Prevalence of Parainfluenza Viruses in Seoul, 2021 - 2022.

BACKGROUND: Parainfluenza virus (PIV) is a significant etiological agent of acute lower respiratory tract infections (ALRIs) in infants and young children. The present study has been conducted to investigate the prevalence of recently identified respiratory viruses. METHODS: In total, 543 oropharyngeal or nasopharyngeal swab samples collected from hospitalized patients with acute respiratory symptoms (ARS) between January and December 2021 (5,653 females and 4,950 males) were tested for respiratory viruses using RT-PCR. RESULTS: At least one respiratory virus was detected by RT-PCR in 119 out of 175 samples (68%). The most frequently detected virus was human rhinovirus (HRV) (34, 6.5%), followed by human parainfluenza viruses (HPIVs) (19, 3.6%), human bocavirus (HBoV) (8, 1.5%), human adenovirus (HAdV) (7, 1.3%), and human respiratory syncytial virus (HRSV) (4, 0.8%). HPIV-3 accounted for 3.6% (19/175) of all viral pathogens and was the second most frequently detected viral pathogen in our study. HPIV-3 infections peaked in the fall (November) of 2021. Phylogenetic analysis of the coding region of the viral protein HA revealed that all 35 (100%) of 35 HPIV-infected patients were infected with HPIV-3. CONCLUSIONS: HPIV was an important causative pathogen associated with ALRI in children hospitalized in Korea in the late fall of 2021, as the social distancing rules for COVID-19 were relaxed. These findings highlight the im-portance of HPIV as a cause of ALRI.

Quantitative differential analysis of norovirus outbreak samples using RT-ddPCR.

Noroviruses cause acute gastroenteritis with symptoms of diarrhoea and vomiting, and their high infectivity allows outbreaks to readily occur. Quickly identifying and isolating potential contaminants is an effective method to prevent the spread of outbreaks. A total of 376 samples collected from nine outbreaks were categorized as either patient, asymptomatic individual, cook or environmental samples, according to the source of contamination. Using real-time PCR and sequencing analysis, norovirus GII genotypes were detected in 34·9% of samples from patients, 19·2% from asymptomatic individuals, 2·4% from the environment and 1·4% from cooks. Our findings showed contrasting results in samples categories quantified based on the limit of blank and detection limit by reverse transcription droplet digital PCR, which is a more sensitive testing method than real-time-PCR.

Field study of the indoor environments for preventing the spread of the SARS-CoV-2 in Seoul.

Despite the prolonged global spread of COVID-19, few studies have investigated the environmental influence on the spread of SARS-CoV-2 RNA with a metropolitan scale, particularly the detection of SARS-CoV-2 after disinfection at multi-use facilities. Between February 2020 and January 2021, 1,769 indoor air samples and object surfaces were tested at 231 multi-use facilities where confirmed cases were known to have occurred in Seoul, to determine whether SARS-CoV-2 RNA could be detected even after disinfection. Samples were collected by air scanner and swab pipette and detected by real-time RT-PCR. As a result, 10 (0.56%) positive samples were detected despite disinfection. The common environmental features of all 10 were surfaces that contained moisture and windowless buildings. With the aim of preventing the spread of COVID-19, from January to February 2021, we next conducted 643 preemptive tests before the outbreak of infections at 22 multi-use facilities where cluster infections were frequent. From these preemptive inspections, we obtained five (0.78%) positive results from two facilities, which enabled us to disinfect the buildings and give all the users a COVID-19 test. Based on the study purpose of finding and investigating cases of positive detection even after disinfection in the field through long-term environmental detection in a large city, our preemptive investigation results helped to prevent the spread of infectious diseases by confirming the potential existence of an asymptomatic patient.

Phylogenetic characterization of norovirus strains detected from sporadic gastroenteritis in Seoul during 2014-2016.

BACKGROUND: Phylogenetic analysis of norovirus (NoV) is efficient for tracking NoV transmission. To determine the widespread NoV strains in Seoul, we conducted an extensive phylogenetic characterization of NoV-positives from 1659 diarrheal specimens collected in 2014-2016 for the Seoul NoV-surveillance. RESULTS: When the large numbers of NoV partial VP1 genome sequences were analyzed in acute gastroenteritis patients along with the phylogenetic characterization, we could identify molecular epidemiologic patterns based on the genetic characteristics of sporadic NoV strains circulating in Seoul, which could provide a detailed description of the genome-wide and community-wide NoV evolution in each genotype. The average NoV detection rate in our study period was 16.34% that was increased by 7.44% from 13.17% in 2014 to 20.61% in 2016. Prevalence of NoV GI and GII was 4.43% and 93.36%, respectively, and the GII.4, GII.17, and GII.3 were found to be the major type among 17 genotypes of NoV. The most prevalent one was GII.4 (50.92%) that was followed by GII.17 (18.08%) and GII.3 (9.96%). According to an extensive phylogenetic analysis based on partial VP1 sequences of 1008 NoV (276 sporadic, 518 outbreak and 214 reference), pandemic strains of GII.17, GII.4 and GII.3 have emerged in succession during the 2014-2016 Seoul NoV-surveillance. GII.17 emerged as GII.17|Kawasaki323 in 2014, and became the predominant genotype in 2015 with GII.17|2014_Kawasaki lineages (CUHK-NS-616/Kawasaki308). The formerly predominant GII.4 remained high-level with GII.4|2012_Sydney in 2014 and internally replaced to GII.4|2016_Kawasaki194 lineage (NOR-2565/NOR-2558/OH16002) that caused the sporadic NoV explosion since December 2015. Sporadically prevalent GII.3|Hu/Aichio334-13/2013 failed to develop any outbreaks, whereas sporadic GII.3|Hu/3-28/2015/HNZZ/CHN caused heavy outbreaks in Seoul without preparation time since November 2016. CONCLUSIONS: This is the first extensive phylogenetic study revealing the important events of NoV strains circulating in Seoul. Particularly, our study period from 2014 to 2016 was very dynamic with the emergences of the three main NoV strains (GII.17|2014_Kawasaki, GII.4|2016_Kawasaki194 and GII.3|Hu/3-28/2015/HNZZ/CHN) every year. We are sure that it is hard to detect above findings by simple conventional analysis. Our present study reports a future paradigm of the NoV molecular epidemiology, which might be highly valuable to track new strains and predict oncoming outbreaks.

Detection of Pathogenic Viruses in the Ambient Air in Seoul, Korea.

The possible transport of pathogenic microorganisms during Asian dust events could be an important concern for health workers; however, this is still uncertain owing to a lack of supporting evidence. The present study aimed to investigate the presence of pathogenic microorganisms in air samples collected during the Asian and non-Asian dust periods. Between March and September 2016, air samples were collected at three weather observation stations in Seoul using a high-volume air sampler. Multiplex PCR was performed using the Allplex™ respiratory and gastrointestinal panel assay kits to detect 46 microorganisms. RT-PCR was performed for klassevirus, Aichivirus, and human parechovirus (HPeV) detection. In total, 71 air samples were collected during the Asian (8 samples) and non-Asian (63 samples) dust events. During an Asian dust event, only one human rhinovirus (HRV)-positive air sample was collected on April 23. During the non-Asian dust period, HRV, HPeV, norovirus (NoV), enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), and Blastocystis hominis were detected in four, two, one, one, one, and one air samples, respectively. Pathogenic viruses were mostly detected in ambient air samples during the non-Asian dust period, which suggests a possible air-borne transmission of viral pathogens; however, the role of Asian dust in epidemics caused by pathogenic viruses is unclear.

Comparison of different methods to quantify fat classes in bakery products.

The definition of fat differs in different countries; thus whether fat is listed on food labels depends on the country. Some countries list crude fat content in the 'Fat' section on the food label, whereas other countries list total fat. In this study, three methods were used for determining fat classes and content in bakery products: the Folch method, the automated Soxhlet method, and the AOAC 996.06 method. The results using these methods were compared. Fat (crude) extracted by the Folch and Soxhlet methods was gravimetrically determined and assessed by fat class using capillary gas chromatography (GC). In most samples, fat (total) content determined by the AOAC 996.06 method was lower than the fat (crude) content determined by the Folch or automated Soxhlet methods. Furthermore, monounsaturated fat or saturated fat content determined by the AOAC 996.06 method was lowest. Almost no difference was observed between fat (crude) content determined by the Folch method and that determined by the automated Soxhlet method for nearly all samples. In three samples (wheat biscuits, butter cookies-1, and chocolate chip cookies), monounsaturated fat, saturated fat, and trans fat content obtained by the automated Soxhlet method was higher than that obtained by the Folch method. The polyunsaturated fat content obtained by the automated Soxhlet method was not higher than that obtained by the Folch method in any sample.

Antimicrobial resistance and resistance genes in Escherichia coli strains isolated from commercial fish and seafood.

The purpose of this study was to investigate the antimicrobial resistance and to characterize the implicated genes in Escherichia coli isolated from commercial fish and seafood. Fish and seafood samples (n=2663) were collected from wholesale and retail markets in Seoul, Korea between 2005 and 2008. A total of 179 E. coli isolates (6.7%) from those samples were tested for resistance to a range of antimicrobial agents. High rates of resistance to the following drugs were observed: tetracycline (30.7%), streptomycin (12.8%), cephalothin (11.7%), ampicillin (6.7%) and ticarcillin (6.1%). No resistances to amikacin, amoxicillin/clavulanic acid and cefoxitin were observed. Seventy out of 179 isolates which were resistant to one or more drugs were investigated by PCR for the presence of 3 classes of antimicrobial resistance genes (tetracycline, aminoglycosides and beta-lactams), class 1, 2 and 3 integrons. Gene cassettes of classes 1 and 2 integrons were further characterized by amplicon sequencing. The tetracycline resistance genes tetB and tetD were found in 29 (41.4%) isolates and 14 (20%) isolates, respectively. The beta-lactam resistance gene, bla(TEM) was found in 15 (21.4%) isolates. The aminoglycoside resistance gene, aadA was found in 18 (25.7%) isolates. Class 1 integron was detected in 41.4% (n=29) of the isolates, while only 2.9% (n=2) of the isolates were positive for the presence of class 2 integron. Two different gene cassettes arrangements were identified in class 1 integron-positive isolates: dfrA12-aadA2 (1.8 kb, five isolates) and aadB-aadA2 (1.6 kb, four isolates). One isolate containing class 2 integron presented the dfrA1-sat-aadA1 gene cassette array. These data suggest that commercial fish and seafood may act as the reservoir for multi-resistant bacteria and facilitate the dissemination of the resistance genes.

Enantioselective hydrolysis of racemic epichlorohydrin using an epoxide hydrolase from Novosphingobium aromaticivorans.

Previously we reported that an epoxide hydrolase (EHase) from Novosphingobium aromaticivorans could preferentially hydrolyze (R)-styrene oxide. In this study, we demonstrate that the purified NEH could be also effective in chiral resolution of racemic epichlorohydrin (ECH). Particularly, the purified NEH showed excellent hydrolyzing activity toward ECH to complete the reaction at a short period of incubation time. Enantiopure (S)-ECH could be obtained with a high enantiopurity of more than 99.99% enantiomeric excess (ee) and yield of 20.7% (theoretical, 50%). The chiral resolution of the purified NEH toward ECH was not susceptible to substrate inhibition by 500 mM racemic ECH.

Biocatalytic resolution of glycidyl phenyl ether using a novel epoxide hydrolase from a marine bacterium, Maritimibacter alkaliphilus KCCM 42376 [corrected].

As a continuous effort of developing highly enantioselective epoxide hydrolase from marine microorganisms, it was found that Maritimibacter alkaliphilus KCCM 42376 [corrected] was highly enantioselective toward racemic glycidyl phenyl ether (GPE). An open reading frame (ORF) encoding a putative epoxide hydrolase (EHase) was cloned from the genome of Maritimibacter alkaliphilus KCCM 42376 [corrected], followed by expression and purification in Escherichia coli. The purified EHase (REH) hydrolyzed (S)-GPE preferentially over (R)-GPE. Enantiopure (R)-GPE from kinetic resolution of 29.2 mM racemic GPE using the purified REH could be obtained with enantiopurity of more than 99.9% enantiomeric excess (ee) and 38.4% yield (theoretical, 50%) within 20 min (enantiomeric ratio (E-value): 38.4). The enantioselective activity of REH toward GPE was also confirmed by the analysis of the vicinal diol, 3-phenoxy-1,2-propanediol. To our knowledge, this study demonstrates the highest enantioselective resolution of racemic GPE using a purified biocatalyst among the known native EHases.

Cloning and characterization of an epoxide hydrolase from Novosphingobium aromaticivorans.

A gene encoding a putative epoxide hydrolase (EHase) was identified by analyzing an open reading frame of the genome sequence of Novosphingobium aromaticivorans, retaining the conserved catalytic residues such as the catalytic triad (Asp177, Glu328, and His355) and the oxyanion hole. The enantioselective EHase gene (neh) was cloned, and the recombinant EHase could be purified to apparent homogeneity by one step of metal affinity chromatography and further characterized. The purified N. aromaticivorans enantioselective epoxide hydrolase (NEH) showed enantioselective hydrolysis toward styrene oxide, glycidyl phenyl ether, epoxybutane, and epichlorohydrin. The optimal EHase activity toward styrene oxide occurred at pH 6.5 and 45 degrees C. The purified NEH could preferentially hydrolyze (R)-styrene oxide with enantiomeric excess of more than 99% and 11.7% yield after 20-min incubation at an optimal condition. The enantioselective hydrolysis of styrene oxide was also confirmed by the analysis of the vicinal diol, 1-phenyl-1,2-ethanediol. The hydrolyzing rates of the purified NEH toward epoxide substrates were not affected by as high as 100 mM racemic styrene oxide.

A cold-adapted epoxide hydrolase from a strict marine bacterium, Sphingophyxis alaskensis.

An open reading frame (ORF) encoding a putative epoxide hydrolase (EHase) was identified by analyzing the genome sequence of Sphingophyxis alaskensis. The EHase gene (seh) was cloned and expressed in E. coli. To facilitate purification, the gene was fused in-frame to 6x histidine at the C-terminus. The recombinant EHase (rSEH) was highly soluble and could be purified to apparent homogeneity by one step of metal affinity chromatography. The purified SEH displayed hydrolyzing activities toward various epoxides such as styrene oxide, glycidyl phenyl ether, epoxyhexane, epoxybutane, epichlorohydrin, and epifluorohydrin. The optimum activity toward styrene oxide was observed at pH 6.5 and 35 degrees . The purified SEH showed a cold-adapted property, displaying more than 40% of activity at low temperature of 10 degrees compared with the optimum activity. Despite the catalytic efficiency, the purified SEH did not hydrolyze various epoxides enantioselectively. Km and kcat of SEH toward (R)-styrene oxide were calculated as 4+/-0.3 mM and 7.42 s(-1), respectively, whereas Km and kcat of SEH toward (S)-styrene oxide were 5.25+/-0.3 mM and 10.08 s(-1), respectively.

Screening enantioselective epoxide hydrolase activities from marine microorganisms: detection of activities in Erythrobacter spp.

To develop an enantioselective epoxide hydrolase (EHase) from marine microorganisms, marine samples were collected from a variety of marine environments. Strains isolated by the capability of living on styrene oxide (SO) were screened for retaining enantioselective EHase activities toward SO by combining spectrophotometric, GC, and HPLC analysis. Consequently, one strain, JCS358, was selected, and the sequence analysis of 16S rRNA gene showed that the strain belonged to Erythrobacter cluster. Twelve additional Erythrobacter strains from this study or acquired from culture collections were thereby tested for displaying EHase activities, and most of tested strains showed enantioselective hydrolysis toward SO and glycidyl phenyl ether. Kinetic resolution of racemic SO using whole cell of Erythrobacter sp. JCS358 was performed. Enantiopure (S)-SO could be obtained with an enantiomeric excess (ee) higher than 99% after 15 h incubation. The determination of 1-phenyl-1,2-ethanediol configuration derived from racemic SO confirmed the enantioselective hydrolyzing activity of Erythrobacter sp. JCS358.

Cloning and characterization of three epoxide hydrolases from a marine bacterium, Erythrobacter litoralis HTCC2594.

Previously, we reported that ten strains belonging to Erythrobacter showed epoxide hydrolase (EHase) activities toward various epoxide substrates. Three genes encoding putative EHases were identified by analyzing open reading frames of Erythrobacter litoralis HTCC2594. Despite low similarities to reported EHases, the phylogenetic analysis of the three genes showed that eeh1 was similar to microsomal EHase, while eeh2 and eeh3 could be grouped with soluble EHases. The three EHase genes were cloned, and the recombinant proteins (rEEH1, rEEH2, and rEEH3) were purified. The functionality of purified proteins was proved by hydrolytic activities toward styrene oxide. EEH1 preferentially hydrolyzed (R)-styrene oxide, whereas EEH3 preferred to hydrolyze (S)-styrene oxide, representing enantioselective hydrolysis of styrene oxide. On the other hand, EEH2 could hydrolyze (R)- and (S)-styrene oxide at an equal rate. The optimal pH and temperature for the EHases occurred largely at neutral pHs and 40-55 degrees C. The substrate selectivity of rEEH1, rEEH2, and rEEH3 toward various epoxide substrates were also investigated. This is the first representation that a strict marine microorganism possessed three EHases with different enantioselectivity toward styrene oxide.