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

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

Caliciviridae Infections ; epidemiology ; genetics ; virology ; China ; epidemiology ; Gastroenteritis ; virology ; Humans ; Molecular Epidemiology ; Norovirus ; genetics ; RNA, Viral ; isolation & purification

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

A simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established to detect norovirus genotype GII. The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for 60 minutes. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP was validated by detecting several different diarrhea viruses including norovirus genotype GII. The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection. The assay was further evaluated with 93 clinical specimens of diarrhea patients. The results showed that the sensitivity of RT-LAMP was 1 000 copies/microL with a high specificity and the relative sensitivity was at the same level as that of conventional RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of conventional RT-PCR as well. This colorimetric RT-LAMP assay was potential for rapid detection of norovirus genotype GII on spot due to the observation of visual result with high specificity and sensitivity, time-saving and cost benefit.
Caliciviridae Infections ; diagnosis ; virology ; Colorimetry ; methods ; Feces ; virology ; Genotype ; Humans ; Norovirus ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods

We set up an SYBR Green I real-time RT-PCR method for the detection of genogroup II Norovirus, and this method's primers were encompassed the conservative region of Norovirus II. The limit of the detection was 10(2) copies. The standard curve's linear range was 10(2)-10(6) copies, correlation coefficient was 0.9952, the slope was -2.982, and the intercept was 35.84. This method possessed specificity for genogroup II Norovirus, without any cross-reaction with rotavirus, adenovirus, hepatitis A virus or astrovirus. The coefficients of variation (CV) of the C(t) values of the standard plasmid were 0.95%-1.69% (n = 5) in intra-assay and 0.87%-1.24% (n = 3) in inter-assay. We used this method to detect 30 shellfish samples, and found 3 samples were positive. This method is sensitive, specific and reliable for Norovirus II. It can be used to detect the Norovirus II in the shellfish rapidly.
Animals ; Bivalvia ; virology ; Norovirus ; isolation & purification ; Organic Chemicals ; RNA, Viral ; analysis ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Shellfish ; virology ; Species Specificity

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

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

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

Objective: To understand the epidemiologic characteristics of outbreaks, caused by norovirus-GⅡ.2、GⅡ.17 and GⅡ.4/Sydney in Guangdong Province from 2013 to 2017 and to provide scientific evidence for epidemic prevention and control. Methods: Incidence data of norovirus outbreaks in Guangdong from January 1(st) 2013 to November 30(th) 2017 were collected from Public Health Emergency Management Information System. RT-PCR was performed for every case of each outbreak to detect norovirus nucleic acid and gene sequencing was conducted to identify the genotype of norovirus. Characteristics of norovirus GⅡ.2, GⅡ.17 and GⅡ.4/Sydney outbreaks were analyzed. Directly standardized method was used to calculate the proportion of symtoms as diarrhea and vomitting. Results: From January 1(st) 2013 to November 30(th) 2017, a total of 167 norovirus outbreaks were reported in Guangdong, and 115 outbreaks were caused by norovirus GⅡ.2, GⅡ.17 and GⅡ.4/Sydney respectively. The outbreaks caused by norovirus GⅡ.2 accounted for 39.68% (25/63) in primary schools, 28.57% (18/63) in child care settings, 25.40% (16/63) in middle schools and 6.35% (4/63) in universities. Outbreaks caused by norovirus GⅡ.17 accounted for 41.03% (16/39) in middle schools, 20.51% (8/39) at workplaces, 15.38% (6/39) in primary schools, 12.82% (5/39) in universities, 5.13% (2/39) in communities and child care settings respectively. The outbreaks caused by norovirus GⅡ.4/Sydney accounted for 53.85% (7/13) in universities, 15.38% (2/13) in child care settings and at workplaces respectively, 7.69%(1/13) in primary schools and middle schools respectively. The outbreaks caused by norovirus GⅡ.2 had 77.78% (49/63) of contact transmission, 17.46% (11/63) of food-borne transmission. The outbreaks caused by norovirus GⅡ.17 showed 53.85% (21/39) of food-borne transmission, 15.38% (6/39) of contract transmission, 12.82% (5/39) of water-borne transmission. The outbreaks caused by norovirus GⅡ.4/Sydney had 53.85% (7/13) of food-borne transmission, 38.46% (5/13) of the contact transmission. In terms of the clinical manifestations, the standardized proportion of vomit was 73.76% and the proportion of diarrhea was 42.85% in cases infected with norovirus GⅡ.2, the proportion of standardized of vomit was 76.37% and the proportion of diarrhea was 51.40% in cases infected with norovirus GⅡ.17, with the standardized proportion of vomit was 54.10% and the proportion of diarrhea was 55.95% in cases infected with norovirus GⅡ.4/Sydney. Conclusions: The outbreaks caused by norovirus GⅡ.2 through contact transmission mainly occurred in primary schools, child care settings and middle schools. The outbreaks caused by norovirus GⅡ.17 through food-borne transmission mainly occurred in middle schools and at workplaces. The outbreaks caused by norovirus GⅡ.4/Sydney food-borne transmission and contact mainly occurred in universities.
Adolescent ; Caliciviridae Infections/epidemiology* ; Child ; Child, Preschool ; Diarrhea/etiology* ; Disease Outbreaks ; Epidemics ; Gastroenteritis/epidemiology* ; Genotype ; Humans ; Norovirus/isolation & purification* ; Reverse Transcriptase Polymerase Chain Reaction ; Sentinel Surveillance ; Vomiting/etiology*