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Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates

Featured protocol,  Authors: Satoru Watanabe
Satoru Watanabe Affiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: satoru.watanabe@duke-nus.edu.sg
Bio-protocol author page: a3270
Kitti Wing Ki Chan
Kitti Wing Ki ChanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
Bio-protocol author page: a3810
 and Subhash G. Vasudevan
Subhash G. VasudevanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: subhash.vasudevan@duke-nus.edu.sg
Bio-protocol author page: a3811
date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2040.

Brief version appeared in Antiviral Res, Mar 2016
Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Most cases of DHF/DSS are associated with secondary heterotypic infections through a phenomenon that is described as antibody-dependent enhancement of infection (ADE). There are an estimated 400 million human infections and several hundred thousand cases of severe dengue occurring yearly. At present, however, there are no approved antiviral drugs against DENV infection. The lack of a suitable animal model has hampered the evaluation of novel antiviral candidates for DENV infection. Since DENV poorly establishes infection in immunocompetent mice, AG129 mice (lacking type I and II IFN [interferon] receptors) and mouse-adapted DENV2 strains have been applied to dengue animal models that enable to reproduce several of the major pathologies of human infection. Recently, we developed new mouse models with clinical isolates DENV1 and DENV2 that would be useful for drug testing and dengue pathogenesis studies (Watanabe et al., 2016). Here we describe the details to establish dengue mouse models of clinical isolates; from in vitro preparation of the materials to in vivo virus infection. Of note, since infectivity of DENV in mice differs among virus strains, not all clinical isolates can induce severe dengue.

Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs)

Featured protocol,  Authors: Mickaël M. Ménager
Mickaël M. MénagerAffiliation: The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, USA
For correspondence: mickael.menager@med.nyu.edu
Bio-protocol author page: a3703
 and Dan R. Littman
Dan R. LittmanAffiliation 1: The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, USA
Affiliation 2: Howard Hughes Medical Institute, Washington, USA
Bio-protocol author page: a3704
date: 11/20/2016, 154 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2004.

Brief version appeared in Cell, Feb 2016
The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells

Featured protocol,  Authors: Gayatri Subramanian
Gayatri SubramanianAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3735
Karen Pan
Karen PanAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3736
Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3737
 and Saurabh Chattopadhyay
Saurabh ChattopadhyayAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: Saurabh.Chattopadhyay@UToledo.edu
Bio-protocol author page: a3738
date: 11/20/2016, 145 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2018.

Brief version appeared in J Biol Chem, Sep 2011
Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) – stimulator of interferon genes (STING), which are sensors of viral components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus-infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al., 2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010; Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase-8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro-viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the specific role of caspase-8 in this process.

Mouse Model of Dengue Virus Infection with Serotypes 1 and 2 Clinical Isolates

Authors: Satoru Watanabe
Satoru Watanabe Affiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: satoru.watanabe@duke-nus.edu.sg
Bio-protocol author page: a3270
Kitti Wing Ki Chan
Kitti Wing Ki ChanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
Bio-protocol author page: a3810
 and Subhash G. Vasudevan
Subhash G. VasudevanAffiliation: Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore
For correspondence: subhash.vasudevan@duke-nus.edu.sg
Bio-protocol author page: a3811
date: 12/5/2016, 33 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2040.

[Abstract] Dengue is a global public health threat caused by infection with any of the 4 related dengue virus serotypes (DENV1-4). Clinical manifestations range from self-limiting febrile illness, known as dengue fever (DF), to life-threatening severe diseases, such as dengue hemorrhagic fever (DHF) or dengue ...

Quantitative Measurements of HIV-1 and Dextran Capture by Human Monocyte-derived Dendritic Cells (MDDCs)

Authors: Mickaël M. Ménager
Mickaël M. MénagerAffiliation: The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, USA
For correspondence: mickael.menager@med.nyu.edu
Bio-protocol author page: a3703
 and Dan R. Littman
Dan R. LittmanAffiliation 1: The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, USA
Affiliation 2: Howard Hughes Medical Institute, Washington, USA
Bio-protocol author page: a3704
date: 11/20/2016, 154 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2004.

[Abstract] The aim of this protocol is to describe how to measure and quantify the amount of HIV-1 particles and dextran molecules internalized in human monocyte derived dendritic cells (MDDCs), using three different techniques: flow cytometry, quantitative PCR and confocal microscopy.

[Background] This protocol ...

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells

Authors: Gayatri Subramanian
Gayatri SubramanianAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3735
Karen Pan
Karen PanAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3736
Ritu Chakravarti
Ritu ChakravartiAffiliation: Department of Surgery, University of Toledo College of Medicine and Life Sciences, Toledo, USA
Bio-protocol author page: a3737
 and Saurabh Chattopadhyay
Saurabh ChattopadhyayAffiliation: Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, USA
For correspondence: Saurabh.Chattopadhyay@UToledo.edu
Bio-protocol author page: a3738
date: 11/20/2016, 145 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.2018.

[Abstract] Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like ...

Sequencing of Ebola Virus Genomes Using Nanopore Technology

Author: Thomas Hoenen
Thomas HoenenAffiliation: Friedrich-Loeffler-Institut, Greifswald – Isle of Riems, Germany
For correspondence: thomas.hoenen@fli.de
Bio-protocol author page: a3685
date: 11/5/2016, 304 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1998.

[Abstract] Sequencing of virus genomes during disease outbreaks can provide valuable information for diagnostics, epidemiology, and evaluation of potential countermeasures. However, particularly in remote areas logistical and technical challenges can be significant. Nanopore sequencing provides an alternative ...

Mungbean Yellow Mosaic India Virus (MYMIV)-infection, Small RNA Library Construction and Deep Sequencing for MicroRNA Identification in Vigna mungo

Authors: Anirban Kundu
Anirban KunduAffiliation: Division of Plant Biology, Bose Institute, Kolkata, West Bengal, India
Bio-protocol author page: a2047
Sujay Paul
Sujay PaulAffiliation: Division of Plant Biology, Bose Institute, Kolkata, West Bengal, India
Bio-protocol author page: a2046
Amita Pal
Amita PalAffiliation: Division of Plant Biology, Bose Institute, Kolkata, West Bengal, India
For correspondence: amita@jcbose.ac.in
Bio-protocol author page: a2048
 and Genotypic Technology
Genotypic TechnologyAffiliation: Genotypic Technology Private Limited, #2/13, Balaji Complex, Bangalore, India
Bio-protocol author page: a3589
date: 10/20/2016, 219 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1961.

[Abstract] This protocol describes small RNA library preparation from Vigna mungo total RNA followed by deep sequencing and analysis for microRNA identification.​...

Purification and Identification of Novel Host-derived Factors for Influenza Virus Replication from Human Nuclear Extracts

Authors: Kenji Sugiyama
Kenji SugiyamaAffiliation: Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
For correspondence: kenjisugiyamascience@gmail.com
Bio-protocol author page: a3516
 and Kyosuke Nagata
Kyosuke NagataAffiliation: Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan
Bio-protocol author page: a3517
date: 9/20/2016, 351 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1934.

[Abstract] Recently, we identified two host cell-derived proteins as novel stimulatory factors of influenza virus RNA replication process, termed “Influenza virus REplication Factor-2 (IREF-2)”, from human nuclear extracts (NEs) by employing biochemical complementation assays (Sugiyama et al., 2015). Herein, we ...

Reporter Assay for Semen-mediated Enhancement of HIV-1 Infection

Authors: Janis A. Müller
Janis A. MüllerAffiliation: Institute of Molecular Virology, Ulm University Medical Center, Germany
Bio-protocol author page: a3323
 and Jan Münch
Jan MünchAffiliation: Institute of Molecular Virology, Ulm University Medical Center, Germany
For correspondence: Jan.muench@uni-ulm.de
Bio-protocol author page: a3324
date: 7/20/2016, 464 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1871.

[Abstract] Semen contains amyloid fibrils that enhance HIV-1 infection (Münch et al., 2007; Kim et al., 2010; Roan et al., 2011; Arnold et al., 2012; Usmani et al., 2014; Roan et al., 2014). Positively charged semen amyloids capture negatively charged viral particles and increase their attachment rates to the ...

Haemagglutination Inhibition (HI) Assay of Influenza Viruses with Monoclonal Antibodies

Authors: Ying Wu*
Ying WuAffiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3175
MyungSam Cho*
MyungSam ChoAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3176
David Shore*
David ShoreAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3177
Manki Song
Manki SongAffiliation: International Vaccine Institute, Seoul, Korea
Bio-protocol author page: a3178
JungAh Choi
JungAh ChoiAffiliation: International Vaccine Institute, Seoul, Korea
Bio-protocol author page: a3179
Tao Jiang
Tao JiangAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
Bio-protocol author page: a3180
Yong-Qiang Deng
Yong-Qiang DengAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
Bio-protocol author page: a3181
Melissa Bourgeois
Melissa BourgeoisAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3182
Lynn Almli
Lynn AlmliAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3183
Hua Yang
Hua YangAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3184
Li-Mei Chen
Li-Mei ChenAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3185
Yi Shi
Yi ShiAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: Research Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3186
Jianxu Qi
Jianxu QiAffiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3187
An Li
An LiAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: College of Veterinary Medicine, Guangxi University, Nanning, China
Bio-protocol author page: a3188
Kye Sook Yi
Kye Sook YiAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3189
MinSeok Chang
MinSeok ChangAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3190
Jin Soo Bae
Jin Soo BaeAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3191
HyunJoo Lee
HyunJoo LeeAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3192
JiYoung Shin
JiYoung ShinAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3194
James Stevens
James StevensAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3195
SeoungSuh Hong
SeoungSuh HongAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3196
Cheng-Feng Qin
Cheng-Feng QinAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
For correspondence: qincf@bmi.ac.cn
Bio-protocol author page: a3197
George F. Gao
George F. GaoAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: Research Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
Affiliation 3: Office of Director-General, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China
For correspondence: gaof@im.ac.cn
Bio-protocol author page: a3198
Shin Jae Chang
Shin Jae ChangAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
For correspondence: ShinJae.Chang@celltrion.com
Bio-protocol author page: a3199
 and Ruben O. Donis
Ruben O. DonisAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
For correspondence: rvd6@cdc.gov
Bio-protocol author page: a3200
 (*contributed equally to this work) date: 6/5/2016, 742 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1828.

[Abstract] Heamagglutination is inhibited when antibodies are present because antibodies to influenza virus will prevent attachment of the virus to red blood cells. The highest dilution of antibody that prevents hemagglutination is called the HI titer. The human monoclonal antibodies generated from single human ...

Micro Neutralization (MN) Assay of Influenza Viruses with Monoclonal Antibodies

Authors: Ying Wu*
Ying WuAffiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3175
MyungSam Cho*
MyungSam ChoAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3176
David Shore*
David ShoreAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3177
Manki Song
Manki SongAffiliation: International Vaccine Institute, Seoul, Korea
Bio-protocol author page: a3178
JungAh Choi
JungAh ChoiAffiliation: International Vaccine Institute, Seoul, Korea
Bio-protocol author page: a3179
Tao Jiang
Tao JiangAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
Bio-protocol author page: a3180
Yong-Qiang Deng
Yong-Qiang DengAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
Bio-protocol author page: a3181
Melissa Bourgeois
Melissa BourgeoisAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3182
Lynn Almli
Lynn AlmliAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3183
Hua Yang
Hua YangAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3184
Li-Mei Chen
Li-Mei ChenAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3185
Yi Shi
Yi ShiAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: Research Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3186
Jianxu Qi
Jianxu QiAffiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3187
An Li
An LiAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: College of Veterinary Medicine, Guangxi University, Nanning, China
Bio-protocol author page: a3188
Kye Sook Yi
Kye Sook YiAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3189
MinSeok Chang
MinSeok ChangAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3190
Jin Soo Bae
Jin Soo BaeAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3191
HyunJoo Lee
HyunJoo LeeAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3192
JiYoung Shin
JiYoung ShinAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3194
James Stevens
James StevensAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3195
SeoungSuh Hong
SeoungSuh HongAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3196
Cheng-Feng Qin
Cheng-Feng QinAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
For correspondence: qincf@bmi.ac.cn
Bio-protocol author page: a3197
George F. Gao
George F. GaoAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: Research Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
Affiliation 3: Office of Director-General, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China
For correspondence: gaof@im.ac.cn
Bio-protocol author page: a3198
Shin Jae Chang
Shin Jae ChangAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
For correspondence: ShinJae.Chang@celltrion.com
Bio-protocol author page: a3199
 and Ruben O. Donis
Ruben O. DonisAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
For correspondence: rvd6@cdc.gov
Bio-protocol author page: a3200
 (*contributed equally to this work) date: 6/5/2016, 498 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1829.

[Abstract] The human monoclonal antibodies generated from single human B cells were tested to characterize their ability to neutralize virus infectivity. The microneutralization assay is a highly sensitive and specific assay for detecting virus-specific neutralizing antibodies to influenza viruses. This protocol ...

Respiratory Syncytial Virus Infection in Mice and Detection of Viral Genomes in the Lung Using RT-qPCR

Authors: Yan Sun
Yan SunAffiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, USA
Bio-protocol author page: a2319
 and Carolina B. López
Carolina B. LópezAffiliation: Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, USA
For correspondence: lopezca@vet.upenn.edu
Bio-protocol author page: a3156
date: 5/20/2016, 569 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.1819.

[Abstract] Respiratory syncytial virus (RSV) is a single-stranded negative sense RNA virus that belongs to the paramyxovirus family. RSV infections lead to a variety of clinical outcomes ranging from a mild “cold-like disease” to death. Infection is usually more severe in infants and the elderly. RSV is associated ...
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[Bio101] Lentivirus Production

Author: Nabila Aboulaich date: 3/5/2011, 21605 views, 6 Q&A, How to cite
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, 16532 views, 1 Q&A, How to cite
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 ...

[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, 8144 views, 0 Q&A, How to cite
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....

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, 7841 views, 0 Q&A, How to cite
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, 7659 views, 0 Q&A, How to cite
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 ...

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, 7051 views, 0 Q&A, How to cite
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, 5626 views, 0 Q&A, How to cite
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, 5380 views, 0 Q&A, How to cite
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, 4968 views, 0 Q&A, How to cite
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 ...

Viral Immunofluorescence with Rift Valley Fever Virus Infected MEFs in a 96 Well Plate

Authors: Theresa S. Moser
Theresa S. MoserAffiliation: Department of Microbiology, Penn Genome Frontiers Institute, The University of Pennsylvania School of Medicine, Philadelphia, USA
Bio-protocol author page: a171
 and Sara Cherry
Sara CherryAffiliation: Department of Microbiology, Penn Genome Frontiers Institute, The University of Pennsylvania School of Medicine, Philadelphia, USA
For correspondence: cherrys@mail.med.upenn.edu
Bio-protocol author page: a172
date: 12/5/2012, 4215 views, 0 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.297.

[Abstract] Immunofluorescence is a method to detect viral infection in multiple types of host cells. This procedure can be adapted for both high-throughput and low-throughput assays for any virus for which there are antibodies available. Time of infection and virus multiplicity of infection (MOI) vary and should ...
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