Detection of waterborne norovirus genogroup I strains using an improved real time RT-PCR assay.

Noroviruses (NoVs) are the major global source of acute gastroenteritis (AGE) outbreaks. To detect NoVs, real-time reverse transcription-quantitative PCR (RT-qPCR) assays have been widely employed since the first decade of the 21st century. We developed a redesigned probe, JJV1PM, for RT-qPCR assay detection of NoV genogroup (G) I strains. The new RT-qPCR assay using the JJV1PM-probe showed broader strain reactivity for 10 NoV GI genotypes, while the old method, using the JJV1PT-probe assay, detected only 7 NoV GI genotypes in a validation panel using human fecal specimens. The improved RT-qPCR assay was also successfully applied to water samples. The JJV1PM-probe assay identified 7 NoV GI genotypes, whereas the JJV1PT-probe assay detected only 2 NoV GI genotypes from water samples. Notably, groundwater-borne NoV GI strains detected by the improved JJV1PM-probe assay were associated with groundwater-borne AGE outbreaks in South Korea. The results of this study underscore the importance of the evaluation of RT-qPCR assays using recently circulating NoV strains prior to field application.

High incidence of group A rotaviruses G4P[6] strains among children in Gyeonggi province of South Korea, from 2009 to 2012.

The genotype distribution of group A rotaviruses (RVAs) circulating in Gyeonggi province, South Korea between 2009 and 2012 was investigated. A total of 2619 stool specimens from sporadic acute gastroenteritis cases and 117 acute gastroenteritis outbreaks were analyzed. Among them, RVAs were detected from 263 (10.0%) sporadic cases and 3 (2.6%) outbreaks. The G4P[6] strains predominated (29.7%), followed by G1P[8] (19.4%), G2P[4] (15.6%), G3P[8] (13.3%) and G9P[8] (6.5%) strain. Especially 96.2% of the genotype G4P[6] strains were isolated from children<1year of age. Phylogenetic analysis revealed that genotype G4P[6] strains were members of sub-lineage Ie(G4) and Ia(P[6]). Intensified monitoring of RVAs, especially G4P[6] strains among young children, is essential to control RVA infections.

Emergence of Norovirus GII.4 variants in acute gastroenteritis outbreaks in South Korea between 2006 and 2013.

BACKGROUND: New emerging strains of noroviruses (NoVs) often increase acute gastroenteritis (AGE) outbreaks worldwide. OBJECTIVE: We analyzed the epidemiological features and genotypic patterns of NoVs in AGE outbreaks. STUDY DESIGN: To elucidate the public health impact of NoVs during AGE outbreaks in South Korea, a molecular and epidemiological investigation was performed with 318 AGE outbreaks reported from the Gyeonggi province of South Korea during the period from 2006 to 2013. RESULTS: NoVs were associated with 102 (32.1%) of the AGE outbreaks. Epidemiological data revealed that the majority of NoV outbreaks were in the student group (47.1%), and the majority of AGE patients were identified in schools (68.8%). NoV genogroup (G) II strains were associated with 94 (92.2%) of the NoV outbreaks, and GII.4 strains were predominantly associated with 57.6% (n=49) of NoV GII outbreaks. Four GII.4 variants (2006b, 2007, 2009 and 2012 variants) emerged and showed different contributions to NoV outbreak activity. The 2006b variant was predominantly associated with NoV outbreaks during the early years of the study period, and was subsequently displaced by the New Orleans 2009 variant, and most recently by the Sydney 2012 variant. In addition, the GII.2, GII.14, and GII.17 strains have recently been often associated with NoV AGE outbreaks. CONCLUSIONS: The emergence of new NoV GII.4 variants significantly affected the NoV outbreak activity in South Korea during the period from 2006 to 2013. The surveillance for new emerging strains affecting NoV outbreak activity should be intensified to develop an adequate policy to prevent further NoV outbreaks.

Expression of cysteine proteinase of Clonorchis sinensis and its use in serodiagnosis of clonorchiasis.

A gene encoding cysteine proteinase from Clonorchis sinensis has been cloned and expressed in Escherichia coli. The cysteine proteinase cDNA fragment was amplified by reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers derived from conserved active site of cysteine proteinases. The 5' and 3' regions of the gene were amplified using rapid amplification of cDNA ends. The cloned gene has an open reading frame of 696 bp and deduced amino acid sequence of 232. Sequence analysis and alignment showed significant homologies with the eukaryotic cysteine proteinases and conservation of the Cys, His, and Asp residues that form the catalytic triad. Analysis of the expressed protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the molecular weight of the protein was approximately 28.5 kDa. Proteolytic activity of the expressed protein was inhibited by cysteine proteinase inhibitors such as L-trans-epoxysuccinyl-leucylamide-(4-guanidino)-butane, iodoacetic acid, and leupeptin. The expressed protein showed biochemical properties similar to those of cysteine proteinases of other parasites. The expressed protein strongly reacted with the sera from patients with clonorchiasis but not with the sera from patients with paragonimiasis, fascioliasis, cysticercosis, and sparganosis, or with sera from normal human controls. These results suggest that the expressed protein may be valuable as a specific diagnostic material for the immunodiagnosis of clonorchiasis.

Molecular epidemiology of norovirus GII.4 variants in children under 5 years with sporadic acute gastroenteritis in South Korea during 2006-2013.

BACKGROUND: The global emergence of norovirus (NoV) GII.4 variants has raised public concerns in the world including South Korea since 1996. OBJECTIVE: We analyzed seasonality and genotypic pattern for sporadic cases by norovirus GII-4 variants. STUDY DESIGN: To determine the epidemic status of GII.4 variants in South Korea during 2006-2013, 7301 fecal specimens were collected from children who were younger than 5 years and had sporadic acute gastroenteritis (AGE). RESULTS: During the study period, NoVs were the most prevalent viral agent, detected in 877 (12.0%) of the 7301 fecal specimens from children with sporadic AGE. NoV GII strains predominantly accounted for 97.6% of all sporadic NoV infections. NoV GII.4 was the most prevalent genotype and comprised 67.6% of the NoV GII strains. However, seasonal prevalence of GII.4 strains varied depending on the spread of GII.4 variants. GII.4-2006b variant most predominantly circulated from 2006-2007 to 2009-2010 and persisted during other seasons. GII.4-2009 variant was first detected in January 2010 and predominant in 2011-2012. However, it was rapidly displaced by GII.4-2012 variant, which emerged in May 2012 and substantially circulated in 2012-2013. CONCLUSIONS: The frequent emergence and rapid spread of GII.4 variants significantly affect the magnitude of sporadic NoV infections in children. Hence, to minimize the disease burden of NoV infections, GII.4 strains should be considered as a primary target for vaccine development against NoVs.