Browsing by Author "Somchai Thiemmeca"

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    Application of one-step reverse transcription droplet digital pcr for dengue virus detection and quantification in clinical specimens
    (2021-04-01) Dumrong Mairiang; Adisak Songjaeng; Prachya Hansuealueang; Yuwares Malila; Paphavee Lertsethtakarn; Sasikorn Silapong; Yongyuth Poolpanichupatam; Chonticha Klungthong; Kwanrutai Chin-Inmanu; Somchai Thiemmeca; Nattaya Tangthawornchaikul; Kanokwan Sriraksa; Wannee Limpitikul; Sirijitt Vasanawathana; Damon W. Ellison; Prida Malasit; Prapat Suriyaphol; Panisadee Avirutnan; Siriraj Hospital; Songkhla Hospital; Armed Forces Research Institute of Medical Sciences, Thailand; Khon Kaen Regional Hospital; Thailand National Center for Genetic Engineering and Biotechnology
    Detection and quantification of viruses in laboratory and clinical samples are standard assays in dengue virus (DENV) studies. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) is considered to be the standard for DENV detection and quantification due to its high sensitivity. However, qRT-PCR offers only quantification relative to a standard curve and consists of several “in-house” components resulting in interlaboratory variations. We developed and optimized a protocol for applying one-step RT-droplet digital PCR (RT-ddPCR) for DENV detection and quantification. The lower limit of detection (LLOD95) and the lower limit of quantification (LLOQ) for RT-ddPCR were estimated to be 1.851 log10-copies/reaction and 2.337 log10-copies/reaction, respectively. The sensitivity of RT-ddPCR was found to be superior to qRT-PCR (94.87% vs. 90.38%, p = 0.039) while no false positives were detected. Quantification of DENV in clinical samples was independently performed in three laboratories showing interlaboratory variations with biases <0.5 log10-copies/mL. The RT-ddPCR protocol presented here could help harmonize DENV quantifi cation results and improve findings in the field such as identifying a DENV titer threshold correlating with disease severity.
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    Immortalized stem cell-derived hepatocyte-like cells: An alternative model for studying dengue pathogenesis and therapy
    (2020-11-01) Kessiri Kongmanas; Nuntaya Punyadee; Kasima Wasuworawong; Adisak Songjaeng; Tanapan Prommool; Yongyut Pewkliang; Siriphan Manocheewa; Somchai Thiemmeca; Khanit Sa-Ngiamsuntorn; Chunya Puttikhunt; Kym Francis Faull; Suradej Hongeng; Panisadee Avirutnan; Faculty of Medicine, Ramathibodi Hospital, Mahidol University; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Faculty of Medicine, Siriraj Hospital, Mahidol University; Jane & Terry Semel Institute for Neuroscience & Human Behavior
    Suitable cell models are essential to advance our understanding of the pathogenesis of liver diseases and the development of therapeutic strategies. Primary human hepatocytes (PHHs), the most ideal hepatic model, are commercially available, but they are expensive and vary from lot-to-lot which confounds their utility. We have recently developed an immortalized hepatocyte-like cell line (imHC) from human mesenchymal stem cells, and tested it for use as a substitute model for hepatotropic infectious diseases. With a special interest in liver pathogenesis of viral infection, herein we determined the suitability of imHC as a host cell target for dengue virus (DENV) and as a model for anti-viral drug testing. We characterized the kinetics of DENV production, cellular responses to DENV infection (apoptosis, cytokine production and lipid droplet metabolism), and examined anti-viral drug effects in imHC cells with comparisons to the commonly used hepatoma cell lines (HepG2 and Huh-7) and PHHs. Our results showed that imHC cells had higher efficiencies in DENV replication and NS1 secretion as compared to HepG2 and Huh-7 cells. The kinetics of DENV infection in imHC cells showed a slower rate of apoptosis than the hepatoma cell lines and a certain similarity of cytokine profiles to PHHs. In imHC, DENV-induced alterations in levels of lipid droplets and triacylglycerols, a major component of lipid droplets, were more apparent than in hepatoma cell lines, suggesting active lipid metabolism in imHC. Significantly, responses to drugs with DENV inhibitory effects were greater in imHC cells than in HepG2 and Huh-7 cells. In conclusion, our findings suggest superior suitability of imHC as a new hepatocyte model for studying mechanisms underlying viral pathogenesis, liver diseases and drug effects.
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    Interaction of dengue virus envelope protein with endoplasmic reticulum-resident chaperones facilitates dengue virus production
    (2009-02-06) Thawornchai Limjindaporn; Wiyada Wongwiwat; Sansanee Noisakran; Chatchawan Srisawat; Janjuree Netsawang; Chunya Puttikhunt; Watchara Kasinrerk; Panisadee Avirutnan; Somchai Thiemmeca; Rungtawan Sriburi; Nopporn Sittisombut; Prida Malasit; Pa thai Yenchitsomanus; Mahidol University; Chiang Mai University; Thailand National Center for Genetic Engineering and Biotechnology
    Dengue virus infection is an important mosquito-borne disease and a public health problem worldwide. A better understanding of interactions between human cellular host and dengue virus proteins will provide insight into dengue virus replication and cellular pathogenesis. The glycosylated envelope protein of dengue virus, DENV E, is processed in the endoplasmic reticulum of host cells and therefore reliant on host processing functions. The complement of host ER functions involved and nature of the interactions with DENV E has not been thoroughly investigated. By employing a yeast two-hybrid assay, we found that domain III of DENV E interacts with human immunoglobulin heavy chain binding protein (BiP). The relevance of this interaction was demonstrated by co-immunoprecipitation and co-localization of BiP and DENV E in dengue virus-infected cells. Using the same approach, association of DENV E with two other chaperones, calnexin and calreticulin was also observed. Knocking-down expression of BiP, calnexin, or calreticulin by siRNA significantly decreased the production of infectious dengue virions. These results indicate that the interaction of these three chaperones with DENV E plays an important role in virion production, likely facilitating proper folding and assembly of dengue proteins. © 2008 Elsevier Inc. All rights reserved.
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    Microparticles provide a novel biomarker to predict severe clinical outcomes of dengue virus infection
    (2015-01-01) Nuntaya Punyadee; Dumrong Mairiang; Somchai Thiemmeca; Chulaluk Komoltri; Wirichada Pan-ngum; Nusara Chomanee; Komgrid Charngkaew; Nattaya Tangthawornchaikul; Wannee Limpitikul; Sirijitt Vasanawathana; Prida Malasit; Panisadee Avirutnan; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Songkhla Hospital; Khon Kaen Regional Hospital
    © 2015, American Society for Microbiology. Shedding of microparticles (MPs) is a consequence of apoptotic cell death and cellular activation. Low levels of circulating MPs in blood help maintain homeostasis, whereas increased MP generation is linked to many pathological conditions. Herein, we investigated the role of MPs in dengue virus (DENV) infection. Infection of various susceptible cells by DENV led to apoptotic death and MP release. These MPs harbored a viral envelope protein and a nonstructural protein 1 (NS1) on their surfaces. Ex vivo analysis of clinical specimens from patients with infections of different degrees of severity at multiple time points revealed that MPs generated from erythrocytes and platelets are two major MP populations in the circulation of DENV-infected patients. Elevated levels of red blood cell-derived MPs (RMPs) directly correlated with DENV disease severity, whereas a significant decrease in platelet-derived MPs was associated with a bleeding tendency. Removal by mononuclear cells of complement-opsonized NS1-anti-NS1 immune complexes bound to erythrocytes via complement receptor type 1 triggered MP shedding in vitro, a process that could explain the increased levels of RMPs in severe dengue. These findings point to the multiple roles of MPs in dengue pathogenesis. They offer a potential novel biomarker candidate capable of differentiating dengue fever from the more serious dengue hemorrhagic fever.
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    Pathologic highlights of dengue hemorrhagic fever in 13 autopsy cases from Myanmar
    (2014-01-01) Khin Saw Aye; Komgrid Charngkaew; Ne Win; Kyaw Zin Wai; Kyaw Moe; Nuntaya Punyadee; Somchai Thiemmeca; Aroonroong Suttitheptumrong; Sanya Sukpanichnant; Malasit Prida; Scott B. Halstead; Department of Medical Research (Lower Myanmar); Mahidol University; National Health Laboratory; Yangon Children's Hospital; Thailand National Center for Genetic Engineering and Biotechnology; International Vaccine Institute, Seoul
    Vascular permeability, thrombocytopenia, liver pathology, complement activation, and altered hemostasis accompanying a febrile disease are the hallmarks of the dengue hemorrhagic fever/dengue shock syndrome, a major arthropod-borne viral disease that causes significant morbidity and mortality throughout tropical countries. We studied tissues from 13 children who died of acute dengue hemorrhagic fever/dengue shock syndrome at the Childrens' Hospital, Yangon, Myanmar. Dengue viral RNA from each of the 4 dengue viruses (DENVs) was detected by reverse transcriptase polymerase chain reaction in 11 cases, and dengue viral proteins (envelope, NS1, or NS3) were detected in 1 or more tissues from all 13 cases. Formalin-fixed and frozen tissues were studied for evidence of virus infection using monoclonal antibodies against DENV structural and nonstructural antigens (E, NS1, and nonsecreting NS3). In the liver, DENV infection occurred in hepatocytes and Kupffer cells but not in endothelial cells. Liver damage was associated with deposition on hepatocytes of complement components of both classical and alternative pathways. Evidence of dengue viral replication was observed in macrophage-like cells in spleens and lymph nodes. No dengue antigens were detected in endothelial cells in any organ. Germinal centers of the spleen and lymph nodes showed a marked reduction in the number of lymphocytes that were replaced by eosinophilic deposits, which contained dengue antigens as well as immunoglobulins, and complement components (C3, C1q, and C9). The latter findings had previously been reported but overlooked as a diagnostic feature. © 2014 Elsevier Inc.
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    Secreted NS1 protects dengue virus from mannose-binding lectin-mediated neutralization
    (2016-11-15) Somchai Thiemmeca; Chamaiporn Tamdet; Nuntaya Punyadee; Tanapan Prommool; Adisak Songjaeng; Sansanee Noisakran; Chunya Puttikhunt; John P. Atkinson; Michael S. Diamond; Alongkot Ponlawat; Panisadee Avirutnan; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Washington University in St. Louis; Washington University School of Medicine in St. Louis; Armed Forces Research Institute of Medical Sciences, Thailand
    © Copyright 2016 by The American Association of Immunologists, Inc. All rights reserved. Flavivirus nonstructural protein 1 (NS1) is a unique secreted nonstructural glycoprotein. Although it is absent from the flavivirus virion, intracellular and extracellular forms of NS1 have essential roles in viral replication and the pathogenesis of infection. The fate of NS1 in insect cells has been more controversial, with some reports suggesting it is exclusively cell associated. In this study, we confirm NS1 secretion from cells of insect origin and characterize its physical, biochemical, and functional properties in the context of dengue virus (DENV) infection. Unlike mammalian cell-derived NS1, which displays both high mannose and complex type N-linked glycans, soluble NS1 secreted from DENV-infected insect cells contains only high mannose glycans. Insect cell-derived secreted NS1 also has different physical properties, including smaller and more heterogeneous sizes and the formation of less stable NS1 hexamers. Both mammalian and insect cell-derived NS1 bind to complement proteins C1s, C4, and C4-binding protein, as well as to a novel partner, mannose-binding lectin. Binding of NS1 to MBL protects DENV against mannosebinding lectin-mediated neutralization by the lectin pathway of complement activation. As we detected secreted NS1 and DENV together in the saliva of infected Aedes aegypti mosquitoes, these findings suggest a mechanism of viral immune evasion at the very earliest phase of infection.
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    Vascular leakage in severe dengue virus infections: A potential role for the nonstructural viral protein NS1 and complement
    (2006-04-15) Panisadee Avirutnan; Nuntaya Punyadee; Sansanee Noisakran; Chulaluk Komoltri; Somchai Thiemmeca; Kusuma Auethavornanan; Aroonroong Jairungsri; Rattiyaporn Kanlaya; Nattaya Tangthawornchaikul; Chunya Puttikhunt; Sa Nga Pattanakitsakul; Pa Thai Yenchitsomanus; Juthathip Mongkolsapaya; Watchara Kasinrerk; Nopporn Sittisombut; Matthias Husmann; Maria Blettner; Sirijitt Vasanawathana; Sucharit Bhakdi; Prida Malasit; Mahidol University; Thailand National Center for Genetic Engineering and Biotechnology; Chiang Mai University; Khon Kaen Regional Hospital; Klinikum der Johannes-Gutenberg-Universitat und Fachbereich Medizin; Inst. of Med. Microbiol. and Hygiene
    Background. Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. Methods. The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed. Results. Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS. Conclusions. Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS. © 2006 by the Infectious Diseases Society of America All rights reserved.