Objective: To analyze the genetic characterization of norovirus isolated in an outbreak of gastroenteritis in Jiangsu province. Methods: Extracted viral RNA from the swab samples of cases of acute gastroenteritis outbreak in Jiangsu province on December 16-27, 2016 was reversely transcribed to cDNA, and partial RNA-dependent RNA polymerase sequence and complete capsid sequence (VP1) were amplified by RT-PCR. Amplification products were sequenced for the analysis of genetic characteristics. Results: Based on sequence alignment, the variant shared a high level of identity with the strain GⅡ.g isolated in Spain and Finland (98.7%) in the RNA-dependent RNA polymerase region, and with the strain GⅡ.1 isolated in American (99.4%) in the VP1. The recombination was determined by using software Simplot, and the breakpoint of recombination was located in the ORF1/2 overlap region at position 5 106 of VP1. The result of amino acids alignment in capsid region showed that there were no mutations in the amino acids of the predicted epitopes and receptor binding site Ⅰ-Ⅲ, but a unique amino acid change was detected at position 132 (N-S). Conclusion: The norovirus isolated in the outbreak of gastroenteritis in Jiangsu province was a rare recombinant norovirus variant GⅡ.g-GⅡ.1.
Caliciviridae Infections/epidemiology* ; Capsid Proteins ; Disease Outbreaks ; Gastroenteritis/epidemiology* ; Genotype ; Humans ; Norovirus/isolation & purification* ; Phylogeny ; RNA, Viral/genetics* ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

This article reviewed the researches proceeding on the contamination and detection of the foodborne pathogen noroviruses (NoVs) in fresh produce, which involved the NoVs contaminations in fresh produce, the special attachment of NoVs in fresh produce, the NoVs outbreaks associated with fresh produce and the NoVs detection in fresh produce. There had been an increase in reported infectious disease risks associated with the consumptions of fresh produce for recent 30 years. Because the NoVs, as a primary cause of viral gastroenteritis thoughout the world, were highly contagious, had a low infectious dose, and were persistent in the environment. And also the methods for NoVs detection in food had significantly developed over the last 15 years. Currently NoVs were the most common pathogen accounting for 40% of outbreaks associated with fresh produce (i. e., fruits and vegetables). Data from outbreaks investigations verified fresh produce as the high risk food products for NoVs. The fresh produce were typically eaten raw with no thermal processing, can be contaminated at any step during production and processing from faecally polluted water and fertilizers, the poor hygiene practices by food handlers and the cross-contamination. The attachment of NoVs to the fresh produce was due to the physio-chemical factors of virus protein coat, the special attachment to different fresh produce, and the possibility for internalization of NoVs. It might provide answers to why those high risk foods were more frequently implicated (i. e., lettuce and raspberries). According to the data of foodborne NoVs outbreaks which were associated with fresh produce from EU countries and the USA, the outbreaks in EU countries were mainly associated with NoVs contaminated raspberries and lettuce, while in USA which were associated with NoVs contaminated lettuce. Unfortunately, there were no NoVs detection methods for fresh produce or the data of foodborne NoVs outbreaks which were associated with fresh produce in China. That made it difficult to analyze the NoVs contamination situation in China. The heterogeneous distributions of presumably low levels of virus, which presented in contaminated fresh produce, also made it difficult to detect NoVs. To solve this problem, different sampling methods, viral elution methods and RT-qPCR methods were chosen. For example, according to the isoelectric point of NoVs particles, high pH and high ionic strength solution could be used as means for releasing NoVs. For the elution from acidic fruit, the buffer capacity and the virus recovery could be increased by the addition of tris-HCl. When analyzing pectin containing raspberries or strawberries, the viral elution usually incubated with pectinase at neutral pH to avoid from foaming jelly. In this paper, the latest ISO standard for NoV detection in food and the new approaches for NoV detection were also reviewed to provide references for domestic researches. It was necessary to establish and develop domestic methods for NoV detection in fresh produce, especially the different NoV conventional molecular detection methods with corresponding NoV extraction methods, which targeted to the different adsorption characteristics of different fruits and vegetables, in order to strengthen the national food safety monitoring.
Food Analysis ; methods ; Food Contamination ; analysis ; Foodborne Diseases ; virology ; Fruit ; virology ; Gastroenteritis ; virology ; Humans ; Norovirus ; classification ; genetics ; isolation & purification ; physiology ; Vegetables ; virology

Noroviruses (NoVs) are one of the most important foodborne viral pathogens worldwide. Shellfish are the most common carriers of NoVs as they can concentrate and accumulate large amounts of the virus through filter feeding from seawater. Shellfish may selectively accumulate NoVs with different genotypes, and this bioaccumulation may depend on the season and location. Our previous studies found various histo-blood group antigens (HBGAs) in shellfish tissues. While HBGAs might be the main reason that NoVs are accumulated in shellfish, the detailed mechanism behind NoV concentration and bioaccumulation in shellfish is not clear. Here we review current research into NoV bioaccumulation, tissue distribution, seasonal variation, and binding mechanism in shellfish. This paper may provide insight into controlling NoV transmission and decreasing the risks associated with shellfish consumption.
Animals ; Caliciviridae Infections ; transmission ; virology ; Food Contamination ; analysis ; Foodborne Diseases ; virology ; Humans ; Norovirus ; classification ; genetics ; isolation & purification ; physiology ; Shellfish ; virology

Noroviruses are the leading cause of epidemic gastroenteritis, including foodborne outbreak, in Korea. The prevalence of human noroviruses was studied in diarrheal stool samples of patients with acute gastroenteritis by conventional duplex reverse transcription (RT)-PCR. Diarrheal stool samples were collected from 1,685 patients from the local hospitals in Seoul. The prevalence of the noroviruses was 22.8% (222/972 patients) in 2012 and 11.2% (80/713 patients) in 2013, with a total of 17.9% (302/1,685 patients). Genotyping was performed on 302 norovirus-positive stool samples to reveal 5.6% prevalence of genogroup I (GI) (17/302) and 94.4% prevalence of genogroup II (GII) (285/302). The patients with norovirus-associated acute gastroenteritis mostly showed prevalence of GII norovirus, especially GII.4 (64.6%; 195/302).
Acute Disease ; Feces/virology ; Gastroenteritis/*diagnosis/epidemiology/virology ; Genotype ; Humans ; Norovirus/*genetics/isolation & purification ; Prevalence ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction

OBJECTIVETo monitor the epidemiology of norovirus infection in diarrheal children in Shanghai between 2009 and 2011 and characterize the genotypes of norovirus strains.

METHODThe stool samples were collected from children visiting outpatient clinic for acute non-dysenteric diarrhea between 2009 and 2011.One step real-time RT-PCR was used for screening norovirus genogroups GI and GII. The genotypes of norovirus genogroup GII were classified based on the nucleotide sequences of both partial capsid and polymerase fragments.

RESULTA total of 2 288 outpatient children with acute diarrhea were included in this study, out of whom, 531 (23.1%) were positive for norovirus in the fecal specimens based on real-time RT-PCR test.Norovirus was prevalent throughout the year and an increased activity of norovirus infection was usually observed between July and October. Children <4 years of age accounted for 95.2% of norovirus-infected cases, and the detection rate of norovirus was significantly higher in diarrheal children <4 years than in those ≥ 4 years (24.4% vs. 10.7%,χ(2) = 10.66, P < 0.05).Of 531 norovirus-positive specimens, 4 (1.7%) were positive for genogroup GI and 527 (98.3%) positive for genogroup GII. Seven distinct capsid genotypes were identified in 234 norovirus strains, including 153 (64.4%) GII.4 (9 belonging to 2010 variants and 145 belonging to 2006b variants), 66 (27.6%) GII.3, 7 (2.9%) GII.2, 6 (2.5%) GII.6, 4 (1.7%) GII.12, 1 (0.4%) GII.7 and GII.14 in each. Seven polymerase genotypes were identified in 244 norovirus strains, including 189 (77.5%) GII.4 (14 belonging to 2010 variants and 175 belonging to 2006b variants), 47 (19.3%) GII.12, 2 (0.8%) GII.16, GII.b and GII.g in each, 1 (0.4%) GII.2 and GII.6 in each. A new GII.4-2010 (New Orleans) variant was first detected in June 2010 and sporadically circulated afterwards.Of 198 norovirus strains in which both polymerase and capsid genotypes were determined, 56 showed discordant results, indicating potential norovirus recombinants. The common discordant combinations of the polymerase and capsid genotypes were GII.12/GII.3 (69.6%) and GII.4/GII.3 (8.9%).

CONCLUSIONNorovirus is a common causative agent responsible for diarrhea in Shanghai children over the three years and norovirus-associated diarrhea was epidemic year round with high activity in late summer and autumn in Shanghai.Infants and young children are susceptible to norovirus infection. The circulating norovirus showed genetic diversity. The GII.4-2006b variant continued to predominate in Shanghai during the period of 2009-2011 despite the emergence of the novel GII.4-2010 (New Orleans) variant.

Adolescent ; Caliciviridae Infections ; epidemiology ; virology ; Capsid Proteins ; genetics ; Child ; Child, Preschool ; China ; epidemiology ; Diarrhea ; epidemiology ; virology ; Feces ; virology ; Female ; Gastroenteritis ; epidemiology ; virology ; Genetic Variation ; Genotype ; Humans ; Infant ; Male ; Molecular Epidemiology ; Norovirus ; classification ; genetics ; isolation & purification ; Prevalence ; RNA, Viral ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

Murine norovirus (MNV) was first discovered in mice in 2003. MNV is a member of the genus Norovirus in the family Caliciviridae. It is one of the most important and prevalent pathogens of laboratory mice, and almost all mouse strains are susceptible to MNV infection. In this study, a MNV strain was isolated from the cecal contents of infected mice and identified by the cytopathic effect (CPE) assay, virus plaque assay, 50% tissue culture infectious dose (TCID50) assay, electron microscopy, indirect immunofluorescence assay (IFA) and nucleotide sequencing. On infection, the RAW264.7 cell line showed obvious cytopathic effects within 24 to 48 hours post-inoculation, as infected cells became rounded, bright and shrunken, with ultimate disintegration of the cell sheet. After the isolation of the MNV virus, the virus was plaque-purified in RAW264.7 cells. The TCID50 of the virus was 10(5.25/0.1 mL. Electron microscopic observations of the purified virus showed the presence of spherical and non-enveloped viral particles that were 30 to 35 nm in diameter. According to the identification results, the isolate was named as MNV Guangzhou/K162/09/CHN. Thereafter, five overlapping gene fragments that covered the entire open reading frame (ORF) were amplified by RT-PCR, and the 3'-untranslated region (UTR) and 5'-UTR were amplified using the 3'-rapid amplification of cDNA ends (RACE) and the 5'-RACE method, respectively. Each of the gene fragments were cloned and sequenced, and whole genome sequences of the strain were obtained by assembling the cDNA fragment sequences. The results showed that the length of the complete genome was 7 380 nucleotides (GenBank accession number: HQ317203). The comparison of nucleotide and deduced amino acid sequences of the isolate was performed against other MNV strains in the GenBank database. A phylogenetic tree based on VP1 nucleotide sequences was constructed using MEGA5.0 software. The homology of nucleotides between the MNV Guangzhou/K162/09/CHN strain and other MNV isolates ranged from 87.4% to 89.7%. Phylogenetic analysis showed that there was a close genetic relationship between the Guangzhou/K162/09/CHN strain and MNV strains isolated from Japan (S7-P2 and S7-PP3 isolates), Korea (K4 isolate), and Germany (Berlin/04/06/DE and Berlin/05/06/DE isolates). This is the first report of the isolation and identification of MNV in China, and the first report of the genetic analysis of its complete genome.
Animals ; Caliciviridae Infections ; veterinary ; virology ; Mice ; Molecular Sequence Data ; Norovirus ; classification ; genetics ; isolation & purification ; physiology ; Open Reading Frames ; Phylogeny ; Rodent Diseases ; virology ; Sequence Homology, Amino Acid ; Viral Proteins ; chemistry ; genetics

To describe the epidemiological characteristics of norovirus (NOV) associated acute gastroenteritis in Shanghai and characterize the evolution pattern of circulating strains. From March 2012 to February 2013, 502 stool specimens were collected from adult (> or = 16 years) outpatients who visited either of the two sentinel hospitals in Shanghai for acute gastroenteritis. Molecular detection and genotyping of NoV were performed and the phylogenetic relationship of the circulating strains has also been comprehensively analyzed. The epidemics level of GI NoV was low throughout the surveillance period, with the positive rate of 3.78% (19 cases), and no seasonality of GI NoV infection could be distinguished. For GII genogroup, higher epidemics in adults in Shanghai, with the detection rate of 17.13% (86 cases), were observed. And relatively high epidemics of GII NoV infection were spotted between October and December in 2012. The frequency of NoV associated acute gastroenteritis in older people is significantly higher than that in young individuals (P < 0.05). Sequencing and genotyping analysis revealed that the high epidemics of GII NoV infection between October and December in 2012 is associated with the emergence of a novel GII.4 norovirus strain, termed Sydney_2012. Sequence analysis also demonstrated that this was a recombinant virus between a GII.e polymerase and GII.4 capsid, which has also been the dominant circulating strain in Shanghai. In 2012, a new GII.4 variant, termed Sydney_2012, emerged in Shanghai and caused high epidemics of acute gastroenteritis during late autumn and winter.
Adolescent ; Adult ; Aged ; Caliciviridae Infections ; epidemiology ; virology ; China ; epidemiology ; Disease Outbreaks ; Female ; Gastroenteritis ; epidemiology ; virology ; Genotype ; Humans ; Male ; Middle Aged ; Norovirus ; chemistry ; classification ; genetics ; isolation & purification ; Phylogeny ; Viral Envelope Proteins ; chemistry ; genetics ; Young Adult

The object of this study is to develop a duplex fluorescent quantitative one-step RT-PCR assay for detection and quantitation of GI and GII norovirus. The specific primers, Taqman probes, optimized reaction solution and condition were used to develop the duplex fluorescent quantitative one-step RT-PCR assay. The sensitivity, specificity and reproducibility of the assay were evaluated. The assay was evaluated by testing the 100 specimen samples and compared with the reference assay conventional RT-PCR. The assay possessed high specificity for norovirus detection without any evident cross-reaction with enteric adenovirus, rotavirus or astrovirus. The detection limit of the real-time RT-PCR assay, for GI and GII norovirus was up to 10(3) copy/microL respectively. Compared with the conventional RT-PCR assay, the assay in this study had higher sensitivity with higher detection rate of norovirus in stool specimens. The duplex fluorescent quantitative one-step RT-PCR assay provides rapid, sensitive and reliable detection of GI and GII norovirus, and could be used as a laboratory diagnosis of norovirus in acute gastroenteritis patients.
DNA Primers ; genetics ; Feces ; virology ; Gastroenteritis ; diagnosis ; virology ; Genotype ; Humans ; Norovirus ; classification ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; instrumentation ; methods

OBJECTIVETo establish a one-step four multiplex reverse transcription polymerase chain reaction (RT-PCR) method based on Homo-Tag Assisted Non-Dimer System (HAND) system for simultaneous detection of 4 diarrhea viruses of rotavirus, astrovirus, norovirus and sapovirus.

METHODSPrimers were designed according to the conserved genome sequence of the 4 viruses and the homologous tail sequences were added to the 5' end. The multiplex RT-PCR system was constructed by optimizing the PCR parameters such as the concentration of universal tag primer and genome-specific Homo-Tailed primers. The specificity, stability and sensitivity of the method were evaluated systematically.

RESULTSThe 4 multiplex RT-PCR methods based on HAND system was established successfully. Specificity analysis showed no cross reaction between the 4 diarrhea viruses. The sensitivity analysis showed detection limits for rotavirus, astrovirus, norovirus and sapovirus of 48, 1.92, 9.6 and 48 pg per reaction, respectively.

CONCLUSIONThe established HAND system-based multiplex RT-PCR assay allows simple, rapid, specific, sensitive, and stable for detection of the 4 common diarrhea viruses at low costs and is suitable for application in general medical laboratories.

Astroviridae ; genetics ; isolation & purification ; Diarrhea ; virology ; Feces ; virology ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Norovirus ; genetics ; isolation & purification ; RNA, Viral ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rotavirus ; genetics ; isolation & purification ; Sapovirus ; genetics ; isolation & purification

OBJECTIVETo explore a convenient and effective method for norovirus nucleic acid extraction from oysters suitable for long-term viral surveillance.

METHODSTwo methods, namely method A (glycine washing and polyethylene glycol precipitation of the virus followed by silica gel centrifugal column) and method B (protease K digestion followed by application of paramagnetic silicon) were compared for their performance in norovirus nucleic acid extraction from oysters. Real-time RT-PCR was used to detect norovirus in naturally infected oysters and in oysters with induced infection.

RESULTSThe two methods yielded comparable positive detection rates for the samples, but the recovery rate of the virus was higher with method B than with method A.

CONCLUSIONMethod B is a more convenient and rapid method for norovirus nucleic acid extraction from oysters and suitable for long-term surveillance of norovirus.

Animals ; Centrifugation ; methods ; Norovirus ; genetics ; isolation & purification ; Ostreidae ; virology ; RNA, Viral ; isolation & purification