A new viral sampling concentration device was designed which was equipped with a new cationic filter membrane-Nanoceram suitable for field sampling. Norovirus Genegroup II was detected from environmental water with the aid of this device. The effects on virus recovery of prefiltration, various second-concentration methods, and different eluants were investigated through pre-experiment. The concentration optimized process, and the optimal concentration process were then determined. The results showed that the prefiltration had a profound effect on virus recovery, and two second-concentration method: PEG-NaC1 precipitation and celite adsorption, had almost the same concentration effects. The Na2 HPO4 solution of 0.15 mol/L was selected as the final eluant to elute the adsorbed Nuorovirus from the celite. The virus recovery of Nanoceram was determined to be 3.02%. Finally, successful detection of Norovirus GII in sewage from Yangqiao River, Fengtai District, Beijing was acheived. All these data had shown that the Naneceram filter concentration method could concentrate Norovirus from environmental water with a steady effects.
Filtration ; instrumentation ; methods ; Fractional Precipitation ; instrumentation ; methods ; Genotype ; Norovirus ; classification ; genetics ; isolation & purification ; Rivers ; virology ; Water Microbiology

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

OBJECTIVETo determine the pathogen of an unexplained epidemic event of infectious diarrhea by laboratory diagnosis of suspected cases samples.

METHODS28 samples from 28 suspected cases (22 fecal samples, 3 vomitus samples, 3 anus swab samples) were tested for Norovirus by RT-PCR. Sequencing and phylogenetic analysis were acomplished of 5 positive samples.

RESULTS160 of 5694 population were ill with an attack rate of 2.81%. The peak period was 7-9, March. 14 of 28 samples were tested Norovirus positive.Sequencing and phylogenetic analysis showed Norovirus type GII/4 was the causative agent and it had highest identity (97. 9%) with epidemic strain 2006b.

CONCLUSIONThe epidemic event ofinfectious diarrhea were caused by GII/4 Norovirus strains.

Disease Outbreaks ; Dysentery ; diagnosis ; epidemiology ; genetics ; Feces ; virology ; Gastroenteritis ; epidemiology ; virology ; Humans ; Molecular Epidemiology ; Norovirus ; classification ; genetics ; isolation & purification ; Phylogeny ; RNA, Viral ; analysis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVEThe purpose of this study was to develop RT- PCR assay for Rapidly detect and distinguish between Norovirus genogroup I and genogroup II with a pair of primers.

METHODSA pairs of primers specific to capsid prote in ORF2 gene of G I and G II Norovirus were dsigned according to the published complete genome sequence, with which the RNA of Norovirus was extracted and RT-PCR amplification. The sensitivity, specificity of the RT- PCR assay was estimated and apply it to the detection of Norovirus in clinical specimens.

RESULTSThe results showed that the assay possessed high specificity for Norovirus detection and without any evident cross-reaction with other viruses, including rotavirus, enteric adenovirus and hepatitis A virus. The detection limit of RT-PCR assay for Norovirus G I and G II were up to 100 pg/ml and 10 pg/ml respectively.

CONCLUSIONThe RT- PCR assay provide rapid and sensitive detection of Norovirus G I and G II and should prove to be useful for Norovirus diagnosis in the outbreaks of acute gastroenteritis.

Caliciviridae Infections ; diagnosis ; virology ; DNA Primers ; genetics ; Gastroenteritis ; diagnosis ; virology ; Humans ; Norovirus ; classification ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; instrumentation ; methods

OBJECTIVETo investigate the molecular epidemiological characteristics of norovirus in Guangzhou from 2009 to 2011.

METHODSA total of 183 water samples, 1162 seafood samples and 1066 diarrhea stool specimens were collected from January 2010 to May 2011, June 2009 to June 2011 and July 2009 to December 2010 respectively in Guangzhou. Norovirus was detected by real time reverse transcript-PCR (qRT-PCR). The partial polymerase gene was amplified from norovirus positive samples, then sequenced and compared with the sequences of norovirus in GenBank. The phylogenetic tree was created.

RESULTSThe positive rate was 19.67% (36/183), 8.26% (96/1162) and 37.05% (395/1066) in water samples, seafood and diarrhea patients respectively. Noroviruses from positive samples could be divided into 10 representative strains, in which 7 representative strains of genotype of 208 samples was type G2-4. The sequences from water, seafood and stool specimens were highly homologous with the similarity of 94% - 100%.

CONCLUSIONIn Guangzhou, the predominant Norovirus genotype was G2-4 and the positive rate of samples was high.

Base Sequence ; Caliciviridae Infections ; epidemiology ; virology ; China ; epidemiology ; Diarrhea ; virology ; Genotype ; Humans ; Molecular Epidemiology ; Norovirus ; classification ; genetics ; isolation & purification ; Phylogeny ; RNA, Viral ; genetics ; Seafood ; virology ; Water Microbiology

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 analyze the epidemiological characteristics of an acute gastroenteritis outbreak in a nursing home caused by norovirus (NoV) and the genotype of the pathogen.

METHODSOn January 29th 2013, a total of 26 acute gastroenteritis patients infected by norovirus were reported in the nursing home of Jinshan, Shanghai. A questionnaire was used to acquire information of patients involved in the outbreak, 9 stool or anal swab samples were collected from 9 patients without treatment by simple random sampling method, and 4 environmental samples from the surface of doorknobs or toilets were collected. The samples were detected by Real-time PCR for NoV, and positive samples were then amplified by routine RT-PCR. The PCR products were purified, sequenced, and aligned by comparing sequences in GenBank. Phylogenetic trees were constructed by using Clustal X, employing MEGA 5.1 program package.

RESULTSFor the 26 patients, 7 were men and 19 were women.8 samples were found NoV positive among the 13 samples when detected by real-time PCR. The sequence alignment showed that the nucleotide sequence homology between Jinshan08 and Jinshan12 strain which obtained sequencing signal was 100%. The phylogenetic analysis revealed that Jinshan08, Jinshan12 and GII.e/NV2634/BCN/Spain/2008 strains in the RdRp region were on the the same branch of evolutionary tree, the confidence level was 99%, and in the N/S region of the Capsid, 2 other strains and Lordsdal strain were in the same branch, the confidence level was 97%.

CONCLUSIONIt was confirmed that the acute gastroenteritis outbreak was caused by the new GII.4 NoV recombinant.

Aged ; Aged, 80 and over ; China ; epidemiology ; Disease Outbreaks ; Female ; Gastroenteritis ; epidemiology ; genetics ; virology ; Genetic Variation ; Humans ; Male ; Middle Aged ; Molecular Epidemiology ; Norovirus ; classification ; genetics ; isolation & purification ; Phylogeny

To describe epidemiologic characteristics of norovirus infection and its genotype in Shenzhen area of 2010. Stool specimens were collected from four monitoring point and detected by reverse transcription polymerase chain reaction (PT-PCR). Positive PCR products were purified and sequenced, and the sequences were performed by Clustal W and MEGA 4.0 programs, then genotype was identified and phylogenetic tree was constructed. Nucleotide sequence analysis revealed that 79 strains of NV belonged to norovirus genogroup II and 6 belonged to genogroup 1 of all 85 positive products. While 55 strains belonged to G II/4(2006b), 16 strains belonged to G II/4(2008 variant), 2 strains belonged to G II /1, 4 strains belonged to G II/5, 2 strains belonged to G II/11, 1 strain belonged to GII/4, 2 strains belonged to GII/5, 3 strains belonged to GI/6. The main genogroups of norovirus in Shenzhen ware GI and G II. G II /4 was one of the most major genotype of norovirus , while G II /4(2006b) variant was identified as the predominant strain in Shenzhen area.
Adolescent ; Adult ; Caliciviridae Infections ; virology ; Child ; Child, Preschool ; China ; Female ; Genotype ; Humans ; Infant ; Male ; Molecular Sequence Data ; Norovirus ; classification ; genetics ; isolation & purification ; Phylogeny ; Young Adult

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

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