Detection of human cytomegalovirus infection in paraffin sections by modified Steiner silver stain for LM and TEM

1991 ◽  
Vol 49 ◽  
pp. 82-83
Author(s):  
K. Chien ◽  
Y.K. Kim ◽  
I.P. Shintaku ◽  
R.L. Van de Velde ◽  
R.C. Heusser ◽  
...  
Keyword(s):  
Epoxy Resin ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Paraffin Sections ◽  
Silver Stain ◽  
Immunosuppressed Patients ◽  
Infected Cells ◽  
Methenamine Silver ◽  
Methenamine Silver Stain

Human cytomegalovirus (HCMV) infection is encountered more frequently in surgical specimens due to the increased number of immunosuppressed patients (organ transplants or HIV positive). Previous studies showed that HCMV infected cells in paraffin sections can be precisely labelled by in situ hybridization or by Grocott's methenamine silver stain for LM and subsequently confirmed by TEM. However, further studies indicate HCMV infection can also be identified by modified Steiner silver stain3 but in a different manner.Modified Steiner silver stained paraffin sections can be processed for TEM as follows: Coverslips are removed by immersion in xylene. A few drops of 1% OsO4, freshly dissolved in xylene, is then pipetted onto each slide for 5 minutes. Aqueous OsO4 can not be used as it will destain the sections instantly. Slides are rinsed in xylene, xylene/acetone and then 100% acetone. The sections are infiltrated with epoxy resin/acetone, embedded in epoxy resin, polymerized and subsequently separated from slides by heat as previously described.

Detection of human cytomegalovirus infection in paraffin sections by Grocott’s methenamine silver stain for light and electron microscopy

1990 ◽  
Vol 48 (3) ◽  
pp. 808-809
Author(s):  
K. Chien ◽  
Y.K. Kim ◽  
I.P. Shintaku ◽  
R.L. Van de Velde ◽  
R.C. Heusser ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Fungal Infections ◽  
Pneumocystis Carinii ◽  
Light And Electron Microscopy ◽  
Paraffin Sections ◽  
Silver Stain ◽  
Infected Cells ◽  
Methenamine Silver ◽  
Methenamine Silver Stain ◽  
Light Green

Grocott's methenamine silver stain (GMS) has been used histologically for identifying Pneumocystis carinii and fungal infections in paraffin sections of surgical specimens. However, current studies indicate that aggregates of human cytomegalovirus (HCMV) in the cytoplasm of infected cells are also stained by GMS and can be detected by LM. Since precipitated silver particles are electron dense, the presence of silver labelled HCMV virions can be easily located and confirmed by TEM.GMS and light green stained paraffin sections positive for HCMV can be processed for TEM either by conventional procedures or as follows: Coverslips are removed by immersion in xylene. A few drops of 1% 0s04, freshly dissolved in xylene, are then pipetted onto each slide for 5 minutes. Prolonged osmication is to be avoided as it will cause reduction of the silver deposits. Slides are then rinsed in xylene and changed to 100% acetone. Substitution of acetone for xylene prior to resin infiltration will produce a better quality block for sectioning. The sections are embedded, polymerized and subsequently separated from slides as previously described. The light green staining is retained in the epoxy block, which facilitates tissue orientation and selection of areas for thin-sectioning.

scholarly journals The human cytomegalovirus protein UL147A downregulates the most prevalent MICA allele: MICA*008, to evade NK cell-mediated killing

2021 ◽  
Vol 17 (5) ◽  
pp. e1008807
Author(s):  
Einat Seidel ◽  
Liat Dassa ◽  
Corinna Schuler ◽  
Esther Oiknine-Djian ◽  
Dana G. Wolf ◽  
...  
Keyword(s):  
Nk Cells ◽  
Human Cytomegalovirus ◽  
Cell Activation ◽  
Hcmv Infection ◽  
Nk Cell ◽  
Innate Immune ◽  
Target Cells ◽  
Histocompatibility Complex ◽  
Immunosuppressed Patients ◽  
Infected Cells

Natural killer (NK) cells are innate immune lymphocytes capable of killing target cells without prior sensitization. One pivotal activating NK receptor is NKG2D, which binds a family of eight ligands, including the major histocompatibility complex (MHC) class I-related chain A (MICA). Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus causing morbidity and mortality in immunosuppressed patients and congenitally infected infants. HCMV encodes multiple antagonists of NK cell activation, including many mechanisms targeting MICA. However, only one of these mechanisms, the HCMV protein US9, counters the most prevalent MICA allele, MICA*008. Here, we discover that a hitherto uncharacterized HCMV protein, UL147A, specifically downregulates MICA*008. UL147A primarily induces MICA*008 maturation arrest, and additionally targets it to proteasomal degradation, acting additively with US9 during HCMV infection. Thus, UL147A hinders NKG2D-mediated elimination of HCMV-infected cells by NK cells. Mechanistic analyses disclose that the non-canonical GPI anchoring pathway of immature MICA*008 constitutes the determinant of UL147A specificity for this MICA allele. These findings advance our understanding of the complex and rapidly evolving HCMV immune evasion mechanisms, which may facilitate the development of antiviral drugs and vaccines.

scholarly journals The Human Cytomegalovirus Protein UL148A Downregulates the NK Cell-Activating Ligand MICA To Avoid NK Cell Attack

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Liat Dassa ◽  
Einat Seidel ◽  
Esther Oiknine-Djian ◽  
Rachel Yamin ◽  
Dana G. Wolf ◽  
...  
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Human Cytomegalovirus ◽  
Innate Immune System ◽  
Hcmv Infection ◽  
Nk Cell ◽  
Innate Immune ◽  
Immunosuppressed Patients ◽  
Infected Cells ◽  
Complications And Mortality

ABSTRACT Natural killer (NK) cells are lymphocytes of the innate immune system capable of killing hazardous cells, including virally infected cells. NK cell-mediated killing is triggered by activating receptors. Prominent among these is the activating receptor NKG2D, which binds several stress-induced ligands, among them major histocompatibility complex (MHC) class I-related chain A (MICA). Most of the human population is persistently infected with human cytomegalovirus (HCMV), a virus which employs multiple immune evasion mechanisms, many of which target NK cell responses. HCMV infection is mostly asymptomatic, but in congenitally infected neonates and in immunosuppressed patients it can lead to serious complications and mortality. Here we discovered that an HCMV protein named UL148A whose role was hitherto unknown is required for evasion of NK cells. We demonstrate that UL148A-deficient HCMV strains are impaired in their ability to downregulate MICA expression. We further show that when expressed by itself, UL148A is not sufficient for MICA targeting, but rather acts in concert with an unknown viral factor. Using inhibitors of different cellular degradation pathways, we show that UL148A targets MICA for lysosomal degradation. Finally, we show that UL148A-mediated MICA downregulation hampers NK cell-mediated killing of HCMV-infected cells. Discovering the full repertoire of HCMV immune evasion mechanisms will lead to a better understanding of the ability of HCMV to persist in the host and may also promote the development of new vaccines and drugs against HCMV. IMPORTANCE Human cytomegalovirus (HCMV) is a ubiquitous pathogen which is usually asymptomatic but that can cause serious complications and mortality in congenital infections and in immunosuppressed patients. One of the difficulties in developing novel vaccines and treatments for HCMV is its remarkable ability to evade our immune system. In particular, HCMV directs significant efforts to thwarting cells of the innate immune system known as natural killer (NK) cells. These cells are crucial for successful control of HCMV infection, and yet our understanding of the mechanisms which HCMV utilizes to elude NK cells is partial at best. In the present study, we discovered that a protein encoded by HCMV which had no known function is important for preventing NK cells from killing HCMV-infected cells. This knowledge can be used in the future for designing more-efficient HCMV vaccines and for formulating novel therapies targeting this virus.

scholarly journals The Human Cytomegalovirus, from Oncomodulation to Oncogenesis

Viruses ◽  
2018 ◽  
Vol 10 (8) ◽  
pp. 408 ◽  
Author(s):  
Georges Herbein
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Direct Transformation ◽  
Oncogenic Pathways ◽  
Immunosuppressed Patients ◽  
Infected Cells ◽  
Per Se

Besides its well-described impact in immunosuppressed patients, the role of human cytomegalovirus (HCMV) in the pathogenesis of cancer has been more recently investigated. In cancer, HCMV could favor the progression and the spread of the tumor, a paradigm named oncomodulation. Although oncomodulation could account for part of the protumoral effect of HCMV, it might not explain the whole impact of HCMV infection on the tumor and the tumoral microenvironment. On the contrary cases have been reported where HCMV infection slows down the progression and the spread of the tumor. In addition, HCMV proteins have oncogenic properties per se, HCMV activates pro-oncogenic pathways in infected cells, and recently the direct transformation of cells following HCMV infection has been described, which gave rise to tumors when injected in mice. Thus, beyond the oncomodulation model, this review will assess the direct transforming role of HMCV-infected cells and the potential classification of HCMV as an oncovirus.

Distinctive, rapid, and easy labeling of diffuse plaques in the Alzheimer brains by a new methenamine silver stain

1990 ◽  
Vol 79 (5) ◽  
pp. 569-572 ◽  
Author(s):  
H. Yamaguchi ◽  
C. Haga ◽  
S. Hirai ◽  
Y. Nakazato ◽  
K. Kosaka
Keyword(s):  
Silver Stain ◽  
Methenamine Silver ◽  
Methenamine Silver Stain ◽  
Diffuse Plaques

scholarly journals The Carboxy-Terminal Tail of Human Cytomegalovirus (HCMV) US28 Regulates both Chemokine-Independent and Chemokine-Dependent Signaling in HCMV-Infected Cells

2009 ◽  
Vol 83 (19) ◽  
pp. 10016-10027 ◽  
Author(s):  
Melissa P. Stropes ◽  
Olivia D. Schneider ◽  
William A. Zagorski ◽  
Jeanette L. C. Miller ◽  
William E. Miller
Keyword(s):  
Human Cytomegalovirus ◽  
Calcium Release ◽  
Hcmv Infection ◽  
Calcium Signal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Domain Truncation ◽  
Infected Cells ◽  
Heterologous Expression Systems ◽  
Carboxy Terminal

ABSTRACT The human cytomegalovirus (HCMV)-encoded G-protein-coupled receptor (GPCR) US28 is a potent activator of a number of signaling pathways in HCMV-infected cells. The intracellular carboxy-terminal domain of US28 contains residues critical for the regulation of US28 signaling in heterologous expression systems; however, the role that this domain plays during HCMV infection remains unknown. For this study, we constructed an HCMV recombinant virus encoding a carboxy-terminal domain truncation mutant of US28, FLAG-US28/1-314, to investigate the role that this domain plays in US28 signaling. We demonstrate that US28/1-314 exhibits a more potent phospholipase C-β (PLC-β) signal than does wild-type US28, indicating that the carboxy-terminal domain plays an important role in regulating agonist-independent signaling in infected cells. Moreover, HMCV-infected cells expressing the US28/1-314 mutant exhibit a prolonged calcium signal in response to CCL5, indicating that the US28 carboxy-terminal domain also regulates agonist-dependent signaling. Finally, while the chemokine CX3CL1 behaves as an inverse agonist or inhibitor of constitutive US28 signaling to PLC-β, we demonstrate that CX3CL1 functions as an agonist with regard to US28-stimulated calcium release. This study is the first to demonstrate that the carboxy terminus of US28 controls US28 signaling in the context of HCMV infection and indicates that chemokines such as CX3CL1 can decrease constitutive US28 signals and yet simultaneously promote nonconstitutive US28 signals.

scholarly journals Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

2006 ◽  
Vol 50 (8) ◽  
pp. 2806-2813 ◽  
Author(s):  
T. Ueno ◽  
Y. Eizuru ◽  
H. Katano ◽  
T. Kurata ◽  
T. Sata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Line ◽  
Real Time ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Fluorescent Protein ◽  
Clinical Isolates ◽  
Pml Bodies ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

P2Y2 purinergic receptor is induced following human cytomegalovirus infection and its activity is required for efficient viral replication

2016 ◽  
Author(s):  
Saisai Chen ◽  
Thomas Shenk ◽  
Maciej T. Nogalski
Keyword(s):  
Viral Replication ◽  
Host Cell ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Purinergic Receptor ◽  
Purinergic Signaling ◽  
Receptor Expression ◽  
Receptor Activity ◽  
P2y2 Receptor ◽  
Infected Cells

AbstractHuman cytomegalovirus (HCMV) manipulates many aspects of host cell biology to create an intracellular milieu optimally supportive of its replication and spread. The current study reveals a role for purinergic signaling in HCMV infection. The levels of several components of the purinergic signaling system, including the P2Y2 receptor, were altered in HCMV-infected fibroblasts. P2Y2 receptor RNA and protein are strongly induced following infection. Pharmacological inhibition of receptor activity or knockdown of receptor expression markedly reduced the production of infectious HCMV progeny. When P2Y2 activity was inhibited, the accumulation of most viral RNAs tested and viral DNA was reduced. In addition, the level of cytosolic calcium within infected cells was reduced when P2Y2 signaling was blocked. The HCMV-coded UL37x1 protein was previously shown to induce calcium flux from the smooth endoplasmic reticulum to the cytosol, and the present study demonstrates that P2Y2 function is required for this mobilization. We conclude that P2Y2 supports the production of HCMV progeny, possibly at multiple points within the viral replication cycle that interface with signaling pathways induced by the purinergic receptor.ImportanceHCMV infection is ubiquitous and can cause life-threatening disease in immunocompromised patients, debilitating birth defects in newborns, and has been increasingly associated with a wide range of chronic conditions. Such broad clinical implications result from the modulation of multiple host cell processes. This study documents that cellular purinergic signaling is usurped in HCMV-infected cells and that the function of this signaling axis is critical for efficient HCMV infection. Therefore, we speculate that blocking P2Y2 receptor activity has the potential to become an attractive novel treatment option for HCMV infection.

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