Viruses as Therapeutics: Methods and Protocols

Preface
	Viruses as Therapeutics: Fighting Fire with Fire
Acknowledgments
Contents
Contributors
Chapter 1: Viruses for Landscape-Scale Therapy: Biological Control of Rabbits in Australia
	1 Background to Biological Control
	2 The European Rabbit in Australia
	3 Viruses
		3.1 Myxoma Virus and Myxomatosis
		3.2 Rabbit Hemorrhagic Disease Virus and Rabbit Hemorrhagic Disease
		3.3 Testing of MYXV and RHDV as Biological Controls
	4 The Early Spread of MYXV and  RHDV
	5 The Impact of Biological Control
	6 Host-Pathogen Coevolution: Biological Arms Race or Detente?
	7 Genomic Evolution of RHDV and  MYXV
	8 Attempts to Enhance Biocontrol
	9 Conclusions and Future Prospects
	References
Chapter 2: Development and Expression of Subunit Vaccines Against Viruses in Plants
	1 Introduction
	2 Materials
		2.1 Plant Growth Materials
		2.2 Bacterial Culture
		2.3 Infiltration
		2.4 Harvesting
		2.5 Immobilized Metal Affinity Chromatography
	3 Methods
		3.1 Plant Growth
		3.2 A. tumefaciens Culturing and Preparation for Syringe Infiltration
		3.3 Syringe Agroinfiltration
		3.4 Harvesting, Isolation, and Characterization of Recombinant Zika Envelope Protein
	4 Notes
	References
Chapter 3: Development of Antiviral Vaccine Utilizing Self-Destructing Salmonella for Antigen and DNA Vaccine Delivery
	1 Introduction
		1.1 Self-Destructing GMS for Antigen Delivery
		1.2 Self-Destructing GMS Enabling Immediate Escape from the Endosome Compartment into the Cytosol
		1.3 Self-Destructing GMS for DNA Vaccine Delivery
		1.4 Hyper-Invasive Self-Destructing GMS Displaying Reduced Pyroptosis/Apoptosis-Inducing Phenotype
	2 Materials
	3 Methods
		3.1 Development of GMS for Protective Antigen Delivery to Induce Cellular Immune Responses Against Influenza Virus
			3.1.1 Bacterial Cultures and General DNA Procedures
			3.1.2 Strain Construction and Characterization
			3.1.3 Codon Optimization of the NP Gene and Synthesis of Peptide
			3.1.4 Vectors for Antigen Delivery via Regulated Delayed Lysis System
			3.1.5 SDS-PAGE and Immunoblots
			3.1.6 Immunization of Mice
			3.1.7 Experimental Design
			3.1.8 ELISA
			3.1.9 Cell Proliferation
			3.1.10 Virus Strain Propagation, Purification, and Titration
			3.1.11 Virus Challenge
			3.1.12 Statistical Analyses
		3.2 Development of GMS for DNA Vaccine Delivery to Induce Systemic Immunity Against Influenza Virus
			3.2.1 Bacterial Cultures, General DNA Procedures, and Strain Characterization
			3.2.2 Construction of the Self-Destructing S. Typhimurium Vaccine Host-Vector System
			3.2.3 Construction of Hyper-invasive GMS Strains
			3.2.4 Construction of RASV Strains to Reduce Host Cell Pyroptosis/Apoptosis
			3.2.5 Construction of Improved DNA Vaccine Vector
			3.2.6 Construction of Improved DNA Vaccine Vector Encoding WSN HA
			3.2.7 Examination of Cell Lysis In Vitro
			3.2.8 Colonization of Mice with the Salmonella DNA Vaccine Strains
			3.2.9 Immunization of Mice and Sample Collection
			3.2.10 ELISA, Virus Strain Propagation, Purification, and Titration, and Virus Challenge
	4 Notes
	References
Chapter 4: Methods for the Construction of Recombinant Oncolytic Myxoma Viruses
	1 Introduction
	2 Materials
		2.1 Tissue Culture
		2.2 Cloning and Virus Construction
		2.3 Vectors
		2.4 Western Blot and Immunocytochemistry
	3 Methods
		3.1 Construction of Recombinant Oncolytic MYXV-Expressing Transgene and/or Reporter Fluorescent Protein(s)
			3.1.1 Molecular Cloning of Recombinant Transgenes
			3.1.2 Construction of Recombinant MYXV by Infection and Transfection of the Recombination Plasmid
			3.1.3 Recombinant Virus Isolation by Foci Purification (Screening for Fluorescent Reporters)
			3.1.4 Upscaling of Virus Stocks from Single  Foci
			3.1.5 Virus Titration
		3.2 Quality Control of Recombinant MYXV
			3.2.1 Multistep Growth Analysis of Recombinant  MYXV
			3.2.2 DNA Purification and Characterization of Recombinant MYXV Genome by  PCR
			3.2.3 Expression of Recombinant Protein Assessed by Western  Blot
			3.2.4 Expression of Recombinant Protein Assessed by Immunocytochemistry
			3.2.5 In Vitro Cytotoxicity Assay
	4 Notes
	References
Chapter 5: Molecular Design and Production of AAV Viral Vectors for Gene Therapy
	1 Introduction
	2 Materials
		2.1 Polymerase Chain Reaction (PCR)
		2.2 Gel Purification
		2.3 Ligation
		2.4 Transformation
		2.5 Plating Cells
		2.6 Picking Colonies and DNA Isolation
		2.7 Screening Colonies
		2.8 Packaging AAV
	3 Methods
		3.1 Creating a Gene of Interest PCR Product
		3.2 Gel Purification
		3.3 Ligation
		3.4 Ligation Cleanup
		3.5 Transformation
		3.6 Plating Cells
		3.7 Picking Colonies and Isolation of DNA
		3.8 Screening Clones for GOI Insert
		3.9 Packaging AAV
	4 Notes
	References
Chapter 6: Murine Model of Thermal Burn Injury for Evaluating Protein Therapeutics Derived from Viruses
	1 Introduction
	2 Materials
		2.1 Experimental Animals
		2.2 Equipment Required for Surgery
		2.3 Medicines Required for Surgery
		2.4 Equipment Required for Debridement
		2.5 Preparation of Treatments
		2.6 Equipment for Digital Analysis of Wound and Scar  Area
		2.7 Equipment for Animal Euthanasia and Harvesting of Tissue
	3 Methods
		3.1 Surgical Preparation and Burn Injury
		3.2 Monitoring of Animal Welfare
		3.3 Debridement and Treatment
		3.4 Digital Analysis of Wound and Scar  Area
		3.5 Harvesting of Tissue for Histological Analysis
	4 Notes
	References
Chapter 7: Deriving Immune-Modulating Peptides from Viral Serine Protease Inhibitors (Serpins)
	1 Introduction
	2 Materials
		2.1 Identification of Predicted Peptide Metabolites in the Serpin-Reactive Center Loop (RCL)
			2.1.1 Online Peptide Identification Programs
			2.1.2 Calculation of Peptide Physical Characteristics
		2.2 Peptide Synthesis
		2.3 Protease and Serpin Inhibition Assays
			2.3.1 Background
			2.3.2 Materials
		2.4 Animal Models of Vasculitis
			2.4.1 Mouse Gamma Herpesvirus 68 (MHV68) Infection in IFNγR-/-  Mice
			2.4.2 Aortic Allograft Transplants: C57Bl/6 Donor to BALB/C Recipient  Mice
			2.4.3 Histological Analysis of Lung Consolidation, Gut Dilatation, and Vascular Inflammation in Animal Models
	3 Methods
		3.1 Protease and Serpin Inhibition Assays
			3.1.1 Gel Shift Assay
			3.1.2 Protease Inhibition Assay
		3.2 Mouse Gamma Herpesvirus 68 (MHV68) Infection in Interferon Gamma Receptor Knockout (IFNγR-/-) Mice (See Note 3)
			3.2.1 MHV68 Infection in IFNγR-/- Mice, Inflammatory Vasculitis Model
			3.2.2 MHV68 Virus Culture
		3.3 Aortic Allograft Transplants C57Bl/6 Donor to BALB/C Recipient  Mice
		3.4 Histological Analysis of Lung Consolidation, Gut Dilatation, and Vascular Inflammation in Animal Models
			3.4.1 MHV68 Inflammatory Vasculitis Model: Harvest of Tissues for Histology Analysis
			3.4.2 For the Aortic Allograft Model: Harvest of Tissues for Histology Analysis
			3.4.3 Staining Elastic Fibers by Verhoeff-Van Gieson (VVG) Method
			3.4.4 Routine and Immunohistochemical Staining
	4 Notes
	References
Chapter 8: Methods for Crystallization and Structural Determination of M-T7 Protein from Myxoma Virus
	1 Introduction
	2 Structural Predictions
		2.1 Predictions Based on Homology
	3 X-Ray Crystallography
		3.1 Protein Crystallization (M-T7 Protein as a Practice Example)
			3.1.1 General Aspects on Macromolecular Crystal Growth: A Quick Overview
			3.1.2 Crystallizing M-T7 Protein for the First Time: Initial Crystallization Screening
			3.1.3 Improving the Diffraction Quality of M-T7 Crystals
			3.1.4 A Practical Guide to Micro-Seeding Technique for Improving Crystal Quality
		3.2 X-Ray Diffraction, Data Collection and Processing
			3.2.1 Introduction to X-Ray Diffraction
			3.2.2 Traditional X-Ray Crystallography
			3.2.3 X-Ray Free Electron Lasers (XFEL)
		3.3 X-Ray Data Collection and Data Processing
			3.3.1 X-Ray Data Collection
			3.3.2 X-Ray Data Processing
	4 A Practical Guide for Preparation for an X-Ray Diffraction Experiment
	5 Model Building, Refinement, and Validation
		5.1 Model Building and Refinement
			5.1.1 A Practical Guide to Phasing and Structure Determination Methods for Novel Proteins
			5.1.2 Structure Validation and Analysis, Deposition, and Publication
	6 The Future of Structural Biology and Impacts on the Understanding of M-T7
		6.1 X-Ray Crystallography Advances
		6.2 Cryo-EM and Micro-Electron Diffraction (Micro-ED)
		6.3 Small-Angle X-Ray Scattering (SAXS)
	References
Chapter 9: Microscopic Analysis of Viral Infection
	1 Introduction
	2 Materials
	3 Methods
		3.1 Imaging Fluorescent Protein Reporters for Viral Infection
			3.1.1 Infection of Adherent Cells
			3.1.2 Infection of Suspension Cells
			3.1.3 Live-Cell Imaging with Fluorescent Protein Reporters
			3.1.4 Image Analysis of Fluorescent Protein Reporters
		3.2 Imaging Viral Particles with Fluorescent Protein Label
		3.3 Imaging Cell-to-Cell Spread of Viral Infection
			3.3.1 Imaging Spread of Viral Infection from Infected Donor Cells to Target Cells
			3.3.2 Imaging Cell-to-Cell Transfer of Bound Virions
		3.4 Imaging Physiological Probes During Viral Infection
			3.4.1 Prepare Adherent Cells for Cell Death Imaging with Propidium Iodide
			3.4.2 Prepare Suspension Cells for Cell Death Imaging with Propidium Iodide
			3.4.3 Imaging Cell Death Using Propidium Iodide
			3.4.4 Prepare Adherent Cells for Apoptosis Imaging with CellEvent
			3.4.5 Prepare Suspension Cells for Apoptosis Imaging with CellEvent
			3.4.6 Imaging Apoptosis Using CellEvent
	4 Notes
	References
Chapter 10: Metabolomics Analysis of Viral Therapeutics
	1 Introduction
	2 Materials
		2.1 Preparation of Tissue Samples
		2.2 LC-MS/MS Targeted Analysis
	3 Methods
		3.1 Preparation of Tissue Samples
		3.2 LC-MS/MS Targeted Analysis
	4 Notes
	References
Chapter 11: Detecting Necroptosis in Virus-Infected Cells
	1 Introduction
	2 Materials
		2.1 Cell Viability by Dye Exclusion with Inhibitors
		2.2 MLKL Serine Phosphorylation
		2.3 MLKL Trimerization
		2.4 Co-Immunoprecipitation
		2.5 Necrosome Cross-Linking
	3 Methods
		3.1 Cell Viability by Dye Exclusion
		3.2 MLKL Serine Phosphorylation
		3.3 MLKL Trimerization
		3.4 Co-Immunoprecipitation
		3.5 Necrosome Cross-Linking
	4 Notes
	References
Chapter 12: Topical Application of Virus-Derived Immunomodulating Proteins and Peptides to Promote Wound Healing in Mouse Mode...
	1 Introduction
	2 Materials
		2.1 Materials for Mouse Skin Wound Surgery
		2.2 Biochemical Reagents
		2.3 Animals
	3 Methods
		3.1 Splint Preparation
		3.2 Chitosan Hydrogel Preparation
		3.3 Wound Procedure and Treatments
		3.4 Observation (Measurement and Intermediate Tissue Collection)
		3.5 Pre-Histology Tissue Processing
		3.6 Hematoxylin and Eosin (H&E) Staining
		3.7 Immunohistochemistry (IHC) Staining
		3.8 Masson´s Trichrome Staining
		3.9 Herovici´s Polychrome Staining
	4 Notes
	References
Chapter 13: Neurologic and Histologic Tests Used to Measure Neuroprotective Effectiveness of Virus-Derived Immune-Modulating P...
	1 Introduction
	2 Materials
		2.1 Model Animals
			2.1.1 Lab Animal  Care
		2.2 Preoperative Procedures
		2.3 Surgical Supplies, Sterile
		2.4 Pathology Supplies
	3 Methods
		3.1 The Clinical Endpoint
		3.2 Spinal Cord Injury
	4 Notes
	References
Chapter 14: Preclinical Testing of Viral Therapeutic Efficacy in Pristane-Induced Lupus Nephritis and Diffuse Alveolar Hemorrh...
	1 Introduction
	2 Materials
		2.1 Reagents and Equipment
		2.2 Animal Models
	3 Methods
		3.1 Methods of Nephritis
			3.1.1 Administration of Viral Therapeutic Protein
			3.1.2 Monitoring Proteinuria
			3.1.3 Blood Collection
			3.1.4 Detection of Cytokines and Chemokines
			3.1.5 Detection of Autoantibodies
			3.1.6 Renal Pathological Examinations
		3.2 Methods of DAH
			3.2.1 Administration of Viral Therapeutic Protein
			3.2.2 Collection and Analysis of Bronchial Alveolar Lavage Fluid (BAL)
			3.2.3 Lung Pathological Evaluation
	4 Notes
	References
Chapter 15: Kidney Subcapsular Allograft Transplants as a Model to Test Virus-Derived Chemokine-Modulating Proteins as Therape...
	1 Introduction
	2 Materials
		2.1 Expression and Purification of M-T7
		2.2 Mice
		2.3 Surgery
		2.4 Materials for Histology
	3 Methods
		3.1 Generation of M-T7 Baculovirus
		3.2 Expression and Purification of M-T7 from High Five Cells
		3.3 Pretreatments with Virus-Derived Immune-Modulating Protein
		3.4 Preparation of Mice for Surgery
		3.5 Surgical Procedure
		3.6 Collection of Tissues for Histological Analysis
		3.7 Histology and Immunohistology
	4 Notes
	References
Chapter 16: A Mouse Model of Acute Liver Injury by Warm, Partial Ischemia-Reperfusion for Testing the Efficacy of Virus-Derive...
	1 Introduction
	2 Materials
		2.1 Ischemia-Reperfusion Surgical Procedure
		2.2 Pre-histology Tissue Processing
		2.3 Hematoxylin and Eosin (H&E) Staining
		2.4 Periodic Acid-Schiff (PAS) Staining
		2.5 Suzuki Scoring
		2.6 Liver Enzyme Assays
	3 Methods
		3.1 Ischemia-Reperfusion Surgical Procedure
		3.2 Pre-histology Tissue Processing
		3.3 Hematoxylin and Eosin (H&E) Staining
		3.4 Periodic Acid-Schiff (PAS) Staining
		3.5 Suzuki Scoring
		3.6 Liver Enzyme Assays
	4 Notes
	References
Index