Dengue Virus: Methods and Protocols

Preface
Contents
Contributors
Part I: Virus and Virus Protein Production
	Chapter 1: Virus Propagation and Plaque Assay for Dengue Virus
		1 Introduction
		2 Materials
			2.1 Propagation
			2.2 Plaque Assay
		3 Methods
			3.1 Propagation
			3.2 Plaque Assay
		4 Notes
		References
	Chapter 2: Transmission Electron Microscopy Studies of Dengue Viruses
		1 Introduction
		2 Materials
		3 Methods
			3.1 Fixation of Biological Samples
				3.1.1 Cell Culture (in 25 cm2 Flasks)
				3.1.2 Animal Tissue Samples (Fixation by Perfusion)
				3.1.3 Animal Tissue Samples (Fixation by Immersion)
			3.2 Post-fixation, Dehydration, and Embedding of Biological Samples
				3.2.1 Cell Culture
				3.2.2 Animal Tissue Samples
			3.3 Ultramicrotomy
			3.4 Positive Staining Technique
			3.5 Negative Staining Technique
		4 Notes
		References
	Chapter 3: Production of Properly Folded NS1 Protein in Bacterial Cells
		1 Introduction
		2 Materials
			2.1 Recombinant Expression in E. coli Cells
			2.2 Extraction from Inclusion Bodies, Purification, and Refolding
		3 Methods
			3.1 Expression of DENV NS1 Protein in E. coli
			3.2 Extraction of DENV NS1 from Inclusion Bodies
			3.3 Purification of DENV NS1 in a Ni2+NTA Affinity Column
			3.4 DENV NS1 Refolding and Concentration
		4 Notes
		References
	Chapter 4: Expression and Purification of Dengue NS1 Protein in Sf9-Baculovirus System
		1 Introduction
		2 Materials
			2.1 Sf9 Cell Transfection and Baculovirus Stock Preparation
			2.2 Purification
		3 Methods
			3.1 Sf9 Cell Transfection and Baculovirus Stock Preparation
			3.2 Large Batch Expression
			3.3 Purification
				3.3.1 Ni2+NTA Affinity Purification
				3.3.2 Size Exclusion Chromatography
				3.3.3 Detergent Removal
		4 Notes
		References
	Chapter 5: Purification of Dengue and Zika Virus Non-structural Protein 5 for Crystallization and Screening of Antivirals
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Plasmids
			2.3 Buffers
			2.4 Instruments
		3 Methods
			3.1 ZIKV NS5 RdRp and DENV2 NS5 FL
				3.1.1 Expression and Protein Purification
				3.1.2 Cell Lysis
				3.1.3 Metal Affinity Chromatography
				3.1.4 Heparin-Affinity Chromatography
				3.1.5 Size Exclusion Chromatography
				3.1.6 Preparation of Protein Crystals
				3.1.7 Soaking of Compounds
			3.2 DENV3 NS5 RdRp
				3.2.1 Expression and Protein Purification
				3.2.2 Cell Lysis
				3.2.3 Metal Affinity Chromatography
				3.2.4 Cation-Exchange Chromatography
				3.2.5 Size Exclusion Chromatography
				3.2.6 Preparation of Protein Crystals
				3.2.7 Co-crystallization
			3.3 DENV3 NS5 FL
				3.3.1 Protein Expression
				3.3.2 Cell Lysis for Protein Purification
				3.3.3 Metal Affinity Chromatography
				3.3.4 Size Exclusion Chromatography
				3.3.5 Preparation of Protein Crystals
		4 Notes
		References
	Chapter 6: Obtention of Dengue Virus Membrane Proteins and Role for Virus Assembly
		1 Introduction
		2 Materials
			2.1 Transfection
			2.2 Sucrose Cushion Ultracentrifugation
			2.3 Sucrose Gradient Ultracentrifugation for VLPs
			2.4 Sucrose Gradient Ultracentrifugation for Cell Lysates
			2.5 Membrane Flotation Assay
			2.6 Subcellular Fractionation Assay
			2.7 Glycosidase Digestion Assay
		3 Methods
			3.1 Transfection
			3.2 Sucrose Cushion Ultracentrifugation
			3.3 Sucrose Gradient Ultracentrifugation for VLPs
			3.4 Sucrose Gradient Ultracentrifugation for Cell Lysate
			3.5 Membrane Flotation Assay
			3.6 Subcellular Fractionation Assay
			3.7 Glycosidase Digestion Assay
		4 Notes
		References
	Chapter 7: Construction of Genomic and Sub-Genomic Dengue Infectious Replicons
		1 Introduction
		2 Materials
			2.1 Plasmids
			2.2 Cloning Reagents
			2.3 Transformation Cell
			2.4 Cloning Kits
			2.5 In Vitro Transcription Reagents
			2.6 Mammalian Cell Culture
			2.7 Electroporation Reagents and Equipment
			2.8 Plaque Assay Reagents
			2.9 Luciferase Assay
		3 Methods
			3.1 Construction of the DENV2-Luc-Sub-Genomic Replicon
				3.1.1 Deletion of the Viral Structural Genes and Insertion of a Unique Restriction Site
				3.1.2 Amplification of the Reporter Gene and the Cleavage Site
				3.1.3 Cloning the Reporter Gene and Cleavage Site to Generate the DENV-Luc-Sub-Genomic Replicon
			3.2 Construction of the DENV2-Luc-Genomic Replicon
				3.2.1 Insertion of a Unique Restriction Site, NgoMIV to the Parental Clone
				3.2.2 Generating Mutations on the Cyclization Sequence on Capsid Gene
				3.2.3 Amplification of the Intact C20, Reporter Gene, and Cleavage Site
				3.2.4 Cloning to Generate the DENV-Luc-Genomic-Replicon
			3.3 Plasmid Minipreparation
			3.4 In Vitro Transcription
			3.5 Electroporation
			3.6 Plaque Assay
			3.7 Luciferase Assay
		4 Notes
		References
Part II: Interaction Between Dengue Virus and Cellular Proteins
	Chapter 8: Dengue Virus Capsid-Protein Dynamics in Live Infected Cells Studied by Pair Correlation Analysis
		1 Introduction
			1.1 2D-pCF Analysis Applied to DENV-Infected Cells
		2 Materials
			2.1 Reagents
			2.2 Equipment
		3 Methods
			3.1 Before Starting
				3.1.1 Calibration of the Observation Volume
				3.1.2 FFS Controls
				3.1.3 FFS Experiment for Calibration
			3.2 FFS and Pair Correlation Experiments on Cells
				3.2.1 Sample Preparation
				3.2.2 Imaging
				3.2.3 Imaging: Capture of the Time Series
				3.2.4 Quality Check of the Image Stack for FFS Analysis
			3.3 2D-pCF Analysis Procedure
		4 Notes
		Appendix
			The (Very) Basics of Fluorescence Fluctuation Spectroscopy Techniques: History and New Challenges
		References
	Chapter 9: Yeast Two-Hybrid System for Mapping Novel Dengue Protein Interactions
		1 Introduction
			1.1 Yeast Two-Hybrid for Viral infections
			1.2 Yeast Growth Selection
			1.3 Y2H Vectors
			1.4 False Positives
			1.5 False Negatives
			1.6 Y2H Approaches for Dengue Proteins
		2 Materials
			2.1 Plasmids and Yeast Strains
			2.2 Yeast Growth and Maintenance
			2.3 Yeast Transformation
			2.4 β-Galactosidase Assays
			2.5 Plasmid Isolation and Sequencing
		3 Methods
			3.1 Construct Fusion Genes
			3.2 Construction of an AD Fusion Library
			3.3 Yeast Initial Transformation (Small-Scale)
			3.4 Bait Toxicity Test
			3.5 HIS3 Self-Activation Test
			3.6 Two-Hybrid Library-Scale Screening
			3.7 Retest Phenotypes
			3.8 β-galactosidase Assay
			3.9 Isolate Plasmid DNA from Yeast and Bacteria
			3.10 Sequence AD/Library Inserts
		4 Notes
		References
	Chapter 10: Isolation and Identification of Dengue Virus Interactome with Human Plasma Proteins by Affinity Purification-Mass ...
		1 Introduction
			1.1 High-Throughput Screening on Virus Interactome Studies
			1.2 Human Plasma as a Source of Interacting Proteins
			1.3 Identification of DENV Binding Proteins by Mass Spectrometry
		2 Materials
			2.1 Stock Solutions
			2.2 Anion Exchange Chromatography
				2.2.1 Equipment
				2.2.2 Buffers and Solutions
			2.3 Protein Pull-Down Assay
				2.3.1 Equipment and Consumables
				2.3.2 Buffers and Solutions
			2.4 Protein Digestion
				2.4.1 Equipment and Consumables
				2.4.2 Buffers and Solutions
			2.5 Mass Spectrometry
				2.5.1 Equipment and Consumables
				2.5.2 Buffers and Solutions
		3 Methods
			3.1 Human Plasma
			3.2 Plasma Conditioning
			3.3 Anion Exchange Chromatography
			3.4 Protein Pull-Down Assay
			3.5 Protein Digestion
				3.5.1 In-Solution Digestion
				3.5.2 ``On-Matrix´´ Digestion
			3.6 Mass Spectrometry
			3.7 MS Data Analysis
			3.8 Selected Reaction Monitoring (SRM)
			3.9 SRM Data Processing
		4 Notes
		References
Part III: Immunopathogensis and Diagnosis
	Chapter 11: Molecular Diagnosis of Dengue
		1 Introduction
		2 Materials
			2.1 RNA Extraction Protocol
			2.2 Conventional Reverse Transcription Followed by RT-PCR
			2.3 Real-Time Reverse Transcription Followed by Polymerase Chain Reaction (qRT-PCR)
		3 Methods
			3.1 RNA Extraction Protocol (QIAamp Viral RNA Mini Kit, QIAGEN)
			3.2 Conventional Reverse Transcription Followed by Polymerase Chain Reaction (RT-PCR)
				3.2.1 RT-PCR (First Stage)
				3.2.2 Semi-Nested PCR (Second Stage)
				3.2.3 Agarose Electrophoresis
			3.3 Real-Time Reverse Transcription Followed by Polymerase Chain Reaction (qRT-PCR)
				3.3.1 qRT-PCR
				3.3.2 Result Analysis
		4 Notes
		References
	Chapter 12: Serological Diagnosis of Dengue
		1 Introduction
		2 Materials
			2.1 Anti-Dengue IgM and IgG Antibody Enzyme-Linked Immunosorbent Assay (ELISA)
			2.2 NS1 Antigen Capture Enzyme-Linked Immunosorbent Assay
		3 Methods
			3.1 Anti-Dengue IgM Antibody Capture Enzyme-Linked Immunosorbent Assay MAC-ELISA
			3.2 Anti-Dengue IgG Antibody Enzyme-Linked Immunosorbent Assay IgG-ELISA
			3.3 Anti-Dengue IgG Antibody Enzyme-Linked Immunosorbent Assay Quantitative IgG-ELISA
			3.4 NS1 Antigen Capture Enzyme-Linked Immunosorbent Assay (NS1 Ag-ELISA)
		4 Notes
		References
	Chapter 13: Measuring Transendothelial Electrical Resistance (TEER) for Dengue Infection Studies
		1 Introduction
		2 Materials
		3 Methods
			3.1 Culturing ECs
			3.2 Growing Polarized ECs in Transwells
			3.3 Evaluating the Transendothelial Electrical Resistance (TEER)
			3.4 FITC-Dextran Permeability Analysis
			3.5 Infecting Polarized ECs with Dengue Virus
		4 Notes
		References
	Chapter 14: Evaluation of DENV-Induced Endothelial Cell Permeability by Measurements of Transendothelial Electrical Resistance...
		1 Introduction
		2 Materials
			2.1 DENV Propagation
			2.2 DENV Titration
			2.3 Culture of Human Brain Microvascular Endothelial Cells (HBMEC)
			2.4 Measurement of Transendothelial Electrical Resistance (TEER)
			2.5 BSA-FITC Extravasation
			2.6 Virus Extravasation
		3 Methods
			3.1 DENV Maintenance and Propagation
			3.2 DENV Titration
				3.2.1 BHK-21 Cell Culture
				3.2.2 Virus Titration
			3.3 Infection of HBMEC and TEER Measurement
			3.4 Measurement of BSA-FITC Extravasation
			3.5 Analysis of DENV Extravasation Through the Endothelial Monolayer
		4 Notes
		References
	Chapter 15: In Vitro Cytokine Production by Dengue-Infected Human Monocyte-Derived Dendritic Cells
		1 Introduction
		2 Materials
			2.1 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
			2.2 CD14+ Monocytes Isolation
			2.3 CD14+ Monocytes Differentiation
			2.4 Dengue Virus Infection of CD14-/CD11c+ Human Monocyte-Derived Dendritic Cells
			2.5 Cytokine Quantification
		3 Methods
			3.1 Human Peripheral Blood Mononuclear Cells Isolation
			3.2 CD14+ Monocyte Isolation
			3.3 CD14+ Monocytes Differentiation into CD14-/CD11c+ Human Monocyte-Derived Dendritic Cells
			3.4 Dengue Virus Infection of CD14-/CD11c+ Human Monocyte-Derived Dendritic Cells
			3.5 Cytokine Quantification with BD CBA  Kits
		4 Notes
		References
	Chapter 16: In Vitro Generation of Human Antibody-Secreting Cells Through the Stimulation of PBMCs with Dengue Virus Particles
		1 Introduction
		2 Materials
			2.1 Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
			2.2 In Vitro Stimulation of PBMCs
		3 Methods
			3.1 Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
			3.2 In Vitro Stimulation of PBMCs
			3.3 PBMC Stimulation with a Mitogen Cocktail
			3.4 Cell Culture Maintenance
			3.5 Cell and Supernatant Recovery for ASC Functional Assays
		4 Notes
		References
	Chapter 17: Isolation of Microvesicles from Plasma Samples Avoiding Lipoprotein Contamination
		1 Introduction
		2 Materials
			2.1 Isolation of Plasma
			2.2 Gradient Assembly and Handling
			2.3 Dialysis and MVs Precipitation
			2.4 Colorimetric Assays
			2.5 Flow Cytometry
			2.6 Western Blot
		3 Methods
			3.1 Plasma Isolation
			3.2 Gradient Assembly
			3.3 Quantification of Plasma Lipoproteins and Albumin
			3.4 Quantification of MVs in Plasma Through Flow Cytometry
			3.5 Sample Dialysis
			3.6 MVs Isolation from Plasma
			3.7 Detection of Apo B100 Through Western Blot
				3.7.1 MVs Samples Preparation
				3.7.2 Protein Separation by Gel Electrophoresis
				3.7.3 Protein Transfer to PVDF Membrane
				3.7.4 Membrane Blocking and Immuno-Detection
		4 Notes
		References
Part IV: New Advances in Animal Models for Dengue
	Chapter 18: Evaluating Dengue Virus Pathogenesis in Mice and Humans by Histological and Immunohistochemistry Approaches
		1 Introduction
		2 Materials
			2.1 Anesthesia
			2.2 Fixing Solution
			2.3 Staining Solutions
			2.4 Histology
			2.5 Immunohistochemistry by Peroxidase
			2.6 Immunohistochemistry by Fluorescence
			2.7 Analysis Equipment
		3 Methods
			3.1 Ethical Procedures
			3.2 Personal Protective Equipment and Collective Protection Equipment
			3.3 Organs Collection and Histological Processing
			3.4 Histological Procedures
			3.5 Microtomy
			3.6 Hematoxylin and Eosin (H.E.) Staining
			3.7 Immunohistochemistry by Peroxidase Protocol (Using a Polymer Detection System)
			3.8 Immunohistochemistry by Fluorescence Protocol
		4 Notes
		References
	Chapter 19: Humanized Mice for the Study of Dengue Disease Pathogenesis: Biological Assays
		1 Introduction
		2 Materials
			2.1 Facilities and Animals
			2.2 Mouse Human Engraftment
			2.3 Quantitative Human Albumin ELISA  Kit
			2.4 Mouse Dengue Virus Infection
			2.5 Plasma Viral Load Measured by RT-qPCR
			2.6 Plasma Viral Load Measured by Plaque Assay
			2.7 Laparotomy
			2.8 Screening of CD34+ Human Stem Cells and Human Hepatocytes Mouse Humanization
			2.9 Blood Sampling
			2.10 Euthanasia
		3 Methods
			3.1 Humanized NSG Mice Single Engraftment Model: Humanization of NSG Mice Using CD34+ Human Stem Cells Via Hepatic Injection
			3.2 Blood Sample Collection Via a Retro-Orbital Vein in NSG  Mice
			3.3 Screening for Human CD45+ Cells in NSG Mice Blood Samples by Flow Cytometry
			3.4 Humanized NSG Mice Dual Engraftment Model: Engraftment of huNSG Mice by Splenic Injection of Human Primary Hepatic Cells T...
			3.5 Screening for Human Hepatic Cells Engraftment by Human Albumin ELISA
			3.6 Dengue Infection
			3.7 Evaluation of Plasma Viral Load by RT-qPCR
			3.8 Evaluation of Plasma Viral Load by Plaque Assay
			3.9 Euthanasia
			3.10 Tissue Collection
		4 Notes
		References
Index