Preface
Contents
Contributors
Chapter 1: Overview of Experimental Vaccines and Antiviral Therapeutics for Henipavirus Infection
1 Introduction
2 Preclinical Vaccine Studies
2.1 Subunit Vaccines
2.2 Rhabdovirus Vectors
2.3 Paramyxovirus Vectors
2.4 Poxvirus Vectors
2.5 VEEV Vector
2.6 AAV Vector
2.7 ChAd Vector
2.8 BoHV Vector
2.9 Nucleic Acid-Based Vaccines
2.10 VLP
2.11 Attenuated Live Virus
2.12 Wild-Type Live Virus
2.13 Summary of Henipavirus Vaccine Candidates
3 Domesticated Animal Vaccines
3.1 Horse Vaccines
3.2 Swine Vaccines
4 Experimental Therapeutics
4.1 Ribavirin
4.2 Chloroquine
4.3 Antibodies
4.4 Purine Analogs
4.5 Fusion Inhibitors
4.6 Interferon Inducers
4.7 Inhibitors of Pyrimidine Nucleotides
4.8 Summary of Therapeutics
5 Concluding Remarks
References
Part I: Studying Henipaviruses Under Biosafety Level 2 Conditions
Chapter 2: A Revised Diagnostic Quantitative RT-PCR for the Detection of Nipah Virus Infection
1 Introduction
2 Samples, Materials, Reagents, and Equipment
2.1 Patient Sample Handling
2.2 RNA Extraction
2.3 qRT-PCR
2.4 Reagent Preparation and Stability
3 Methods
3.1 Sample Inactivation
3.2 RNA Extraction
3.3 NiV-Specific One-Step qRT-PCR
4 Notes
References
Chapter 3: Recombinant Soluble Henipavirus Glycoprotein Preparation
1 Introduction
2 Materials
2.1 Cell Culture
2.2 DNA Transfection
2.3 Affinity Precipitations
2.4 Henipavirus Soluble G (sG)
2.5 Henipavirus Soluble F (sF)
2.6 Size Exclusion Chromatography
2.7 Polyacrylamide Gel Electrophoresis (PAGE) and Western Blotting
2.7.1 Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) (Invitrogen)
2.7.2 Western Blotting and Immunodetection
2.8 Cross-Linking
2.9 Removing S-Tag for Soluble F (sF)
2.10 Multiplex Microsphere Immunoassay
3 Methods
3.1 Constructions of the Plasmids That Express Henipaviruses Surface Proteins (HeV, NiV CedV, and GhV sF or sG)
3.2 Transient Transfection of FreeStyle 293F Cells
3.3 Supernatant Harvest and Affinity Precipitation
3.4 Stable Cell Line Production
3.4.1 Transfected Cell Selection
3.4.2 Single Cell Isolation
3.5 Expression and Collection of Protein in Suspension
3.5.1 Expression of Soluble Proteins
3.5.2 Harvesting Supernatant from Erlenmeyer Flasks
3.6 Affinity Purification of S-Tag Proteins
3.7 Size Exclusion Chromatography
3.8 Analysis of Proteins by Native PAGE System
3.8.1 Electrophoresis
3.8.2 Coomassie Stain
3.8.3 Western Blotting
3.9 Determination of Glycoprotein Oligomerization Status by Cross-Linking
3.10 Removal of S-Tag for Soluble F Proteins
3.10.1 Factor Xa Cleavage of Recombinant Protein
3.10.2 Removal of Factor Xa Enzyme
3.10.3 Removal of Residual S-Tag
3.11 Henipavirus Multiplex Microsphere Immunoassay
4 Notes
References
Chapter 4: Cell-Cell Fusion Assays to Study Henipavirus Entry and Evaluate Therapeutics
1 Introduction
2 Materials
2.1 Syncytia Counting and Cell-Cell Fusion Inhibition Assays
2.1.1 Cell Culture and Transfection
2.1.2 Counting
2.1.3 Inhibition
2.2 Heterologous Fusion Assay
2.2.1 Transfection and Seeding
2.2.2 Labeling and Overlaying Veros
2.2.3 Microscopy
3 Methods
3.1 Syncytia Counting and Cell-Cell Fusion Inhibition Assays
3.1.1 Transfection
3.1.2 Syncytia Counting
3.1.3 Inhibition Assays
3.2 Heterologous Fusion Assay
3.2.1 Cell Seeding and Coverslip Preparation
3.2.2 Transfection and Labeling
3.2.3 Overlay and Slide Preparation
3.2.4 Microscopy and Imaging
4 Notes
References
Part II: Studying Infectious Henipaviruses In Vitro
Chapter 5: Recombinant Cedar Virus: A Henipavirus Reverse Genetics Platform
1 Introduction
2 Materials
2.1 Cells, Medium, Transfection Reagents, and Culture Vessels
2.2 Plasmids
3 Methods
3.1 Recovery of Replication-Competent rCedV from Plasmids
3.2 Virus Purification (See Notes 6 and 7)
3.3 Titration of rCedV by Fluorescence
3.4 Titration of rCedV by Plaque Assay
4 Notes
References
Chapter 6: Utilizing Recombinant Reporter Henipaviruses to Conduct Antiviral Screening
1 Introduction
2 Materials and Equipment
2.1 Cell Lines
2.2 Cell Culture Media
2.3 Cell Culture Plasticware
2.4 Assay Reagent
2.5 Reporter Measurement Device
3 Methods
3.1 Fluorescence-Based Reporter Assay (ZsGreen1 Fluorescence Protein)
3.2 Luminescence-Based Reporter Assay (Non-Secreted Renilla luciferase)
4 Notes
References
Chapter 7: In Vitro Antiviral Screening for Henipaviruses at BSL4
1 Introduction
2 Materials
2.1 Viruses
2.2 Cells
2.3 Media
2.4 Antibodies and Conjugates
2.5 Other Reagents and Materials
3 Methods
3.1 Cell and Compound Preparation (BSL2)
3.2 Antiviral Screening (BSL4)
3.3 Hendra and Nipah Virus Infection of Cells (BSL4)
3.4 Assay Termination and Decontamination (BSL4)
3.5 Immunolabeling Assay for Viral Antigen (BLS2)
3.6 Cytotoxicity Assay (BSL2)
4 Notes
References
Chapter 8: Isolation of Primary Porcine Bronchial Epithelial Cells for Nipah Virus Infections
1 Introduction
2 Materials
2.1 Lung Tissue Dissection, Cell Seeding, Subculturing, and Cryopreservation
2.2 Collagen Coating of Cell Culture Dishes
2.3 Immunostaining of Epithelial Cell Markers
2.4 NiV Infection (Kept or Transferred to the BSL-4 Containment)
3 Methods
3.1 Isolation of Primary Bronchial Epithelial Cells (PBEpC)
3.1.1 Collagen Coating of Cell Culture Materials
3.1.2 Preparation of Bronchi Segments and Removal of Mucus (Day 1)
3.1.3 Protease Digestion of Bronchi Segments (Day 2), Cell Isolation and Seeding (Day 3), and PBEpC Cultivation (Days 4-9)
3.2 PBEpC Cryopreservation and Subculturing
3.2.1 Cell Detachment Using Passage Kit 4
3.2.2 Cell Counting
3.2.3 Cryopreservation: Freezing and Thawing of PBEpC
3.2.4 Subculturing PBEpC on 6-Well Plates
3.2.5 Subculturing PBEpC on Transwell Membrane Inserts
3.3 Epithelial Cell Marker Staining in PBEpC Grown on Transwell Membrane Inserts
3.4 NiV Infection of PBEpC
3.4.1 NiV Infection
3.4.2 Sampling Virus Supernatants
3.4.3 Harvesting NiV-Infected Cell Lysates for RNA Isolation
4 Notes
References
Chapter 9: Primary Culture of the Human Olfactory Neuroepithelium and Utilization for Henipavirus Infection In Vitro
1 Introduction
2 Materials
2.1 Human Olfactory Epithelial Biopsy
2.2 Cell Culture
2.3 Immunocytochemistry
2.4 Calcium Imaging
2.5 Henipavirus Infection
3 Methods
3.1 Harvest, Isolation, Culture, and Maintenance of Human Olfactory Cells
3.2 Propagation of Human Olfactory Cells
3.3 Freezing and Thawing Cultured Human Olfactory Cells
3.4 Confocal Immunofluorescence for Olfactory Cell Markers
3.5 Calcium Imaging for Functional Assays
3.6 Henipavirus Infection of hOEC
4 Notes
References
Part III: Studying Infectious Henipaviruses In Vivo
Chapter 10: Mouse Models of Henipavirus Infection
1 Introduction
2 Materials
3 Methods
3.1 Manipulation of Mice in BSL4
3.2 Anesthesia
3.3 Infection
3.4 Blood Collection and Serum Preparation
3.5 Virus Titration from Murine Tissues
3.6 Seroneutralization
3.7 Euthanasia and Collection of Organs for RNA Isolation and Immunocytochemistry
4 Notes
References
Chapter 11: In Vivo Imaging of Nipah Virus Infection in Small Animal Rodent Models
1 Introduction
2 Materials
2.1 IVIS Equipment
2.2 Materials and Supplies
3 Methods
3.1 In Vivo Bioluminescence Imaging
3.2 In Vivo Fluorescence Imaging
3.3 Ex Vivo Bioluminescent Imaging
3.4 Analysis of Data
4 Notes
References
Chapter 12: Nonhuman Primate Models for Nipah and Hendra Virus Countermeasure Evaluation
1 Introduction
2 Materials
2.1 Requirements to Work with Henipaviruses in AGMs
2.2 General
2.3 Inoculation of AGMs with Henipaviruses
2.4 Vaccination, Treatment, and Sampling of AGMs
2.5 Euthanasia of AGMs at Humane and Scientific Endpoints
3 Methods
3.1 Vaccination
3.2 Henipavirus Exposure in AGMs
3.3 Post-Exposure Treatment
3.4 Clinical Samples, Clinical Observations, Humane Endpoint Scoring, and Euthanasia
4 Notes
References
Chapter 13: Nipah Virus Aerosol Challenge of Three Distinct Particle Sizes in Nonhuman Primates
1 Introduction
2 Materials
2.1 Aerosol Equipment
2.2 Aerosol Consumables
3 Methods
3.1 Class III BSC Preparation
3.2 Aerosol Equipment Assembly and Nipah Virus Preparation of a Nonhuman Primate Head-Only Exposure Chamber
3.3 Plethysmography Section
3.4 NHP Nipah Virus Head-Only Aerosol Exposure Using a Small Particle Generator
3.5 NHP Nipah Virus Head-Only Aerosol Exposure Using an Intermediate/Large Particle Generator
4 Notes
References
Chapter 14: Generation and Characterization of a Humanized Lung Xenograft Mouse Model for Studying Henipavirus Pathogenesis
1 Introduction
2 Material
2.1 Processing of Tissues for Grafting onto NSG Mice
2.2 Tissue Grafting onto NSG Mice and Care of NSG Mice
2.3 NiV Infection of NSG Mice
2.4 Euthanasia and Necropsy of NSG Mice
2.5 Processing of Human Lung Grafts
3 Method
3.1 Preparation of OKT3 Antibody Solution
3.2 Processing of Human Thymus
3.3 Processing of Human Liver
3.4 Processing of Human Bone
3.5 Processing of Human Lung
3.6 Pre-surgical Procedures
3.7 Surgical Grafting of Tissues onto NSG Mice
3.8 Analysis of HSC Grafting
3.9 NiV Infection
3.10 Euthanasia and Necropsy of Animals
3.11 Graft Sampling and Processing
4 Notes
References
Chapter 15: Ferret Models for Henipavirus Infection
1 Introduction
2 Materials
2.1 Requirements to Work with Henipaviruses in Ferrets
2.2 General
2.3 Inoculation of Ferrets
2.4 Vaccination, Treatment, Sampling of Ferrets
2.5 Euthanasia of Ferrets at Humane and Scientific Endpoints
3 Methods
3.1 Vaccination
3.2 Henipavirus Inoculation
3.3 Post-exposure Treatment
3.4 Clinical Samples, Clinical Observations, Humane Endpoint Scoring, and Euthanasia
4 Notes
References
Chapter 16: Syrian Golden Hamster Model for Nipah Virus Infection
1 Introduction
2 Materials
2.1 General Materials and Supplies
2.2 Specialized Supplies for Biosampling, Vaccination, or Therapeutic Administration
3 Methods
3.1 Nipah Virus Inoculation
3.2 Evaluation of Therapeutics
3.3 Evaluation of Vaccines
3.4 Biosampling, Euthanasia, and Necropsy
4 Notes
References
Part IV: Immune Assays for Henipaviruses
Chapter 17: Anti-Nipah Virus Enzyme-Linked Immunosorbent Assays with Non-human Primate and Hamster Serum
1 Introduction
2 Materials
2.1 Nipah Virus Preparation
2.2 Plate Coating
2.3 ELISA Procedure
3 Methods
3.1 Nipah Virus Procedure
3.2 NiV Slurry Preparation
3.3 Indirect IgG ELISA Procedure
3.4 Indirect IgA (NHP Only) ELISA Procedure
3.5 IgM, IgG2, and IgG1 Capture ELISA Procedure
4 Notes
References
Chapter 18: Assays for Detecting Henipavirus Antibodies
1 Introduction
2 Materials
2.1 General
2.2 Gene Cloning
2.3 Luciferase Immunoprecipitation System (LIPS)
2.3.1 Primers
2.3.2 LIPS Assay
2.4 Luminex
2.5 Pseudovirus
3 Methods
3.1 LIPS
3.1.1 Cloning of Target Gene
3.1.2 Production of Recombinant Luc-Fusion Proteins
3.1.3 Antibody Detection Using LIPS
3.2 Luminex
3.2.1 Cloning and Expression of Target Genes
3.2.2 Purification of Recombinant Proteins
3.2.3 Conjugation of Proteins to Luminex Beads
3.2.4 Antibody Detection Using Luminex
3.3 Pseudotyped Virus
3.3.1 Cloning of Henipavirus G and F Genes into pCAGGS Mammalian Expression Vector
3.3.2 Production of Pseudotyped Virus
3.3.3 Antibody Detection Using Pseudovirus (Virus Neutralization Test)
4 Notes
References
Part V: Host Responses to Henipavirus Infection
Chapter 19: Profiling Host MicroRNA Responses to Henipavirus Infection
1 Introduction
2 Materials
3 Methods
3.1 miR Harvesting and Purification
3.2 cDNA Library Preparation
3.2.1 3โฒ Ligation
3.2.2 5โฒ Ligation
3.2.3 Reverse Transcription (RT)
3.2.4 Library Amplification
3.3 Next-Generation Sequencing
3.4 MicroRNA Identification via Bioinformatics
3.5 cDNA Synthesis for qPCR Validation
4 Notes
References
Chapter 20: Host Transcriptome Analysis of Ferret Tissues Following Henipavirus Infection
1 Introduction
2 Materials
2.1 RNA Extraction, Library, and Sequencing
2.2 Sequencing Analysis
3 Methods
3.1 RNA extraction was performed on lung tissue samples from Days 3 and 5 post-infection with HeV and NiV-B. Infections were c...
3.2 Library Construction
3.3 Data Analysis
3.3.1 Quality Control and Adapter Removal
3.3.2 Reads Mapping to the Reference Genome
3.3.3 Quantification of Gene Expression Levels
3.3.4 Differential Gene Expression Analysis
3.3.5 KEGG Gene Set Enrichment Analysis. (See Note 20)
3.3.6 Determination of Transcription Factor Target Enrichment
3.3.7 Transcription factor annotation based on GO ontology. (See Note 23).
3.3.8 PPI Network Analysis
3.3.9 Identification of Cytokine-Cytokine Receptor Interaction Pathways
4 Notes
References
Index