Preface
Contents
Contributors
Chapter 1: Overview of Human Cytomegalovirus Pathogenesis
1 Introduction
2 Pathogenesis in Immunocompetent Hosts
2.1 Infectious Mononucleosis
2.2 Viral Role in Vascular Disease
2.3 Possible Viral Role in Oncogenesis
3 Pathogenesis in Immunocompromised Hosts
3.1 Congenital Infection
3.2 Infection of Infants
3.3 Infection of Immunocompromised Hosts
3.3.1 Infection of Transplant Recipients
3.3.2 Infection in AIDS Patients
4 Conclusions
References
Chapter 2: Distinct Properties of Human Cytomegalovirus Strains and the Appropriate Choice of Strains for Particular Studies
1 Introduction
2 Materials
2.1 Plasticware
2.2 Solutions and Culture Media
2.3 Other Chemicals
2.4 Cells
2.5 Equipment
3 Methods
3.1 HCMV Strains
3.1.1 Clinical Isolates
3.1.2 Established Laboratory Strains with Restricted Tropism
3.1.3 Established Laboratory Strains with Extended Tropism
3.1.4 BAC-Cloned Strains
3.1.5 Genetically Repaired BAC-Derived Strains
3.2 Choice of Strains for Particular Projects
3.2.1 Experiments Addressing Genetic Variability Between HCMV Isolates
3.2.2 Experiments Depending on High Infection Multiplicities
3.2.3 Infection of Endothelial Cells, Epithelial Cells, or Professional Antigen Presenting Cells
3.2.4 Genetic Manipulation of HCMV Genomes
3.2.5 Virus-Host Defense Interactions
3.2.6 Cell-Associated Spread
3.3 Conclusions
4 Notes
References
Chapter 3: Using Diploid Human Fibroblasts as a Model System to Culture, Grow, and Study Human Cytomegalovirus Infection
1 Introduction
2 Materials
2.1 Cells and Culture Media
2.2 Additional Solutions
2.3 Plasticware
2.4 Other Necessary Equipment
3 Methods
3.1 Growing Viral Stocks on Fibroblasts
3.2 Titration of Virus Stocks
4 Notes
References
Chapter 4: Using Primary Human Cells to Analyze Human Cytomegalovirus Biology
1 Introduction
2 Materials
2.1 Mononuclear Cell Isolation
2.2 Magnetic Isolation of CD34+ and CD14+ Cells
2.3 Dermal Fibroblast Isolation
2.4 Infection and Differentiation
2.5 Flow Cytometry Validation of Cell Purity and Differentiation Phenotype
2.6 Validation of Infection and Reactivation
2.7 Detection of Naturally Latent Cells
3 Methods
3.1 Mononuclear Cell Isolation
3.1.1 Peripheral Venous Blood Preparation
3.1.2 Apheresis Cone Preparation
3.1.3 Peripheral Blood Mononuclear Cell (PBMC) Isolation
3.2 CD34+ and CD14+ Cell Magnetic Isolation
3.2.1 Manual Magnetic Separation Protocol for CD34+ Cells
3.2.2 Automated Magnetic Separation of CD34+ Cells
3.2.3 Isolation of CD14+ Mononuclear Cells by Manual Magnetic Separation
3.2.4 Automatic Magnetic Separation of CD14+ Mononuclear Cells
3.3 Dermal Fibroblast Isolation
3.4 Differentiation and Infection of Primary Human Cells
3.4.1 CD34+ Differentiation into CD34+-Derived Langerhans DCs
3.4.2 Infection of CD34+ Cells and CD34+-Derived Langerhans DCs
3.4.3 Monocyte Differentiation into Monocyte-Derived DCs and Macrophages
3.4.4 Infection of Monocytes and Monocyte-Derived DCs and Macrophages
3.4.5 Infection of Dermal Fibroblasts
3.5 Flow Cytometry Analysis of Purity and Differentiation Phenotypes
3.5.1 Purity Staining of Separated Cell Populations
3.5.2 Myeloid Differentiation Phenotyping
3.6 Validation of Experimental HCMV Latency and Reactivation from CD34+ and Monocyte Models
3.6.1 DNA Preparation
3.6.2 Droplet PCR of gDNA
3.6.3 qPCR of gDNA
3.6.4 HCMV Infection Transcriptional Profiling
3.6.5 RNA Isolation
3.6.6 RT-PCR
3.6.7 RT-qPCR-SYBR Green
3.6.8 RT-qPCR-Taqman Probe
3.6.9 Reactivation
3.6.10 Validation of Experimental HCMV Fibroblast Infection
3.7 Validation of Natural HCMV Latency and Reactivation
4 Notes
References
Chapter 5: Human Hematopoietic Long-Term Culture (hLTC) for Human Cytomegalovirus Latency and Reactivation
1 Introduction
2 Materials
2.1 Human Hematopoietic Long-Term Culture Components
2.2 Reactivation Components
3 Methods
3.1 Purification of CD34+ Cells from Fresh Cord Blood or Bone Marrow
3.2 Thaw CD34+ Cells for Infection
3.3 Infection of CD34+ Cells
3.4 Irradiation of Stromal Cells (See Note 7)
3.5 Purify Infected CD34+ Cells
3.6 Human CD34+ Cells Long-Term Culture
3.7 Reactivation
4 Notes
References
Chapter 6: Collection and Isolation of CD14+ Primary Human Monocytes Via Dual Density Gradient Centrifugation as a Model Syste...
1 Introduction
2 Materials
2.1 Blood Donation
2.2 Monocyte Isolation
3 Methods
3.1 Donation Preparation and Venipuncture Blood Donation
3.2 Ficoll-Hypaque Density Gradient 1 (See Figs. 1 and 2)
3.3 Percoll Density Gradient 2: (See Note 7)
3.4 Monocyte Collection and Cell Counting
3.5 Conclusion
4 Notes
References
Chapter 7: Stable and Inducible Gene Knockdown in Primary Human Fibroblasts: A Versatile Tool to Study the Role of Human Cytom...
1 Introduction
2 Materials
2.1 Chemicals, Buffers and Solutions
2.2 Cell Culture
2.3 Plasmids
2.4 Oligonucleotides and Primers
2.5 Antibodies
2.6 Ready-to-Use Products
3 Methods
3.1 Stable Knockdown of a Cellular Gene
3.1.1 Design of an shRNA
3.1.2 Cloning
3.1.3 Generation of Transgenic Lentiviral Particles
3.2 Inducible Knockdown of a Cellular Gene
3.2.1 Design of a mir30-Based shRNA
3.2.2 Cloning
3.2.3 Generation of Transgenic Lentiviral Particles
3.3 Transduction
3.4 Validation of a Successful Knockdown of the Target Protein
3.4.1 Western Blot Analysis of Transduced Cells with a Stable shRNA Expression (e.g., pHM3715)
3.4.2 Western Blot Analysis of Transduced Cells with an Inducible shRNA Expression (e.g., pHM4703)
3.4.3 Quantitative Reverse Transcriptase PCR (qRT-PCR, e.g., pHM4703)
4 Notes
References
Chapter 8: Construction of Human Cytomegalovirus Mutants with Markerless BAC Mutagenesis
1 Introduction
2 Materials
2.1 Cloning of HCMV Isolates as Bacterial Artificial Chromosomes (BACs)
2.2 En Passant Mutagenesis of HCMV Genomes
2.3 Transfection of HCMV BACs into Human Cells and Reconstitution of HCMV Infection
3 Methods
3.1 Cloning of HCMV Isolates as Bacterial Artificial Chromosomes (BACs)
3.1.1 Generation of a Recombinant CMV Carrying the BAC Vector Backbone
3.1.2 Enrichment of the CMV Recombinant Carrying the BAC Vector
3.1.3 Transfer of the Circular Virus Genomes to E. coli
3.2 En Passant Mutagenesis of HCMV Genomes
3.2.1 Generation of Insertion Construct for Large Sequence Insertion (Gibson Assembly)
3.2.2 Insertion Construct Amplification
3.2.3 Preparation of Recombination and Electroporation Competent GS1783 Bacteria
3.2.4 Electroporation and First Red Recombination Step
3.2.5 Resolution of Cointegrase
3.3 Transfection of HCMV BAC into Human Cells and HCMV Reconstitution
4 Notes
References
Chapter 9: Methods for Studying the Function of Cytomegalovirus GPCRs
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Assessing PLC-ฮฒ Activity by Measuring IP3 Accumulation
2.3 Cell Lysis
2.4 Western Blotting
2.5 Luciferase Reporter Assays
2.6 BAC Recombineering
2.7 Purification of BAC DNA
2.8 Plasmids
2.9 Miscellaneous
3 Methods
3.1 Measuring Viral GPCR-Stimulated Inositol Triphosphate (IP3) Accumulation
3.2 Packaging of pSLIK-Based Lentiviral Constructs
3.3 Measuring Viral GPCR-Stimulated Protein Kinase Activation
3.4 Measuring vGPCR-Stimulated Transcription Factor Activity
3.5 Generating and Analyzing Recombinant Cytomegaloviruses with Mutant GPCRs
3.5.1 BAC Recombineering of GPCR Genes
3.5.2 Reconstitution of Infectious Virus
3.5.3 Assessment of Viral Growth Properties
3.6 Detecting Viral GPCR Proteins in Infected Cells
3.6.1 Detection and Localization of Viral GPCRs by Immunofluorescence Assay (IFA)
3.6.2 Detection and Localization of Viral GPCRs (and Interacting Partners) by FLAG Immunoprecipitation/Western Blot
3.6.3 Detection and Localization of Viral GPCRs by Fluorescence Activated Cell Sorting (FACS)
3.6.4 Detecting vGPCRs During Latency by RT-qPCR
3.6.5 Detecting vGPCRs During Latency by Immunoblot
3.6.6 Other Potential Methodologies for the Detection and Localization of Viral GPCRs
3.7 Methods for Studying vGPCR Function in Animal Models
3.8 Conclusions and Discussion of Current State of the Art Techniques Useful for Studying vGPCR Signaling/Function
4 Notes
References
Chapter 10: Analysis of Cytomegalovirus Glycoprotein and Cellular Receptor Interactions
1 Introduction
2 Materials
2.1 Purification of Virions
2.2 Purification of Soluble Glycoproteins gB and gH (sgB and sgH)
2.2.1 Isolation of HCMV Bacterial Artificial Chromosome (BAC)
2.2.2 Cloning HCMV sgB and sgH
2.2.3 Generating Stably sgB and sgH Expressing Expi293F Cells
2.2.4 Purifying sgB and sgH
2.3 Detection of Cellular Receptor Activation by Western Blot
2.4 Coimmunoprecipitation of HCMV Glycoproteins and Cellular Receptors
3 Methods
3.1 Purification of HCMV
3.2 Purification of sgB and sgH
3.2.1 Purify HCMV Bacterial Artificial Chromosome (BAC)
3.2.2 Cloning sgB and sgH into pQCXIN Retroviral Plasmid
3.2.3 Generating Retroviral Vectors Containing sgB and sgH
3.2.4 Affinity Chromatography Purification of sgB and sgH
3.3 Analysis of Cellular Receptor Activation During HCMV Entry by Western Blot
3.4 Coimmunoprecipitation HCMV Glycoproteins and Cellular Receptors
4 Notes
References
Chapter 11: Antibody-Independent Quantification of Cytomegalovirus Virion Protein Incorporation Using HiBiT
1 Introduction
2 Materials
2.1 RCMV BAC Recombineering
2.2 Virus Rescue
2.3 RCMV Growth and Purification
2.4 Western Blotting
2.5 HiBiT In-Solution Detection
2.6 RCMV Viral Genome Quantification
3 Methods
3.1 Generation of RCMV Containing R131 or R129(short) HiBiT Fusion Tags
3.1.1 Generate PCR Fragments for Homologous Recombination
3.1.2 First Recombination Step
3.1.3 Second Recombination Step
3.2 Transfection of BAC DNA and Rescue of RCMV-R131 and -R129(short) HiBiT
3.3 RCMV Purification Protocol
3.4 Virion Protein Detection
3.5 Trypsin Sensitivity of Virion-Associated R131 and R129(short) HiBiT
3.6 Quantification of Virion-Associated R131 and R129(short) HiBiT-Tagged Molecules Relative to Viral Genome Copy Number
3.6.1 In-Solution Detection of HiBiT
3.6.2 Quantification of Viral DNA Genomes
3.7 Observations
4 Notes
References
Chapter 12: Using a Phosphoproteomic Screen to Profile Early Changes During HCMV Infection of Human Monocytes
1 Introduction
2 Materials
2.1 HCMV Culture and Infection (See Note 1)
2.2 Isolation of Human Peripheral Blood monocytes (See Note 2)
2.3 Monocyte Infection
2.4 Protein Extraction
2.5 Protein Binding to Antibody Arrays and Array Processing
3 Methods
3.1 HCMV Culture and Infection (the Step Takes About 3 Weeks (See Chapter 3 for Additional Detail)
3.2 Isolation of Human Peripheral Monocytes (Takes 5-6 h). See Note 2 and Chapter 6 for Detailed Instructions
3.3 Monocyte Infection (Takes Less Than 3 h)
3.4 Protein Extraction (~1.5 h): After This Step, the Sample Can Be Stored at -80 C for up to 2 Weeks
3.5 Buffer Exchange and Protein Determination (This Step Takes ~30 min): After This Step, the Sample Can Be Stored at -80 C fo...
3.6 Unpacking, and Blocking the Array Slides Prior to the Protein Binding (the Step Takes About 3 h and May Be Done at the Sam...
3.7 Protein Biotinylation. (The Process Takes about 3 h and Could Be Done at the Same Time as the Array Slides Are Being Equil...
3.8 Binding of Biotinylated Proteins to the Arrays (~4 h)
3.9 Detection of Bound Biotinylated Proteins with Cy3-Streptavidin (~1 h)
3.10 Array Quick Drying (~15 min)
3.11 Raw Data Analysis (See Note 6)
4 Notes
References
Chapter 13: A Generally Applicable CRISPR/Cas9 Screening Technique to Identify Host Genes Required for Virus Infection as Appl...
1 Introduction
2 Materials
2.1 Common Materials
2.2 Chemical Reagents and Supplies
2.3 Significant Equipment
2.4 Deep Sequencing Reagents (Life Technologies)
3 Methods
3.1 Production for Cas9 Lentivirus
3.2 Titering of Cas9 Lentivirus
3.3 Production of ARPE19-Cas9 Stable Cells
3.4 Production for Lentiviral sgRNA-Library A and Lentiviral sgRNA-Library B Virus
3.5 Generation of ARPE19-Cas9-sgRNA Library A and ARPE19-Cas9-sgRNA Library B Stable Cells
3.6 TB40E-GFP Virus Amplification in Fibroblasts
3.7 TB40E-GFP Virus Titer Is Determined in HEL Cells by Plaque Assay
3.8 TB40E-GFP Adaptation to Epithelial Cells (See Note 7)
3.9 Infection of ARPE19-Cas9-sgRNA Library A and ARPE19-Cas9-sgRNA Library B Stable Cells with TB40E-GFP Virus
3.10 Monitor for Cell Death (See Note 11)
3.11 Isolation of Surviving Cells
3.12 Isolation of Genomic DNA Using the DNeasy Blood and Tissue Kit (QIAGEN)
3.13 sgRNA Sequences Are Amplified from Integrated Proviruses
3.14 Barcode and Adapter Was Added to the PCR Fragment (See Note 13)
3.15 Size-Selection of the Libraries Using a Pippin Prep Instrument (See Note 13)
3.16 Deep Sequencing of Samples from Subheading 3.15 step 4
3.17 Data Analysis
4 Notes
References
Chapter 14: Quantitative Electron Microscopy to Study HCMV Morphogenesis
1 Introduction
1.1 Nuclear Stages of HCMV Morphogenesis
1.2 Cytoplasmic Stages of HCMV Morphogenesis
1.3 Optimized Electron Microscopy Sample Preparation and Imaging Techniques
2 Materials
2.1 Cell Culture
2.2 Electron Microscopy Sample Preparation
2.2.1 High Pressure Freezing
2.2.2 Freeze Substitution and Embedding
2.2.3 TEM
2.2.4 STEM Tomography
2.3 Image Acquisition and Analysis
2.3.1 TEM
2.3.2 STEM Tomography
3 Methods
3.1 Cell Culture
3.1.1 Preparation of Sapphire Disks
3.1.2 Cell Culture and Infection
3.2 Electron Microscopy Sample Preparation
3.2.1 High Pressure Freezing
3.2.2 Freeze Substitution and Embedding
3.2.3 Ultrathin Sectioning for TEM
3.2.4 Preparation of STEM Tomography Samples (See Note 27)
3.3 Selection of Infected Cells and Data Acquisition
3.3.1 TEM
3.3.2 STEM Tomography
3.4 Image Analysis/Quantification
3.4.1 TEM
3.4.2 STEM Tomography
3.5 Conclusions
4 Notes
References
Chapter 15: Detection of Cytomegalovirus Interleukin 10 (cmvIL-10) by Enzyme-Linked Immunosorbent Assay (ELISA)
1 Introduction
2 Materials
2.1 Buffers
2.2 Antibodies and Protein Standards
2.3 Other Reagents and Consumables
2.4 Equipment
3 Methods
3.1 Day 1: Coat the Plate
3.2 Day 2: The Assay
4 Notes
References
Chapter 16: Techniques for Characterizing Cytomegalovirus-Encoded miRNAs
1 Introduction
2 Materials
2.1 Expression of miRNAs in Cells
2.1.1 Expressing miRNAs Using an Expression Vector (pSIREN)
2.1.2 Expressing miRNAs Using GFP-Expressing Adenovirus
2.2 Detecting miRNAs
2.2.1 miRNA Northern Blot
Urea-Acrylamide Gel Components
Transfer Components
Probe Labeling Components
Hybridization Components
Detection Components
2.2.2 Stem-Loop RT-PCR for Detection of miRNAs
Reverse Transcription Components
Taqman Assay Components
2.3 Identifying mRNA Targets of Viral miRNAs
2.3.1 Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP)
Immunoprecipitation Components
RNA Extraction and cDNA Library Components
2.3.2 RNA-Induced Silencing Complex (RISC) Immunoprecipitation (IP)
Immunoprecipitation and Pull-Down Components
RNA Isolation Components
2.3.3 Reporter Assays for Identification of Viral miRNA Targets
Transfection Components
Luciferase Assay Components
2.4 Inhibition of HCMV miRNAs Using Locked Nucleic Acids (LNA)
3 Methods
3.1 Expression of miRNAs in Cells
3.1.1 Expressing miRNAs Using a pSIREN Expression Vector
Cloning into pSIREN Vector
Transfection
Nucleofection
3.1.2 Expressing miRNAs Using GFP-Expressing Adenovirus
Cloning into the pAdTrack-CMV Vector
Adenovirus Production
3.1.3 Designing and Expressing miRNA Mimics
Design miRNA Mimics
Transfection of miRNA Mimics
3.2 Detecting miRNAs
3.2.1 miRNA Northern Blot
Urea-Acrylamide Gel Electrophoresis
Electrophoretic Transfer
Prehybridization
Label Probe
Hybridization and Washes
Film Development
3.2.2 Stem-Loop RT-PCR for Detection of miRNAs
Anneal the RT Primer (See Note 35)
Reverse Transcription
Taqman qPCR Assay
3.3 Identifying mRNA Targets of Viral miRNAs
3.3.1 Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP)
Preparing Cell Pellets
Cell Lysis and Preparation of Magnetic Beads
RNase T1 Treatment and Immunoprecipitation
Dephosphorylation, Labeling, and Phosphorylation
SDS-PAGE and Electroelution
RNA Extraction
Generation of cDNA Libraries
cDNA Library purification (See Note 47)
3.3.2 RNA-Induced Silencing Complex (RISC) Immunoprecipitation (IP)
Preparing the Cell Lysate
Preparing the Streptavidin-Agarose, ANTI-c-myc Agarose Conjugate, or Protein A Beads
Immunoprecipitating
Pull-Down
RNA Isolation from Pull-Downs
Analysis
3.3.3 Reporter Assays for Identification of Viral miRNA Targets
Transfection
Luciferase Assay
3.4 Inhibition of HCMV miRNAs Using Locked Nucleic Acids (LNA)
4 Notes
References
Chapter 17: Development of a huBLT Mouse Model to Study HCMV Latency, Reactivation, and Immune Response
1 Introduction
2 Materials
2.1 Tissue Preparation
2.2 Immunodeficient Mouse Supplies
2.3 Generation of huBLT Mice
2.4 Cell Culture
2.5 Collection of Blood Samples to Monitor Human Cell Engraftment
2.6 Necropsy and Sample Collection
2.7 Extraction of DNA/RNA from Tissue
2.8 qPCR for Viral Genomes
2.9 Flow Cytometry
2.10 Immunological Analysis of T-Cells
2.10.1 Production of Cytokines by T-Cells Analyzed by ELISPOT
2.10.2 Production of Cytokines by T-Cells Analyzed by Intracellular Staining
2.10.3 Additional Isolation of T-Cell Subsets
2.10.4 Generation of Autologous LCLs
2.11 HCMV Antibody Analysis by ELISA
3 Methods
3.1 Preparation of Human Fetal Tissue for Transplant and Isolation of Primary CD34+ HPCs
3.2 Generation of huBLT Mice
3.2.1 Preoperative Preparation
3.2.2 Surgical Implantation of Human Tissue into Mice
3.2.3 Postoperative Monitoring
3.2.4 Irradiation and Injection of Autologous CD34+ HPCs
3.3 Monitoring Human Cell Engraftment by Survival Bleed and Flow Cytometry
3.4 Infection of huBLT Mice with HCMV and Reactivation In Vivo
3.4.1 Preparation of HCMV-Infected Human fibroblasts and Infection of huBLT Mice
3.4.2 Reactivation of HCMV In Vivo
3.5 Analysis of HCMV Infection and Immune Responses
3.5.1 Necropsy of HCMV-Infected huBLT Mice
3.5.2 Analysis of Viral DNA in Tissues (DNA Extraction and qPCR)
3.5.3 Flow Cytometry of Human Cell Populations in Lymphoid Organs
3.5.4 Analysis of Cytokines Produced by T-Cells
3.5.5 Analysis of Antibody Production
3.6 Conclusions
4 Notes
References
Chapter 18: Rodent Models of Congenital Cytomegalovirus Infection
1 Introduction
1.1 HCMV and Congenital Infection
1.2 Developmental Abnormalities in the Brains of MCMV-Infected Newborn Mice
1.3 Immune Response in MCMV-Infected Newborn Mice
1.4 Role of Antibodies in the Control of MCMV Infection in Newborn Mice
2 Materials
3 Methods
3.1 Analysis of the MCMV Pathogenesis in Newborn Mice
3.1.1 Production of Cell Culture-Derived Virus and Preparation of Virus Stocks
3.1.2 Infection of Newborn Mice
3.1.3 Determination of Virus Titers in Organs and Virus Stock Solutions Using Standard Plaque-Forming Assay
3.1.4 Extensive Titration
3.1.5 Determination of Virus DNA by Quantitative PCR
3.2 Immunohistochemical Detection of MCMV-Infected Cells and Immune Cells in Tissues
3.2.1 Preparation of Paraffin-Embedded Tissue Sections for IHC
3.2.2 Preparation of Frozen Tissue Sections for IHC
3.3 Assessment of Developmental Abnormalities in Brains of MCMV-Infected Newborn Mice and Consequent Neurological Impairments
3.3.1 Histomorphometrical Analyses of the Brain-Cresyl
Cresyl Violet Staining
Measurement of Cerebellar Area and External Granular Layer Thickness
3.3.2 Balance Beam Test
3.4 Assessment of Inflammatory Response in MCMV-Infected Newborn Brain
3.4.1 Isolation of Mononuclear Cells from Brain and Similar Soft Tissues (e.g., Liver)
3.4.2 Isolation of Mononuclear Cells from Spleen
3.4.3 Isolation of Mononuclear Cells from Blood
3.4.4 Isolation of Mononuclear Cells from Lungs
3.4.5 Labeling of Surface Targets for Flow Cytometry Analysis
3.4.6 Fixation of Cells for Flow Cytometry
Fixation of Cells for Flow cytometry Using Formaldehyde
3.4.7 Permeabilization of Cells and Intracellular Staining for Flow Cytometry
Permeabilization of Cells Using Methanol
Permeabilization of Cells Using Saponin and Staining of Intracellular Targets
3.4.8 Measuring Cytokines in Flow Cytometry
3.4.9 Mononuclear Cell Adoptive Transfer (Via Cell Sorting)
Adoptive Cell Transfer by Cell Sorting
Adoptive Cell Transfer of all Cells Normalized to Target Cell Numbers
3.4.10 Depletion of Immune Cell Subsets in the Brain
3.5 Assessment of Antiviral Antibody-Mediated Protection of MCMV Infection of Developing Brain
3.5.1 Preparation of Hyperimmune Serum
3.5.2 In vivo Administration of Immune Serum or Monoclonal Antibodies to Newborn Mice
4 Notes
References
Chapter 19: Recent Approaches and Strategies in the Generation of Anti-human Cytomegalovirus Vaccines
1 Epidemiology of HCMV infections
1.1 Transmission of HCMV
1.2 Vertical transmission
1.3 Congenital HCMV Infection Following Maternal Nonprimary Infections
2 HCMV-Specific Immunity
2.1 Introduction
2.2 Maternal and Congenital Infections
2.2.1 The Importance of Antiviral Antibodies in the Natural History of cCMV Infections
2.2.2 HCMV Specific T Lymphocyte Responses and cCMV Infections
2.3 Transplant Recipients
2.3.1 Antiviral Antibodies and the Outcome of HCMV Infection in Allograft Recipients
3 Animal Models of Protective Immunity
3.1 Introduction
3.2 Animal Models Using Immunocompetent Animals to Investigate Responses Associated with Control of Virus Replication and Shed...
3.3 Models Utilizing Immune-Compromised Animals to Characterize Immune Responses Responsible for Control of Virus Replication ...
3.4 Animal Models of Congenital HCMV Infection That Include Both Intrauterine Transmission to the Developing Embryo or Infecti...
4 Approaches for Vaccine Induced Protective Immunity
4.1 Introduction
4.2 Generation of Protective Immunity by Induction of Adaptive Immunity to HCMV
4.3 Vaccine Strategies to Overcome Viral Immunoevasion Functions
5 Target Populations for Evaluating HCMV Vaccines
5.1 Women of Childbearing Age
5.2 Adolescents
5.3 Early Childhood
6 Prevention of Infection/Transmission/Disease
7 Roadblocks in the Development of Efficacious HCMV Vaccines
7.1 Introduction
References
Index