• Journal of fish pathology
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• v.24 no.3
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• pp.255-268
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• 2011

For detection of noroviruses (NVs), we compared various PCR primer sets based on reverse transcription nested PCR (RT-nested PCR) in the water samples from Dong brook in Busan, South Korea. We designed various new primer sets based on the most conserved sequences of the capsid protein gene that react with diverse NVs found in Korea. Designed primer sets (KG1F/KG1R and KG2F/KG2R, named as PNK) for the respective genogroups of NVs, genogroup I and II (GI and GII), were applied to detect NVs in the water samples from Dong brook concentrated with ultracentrifugation. In the application to the water samples, proportion of GI (76.47%) and GII (70.59%) in water samples of Dong brook in RT-nested PCR with the primer sets of this study. However, no significant differences of the proportion of the positive samples were not found between RT-nested PCRs with reported and newly designed primer sets. From the nucleotide sequencing, GI and GII of NVs present in Dong brook were appeared to be the members of 1/2/4/5/9/10 genotypes, and 3/4/5/11/13 genotypes respectively. Appeared genotype 4 of GII known as an one of main genotype found in patients of many Asian countries warned us to consider the risks of norovirus in aquatic environments in southern part of Korea.

• Biomedical Science Letters
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• v.17 no.3
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• Korean Journal of Food Science and Technology
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• v.39 no.1
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• pp.71-76
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• 2007

Foodborne illness caused by Noroviruses (NVs) is increasing rapidly in Korea. This study developed an effective detection protocol for NVs found in contaminated oysters and lettuce through an investigation using the major steps of virus particle separation, concentration and RT-PCR. As a surrogate model for NVs, the cultivable feline calicivirus (FCV) that belongs to the same Caliciviridae family was used. Instead of using a time-consuming ultracentrifugation method, efficient methods based on solvent extraction and PEG precipitation procedure were applied. Direct homogenization of a 25g sample of whole oyster and lettuce in 175mL PBS provided the simplicity that would be needed in the actual field of food product examination. The overnight PEG precipitation step at $4^{\circ}C$ was reduced to 3 h by placing the reaction tube in ice and by adjusting the PEG concentrations. The application of the use of chloroform and 0.2 ${\mu}m$ syringe filtration together showed a better detection efficiency than the use of chloroform alone in removing PCR inhibitors for both oyster and lettuce samples. Also, dilution of the extracted RNA solution before PCR provided increased sensitivity. The improved detection protocol developed in this study could be efficiently applied to detect FCV and most likely NVs from oysters and lettuce.

• Journal of Life Science
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• v.21 no.6
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• pp.819-828
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• 2011

To inspect norovirus contamination of groundwater in south eastern areas of Korea, a systematic survey of groundwater in Busan, Ulsan, and Gyeongsangnam-do was performed for two years from 2009 to 2010. For this purpose, we first optimized the nested reverse transcription-PCR condition by designing two sets of primers for the detection of norovirus genogroups, GI and GII. Of 145 samples, 21 (25.9%) and 15 (23.4%) were norovirus positive in the dry season (April to June) and wet season (July to August), respectively. The detection frequencies of norovirus in Busan, Ulsan, and Gyeongsangnam-do were 15%, 7%, and 32%, respectively, reflecting a geographical difference in their distribution. The GI and GII isolates were 5 and 31, respectively, indicating the prevalence of GII in the tested areas. According to phylogenetic analysis of their nucleotide sequences, all of the GI isolates were identified to genotype GI.5 whilst the GII isolates were divided into two genotypes, GII.3 and GII.4. Neither physical-chemical parameters such as pH, temperature, oxidation-reduction potential, and dissolved oxygen, nor microbial indicators of water quality such as total bacteria, total coliforms, and Escherichia coli were statistically correlated with contamination of norovirus in the groundwater. Interestingly, however, the presence of norovirus was closely correlated with low turbidity (<0.50 NTU). The present study suggests that periodical monitoring of norovirus in groundwater is necessary to prevent epidemic waterborne diseases and to secure better sanitary conditions for public health.

• Journal of Microbiology and Biotechnology
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• v.30 no.9
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• pp.1412-1419
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• 2020

Human noroviruses (HuNoVs) are a leading cause of gastroenteritis outbreaks worldwide. However, the paucity of appropriate cell culture models for HuNoV replication has prevented developing effective anti-HuNoV therapies. In this study, first, the replication of the virus at various temperatures in different cells was compared, which showed that lowering the culture temperature from 37℃ significantly increased virus replication in Madin-Darby canine kidney (MDCK) cells. Second, the expression levels of autophagy-, immune-, and apoptosis-related genes at 30℃ and 37℃ were compared to explore factors affecting HuNoV replication. HuNoV cultured at 37℃ showed significantly increased autophagy-related genes (ATG5 and ATG7) and immune-related genes (IFNA, IFNB, ISG15, and NFKB) compared to mock. However, the virus cultured at 30℃ showed significantly decreased expression of autophagy-related genes (ATG5 and ATG7), but not significantly different major immune-related genes (IFNA, ISG15, and NFKB) compared to mock. Importantly, expression of the transcription factor FOXO1, which controls autophagy- and immune-related gene expression, was significantly lower at 30℃. Moreover, FOXO1 inhibition in temperature-optimized MDCK cells enhanced HuNoV replication, highlighting FOXO1 inhibition as an approach for successful virus replication. In the temperature-optimized cells, various HuNoV genotypes were successfully replicated, with GI.8 showing the highest replication levels followed by GII.1, GII.3, and GII.4. Furthermore, ultrastructural analysis of the infected cells revealed functional HuNoV replication at low temperature, with increased cellular apoptosis and decreased autophagic vacuoles. In conclusion, temperature-optimized MDCK cells can be used as a convenient culture model for HuNoV replication by inhibiting FOXO1 and providing adaptability to different genotypes.

• Korean Journal of Microbiology
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• v.54 no.1
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• pp.24-30
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• 2018

Norovirus infection is a leading cause of nonbacterial gastroenteritis outbreaks. New variants of GII.4 have emerged approximately every 2~3 years and have caused norovirus gastroenteritis pandemics globally. In this study, analysis and molecular genetic characteristics of the norovirus gastroenteritis outbreaks 2,917 samples in Gyeonggi-Do from 2014 to 2015. As a result, 247 samples out of 2,917 samples are positive for norovirus. Norovirus molecular genetic characteristics of the GI 8 types (GI-1, 2, 3, 4, 5, 6, 12, 14), GII 10 types (GII - 2, 3, 4, 5, 6, 11, 12, 14, 16, 17). Genome sequences of isolated noroviruses were similar to those of new GII.17 Kawasaki 2014 variants with 96.6 identity, suggesting that these viruses were imported from overseas. 44% of virus incidence was originated from school meal service. Therefore, a continuous monitoring and school sanitation should be required for preventing a massive virus outbreak.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• v.12 no.2
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• pp.183-193
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• 2009

Purpose: Rotaviruses, noroviruses, astroviruses, and enteric adenoviruses cause acute gastroenteritis (AGE) in children. Some children with AGE have afebrile convulsions associated with viral gastroenteritis. The purpose of this study was to detect and genotype viruses from children with AGE or benign infantile seizures associated with mild gastroenteritis (BIS-MG). Methods: Between August 2004 and June 2005, 311 children with AGE were included. Four viral agents, including rotavirus, norovirus, astrovirus, and adenovirus, were analyzed from stool specimens of each patient using the latex agglutination method, enzyme immunoassay, and reverse transcriptase polymerase chain reaction. Genotyping of each virus was performed in 217 of the 311 children. Results: Among 217 children (male, 121; female, 96; mean age, 20.6${\pm}$15.4 months), rotavirus was detected in 109 (50.2%), norovirus in 28 (12.9%), adenovirus in 13 (6.0%), and astrovirus in 2 children (0.9%). Genotyping of rotavirus revealed positive results in 97 children; P[8]G3 in 36, P[4]G2 in 21, P[6]G4 in 10, P[4]G4 in 9, P[8]G9 in 6, P[8]G1 in 6, P[4]G3 in 4, P[4]G9 in 3, and P[6]G2 in 2. Genotyping of norovirus showed GII-4 in 27 of 28 children and GII-6 in 1 child. Sixteen children were diagnosed with BIS-MG. Rotavirus was detected in 13 of 16 children with BIS-MG, and norovirus in 2 children. Genotyping of rotavirus detected in children with BIS-MG revealed P[8]G3 in 6 children, P[4]G2 in 2 children, and P[4]G9 in 1 child. Conclusion: Analysis of viruses from stool specimens indicates that both rotavirus and norovirus are the main viruses related to BIS-MG in children.