year with an estimated $60 billion medically and socioeconomically. This overwhelming global burden drives the need for identification and characterization of potential norovirus drug targets to exploit for design and development. Specifically, the GII.4 norovirus genotype accounts for more than 50% of all outbreaks worldwide. Up until now, the only GII.4 model was based on an alignment of the GI.1 and GII.4 protease sequences which resulted in a sequence identity of only 67%. This high genetic diversity among human noroviruses enhances the need for an improved model of the GII.4 protease. We report the crystallization and structure elucidation of a 2.3Å resolution structure of the GII.4 norovirus protease (GII.4 NoV PR) through X‐ray crystallography. Additionally, extensive investigation and characterization of the GII.4 norovirus protease structure and mechanism were carried out through computational studies and enzyme kinetics. With the elucidation of the GII.4 norovirus protease structure, we hope to significantly improve the structure‐based design of both potent therapeutic inhibitors and potential vaccine strategies for future studies.Support or Funding InformationWayne State University School of MedicineThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Emerging recombinant noroviruses identified by clinical and waste water screening

Norovirus is estimated to cause 677 million annual cases of gastroenteritis worldwide, resulting in 210,000 deaths. As viral gastroenteritis is generally self-limiting, clinical samples for epidemiological studies only partially represent circulating noroviruses in the population and is biased towards severe symptomatic cases. As infected individuals from both symptomatic and asymptomatic cases shed viruses into the sewerage system at a high concentration, waste water samples are useful for the molecular epidemiological analysis of norovirus genotypes at a population level. Using Illumina MiSeq and Sanger sequencing, we surveyed circulating norovirus within Australia and New Zealand, from July 2014 to December 2016. Importantly, norovirus genomic diversity during 2016 was compared between clinical and waste water samples to identify potential pandemic variants, novel recombinant viruses and the timing of their emergence. Although the GII.4 Sydney 2012 variant was prominent in 2014 and 2015, its prevalence significantly decreased in both clinical and waste water samples over 2016. This was concomitant with the emergence of multiple norovirus strains, including twoGII.4 Sydney 2012 recombinant viruses, GII.P4 New Orleans 2009/GII.4 Sydney 2012 and GII.P16/GII.4 Sydney 2012, along with three other emerging strains GII.17, GII.P12/GII.3 and GII.P16/GII.2. This is unusual, as a single GII.4 pandemic variant is generally responsible for 65–80% of all human norovirus infections at any one time and predominates until it is replaced by a new pandemic variant. In sumary, this study demonstrates the combined use of clinical and wastewater samples provides a more complete picture of norovirus circulating within the population.

GII.4 norovirus recombinant causes gastroenteritis epidemic in Eastern Australia, winter 2017.

In Victoria, Australia, 160 gastroenteritis outbreaks were norovirus positive for the period January-September 2017. A distinctive peak in norovirus outbreaks was seen May-August, with 118 positive outbreaks occurring in the peak period. The peak was primarily due to the emergence of a GII.P4_NewOrleans_2009/GII.4_Sydney_2012 recombinant that had genetically changed sufficiently to escape herd immunity. This recombinant was also identified elsewhere in Australia, with highly similar sequences identified in Queensland during the same time period. The recombinant GII.P4_NewOrleans_2009/GII.4_Sydney_2012 has not been reported to cause norovirus epidemics outside Australia, suggesting regional factors play a role in determining norovirus genotype incidence.

Detection of noroviruses in free-ranging jaguars (Panthera onca) in the Pantanal, Mato Grosso, Brazil.

Nine free-ranging jaguars (Panthera onca) were captured, and rectal swabs were collected in the Pantanal of Cáceres, Mato Grosso, Brazil. Reverse transcription polymerase chain reaction specific for noroviruses was performed. Six jaguars (66.6%) tested positive for norovirus genotype GII.11.

The dynamics of norovirus genotypes and genetic analysis of a novel recombinant GII.P12-GII.3 among infants and children in Bangkok, Thailand between 2014 and 2016

Norovirus (NoV) is the leading cause of viral acute gastroenteritis among all age groups in the world. We performed a molecular epidemiological study of the NoVs prevalent in Bangkok between November 2014 and July 2016 to investigate the emergence of new NoV variants in Thailand. A total of 332 stool specimens were collected from hospitalized pediatric patients with acute gastroenteritis in Bangkok, Thailand. NoVs were detected by real-time PCR. The genome of the N-terminal/shell domain was amplified, the nucleotide sequence was determined, and phylogenetic analyses were performed. GII NoV was detected in 58 (17.5%) of the 332 specimens. GII.17, a genotype strain prevalent from 2014 to mid-2015, was hardly detected and replaced by the GII.3 genotype strain. Entire genome sequencing followed by phylogenetic analysis of the GII.3 genotype strains indicated that they are new recombinant viruses, because the genome encoding ORF1 is derived from a GII.12 genotype strain, whereas that encoding ORF2-3 is from a GII.3 genotype strain. The putative recombination breakpoints with the highest statistical significance were located around the border of 3Dpol and ORF2. The change in the prevalent strain of NoV seems to be linked to the emergence of new forms of recombinant viruses. These findings suggested that the swapping of the structural and non-structural proteins of NoV is a common mechanism by which new epidemic variants are generated in nature.

Conformational Occlusion of Blockade Antibody Epitopes, a Novel Mechanism of GII.4 Human Norovirus Immune Evasion.

Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach for identifying conserved GII.4 norovirus epitopes. Utilizing a unique set of virus-like particles (VLPs) representing the in vivo-evolved sequence diversity within an immunocompromised person, we identify key residues within epitope F, a conserved GII.4 blockade antibody epitope. The residues critical for antibody binding are proximal to evolving blockade epitope E. Like epitope F, antibody blockade of epitope E was temperature sensitive, indicating that particle conformation regulates antibody access not only to the conserved GII.4 blockade epitope F but also to the evolving epitope E. These data highlight novel GII.4 mechanisms to protect blockade antibody epitopes, map essential residues of a GII.4 conserved epitope, and expand our understanding of how viral particle dynamics may drive antigenicity and antibody-mediated protection by effectively shielding blockade epitopes. Our data support the notion that GII.4 particle breathing may well represent a major mechanism of humoral immune evasion supporting cyclic pandemic virus persistence and spread in human populations. IMPORTANCE In this study, we use norovirus virus-like particles to identify key residues of a conserved GII.4 blockade antibody epitope. Further, we identify an additional GII.4 blockade antibody epitope to be occluded, with antibody access governed by temperature and particle dynamics. These findings provide additional support for particle conformation-based presentation of binding residues mediated by a particle "breathing core." Together, these data suggest that limiting antibody access to blockade antibody epitopes may be a frequent mechanism of immune evasion for GII.4 human noroviruses. Mapping blockade antibody epitopes, the interaction between adjacent epitopes on the particle, and the breathing core that mediates antibody access to epitopes provides greater mechanistic understanding of epitope camouflage strategies utilized by human viral pathogens to evade immunity.

Evolutionary dynamics of GII.17 norovirus.

BackgroundDuring the winter of 2014–2015, a rarely reported norovirus (NoV) genotype GII.17 was found to have increased its frequency in norovirus outbreaks in East Asia, surpassing the GII.4 NoV infections. GII.17 genotype has been detected for over three decades in the world. The aim of this study is to examine the evolutionary dynamics of GII.17 over the last four decades.MethodsNoV GII.17 sequences with complete or nearly complete VP1 were downloaded from GenBank and the phylogenetic analyses were then conducted.ResultsThe maximum likelihood analysis showed that GII.17 genotype could be divided into four different clades (Clades A–D). The strains detected after 2012, which could be the cause of the outbreaks, were separated into Clades C–D with their mean amino acid distance being 4.5%. Bayesian Markov chain Monte Carlo analyses indicated that the rate of nucleotide substitution per sites was 1.68 × 10−3 nucleotide substitutions/site/year and the time of the most recent common ancestor was 1840. The P2 subdomain of GII.17 was highly variable with 44% (56/128) amino acids variations including two insertions at positions 295–296 and one deletion at position 385 (Clades C and D) and one insertion at position 375 (Clade D). Variations existed in Epitopes A, B and D corresponding to GII.4 and human histo-blood group antigens binding site I in P2 subdomain.ConclusionThe novel GII.17 strains that caused outbreaks in 2013–2015 may have two new variants. The evolvement of HBGAs binding site and epitopes in P2 subdomain might contribute to the novel GII.17 strains predominance in some regions.

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Norovirus is the leading cause of gastroenteritis worldwide. Little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses.

Risk factors for hospital norovirus outbreaks: impact of vomiting, genotype, and multi-occupancy rooms

Norovirus is frequently introduced to the hospital and is a frequent cause of hospital outbreaks. Recognition of the factors that facilitate or impede norovirus transmission is an important step to effectively prevent hospital outbreaks. To investigate risk factors for norovirus outbreaks in hospital settings. Clinical data, ward setting, and norovirus genotype were collected from all 65 norovirus-positive index cases in outbreaks and all 186 sporadic norovirus cases at 192 wards in southern Sweden during 2010-2012 in a nested case-control study. Uni- and multivariate statistical analyses were conducted. Outbreak was independently associated with the number of patients sharing a room with the norovirus case (odds ratio (OR): 1.9 per additional patient in the room; P<0.01), vomiting (OR: 2.6; P=0.04), age >80 years (OR: 3.2; P<0.01), comorbidity (OR: 2.3; P=0.05), and onset of symptoms after admission to the ward (OR: 3.5; P<0.01) in the multivariate analysis. Infection with genotype GII.4 was found to be strongly associated with outbreak in the univariate analysis (OR: 5.7; P<0.01). Moreover, associations between GII.4 and vomiting (OR: 2.5; P=0.01) and old age (OR: 4.3: P<0.01) were found. This is the first study to investigate clinical, ward and genotype risk factors for norovirus hospital outbreaks. Recognition of these factors may help direct and prioritize infection control actions based on the outbreak risk. The results also suggest that the outbreak association with GII.4 partly may be explained by an enhanced ability to induce vomiting.

Epidemiology of Foodborne Norovirus Outbreaks - United States, 2009-2015.

Noroviruses are the leading cause of acute gastroenteritis and foodborne disease in the United States (U.S.). About 1 in 5 reported norovirus outbreaks are spread through foodborne transmission, presenting opportunities for prevention. We describe the epidemiology of U.S. foodborne norovirus outbreaks reported to national surveillance systems, including differences between genotypes. Foodborne outbreaks that occurred during August 2009-July 2015 with norovirus reported as a single confirmed etiology to the National Outbreak Reporting System (NORS) were matched with outbreaks reported to CaliciNet, a U.S. laboratory norovirus outbreak surveillance network. We analyzed these matched outbreaks stratified by genotype for epidemiologic characteristics, including setting, size and duration, health outcomes of case-patients, implicated food, and outbreak contributing factors. Four hundred ninety-three confirmed foodborne norovirus outbreaks were reported in both NORS and CaliciNet. The most common norovirus genotypes reported were GII.4 (52%), GII.6 (9%), and GI.3 (8%). Compared to non-GII.4 outbreaks, GII.4 outbreaks had higher hospitalization rates (12.8 vs. 4.8 per 1,000 cases, P < 0.01). While contaminated foods were identified and reported in only 35% of outbreaks, molluscan shellfish (4% overall) were more often implicated in non-GII.4 outbreaks than in GII.4 outbreaks (7% vs. 1%, P = 0.04). Of the 240 outbreaks reporting at least one contributing factor, food workers were implicated as the source of contamination in 182 (76%), with no difference between GII.4 and non-GII.4 (73% vs 79%, P = 0.3). Foodborne norovirus outbreaks are frequently reported in the U.S., most of which are caused by GII.4 noroviruses. Viruses of this genotype are associated with higher rates of hospitalization; non-GII.4 noroviruses are more frequently associated with contaminated molluscan shellfish. These surveillance data highlight the diversity of noroviruses causing foodborne disease and can help guide appropriate food safety interventions, including worker hygiene, improved food handling and preparation, and further development of norovirus vaccines.

Emergence of GI.6 Outbreaks in a High School in Fangshan District, Beijing, China

[Background] An outbreak of gastroenteritis happened in a high school in Fangshan District, Beijing, China in March 2016，in which 44 students developed the symptoms of vomiting, nausea, diarrhea and abdominal pain. Epidemiological investigation, laboratory investigation and Statistical analysis were conducted to identify the agent and source of this outbreak.[Methods] A case–control study was performed to discover the factors associated with this infection. Questionnaires and clinical data from those infection-exposed people were assessed. Here an outbreak case was defined as a student who developed at least three following symptoms: diarrhea, vomiting, abdominal pain and nausea in 72 hours after the lunch on 2nd March., and controls were those who study or work in the school but did not have lunch on the day. Samples (Stool samples, rectal swabs and vomit samples) from infection-exposed students and controls, as well as environmental samples like food were collected to test for the existence of any suspicious bacteria or viruses. Detection of norovirus wasdone by real-time TaqMan RT-PCR and sequence analysis. Chi-square test was used to decide whether those differences could be of any statistical significance. [Results] The total number of respondents with complete data was 44. An epidemiological data combined with the epidemic curve indicated that the outbreak started initially from a point source type, and was followed by a secondary transmission. Five stool samples, three rectal swabs and one vomit sample from case group turned out to be positive for norovirus genotype I by real-time PCR, and two asymptomatic food handlers in control group were positive for NoV GI. Sequence analysis of GI positive sample confirmed that the norovirus GI.6 variant was the etiological agent of the outbreak. Comparasion between the tested results from samples of those students who had lunch in school canteen and those who did notindicates that the difference of incidence was of statistical significance (P<0.01, 95% CI:4.22-324.41). Food and environmental samples were tested to be bacteria-negative. [Conclusions] Our result suggested that the etiological agent of the outbreak was norvirus GI.6, which leads to gastroenteritis. We identified that asymptomatic infected food handlers were most likely to be the source of the outbreak. Therefore，hand hygiene practices strict adherence to regulations and access to hand washing facilities should be strengthened.

Parenterally Administered Norovirus GII.4 Virus-Like Particle Vaccine Formulated with Aluminum Hydroxide or Monophosphoryl Lipid A Adjuvants Induces Systemic but Not Mucosal Immune Responses in Mice.

Norovirus (NoV) is a main cause of acute gastroenteritis across all ages worldwide. NoV vaccine candidates currently in clinical trials are based on noninfectious highly immunogenic virus-like particles (VLPs) delivered intramuscularly (IM). Since NoV is an enteric pathogen, it is likely that mucosal immunity has a significant role in protection from infection in the intestine. Due to the fact that IM delivery of NoV VLPs does not generate mucosal immunity, we investigated whether NoV genotype GII.4 VLPs coadministered with aluminum hydroxide (Al(OH)3) or monophosphoryl lipid A (MPLA) would induce mucosal antibodies in mice. Systemic as well as mucosal IgG and IgA antibodies in serum and intestinal and nasal secretions were measured. As expected, strong serum IgG, IgG1, and IgG2a antibodies as well as a dose sparing effect were induced by both Al(OH)3 and MPLA, but no mucosal IgA antibodies were detected. In contrast, IN immunization with GII.4 VLPs without an adjuvant induced systemic as well as mucosal IgA antibody response. These results indicate that mucosal delivery of NoV VLPs is needed for induction of mucosal responses.