Increased Complement-Associated Inflammation in Cytomegalovirus-Positive Hypertensive Anterior Uveitis Patients Based on the Aqueous Humor Proteomics Analysis.

Herpetic anterior uveitis-associated ocular inflammation is commonly manifested with ocular hypertension and glaucoma. Relative to other viruses, cytomegalovirus (CMV) positive hypertensive anterior uveitis is associated with high recurrences of uveitis, as well as with uncontrolled intraocular pressure (IOP) and a subsequent higher requirement for future glaucoma surgery. To gain novel insights into the pathogenesis of ocular hypertension in these patients, we investigated the proteome changes of the aqueous humor (AH) derived from the CMV hypertensive anterior uveitis (CMV-HAU; n = 10) patients and non-glaucoma (cataract; n = 10) patients using liquid chromatography with tandem mass spectrometry. Among a total of 562 proteins identified, fifty and fifteen proteins were significantly elevated and decreased, respectively, in the AH of CMV-HAU patients compared to the control subjects by ≥2 fold. Gene ontology (GO) enrichment and network analyses of elevated proteins revealed that the enrichment of protein was involved in the complement activation, the humoral immune response mediated by the circulating immunoglobulins, proteolysis, and platelet degranulation. In the AH of CMV-HAU, GDF (growth/differentiation factor)-15, the inflammatory marker belonging to the TGF-ß superfamily proteins, was significantly increased, while vasorin, an anti-TGF-ß protein, levels were decreased. The trabecular meshwork cells infected with CMV exhibited a significantly increased expression of inflammatory markers. Collectively, these data indicate increased complement factor associated inflammation and humoral immunity in CMV-HAU associated ocular hypertension.

Cytokine profile and cytoskeletal changes after herpes simplex virus type 1 infection in human trabecular meshwork cells.

Uveitis caused by herpes simplex virus (HSV)-1 is characterized by increased intraocular pressure (IOP) in the presence of anterior chamber inflammation. Despite their clinical significance, the pathogenic changes associated with HSV-1 infection in trabecular meshwork (TM) cells, the key cell type regulating IOP, have not been completely elucidated. In this study, cytokine array analyses showed a significant stepwise increase in monocyte chemoattractant protein (MCP)-1 expression upon HSV-1 infection in TM cells (p < 0.05). HSV-1 infection led to downregulation of fibrogenic molecules (fibronectin, α-smooth muscle actin, connective tissue growth factor and TGF-ß1). Notably, HSV-1 infection caused a significant increase in actin stress fibres, with a twofold increase in active RhoA, which was enhanced by treatment with TGF-ß1 and inhibited by treatment with the Rho-kinase inhibitor, Y-27632. TM cells treated with MCP-1 exhibited a dose-dependent increase in actin stress fibres compared to untreated TM cells. Our study suggests that HSV-1 infection in TM cells increases cell contractile activity rather than fibrotic changes in the extracellular matrix (ECM) components. Taken together, these observations demonstrate the enhanced expression of MCP-1 and TM cell contractile activity upon HSV-1 infection and events with potential implications for the pathobiology of abrupt IOP elevation in HSV-1 anterior uveitis.

Role of MCP-1 and IL-8 in viral anterior uveitis, and contractility and fibrogenic activity of trabecular meshwork cells.

The inflammatory chemokines, monocyte chemoattractant protein (MCP)-1 and IL-8, are produced by normal trabecular meshwork cells (TM) and elevated in the aqueous humor of primary open angle glaucoma (POAG) and hypertensive anterior uveitis associated with viral infection. However, their role in TM cells and aqueous humor outflow remains unclear. Here, we explored the possible involvement of MCP-1 and IL-8 in the physiology of TM cells in the context of aqueous outflow, and the viral anterior uveitis. We found that the stimulation of human TM cells with MCP-1 and IL-8 induced significant increase in the formation of actin stress fibers and focal adhesions, myosin light chain phosphorylation, and the contraction of TM cells. MCP-1 and IL-8 also demonstrated elevation of extracellular matrix proteins, and the migration of TM cells. When TM cells were infected with HSV-1 and CMV virus, there was a significant increase in cytoskeletal contraction and Rho-GTPase activation. Viral infection of TM cells revealed significantly increased expression of MCP-1 and IL-8. Taken together, these results indicate that MCP-1 and IL-8 induce TM cell contractibility, fibrogenic activity, and plasticity, which are presumed to increase resistance to aqueous outflow in viral anterior uveitis and POAG.

Determining the efficacy of 27 commercially available disinfectants against human noroviruses.

BACKGROUND: Recently, monoclonal-antibody-conjugated immunomagnetic separation (IMS) procedure combined with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) has been used for quantifying non-cultivated human noroviruses (HuNoVs). METHODS: We examined the efficacy of 27 commercially available disinfectants and a prototype against GII.4 strain HuNoV through the IMS/qRT-PCR assay. RESULTS: The average log reduction in viral titer in vitro varied among the disinfectants. The prototype was the most effective with an average log reduction of 6.86 log. CONCLUSIONS: The IMS/RT-qPCR assay is an effective method to evaluate the activities of disinfectants against GII.4 HuNoV in vitro. Further work is needed to enhance the virucidal activity of the prototype disinfectant against more resistant HuNoV strains.

Development of a real-time loop-mediated isothermal amplification method for the detection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is being reported annually in South Korea since its first detection there in 2010. The causal agent is a negative-strand RNA virus 80-100 nm in diameter. It causes fever, thrombocytopenia, leukocytopenia, gastrointestinal symptoms, and neural symptoms. The mortality rate of SFTS was 32.6% among 172 cases reported from 2012 to 2015 in South Korea. Thus, is necessary to develop an effective diagnostic method that selectively identifies the isolates circulating in South Korea. The real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay is a simple, rapid, and sensitive approach for molecular diagnosis. Here, we designed novel primers for this assay and found that the technique had very high specificity, sensitivity, and efficiency. This real-time RT-LAMP approach using the novel primers developed herein can be applied for early diagnosis of SFTSV strains in South Korea to reduce the mortality rate of SFTS.

Correction: The effects of losartan on cytomegalovirus infection in human trabecular meshwork cells.

[This corrects the article DOI: 10.1371/journal.pone.0218471.].

The effects of losartan on cytomegalovirus infection in human trabecular meshwork cells.

BACKGROUND: Human cytomegalovirus (CMV) has been emerged as one of the causes of acute recurrent or chronic hypertensive anterior uveitis in immunocompetent. In hypertensive anterior uveitis, human trabecular meshwork (TM) cells are considered a focus of inflammation. We investigated the effects of losartan, a selective angiotensin II receptor antagonist, on CMV infection in human TM cells. METHODS: Human TM cells were infected with CMV AD169. Virus infected and mock-infected cells were treated with losartan or dexamethasone or ganciclovir with or without transforming growth factor (TGF)-ß1. Viral DNA accumulation and host cell response were analyzed using real-time PCR. Levels of secreted TGF-ß1 were measured by determining its concentration in conditioned medium using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: CMV infection significantly increased the concentrations of the secreted TGF-ß1 at 3, 5, and 7 day post infection in TM cells. Treatment with dexamethasone or losartan significantly decreased the levels of TGF-ß1, whereas treatment with ganciclovir did not affect TGF-ß1 levels. TM cells treated with TGF-ß1 along with the presence of losartan for 48 hours showed marked decrease in the expression of α-smooth muscle actin (SMA), lysyl oxidase (LOX), connective tissue growth factor (CTGF), fibronectin and collagen-1A, compared with cells treated with TGF-ß1 alone. CMV-infected TM cells stimulated by TGF-ß1 significantly increased the expression of α-SMA and CTGF, which were attenuated by additional treatment with losartan. CONCLUSION: Losartan inhibited the expression of TGF-ß1 and fibrogenic molecules in human TM cells. Thus, losartan has the potential to decrease TM fibrosis in patients with CMV-induced hypertensive anterior uveitis.

Transcriptional changes after herpes simplex virus type 1 infection in human trabecular meshwork cells.

BACKGROUND: Herpes simplex virus type 1 (HSV-1) is causative for hypertensive anterior uveitis. Trabecular meshwork (TM) cells, which are the key cells regulating intraocular pressure (IOP), is considered to be the site of inflammation. We explored the profiles of genes expressed in human TM primary cells upon HSV-1 infection. METHODS: Human TM cells were infected with HSV-1 and total RNA was isolated. The global transcriptional gene network analyses were performed in mock-infected and HSV-1 infected TM cells. Using ingenuity pathway analysis, we determined the key biological networks upon HSV-1 infection. The results of microarray analyses were validated using quantitative PCR. RESULTS: TM cells had a high susceptibility to HSV-1 infection. HSV-1 induced transcriptional suppression of many components related to fibrosis in TM cells. The top biological network related to the genes which were significantly altered upon HSV-1 infection was organismal injury and abnormalities involving TGF-ß1 and PDGF-BB. The results of PCR analyses for selected molecules were found to be in good agreement with the microarray data. HSV-1-infected TM cells showed an 80-fold increase in the expression of PDGF-BB, which was further increased by treatment with TGF-ß1. HSV-1 also induced a 4-fold increase in the expression of the monocyte chemoattractant protein (MCP)-1, the downstream molecules of PDGF-BB. CONCLUSIONS: In human TM cells, HSV-1 induced transcriptional suppression of many components related to fibrosis and enhanced expression of both PDGF-BB and MCP-1. Our study may provide a novel mechanism for the pathogenesis of HSV-1 infection in TM cells.

Gene Expression Analysis of Inflammatory Cytokines in Korean Psoriatic Patients.

BACKGROUND: Although phenotypic heterogeneity of psoriasis is suggested by the alternate activation of either T-helper (Th)1-related or Th17-related cytokines, little is known about the mRNA levels of inflammatory cytokines. OBJECTIVE: To investigate whether there is differential expression of Th1-related and Th17-related inflammatory cytokine genes 1) between psoriatic patients and healthy controls, and 2) between patients with different psoriasis phenotypes. METHODS: Twenty-five patients with psoriasis (10 with guttate psoriasis and 15 with plaque psoriasis) and 5 healthy volunteers were enrolled in this study. The mRNA levels of circulating cytokines (interleukin [IL]-2, IL-12p40, interferon-γ, IL-17A, IL-22, and IL-23R) were measured by real-time reverse transcription polymerase chain reaction. RESULTS: The comparison between psoriatic and healthy control samples revealed that IL-12p40, IL-17A, and IL-22 mRNA levels were significantly higher (approximately 4∼6 folds) in the patients with psoriasis. The mRNA levels of these six cytokines in the blood did not differ between the guttate and plaque psoriasis groups. CONCLUSION: We found that the mRNA levels of blood inflammatory cytokines (IL-12p40, IL-17A, and IL-22) were significantly elevated in patients with psoriasis compared to the levels in healthy controls, but they did not significantly differ between patients with guttate and plaque type psoriasis.

Enhanced cytomegalovirus infection in human trabecular meshwork cells and its implication in glaucoma pathogenesis.

Cytomegalovirus (CMV) is one of the infectious causes of hypertensive anterior uveitis, which is characterized by recurrent episodes of elevated intraocular pressure (IOP) and mild anterior uveitis. Despite the potentially vision-threatening complications of this disease, the underlying mechanisms remain largely undefined. We aimed to investigate whether human trabecular meshwork (TM) cells, the key cell type that regulates IOP, could support CMV replication, as well as demonstrate the relevant pathological changes in TM. When human TM cells were infected with CMV AD169, immediate early antigens were detected 1 day post-infection (dpi); cytopathic changes including rounding, a ballooned appearance with disorganization, and a decreased number of stress fibers were noted in TM cells. The marked increase in viral DNA accumulation was observed most notably at 5 and 7 dpi, suggesting that the active viral infection in human TM cells could be the key mechanism underlying the elevation of IOP in anterior viral uveitis. Notably, CMV infection enhanced the production of transforming growth factor (TGF)-ß1, an upstream molecule that increases the resistance of the outflow pathway in human TM cells. The increase of TGF-ß1 was countervailed by additional treatment with corticosteroids. Our results provide a pathogenic mechanism for IOP elevation in viral anterior uveitis.

Isolation and Genome Characterization of the Virulent Staphylococcus aureus Bacteriophage SA97.

A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus.

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.

Whole-Genome Sequencing Analysis of Sapovirus Detected in South Korea.

Sapovirus (SaV), a virus residing in the intestines, is one of the important causes of gastroenteritis in human beings. Human SaV genomes are classified into various genogroups and genotypes. Whole-genome analysis and phylogenetic analysis of ROK62, the SaV isolated in South Korea, were carried out. The ROK62 genome of 7429 nucleotides contains 3 open-reading frames (ORF). The genotype of ROK62 is SaV GI-1, and 94% of its nucleotide sequence is identical with other SaVs, namely Manchester and Mc114. Recently, SaV infection has been on the rise throughout the world, particularly in countries neighboring South Korea; however, very few academic studies have been done nationally. As the first whole-genome sequence analysis of SaV in South Korea, this research will help provide reference for the detection of recombination, tracking of epidemic spread, and development of diagnosis methods for SaV.

Complete nucleotide sequence analysis of the norovirus GII.4 Sydney variant in South Korea.

Norovirus is the primary cause of acute gastroenteritis in individuals of all ages. In Australia, a new strain of norovirus (GII.4) was identified in March 2012, and this strain has spread rapidly around the world. In August 2012, this new GII.4 strain was identified in patients in South Korea. Therefore, to examine the characteristics of the epidemic norovirus GII.4 2012 variant in South Korea, we conducted KM272334 full-length genomic analysis. The genome of the gg-12-08-04 strain consisted of 7,558 bp and contained three open reading frame (ORF) composites throughout the whole genome: ORF1 (5,100 bp), ORF2 (1,623 bp), and ORF3 (807 bp). Phylogenetic analyses showed that gg-12-08-04 belonged to the GII.4 Sydney 2012 variant, sharing 98.92% nucleotide similarity with this variant strain. According to SimPlot analysis, the gg-12-08-04 strain was a recombinant strain with breakpoint at the ORF1/2 junction between Osaka 2007 and Apeldoorn 2008 strains. This study is the first report of the complete sequence of the GII.4 Sydney 2012 strain in South Korea. Therefore, this may represent the standard sequence of the norovirus GII.4 2012 variant in South Korea and could therefore be useful for the development of norovirus vaccines.

Development of enhanced primer sets for detection of norovirus.

Norovirus (NV) is a major viral pathogen that causes nonbacterial acute gastroenteritis and outbreaks of food-borne disease. The genotype of NV most frequently responsible for NV outbreaks is GII.4, which accounts for 60-80% of cases. Moreover, original and new NV variant types have been continuously emerging, and their emergence is related to the recent global increase in NV infection. In this study, we developed advanced primer sets (NKI-F/R/F2, NKII-F/R/R2) for the detection of NV, including the variant types. The new primer sets were compared with conventional primer sets (GI-F1/R1/F2, SRI-1/2/3, GII-F1/R1/F2, and SRII-1/2/3) to evaluate their efficiency when using clinical and environmental samples. Using reverse transcription polymerase chain reaction (RT-PCR) and seminested PCR, NV GI and GII were detected in 91.7% (NKI-F/R/F2), 89.3% (NKII-F/R/R2), 54.2% (GI-F1/R1/F2), 52.5% (GII-F1/R1/F2), 25.0% (SRI-1/2/3), and 32.2% (SRII-1/2/3) of clinical and environmental specimens. Therefore, our primer sets perform better than conventional primer sets in the detection of emerged types of NV and could be used in the future for epidemiological diagnosis of infection with the virus.

Molecular epidemiology of norovirus in South Korea.

Norovirus is a major cause of viral gastroenteritis and a common cause of foodborne and waterborne outbreaks. Norovirus outbreaks are responsible for economic losses, most notably to the public health and food industry field. Norovirus has characteristics such as low infectious dose, prolonged shedding period, strong stability, great diversity, and frequent genome mutations. Besides these characteristics, they are known for rapid and extensive spread in closed settings such as hospitals, hotels, and schools. Norovirus is well known as a major agent of food-poisoning in diverse settings in South Korea. For these reasons, nationwide surveillance for norovirus is active in both clinical and environmental settings in South Korea. Recent studies have reported the emergence of variants and novel recombinants of norovirus. In this review, we summarized studies on the molecular epidemiology and nationwide surveillance of norovirus in South Korea. This review will provide information for vaccine development and prediction of new emerging variants of norovirus in South Korea.

Evaluation of antimicrobial effects of commercial mouthwashes utilized in South Korea.

Streptococcus mutans is frequently associated with dental caries. Bacterial fermentation of food debris generates an acidic environment on the tooth surface, ultimately resulting in tooth deterioration. Therefore, various mouthwashes have been used to reduce and prevent Streptococcus mutans. The aim of this study was to evaluate the antimicrobial activities of 4 commercial mouthwashes and those of 10% and 20% ethanol solutions (formula A, B, C, D, E and F) against Streptococcus mutans using biofilm and planktonic methods. The range of reduction in the viable cell count of Streptococcus mutans as estimated by the biofilm and planktonic methods was 0.05-5.51 log (P ≤ 0.01) and 1.23-7.51 log (P ≤ 0.001) compared with the negative control, respectively, indicating that the planktonic method had a stronger antibacterial effect against S. mutans. Among the tested formulations, formula A (Garglin regular® mouthwash) was the most effective against Streptococcus mutans (P ≤ 0.001).

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.

DNA looping-dependent autorepression of LEE1 P1 promoters by Ler in enteropathogenic Escherichia coli (EPEC).

Ler, a homolog of H-NS in enteropathogenic Escherichia coli (EPEC), plays a critical role in the expression of virulence genes encoded by the pathogenic island, locus of enterocyte effacement (LEE). Although Ler acts as an antisilencer of multiple LEE operons by alleviating H-NS-mediated silencing, it represses its own expression from two LEE1 P1 promoters, P1A and P1B, that are separated by 10 bp. Various in vitro biochemical methods were used in this study to elucidate the mechanism underlying transcription repression by Ler. Ler acts through two AATT motifs, centered at position -111.5 on the coding strand and at +65.5 on the noncoding strand, by simultaneously repressing P1A and P1B through DNA-looping. DNA-looping was visualized using atomic force microscopy. It is intriguing that an antisilencing protein represses transcription, not by steric exclusion of RNA polymerase, but by DNA-looping. We propose that the DNA-looping prevents further processing of open promoter complex (RPO) at these promoters during transcription initiation.

Genetic analysis of the capsid region of norovirus GII.4 variants isolated in South Korea.

Norovirus is one of the major causes of non-bacterial gastroenteritis in humans. The aim of this study was to analyze the amino acid variation of open reading frame 2 of GII.4 variants in South Korea during the period from November 2006 to December 2012. Sixty-nine complete nucleotide sequences of open reading frame 2 were obtained from 113 GII.4 strains. The GII.4 2006b variants were detected predominantly between 2006 and 2009; however, new GII.4 variants, which were termed the 2010 variant and the 2012 variant, emerged in 2010 and 2012, respectively. The number of GII.4 2006b variants steadily decreased until 2012, whereas the number of gastroenteritis cases caused by the new variants increased between 2010 and 2012. The amino acid sequence in the ORF2 region obtained in this study was compared with other GII.4 variants isolated in various countries. Amino acid variations were observed primarily at epitope sites and the surrounding regions. Amino acids 294, 359, 393, and 413 of the P2 subdomain were the most variable sites among the GII.4 variants. The information in this study can be useful in basic research to predict the emergence and determine the genetic functions of new GII.4 variants.