Virus-Host Interactions: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Genetic Tools for Studying the Roles of Sphingolipids in Viral Infections
	1 Introduction
	2 Considerations When Ablating Lipid-Metabolizing Enzymes
	3 The Early Sphingolipid Synthesis Cascade
	4 Terminal Branches of the Sphingolipid Synthesis Pathway
	5 SL Degradation and Salvage
	6 Discussion/Conclusion
	References
Chapter 2: Detection of Serum Antibody Responses in Nipah Virus-Infected Pigs
	1 Introduction
	2 Materials
		2.1 Materials Including Devices, Antigens, and Conjugates for Indirect ELISA
		2.2 Materials Including Devices for VNT
	3 Methods
		3.1 ELISA
		3.2 Virus Neutralization Test (VNT)
	4 Notes
	References
Chapter 3: Computational Integration of HSV-1 Multi-omics Data
	1 Introduction
	2 Methods
		2.1 Preprocessing of Raw Sequencing Data
		2.2 Identification of Read-Through Transcription
		2.3 Chromatin Accessibility, Histone Modifications, and Read-Through
	3 Notes
	References
Chapter 4: A Cell Membrane Fusion Assay for the Fish Pathogen Spring Viremia of Carp Virus (SVCV)
	1 Introduction
	2 Materials
		2.1 Cell Culture and Virus
		2.2 Virus-Cell Fusion Assay
	3 Methods
		3.1 Production of SVCV
		3.2 Syncytia Formation Assay (Fig. 1)
		3.3 Virus Titer Determination by Plaque Assay
	4 Notes
	References
Chapter 5: Viral Quantification in Bee Samples Using Synthetic DNA Sequences with Real-Time PCR (qPCR)
	1 Introduction
	2 Materials
	3 Methods
		3.1 RNA Extraction and cDNA Synthesis
		3.2 Synthetic DNA Sequence Stock Solution
		3.3 Tenfold Serial Dilutions of Synthetic DNA Sequence
		3.4 qPCR Assay
	4 Notes
	References
Chapter 6: Inhibition of White Spot Syndrome Virus (WSSV) in Pacific White Shrimp (Litopenaeus vannamei) Using Polyamine-Modif...
	1 Introduction
	2 Materials
		2.1 Polyamine CQD Preparation
		2.2 WSSV Purification
		2.3 In Vivo Experiments
	3 Methods
		3.1 Experimental Animal Handling
		3.2 Synthesis of Polyamine CQDs
		3.3 Preparation of Test Diet
		3.4 WSSV Inoculum Preparation
		3.5 In Vivo Experiments
	4 Notes
	References
Chapter 7: Computational Modeling of IN-CTD/TAR Complex to Elucidate Additional Strategies to Inhibit HIV-1 Replication
	1 Introduction
	2 Materials
		2.1 Molecular Structures for Complex Structure Predictions
		2.2 Online Server MDockPP
		2.3 Structure Optimization and Visualization
		2.4 Experimental Data for Screening of Docking Poses
	3 Methods
		3.1 Input to MDockPP Online Server
		3.2 Add Experimental Constraints for Screening of the Docking Outputs (Optional)
		3.3 Job Submission and Result Retrieval
		3.4 Results
	4 Notes
	References
Chapter 8: Protein-Protein and Protein-RNA Interaction Assays to Determine Similarity of INI1/SMARCB1 and TAR RNA in Binding t...
	1 Introduction
	2 Materials
		2.1 IN-CTD/INI1-Rpt1 Interaction
		2.2 IN-CTD/TAR RNA Interaction
		2.3 To Test Competition of TAR with INI1-Rpt1 to Bind to IN
		2.4 To Test Competition of INI1-Rpt1 with TAR to Bind to IN
		2.5 The Reaction Buffer
	3 Methods
		3.1 Two-Component Protein-Protein Interaction Assay (Example: GST-IN-CTD/6His-SUMO-INI1-Rpt1 Interaction)
		3.2 Two-Component Protein/RNA Interaction Assay (Example: GST-IN-CTD/TAR RNA Interaction)
		3.3 Three-Component Competition Assays: To Test Competition of TAR RNA with INI1-Rpt1 to bind to IN-CTD
		3.4 Three-Component Competition Assays: To Test Competition of INI1-Rpt1 with TAR to Bind to IN-CTD
	4 Notes
	References
Chapter 9: 4C Analysis of EBV-Host DNA Interactome
	1 Introduction
	2 Materials
		2.1 Cell Fixation
		2.2 Preparation of 4C DNA
		2.3 Preparation of Sequencing Library
		2.4 Equipment
	3 Methods
		3.1 Cell Cross-Linking
		3.2 Preparation of 3C-DNA (See Note 1)
		3.3 Preparation of 4C-DNA
		3.4 Construction of Sequencing Library
	4 Notes
	References
Chapter 10: Detection Methods for H1N1 Virus
	1 Introduction
	2 Traditional Methods
		2.1 Virus Culture
		2.2 RIDT
	3 Molecular Assays
		3.1 Rolling Circle Amplification
		3.2 Multi-fluorescent-Based Digital PCR
		3.3 Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)
		3.4 Real-Time PCR
		3.5 Reverse Transcriptase PCR
	4 Serological Test
		4.1 Immunofluorescence Tests
		4.2 Complement Fixation
		4.3 Double Immunodiffusion Test
		4.4 Single Radial Immunodiffusion Test
		4.5 Enzyme-Linked Immunosorbent Assay (ELISA)
		4.6 Hemagglutination Inhibition
	5 Biosensors
		5.1 DNA Probe-Based Sensor
		5.2 Boron-Doped Diamond Biosensor
		5.3 Aptamer-Based Sensor
		5.4 Immunosensor
	6 Conclusion
	References
Chapter 11: Detection of SARS-CoV-2 in Human Breast Milk
	1 Introduction
	2 Materials
		2.1 Collection of Human Breast Milk
		2.2 Detection of Viral RNA by qRT-PCR
		2.3 Virus Isolation in Cell Culture
	3 Methods
		3.1 Collection of Human Breast Milk
		3.2 RNA Isolation
		3.3 qRT-PCR
		3.4 Virus Isolation in Cell Culture
	4 Notes
	References
Chapter 12: The Rhizopus Holobiont: A Model to Decipher Fungal-Bacterial-Viral Symbioses
	1 Introduction
	2 Materials
	3 Methods
		3.1 Harvesting of Fresh Fungal Sporangiospores of R. microsporus
		3.2 Protoplast Generation
		3.3 Chemotherapy
		3.4 Genotyping the Recovered Fungal Strains by RT-PCR
		3.5 cDNA Synthesis
		3.6 Genotyping Fungal Strains
	4 Notes
	References
Chapter 13: Combining Metabolic Pulse Labeling and Quantitative Proteomics to Monitor Protein Synthesis Upon Viral Infection
	1 Introduction
	2 Materials
		2.1 Cell Labeling, Infection, and Pulse Labeling
		2.2 Enrichment and Sample Preparation
		2.3 Desalting, LC-MS/MS, and Data Analysis
	3 Methods
		3.1 Experimental Design, Cell Labeling, Infection, and Pulse Labeling
		3.2 Enrichment of Newly Synthesized Proteins
		3.3 Preparation of the Input for LC-MS/MS
		3.4 Sep-Pak Desalting, LC-MS/MS, and Processing of Raw Files
		3.5 Comparative Quantification of Newly Synthesized Host and Viral Proteins
	4 Notes
	References
Chapter 14: Three-Dimensional Human Brain Organoids to Model HIV-1 Neuropathogenesis
	1 Introduction
	2 Materials
		2.1 Viruses and Cell Lines
		2.2 Cell Culture Media, Supplements, and Reagents
		2.3 Fabrication of Hydrogel Devices
		2.4 Brain Organoids and Triculture
	3 Methods
		3.1 Hydrogel Device Manufacturing
		3.2 Generation of 3D Organoids
		3.3 HIV-1 Infection and Triculture Assembly
	4 Notes
	References
Chapter 15: Characterization of SARS-CoV-2 Glycoprotein Using a Quantitative Cell-Cell Fusion System
	1 Introduction
	2 Materials
		2.1 Cell Culture and Transfection
		2.2 Detection of Fusion-Activated Luciferase Activity
	3 Methods
		3.1 Generation of the Effector Cells
		3.2 Generation of the Target Cells
		3.3 Co-culture of the Effector and Target Cells
		3.4 Detection of Fusion-Activated Luciferase Activity
	4 Notes
	References
Chapter 16: A Lentiviral Pseudotype System to Characterize SARS-CoV-2 Glycoprotein
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 General Materials
		2.3 Materials for Making and Titrating SARS-CoV-2 pp
		2.4 Materials for Western Blot Analysis of SARS-CoV-2 S Pseudovirions
		2.5 Materials for Inhibition of Entry of SARS-CoV-2 S Pseudovirus by Bafilomycin A
		2.6 Materials for Neutralization Assay
	3 Methods
		3.1 Production of Lentiviral Particles Pseudotyped with SARS-CoV-2 S Proteins
		3.2 Titration of Pseudovirions with SARS-CoV-2 S
		3.3 Western Blot Analysis of Incorporation of SARS-CoV-2 S Protein in Pseudovirons (Fig. 3)
		3.4 Inhibition of Entry of SARS-CoV-2 S Pseudovirions by Bafilomycin A1 (Fig. 4)
		3.5 Neutralization (MN) Assay with SARS-CoV-2 S Pseudovirions (Fig. 5)
	4 Notes
	References
Index