Human Respiratory Syncytial Virus: Methods and Protocols (Methods in Molecular Biology, 1442)

✍ Scribed by Ralph A. Tripp (editor), Patricia A. Jorquera (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 253
• Category: Library

✦ Synopsis

This volume summarizes current techniques; ranging from protocols for virus growth, isolation, quantification and generation of recombinant RSV virus to procedures for the efficient characterization of the host immune response to RSV infection. These techniques are used in numerous laboratories around the world and are the building blocks that support the majority of RSV virus research. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Humana Respiratory Syncytial Virus: Methods and Protocols aims to ensure successful results in the further study of this vital field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Human Respiratory Syncytial Virus: An Introduction
1 Introduction
1.1 Classification
1.2 Genome Organization and Viral Proteins
1.3 RSV Replication
1.4 RSV Pathogenesis
1.5 Host Immune Response to RSV
1.6 Control and Prevention of RSV Infection
References
Chapter 2: RSV Growth and Quantification by Microtitration and qRT-­PCR Assays
1 Introduction
2 Materials
2.1 Tissue Culture
2.2 Microtitration by Plaque Assay
2.3 Immunostaining
2.4 Quantitative Real-time PCR (qRT-PCR)
3 Methods
3.1 Preparation of Cell Culture for Limiting Dilution
3.2 Preparation of Virus Seed Stocks by Limiting Dilution
3.3 Preparation of RSV Working Stock
3.4 Microtitration of RSV by Plaque Assay
3.5 Determination of the Viral Titer in RSV Stocks by qRT-PCR)
4 Notes
References
Chapter 3: Quantification of RSV Infectious Particles by Plaque Assay and Immunostaining Assay
1 Introduction
2 Materials
2.1 RSV Immunostaining Assay
2.2 RSV Plaque Assay
3 Methods
3.1 RSV Immunostaining Assay
3.2 RSV Plaque Assay
4 Notes
References
Chapter 4: Detection of RSV Antibodies in Human Plasma by Enzyme Immunoassays
1 Introduction
2 Materials
2.1 Production of RSV Virus and HEp-2 Lysate Antigens
2.2 Determining the Dilution of RSV Lysate Antigen to be Used in the RSV Lysate EIA
2.3 Detection of Anti-RSV Antibodies in Human Plasma or Serum Using RSV Lysate EIA
2.4 Detection of Anti-RSV G or F Protein Antibodies in Human Plasma or Serum
2.5 Detection of Anti-RSV Antibodies in Human Plasma or Serum Using RSV Lysate EIA
3 Methods
3.1 Production of RSV Virus and HEp-2 Lysate Antigens
3.2 Determining the Dilution of RSV Virus Lysate Antigen to be Used in the RSV Lysate EIA
3.3 Detection of Anti-RSV Antibodies in Human Plasma or Serum Using RSV Lysate EIA
3.4 Detection of Anti-RSV G or F Protein Antibodies in Human Plasma or Serum
3.5 Detection of Antibodies to Central Conserved Region of the RSV G Protein (see Note 9)
4 Notes
Chapter 5: Secretory Expression and Purification of Respiratory Syncytial Virus G and F Proteins in Human Cells
1 Introduction
2 Materials
2.1 Transfection of 293F Human Cells for Secretion of RSV F and G Proteins
2.2 Purification of RSV G or F Protein
2.3 Detection of RSV G and F Proteins by Western Blotting
3 Methods
3.1 Transfection of FreeStyle 293-F Cells with pcDNA-G-­His and pcDNA-F-His
3.2 Ni Sepharose Column Packing
3.3 Purification of RSV G or F Protein
3.4 Detection of RSV G and F Proteins by Western Blotting
4 Notes
References
Chapter 6: Development of Human Monoclonal Antibodies Against Respiratory Syncytial Virus Using a High Efficiency Human Hybridoma Technique
1 Introduction
2 Materials
2.1 Expression of RSV FECTO and GECTO
2.2 Generation of Epstein-Barr Virus (EBV)
2.3 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
2.4 Isolation of Peripheral Blood Mononuclear Cells (PBMCs) for Feeder Layers
2.5 EBV Transformation of B Cells from RSV-
2.6 Growth and Maintenance of HMMA 2.5 Cell Line
2.7 Cytofusion of EBV Transformed B Cells and HMMA 2.5 Cells
2.8 Subcloning Hybridomas by Flow Cytometry
2.9 Hybridoma Expansion
2.10 Plaque Reduction Assay
3 Methods
3.1 Cloning, Expression, and Purification of RSV FECTO and GECTO
3.2 Generation of Epstein-Barr Virus (EBV) Stock for Transformation
3.3 Isolation of Peripheral Blood Mononuclear Cells (PBMCs) from Respiratory Syncytial Virus (RSV) Immune Subject Blood Samples
3.4 Preparation of Peripheral Blood Mononuclear Cells (PBMCs) from Human Blood for Feeder Layers
3.5 EBV Transformation of B Cells from RSV-­Immune Subject PBMCs
3.6 Growth and Maintenance of HMMA 2.5 Cells
3.7 Fusion of EBV-­Transformed B Cells with HMMA 2.5 Cells
3.8 Subcloning Hybridoma Cell Lines by Flow Cytometric Sorting
3.9 Expanding Hybridoma Clones
3.10 Characteri
4 Notes
Chapter 7: Respiratory Syncytial Virus (RSV): Neutralizing Antibody, a Correlate of Immune Protection
1 Introduction
1.1 Key Elements in the Microneutralization Assay
2 Materials
2.1 Hardware
2.2 Tissue Culture
2.3 Solutions and Reagents
3 Methods
3.1 RSV Microneutralization Assay (See Note 2)
4 Notes
References
Chapter 8: Host Factors Modulating RSV Infection: Use of Small Interfering RNAs to Probe Functional Importance
1 Introduction
2 Materials
2.1 Growth of A549 Cells
2.2 Preparation of siRNA Oligos and Transfection
2.3 Bradford Assay
2.4 SDS Polyacrylamide Gel
2.5 Western Blotting
2.6 Quantitative RT-PCR (qRT-PCR)
2.7 Treating RSV Infected Cells with the CK2α Inhibitor TBB
3 Methods
3.1 Maintenance and Preparation of A549 Lung Epithelial Cells
3.2 Preparation of A549 Cells for siRNA Transfection
3.3 Preparation of siRNAs for Your Gene(s) of Interest
3.4 Transfection of siRNA Using DharmaFECT1
3.5 Optimizing siRNA Transfection (See Note 5)
3.6 Determining Protein Concentration Using Bradford Assay
3.7 Using SDS-PAGE Gel Separation and Western Analysis to Confirm siRNA Gene Knockdown (See Note 8)
3.8 Infecting siRNA Transfected Cells
3.9 Harvesting Virus from siRNA Transfected and RSV Infected Cells
3.10 Analyzing the Effect of Host Gene Knockdown on RSV Viral Replication by qRT-PCR Amplification of the RSV N Gene
3.11 Analyzing Infectious Viral Titers by Plaque Assay
3.12 Using a Selective Inhibitor to Confirm Results Obtained in siRNA Experiments
4 Notes
References
Chapter 9: In Vitro Modeling of RSV Infection and Cytopathogenesis in Well-Differentiated Human Primary Airway Epithelial Cells (WD-PAECs)
1 Introduction
2 Materials
2.1 Hardware and Consumables
2.2 Patient Sample Collection
2.3 Tissue Culture Media and Solutions
2.4 Cytospin and Cell Staining
3 Methods
3.1 Collagen Coating of Tissue Culture Flasks
3.2 Collagen Coating of Transwells (See Note 2)
3.3 Obtaining Fresh Bronchial Epithelial Cells from Patients (Non-­bronchoscopic Method) (See Note 7)
3.4 Obtaining Fresh Nasal Epithelial Cells from Patients (See Note 7)
3.5 Processing Bronchial or Nasal Brushes from Patients
3.6 Passage of Human Airway Epithelial Cells (PAECs)
3.7 Cell Counting of PAECs
3.8 Freezing of PAECs
3.9 Defrosting PAECs
3.10 Differentiation of PAECs
3.10.1 Submerged Culturing in Transwells (for 6.5 mm Diameter Transwell)
3.10.2 Air–Liquid Interface (ALI) Culturing (See Note 11)
3.11 RSV Infection of Well-­Differentiated PAECs (WD-PAECs)
3.12 Apical Rinsing and Basal Medium Harvesting from RSV-­Infected Cultures
3.13 Cytospin
3.14 Fixation of WD-PAECs
3.15 Removal of Membrane from Transwell
3.16 Staining of WD-PAECs for Immunofluorescence
4 Notes
References
Chapter 10: Reverse Genetics of Respiratory Syncytial Virus
1 Introduction
2 Materials
2.1 Transformation and Expansion of BAC Constructs
2.2 RSV Recovery Components
2.3 Plaque Purification and Virus Stock Generation Components
3 Methods
3.1 Transformation and Preparation of RSV BAC Constructs for Transfection
3.2 Recovery of Recombinant RSV A2-Line19F
3.2.1 Day-1: Preparation of BSR-T7/5 Cells for Transfection
3.2.2 Day 0: Transfection for RSV Recovery
3.2.3 Day 1: Expansion and Recovery of RSV
3.2.4 Day 2: Transfer of Transfected Cells into Recovery Flask
3.2.5 Day 3–Day 6: Passaging of RSV for Recovery
3.2.6 Day 7: Harvest of Virus BSR Stock
3.3 Plaque Purification of RSV
3.3.1 Day-1: Preparation of HEp-2 Cells for Plaque Purification
3.3.2 Day 0: Infection for Plaque Purification of Virus Stock
3.3.3 Day 1–Day ~5: Formation of Visible Plaques
3.3.4 Day 6: Picking Plaques
3.4 Generation of Master/Working Stocks
3.4.1 Day-1: Preparation of Cells for Virus Recovery
3.4.2 Day 0: Infection for Virus Recovery
3.4.3 Day ~5: Harvest of Virus Stock
4 Notes
References
Chapter 11: Use of Minigenome Systems to Study RSV Transcription
1 Introduction
2 Materials
2.1 Transfection
2.2 Luciferase Assay
2.3 Real-Time Quantitative PCR (QPCR) Assay
3 Methods
3.1 Transfection (See Note 1)
3.2 Luciferase Assay (Dual Luciferase Reporter Assay System Method)
3.3 General Injector Wash Protocol (See Note 12)
3.4 RNA Isolation (See Note 13)
3.5 First Strand cDNA Synthesis (iScript Method) (See Note 15)
3.6 Real-Time Quantitative PCR Protocol (See Note 17)
4 Notes
References
Chapter 12: Screening for Host Factors Directly Interacting with RSV Protein: Microfluidics
1 Introduction
2 Materials
2.1 Microfluidics
2.2 Antibodies
2.3 Co-precipitation
3 Methods
3.1 Microfluidics
3.1.1 DNA Arraying and Device Alignment
3.1.2 Surface Chemistry
3.1.3 Protein Expression and Labeling
3.2 Small Evaluation Microfluidics Screen
3.3 Protein Network Interaction Generator (PING)
3.4 Validation of Interactions by Co-precipitation
4 Notes
References
Chapter 13: A Proteomic-Based Workflow Using Purified Respiratory Syncytial Virus Particles to Identify Cellular Factors as Drug Targets
1 Introduction
2 Materials
2.1 RSV Production and Purification
2.2 RSV Protein Analysis by SDS-PAGE
2.3 One Dimensional LC-MS/MS (1D LC MS/MS)
2.4 Western Blot
2.5 Immunostaining
2.6 Transfection
3 Methods
3.1 RSV Production
3.2 RSV Purification
3.3 Morphological Analysis of Purified RSV Preparation Using Transmission Electron Microscopy (See Note 6)
3.4 Protein Analysis to Assess Level of Purity (See Note 7)
3.4.1 Coomassie Brilliant Blue
3.4.2 SYPRO Ruby Red
3.5 One Dimensional LC-MS/MS (1D LC MS/MS)
3.5.1 Preparation of Peptide Digest from Virus Preparation (See Note 10)
3.5.2 1D LC MS/MS Analysis (See Note 11)
3.5.3 Database Search (See Table 1)
3.6 Confirmation That the Proteins Detected in the 1D LC MS/MS Analysis Are Present in the Virus Preparation (See Note 12)
3.6.1 Distribution of the Cellular Proteins in the Continuous Sucrose Gradient to Confirm Their Co-migration with Virus Particles
3.6.2 Detection of Virus and Cellular Proteins in the Purified Virus Preparation (As Indicated in Subheading 3.2)
3.7 Demonstration of Relevance of Cellular Proteins Identified in the Proteomic Analysis to Virus Replication in RSV-Infected Cells (See Note 13)
3.7.1 Immunolocalization of HSP90 in Virus Filaments by Imaging Co-stained Virus-Infected Cells (See Note 14)
3.7.2 Effect of HSP90 Gene Silencing on RSV Morphogenesis (See Note 16)
3.7.3 The Effect of the HSP90 Inhibitor 17-Allyaminogel-danamycin. (17AAG) on RSV Filament Formation (See Note 18)
3.7.4 The Effect of the HSP90 Inhibitor 17-Allyaminogeldanamycin (17AAG) on RSV Transmission in a HEp-2 Cell Monolayer
4 Notes
References
Chapter 14: MicroRNA Profiling from RSV-Infected Biofluids, Whole Blood, and Tissue Samples
1 Introduction
2 Materials
2.1 Exosome Isolation from Biofluids
2.2 Plasma Separation and Storage from Total Blood
2.3 Serum Separation and Storage from Whole Blood
2.4 RNA Isolation
2.5 RNA Quantification
2.6 RT-qPCR
3 Methods
3.1 Exosome Isolation from Biofluid Samples
3.2 Plasma Separation from Whole Blood Samples
3.3 Serum Separation from Whole Blood Samples
3.4 RNA Isolation
3.4.1 Purified Exosome Samples
3.4.2 Total Blood Samples
3.4.3 Tissue Samples
3.5 RNA Quantification
3.6 RT q-PCR
3.6.1 Polyadenylation Reaction
3.6.2 cDNA Synthesis
3.6.3 qPCR
3.7 Data Analysis
4 Notes
References
Chapter 15: Mouse and Cotton Rat Models of Human Respiratory Syncytial Virus
1 Introduction
2 Materials
2.1 Animals
2.2 Anesthetics
2.3 Infection of Mice and Cotton Rats with RSV
3 Methods
3.1 RSV Infection of Adult and Neonate Mice (Mus musculus)
3.2 RSV Infection in Cotton Rats (Sigmodon hispidus) (see Note 9)
4 Notes
References
Chapter 16: In Vivo Assessment of Airway Function in the Mouse Model
1 Introduction
2 Materials
2.1 Equipment
2.1.1 Noninvasive, Unrestrained Whole-Body Plethysmography
2.1.2 Invasive Airway Physiology System
2.1.3 Small Equipment
2.2 Reagents and Solutions
3 Methods
3.1 Noninvasive, Unrestrained Whole-Body Plethysmography
3.2 Invasive Airway Physiology Measurements
3.3 Output Parameters and Interpretation
3.3.1 Noninvasive, Unrestrained Whole-Body Plethysmography
3.3.2 Invasive Airway Physiology Measurements
4 Notes
References
Chapter 17: Evaluation of the Adaptive Immune Response to Respiratory Syncytial Virus
1 Introduction
2 Materials
2.1 Intravenous Labeling
2.2 Cell Isolation, Stimulation, and Staining
3 Methods
3.1 Intravascular Labeling of Immune Cells (See Note 4)
3.2 Cell Collection and Preparation: Bronchoalveolar Lavage (BAL)
3.3 Cell Preparation: Draining Lymph Node (Mediastinal)
3.4 Cell Preparation: Lungs
3.5 Processing Cells: Peripheral Blood Leukocytes (PBL)
3.6 In Vitro Stimulation (If Not Performing a Stimulation, Advance to Subheading 3.7)
3.7 Cell Staining
4 Notes
References
Index