Hepatitis C Methods and Protocols

About this book

With the development of robust cell-culturing systems capable of Hepatitis C Virus replication and infection, the past decade has witnessed an explosion of innovative research directed at understanding HCV biology in the context of the authentic replicative cycle of the virus. By providing a compilation of cutting-edge research techniques currently in use in HCV labs worldwide, Hepatitis C: Methods and Protocols, Second Edition fully updates the previous edition to reflect this progress which promises to unlock the remaining vital information regarding this human pathogen and its interactions. Written in the highly successful Methods in Molecular Biology™ series format, chapters include brief introductions to the topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.

Fully updated and comprehensive, Hepatitis C: Methods and Protocols, Second Edition serves as a valuable reference guide for basic and clinical researchers using HCV as a model and striving to better understand this critical disease.

Written for: Basic and clinical scientists, hepatologists, virologists, infectious disease researchers, immunologists, molecular and cellular biologists

Keywords:

• Genotypes
• HCV research
• Infection
• Quasispecies
• RNA replication

Table of contents

PART I: DETECTION AND QUANTIFICATION OF HCV

1. Detection and Quantification of Serum or Plasma HCV RNA: Mini Review of Commercially Available Assays
Hélène Le Guillou-Guillemette and Françoise Lunel-Fabiani
2. Simultaneous Detection of Anti-HCV Antibody and HCV Core Antigen
Filippo Ansaldi and Giancarlo Icardi
3. Immunohistochemical Detection of HCV Proteins in Liver Tissue
Oivier Galy, Isabelle Vincent, Philippe Chevallier, Nicole Lyandrat, and Isabelle Chemin

PART II: HCV NONMENCLATURE, GENOTYPING, AND QUASISPECIES

4. Nomenclature and Numbering of the Hepatitis C Virus
Carla Kuiken and Peter Simmonds
5. A Real-Time Taqman® Method for Hepatitis C Virus Genotyping and Methods for Further Subtyping of Isolates
Kathryn J. Rolfe, Tim G. Wreghitt, Graeme J.M. Alexander, and Martin D. Curran
6. Characterization of HCV Quasispecies in the Hypervariable Region 1 (HVR1) by In Vitro Translation and Mass Spectrometry
Carmen Yea, Melissa Ayers, and Raymond Tellier

PART III: HCV REPLICATION IN VITRO

7. Purification and Crystallization of NS5A Domain I of Hepatitis C Virus
Joseph Marcotrigiano and Timothy L. Tellinghuisen
8. NS5A Phosphorylation and Hyperphosphorylation
Petra Neddermann
9. Preparation and Handling of Hepatitis C Viral Proteins NS3 and NS5B for Structural Studies
Stefania Di Marco, Edwin Rydberg, Matthew Bottomley, and Andrea Carfi
10. Structural and Functional Analysis of the HCV p7 Protein
Nathalie Saint, Roland Montserret, Christophe Chipot, and François Penin
11. HCV Replicons: Overview and Basic Protocols
Volker Lohmann
12. Reverse Transcription PCR Based Sequence Analysis of Hepatitis C Virus Replicon RNA
Timothy L. Tellinghuisen and Brett D. Lindenbach
13. Studying HCV RNA Synthesis In Vitro with Replication Complexes
Wengang Yang and Mingjun Huang
14. Proteomics Study of the Hepatitis C Virus Replication Complexes
Kyungsoo Chang, Tianyi Wang, and Guangxiang Luo
15. Investigation of the Hepatitis C Virus Replication Complex
Volker Brass, Rainer Gosert, and Darius Moradpour
16. Regulation of Interferon Regulatory Factor-3-dependent Innate Immunity by the HCV NS3/4A Protease
Kui Li
17. Selection and Characterization of Drug Resistant HCV Replicons In Vitro with a Flow Cytometry–Based Assay
Jason M. Robotham, Heather B. Nelson, and Hengli Tang
18. High Throughput Screening of HCV RNA Replication Inhibitors by Means of a Reporter Replicon System
Weidong Hao and Rohit Duggal
19. Inhibition of HCV Replication by Small Interfering RNA
Ratna B. Ray and Tastuo Kanda

PART IV: HCV ENTRY AND INFECTION

20. Cellular Receptors and HCV Entry
Mike Flint and Donna M. Tscherne
21. Studying HCV Cell Entry with HCV Pseudoparticles (HCVpp)
Birke Bartosch and Francois-Loic Cosset
22. Screening of Small-molecule Compounds as Inhibitors of HCV Entry
Jian-Ping Yang, Demin Zhou and Flossie Wong-Staal
23. Isolation of the JFH-1 Strain and Development of an HCV Infection System
Takaji Wakita
24. Measuring HCV Infectivity Produced in Cell Culture and In Vivo
Brett D. Lindenbach
25. Genotype 1a HCV (H77S) Infection System
MinKyung Yi and Stanley M. Lemon
26. Full Length Infectious HCV Chimeras
Thomas Pietschmann
27. Adaptation of the Hepatitis C Virus to Cell Culture
Artur Kaul, Ilka Wörz and Ralf Bartenschlager
28. Primary Human Hepatocyte Culture for HCV Study
Haizhen Zhu, John Elyar, Robin Foss, Alan Hemming, Eric Hall, Edward L. LeCluyse, and Chen Liu
29. In Vivo Study of HCV in Mice with Chimeric Human Livers
Norman M. Kneteman and Christian Toso

PART V: IMMUNITY AND MODELING

30. Determination of HCV-Specific T Cell Activity
Joo Chun Yoon and Barbara Rehermann
31. Analysis of HCV-Specific T Cells By Flow Cytometry
Masaaki Shiina and Barbara Rehermann
32. Detection of Neutralizing Antibodies Using HCV Pseudo-Particles (HCVpp)
Marlène Dreux and François-Loïc Cosset
33. Mathematical Modeling of HCV Infection and Treatment
Harel Dahari, Emi Shudo, Ruy M. Ribeiro, and Alan S. Perelson