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Automated Analysis of siRNA Screens of Virus Infected Cells Based on Immunofluorescence Microscopy [chapter]

Petr Matula, Anil Kumar, Ilka Wörz, Nathalie Harder, Holger Erfle, Ralf Bartenschlager, Roland Eils, Karl Rohr
Informatik aktuell  
We present an image analysis approach as part of a highthroughput microscopy screening system based on cell arrays for the identification of genes involved in Hepatitis C and Dengue virus replication.  ...  Our approach comprises: cell nucleus segmentation, quantification of virus replication level in cells, localization of regions with transfected cells, cell classification by infection status, and quality  ...  of the level of viral protein expression in channel 2 stained by immunofluorescence(Fig. 1c), (3) detection of siRNA spot areas with transfected cells(Fig. 1d), (4) classification of cells based on their  ... 
doi:10.1007/978-3-540-78640-5_91 dblp:conf/bildmed/MatulaKWHEBER08 fatcat:oanrkwqj2faurbpredu7s6n4vu

Shedding Light on Filovirus Infection with High-Content Imaging

Gianluca Pegoraro, Sina Bavari, Rekha G. Panchal
2012 Viruses  
Major advances in high-throughput fluorescence microscopy and automated, high-content image analysis tools are paving the way to the systematic and quantitative study of the molecular properties of cellular  ...  Finally, we discuss possible novel applications of HCI in the context of filovirus research with particular emphasis on the identification of possible cellular biomarkers of virus infection.  ...  The content of this publication does not necessarily reflect the views or policies of the US Department of Defense, the US Department of the Army, the US Department of Health and Human Services or of the  ... 
doi:10.3390/v4081354 pmid:23012631 pmcid:PMC3446768 fatcat:lthst5k53ffprbn7ijnbcz7cu4

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication

Alexander Karlas, Nikolaus Machuy, Yujin Shin, Klaus-Peter Pleissner, Anita Artarini, Dagmar Heuer, Daniel Becker, Hany Khalil, Lesley A. Ogilvie, Simone Hess, André P. Mäurer, Elke Müller (+3 others)
2010 Nature  
Members of ribosomal and spliceosomal multi-protein complexes are enclosed in larger circles. Thick grey border indicates hits identified in Reactome analysis (see Supplementary Fig. 6 ).  ...  Figure 4 | 4 In-depth analysis of the impact of p27 and CLK1 on influenza A virus infection. a, Quantification of virus replication in primary NHBE cells after siRNA-mediated target knockdown using the  ...  For identification of primary hits, three parameters were included: luciferase expression, the percentage of infected cells, as determined by immunofluorescence microscopy, and the total number of infected  ... 
doi:10.1038/nature08760 pmid:20081832 fatcat:3cty47q3xvg53ft3zhdnbbdu24

c-Src protein kinase inhibitors block assembly and maturation of dengue virus

J. J. H. Chu, P. L. Yang
2007 Proceedings of the National Academy of Sciences of the United States of America  
In this study, we report an immunofluorescence imagebased assay suitable for identification of small molecule inhibitors of dengue virus infection and replication.  ...  Given the simplicity and excellent reproducibility of the assay, it should be useful in high-throughput screens of both small molecule and RNAi libraries when implemented on a robotic image-based highthroughput  ...  The primary screens were carried out with the technical assistance and instrumentation of the ICCB-Longwood Screening Facility.  ... 
doi:10.1073/pnas.0611681104 pmid:17360676 pmcid:PMC1805510 fatcat:wn2ahsfoorgchjgnen7w3kprci

Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

R. Meier, A. Franceschini, P. Horvath, M. Tetard, R. Mancini, C. von Mering, A. Helenius, P.-Y. Lozach
2014 Journal of Virology  
., and from the Natural Sciences and Engineering Research Council of Canada and the Banting Research Foundation to P.Y.L. We thank M. Krzyzaniak, J. Mercer, L. Burleigh, S. Noerrelykke, and N.  ...  Automated transfection, infection, and immunostaining against newly synthesized nucleoprotein N as well as automated fluorescence microscopy and image analysis were employed to analyze cell factors required  ...  (C) Entry of prebound AF647-labeled UUKV (UUKV-AF647; MOI, ϳ10) into cells expressing EGFP-VAMP3 and RFP-LAMP1, based on confocal microscopy. Viruses were internalized at 37°C for 30 min.  ... 
doi:10.1128/jvi.00388-14 pmid:24850728 pmcid:PMC4135934 fatcat:pvair6waqzd37er6iwjxwhqq4m

RNAi Screening Reveals Proteasome- and Cullin3-Dependent Stages in Vaccinia Virus Infection

Jason Mercer, Berend Snijder, Raphael Sacher, Christine Burkard, Christopher Karl Ernst Bleck, Henning Stahlberg, Lucas Pelkmans, Ari Helenius
2012 Cell Reports  
A two-step, automated, high-throughput RNAi silencing screen was used to identify host cell factors required during vaccinia virus infection.  ...  The list of cell functions required during poxvirus infection will, moreover, provide a resource for future virus-host cell interaction studies and for the discovery of antivirals.  ...  Byrde for data storage and cluster computing, Pauli Rä mö for statistical and image analysis, Karin Mench for technical assistance, and Jacomine Krijnse-Locker for anti-I3L.  ... 
doi:10.1016/j.celrep.2012.09.003 pmid:23084750 fatcat:tzc6ldtpvff3xoyxlzyq63yjge

RNASEK is required for internalization of diverse acid-dependent viruses

Brent A. Hackett, Ari Yasunaga, Debasis Panda, Michael A. Tartell, Kaycie C. Hopkins, Scott E. Hensley, Sara Cherry
2015 Proceedings of the National Academy of Sciences of the United States of America  
S.C. is a recipient of the Burroughs Wellcome Investigators in the Pathogenesis of Infectious Disease Award.  ...  We thank members of the S.C. laboratory for helpful discussions and technical advice throughout. We thank BEI.  ...  (H) Human U2OS cells were transfected with either the indicated siRNAs for 3 d and subsequently infected with VACV (MOI 0.5, 8 h) and processed for automated microscopy with the percentage of infected  ... 
doi:10.1073/pnas.1424098112 pmid:26056282 pmcid:PMC4485095 fatcat:g36n3l5yhvhffeygoaruz3cinm

The Essential Role of Clathrin-mediated Endocytosis in the Infectious Entry of Human Enterovirus 71

Khairunnisa' Mohamed Hussain, Kim Lian Janet Leong, Mary Mah-Lee Ng, Justin Jang Hann Chu
2010 Journal of Biological Chemistry  
Screen evaluation was conducted via the expression of well characterized dominant-negative mutants, bioimaging studies (double-labeled immunofluorescence assays, transmission electron microscopy analysis  ...  ), secondary siRNA-based dosage dependence studies, and drug inhibition assays.  ...  The immunofluorescence screening assay was based on the detection of HEV71 capsid (VP1) protein in HEV71-infected RD cell monolayers.  ... 
doi:10.1074/jbc.m110.168468 pmid:20956521 pmcid:PMC3012988 fatcat:h3lf73hbo5g3tfem427gowdnfm

Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

Firzan Ang, Andrew Wong, Mary Ng, Justin Chu
2010 Virology Journal  
The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites.  ...  The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis.  ...  The human isolate of dengue virus serotypes used in this study was a kind gift from the Department of Pathology, Singapore General Hospital.  ... 
doi:10.1186/1743-422x-7-24 pmid:20122152 pmcid:PMC2825209 fatcat:at6tmoxpvfh6pefpasojlm3zcq

Automated microscopy for high-content RNAi screening

Christian Conrad, Daniel W. Gerlich
2010 Journal of Cell Biology  
In this screen for human genes associated with West Nile virus infection, cell nuclei were labeled with DAPI (blue) and stained by immunofluorescence against a viral epitope (red).  ...  In this review, we provide an overview of imaging-based screening strategies. We focus on biological assay design, automated image acquisition, and computational analysis.  ... 
doi:10.1083/jcb.200910105 pmid:20176920 pmcid:PMC2828931 fatcat:ldlktrjnbzfa5lzbog7wlsb76i

Identification of Essential Filovirion-associated Host Factors by Serial Proteomic Analysis and RNAi Screen

Kevin B. Spurgers, Tim Alefantis, Brian D. Peyser, Gordon T. Ruthel, Alison A. Bergeron, Julie A. Costantino, Sven Enterlein, Krishna P. Kota, R. C. Dutch Boltz, M. Javad Aman, Vito G. DelVecchio, Sina Bavari
2010 Molecular & Cellular Proteomics  
Notably, select siRNAs inhibited ebolavirus, but enhanced marburgvirus infection, suggesting important differences between the two viruses.  ...  Significant overlap exists between this data set and proteomic studies of disparate viruses, including HIV-1 and influenza A, generated in multiple cell types.  ...  Acknowledgments-We thank Jens Kuhn for a critical review of this manuscript. We thank Lian Dong for performing the qRT-PCR viral titer measurements.  ... 
doi:10.1074/mcp.m110.003418 pmid:20702783 pmcid:PMC3101857 fatcat:imd7gbka55adjfh2ods6vvrsqm

An RNA replication-center assay for high content image-based quantifications of human rhinovirus and coxsackievirus infections

Andreas Jurgeit, Stefan Moese, Pascal Roulin, Alexander Dorsch, Mark Lötzerich, Wai-Ming Lee, Urs F Greber
2010 Virology Journal  
We optimized the procedure for detection of virus replication in settings for high content screening with automated fluorescence microscopy and single cell analysis.  ...  This protocol also allows to directly compare results from small compound or siRNA infection screens for different serotypes without the risk of assay specific artifacts.  ...  Kaiser for advice in diagnostic sequencing and providing HRV2, 14, 37 and HeLa-Ohio cells, and Qian Feng (Department of Medical  ... 
doi:10.1186/1743-422x-7-264 pmid:20937137 pmcid:PMC2958916 fatcat:ewnanou2y5f3xe2ctt24zk2apq

High-Content Analysis of Sequential Events during the Early Phase of Influenza A Virus Infection

Indranil Banerjee, Yohei Yamauchi, Ari Helenius, Peter Horvath, Paul Digard
2013 PLoS ONE  
Acknowledgments The authors are grateful to the Light Microscopy and Screening Centre (LMSC) at ETH Zurich for support in high-throughput microscopy.  ...  Typical images acquired with automated microscopy are shown in Figure S4 . All the results were based on at least three experiments performed on separate days.  ...  To be able to monitor the passage of incoming viruses through steps of the entry program, we developed fluorescence microscopy-based methods.  ... 
doi:10.1371/journal.pone.0068450 pmid:23874633 pmcid:PMC3709902 fatcat:6vvmlrheuvefdap7oybg264kh4

High-Throughput Small Interfering RNA Screening Identifies Phosphatidylinositol 3-Kinase Class II Alpha as Important for Production of Human Cytomegalovirus Virions

William S. Polachek, Hanan F. Moshrif, Michael Franti, Donald M. Coen, Vattipally B. Sreenu, Blair L. Strang, R. M. Sandri-Goldin
2016 Journal of Virology  
Here we utilized a high-throughput siRNA screen based on detection of a viral antigen by microscopy to interrogate cellular protein kinases and phosphatases for their importance during human cytomegalovirus  ...  Analysis of siRNA-treated cells by electron microscopy and Western blotting indicated that PI3K-C2A was not required for the production of viral capsids but did lead to increased numbers of enveloped capsids  ...  Our thanks also go to Ken Laing and Eugen Buehler for support with quantitative PCR and siRNA binding analysis, to several colleagues (Jeremy Kamil, Wade Gibson, Nathanael Gray, and Richard Stanton) and  ... 
doi:10.1128/jvi.01134-16 pmid:27412598 pmcid:PMC5008103 fatcat:jsdgrmne4vg7hc6zycic7zxnme

Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication

Giuseppe Balistreri, Johanna Viiliäinen, Mikko Turunen, Raquel Diaz, Lauri Lyly, Pirita Pekkonen, Juha Rantala, Krista Ojala, Grzegorz Sarek, Mari Teesalu, Oxana Denisova, Karita Peltonen (+9 others)
2016 PLoS Pathogens  
Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression.  ...  A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation.  ...  of Nutlin for 24 h, and measured the levels of RFP (virus reactivation) by automated microscopy.  ... 
doi:10.1371/journal.ppat.1005424 pmid:26891221 pmcid:PMC4758658 fatcat:3jbvhjzc7jh5xcinzosvrihsbu
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