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.

Sequence analysis of the first B5 subgenogroup strain of enterovirus 71 isolated in Korea.

Enterovirus A71 (EV71), the main etiological agent of handfoot- mouth disease (HFMD), circulates in many areas of the world and has caused large epidemics since 1997, especially in the Asia-Pacific region. In this study, we determined the full-genome sequence of CMC718, a newly isolated EV71 strain in Korea. The CMC718 genome was 7,415 nucleotides in length and was confirmed by whole-genome phylogenetic analysis to belong to the B5 genotype. In particular, CMC718 demonstrated maximum identity with strain M988 of the B5 genotype and numerous amino acid variants were detected in the 3D domain of the viral protein P3, which is consistent with the mutation pattern of a B5 strain isolated in 2012-2013. Comparison of the CMC718 sequence with other EV71 reference strains confirmed the relationship and genetic variation of CMC718. Our study was a full-genome sequence analysis of the first EV71 strain of the B5 genotype isolated in South Korea. This information will be a valuable reference for the development of methods for the detection of recombinant viruses, the tracking of infections, and the diagnosis of EV71.

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

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

Molecular genomic characterization of severe fever with thrombocytopenia syndrome virus isolates from South Korea.

Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne emerging infectious disease caused by the SFTS virus (SFTSV) and is a threat to public health due to its high fatality rate. However, details on tick-to-human transmission of SFTSV are limited. In this study, we determined the whole-genome sequence of a South Korean SFTSV strain (CUK-JJ01), compared it to those of other recent human SFTSV isolates, and identified the genetic variations and relationships among the SFTSV strains. The genome of CUK-JJ01 was consistent with the genome of other members of the genus Phlebovirus, including the large (L), medium (M), and small (S) segments of 6368, 3378, and 1744 nucleotides, respectively. Based on amino acid sequences of the M and S segments, which are used to distinguish the six SFTSV genotypes, CUK-JJ01 was classified as genotype B. Segment analysis revealed that the L, M, and S segments were 97.49%, 97.18%, and 97.94% similar to those of KAJNH2/2013/Korea, ZJZHSH-FDE/2012/China, and KADGH/2013/Korea, respectively. Currently, only few studies on SFTSV have been conducted in Korean population and most were limited to serological analysis. Although the present study has limitations in terms of number of sample analyzed, the findings may serve as basis to understand the transmission and spread of SFTSV, as well as for the development of diagnostic and detection methods for viral recombinants by comparing the whole genome sequence of SFTSV isolates from South Korea and that of foreign isolates.

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.

Long-term Efficacy of Tenofovir Disoproxil Fumarate Monotherapy for Multidrug-Resistant Chronic HBV infection.

BACKGROUND & AIMS: There are no globally agreed upon treatment guidelines for patients with chronic hepatitis B virus (HBV) with multidrug resistance (MDR). We conducted a multicenter, prospective, real-world cohort study of effects of tenofovir disoproxyl fumarate (TDF) monotherapy and TDF-based combination therapy, as rescue therapy, in patients with multidrug-resistant chronic HBV infections. METHODS: We recruited patients with chronic HBV infection with resistance to antivirals from 8 tertiary hospitals in Korea. Patients (n=423) received rescue therapy with TDF monotherapy (n=174) or TDF-based combination therapy (n=249). The median follow-up period was 180 weeks. A virologic response was defined as a serum HBV DNA level of <20 IU/mL. RESULTS: Cumulative rates of virologic response did not differ significantly between the groups that received TDF monotherapy vs combination therapy at 48 weeks (71.7% vs 68.9%), 96 weeks (85.1% vs 84.2%), 144 weeks (92.1% vs 92.7%), 192 weeks (93.4% vs 95.7%), or 240 weeks (97.7% vs 97.2%). Serum levels of HBV DNA below 4.0 log10 IU/mL (odds ratio, 2.478; 95% CI 1.959-3.135; P < .001) and the absence of mutations associated with resistance to adefovir (odds ratio, 1.570; 95% CI 1.279-1.926; P < .001) were associated with virologic response in patients with MDR. There was no significant difference of virologic response among patients of different ages, sex, patients with vs without cirrhosis, positivity for hepatitis B e antigen, or renal function (all P > .05). CONCLUSION: In a multicenter, real-world cohort study, long-term use of TDF monotherapy showed non-inferior antiviral efficacy compared with that of TDF-based combination therapy in patients with MDR.

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

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

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.

Complete genome sequencing of a recombinant strain between human astrovirus antigen types 2 and 8 isolated from South Korea.

Human astroviruses (HAstVs) occur worldwide and are known to the causative agents of diarrhea in infants and elderly patients with immune dysfunction. This study aimed to identify recombinant HAstV strains and characterize rare genotypes. The full-length genome of a recombinant HAstV strain isolated from the stool sample of a patient with acute gastroenteritis from South Korea was amplified using three pairs of previously designed primers and seven newly designed primers. The recombinant HAstV was 6757-bp long and contained three sequential open reading frames (ORFs), designated as ORF1a (2781 bp), ORF1b (1548 bp), and ORF2 (2349 bp). Our findings suggested that a recombination event had occurred between ORF1b and ORF2 of the isolated strain, with a recombination breakpoint at 4081 bp. To our knowledge, this is the first study to reveal the complete nucleotide sequence of a recombinant HAstV strain from South Korea. Our study findings might be useful for identifying other recombinant HAstV strains and for developing vaccines against this pathogenic virus.

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.