Acute gastroenteritis outbreaks associated with ground-waterborne norovirus in South Korea during 2008-2012.

Epidemiological and virological studies indicate that noroviruses-contaminated groundwater was the primary source of four acute gastroenteritis outbreaks in South Korea between 2008 and 2012. Furthermore, cabbage kimchi was first identified as the vehicle of transmission between groundwater and infected patients in an outbreak in 2011. The proper treatment of groundwater sources prior to use for drinking or in food preparation is necessary to prevent further outbreaks.

Foxc2 induces Wnt4 and Bmp4 expression during muscle regeneration and osteogenesis.

Proliferation and fusion of myoblasts is a well-orchestrated process occurring during muscle development and regeneration. Although myoblasts are known to originate from muscle satellite cells, the molecular mechanisms that coordinate their commitment toward differentiation are poorly understood. Here, we present a novel role for the transcription factor Forkhead box protein C2 (Foxc2) in regulating proliferation and preventing premature differentiation of activated muscle satellite cells. We demonstrate that Foxc2 expression is upregulated early in activated mouse muscle satellite cells and then diminishes during myogenesis. In undifferentiated C2C12 myoblasts, downregulation of endogenous Foxc2 expression leads to a decrease in proliferation, whereas forced expression of FOXC2 sustains proliferation and prevents differentiation into myotubes. We also show that FOXC2 induces Wnt signaling by direct interaction with the Wnt4 (wingless-type MMTV integration site family member-4) promoter region. The resulting elevated expression of bone morphogenetic protein-4 (Bmp4) and RhoA-GTP proteins inhibits the proper myoblast alignment and fusion required for myotube formation. Interestingly, continuous forced expression of FOXC2 alters the commitment of C2C12 myoblasts toward osteogenic differentiation, which is consistent with FOXC2 expression observed in patients with myositis ossificans, an abnormal bone growth within muscle tissue. In summary, our results suggest that (a) Foxc2 regulates the proliferation of multipotent muscle satellite cells; (b) downregulation of Foxc2 is critical for myogenesis to progress; and (c) sustained Foxc2 expression in myoblast cells suppresses myogenesis and alters their lineage commitment toward osteogenesis by inducing the Wnt4 and Bmp4 signaling pathways.

Genotypes of the circulating rotavirus strains in the seven prevaccine seasons from September 2000 to August 2007 in South Korea.

A Korean nationwide surveillance on circulating rotavirus strains was conducted from September 2000 to August 2007 aiming to obtain prevaccine data for predicting vaccine effectiveness. The predominant strains among the 2779 strains analyzed varied annually and only approximately 50% had either a G or a P antigen present in both RotaTeq (Merck & Co. Inc., Whitehouse Station, NJ, USA) and Rotarix (GlaxoSmithKline, Brentford, UK).

Molecular epidemiology and genetic diversity of human astrovirus in South Korea from 2002 to 2007.

The present study was conducted to survey the prevalence and genotypic distribution of human astrovirus (HAstV) circulating in South Korea. Of 160,027 patients with acute gastroenteritis, 2,057 (1.3%) were positive for HAstV antigen. We determined the genotypes of 187 HAstV strains collected from laboratories across the country. Genetic analysis revealed genotype 1 to be the most prevalent, accounting for 72.19% of the strains, followed by genotypes 8 (9.63%), 6 (6.95%), 4 (6.42%), 2 (3.21%) and 3 (1.60%). Our findings indicate that HAstV is less common but, even so, a potentially important viral agent of gastroenteritis in South Korea, with significant genetic diversity among circulating HAstV strains.

Detection and characterization of enterovirus associated with herpangina and hand, foot, and mouth disease in Seoul, Korea.

BACKGROUND: Human enteroviruses (HEVs) are a major cause of herpangina, HFMD (hand, foot, and mouth disease), and other neurological diseases in Seoul, Korea. METHODS: A total of 56 specimens from hospitalized patients collected from February to December 2009 (37 females and 19 males) in Seoul were tested for HEV from stool, throat swab, and vesicle swab samples taken from patients with herpangina or HFMD using cell culture and RT-PCR in 2009. By the 1D gene, encoding the VP1 capsid protein, seven different HEV genotypes were detected with Coxsackievirus A2, A4, A5, A9, A16 (CA), Coxsackievirus B1 (CB), and Enterovirus 71 (EV71). The most prevalent genotype was CA16 (6, 10.7%), followed by CA2 (4, 7.1%), CA5 (4, 7.1%), EV71 (2, 3.6%), CA4 (1, 1.8%), CA9 (1, 1.8%), and CB1 (1, 1.8%). The 1D gene sequences of two EV71 strains were closely related with one another (98.5% nucleotide similarity) and belonged to the C4 genotype. CONCLUSIONS: It is important to continuously survey the genetic characteristics of EV71 and CA16 from patients, which will provide useful data that aids in our understanding of HFMD infections in Seoul, Korea and may contribute to future control.

Genetic analysis of norovirus GII.4 variants circulating in Korea in 2008.

Noroviruses are the enteric pathogens most commonly responsible for infectious gastroenteritis and outbreaks of foodborne illness. The GII.4 norovirus, in particular, is responsible for the majority of epidemics. Here, we present data on the distribution of norovirus genotypes in Chungnam, Korea, in 2008, measure genetic variation among GII.4 strains, and compare Korean GII.4 variants with reference strains based on the 237-bp junction of ORF1 and ORF2. We detected 139 different strains, which formed two distinct genetic clusters with significant sequence diversity. One Korean cluster (2008-Korea_a) showed high similarity to the Sakai cluster that appeared in Japan and Europe in 2006. The other cluster (2008-Korea_b) was unique and unrelated to previously reported clusters. Genotype GII.4 was confirmed as the predominant cause of norovirus epidemics in Korea. Foodborne norovirus infections, on the other hand, were generally caused by emerging GII.4 genetic variants similar to those responsible for global epidemics.

Detection of hepatitis A virus from clotting factors implicated as a source of HAV infection among haemophilia patients in Korea.

To investigate the causal relationship of blood clotting factors and hepatitis A virus (HAV) infection in haemophilia patients during 1998-1999 in Korea, we performed a 1:3 matched case-control study and molecular detection of HAV from clotting factors and patients. The epidemiological investigation showed that one lot of clotting factor VIII was related epidemiologically to patients with hepatitis A with an odds ratio of 35.0, or 38.4 when adjusted for the interval between injections. We examined 17 sera collected from seven patients and 124 lots of blood clotting factors (factor VIII and factor IV) by HAV reverse transcriptase-polymerase chain reaction (RT-PCR). HAV RNA was detected in five clotting factors and six sera. The HAV sequence of one of the factor VIII samples was identical to the sequences found in three patients' sera. Findings from the laboratory and epidemiological studies suggested that the clotting factor was causally related to HAV infection in three haemophilia patients.

A seroprevalence study of poliovirus antibody among primary schoolchildren in Korea.

We aimed to determine the seroprevalence of poliovirus antibody in Korea by using the cell culture neutralization method recommended by the WHO. A total of 500 sera collected from children at eight primary schools in Kyunggi province were used for this study. We found that 82.2% of children were positive for all three types of poliovirus and antibody-positive rates for types I, II and III were 94.4, 96.6 and 86.8% respectively, indicating that seropositive rates for types I and II were considerably higher than for type III (P<0.0001). This result implies that the type III component of the oral polio vaccine should be evaluated further. Although a greater number of children, including young infants, need to be tested for seroprevalence, this study still provides us with valuable information on the effectiveness of vaccination against polioviruses in Korea.

Comparison of the pathogenicity of two strains (wild type and vaccine-like) of porcine reproductive and respiratory syndrome virus (PRRSV) in experimentally infected sows.

The objective of this study was to compare two Korean strains of porcine reproductive and respiratory syndrome virus (PRRSV), namely a wild type (WT) strain and a vaccine-like (VL) strain, in respect of pathogenicity and viral distribution in the tissues. Both strains were of the North American genotype. Two groups of five pregnant sows were infected with either the WT or the VL strain 2 weeks before their expected farrowing date. The WT strain-inoculated sows showed abortion and premature farrowing, whereas the VL strain-inoculated sows remained clinically normal and did not farrow prematurely. Of the 18 liveborn piglets from the WT strain-inoculated sows, 14 had interstitial pneumonia. Of the 60 liveborn piglets from the VL strain-inoculated sows, only six had interstitial pneumonia. PRRSV antigen or nucleic acid was detected in 48/65 (73.8%) of stillborn and liveborn piglets from the WT strain-inoculated sows, but in only 12/64 (18.8%) of stillborn and liveborn piglets from the VL strain-inoculated sows.

Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Korea during 2000.

We have isolated Human enterovirus 71 (EV71) from stool and CSF samples taken from patients with acute flaccid paralysis, herpangina, or hand, foot and mouth disease in 2000. Both the cell culture-neutralization test and RT-PCR were used to detect enteroviruses. Rhabdomyosarcoma (RD), HEP2c, and BGM cells were used for the isolation of viruses, and serotypes were determined by the neutralization test using EV71-specific antiserum. For genomic analysis, we amplified a 437-bp fragment of the 5'-noncoding region of the enterovirus genome and a 484-bp fragment of the VP3/VP1 region of EV71 by RT-PCR, with positive results. Products amplified using an EV71-specific primer pair were sequenced and compared with other isolates of EV71. Analysis of the nucleotide sequences of the amplified fragments showed that the EV71 isolates from patients were over 98% homologous and belonged to the genotype C.

PCR-RFLP based molecular typing of enteroviruses isolated from patients with aseptic meningitis in Korea.

We have evaluated PCR-RFLP as a practical method for rapid typing of enteroviruses causing aseptic meningitis in Korea. Through blind examination of 80 clinical isolates from patients with aseptic meningitis, we have compared the results of conventional serotyping with PCR-RFLP based genotyping, which was developed for this study. Among the 80 case isolates, which had been previously typed by routine neutralization test, only 42 cases (52.5%) were matched with typing by PCR-RFLP. The result clearly demonstrated that the enterovirus serotype does not coincide with the genotype. Therefore, the classification of enteroviruses by genotyping with PCR-RFLP, although rapid and simple, may be complicated by regional or seasonal differences. However, the PCR-RFLP method developed in this study is applicable to the epidemiological study of enteroviruses when regional or seasonal differences exist, and is useful in identifying the source of an infection.

Distribution of porcine reproductive and respiratory syndrome virus in stillborn and liveborn piglets from experimentally infected sows.

Four pregnant sows were infected 3 weeks before their expected farrowing date with a Korean isolate (North American genotype) of porcine reproductive and respiratory syndrome virus (PRRSV). The distribution of virus in their stillborn and liveborn (killed 7 days after birth) offspring was assessed immunohistochemically and by in-situ hybridization. PRRSV antigen and nucleic acid were detected in lung, thymus, liver, tonsil, spleen, heart, kidney and lymph nodes from both stillborn and liveborn piglets. Positive cells typically exhibited a red (immunohistochemistry) or dark brown (in-situ hybridization) reaction product in the cytoplasm, without background staining. The most consistent labelling for PRRSV was in the thymus, tonsil and lymph nodes. The experiment suggested that, in prenatal piglets, PRRSV replicates primarily in lymphoid tissues, having gained access to them from the placenta via the bloodstream.

Polymerase chain reaction-based restriction fragment length polymorphism pattern of porcine reproductive and respiratory syndrome virus directly from lung tissues without virus isolation in Korea.

Polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis was developed for directly typing porcine reproductive and respiratory syndrome virus (PRRSV) from lung specimens without virus isolation. Twenty nine lung specimens collected from postweaning pigs were isolated for PRRSV. When the PCR products from the 29 lung specimens were digested by the restriction enzymes MluI, HincII, SacII and HaeIII, the RFLP patterns from the 29 lung specimens matched with those from the corresponding PRRSV isolates from each pig. The results suggest that the PCR-based RFLP analysis method may be useful to distinguish PRRSV isolates directly from lung specimens without virus isolation.

Antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolated from pigs in Korea using new standardized procedures.

The in vitro susceptibilities of 76 isolates of Actinobacillus pleuropneumoniae collected from pigs with pleuropneumonia were tested with 12 commonly used antimicrobial drugs by an agar dilution minimal inhibitory concentration procedure according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. Field isolates had low MICs for ceftiofur, danofloxacin and penicillin. No correlation of antimicrobial resistance was related to serotype.

Comparison of virus isolation, reverse transcription-polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine reproductive and respiratory syndrome virus from naturally aborted fetuses and stillborn piglets.

Virus isolation, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and in situ hybridization methods were compared for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Seven aborted fetuses and 6 stillborn piglets naturally infected with PRRSV were used in the study. Viral antigen and viral nucleic acid were detected in macrophages and dendritic cells in the spleen, tonsil, lymph nodes, and thymus; in macrophages of liver, heart, and lung; and in endothelial cells and myocytes of the heart. Viral antigen and viral nucleic acid were most consistently detected in the spleen. Of the 13 samples, 6 were positive for PRRSV by all 4 techniques. Four (31%) samples were positive for PRRSV by RT-PCR, in situ hybridization, and virus isolation. Two (15%) samples were positive for PRRSV by virus isolation, RT-PCR, and in situ hybridization. One (8%) was positive for PRRSV by virus isolation and RT-PCR. The RT-PCR identified the presence of PRRSV more frequently than the other methods. However, when only formalin-fixed tissues are submitted, immunohistochemistry and in situ hybridization would be useful methods for the detection of PRRSV antigen and nucleic acid.

Antigenic variation and genotype of isolates of porcine reproductive and respiratory syndrome virus in Korea.

A panel of three anti-glycoprotein 5 (gp5) protein monoclonal antibodies (mAbs) (15, 28 and 246) and three anti-nucleocapsid (N) protein mAbs (SDOW17, VO17 and EP147) was used to investigate, by an indirect fluorescent antibody test, the antigenic variations of 50 Korean isolates of porcine reproductive and respiratory syndrome virus (PRRSV), and compare them with a us ATCC vR2332-derived attenuated vaccine strain and the reference European Lelystad strain of PRRSV. A multiplex PCR assay for the differentiation of European and North American genotypes of PRRSV was used to determine the genotype of the 50 Korean isolates. Forty-six (92 per cent) of the 50 Korean isolates shared the epitopes recognised by the anti-N protein mAb SDOW17. No reactivity to the anti-gp5 and anti-N protein mAbs was observed with the other four isolates. Six distinct patterns could be identified on the basis of their reactivities with the anti-PRRSV mAbs. All 50 isolates were identified as North American genotypes by the differential PCR.

Restriction fragment length polymorphism analysis of open reading frame 5 gene of porcine reproductive and respiratory syndrome virus isolates in Korea.

The genetic variability of porcine reproductive and respiratory syndrome virus (PRRSV) was studied by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments among 50 Korean isolates from open reading frame 5. All Korean PRRSVs were isolated from the field cases after the marketing of an U.S. ATCC VR2332-derived modified live PRRSV vaccine. Combining the restriction enzyme digestion patterns obtained with MluI, HincII, SacII, and HaeIII, we observed 19 distinct RFLP patterns. Seventeen out of 50 PRRSV isolates (34%) exhibited the modified live PRRSV vaccine RFLP pattern. The genomic variations that have been identified in the present study seemed to represent characteristic features of the Korean PRRSV isolates. PCR-based RFLP analysis using several restriction enzymes provides a good genetic estimate for isolate differentiation.

Distribution of a Korean strain of porcine reproductive and respiratory syndrome virus in experimentally infected pigs, as demonstrated immunohistochemically and by in-situ hybridization.

In an experiment with 40 specific pathogen-free pigs aged 3 days, the distribution of a Korean isolate of porcine reproductive and respiratory syndrome virus (PRRSV) was assessed immunohistochemically and by in-situ hybridization for a period of 28 days after intranasal inoculation. The most consistent and intense labelling for PRRSV was in the lung, the virus persisting in pulmonary macrophages for at least 28 days. The middle lobe of the lung was the optimum site for the detection of PRRSV antigens and nucleic acids, and the interstitial macrophage was the main cell type in which PRRSV was identified. Other tissues and cells in which the virus was detected included macrophages and dendritic cells in the tonsil, lymph nodes, spleen and Peyer's patches, and macrophages in the hepatic sinusoids and adrenal gland. The experiment suggested that the pathogenesis of PRRSV infection may be summarized thus: initial entry of virus through tonsillar and pulmonary macrophages, followed within 3 days by viraemia and subsequent interstitial pneumonia.

In situ hybridization for the detection and localization of swine Chlamydia trachomatis.

Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4-7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were not detected in tissue sections from an uninfected control piglet or in C. psittaci-infected sheep placenta. The morphology of host cells was preserved despite the relatively high temperature required in parts of the incubation procedure. The data indicate that in situ hybridization can be used to detect swine C. trachomatis in formalin-fixed, paraffin-embedded tissue specimens.