Comparative study of the geographical spread of genogroup II porcine norovirus and human norovirus.

Livestock pigs and porcine norovirus could be candidate tools for future studies on the geographic isolation of norovirus. In this study, we provide the first evidence for geographic isolation of the host as a determinant of the distribution of subgenotypes of the porcine norovirus genogroup II (GII) genotype 11. Environmental water samples were collected from peri-urban streams and estuaries in South Korea between 2014 and 2020. In total, 488 GII region C sequences of norovirus open reading frame 2 were isolated. A total of 14 genotypes were detected, two of which (GII.11 and GII.18) corresponded to porcine norovirus. Five human norovirus genotypes (GII.2, GII.3, GII.4, GII.6, and GII.17) and one porcine norovirus genotype (GII.11) comprised the subgenotypes. Integrated analysis of seasonal and geographical factors revealed that the possibility of the co-emergence of different GII.11 subgenotypes in the same province was lower than that of human norovirus subgenotypes in the same province. Additional algorithms designed to eliminate potential biases further supported the estimated restricted geographical spread of the GII.11 subgenotypes. Fecal contamination source tracking revealed low detection rates of porcine norovirus in the absence of upstream pig farms. These results suggest that a one-sided viral transmission route, mainly dependent on indirect contact owing to the limited chance of direct contact between geographically separated livestock pig populations, may be responsible for the restricted geographical spread of the GII.11 subgenotypes.

Pairing of Parental Noroviruses with Unequal Competitiveness Provides a Clear Advantage for Emergence of Progeny Recombinants.

Genetic recombination plays a pivotal role in the appearance of human norovirus recombinants that cause global epidemics. However, the factors responsible for the appearance of these recombinants remains largely unknown. In this study, we revealed a selective pressure that restricts parental combinations leading to the emergence of norovirus recombinants. To investigate traces of emerging novel recombinants and their parents in the human population, we isolated mass nucleotide sequence clones of human norovirus genogroups I and II in sewage-affected waters over a 4-year sampling period. Fourteen different phylogenetic combinations of recombinants and their parents were defined from the dozens of phylogenetic lineages circulating in the human population. To evaluate the probability of these combinations, parental lineages of each recombinant were categorized into two groups as HP (relatively higher-competitiveness parents) and LP (relatively lower-competitiveness parents), according to their relative detection frequency. Strong categorization of HP and LP was confirmed by tests with modified data and additional variables. An algorithm that was developed in this study to visualize the chance of mixed infection between parents revealed that HP lineages have a higher chance of mixed infection than LP lineages in the human population. Three parental pairing types in recombinants were defined: HP-HP, HP-LP, and LP-LP. Among these, most recombinants were identified as HP-LP, despite the prediction of dominant emergence of HP-HP-type recombinants. These results suggest that nature favors recombinants of human norovirus that originate from parental pairing of heterogeneous competitiveness.IMPORTANCE Novel recombinants, generated from inter- and intraspecies recombination of norovirus lineages, often emerge and pose a threat to public health. However, the factors determining emergence of these particular recombinants from all possible combinations of parental lineages remain largely unknown. Therefore, current investigations on these recombinants are inevitably limited to postepidemic analyses, which merely identify genetic or phenotypic changes in the newly emerged recombinants compared to their parents. Here, we provide a new theoretical concept that emergence of novel recombinants could be explained by a combination of parental noroviruses thriving in the human population and those circulating at lower levels. This study could provide an additional and important rationale for the proactive environmental monitoring of potential future epidemics due to viral recombinants.

Male-Specific and Somatic Coliphage Profiles from Major Aquaculture Areas in Republic of Korea.

Human and animal feces are important sources of various types of microbial contamination in water. Especially, enteric viruses, the major agents of waterborne infection, can attain long-term survival in water environments due to their strong resistance to various environmental factors including pH, salinity, and temperature. Coliphages are promising viral indicators for fecal contamination in water environments. Here, we investigated the seasonal and spatial distribution of male-specific and somatic coliphages in surface water and seawater at three major aquaculture areas, including Goseong Bay, Aphae Island, and Gomso Bay, in Republic of Korea over a period of 1 year. We selected 6 surface water and 14 seawater sampling sites for each study area and collected a total of 480 water samples from March 2014 to February 2015. Overall, surface water samples contained higher occurrences of coliphages than seawater samples. The high coliphage concentrations were detected in spring (March to May 2014). The differences in geographical features and patterns in land usage of the three aquaculture areas may have affected the coliphage concentration and occurrence. Moreover, environmental factors such as cumulative precipitation were strongly correlated with coliphage concentrations. Therefore, we suggest that further longitudinal studies on coliphage concentrations and distributions should be performed to support the application of coliphages in tracking fecal contamination in water.

Peroxiredoxin 6 Inhibits Osteogenic Differentiation and Bone Formation Through Human Dental Pulp Stem Cells and Induces Delayed Bone Development.

Aims: Peroxiredoxins (PRDXs) are thiol-specific antioxidant enzymes that regulate redox balance that are critical for maintaining the cellular potential for self-renewal and stemness. Stem cell-based regenerative medicine is a promising approach in tissue reconstruction. However, to obtain functional cells for use in clinical applications, stem cell technology still requires improvements. Results: In this study, we found that PRDX6 levels were decreased during osteogenic differentiation in human dental pulp stem cells (hDPSCs). hDPSCs stably expressing Myc-PRDX6 (hDPSC/myc-PRDX6) inhibited cell growth in hDPSCs during osteogenic differentiation and impaired osteogenic phenotypes such as alkaline phosphatase (ALP) activity, mineralized nodule formation, and osteogenic marker genes [ALP and osteocalcin (OCN)]. hDPSC cell lines stably expressing mutant glutathione peroxidase (PRDX6(C47S)) and independent phospholipase A2 (PRDX6(S32A)) were also generated. Each mutant form of PRDX6 abolished the impaired osteogenic phenotypes, the transforming growth factor-ß-mediated Smad2 and p38 pathways, and RUNX2 expression. Furthermore, in vivo experiments revealed that hDPSC/myc-PRDX6 suppressed hDPSC-based bone regeneration in calvarial defect mice, and newborn PRDX6 transgenic mice exhibited delayed bone development and reduced RUNX2 expression. Innovation and Conclusion: These findings illuminate the effects of PRDX6 during osteogenic differentiation of hDPSCs, and also suggest that regulating PRDX6 may improve the clinical utility of stem cell-based regenerative medicine for the treatment of bone diseases. Antioxid. Redox Signal. 30, 1969-1982.

Use of coliphages to investigate norovirus contamination in a shellfish growing area in Republic of Korea.

A number of severe norovirus outbreaks due to the consumption of contaminated shellfish have been reported recently. In this study, we evaluated the distribution of coliphage densities to determine their efficacy as fecal indicators of enteric viruses, including noroviruses, in water samples collected from a shellfish growing area in Republic of Korea over a period of approximately 1 year. Male-specific and somatic coliphages in water samples were analyzed using the single agar layer method, and norovirus genogroups I and II, which infect mainly humans, were analyzed using duplex reverse transcription quantitative PCR. Male-specific and somatic coliphages were detected widely throughout the study area. Several environmental parameters, including salinity, precipitation, temperature, and wind speed were significantly correlated with coliphage concentrations (P < 0.05). Moreover, the concentrations of male-specific coliphages were positively correlated with the presence of human noroviruses (r = 0.443; P < 0.01). The geospatial analysis with coliphage concentrations using a geographic information system revealed that densely populated residential areas were the major source of fecal contamination. Our results indicate that coliphage monitoring in water could be a useful approach to prevent norovirus contamination in shellfish.

Host-Specific Bacteroides Markers-Based Microbial Source Tracking in Aquaculture Areas.

Various waterborne pathogens originate from human or animal feces and may cause severe gastroenteric outbreaks. Bacteroides spp. that exhibit strong host- or group-specificities are promising markers for identifying fecal sources and their origins. In the present study, 240 water samples were collected from two major aquaculture areas in Republic of Korea over a period of approximately 1 year, and the concentrations and occurrences of four host-specific Bacteroides markers (human, poultry, pig, and ruminant) were evaluated in the study areas. Host-specific Bacteroides markers were detected widely in the study areas, among which the poultry-specific Bacteroides marker was detected at the highest concentration (1.0-1.2 log10 copies L-1). During the sampling period, high concentrations of host-specific Bacteroides markers were detected between September and December 2015. The host-specific Bacteroides marker-combined geospatial map revealed the up-to-downstream gradient of fecal contamination, as well as the effects of land-use patterns on host-specific Bacteroides marker concentrations. In contrast to traditional bacterial indicators, the human-specific Bacteroides marker correlated with human specific pathogens, such as noroviruses (r=0.337; P<0.001). The present results indicate that host-specific Bacteroides genetic markers with an advanced geospatial analysis are useful for tracking fecal sources and associated pathogens in aquaculture areas.

Occurrence of novel GII.17 and GII.21 norovirus variants in the coastal environment of South Korea in 2015.

Human norovirus (HNoV), a positive-sense RNA virus, is the main causative agent of acute viral gastroenteritis. Multiple pandemic variants of the genogroup II genotype 4 (GII.4) of NoV have attracted great attention from researchers worldwide. However, novel variants of GII.17 have been overtaking those pandemic variants in some areas of East Asia. To investigate the environmental occurrence of GII in South Korea, we collected water samples from coastal streams and a neighboring waste water treatment plant in North Jeolla province (in March, July, and December of 2015). Based on capsid gene region C analysis, four different genotypes (GII.4, GII.13, GII.17, and GII.21) were detected, with much higher prevalence of GII.17 than of GII.4. Additional sequence analyses of the ORF1-ORF2 junction and ORF2 from the water samples revealed that the GII.17 sequences in this study were closely related to the novel strains of GII.P17-GII.17, the main causative variants of the 2014-2015 HNoV outbreak in China and Japan. In addition, the GII.P21-GII.21 variants were identified in this study and they had new amino acid sequence variations in the blockade epitopes of the P2 domain. From these results, we present two important findings: 1) the novel GII.P17-GII.17 variants appeared to be predominant in the study area, and 2) new GII.21 variants have emerged in South Korea.

Re-emergence of a GII.4 Norovirus Sydney 2012 Variant Equipped with GII.P16 RdRp and Its Predominance over Novel Variants of GII.17 in South Korea in 2016.

Noroviruses are major causative pathogen of nonbacterial acute gastroenteritis worldwide. Of the seven genogroups of noroviruses suggested recently, genogroup II genotype 4 (GII.4) had been the most common genotype identified in hospitalized patients in the last few decades. However, since the latter half of 2014, new variants of GII.17 have been reported as the main causes of outbreaks over GII.4 in East Asia and have also occurred in America and Europe. In this study, we monitored norovirus GII in coastal streams at South Gyeongsang province and South Jeolla province of South Korea from March 2015 to May 2016. Norovirus GII.17 capsid sequences were predominantly detected until September 2015 in water samples. However, we found that the number of positive cases of the norovirus GII.4 Sydney 2012 capsid sequence has been increasing since December 2015, overtaking that of GII.17 in 2016. The RdRp genotype of this predominant GII.4 variant in 2016 was identified as GII.P16. The emergence and predominance of the GII.4 pandemic capsid sequence harboring a different RdRp genotype suggested the potential for a future pandemic.

Distribution of Human Norovirus in the Coastal Waters of South Korea.

The presence of human norovirus in the aquatic environment can cause outbreaks related to recreational activities and the consumption of norovirus-contaminated clams. In this study, we investigated the prevalence of norovirus genogroups I (GI) and II (GII) in the coastal aquatic environment in South Korea (March 2014 to February 2015). A total of 504 water samples were collected periodically from four coastal areas (total sites = 63), of which 44 sites were in estuaries (clam fisheries) and 19 were in inflow streams. RT-PCR analysis targeting ORF2 region C revealed that 20.6% of the water samples were contaminated by GI (13.3%) or GII (16.6%). The prevalence of human norovirus was higher in winter/spring than in summer/fall, and higher in inflow streams (50.0%) than in estuaries (7.9%). A total of 229 human norovirus sequences were identified from the water samples, and phylogenetic analysis showed that the sequences clustered into eight GI genotypes (GI.1, 2, 3, 4, 5, 6, 7, and 9) and nine GII genotypes (GII.2, 3, 4, 5, 6, 11, 13, 17, and 21). This study highlighted three issues: 1) a strong correlation between norovirus contamination via inflow streams and coastal areas used in clam fisheries; 2) increased prevalence of certain non-GII.4 genotypes, exceeding that of the GII.4 pandemic variants; 3) seasonal shifts in the dominant genotypes of both GI and GII.

Epidemiology and virologic investigation of human enterovirus 71 infection in the Republic of Korea from 2007 to 2012: a nationwide cross-sectional study.

BACKGROUND: Enterovirus (EV) 71 is the main pathogen associated with hand, foot and mouth disease (HFMD) or herpangina. Outbreaks of HFMD caused by EV71 infection are associated severe neurological disease and high mortality rates in children. Several sporadic cases of EV71 infection occurred in the Republic of Korea (ROK) in 2000, and EV71 infections were not reported thereafter until 2006. In this prospective study, we report the epidemic and virologic characteristics of the EV71 endemic from 2007 to 2012 in the Republic of Korea. METHODS: We analyzed characteristics of the EV71 infection-associated epidemic from collected specimens and clinical information from 9987 patients with suspected EV infection from the National EV Surveillance System in ROK. To identify the EV71 subgenotype, the homology of viral protein 1 sequences obtained using reverse transcription polymerase chain reaction was compared with the sequences on other countries available from GenBank database. RESULTS: EV71 was detected in 585 (16.7 %) specimens (cerebrospinal fluid, stool or rectal swabs, throat swabs and blood) during study period and was most frequently observed during epidemic seasons in 2009-2012. Major manifestations due to EV71 infection were HFMD (62.2 %) and HFMD with severe neurological complications (28.4 %). Five deaths (0.9 %) due to EV71 infection occurred, with an increased mortality rate during the period after 2009. Most patients (476; 81.4 %) were less than 5 years of age. Analysis of the monthly distribution showed that there was an obvious seasonal pattern to the epidemics, with infections appearing from June to August. The major subgenotype of EV71 isolates circulating in ROK was the C4a strain, which has also appeared in China, Japan and Vietnam. CONCLUSIONS: This surveillance provided valuable data on the epidemic characteristics of EV71 infections in ROK during a 6-year period. Our findings provide data to assist during future outbreaks of EV71 and associated acute neurologic disease.

Constant up-regulation of BiP/GRP78 expression prevents virus-induced apoptosis in BHK-21 cells with Japanese encephalitis virus persistent infection.

BACKGROUND: Persistent infection of the Japanese Encephalitis Virus (JEV) has been reported in clinical cases, experimental animals, and various cell culture systems. We previously reported the establishment of spontaneous JEV persistent infection, assisted by defective interfering particle accumulation and/or attenuated helper viruses, in BHK-21 cells devoid of virus-induced apoptosis, cBS6-2 and cBS6-3. However, cell-specific factors may play important roles in controlling JEV replication and have never been assessed for this specific phenomenon. Recent evidence suggests that viruses have evolved various mechanisms to cope with endoplasmic reticulum stress signaling pathways for their efficient amplification and transmission, including the unfolded protein response (UPR). RESULTS: To identify the host cell factors that affect JEV persistence, we investigated the expression of essential UPR factors in cBS6-2 and cBS6-3 cells. Of the selected UPR factors tested, the most noticeable deviations from those of the normal BHK-21 cells with JEV acute infection were as follows: the suppression of C/EBP homologous binding protein (CHOP) and the constant up-regulation of immunoglobulin binding protein (BiP) expression in cBS6-2 and cBS6-3 cells. In JEV acute infection on normal BHK-21 cells, silencing CHOP expression through specific siRNA blocked cell death almost completely. Meanwhile, depletion of BiP by specific siRNA unlocked CHOP expression in cBS6-2 and cBS6-3 cells, resulting in massive cell death. Fulminant apoptotic cell death for both cell clones on tunicamycin treatment revealed that the JEV persistently infected cells still contained functional arms for cell fate decisions. CONCLUSIONS: BHK-21 cells with JEV persistent infection strive against virus-induced apoptosis through constant up-regulation of BiP expression, resulting in the complete depletion of CHOP even with apparent virus amplification in the cells. Accordingly, the attenuation of virus replication as well as the modifications to cell metabolism could be additional factors contributing to the development of JEV persistent infection in mammalian cells.

A national cross-sectional study for poliovirus seroprevalence in the Republic of Korea in 2012: implication for deficiency in immunity to polio among middle-aged people.

BACKGROUND: A worldwide poliomyelitis eradication program was initiated in 1988; however, strains of wild poliovirus (WPV) are still endemic in some countries. Until WPV transmission is eradicated globally, importation and outbreaks of WPV are alarming possibilities. This study is the first report to document the polio immunity after 2004, when an inactivated polio vaccine (IPV) was introduced in the Republic of Korea. METHODS: A total of 745 serum samples from randomly selected patients ranging from 6 to 84 years of age were used for neutralization tests, performed in the World Health Organization polio national reference laboratory. RESULTS: Among the 745 tested sera, 439 (58.9%) were seropositive and 19 (2.6%) were seronegative to all PV serotypes. In all age groups, PV3 showed the lowest level of seroprevalence, at 509 cases (68.3%), compared to 616 (82.7%) for PV1 and 685 (91.9%) for PV2. In the 6-10-year age group, which included IPV-immunized children, the highest seropositive rate was observed and the difference in seroprevalence between PV3 and other serotypes was the lowest compared to the other age groups immunized with oral PV vaccines (OPV). In addition, the seronegative rates of all three PV types in children aged 6-10 in this study were found to be lower than those in OPV-immunized children reported in a previous study from the Republic of Korea. Meanwhile, middle-aged subjects (41-60 years) had the lowest seroprevalence and geometric mean titer. CONCLUSIONS: This study indicates a deficiency in immunity to PV in middle-aged individuals, and low seroprevalence to PV3 in all age groups. In addition, due to the ongoing risk of importing PV, middle-aged people should consider PV vaccination before visiting a PV-endemic country. Our findings provide data to assist those involved in deciding future national polio vaccination strategies for the maintenance of a polio-free status in Korea.

A metal-free tandem C-C/C-O bond formation approach to densely functionalized indolyl 4H-chromenes catalyzed by polystyrene-supported p-toluenesulfonic acid under solvent-free conditions.

A new environmentally benign and highly convergent protocol for the synthesis of indolyl 4H-chromene derivatives has been developed. This one-pot three-component condensation reaction of salicylaldehyde, cyclic 1,3-diketones, and indole is promoted by PS-PTSA as a reusable heterogeneous acid catalyst under solvent-free conditions. This protocol demonstrates several notable advantages such as that the catalyst is readily available and can be recovered and reused for at least five runs without any significant impact on product yields, high atom economy, excellent yields, and efficiency of producing three new bonds (two C-C and one C-O) and one stereo center in a single operation.

Extended stability of cyclin D1 contributes to limited cell cycle arrest at G1-phase in BHK-21 cells with Japanese encephalitis virus persistent infection.

There is increasing evidence that many RNA viruses manipulate cell cycle control to achieve favorable cellular environments for their efficient replication during infection. Although virus-induced G0/G1 arrest often delays early apoptosis temporarily, a prolonged replication of the infected virus leads host cells to eventual death. In contrast, most mammalian cells with RNA virus persistent infection often escape cytolysis in the presence of productive viral replication. In this study, we demonstrated that the extended endurance of cyclin D1 was clearly associated with the suppression of glycogen synthase kinase-3ß (GSK-3ß) expression in BHK-21 cells that are persistently infected with Japanese encephalitis virus (JEV). The G0/G1 arrest of these cells turned much loose compared to the normal BHK-21 cells with JEV acute infection. After cycloheximide treatment, cyclin D1 in the persistently infected cells lasted several hours longer than those in acutely infected cells. Furthermore, both p21(Cip1) and p27(Kip1), positive regulators for cyclin D1 accumulation in the nucleus, were suppressed in their expression, which contrasts with those in JEV acute infection. Inhibition of the GSK-3ß by lithium chloride treatment rescued a significant number of cells from cytolysis in JEV acute infection, which coincided with the levels of cyclin D1 that escaped from proteolysis. Therefore, the limitation of G1/S arrest in the BHK-21 cells with JEV persistent infection is associated with the suppression of GSK-3ß expression, resulting in the extended duration of cyclin D1.

Clinical and enterovirus findings associated with acute flaccid paralysis in the Republic of Korea during the recent decade.

Acute flaccid paralysis (AFP) is described as sudden onset of flaccid paralysis in one or more limbs in children caused by polioviruses (PVs). PV eradication is achieved through intensive immunization and AFP attentive surveillance, according to the World Health Organization. Since 1998, the Korea Centers for Disease Control and Prevention has conducted surveillance system. This is an overview of surveillance in the Republic of Korea during the 10-year period from 2002 to 2011. The surveillance system for wild PV eradication was conducted through reporting and laboratory testing. Cell culture isolates were identified by neutralization tests using standard polyclonal antisera typing. The molecular methods were used for further characterization to improve specificity. For genotyping, semi-nested RT-PCR was used to amplify part of the viral protein 1 gene. Patients below 5 years of age accounted for the largest proportion of cases, and a positive association between age and incidence was found. In the total 285 cases, Guillain-Barré syndrome was the major leading causes of AFP. Non-polio enterovirus was detected in some AFP patients. EV71 was detected in 21 cases and Coxsackievirus (C) A2, CA6, CA9, CB2, CB3, CB4, CB5, and Echovirus (E) 25, E30, Sabin strain polio 2, polio 1 and 3 were also detected in some patients. The present study represents a comprehensive 10-year country-based survey of AFP in the Republic of Korea. This surveillance could provide better understanding of the epidemiologic pattern, and clinical manifestations associated with specific genotypes of AFP in the Republic of Korea.

Nationwide surveillance for pathogenic microorganisms in groundwater near carcass burials constructed in South Korea in 2010.

Widespread outbreaks of foot-and-mouth disease and avian influenza occurred in South Korea during 2010. In response to the culling of many animals to attenuate the spread of disease, South Korea used mass burial sites to dispose of the large number of carcasses; consequently, concerns about groundwater contamination by leachate from these burial sites are increasing. Groundwater is one of the main sources of drinking water, and its cleanliness is directly related to public health. Thus, this study aimed to evaluate the safety of groundwater around the burial sites (total of 600 sites). A total of 1,200 groundwater samples were collected though the country, and microbial analysis was conducted during two time periods: during the spring (n = 600; April to June 2012) and after rainfall (n = 600; August to October, 2012; fall). Fecal coliform and Escherichia coli were detected in 173 (14.4%) and 85 (7.1%) of the 1,200 samples, respectively. Salmonella spp. and Shigella spp. each were detected only once (0.083%). Clostridium perfringens was detected from 7 groundwater samples (0.583%), and E. coli O157:H7 was not detected. With respect to norovirus, only the GII type was detected from six groundwater samples (0.5%), and enterovirus was detected in 15 groundwater samples (1.25%). The frequency of E. coli that we detected was lower than that found in previous studies conducted in South Korea, but we detected higher frequency of fecal coliform than that observed in a previous report. The contamination frequencies of Salmonella spp. and Shigella spp. were very low, but C. perfringens, which could be an indicator of fecal pollution, was detected in seven regions. Overall, the results of the present study indicate a low possibility of contamination from burial sites. However, consistent monitoring is required to prevent microbial contamination of groundwater near the burial sites.

Integrative effect of defective interfering RNA accumulation and helper virus attenuation is responsible for the persistent infection of Japanese encephalitis virus in BHK-21 cells.

Persistence of RNA viruses is often, but not always, associated with the production of defective interfering (DI) particles. To investigate possible roles of DI particles and helper viruses in RNA virus persistence, persistent infection with Japanese encephalitis virus (JEV) was established in baby hamster kidney (BHK-21) cells. At the 6th and 7th serial undiluted passages of JEV on BHK-21 cells, viral persistence was established spontaneously with DI RNA generation. Seven cell clones exhibiting persistent infection were obtained from the initial BHK-21 cell batches exhibiting JEV persistence, and maintained for over 400 days. Most cell clones produced infectious particles (10(1) -10(5) PFU/ml) continuously, expressed viral proteins, and resisted homologous superinfection. Two helper viruses, chvBS6-3 and chvBS7-1, were isolated from two of the seven cell clones, and characterized to investigate their roles in JEV persistence. While chvBS6-3 was restored to its full cytopathicity in the absence of DI RNA, chvBS7-1 exhibited almost no cytopathicity, regardless of DI RNA co-replication. Attenuation of chvBS7-1 did not appear to be due to inadequate adsorption or genome replication, but due to inefficient egress of the assembled progeny virions, suggesting altered helper virus emergence during JEV persistence in BHK-21 cells. These observations suggest that at least two mechanisms are involved in JEV persistence; a DI RNA-dependent mechanism, where DI RNA co-replication nullifies the helper virus's cytopathicity, or a DI RNA-independent mechanism, where the helper virus is self-attenuated. This study provides a useful in vitro tool for understanding the mechanisms underlying RNA virus persistent infections.

Molecular characteristics of human coxsackievirus B1 infection in Korea, 2008-2009.

This study was performed to analyze epidemiological and molecular characteristics of coxsakievirus (CV) B1 infection associated with severe neonatal illness cases and death in Korea during 2008-2009. Through a nationwide surveillance program, specimens were collected from 104 patients infected with CVB1. The detection of enteroviruses (EVs) from specimens was subjected to a diagnostic real-time polymerase chain reaction (RT-PCR) in the 5'-non-coding region (NCR). A semi-nested PCR was conducted to amplify sequences from the VP1 region and sequence comparison was performed with reference strains registered in Genbank. Male-to-female ratio confirmed approximately 5:4. The major clinical manifestation of patients infected with CVB1 was aseptic meningitis (55.8%). The other clinical symptoms were herpangina or hand-foot-mouth disease (22.1%) and neonatal sepsis (7.7%). The sequences of CVB1 isolates were divided into four genetic clusters (A-D) with at least 15% diversity between the clusters. Almost all the CVB1 isolates in Korea from 2008 to 2009 were in cluster D (except for 2 cases). The homology relationship was also similar between the Korean CVB1 strains and US strain (above 93%). It is possible that Korean CVB1 isolates found during 2008-2009 originated from the US strains found during 2006-2008. The identification of CVB1 in South Korea shows the potential of EVs to cause serious disease in an unpredictable fashion.

Reliability of non-culturable virus monitoring by PCR-based detection methods in environmental waters containing various concentrations of target RNA.

Owing to the lack of practical cell culture system for human noroviruses (HuNoV), various detection methods based on conventional reverse transcription-PCR (RT-PCR) and the quantitative real-time PCR have been major tools for monitoring environmental water safety. In this study, we showed that the proportion of water sample concentrates used for one-step RT-PCR significantly influences false-negative findings of the non-culturable viruses. In total, 59 archived samples of previously analyzed water concentrates were reexamined for HuNoV RNA by the one-step RT-PCR and semi-nested PCR. Using new aliquots for RNA extraction for every trial, up to 20 PCR trials were performed for each archive to determine whether the crosscheck results supported the previous determinations. We reconfirmed that 27.6% (8/29) of the samples were HuNoV-positive samples: 6.7% (1/15) from groundwater, 33.3% (3/9) from river water, and 80% (4/5) from treated sewage effluent (TSE). These results corresponded to the ratio of previously negative HuNoV samples now identified as positive (8/30): 6.7% (1/15) from groundwater, 20% (1/5) from river water, and 60% (6/10) from TSE. To elucidate the cause of these results, 16 different concentrations of murine norovirus (MNV) RNA (from 2×10(2) to 8×10(3) copies, divided into 10 tubes for each concentration) were subjected to one-step RT-PCR. The detection frequency and reproducibility decreased sharply when the number of MNV RNA copies fell below threshold levels. These observations suggest that the proportion of water concentrate used for PCR-based detection should be considered carefully when deciding viral presence in certain types of environmental water, particularly in regard with legal controls.

Outcomes after unrestricted use of everolimus-eluting and sirolimus-eluting stents in routine clinical practice: a multicenter, prospective cohort study.

BACKGROUND: It remains unclear whether there are differences in the safety and efficacy outcomes between everolimus-eluting stents (EES) and sirolimus-eluting stents (SES) in contemporary practice. METHODS AND RESULTS: We prospectively enrolled 6166 consecutive patients who received EES (3081 patients) and SES (3085 patients) between April 2008 and June 2010, using data from the Interventional Cardiology Research In-Cooperation Society-Drug-Eluting Stents Registry. The primary end point was a composite of death, nonfatal myocardial infarction (MI), or target-vessel revascularization (TVR). At 2 years of follow-up, the 2 study groups did not differ significantly in crude risk of the primary end point (12.1% for EES versus 12.4% for SES; HR, 0.97; 95% CI, 0.84-1.12, P=0.66). After adjustment for differences in baseline risk factors, the adjusted risk for the primary end point remained similar for the 2 stent types (HR, 0.96; 95% CI, 0.82-1.12, P=0.60). There were also no differences between the stent groups in the adjusted risks of the individual component of death (HR, 0.93; 95% CI, 0.67-1.30, P=0.68), MI (HR, 0.97; 95% CI, 0.79-1.18, P=0.74), and TVR (HR, 1.10; 95% CI, 0.82-1.49, P=0.51). The adjusted risk of stent thrombosis also was similar (HR, 1.16; 95% CI, 0.47-2.84, P=0.75). CONCLUSIONS: In contemporary practice of percutaneous coronary intervention procedures, the unrestricted use of EES and SES showed similar rates of safety and efficacy outcomes with regard to death, MI, sent thrombosis, and TVR. Future longer-term follow-up is needed to better define the relative benefits of these drug-eluting stents. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01070420.