Preface
Contents
Contributors
Chapter 1: Reverse Genetics Systems for Filoviruses
1 Introduction
2 Materials
2.1 Rescue and Passaging of Viruses
2.2 Sequence Confirmation of Rescued Viruses
3 Methods
3.1 Initial Rescue of Recombinant Viruses
3.2 Passage of Recombinant Viruses
3.3 Harvest of Recombinant Viruses
3.4 Sequence Confirmation of Rescued Virus
4 Notes
References
Chapter 2: Reverse Genetics Systems for the De Novo Rescue of Diverse Members of Paramyxoviridae
1 Introduction
1.1 History and Optimization of Paramyxovirus Reverse Genetics Systems
1.2 Design and Manipulation of Recombinant Paramyxovirus Reverse Genetics Systems
2 Materials
2.1 Transformation and Validation of Full-Length Paramyxovirus cDNA and Accessory Rescue Plasmids
2.2 Cell Preparation
2.3 Preparation of Transfection Complexes and Virus Rescue
2.4 Virus Recovery and Amplification
3 Methods
3.1 Transformation and Validation of Full-Length Paramyxovirus cDNA and Accessory Rescue Plasmids
3.2 Cell Preparation
3.3 Preparation of Transfection Complexes and Virus Rescue
3.4 Virus Recovery and Amplification
4 Notes
References
Chapter 3: Rescue of Recombinant Newcastle Disease Virus Expressing Heterologous Genes
1 Introduction
2 Materials
2.1 Design and Cloning of the Additional Transcription Unit
2.2 Rescue of the Recombinant NDV Expressing a Codon-Optimized Influenza Hemagglutinin
3 Methods
3.1 In Silico Design and Insertion into the NDV Genome of an Additional Transcription Unit Encoding a Chimeric Influenza Hemag...
3.2 Use the Sequence-Confirmed Rescue Plasmid pNDV-H5 to Rescue a rNDV Expressing a Chimeric, Codon-Optimized H5
4 Notes
References
Chapter 4: Use of Reverse Genetics for the Generation of Recombinant Influenza Viruses Carrying Nanoluciferase
1 Introduction
2 Materials
2.1 Influenza A and B Viral RNA (vRNA) Extraction
2.2 Amplification of Influenza A and B Gene Segments from vRNA
2.3 Bidirectional Reverse Genetic pDP2002 Plasmid Vector
2.4 Cloning of Influenza A and B Virus Gene Segments into pDP2002
2.5 Cell Culture Growth and Maintenance
2.6 Generation of Influenza A and B NanoLuc Viruses by Reverse Genetics Using the pDP2002 Plasmid Sets
2.7 Propagation of Rescued Influenza A and B Viruses in MDCK Cells
2.8 Propagation of Rescued Influenza A and B NanoLuc Viruses in Embryonated Chicken Eggs
2.9 Titration of FLUAV-NLuc and FLUBV-NLuc by Tissue Culture Infectious Dose 50 (TCID50)
2.10 Use of FLUAV-NLuc and FLUBV-NLuc Viruses for the Assessment of Neutralizing Antibodies
3 Methods
3.1 vRNA Extraction from Influenza A and B Viruses
3.2 FLUAV or FLUBV RT-PCR Amplification
3.3 Cloning of FLUAV and FLUBV Gene Segments into pDP2002
3.4 Screening of Transformant Colonies by cPCR
3.5 Cell Culture Growth and Maintenance
3.6 Rescue of FLUAV-NLuc and FLUBV-NLuc Using pDP2002 Plasmid Sets
3.7 Propagation of FLUAV-NLuc and FLUBV-NLuc in MDCK Cells
3.8 Propagation of FLUAV-NLuc and FLUBV-NLuc in ECEs
3.9 Titration of FLUAV-NLuc and FLUBV-NLuc by Tissue Culture Infectious Dose 50 (TCID50)
3.10 Use of FLUAV-NLuc and FLUBV-NLuc Viruses for the Assessment of Neutralizing Antibodies
3.11 Analysis of Luciferase Expression and Determination of the Effective Serum Dilution 50 (ESD50)
4 Notes
References
Chapter 5: Reverse Genetics of Bat Influenza A Viruses
1 Introduction
2 Materials
2.1 Reagents and Kits
2.2 Primers
2.3 Cells and Media
3 Methods
3.1 Extraction of Bat Influenza A vRNA
3.2 cDNA Synthesis of Bat Influenza A Virus vRNA
3.3 Cloning of Bat Influenza A Virus cDNAs into pHW2000 Rescue Plasmids and pCAGGS Helper Plasmids
3.4 Rescue of Bat Influenza A Virus by Reverse Genetics
3.5 Propagation of Bat Influenza A Viruses In Vitro
3.6 Titration of Bat Influenza A Viruses
4 Notes
References
Chapter 6: Rescue of Infectious Salmon Anemia Virus (ISAV) from Cloned cDNA
1 Introduction
2 Materials
2.1 Viral RNA Extraction
2.2 cDNA Synthesis and PCR
2.3 Cloning ISAV cDNA
2.4 Cloning of Expression Plasmids
2.5 Transfection of Salmon Cells
2.6 Isavirus Growth
3 Methods
3.1 Viral RNA Extraction
3.2 cDNA Synthesis and PCR
3.3 Cloning of Viral cDNAs
3.4 Cloning of Expression Plasmids
3.5 Transfection of Salmon Cells
3.6 Isavirus Growth in ASK Cells
4 Notes
References
Chapter 7: Reverse Genetics System for Rift Valley Fever Virus
1 Introduction
2 Materials
2.1 RVFV Reverse Genetics Plasmids
2.1.1 Viral RNA Expression Plasmids
2.1.2 Viral Protein Expression Plasmids
2.2 Cell Lines
2.3 Tissue Culture Media
2.4 Reagents and Supplies
3 Methods
3.1 RVFV MP-12 Generation by Reverse Genetics
3.1.1 Preparation of BSR-T7/5 Cells
3.1.2 Transfection Mix
3.1.3 Transfection
3.1.4 Virus Collection and Clarification: P0 Rescue Stock
3.2 Plaque Assay of RVFV MP-12: P0 Rescue Stock
3.3 Amplification of RVFV MP-12: P1 Working Stock
4 Notes
References
Chapter 8: Plasmid-Based Lassa Virus Reverse Genetics
1 Introduction
2 Materials
2.1 LASV Reverse Genetics Plasmids
2.2 Cell Lines for the Generation of rLASV
2.3 Cell Culture Media and Reagents
3 Methods
3.1 Biosafety Conditions to Generate rLASV
3.2 LASV Rescue System Experimental Approach (Fig. 4)
3.3 Confirmation of Successful rLASV Rescue
3.4 Amplification of Rescued rLASV
4 Notes
References
Chapter 9: Bacterial Artificial Chromosome Reverse Genetics Approaches for SARS-CoV-2
1 Introduction
2 Materials
2.1 Biosafety Recommendations
2.2 SARS-CoV-2
2.3 BAC and Plasmid Extraction
2.4 E. coli DH10B Cells
2.5 Culture Media for DH10B Cells
2.6 Cell Line for the Rescue of rSARS-CoV-2
2.7 Cell Culture Media and Reagents for Rescue and Propagation of rSARS-CoV-2
2.8 Reagents for Plaque Assay and Immunostaining
2.9 Reagents for Immunofluorescence
2.10 Laboratory Equipment, Reagents, and Supplies
3 Methods
3.1 Preparation of the Infectious BAC Clone for the Rescue of rSARS-CoV-2
3.2 Rescue of rSARS-CoV-2
3.3 Validation of Viral Rescue and Characterization of rSARS-CoV-2
3.4 Plaque Assay for Viral Titration and Visualization of Plaque Sizes
3.5 Amplification of rSARS-CoV-2 and Generation of Virus Stocks
4 Notes
References
Chapter 10: Construction of Infectious Clones for Human Enteroviruses
1 Introduction
2 Materials
2.1 Viral RNA Extraction
2.2 Reverse Transcription
2.3 PCR Amplification
2.4 Agarose Gel Electrophoresis and Gel Extraction
2.5 DNA Cloning
2.6 Recovery and Generation of Infectious Clone-Derived EV-D68 Virus and Fluorescence Reporter Gene Expression EV-D68 Virus
3 Methods
3.1 EV-D68 Viral RNA Extraction and Viral cDNA Synthesis
3.2 Viral cDNA Fragment Amplification with PCR
3.3 Viral cDNAs Cloning into a Plasmid Vector
3.4 Plasmid Transfection to Recover Infectious Clone-Derived EV-D68 Virus
3.5 Propagation and Characterization of Clone-Derived EV-D68
3.6 Introduction of a Fluorescence Reporter Gene into the Complete Genome cDNA Clone
4 Notes
References
Chapter 11: Reverse Genetics of Hepatitis C Virus Using an RNA Polymerase I-Mediated Transcription
1 Introduction
2 Materials
2.1 HCV
2.2 Cloning Vector
2.3 Plasmid Preparation
2.4 Cell Line and Reagents
3 Methods
3.1 Isolation of Viral RNA
3.2 PCR Amplification of Full-Length HCV Genome
3.3 Assembly of the Full-Length cDNA Clone
3.4 Generation of Infectious HCV from Plasmid
3.5 Determination of the Infectious Titers of HCV
4 Notes
References
Chapter 12: Reverse Genetics of Zika Virus Using a Bacterial Artificial Chromosome
1 Introduction
2 Materials
2.1 Biosafety Recommendation
2.2 Plasmids and Bacterial Strains for the Generation of an Infectious ZIVK Infectious cDNA Clone
2.3 Culture Media for E. coli
2.4 Enzymes, Reagents, and Kits for the Assembly and Manipulation of BAC Clones
2.5 Cell Culture Media and Reagents for Rescue and Propagation of rZIKV
2.6 Cell Line for the Rescue of rZIKV
2.7 Reagents for the Titration and Detection of ZIKV
2.7.1 Plaque Assay and Immunostaining
2.7.2 Immunofluorescence
2.8 Laboratory Equipment
3 Methods
3.1 Assembly of a ZIKV Infectious cDNA Clone in the BAC
3.2 Preparation of Ultrapure pBAC-ZIKV Infectious Clone
3.3 Rescue of rZIKV
3.4 Titration of Recovered rZIKV by Plaque Assay
3.4.1 Plaque Assay and Crystal Violet Staining
3.4.2 Plaque Assay and Immunostaining
3.5 Confirmation of rZIKV Rescue by IFA
3.6 Amplification of ZIKV and Production of Virus Stocks
4 Notes
References
Chapter 13: A Stable Reverse Genetics System of Zika Virus Based on a Self-Splicing Group II Intron
1 Introduction
2 Materials and Methods
2.1 Isolation of ZIKV RNA from Culture Supernatants
2.2 RT-PCR for ZIKV cDNA Fragments
2.3 Molecular Cloning and Assembly of the pACNR-GZ01-Intron-IC
2.4 In Vitro Transcription
2.5 In Vitro Splicing
2.6 RNA Transfection
2.7 Titration
2.8 vRNA Copy Determination
2.9 Immunofluorescent Staining
3 Methods
3.1 Isolation of ZIKV RNA from Culture Supernatants
3.1.1 Propagation of ZIKV in BHK-21 Cells
3.1.2 ZIKV RNA Isolation from Culture Supernatants
3.2 RT-PCR
3.2.1 Reverse Transcription
3.2.2 PCR Amplification
3.3 Generation of the Infectious Clone Plasmid
3.3.1 Construction of an Assemble Backbone
3.3.2 Sub-cloning of ZIKV cDNA Fragments
3.3.3 Design of the Modified P.li.LSUI2 Intron
3.3.4 Assembly of the Full-Length pACNR-GZ01-Intron-IC
3.4 In Vitro Transcription
3.5 In Vitro Splicing
3.6 RNA Transfection
3.7 Titration
3.8 Mutagenesis Analysis of cis-Acting RNA Elements Based on pACNR-GZ01-Intron-IC
3.9 qRT-PCR
3.10 IFA
4 Notes
References
Chapter 14: Reverse Genetics of Dengue Virus
1 Introduction
2 Materials
2.1 Dengue RNA Extraction
2.2 Amplification and Purification of the DENV Genome
2.3 Vector Preparation
2.4 Yeast Transformation
2.5 Yeast Plasmid Isolation
2.6 Full-Length PCR and In Vitro Transcription
2.7 Transfection into BHK-21 Cells and DENV Rescue
2.8 Indirect Immunofluorescence
3 Methods
3.1 DENV RNA Extraction
3.2 Amplification of Genomic Fragments by RT-PCR
3.3 Purification and Quality Control Check of Amplicons Prior to Cloning
3.4 Vector Preparation
3.5 Assembly of the Full-Length cDNA Clone in a Single-Step Cloning (Fig. 1)
3.6 Yeast Plasmid Isolation (for Cloning Screening)
3.7 Yeast Plasmid Isolation (for Full-Length PCR Template)
3.8 Full-Length PCR
3.9 Purification and Precipitation of the Full Amplicon
3.10 In Vitro Transcription
3.11 Cell Transfection
3.12 Viral Rescue Monitoring by Indirect Immunofluorescence
4 Notes
References
Chapter 15: Recovery of Recombinant Rotaviruses by Reverse Genetics
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Cell Culture Media
2.3 Reverse Genetics Plasmids
2.4 Other Reagents
3 Methods
3.1 Maintaining Cell Lines
3.2 Generation of Recombinant Rotavirus
3.3 Detection of Recombinant Rotavirus RNA by Gel Electrophoresis
3.4 Plaque Isolation of Recombinant Rotaviruses
3.5 Amplification of Plaque Isolated Virus
4 Notes
References
Index