High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells

Ibig-Rehm, Yvonne; Götte, Marjo; Gabriel, Daniela; Woodhall, David L.; Shea, Ashley; Brown, Nathan E.; Compton, Teresa; Feire, Adam L.

doi:10.1016/j.antiviral.2011.01.007

Cited by 11 publications

(9 citation statements)

References 43 publications

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“…To induce differentiation of KG-1 and Kasumi-3 cells, cells were treated with 10 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma) and 500 ng/ml ionomycin (Sigma) in X-VIVO-15 medium (Lonza) for 3 days, as previously described for KG-1 cells (55). HCMV viral strains used were AD169 and derivatives expressing IE2 or pp65 fused to green fluorescent protein (GFP) (57,58) and GFP-expressing FIX (59). Cells were infected with HCMV in minimal volume for 60 min, followed by addition of medium to normal culture volumes.…”

Section: Methodsmentioning

confidence: 99%

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34⁺Cell Populations

Albright

Kalejta

2013

J Virol

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Section: Methodsmentioning

confidence: 99%

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34⁺Cell Populations

Albright

Kalejta

2013

J Virol

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mentioning

confidence: 99%

“…Such strains have also been used to examine the kinetics and localization of viral protein expression during CMV and herpes simplex virus 1 (HSV-1) infections (12). Viral strains that express fluorescent proteins alone or as a viral chimera are also useful for measuring virus infectivity, testing the antiviral properties of small molecules and the neutralizing capability of monoclonal anti-CMV antibodies, and elucidating early steps in viral binding and entry (13)(14)(15).In this study, we employed a CMV strain that ectopically expresses a yellow fluorescent protein (YFP) fused to the IE2 transcript, AD169 IE2-YFP , to establish a high-throughput cell-based assay that can quantify viral entry into human cells by measuring fluorescence of infected cells. The high-throughput format of the assay offers an easy and rapid approach for evaluating viral growth kinetics and, as we demonstrate, can be employed to test the virusneutralizing capability of monoclonal antibodies and human serum from CMV-positive patients.…”

mentioning

confidence: 99%

Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Gardner

Bolovan-Fritts

Teng

et al. 2013

Clin Vaccine Immunol

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e Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169 IE2-YFP ) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169 IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.T he coevolution of human herpesviruses with their hosts over the past millions of years has led to the development of complex strategies of immune evasion that allow persistent viral infection despite the presence of an active immune response (1). A comprehensive understanding of cytomegalovirus (CMV) infection is essential to delineate the molecular and cellular interactions necessary for priming a targeted humoral immune response and how a pathogen may coopt these processes to establish a persistent and lifelong infection (2). Furthermore, a more complete comprehension of CMV entry, replication, and immune evasion is paramount in developing strategies to diagnose and alleviate CMV disease in immunocompromised patients such as transplant recipients, AIDS patients, and neonates.Analysis of viral protein expression during CMV infection can be useful in studying viral entry, cellular manipulation, and egress. The replication cycle of CMV is temporally controlled and regulated by different segments of the viral genome. The replicative cycle is divided into immediate-early (IE), early (E), and late (L) phases of replication. CMV IE proteins are produced first and appear within 6 h postinfection (hpi). IE proteins are potent transactivators that stimulate the transcription of E genes (3, 4). IE1 and IE2 are the best-characterized IE gene products and are essential for viral replication a...

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“…30,31 In high-content image-based screening, studies typically focus on one primary readout, such as a target protein signal, while using other readouts as filters to identify treatments that have resulted in overt toxicity. 32 This approach, however, is limited to a small number of readouts and does not take into account the correlation among readouts. To fully leverage the phenotypic knowledge in multiparametric data, one needs to apply multiparametric statistical learning methods.…”

Section: Methods For Analyzing Multiparametric Datamentioning

confidence: 99%

Multiparametric Analysis of Screening Data: Growing Beyond the Single Dimension to Infinity and Beyond

2014

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Advances in instrumentation now allow the development of screening assays that are capable of monitoring multiple readouts such as transcript or protein levels, or even multiple parameters derived from images. Such advances in assay technologies highlight the complex nature of biology and disease. Harnessing this complexity requires integration of all the different parameters that can be measured rather than just monitoring a single dimension as is commonly used. Although some of the methods used to combine multiple measurements, such as principal component analysis, are commonly used for microarray analysis, biologists are not yet using many of the tools that have been developed in other fields to address such issues. Visualization of multiparametric data sets is one of the major challenges in this field, and a depiction of the results in a manner that can be readily interpreted is essential. This article describes a number of assay systems being used to generate such data sets en masse, and the methods being applied to their visualization and analysis. We also discuss some of the challenges of applying methods developed in other fields to biology.

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High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells

Cited by 11 publications

References 43 publications

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34⁺Cell Populations

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34⁺Cell Populations

Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Multiparametric Analysis of Screening Data: Growing Beyond the Single Dimension to Infinity and Beyond

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High-content screening to distinguish between attachment and post-attachment steps of human cytomegalovirus entry into fibroblasts and epithelial cells

Cited by 11 publications

References 43 publications

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+Cell Populations

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+Cell Populations

Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

Multiparametric Analysis of Screening Data: Growing Beyond the Single Dimension to Infinity and Beyond

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Product

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Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34⁺Cell Populations

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34⁺Cell Populations