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Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines

Featured protocol,  Authors: Daniela Boehm
Daniela BoehmAffiliation: Gladstone Institute of Virology and Immunology, San Francisco, CA, USA
Bio-protocol author page: a4533
 and Melanie Ott
Melanie OttAffiliation 1: Gladstone Institute of Virology and Immunology, San Francisco, CA, USA
Affiliation 2: Department of Medicine, University of California, San Francisco, CA, USA
For correspondence: mott@gladstone.ucsf.edu
Bio-protocol author page: a2100
date: 5/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2290.

Brief version appeared in Cell Cycle, Feb 2013
The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). A combination of antiretroviral treatments with latency-purging strategies may accelerate the depletion of latent reservoirs and lead to a cure (Geeraert et al., 2008). Current strategies to reactivate HIV-1 from latency include use of prostratin, a non-tumor-promoting phorbol ester (Williams et al., 2004), BET inhibitors (Filippakopoulos et al., 2010; Delmore et al., 2011), and histone deacetylase (HDAC) inhibitors, such as suberoylanilidehydroxamic acid (i.e., SAHA or Vorinostat) (Kelly et al., 2003; Archin et al., 2009; Contreras et al., 2009; Edelstein et al., 2009). As the mechanisms of HIV-1 latency are diverse, effective reactivation may require combinatorial strategies (Quivy et al., 2002). The following protocol describes a flow cytometry-based method to quantify transcriptional activation of the HIV-1 long terminal repeat (LTR) upon drug treatment. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines that contain a GFP cassette. J-Lats that contain a different reporter, for example Luciferase, can be treated with drugs as described but have to be analyzed differently.

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes

Featured protocol,  Authors: Brendan A. Palmer
Brendan A. PalmerAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
Bio-protocol author page: a4519
Zoya Dimitrova
Zoya DimitrovaAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4520
Pavel Skums
Pavel SkumsAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4521
Orla Crosbie
Orla CrosbieAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4522
Elizabeth Kenny-Walsh
Elizabeth Kenny-WalshAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4523
 and Liam J. Fanning
Liam J. FanningAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
For correspondence: l.fanning@ucc.ie
Bio-protocol author page: a4524
date: 5/20/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2284.

Brief version appeared in J Virol, Dec 2015
Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are unreliable through the application of arbitrary frequency cut offs. Cumulatively this leads to an underestimation of genetic diversity. In the following technique we describe the analysis of Hepatitis C virus (HCV) HVR1 which includes the E1/E2 glycoprotein gene junction. This procedure describes the evolution of HCV in a treatment naïve environment, from 10 samples collected over 10 years, using ultradeep pyrosequencing (UDPS) performed on the Roche GS FLX titanium platform (Palmer et al., 2014). Initial clonal analysis of serum samples was used to inform downstream error correction algorithms that allowed for a greater sequence depth to be reached. PCR amplification of this region has been tested for HCV genotypes 1, 2, 3 and 4.

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting

Featured protocol,  Authors: Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
Bio-protocol author page: a4283
Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Department of Genetics, University College London Institute of Ophthalmology, London, United Kingdom
Bio-protocol author page: a4285
Nelly Jolinon
Nelly JolinonAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4486
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Genetic Medicine Institute, Pfizer Inc., London, United Kingdom
Bio-protocol author page: a4487
Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA
Bio-protocol author page: a4288
 and Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
For correspondence: els.henckaerts@kcl.ac.uk
Bio-protocol author page: a4289
date: 5/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2271.

Brief version appeared in J Virol, Aug 2016
Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems

Featured protocol,  Authors: Hong-Ru Chen
Hong-Ru ChenAffiliation: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Bio-protocol author page: a4488
 and Trai-Ming Yeh
Trai-Ming YehAffiliation 1: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Affiliation 2: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
For correspondence: today@mail.ncku.edu.tw
Bio-protocol author page: a4489
date: 5/5/2017, 160 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2273.

Brief version appeared in PLoS Negl Trop Dis, Jul 2016
Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. Here, we provide transwell permeability assay and electrical impedance sensing assay for studying endothelial permeability in vitro. With these methods, the effect of a molecule on endothelial permeability could be defined.

Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines

Authors: Daniela Boehm
Daniela BoehmAffiliation: Gladstone Institute of Virology and Immunology, San Francisco, CA, USA
Bio-protocol author page: a4533
 and Melanie Ott
Melanie OttAffiliation 1: Gladstone Institute of Virology and Immunology, San Francisco, CA, USA
Affiliation 2: Department of Medicine, University of California, San Francisco, CA, USA
For correspondence: mott@gladstone.ucsf.edu
Bio-protocol author page: a2100
date: 5/20/2017, 108 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2290.

[Abstract] The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). A combination of antiretroviral ...

Ultradeep Pyrosequencing of Hepatitis C Virus to Define Evolutionary Phenotypes

Authors: Brendan A. Palmer
Brendan A. PalmerAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
Bio-protocol author page: a4519
Zoya Dimitrova
Zoya DimitrovaAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4520
Pavel Skums
Pavel SkumsAffiliation: Division of Viral Hepatitis, Centers of Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a4521
Orla Crosbie
Orla CrosbieAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4522
Elizabeth Kenny-Walsh
Elizabeth Kenny-WalshAffiliation: Department of Gastroenterology, Cork University Hospital, Cork, Ireland
Bio-protocol author page: a4523
 and Liam J. Fanning
Liam J. FanningAffiliation: Molecular Virology Diagnostic & Research Laboratory, Department of Medicine, University College Cork, Cork, Ireland
For correspondence: l.fanning@ucc.ie
Bio-protocol author page: a4524
date: 5/20/2017, 91 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2284.

[Abstract] Analysis of hypervariable regions (HVR) using pyrosequencing techniques is hampered by the ability of error correction algorithms to account for the heterogeneity of the variants present. Analysis of between-sample fluctuations to virome sub-populations, and detection of low frequency variants, are ...

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting

Authors: Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom
Bio-protocol author page: a4283
Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Department of Genetics, University College London Institute of Ophthalmology, London, United Kingdom
Bio-protocol author page: a4285
Nelly Jolinon
Nelly JolinonAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4486
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
Present address: Genetic Medicine Institute, Pfizer Inc., London, United Kingdom
Bio-protocol author page: a4487
Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA, USA
Bio-protocol author page: a4288
 and Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King's College London, London, United Kingdom
For correspondence: els.henckaerts@kcl.ac.uk
Bio-protocol author page: a4289
date: 5/5/2017, 163 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2271.

[Abstract] Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates ...

In vitro Assays for Measuring Endothelial Permeability by Transwells and Electrical Impedance Systems

Authors: Hong-Ru Chen
Hong-Ru ChenAffiliation: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Bio-protocol author page: a4488
 and Trai-Ming Yeh
Trai-Ming YehAffiliation 1: The Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
Affiliation 2: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan City, Taiwan
For correspondence: today@mail.ncku.edu.tw
Bio-protocol author page: a4489
date: 5/5/2017, 160 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2273.

[Abstract] Vascular leakage is an important feature in several diseases, such as septic shock, viral hemorrhagic fever, cancer metastasis and ischemia-reperfusion injuries. Thus establishing assays for measuring endothelial permeability will provide insight into the establishment or progression of such diseases. ...

RNA-dependent RNA Polymerase Assay for Hepatitis E Virus

Authors: Vidya P. Nair
Vidya P. NairAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
Bio-protocol author page: a4217
Saumya Anang
Saumya AnangAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
Bio-protocol author page: a4292
Akriti Srivastava
Akriti SrivastavaAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
Bio-protocol author page: a4293
 and Milan Surjit
Milan SurjitAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
For correspondence: milan@thsti.res.in
Bio-protocol author page: a4218
date: 4/5/2017, 362 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2199.

[Abstract] RNA-dependent RNA polymerase (RdRp) is essential for the replication of viral RNA for RNA viruses. It synthesizes the complementary strand of viral genomic RNA, which is used subsequently as a template to generate more copies of viral genome. This assay measures activity of the hepatitis E virus (HEV) ...

RNA Strand Displacement Assay for Hepatitis E Virus Helicase

Authors: Vidya P. Nair
Vidya P. NairAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
Bio-protocol author page: a4217
 and Milan Surjit
Milan SurjitAffiliation: Virology Laboratory, Vaccine and Infectious Disease Research Centre, Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, India
For correspondence: milan@thsti.res.in
Bio-protocol author page: a4218
date: 4/5/2017, 303 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2198.

[Abstract] The hepatitis E virus (HEV) helicase uses ATP to unwind the RNA duplexes. This is an essential step for viral replication. This protocol aims to measure the double strand RNA unwinding activity of the HEV helicase....

Robust Generation of Knock-in Cell Lines Using CRISPR-Cas9 and rAAV-assisted Repair Template Delivery

Authors: Giel Vandemoortele
Giel VandemoorteleAffiliation 1: VIB-UGent Center for Medical Biotechnology, Ghent, Belgium
Affiliation 2: Department of Biochemistry, Ghent University, Ghent, Belgium
Bio-protocol author page: a4318
Delphine De Sutter
Delphine De SutterAffiliation 1: VIB-UGent Center for Medical Biotechnology, Ghent, Belgium
Affiliation 2: Department of Biochemistry, Ghent University, Ghent, Belgium
Bio-protocol author page: a4319
 and Sven Eyckerman
Sven EyckermanAffiliation 1: VIB-UGent Center for Medical Biotechnology, Ghent, Belgium
Affiliation 2: Department of Biochemistry, Ghent University, Ghent, Belgium
For correspondence: sven.eyckerman@vib-ugent.be
Bio-protocol author page: a4320
date: 4/5/2017, 337 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2211.

[Abstract] The programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease 9 (Cas9) technology revolutionized genome editing by providing an efficient way to cut the genome at a desired location (Ledford, 2015). In mammalian cells, DNA lesions trigger the error-prone non-homologous ...

Isolation of Exosomes from Semen for in vitro Uptake and HIV-1 Infection Assays

Authors: Marisa N. Madison
Marisa N. MadisonAffiliation: Department of Mathematics and Natural Sciences, Miami Dade College, Homestead, USA
Bio-protocol author page: a4323
Jennifer L. Welch
Jennifer L. WelchAffiliation: Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, USA
Bio-protocol author page: a4324
 and Chioma M. Okeoma
Chioma M. OkeomaAffiliation 1: Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, USA
Affiliation 2: Interdisciplinary Program in Molecular and Cellular Biology, University of Iowa, Iowa City, USA
For correspondence: chioma-okeoma@uiowa.edu
Bio-protocol author page: a4325
date: 4/5/2017, 302 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2216.

[Abstract] Exosomes are membranous extracellular nanovesicles of endocytic origin. Exosomes are known to carry host and pathogen-derived genomic, proteomic, lipidomic cargos and other extraneous molecules. Exosomes are secreted by diverse cell types into the extracellular milieu and are subsequently internalized ...

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

Authors: Francisco Zarate-Perez
Francisco Zarate-PerezAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4284
Vishaka Santosh
Vishaka SantoshAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
Bio-protocol author page: a4286
Martino Bardelli
Martino BardelliAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4283
Leticia Agundez
Leticia AgundezAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4285
R. Michael Linden
R. Michael LindenAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4287
Els Henckaerts
Els HenckaertsAffiliation: Department of Infectious Diseases, King’s College London, London, United Kingdom
Bio-protocol author page: a4289
 and Carlos R. Escalante
Carlos R. EscalanteAffiliation: Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond VA, USA
For correspondence: carlos.escalante@vcuhealth.org
Bio-protocol author page: a4288
date: 3/20/2017, 369 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2194.

[Abstract] Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated ...

In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68

Authors: Liang Sun
Liang SunAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4263
Leen Delang
Leen DelangAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4264
Carmen Mirabelli
Carmen Mirabelli Affiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
Bio-protocol author page: a4265
 and Johan Neyts
Johan NeytsAffiliation: KU Leuven – University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, Leuven, Belgium
For correspondence: johan.neyts@kuleuven.be
Bio-protocol author page: a1546
date: 3/20/2017, 347 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2183.

[Abstract] In 2014 enterovirus D68 (EV-D68) caused the largest outbreak in the United States since the discovery of the virus. Distinct from before, the 2014 infections were associated with more severe respiratory disease and occasional neurological complications. So far, there are no available vaccines or antivirals ...
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[Bio101] Lentivirus Production

Author: Nabila Aboulaich date: 3/5/2011, 23596 views, 6 Q&A
DOI: https://doi.org/10.21769/BioProtoc.39.

[Abstract] Lentivirus is a common tool used to introduce a gene into mammalian or other animal cells.This protocol is to produce lentivirus stocks from hairpin-pLKO.1 plasmid....

[Bio101] Purification of Adenovirus by Cesium Chloride Density Gradients

Author: Huan Pang
Huan PangAffiliation: Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, USA
For correspondence: pang_huan@hotmail.com
Bio-protocol author page: a48
date: 4/5/2012, 18071 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.201.

[Abstract] Adenovirus are efficient gene delivery systems. The standard method for purification of adenoviral vectors is based on using a cesium chloride (CsCl) density gradient combined with ultracentrifugation. This method is suitable for small-scale purification and is less expensive than column chromatography ...

Infectious Focus Assays and Multiplicity of Infection (MOI) Calculations for Alpha-herpesviruses

Authors: Anna Sloutskin
Anna SloutskinAffiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
Bio-protocol author page: a1808
 and Ronald S. Goldstein
Ronald S. GoldsteinAffiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
For correspondence: ron.goldstein@biu.ac.il
Bio-protocol author page: a1809
date: 11/20/2014, 10612 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1295.

[Abstract] Titration of viral stocks is a critical process before any experimental use of the virus. Here we describe an infectious focus assay for several alphaherpesviruses, a titration method for fluorescently labeled viruses, based on the original plaque assay. In addition, the calculation of multiplicity ...

ADCC Assay Protocol

Authors: Vikram Srivastava
Vikram SrivastavaAffiliation: Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
Bio-protocol author page: a1115
Zheng Yang
Zheng YangAffiliation: Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
Bio-protocol author page: a1116
Ivan Fan Ngai Hung
Ivan Fan Ngai HungAffiliation: Department of Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
Bio-protocol author page: a1117
Jianqing Xu
Jianqing XuAffiliation: Institutes of Biomedical Sciences, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
Bio-protocol author page: a1118
Bojian Zheng
Bojian ZhengAffiliation: Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
Bio-protocol author page: a1119
 and Mei-Yun Zhang
Mei-Yun ZhangAffiliation: Department of Microbiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China
For correspondence: zhangmy@hku.hk
Bio-protocol author page: a1120
date: 1/20/2014, 9491 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1029.

[Abstract] Antibody-dependent cell-mediated cytotoxicity (ADCC) bridges innate and adaptive immunity, and it involves both humoral and cellular immune responses. ADCC has been found to be a main route of immune protection against viral infections and cancers in vivo. Here we developed a flow cytometry based protocol ...

[Bio101] VSVG Psudotyped Retrovirus Production

Author: Jia Li
Jia LiAffiliation: Department of Immunology, Medical Center, Duke University, Durham, North Carolina, USA
For correspondence: jiali.email@gmail.com
Bio-protocol author page: a16
date: 12/5/2011, 8968 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.158.

[Abstract] Retrovirus vector pseudotyped with vesicular stomatitis virus G (VSV-G) protein has been proven to exhibit high efficiency to deliver genes in a variety of cells. Efficiency is affected by relative cell growth rate and phosphatidylserine level on the cell membrane....

Assay to Evaluate Vascular Permeability Induction in Mice

Authors: Henry Puerta-Guardo
Henry Puerta-GuardoAffiliation: Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
Bio-protocol author page: a980
Arturo Raya-Sandino
Arturo Raya-SandinoAffiliation: Department of Physiolology, Biophysics and Neurosciences, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
Bio-protocol author page: a981
Lorenza González-Mariscal
Lorenza González-MariscalAffiliation: Department of Physiolology, Biophysics and Neurosciences, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
Bio-protocol author page: a982
Victor H. Rosales
Victor H. RosalesAffiliation: General Services Laboratory, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
Bio-protocol author page: a983
José Ayala-Dávila
José Ayala-DávilaAffiliation: Department of Physiolology, Biophysics and Neurosciences, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
Bio-protocol author page: a984
Bibiana Chávez-Mungía
Bibiana Chávez-MungíaAffiliation: Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
Bio-protocol author page: a985
Daniel Martínez-Fong
Daniel Martínez-FongAffiliation: Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
Bio-protocol author page: a986
Fernando Medina
Fernando MedinaAffiliation: Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
Bio-protocol author page: a987
Juan E. Ludert
Juan E. LudertAffiliation: Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
For correspondence: jludert@cinvestav.mx
Bio-protocol author page: a988
 and Rosa María del Angel
Rosa María del AngelAffiliation: Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies, Instituto Politécnico Nacional (CINVESTAV-IPN), Mexico City, México
For correspondence: rmangel@cinvestav.mx
Bio-protocol author page: a989
date: 11/20/2013, 8548 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.977.

[Abstract] Dengue virus infection usually courses as a benign self-limited fever, called dengue fever. However, on occasions it can progress to a life-threatening complication known as severe dengue (SD). A hallmark of SD is a sharp increase in vascular permeability. Secondary infections are considered a risk ...

Immunoplaque Assay (Influenza Virus)

Authors: Longping V. Tse
Longping V. TseAffiliation: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, USA
For correspondence: lt273@cornell.edu
Bio-protocol author page: a940
Yueting Zhang
Yueting ZhangAffiliation: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, USA
Bio-protocol author page: a941
 and Gary R. Whittaker
Gary R. WhittakerAffiliation: Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, USA
For correspondence: grw7@cornell.edu
Bio-protocol author page: a942
date: 11/5/2013, 6819 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.959.

[Abstract] Despite developed long time ago, plaque assay is still the gold standard for viral titer quantification in modern virology. The standard crystal violet-based plaque assay relies on virus’ ability to induce cytopathic effect (CPE) which limits the assay to lytic viruses. Alternative viral quantification ...

Amplification of HIV-1 Infectious Virus in BL3 Lab

Author: Xin Wang
Xin WangAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: xinwang73@hotmail.com
Bio-protocol author page: a26
date: 3/5/2012, 6152 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.102.

[Abstract] This method is used for making high titer human immunodeficiency virus type-1 (HIV-1) virus stock for subsequent infection assays. The amplification of T-tropic HIV-1 virus (IIIB strain) uses the CD4+ T cell line H9....

Packaging of Retroviral RNA into Viral Particles Analyzed by Quantitative Reverse Transcriptase-PCR

Authors: Bianca Hoffmann
Bianca HoffmannAffiliation: Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
Bio-protocol author page: a446
 and Bastian Grewe
Bastian GreweAffiliation: Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany
For correspondence: bastian.grewe@rub.de
Bio-protocol author page: a371
date: 4/20/2013, 5882 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.684.

[Abstract] Formation of viral particles and packaging of genomic retroviral RNA into these particles are important steps in the late phase of the viral replication cycle. The efficiency of the incorporation of viral or cellular RNAs into viral particles can be studied using a quantitative Reverse Transcriptase-PCR ...

RNA Isolation and Northern Blot Analysis

Authors: Ying Liao
Ying LiaoAffiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
Bio-protocol author page: a1236
To Sing Fung
To Sing FungAffiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
Bio-protocol author page: a1237
Mei Huang
Mei HuangAffiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
Bio-protocol author page: a1238
Shouguo Fang
Shouguo FangAffiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
Bio-protocol author page: a1239
Yanxin Zhong
Yanxin ZhongAffiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
Bio-protocol author page: a1240
 and Dingxiang Liu
Dingxiang LiuAffiliation: School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
For correspondence: dxliu@ntu.edu.sg
Bio-protocol author page: a1241
date: 3/20/2014, 5077 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1077.

[Abstract] The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe particular gene expression levels during differentiation, morphogenesis, as well as abnormal or diseased conditions. Here, we ...
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