Coronaviruses: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Coronaviruses: An Updated Overview of Their Replication and Pathogenesis
	1 Classification
	2 Genomic Organization
	3 Virion Structure
	4 Coronavirus Life Cycle
		4.1 Attachment and Entry
		4.2 Replicase Protein Expression
		4.3 Replication and Transcription
		4.4 Assembly and Release
	5 Pathogenesis
		5.1 Animal Coronaviruses
		5.2 Human Coronaviruses
	6 Diagnosis, Treatment, and Prevention
	7 Conclusions
	References
Part I: Coronavirus Detection, Discovery, and Evolution
	Chapter 2: Discovery of Novel Coronaviruses in Rodents
		1 Introduction
		2 Materials
			2.1 Sample Acquisition
			2.2 Total RNA Extraction from Mammalian Tissue
			2.3 cDNA Synthesis with Random Hexamers
			2.4 PCR
			2.5 Agarose Gel Electrophoresis
		3 Methods
			3.1 Sample Acquisition
			3.2 RNA Extraction from Mammalian Tissues
			3.3 cDNA Synthesis with Random Hexamers
			3.4 PCR Amplification of the GAPDH ``Housekeeping Gene´´ to Assess cDNA Quality
			3.5 PCR Screening for Coronaviruses
			3.6 Agarose Gel Preparation and Electrophoresis
		4 Notes
		References
	Chapter 3: Detection and Discovery of Coronaviruses in Wild Bird Populations
		1 Introduction
		2 Materials
			2.1 Specimen Collection (See Note 1)
			2.2 RNA Extraction and Amplification of the RNA-Dependent RNA Polymerase (RdRp)  Gene
			2.3 NGS Sequencing
				2.3.1 RNA Extraction Prior to NGS Sequencing
				2.3.2 Quantification and RNA Integrity Check
				2.3.3 cDNA Synthesis and Quantification
				2.3.4 Library Preparation for Sequencing with Illumina MiSeq
		3 Methods
			3.1 Specimen Collection
			3.2 RNA Extraction and Amplification of the RdRp  Gene
			3.3 NGS Sequencing (See Note 11)
				3.3.1 RNA Extraction Prior to NGS Sequencing
				3.3.2 Quantification and RNA Integrity Check
				3.3.3 cDNA Synthesis and Quantification
				3.3.4 Illumina MiSeq Library Preparation-DNA Tagmentation
				3.3.5 Illumina MiSeq Library Preparation-Library Amplification
				3.3.6 Illumina MiSeq Library Preparation-Library Cleanup
				3.3.7 Illumina MiSeq Library Preparation-Library Check and Pooling
				3.3.8 Sequencing of Pooled Libraries with the MiSeq Instrument
		4 Notes
		References
	Chapter 4: Competitive ELISA for the Detection of Serum Antibodies Specific for Middle East Respiratory Syndrome Coronavirus (...
		1 Introduction
		2 Materials
		3 Methods
			3.1 cELISA
			3.2 Evaluate cELISA Results
		4 Notes
		References
	Chapter 5: Whole-Genome Sequencing Protocols for IBV and Other Coronaviruses Using High-Throughput Sequencing
		1 Introduction
		2 Materials
			2.1 Purification of IBV
			2.2 RNA Extraction
			2.3 QC of Extracted Total RNA
			2.4 IBV Sequencing Library Preparation
			2.5 Normalization of Sequencing Libraries
			2.6 Quantification of Sequencing Library Pool
			2.7 Denaturation of Sequencing Pool and Loading of Libraries
		3 Methods
			3.1 Purification of IBV (2 h)
			3.2 RNA Extraction (1 h)
			3.3 Quality Control (QC) of Extracted Total RNA (1 h)
			3.4 Library Preparation (1-2 Days Dependent Upon Kit)
			3.5 Library QC and Normalization of Sequencing Libraries (3 h)
			3.6 Quantitation of Sequencing Pool Using qPCR, Sample Denaturation and Loading onto Illumina MiSeq (1 Day)
		4 Notes
		References
Part II: Propagation and Titration of Coronaviruses
	Chapter 6: Isolation and Tissue Culture Adaptation of Porcine Deltacoronavirus: A Case Study
		1 Introduction
		2 Materials
			2.1 Sample Collection and Preparation
			2.2 Cell Culture, Virus Propagation, and TCID50
			2.3 Plaque Assay for Purification of PDCoV
		3 Methods
			3.1 Isolation and Propagation of PDCoV in LLC-PK or ST Cells
				3.1.1 Passage or Preparation of LLC-PK Cells
				3.1.2 Passage or Preparation of ST Cells
				3.1.3 Isolation of PDCoV in LLC-PK or ST Cells
				3.1.4 Serial Passage of PDCoV in LLC-PK or ST Cells
			3.2 Titration of Infectious PDCoV by TCID50 Assay
			3.3 Titration and Purification of PDCoV in ST Cells by Plaque Assay (See Note 14)
		4 Notes
		References
	Chapter 7: The Preparation of Chicken Kidney Cell Cultures for Virus Propagation
		1 Introduction
		2 Materials
			2.1 Extraction of Kidneys
			2.2 Isolation and Culture of Kidney Cells
		3 Method
			3.1 Extraction of Kidneys
			3.2 Isolation and Culture of Kidney Cells
		4 Notes
		References
	Chapter 8: The Preparation of Chicken Tracheal Organ Cultures and Their Application for Ciliostasis Test, Growth Kinetics Stud...
		1 Introduction
		2 Materials
			2.1 Preparation of Tracheal Section
			2.2 Culture of Tracheal Section and All Applications
		3 Methods
			3.1 Preparation of Tracheal Sections
			3.2 Culture of Tracheal Sections
			3.3 TOC Assessment and Selection
			3.4 Ciliostasis Test During In Vitro Infection
			3.5 Assessment of Ciliary Activity Ex Vivo
			3.6 Growth Kinetics Study
			3.7 Virus Propagation in TOCs
		4 Notes
		References
	Chapter 9: Isolation and Propagation of Coronaviruses in Embryonated Eggs
		1 Introduction
		2 Materials
			2.1 Preparation and Collection of Samples for Egg Inoculation
			2.2 Egg Inoculation and Incubation
			2.3 Collection of Specimens from Inoculated Eggs
		3 Methods
			3.1 Collection of Samples for Egg Inoculation
			3.2 Preparation of Samples for Egg Inoculation
			3.3 Allantoic Sac Inoculation
			3.4 Amniotic Sac Inoculation (Method A)
			3.5 Amniotic Sac Inoculation (Method B)
			3.6 Collection of Allantoic Fluid from Eggs Inoculated by Allantoic Route
			3.7 Collection of Embryo Tissues from Eggs Inoculated by Amniotic Route
		4 Notes
		References
	Chapter 10: Well-Differentiated Primary Mammalian Airway Epithelial Cell Cultures
		1 Introduction
		2 Materials
			2.1 AEC Cultures
			2.2 Propagation of Coronaviruses from Mammalian Origin
			2.3 Immunofluorescence Analysis
		3 Methods
			3.1 AEC Cultures
				3.1.1 Collagen Coating of Cell Culture Flasks for Cell Expansion
				3.1.2 Collagen Type IV Coating of Inserts
				3.1.3 Isolation of Primary Tracheal and/or Bronchial Cells
				3.1.4 Maintenance of AEC on Plastics for Expansion
				3.1.5 Subcultivation of Primary Epithelial Cells on Porous Inserts for Differentiation
			3.2 Virus Propagation
			3.3 Immunofluorescence Analysis
		4 Notes
		References
	Chapter 11: Quantification of Coronaviruses by Titration In Vitro and Ex Vivo
		1 Introduction
		2 Materials
			2.1 Titration of PDCoV by Tissue Culture Infective Dose (TCID50)
			2.2 Titration of IBV by Plaque-Forming Units
			2.3 Titration of IBV by Ciliostatic Dose (CD50) in Tracheal Organ Cultures (TOCs)
		3 Methods
			3.1 Titration of PDCoV by Tissue Culture Infective Dose (TCID50)
			3.2 Titration of IBV by Plaque-Forming Units
				3.2.1 Infection of Cells
				3.2.2 Staining Cells and Determining Titer
			3.3 Titration of IBV by Ciliostatic Dose Method
				3.3.1 Screening and Selection of TOCs
				3.3.2 Infecting TOCs
		4 Notes
		References
Part III: Manipulating the Genomes of Coronaviruses
	Chapter 12: Transient Dominant Selection for the Modification and Generation of Recombinant Infectious Bronchitis Coronaviruses
		1 Introduction
		2 Materials
			2.1 Homologous Recombination and Transient Dominant Selection in Vero Cells
			2.2 Extraction of DNA from Recombinant Vaccinia Virus
			2.3 Production of Large Stocks of Vaccinia Virus
			2.4 Vaccinia Virus Partial Purification
			2.5 Analysis of Vaccinia Virus DNA by Pulse Field Agarose Gel Electrophoresis (PFGE)
			2.6 Preparation of rFPV-T7 Stock Virus
			2.7 Recovery of rIBV and Serial Passage on CK Cells
		3 Methods
			3.1 Infection/Transfection of Vero Cells with Vaccinia Virus
			3.2 Plaque Purification in the Presence of GPT Selection Agents: Selection of MPA-Resistant Recombinant Vaccinia Viruses (GPT+...
			3.3 Plaque Purification in the Absence of GPT Selection Agents: Selection of MPA Sensitive Recombinant Vaccinia Viruses (Loss ...
			3.4 Production of Small Stocks of Recombinant Vaccinia Viruses
			3.5 DNA Extraction from Small Stocks of Recombinant Vaccinia Virus for Screening by  PCR
				3.5.1 DNA Extraction Using Phenol/Chloroform/Isoamyl Alcohol
				3.5.2 Extraction of rVV DNA Using the Qiagen QlAamp DNA Mini  Kit
			3.6 Production of Large Stocks of Vaccinia Virus
			3.7 Vaccinia Virus Partial Purification
			3.8 Extraction of Vaccinia Virus DNA from Large Partially Purified rVV Stocks
			3.9 Analysis of Vaccinia Virus DNA by PFGE
			3.10 Preparation of rFPV-T7 Stock
			3.11 Infection and Transfection of CK Cells for the Recovery of  rIBV
			3.12 Serial Passage of rIBVs in CK Cells
		4 Notes
		References
	Chapter 13: In-Yeast Assembly of Coronavirus Infectious cDNA Clones Using a Synthetic Genomics Pipeline
		1 Introduction
		2 Materials
			2.1 Preparation of Overlapping DNA Fragments and TAR Cloning Vector for Viral Genome Assembly
			2.2 Assembly of Full-Length cDNA Via TAR in Yeast
			2.3 Identification of Yeast Clones Harboring Correctly Assembled Viral cDNA
			2.4 Large-Scale Preparation of Recombinant Plasmids in Yeast: Midiprep
			2.5 Recovery of Infectious Coronaviruses from TAR-Cloned Full-Length Viral cDNA
				2.5.1 Generation of Infectious Full-Length Viral RNA and N Gene RNA by In Vitro Transcription
				2.5.2 Recovery of Infectious MHV
		3 Methods
			3.1 Preparation of Overlapping DNA Fragments and TAR Cloning Vector for Viral Genome Assembly
				3.1.1 Design and Generation of Overlapping Viral DNA Fragments from Viral RNA
				3.1.2 Design and Generation of TAR Cloning Vector
			3.2 Assembly of Full-Length cDNA Via TAR in Yeast
			3.3 Identification of Yeast Clones Harboring Correctly Assembled Viral cDNA
				3.3.1 Growth of Yeast Transformants on Agar Dishes
				3.3.2 Extraction of Yeast DNA with GC Prep Method (Chelex100 Preparation) for Colony Screening
				3.3.3 Screening for the Presence of Desired Construct by Simplex PCR
				3.3.4 Verification of Assembly Junctions by Multiplex PCR
			3.4 Large-Scale Preparation of Recombinant Plasmids in Yeast: Midiprep
			3.5 Recovery of Infectious Coronaviruses from TAR-Cloned Full-Length Viral cDNA
				3.5.1 Generation of Infectious Full-Length Viral RNA and N Gene RNA by In Vitro Transcription (see Note 25)
				3.5.2 Recovery of Infectious MHV
		4 Notes
		References
Part IV: Studying Virus-Host Interactions
	Chapter 14: Proximity Labeling for the Identification of Coronavirus-Host Protein Interactions
		1 Introduction
		2 Materials
			2.1 General Reagents and Equipment
			2.2 BioID and TurboID Reagents
			2.3 APEX Reagents
			2.4 APEX Electron Microscopy Reagents
		3 Methods
			3.1 Generation of Recombinant MHV, General Considerations Prior to Commencing Proximity Labeling
			3.2 Labeling Procedure Using MHV-BirAR118G-nsp2
			3.3 Labeling Procedure Using MHV-TurboID-nsp2
			3.4 Labeling Procedure Using MHV-APEX2-nsp2
			3.5 Controls
			3.6 Immunofluorescence Microscopy Analysis (IFA) of Biotinylated Proteins
			3.7 Western Blot Analysis of Biotinylated Proteins (Analytical Scale)
			3.8 Preparation of Cells Infected with MHV-APEX2-nsp2 for Electron Microscopy
			3.9 Sample Preparation for Mass Spectrometry, Infections with MHV-BirAR118G-nps2
			3.10 Sample Preparation for Mass Spectrometry, Infections with MHV-Turbo-nsp2
			3.11 Sample Preparation of Cells Infected with MHV-APEX2-nsp2 for Mass Spectrometry
		4 Notes
		References
	Chapter 15: Using Yeast to Identify Coronavirus-Host Protein Interactions
		1 Introduction
		2 Materials
			2.1 Yeast Strains
			2.2 Yeast Culture Media
			2.3 Plasmid Transformation
				2.3.1 Small-Scale and Large-Scale Transformation
				2.3.2 Transformation in a 96-Well Format
			2.4 Culturing Yeast
			2.5 NaOH Protein Extraction
			2.6 Genomic DNA Extraction
		3 Methods
			3.1 Yeast Culture
			3.2 Culturing Pooled Collections of Yeast Libraries from 96 Well Arrays
			3.3 Small-Scale Plasmid Transformation
			3.4 Large-Scale Plasmid Transformation
				3.4.1 Large-Scale Plasmid Transformation of Pooled Yeast Library
				3.4.2 Large-Scale Plasmid Transformation in 96-Well Format
			3.5 Protein Extraction
			3.6 Identification of Slow-Growth Phenotypes
				3.6.1 Growth Curve Analysis
				3.6.2 Serial Dilution Drop Cultures
			3.7 Suppressor Screening
			3.8 Identifying ``Hit´´ Genes
				3.8.1 Genomic DNA Extraction
		4 Notes
		References
	Chapter 16: Determining How Coronaviruses Overcome the Interferon and Innate Immune Response
		1 Introduction
		2 Materials
			2.1 Cloning of Coronavirus Genes
			2.2 Luciferase Assays for Innate Immune Signaling
			2.3 Virology Assay
		3 Methods
			3.1 Cloning of Individual Coronavirus Genes into Plasmids
			3.2 Luciferase Assays for Innate Immune Signaling
			3.3 Virology Assays Using Sensitive Viruses
				3.3.1 HIVDeltaVpu Stock Generation
				3.3.2 Virology Assay
			3.4 Coronavirus Infectious Clones
		4 Notes
		References
	Chapter 17: Ribopuromycylation in Coronavirus-Infected Cells
		1 Introduction
		2 Materials
			2.1 RPM to Tag Nascent Polypeptides
			2.2 Detection of Tagged Nascent Polypeptides by Immunofluorescence (IF)
			2.3 Detection of Tagged Nascent Polypeptides by Western Blot (WB)
		3 Methods
			3.1 RPM and Detection of Tagged Nascent Polypeptides by IF
			3.2 Immunofluorescence Quantification
			3.3 RPM and Detection of Tagged Nascent Polypeptides by WB
		4 Notes
		References
Part V: Imaging Coronavirus Infections
	Chapter 18: Visualizing Coronavirus Entry into Cells
		1 Introduction
		2 Materials
			2.1 Purification of a Coronavirus Stock
			2.2 Cell Culture Experiments
				2.2.1 In Vitro Cell Cultures
				2.2.2 Ex Vivo HAE Cultures
				2.2.3 Entry Inhibitors
				2.2.4 Endocytosis Cargo Proteins
			2.3 Immunostaining to Visualize Viral Entry into Cells
			2.4 Image Acquisition, Processing, and Presentation
		3 Methods
			3.1 Purification of a Coronavirus Stock
			3.2 Cell Culture Experiments
				3.2.1 Viral Entry In Vitro into Cell Lines
				3.2.2 Viral Entry into Ex Vivo HAE Cultures
				3.2.3 Synchronized Viral Entry into Cell Lines
			3.3 Immunostaining to Visualize Viral Entry into Cells
				3.3.1 Immunostaining Cell Lines
				3.3.2 HAE Cultures
			3.4 Image Acquisition
				3.4.1 Selection of Fluorescent  Dyes
				3.4.2 Scanning Settings
				3.4.3 3D Imaging
			3.5 Image Analysis
				3.5.1 Image Processing and Presentation
				3.5.2 Colocalization
				3.5.3 Statistics: Particle Counting
		4 Notes
		References
	Chapter 19: Preparation of Cultured Cells Using High-Pressure Freezing and Freeze Substitution for Subsequent 2D or 3D Visuali...
		1 Introduction
		2 Materials
			2.1 Chemical Reagents
			2.2 Hardware
			2.3 Consumables
		3 Methods
			3.1 Preparation of Cells
			3.2 Fixation of Cells by High-Pressure Freezing
			3.3 Freeze Substitution
		4 Notes
		References
Index