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Table of Contents:

 

Part 1. Viral Detection

 

1. Viral Detection 3

2. Quantitative Detection of Epstein-Barr Virus DNA in Clinical Specimens by Rapid Real-Time PCR Targeting a Highly Conserved Region of EBNA-1 15

3. Profiling of Epstein-Barr Virus Latent RNA Expression in Clinical Specimens by Gene-Specific Multiprimed cDNA Synthesis and PCR 27

4. Quantitative Detection of Viral Gene Expression in Populations of Epstein-Barr Virus-Infected Cells in Vivo 39

5. Detection and Quantification of the Rare Latently Infected Cell Undergoing Herpes Simplex Virus Transcriptional Activation in the Nervous System in Vivo 57

6. Reporter Cell Lines for the Detection of Herpes Simplex Viruses 73

 

Part 2. Virus Structure and Imaging

 

1. Unraveling the Architecture of Viruses by High-Resolution Atomic Force Microscopy 85

2. Studying the Structure of Large Viruses with Multiresolution Imaging 109

3. Herpes Simplex Virus-Cell Interactions Studied by Low-Fading Contrasted Immunofluorescence 129

4. Herpes Simplex Virus-Cell Interactions Studied by Immunogold Cryosection Electron Microscopy 143

5. FTIR Microscopy Detection of Cells Infected with Viruses 161

 

Part 3. Virus Entry

 

1. The JC Virus-Like Particle Overlay Assay 175

2. Analysis of Fusion Using a Virus-Free Cell Fusion Assay 187

3. Pseudovirions as Specific Tools for Investigation of Virus Interactions with Cells 197

 

Part 4. Gene Expression

 

1. Simultaneous In Situ Detection of RNA, DNA, and Protein Using Tyramide-Coupled Immunofluorescence 215

2. Identification and Characterization of Herpesviral Immediate-Early Genes 231

 

Part 5. Replication and Genome Maintenance

 

1. Methods for Measuring the Replication and Segregation of Epstein-Barr Virus-Based Plasmids 247

2. DNA Affinity Purification of Epstein-Barr Virus OriP-Binding Proteins 267

 

Part 6. Pathogenesis

 

1. Pre-B-Cell Colony Formation Assay 279

2. Luciferase Real-Time Bioluminescence Imaging for the Study of Viral Pathogenesis 285

 

Part 7. Complex Cell Systems

 

1. Culturing Primary and Transformed Neuronal Cells for Studying Pseudorabies Virus Infection 299

2. Human Papillomavirus Type 31 Life Cycle 317

 

Part 8. Recombinant Genetics

 

1. Molecular Genetics of Herpesviruses 333

2. Molecular Genetics of DNA Viruses 353

3. Genetic Analysis of Cytomegalovirus by Shuttle Mutagenesis 371

4. Construction of a Gene Inactivation Library for Bovine herpesvirs 1 Using Infectious Clone Technology 387

5. Selective Silencing of Viral Gene E6 and E7 Expression in HPV-Positive Human Cervical Carcinoma Cells Using Small Interfering RNAs 401

 

Part 9. Computation/Systems Biology of Viruses

 

1. Design of a Herpes Simplex Virus Type 2 Long Oligonucleotide-Based Microarray 423

2. Real-Time Quantitative PCR Analysis of Viral Transcription 449

3. Rapid Screening of Chemical Inhibitors that Block Processive DNA Synthesis of Herpesviruses 481.

 

Index 493

 

Paul Lieberman of the Wistar Institute at the University of Pennsylvania compiles important methods for manipulating DNA viruses and characterizing their varied biological properties. DNA Viruses: Methods and Protocols describes basic, innovative methodological approaches for the field of DNA virology. Although, not a comprehensive guide, the manual captures the most important techniques for the study of DNA viruses. The scope of this work is limited to DNA virology, but methods can often be generally applied to other biological systems such as RNA viruses and cell biology.

 

DNA Viruses: Methods and Protocols is divided into nine sections including basic techniques for viral detection, computation, and structure/imaging. The manual includes biological techniques to study viral entry, replication, pathogenesis, and cell systems. The manual also outlines genetic, molecular biological techniques for gene expression and recombinant genetics. Methods allow researchers and students to duplicate them in the laboratory. Protocols follow the series format including step-by-step instructions, background on principles, required equipment and reagents, and troubleshooting notes. Each chapter includes supporting bibliographic references.

 

The manual is a useful addition for academic health sciences or science libraries. It is also recommended for microbiologists, virologists, biochemists, and geneticists who need a basic laboratory methods guide to study DNA viruses.